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Introduction {#s1} ============ Copper is a kind of essential trace elements in human beings as well as other species. As a transition trace element, it is a significant component for many enzymes. Besides its essential role involving in iron metabolism ([@B1]), it also participates in a number of biological processes, including oxidative stress ([@B2]), immunological function ([@B3]), and neurotransmitter synthesis ([@B4]). Dietary copper deficiency has been shown to lead to adverse human health outcomes throughout the whole life course ([@B5]). Copper deficiency may result in impaired cardiovascular system development, bone malformation ([@B6]), dyslipidemia ([@B7]), and continuous neurologic abnormalities for both infants and adult ([@B8], [@B9]). Telomere length is involved in biological aging ([@B10]). Telomere length shortening was associated with cardiovascular disease ([@B11]), neurodegenerative disorders ([@B12]--[@B14]), and other metabolic diseases ([@B15]--[@B17]). Studying the determinants of telomere shortening is of paramount importance in disentangling the pathophysiology of major chronic diseases. Previous studies have shown that some nutrients were related to telomere length shortening and maintenance ([@B18]), and found superoxide dismutase could slow telomere length shortening in human fibroblasts ([@B19]) and antioxidant therapy could also attenuate the reduction of telomerase activity in superoxide dismutase-deficient mice ([@B20]). While copper is a major component of superoxide dismutase and diet is one of the main sources for copper, it is thus natural to hypothesize that dietary copper intake could be related to leukocyte telomere length. However, this association of dietary copper intake and leukocyte telomere length has rarely been examined. In this study, we tested this association using a large population based survey data, the National Health and Nutrition Examination Survey (NHANES). Methods {#s2} ======= Study design and participants ----------------------------- The National Center for Health Statistics of the Centers for Disease Control and Prevention has been collecting the NHANES data for decades. Written informed consent was obtained from all study participants. The survey plan and study protocol were approved by the Research Ethics Review Board at the National Center for Health Statistics. The data from NHANES 1999--2000 and 2001--2002 were combined for these analyses because leukocyte telomere length was assessed in these two data collection cycles. The final analytic sample included 7,832 participants who had their 24--h dietary intake data assessed and had telomere length measured. Dietary copper intake --------------------- NHANES survey participants took part in an in-person household interview and underwent a health examination in a mobile examination center, where the participants also provided a 24--h dietary recall data collection session. All study participants were asked to provide data of dietary intake during the 24--h period before the interview date when blood sample was collected. The dietary data collection was performed using the NHANES computer-assisted dietary interview system in the mobile examination center. Based on the study protocol, all participants were randomized to either a morning or an afternoon/evening exam sessions for the data collection. The completed interview data were electronically transferred to the data monitor center. And then data were entered into the University of Texas Food Intake Analysis System and USDA Survey Nutrients Database for coding dietary copper intake values. Telomere length assessment -------------------------- Blood samples were used for leukocyte telomere length assessment. The telomere length assessment experiments were conducted in the laboratory of Prof. Elizabeth Blackburn at the University of California, San Francisco, US. The quantitative polymerase chain reaction (qPCR) method was employed to measure leukocyte telomere length, which was a relative measurement in relation to a standard reference (coded in a T/S ratio scale). The detailed methods used for telomere assessment as described elsewhere previously ([@B21]--[@B23]). In brief, the single-copy gene used for this PCR experiment (human beta-globin) was *hbg1* \[5′GCTTCTGACACAACTGTGTTCACTAGC-3′\] ([@B23]). Blood samples were assayed three times on three different days on duplicate plate wells, generating six data points that were used for the standard curve. Quality control was performed as follows. If the assay had eight or more invalid control wells, then they were considered invalid and were excluded from further analysis (\<1% of experiments failed this criterion). The inter-assay coefficient of variation was 4.4%. The extreme T/S ratio values in the data set were considered as potential outliers. Then the mean of the T/S ratio value was calculated by excluding the potential outliers ([@B24]). Statistical analysis -------------------- We performed all statistical analyses using SAS (version 9.4, SAS Institute Inc, Cary, NC) software. We used sample weights to account for planned oversampling of some groups. We presented continuous variables as mean ± standard deviations, and categorical variables as number and proportions. Dietary copper intake was log-transformed to achieve a normal distribution. Dietary copper intake was used as both a continuous variable and a categorical variable in the regression models. We constructed three linear regression models to assess the association between dietary copper intake and leukocyte telomere length. The first model was a crude estimate of this association, while the second model was further adjusted for age, sex, and ethnicity. The third model was additionally controlled for physical activity, smoking status, hypertension, cardiovascular diseases, and body mass index. We also examined if the association differed between men and women by including an interaction term of sex and dietary copper intake. These models were specified as follows: *Model 1:* $Telomere\ length = \beta_{1}^{*}copper\ intake$*Model 2:* $Telomere\ length = \beta_{1}^{*}copper\ intake + \beta_{2}^{*}Age + \beta_{3}^{*}sex + \beta_{4}^{*}ethnicity$*Model 3:* $Telomere\ length = \beta_{1}^{*}copper\ intake + \beta_{2}^{*}Age + \beta_{3}^{*}sex + \beta_{4}^{*}ethnicity + \beta_{5}^{*}BMI + \beta_{6}^{*}physical\ activity + \beta_{7}^{*}smoking\ status + \beta_{8}^{*}hypertension + \beta_{9}^{*}cardiovascular\ diseases$ Results {#s3} ======= Table [1](#T1){ref-type="table"} describes the basic characteristics of study participants by sex. There are 7,324 participants in the final analytic sample; around 48.0% of them were men. The average age was 50.2 ± 18.1 years for men and 47.9 ± 18.8 years for women. Leukocyte telomere length decreased with increasing age (−0.06 T/S ratio per year increase in age, Figure [1](#F1){ref-type="fig"}), and was longer in women than that in men (1.05 ± 0.26 vs. 1.00 ± 0.26 T/S ratio), while dietary copper intake was less in women that that in men (1.12 ± 0.80 vs. 1.51 ± 1.61 mg). ###### Basic Characteristics of Study Participants. **Variables** **Men (*n* = 3520)** **Women = 3804)** --------------------------------- ---------------------- ------------------- Age (years) 50.2 ± 18.1 47.9 ± 18.8 Weight (kg) 85.0 ± 18.5 74.5 ± 18.9 Height (cm) 174.2 ± 7.9 160.8 ± 7.3 BMI (kg/m2) 27.9 ± 5.4 28.8 ± 6.8 Telomere (T/S ratio) 1.00 ± 0.26 1.05 ± 0.26 Copper Intake (mg) 1.51 ± 1.61 1.12 ± 0.80 Log-transformed copper intake 0.23 ± 0.59 −0.03 ± 0.53 **ETHNICITY, *n*(%)**      Mexican American 845 (24.01) 927 (24.37)      Other Hispanic 175 (4.97) 216 (5.68)      Non-Hispanic White 1819 (51.68) 1896 (49.84)      Non-Hispanic Black 584 (16.59) 645 (16.96)      Other Race 97 (2.76) 120 (3.15) Physical activity, *n*(%) 1222 (34.7) 934 (24.6) Current Smoking, *n*(%) 717 (20.4) 574 (15.1) Hypertension, *n*(%) 999 (28.4) 1157 (30.4) Cardiovascular diseases*, n*(%) 430 (12.2) 306 (8.0) *Mean and standard deviation were presented for continuous variables, number and proportion were presented for categorical variables*. {#F1} Table [2](#T2){ref-type="table"} presents the association between telomere length and dietary copper intake. Compared with participants with the lowest dietary copper intake in the first quantile, telomere length was longer in the fourth quantile of dietary copper intake in the first model (β = 0.05, 95% confidence interval: 0.02, 0.07). After controlling for age, sex, ethnicity, and body mass index in the multiple linear regression models, the association magnitude decreased slightly but remained significant in the third model (β = 0.04, 95% confidence interval: 0.01, 0.06). When dietary copper intake was modeled as a continuous variable, one unit increase of log-transformed dietary copper intake was significantly associated with longer telomere length (β = 0.02, 95% confidence interval: 0.01, 0.04). No significant sex difference was observed for this association. ###### Association of Telomere Length with Copper Intake, β(95% CI). **Copper Intake** **Model 1** **Model 2** **Model 3** ------------------- -------------------- -------------------- -------------------- Quantile 1 reference reference reference Quantile 2 0.01 (−0.02, 0.03) 0.01 (−0.01, 0.03) 0.01 (−0.01, 0.03) Quantile 3 0.03 (0.01, 0.05) 0.02 (0.003, 0.04) 0.02 (0.002, 0.04) Quantile 4 0.05 (0.02, 0.07) 0.04 (0.01, 0.06) 0.03 (0.01, 0.06) Continuous 0.03 (0.02, 0.04) 0.02 (0.01, 0.04) 0.02 (0.01, 0.03) *Model 1 included only the exposure variable, log-transformed copper intake, Model 2 was additionally adjusted for age, sex, and ethnicity. Model 3 was further adjusted for BMI, physical activity, current smoking, hypertension, and cardiovascular disease. The values represent β(95% CI)*. Discussion {#s4} ========== In the present study, we investigated the association of TL with dietary copper intake in a large population-based health and nutrition survey using the NHANES data, and found that dietary copper intake was association with longer telomere length. This reported association remained significant after controlling for various potential covariates including age, sex, ethnicity, but was attenuated only slightly with body mass index adjustment, suggesting that dietary copper might influence telomere through biological pathways beyond obesity. We did not find significant sex differences of these associations. Dietary copper has been reported to be predictive of metabolic related disorders in both observational population studies and animal experiments ([@B25], [@B26]). One study consisting of more than one thousand participants found dietary copper intake was inversely associated with hypertension, blood pressure, glycemic traits, lipid fraction levels, and uric acid ([@B25]). Another study tested the association of dietary copper intake with cognitive ability and found that high dietary intake of copper in conjunction with a diet high in saturated and trans fatty acid may be associated with accelerated cognitive decline in the elderly population ([@B27]). Because telomere length is regarded as the fundamental molecular aspects of biological aging, it is interesting to examine if telomere is correlated to dietary copper intake. However, this association has seldom been investigated. Only a few studies reported a significant association between trace element and telomere length ([@B28], [@B29]), one of them used human hepatocytes and hematoma cell lines and the other used data from a cross-sectional survey. Previous studies have also found that obesity was significantly associated with leukocyte telomere length ([@B30]). Obese people had shorter telomere length compared with their lean peers. Likewise, obesity is one of the most important risk factors for chronic diseases such as cardiovascular and neurodegenerative diseases ([@B31], [@B32]). It is also closely related to dietary nutrients intake ([@B33]). Thus, we hypothesized obesity could confound the association of leukocyte telomere length and dietary copper intake. In this study, we tested this hypothesis and found the results did not change very much. When additionally adjusting for body mass index, the magnitudes of these associations were attenuated only slightly. This means the observed telomere length and dietary copper intake relationship was independent of obesity. In the present study, we found women had longer telomere length than men. This finding is in line with previous studies ([@B34]). The relationship between copper and telomere are not quite clear. Several potential biological mechanisms might explain the observed association. Copper is known to be an essential nutrient and functions as a key element for enzymes. One of the mostly studies enzyme related to copper is superoxide dismutase ([@B35]), which helps cells to break down potentially harmful reactive oxygen ([@B36]) that could accelerate telomere shortening ([@B37]). Besides that, pharmacological level of copper has been found to be able to induce the immune and antioxidant mechanisms *in vivo* ([@B38]). Enhanced immunological function could also slow down telomere shortening induced by infection ([@B39]). Several strengths deserve to be noted for the present study. First, telomere length was measured in a lab using well-established methods. Second, the study samples were very large and participants were representative of the national US population from which study participants were randomly invited to NHANES. The large sample size was also representative of the US population with a broad age interval. These results could thus be generalized. Lastly, statistical regression models took into account of multiple potential confounders including age, sex, and body mass index. The results showed that the association between leukocyte telomere lengths with dietary copper intake was independent of these potential confounders. However, several limitations should also be acknowledged. This study used a national survey that was cross-sectional by design. The nature of this study design makes causal inference based on our analyses difficult. The association between dietary copper intake and telomere length does not imply increasing dietary copper could increase telomere length. Residual confounding could still bias this association and should be considered by other study design or advanced statistical analyses. Additionally, the present study did not include genetic variants or telomerase activity assessment. No information was available regarding the cell types which might affect telomere length assessment. The circulating copper was also unavailable from NHANES. Examining these genetic biomarkers, cell types, and blood or urine copper could probably provide more valuable evidence to assess the present results. In summary, this study demonstrates that dietary copper intake significantly correlates with telomere length in participants of NHANES. Though our findings are derived in a cross-sectional survey, the discovery of dietary copper intake as a prognostic determinant of telomere length, independent of known obesity, may enhance our understanding of disease diagnosis or prognosis and shed light on the disease mechanisms underlying the association between biological aging and other metabolic disorders. Ethics statement {#s5} ================ This study was carried out in accordance with the recommendations of NHANES committee with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the NHANES committee. Author contributions {#s6} ==================== YW and ZL designed the study. All authors drafted and revised 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. **Funding.** The study was supported by the Foundation of Tianjin Health and Family Planning Commission (14KG101). [^1]: Edited by: Yiqiang Zhan, Karolinska Institutet (KI), Sweden [^2]: Reviewed by: Sofia Pavanello, Università degli Studi di Padova, Italy; Qi Wang, Huazhong University of Science and Technology, China [^3]: This article was submitted to Endocrinology of Aging, a section of the journal Frontiers in Endocrinology

###### Strengths and limitations of this study - This study is the largest cohort study based on nationwide follow-up data including all Korean public officers. - We have included a comprehensive set of various health problems potentially related to job as well as cardiovascular disorders. - A limitation of the study is that incidences of some diseases could have been underestimated or overestimated because our study used claims data. Introduction {#s1} ============ The broad category of 'public officers' comprises individuals in a range of government-overseen positions and public sector employment. As a percentage of total employment across the Organisation for Economic Co-operation and Development countries, the employment rate for public officers rose slightly between 2009 and 2013, from 21.1% to 21.3%.[@R1] In Korea and in many developed countries, positions at public offices are regarded as prestigious occupations, although uniquely stressful.[@R2] The work of public officers is fundamental to the maintenance of society, and it is therefore important to understand any afflictions or ailments associated with this category of individuals. The work of public officers involves repeated and long-term exposure to heavy workloads, high job strain or workplace violence, all of which have a potentially negative impact on physical and mental health. This is particularly true for police officers and firefighters who in addition to workplace stress also deal with physical, chemical, biological and psychological hazards while on duty.[@R4] For example, in the USA in particular, studies have shown police officers to have a shorter life expectancy than the general population.[@R9] For the reasons above, research shows that firefighters and police officers have higher mortality and morbidity rates compared with the general population, particularly for cardiovascular diseases (CVD). The incidence of cardiovascular diseases has been reported to be higher among police officers than in the general civilian population (31.4% vs 18.4%).[@R10] Similarly, a study involving American firefighters found that CVD was the primary cause of death in the line of duty, accounting for approximately 45% of the on-duty fatalities.[@R11] Considerable research on the health of public officers has been conducted in many Western countries, focusing mainly on cardiovascular diseases, chronic diseases and mental disorders. However, comparable research in Asian countries is scarce. Using a large, nationwide dataset based on insurance claims, this study aimed to evaluate the differences in the incidence rates of specific diseases among different categories of public officers in Korea, in order to develop an understanding of health risks associated with these occupations. Subjects and methods {#s2} ==================== Data source {#s2a} ----------- The study population consisted of public officers, including police officers, firefighters, public educational officers (PEOs), and national and regional government officers (NRGs), with claims data between 2002 and 2014, which were collected from the National Health Insurance Service (NHIS) using their customised database service. The Korean National Health Insurance programme covers almost 100% of the Korean population and the database contains information on demographic characteristics, hospital admissions, ambulatory care, principal diagnosis, comorbidities (using the International Classification of Disease's, 10th revision (ICD-10)), procedures and prescriptions regarding all inpatients and outpatients. The date of death was ascertained from death certificates collected from the Korean National Statistical Office. Patients who were alive on 31 December 2014 or after were not considered deceased for the purpose of this study. Study subjects and identification {#s2b} --------------------------------- We identified 860 221 public officers as the study population based on occupation codes. We then collected all patient claims data between 2002 and 2014, and dates of death, through the NHIS customised database service. We defined each disease based on its ICD-10 and procedure codes. We identified patients with alcoholic liver disease (ICD-10 K70), peptic ulcer (K25--K28), dyslipidaemia (E78 with prescription), diabetes mellitus (DM) (E10--E14 with prescription), type 2 DM (E11), hypertension (I10--I15 or I30 with prescription), angina pectoris (I20), acute myocardial infarction (I21), cerebrovascular diseases (I63), admission due to injury (S00-T98 and document code of 'hospital admission'), lower back pain (M543--M545 and document code of 'hospital admission'), lumbar disc herniation (M51), soft tissue diseases in shoulder region (M75), mental illness (F00--F99), mood disorders (F30--F39), sleep disorders (G47 or F51) and traumatic stress disorders (F43.0--F43.1). In order to designate new cases of diseases (incidence), we used a 1-year washout period between 1 January 2002 and 31 December 2002. Statistical analysis {#s2c} -------------------- The demographic characteristics of the study subjects were expressed as means and SD for continuous variables, or as percentages for categorical variables. Age-standardised rates (ASRs) were calculated by the direct standardisation method, using the person-years of NRG officers as the standard population. We calculated person-years as the time after the 1-year washout period, 1 January 2003 to the end of observation or death. In order to calculate person-years by age group (10-year intervals), we divided each individual's person-years by age group and then summed up all person-years for each respective age group. We calculated HRs using the Cox proportional hazards regression models with adjustments for age and sex. All analyses were performed using the SAS Enterprise Guide V.9.4 (SAS Institute, Cary, NC, USA). The results were considered statistically significant when the P value was less than 0.05. Results {#s3} ======= Study population {#s3a} ---------------- A total of 860 221 public officers were included in this study and were followed up for a total of 10 017 374 person-years. The overall mean length of follow-up was 11.6 years, and the mean age was 39.55±9.06 years. The total proportion of male to female public officers was 63.7% to 36.3%, respectively. The proportions of different public officers were as follows: police officers, 10.8% (1073 302 person-years); firefighters, 2.7% (272 189 person-years); PEOs, 39.4% (3973 058 person-years); and NRG officers, 47.1% (4698 825 person-years) ([table 1](#T1){ref-type="table"}). ###### Characteristics of public officers Characteristics All officers Person-year ------------------------------------------- ---------------- ------------- Total, n (%) 860 221 10 017 374  Men 547 808 (63.7) 6315 940  Women 312 413 (36.3) 3701 434 Age, mean±SD, years 39.55±9.06 10 017 374 Type of public officers, n (%)  Police officer 92 545 (10.8) 1073 302  Firefighter 23 356 (2.7) 272 189  Public educational officer 338 857 (39.4) 3973 058  National and regional government officer 405 463 (47.1) 4698 825 Incidence rate {#s3b} -------------- ASRs broken down by sex and public officer type are shown in [table 2](#T2){ref-type="table"}. Among men with the alcoholic liver disease, NRG officers showed the highest ASR for incidence with 1180.0 cases per 100 000 person-years, followed by police officers (1177.1), PEOs (1060.1) and firefighters (857.8). Among women with the alcoholic liver disease, firefighters showed the highest ASR with 164.9 cases per 100 000 person-years. Among men with peptic ulcers, the highest ASR was for PEOs (5245.8), followed by police officers (5166.8); among women with peptic ulcers, firefighters showed the highest ASR (4852.4), followed by NRG officers (4847.5). ###### Age-standardised cause-specific incidence rate by public officers Men (per 100 000 person-years) Women (per 100 000 person-years) ------------------------------------------- -------------------------------- ---------------------------------- -------- -------- -------- -------- -------- -------- -------- -------- Alcoholic liver disease 1177.1 857.8 1060.1 1180.8 −3.7 161.5 164.9 141.7 163.9 −2.4 Peptic ulcer 5166.8 4869.6 5245.8 5090.6 76.2 4598.4 4852.4 4804.4 4847.5 −249.1 Dyslipidaemia 2673.1 1955.5 2207.8 2358.9 314.2 1115.1 1087.6 995.3 1030.1 85.0 Diabetes mellitus 915.2 699.4 821.2 942.0 −26.8 237.3 254.9 171.0 233.9 3.4 Type 2 diabetes mellitus 918.5 703.0 822.5 943.2 −24.7 238.1 254.9 170.2 233.1 5.0 Hypertension 2329.6 1924.2 2401.6 2457.8 −128.2 875.0 742.0 753.8 846.8 28.2 Angina pectoris 1648.3 1186.5 1251.5 1256.7 391.6 673.5 668.5 549.1 557.0 116.5 Acute myocardial infarction 342.2 216.8 200.6 208.1 134.1 87.6 98.9 63.8 70.7 16.9 Cerebrovascular diseases 530.5 431.4 477.8 496.7 33.8 239.3 209.5 184.3 213.1 26.2 Admission due to injury 1714.2 1854.2 1322.7 1323.3 390.9 1008.9 1508.6 743.8 854.1 154.8 Lower back pain 377.3 383.4 248.4 249.4 127.9 322.6 446.6 215.7 250.0 72.6 Lumbar disc herniation 2358.1 2552.2 2208.6 2111.6 246.5 2113.3 2662.5 1970.3 1986.8 126.5 Soft tissue diseases in shoulder region\* 3061.6 2987.1 2928.5 2724.8 336.8 2250.8 2761.2 2201.7 2263.3 −12.5 Mental illness 3569.1 3543.4 3627.4 3571.8 −2.7 3648.5 3860.0 3883.7 3828.7 −180.2 Mood disorder 1273.3 1339.2 1215.6 1262.0 11.3 1378.6 1618.9 1375.2 1390.8 −12.2 Sleep disorder 1362.5 1328.0 1285.8 1350.0 12.5 1312.1 1540.7 1275.3 1319.5 −7.4 Traumatic stress disorder 95.1 132.6 98.6 98.5 −3.4 141.6 244.3 169.6 140.0 1.6 \*Shoulder disease including adhesive capsulitis of shoulder, rotator cuff syndrome, bicipital tendinitis, calcific tendinitis of shoulder, impingement syndrome of shoulder, bursitis of shoulder and so on. IRD, incidence rate difference between police and national or regional government officer; NRG, national and regional government officer; PEO, public educational officer. The highest incidence of dyslipidaemia in both sexes was found among police officers (men: 2673.1, women: 1115.1), while the lowest incidence was seen in firefighters among men (1955.5) and PEOs among women (995.3). Among men, the incidence rates for DM and type 2 DM were highest among NRGs (942.0 and 943.2, respectively), followed by police officers. Among women, DM and type 2 DM rates were highest among firefighters, followed by police officers. Hypertension rates in men were highest among NRGs (2457.8) and lowest among firefighters (1924.2), while in women they were highest among police officers (875.0) and lowest among firefighters (742.0). For both sexes, angina pectoris and cerebrovascular diseases were highest among police officers. Acute myocardial infarction in men was highest among police officers, followed by firefighters, while in women the highest rates were observed among firefighters, followed by police officers. Admission due to injury, lower back pain and lumbar disc herniation were highest among firefighters for both sexes, followed by police officers (both sexes). For both sexes, PEOs had the highest rate of mental illness. Finally, for both sexes, firefighters and PEOs had the highest rates of traumatic stress disorders, while firefighters and police officers had the highest rates of mood and sleep disorders. Differences in HRs for incident diseases by public officer type {#s3c} --------------------------------------------------------------- To investigate the difference in HRs for each incident disease by the type of public officer, we conducted a survival analysis using a Cox proportional model adjusted for age and sex. The results of this analysis are shown in [table 3](#T3){ref-type="table"}. ###### Differences in HRs for incident diseases by public officer type HR (95% CI) ----------------------------------------- ------------- --------------------- --------------------- --------------------- Alcoholic liver disease 1(ref.) 1.10 (1.07 to 1.12) 0.80 (0.76 to 0.83) 0.82 (0.81 to 0.83) Peptic ulcer 1(ref.) 1.19 (1.18 to 1.20) 1.13 (1.11 to 1.15) 0.97 (0.96 to 0.98) Dyslipidaemia 1(ref.) 1.25 (1.23 to 1.27) 0.89 (0.86 to 0.92) 0.89 (0.88 to 0.90) Diabetes mellitus 1(ref.) 1.05 (1.02 to 1.08) 0.73 (0.69 to 0.77) 0.77 (0.75 to 0.78) Type 2 diabetes mellitus 1(ref.) 1.05 (1.03 to 1.07) 0.85 (0.82 to 0.88) 0.85 (0.85 to 0.86) Hypertension 1(ref.) 1.05 (1.03 to 1.07) 0.85 (0.82 to 0.88) 0.85 (0.85 to 0.86) Angina pectoris 1(ref.) 1.52 (1.49 to 1.54) 1.06 (1.02 to 1.10) 0.93 (0.92 to 0.94) Acute myocardial infarction 1(ref.) 1.84 (1.77 to 1.92) 1.21 (1.10 to 1.32) 0.89 (0.86 to 0.92) Cerebrovascular diseases 1(ref.) 1.36 (1.31 to 1.40) 0.97 (0.90 to 1.04) 0.87 (0.85 to 0.89) Admission due to injury 1(ref.) 1.41 (1.39 to 1.43) 1.58 (1.53 to 1.63) 0.95 (0.93 to 0.96) Lower back pain 1(ref.) 1.47 (1.41 to 1.52) 1.52 (1.43 to 1.63) 0.96 (0.93 to 0.99) Lumbar disc herniation 1(ref.) 1.20 (1.18 to 1.22) 1.43 (1.39 to 1.46) 1.00 (0.99 to 1.01) Soft tissue diseases in shoulder region 1(ref.) 1.20 (1.18 to 1.21) 1.26 (1.24 to 1.29) 1.00 (0.99 to 1.01) Mental illness 1(ref.) 1.07 (1.06 to 1.09) 1.11 (1.08 to 1.13) 0.98 (0.98 to 0.99) Mood disorder 1(ref.) 1.03 (1.01 to 1.05) 1.12 (1.08 to 1.16) 0.96 (0.95 to 0.97) Sleep disorder 1(ref.) 1.06 (1.04 to 1.08) 1.04 (1.01 to 1.08) 0.94 (0.92 to 0.95) Traumatic stress disorder 1(ref.) 1.00 (0.93 to 1.07) 1.40 (1.26 to 1.56) 1.11 (1.07 to 1.15) All models adjusted for age and sex. NRG, national and regional government officer; PEO, public educational officer; ref, reference. The following results were statistically significant: police officers (HR: 1.25, 95% CI 1.23 to 1.27) had a greater risk of dyslipidaemia compared with NRGs; police officers (HR: 1.19, 95% CI 1.18 to 1.20) and firefighters (HR: 1.13, 95% CI 1.11 to 1.15) had a greater risk of peptic ulcer compared with NRGs, while PEOs (HR: 0.97, 95% CI 0.96 to 0.98) had a lower risk. Compared with NRGs, police officers and firefighters had a greater risk of angina pectoris and acute myocardial infarction (police officer HR: 1.52, 95% CI 1.49 to 1.54; HR: 1.84, 95% CI 1.77 to 1.92 and firefighter HR: 1.06, 95% CI 1.02 to 1.10; HR: 1.21, 95% CI 1.10 to 1.32). For cerebrovascular diseases, police officers had a higher HR compared with NRGs, while firefighters and PEOs had lower HRs relative to NRGs. Both firefighters and police officers had a greater risk of admission due to injury, lower back pain, lumbar disc herniation and soft disease in the shoulder region relative to NRGs, with firefighters having the highest HRs for all these conditions. Finally, both firefighters and police officers had a higher risk of mental illnesses, mood disorders and sleep disorders compared with NRGs, while PEOs had a lower risk of incidence. Discussion {#s4} ========== This is the first Korean population-based analysis of disease incidence among public officers using nationwide data and is based on one of the largest cohorts used for this type of study to date. Overall, we found that police officers and firefighters had higher incidences of a range of diseases compared with NRGs. Police officers also had higher HRs for all measured diseases, except for traumatic stress disorders, when compared with NRGs. Similarly, when compared with NRGs, firefighters had also higher HRs for peptic ulcer, angina pectoris, acute myocardial infarction, admissions due to injury, lower back pain, lumbar disc herniation, soft tissue diseases involving the shoulder region, mental illness, mood disorders, sleep disorders and traumatic stress disorders. Finally, the PEOs had a higher HR for traumatic stress disorders when compared with NRGs. The most prominent HRs in this study were observed among police officers with regard to angina pectoris, acute myocardial infarction and cerebrovascular diseases. Among men, police officers had the highest incidences of all the three diseases. While women police officers had the highest incidences of angina pectoris and cerebrovascular disease, they had the second highest incidence of acute myocardial infarction. Even after adjusting for age and sex, police officers continued to have higher incidences of cerebrocardiovascular ailments with significantly high HRs for all conditions compared with NRGs. A number of Western studies have found that police officers had several risk factors for CVD, including personal factors such as smoking, alcohol consumption, hypertension, obesity, DM or dyslipidaemia, and work-related factors such as night duties, high job stress, workplace violence and long work shifts.[@R12] Our research shows that the higher HRs among police officers compared with NRGs for other diseases such as alcoholic liver disease, DM and dyslipidaemia were also associated with these risk factors suggesting that these are serious risk factors. We did not collect socioeconomic or demographic variables, except for age and sex, and therefore, were unable to ascertain the presence or absence of these risk factors among Korean public officers. In view of the high incidence of cerebrocardiovascular diseases among police officers, it is important to establish preventative measures to reduce their risk for these diseases. We assumed that the distribution of socioeconomic variables such as education level, income or regional characteristics would also be comparable among public officers due to regulations on the working conditions and employment packages for these public officers. Therefore, the higher HR for CVD among police officers may be associated with working conditions, such as long working hours, night work and poor sleep or job-related stress. Several studies have suggested a correlation between shift work and cardiovascular disease or other negative health outcomes.[@R16] A meta-analysis found that longer working hours were associated with cerebrocardiovascular diseases.[@R18] A study involving police officers in Buffalo, New York, found that 28% of police officers worked afternoon shifts and 22% worked night shifts. The study also found that 54% of all police officers suffered from poor sleep quality: 44% for day shifts, 60% for afternoon shifts and 69% for night shifts.[@R19] Furthermore, police officers deal with a variety of civil complaints, increasing their exposure to violent situations which increase the job-related stress. Shift work, night shifts and higher workloads are more prevalent among both police officers and firefighters than among NRGs and education officers. This is consistent with our findings of higher CVD HRs in police officers and firefighters. This study also found higher HRs for lower back pain, lumbar disc herniation and soft tissue diseases in the shoulder region among the firefighters compared with NRGs. These findings coincide with previous research which found that lower back pain was the most common work-related musculoskeletal disorder among firefighters in Korea.[@R20] Additionally, one study suggested that a primary contributing risk factor for lower back pain in firefighters was stress.[@R21] A common hypothesis regarding the association between stress and injury is that the severity of muscle strain, and therefore, the likelihood of injury, increases with stress which in turn can further heighten the awareness of the musculoskeletal symptoms or hamper their management.[@R22] Firefighters are required to move heavy equipment and engage in demanding physical activity as part of their work. This, combined with higher levels of stress, may explain the high levels of back pain among firefighters. Additionally, firefighters are often not fit enough to deal with the physical demands of emergency situations. Some studies have suggested that physical check-up programmes similar to those used for athletes may be necessary to achieve a higher VO~2~ max and to improve the overall health.[@R24] Furthermore, our research found that compared with NRGs, police officers had also higher HRs for lower back pain, lumbar disc herniation and soft tissue diseases of the shoulder. Police officers and firefighters were both found to have significantly high HRs for hospital admissions due to injury, with firefighters having the highest HRs compared with NRGs. This coincides with the findings of a study of American emergency responders which found high rates of injury in both firefighters (7.4 cases per 100 full-time equivalent firefighters) and police officers (8.5 cases per 100 full-time equivalent police officers).[@R28] As previously observed, police officers and firefighters are exposed to a variety of environmental, physical and chemical hazards leading to relatively higher rates of injury.[@R4] Particularly for firefighters, chemicals hazards including the inhalation of fire smoke, asbestos, diesel exhaust and other chemicals are of concern.[@R8] Police work and firefighting are generally regarded as high-risk and high-stress occupations. Firefighters and police officers spend significant time working outside their workplaces and managing unpredictable and urgent situations. These factors contribute to the high-stress work environment reported by a number of studies.[@R29] It is generally accepted that high levels of stress affect all areas of health and that stress can exacerbate pre-existing ailments. An American study found that stress was a potential factor for negative health outcomes among police officers.[@R9] Additionally, several studies have noted that factors, such as a lack of sleep, job insecurity, workplace conflicts, physical environment, levels of alcohol consumption and organisation systems, contributed to the stress of firefighters.[@R30] Other studies have suggested that the inherent risk associated with the job, high workloads, shift work and the police administrative system contributes to the stress experienced by police officers. This is consistent with our finding of high HRs for a considerable number of diseases among police officers and firefighters.[@R9] Both police officers and firefighters were found to have similar high HRs for cerebrocardiovascular and musculoskeletal diseases, as well as for mental, mood and sleep disorders. However, police officers, unlike firefighters, did not have a significantly higher HR for traumatic stress disorders compared with NRGs. This could be due to the organisational culture of police work in Korea, and more research is required in this area. In Korea, candidates for jobs as police officers need to pass a very competitive official examination and mental health check-up, including a clinical psychology test. Once selected, the police officers may feel pressured to maintain their psychological health to maintain their careers and for promotions within the workplace, which can lead to under-reporting of psychological diseases, such as traumatic stress disorders. An anonymous study found that the prevalence of stress-related psychological symptoms, particularly post-traumatic stress disorder (PTSD), remains high among police officers in Korea. According to this study, 41.1% of a study population of 3000 South Korean police officers were at high risk for developing PTSD.[@R7] Finally, another interesting finding of our study was the higher HR for traumatic stress disorders among the PEOs compared with NRGs. For all other diseases measured in our study, the HRs in PEOs were lower than the reference or were not significant. PEOs usually are required to care for students and their parents, and the education culture in Korea is competitive, with suicide among adolescents representing a significant social problem. Therefore, the emotional demands could be severe or distressing for the PEOs, and could potentially produce traumatic effects. There were a number of limitations in our study. First, because our study used only ICD-10 codes and procedure codes to identify each disease, incidences could be overestimated. However, incidences of some diseases such as mental disorders could also be underestimated due to under-reporting in response to workplace culture. For example, some police officers may feel reluctant to receive treatments for a variety of reasons, including fear of losing eligibility for promotions or fear of losing their jobs. Another limitation was that the public officers were divided into four categories and compared. It is likely that there are additional subgroups within each category which may show different rates of incidence. For example, while we compared firefighters and NRGs, we did not compare different types of firefighting work with each other. Likewise, the category 'police officer' includes very different subfields of police work each with different sets of risk factors. For instance, the most common health risk for the majority of police officers is low physical activity due to the sedentary nature of their work, yet this is not true for the special forces.[@R33] Additionally, this study did not account for the regional differences, which may also influence the risk of death or injury.[@R35] Lastly, a final limitation of our study could be its inability to account for a 'healthy worker effect'. Compared with other public officers, firefighters and police officers have to meet more stringent health-related selection criteria due to the physical demands of their job. Therefore, police officers and firefighters may, depending on their subfield of work, be in better general health than the average population, including other public officers. Similarly, comparing firefighters and police officers with other public officers may lead to an underappreciation of the severity of risk factors that they encounter. We recommend more research on the specific characteristics, risk factors and incidences of diseases for specific subgroups within each field. Supplementary Material ====================== ###### Reviewer comments ###### Author\'s manuscript **Contributors:** IK and SH planned the study. MH and SP analysed the data. JHP abstracted the variables and established the data set. MH drafted the article. All authors interpreted the results, critically revised the article and approved the final version. **Funding:** This study was supported by a grant from the Korean national police agency. **Competing interests:** None declared. **Patient consent:** Not required. **Ethics approval:** Ethics approval for this study was obtained from Institutional Review Board (IRB) of Hanyang University (IRB no: HYI-15-213-4). **Provenance and peer review:** Not commissioned; externally peer reviewed. **Data sharing statement:** Extra data are available by emailing Inah Kim.

Competing interest statement ============================ The authors declare no conflict of interests. ***D**iabetes mellitus* is a chronic degenerative endocrine disease that affects millions of people. There are many complications associated with diabetes, such as cardiovascular diseases, peripheral neuropathy, retinopathy, chronic renal failure and impaired mental health.^[@ref1]^ Diabetes is also associated with reduced muscle strength, poor muscle quality, and accelerated loss of muscle mass.^[@ref4]^ Indeed, diabetes mellitus and insulin resistance increase the likelihood of accelerating the aging process and development of frailty syndrome.^[@ref4],\ [@ref7]^ This process may contribute to increase the risk of falls, institutionalization and disability.^[@ref11]^ Recently, it has been reported there is a greater decline in muscle strength in elderly patients with Type 2 diabetes when compared with normoglycemic controls.^[@ref6],[@ref12]^ In addition, Leenders et al.^[@ref5]^ reported that aging patients with Type 2 diabetes presented a greater decline in functional capacity along with the lower-body muscle mass and strength when compared with normoglycemic participants. Thus, besides the metabolic control, effective strategies are needed to prevent the exacerbated loss of strength and functional capacity in aging diabetic patients. Moreover, functional capacity preservation should be especially addressed in aging diabetic patients, because unlike other chronic conditions, diabetes care is dependent on the patients' ability to perform self-care tasks.^[@ref11]^ Exercise programs including resistance training are one of the cornerstones of diabetes management, together with pharmacological and dietary interventions.^[@ref13]^ It has been widely shown that resistance training programs, especially those with low-load high-velocity muscle actions during the concentric phase, are effective interventions to improve muscle strength, power output, rate of force development and functional capacity in elderly participants.^[@ref14]^ In the study Bottaro et al.^[@ref20]^ reported greater increases in functional performance were observed after resistance training performed with explosive muscle actions. In fact, studies have shown that muscle power seems to be a more important predictor of functional performance in elderly adults than muscle strength alone.^[@ref21],[@ref22]^ Despite the effectiveness of low-load high-velocity resistance training on strength performance and functional capacity in the elderly, its effects upon patients with type 2 diabetes remains to be elucidated. Furthermore, although the positive effects of resistance exercises on glycemic control and others disorders associated with diabetes have been often investigated,^[@ref23],[@ref24]^ the effects of resistance training on neuromuscular parameters, such as power output and rate of force development (RFD) and rate of velocity development (RVD) are poorly investigated in participants with diabetic's patients. Therefore, the aim of this study was investigate the effects of low-load high-velocity resistance exercises on neuromuscular and functional outcomes in patients with Type 2 diabetes during the early-phase of resistance training. Our hypothesis was that significant strength and functional capacity gains would be observed in the diabetic patients, even after short period of training (i.e., 6 weeks of training). Material and Methods {#sec1-1} ==================== This study was approved by the University Institutional Review Board (035/11). Participants {#sec2-1} ------------ Thirty inactive Type 2 diabetic participants (diagnosed disease for at least ten years) volunteered to participate in the study. The diabetics were selected at random from responders to advertisements placed in health clinics, hospital, public officers, and by word-of-mouth. The volunteers were randomly assigned to either a low-load high-velocity resistance exercises (LLHV) or recreational activities (RA) group. Due to ethical reasons, the Institutional Review Board suggested that all participants should have some type of exercise intervention. Instead a non-exercise control group, to avoid any major treats of internal validity, we chose to have a recreational physical activities group. The recreational activity was performed with low intensity to avoid any kind of overload that could induce any supercompensation in strength or power. Volunteers were excluded if they were enrolled in another exercise program and those who had less than 85% attendance in the present study. Therefore, 15 participants in the LLHV and 15 in the RA group successfully concluded the study. All participants were required to, with the exception of their T2D, be apparently healthy, have their T2D under control, and to have been given medical clearance to undertake physical activity and exercise. Additionally, participants were all sedentary and did not perform any kind of physical activity more than once a week for the past six months. A clinical examination was conducted by a physician in all participants to check if they were able to participate in the study. Before signing an informed consent, details about the study were explained to the participants, which included a description of the associated risks and benefits of participation. The study was conducted according to Declaration of Helsinki and was approved by an Institutional Review Board. Procedures {#sec2-2} ---------- ### Lower-body strength assessment {#sec3-1} The right knee extension peak torque (PT) was measured on the Biodex system 3 Isokinetic Dynamometer (Biodex Medical, Inc., Shirley, NY). The calibration of the dynamometer was performed according to the manufacturer\'s specifications before every testing session. Participants sat upright with the axis of rotation of the dynamometer oriented with the lateral femoral condyle of the right knee. Belts were used to secure the thigh, pelvis, and trunk to the dynamometer chair to prevent additional body movement. The chair and dynamometer settings were recorded to ensure the same positioning for all tests. The flexor torque produced by the relaxed segment was used for gravity correction. Three sets of four knee extension isokinetic concentric contraction at 60°/s and 1807s. Knee extension range of motion was performed between 10° and 90°, with full extension acting as the reference point. Participants were instructed to fully extend and flex the knee as strong and fast as possible during each set of exercises. In order to assess the rate of force development, three 4-s isometric contractions at 60° of knee extension were performed (0° is the full extension). The participants were instructed to fully extend the knee as strong and fast as possible during each set of exercises and the rest intervals among each isometric and dynamic contractions were 60 s, based on the recommendations of Bottaro et al.^[@ref25]^ Verbal encouragement was given throughout the testing session. The knee strap was released during each rest period to ensure unrestricted blood flow to the quadriceps. The procedures were administered to all participants by the same investigator. ### Rate of force development (RFD) and rate of velocity development (RVD) {#sec3-2} A software tool on MatLab software 6.5 was developed to estimate RFD and RVD from the file generated by the Biodex software where the signals (angular position, velocity, and torque) were sampled.^[@ref26]^ Once the original sample rate of the digitized biomechanical signals was 100 samples/s, cubic spline data interpolation was implemented as a tool feature and it was used in order to improve the accuracy.^[@ref27]^ Thus, the interpolation frequency used was 20,000 samples/s. RFD is usually obtained from the slope of the force-time curve (Δforce/Δtime), whereas, for intact joint actions as isometric contraction, RFD is calculated as the slope of the joint moment-time curve (ΔTorque/Δtime).^[@ref28]^ Possible estimates of RFD include time intervals of 0--100, 0--200, and 0--300 ms as well as the interval needed to achieve peak torque. All of the considered intervals are relative to the onset of contraction. Onset of muscle contraction is defined as the time point at which the moment curve exceeded baseline moment by 7 Nm.^[@ref28]^ Estimates of RVD were obtained from the slope of the velocity-time curve (ΔVelocity/Δtime). According to Brown and Whitehurst,^[@ref29]^ RVD is movement before matching the predetermined velocity (60°/s and 180°/s for the present study). In other words, RVD is the slope from the point of velocity zero to the point matching the predetermined velocity. Functional fitness test {#sec2-3} ----------------------- Three components of the Rikli and Jones functional fitness test 30 were selected as a functional performance measure. This particular battery of tests was considered to be an appropriate measurement of the physiologic parameters that were associated with functional mobility in independent elderly adults and would therefore be targeted in the study's intervention. This included lower body strength and agility/dynamic balance. The following test items were conducted: 1) a 30-s chair stand test (the maximum number of times within 30 s that an individual can rise to a full stand from a seated position, without pushing with the arms) 31, 2) an 8-ft up-and-go test (standing up from a chair, walking 8 ft to and around a cone, and walking back to the original position in the shortest possible time) 32, and 3) 6 minute walk test (walk as fast as possible for 6 minutes around a 50 yard's rectangle -- 20 x 5 yards 33. Resistance training intervention {#sec2-4} -------------------------------- The participants began by performing six-familiarization training sessions over a 2-week period.^[@ref34]^ This familiarization period was included for two reasons: 1) initial muscle strength increases at the beginning of a training program may be associated with learning effects,^[@ref35]^ and 2) a decreased risk of injury would be more likely as most weight training injuries occur during the first 2 weeks.^[@ref36]^ When the familiarization sessions were completed, the tests of muscle strength and power were conducted, followed by the functional performance fitness tests. Participants were given a 2-day recovery period between each test. The exercise protocols were designed in accordance with published guidelines for resistance training of elderly adults.^[@ref37]^ The 6-week training regimen consisted of 18 training sessions, which were divided into three training days/week. For LLHV group the program incorporated 3 sets of 8 repetitions of the following exercises: squat on the Smith machine, lat-pull down, seated knee extension, chest press and seated knee flexion (Righeto®, Free Style e Pro, Campinas/SP, Brazil) which were interspersed by 90 s recovery intervals. The LLHV group performed all of the exercises with contractions as fast as possible in the concentric phase and 1-2 s in the eccentric phase. The rating of perceived exertion OMNI-RES scale was used to determine the training load.^[@ref38]^ On the first 9 sessions the OMNI-RES scale was set on 3 (light weight -- approximately 50% of 1 RM) and on the last 9 sessions it was set on six (moderated weight - approximately 60% of 1 RM). Recreational activities intervention {#sec2-5} ------------------------------------ Same as LLHV group, the RA participants began by performing six-familiarization training sessions over a 2-week period. This familiarization period was included for RA performed the same number of intervention sections as LLHV group. When the familiarization sessions were completed, the tests of muscle strength and power were conducted, followed by the functional performance fitness tests. Participants were given a 2- day recovery period between each test. The RA group performed 18 training sessions alternated one of the followed interventions: 1) 40 min of very low-intensity walk, 2) 40 min of dance class, 3) 40 min of light stretching. The activities performed by the RA group were guided by a trained instructor who was instructed to conduct activities as lightly as possible (low intensity). Participants could not greatly increase their breathing rates (i.e., they could not be panting). Statistical analysis {#sec2-6} -------------------- Descriptive statistics were expressed as means (±SD). The normality of data was tested by Shapiro-Wilk test. The possible effects of training were tested by a 2-way mixed factor ANOVA \[group (LLHV and RA) x time (pre and post-test)\] for all dependent variable followed by the LSD post-hoc procedure whenever necessary. Due to the initial difference, comparison between groups was determined by the analysis of covariance (ANCOVA) in the PT, RFD and RVD variables. The Effect Size (ES) was calculated from Pre to Posttest by the formula (SD = standart deviation).^[@ref39]^ The probability level of statistical significance was set at a \< 0.05. All calculations were performed using SPSS (version 19.0). Results {#sec1-2} ======= Thirty participants were divided in two groups: LLHV (n=15, 62.1 ± 10.5 years, 161.5 ± 8.6 cm, 75.1 ± 16.7 kg -- mean blood glucose 134.2 ± 28.09 mg/dl) and RA (n=15, 56.7 ± 19.4 years, 166.2 ± 7.4 cm, 77.7 ± 18.0 kg - mean blood glucose 163.3 ± 48.05 mg/dl). Strength performance {#sec2-7} -------------------- The strength performance variables are presented in [Table 1](#table001){ref-type="table"} and 2. Before the training period, there were no significant differences between groups in the isokinetic peak torques, as well as in the RFD variables. There were significant increases, but trivial in peak torque at 60°/s (7.6%, P\<0.001, ES=0.27) and 180°/s (12.2%, P\<0.01, 0.36) in the LLHV group, whereas no significant changes were observed in the RA group (3.7% and 2.1%, respectively). After the intervention period, there were no significant differences (P\>0.005). ([Table 1](#table001){ref-type="table"}). Regarding the RFD variables, there were significant increases in the LLHV group in RFD at 0-200ms (25.3%) and 0-300ms (20.9%) intervals (P\<0.05), whereas no changes were observed in the RA group. Regarding the RVD (at 180°/s), there was a significant reduction in the RA (-21.89%, P\<0.05), whereas a no significant increase (8%) in the LLHV was reported. No significant differences between groups in the RFD and RVD variables were observed after training ([Table 2](#table002){ref-type="table"}). Functional capacity {#sec2-8} ------------------- The functional capacity variables are presented in the [Table 3](#table003){ref-type="table"}. Before the training period, there were no significant differences between groups in the 6 min walking test, stand up from a chair ability, and time-up-and-go test. After the exercise intervention, there was significant improvement in the 6 min walking test performance in the LLHV group (8.2% P\<0.01), whereas no significant change was observed in the RA group (2.2%). In addition, only the LLHV group increased the stand up from a chair ability (24.2%, P\<0.001), while no change was observed in the RA group (4.8%, P\>0.05). Moreover, no significant changes were observed in both LLHV (-4.6%, P=0.1) and RA (-0.6%, P=0.78) groups in the time-up-and-go test performance. After the interventions, the LLHV group showed significant greater performance in the 6 min walking test (P\<0.05), and in the stand from a chair ability test (P\<0.05) while no significant change was observed in RA group. Discussion {#sec1-3} ========== The primary findings of the present study was that only 6 weeks of low-load high-velocity resistance exercises improved muscle strength, power output, and functional capacity in Type 2 diabetics participants. In addition, physical activity intervention composed by low-intensity walking, dancing classes and stretching exercises did not induce any changes in the strength, power, and functional capacity. It has been shown that patients with Type 2 diabetes have a greater decline in the muscle strength and functional capacity, as well as accelerated loss of muscle mass when compared with normoglycemic controls.^[@ref4]^,^[@ref12]^. Indeed, diabetes complications such as peripheral vascular diseases and peripheral neuropathy are associated with poor gait ability, impaired balance and greater risk of falls.^[@ref40]^ Thus, preservation of functional capacity should be especially addressed in aging diabetic patients, because these individuals are at greater risk of development of frailty syndrome, institutionalization and disability.^[@ref8],[@ref10],[@ref11]^ Nevertheless, the effects of resistance exercise intervention upon functional outcomes in participants with Type 2 diabetes are poorly investigated. In the present study we choose to test the effectiveness of low-load high-velocity resistance exercises in patients with diabetes, because in elderly, it has been shown that this type of resistance training promotes greater functional outcomes enhancements than traditional resistance training.^[@ref16],[@ref43]^ For example, Bottaro, Machado have shown greater improvement in the sit-to-stand ability after high-velocity resistance training compared with traditional resistance training (42.8 vs. 6.05%, respectively).^[@ref20]^ Recently, Geirsdottir et al.^[@ref44]^ showed that 12 weeks of traditional resistance training improved \~13% the 6 minutes walking performance in diabetic elderly Icelanders. Different from Geirsdottir et al.,^[@ref44]^ using high-velocity instead of traditional resistance training, the present study also reported an improvement (8%) on the 6 minutes walking performance that was achieved in only half time of the training period (i.e. 6 weeks). Thus, our findings suggest that the three times a week low-load high-velocity resistance training is an effective way to promote rapid functional capacity increases in subject with Type 2 diabetes individuals, who are at greater risk of functional and neuromuscular declines during the aging process. A unique finding of the present study was the increase in the capacity of rapid force development (RFD) in the diabetic patients. Along with the increase in peak torque at 60°/s, the present study demonstrates that the patients with diabetes also improved peak torque at 180°/s, as well as RFD in different time intervals (i.e., 0-200ms and 0-300ms). These findings are especially important because RFD seems to be more associated with the functional outcomes than maximal strength *per* se.^[@ref45]^ Functional activities in daily life, as well as the capacity to prevent falls are actions characterized by a limited time to produce force, which is considerably less time than it takes to develop the maximal force.^[@ref28],[@ref46]^ Moreover, muscle power decreases more rapidly than maximal strength during the aging.^[@ref47]^ Furthermore, in aging patients with Type 2 diabetes, besides the exacerbated decline in muscle strength,^[@ref5],[@ref6]^ it has been shown that this population has a lower capacity to improve strength than normoglycemic individuals.^[@ref48]^ Thus, effective strategies to promote neuromuscular gains in diabetics is needed, and the present findings showed that the low-load high-velocity resistance exercises were able to improve not only maximal strength, but also RFD in this population in just six weeks of resistance training intervention. Rate of velocity development (RVD) represents the acceleration of a limb, which refers to the ability to reach top speed quickly, and may be also advantageous to the human performance.^[@ref29],[@ref49]^ In the present study, there was a significant reduction in RVD in the RA group, whereas a non-significant increase was observed in the LLHV group. It can be speculated that maintaining the ability to rapidly increase the velocity of motion (RVD) may allow the aging diabetic participants resume the stability against unbalances. However, although muscle power output and RFD are strongly associated with the functional capacity in elderly,^[@ref21],[@ref28],[@ref46]^ it seems that this the first study to investigate the effects of resistance training on the RVD in patients with Type 2 diabetes. Thus, the relationship between RVD and functional capacity in aging participants need to be further investigated. In addition, future studies are granted in order to compare the effects of low-load high-velocity resistance exercises vs traditional resistance training (i.e., moderate/heavy load and slow concentric phase) in functional capacity outcomes in diabetics. In despite of this limitation, it seems that this is the first study investigating the effects of low-load high velocity resistance exercises in diabetics patients during the early-phase of the resistance training period. Another possible limitation of the present study was the absence of a control group not performing any type of physical activity. In summary, the present study showed that a short-term high-velocity resistance exercises performed with low intensity (i.e., ≥ 60% 1RM) induced improvements in muscle strength and power parameters, as well as in functional capacity of participants type 2 diabetes. Moreover, the recreational physical activity intervention composed of low-intensity walking, dancing classes and stretching exercise were not sufficient stimulus to induce the same benefits. Although the group LLHV obtained significant improvements in PT, it should be noted that the magnitude of these gains are trivial (ES \<0.50). Perhaps longer-lasting interventions (more than 6 weeks) can lead to greater gains. From a practical standpoint, a high-velocity resistance exercises intervention using light intensity (i.e., 50-60% of 1RM) should be include in the exercise programs in order to improve strength, power output, and functional capacity in subject with type 2 diabetes. This study was partially supported by the Council for the Research Development (CNPq) and by the Coordination for the Improvement of Higher Level Personnel (Capes). T2D : type 2 diabetes LLHV : low-load high-velocity resistance exercises RA : recreational activities RFD : rate of force development RVD : rate of velocity development PT : peak torque ###### -- Pre and Post Peak torque (PT) (N.m) on different velocity Groups Pre Post F^[b](#t1fn002){ref-type="table-fn"}^ *p^[b](#t1fn002){ref-type="table-fn"}^* ES ----------------------------------------- -------------- -------------- -------------- --------------------------------------- ----------------------------------------- ------ Isometric LLHV 146.2 ± 32.8 157.1 ± 30.8 5.618 0.027 0.33 RA 149.6 ± 53.6 158.1 ± 44.8 2.633 0.120 0.16 F^[a](#t1fn001){ref-type="table-fn"}^ 0.034 0.004 *p^[a](#t1fn001){ref-type="table-fn"}^* 0.856 0.953 60%s LLHV 127.0 ± 47.0 139.9 ± 51.0 29.213 0.000 0.27 RA 132.6 ± 37.6 137.5 ± 32.4 3.45 0.078 0.13 F^[a](#t1fn001){ref-type="table-fn"}^ 0.093 0.017 *p^[a](#t1fn001){ref-type="table-fn"}^* 0.763 0.897 180%s LLHV 83.2 ± 28.4 93.3 ± 34.6 12.469 0.002 0.36 RA 95.3 ± 28.9 97.3 ± 28.4 0.399 0.535 0.07 F^[a](#t1fn001){ref-type="table-fn"}^ 0.979 0.083 *p^[a](#t1fn001){ref-type="table-fn"}^* 0.334 0.777 RA, recreational activities (n=15); LLHV, low-load high-velocity resistance exercises (n=15). ^a^Difference between groups. ^b^difference between time. ###### -- Rate of force development (RFD) (N.ms^-1^) and rate of velocity development (RVD) (°/s^2^) (Mean ± SD) --------------------------------------------------------------------------------------------------------------------------------------------------------------------- Intervals LLHV RA F^[a](#t2fn001){ref-type="table-fn"}^ *p*^[a](#t2fn001){ref-type="table-fn"}^ ----------------------------------------- ---------------- ---------------- --------------------------------------- ----------------------------------------- ------- RFD 0-100 ms\ Pre 369.3 ± 218.5 596.0 ± 404.9 2.803 0.11 (Isometric) Post 459.2 ± 220.0 508.6 ± 331.9 0.175 0.681 F^[b](#t2fn002){ref-type="table-fn"}^ 2.432 1.916 *p*^[b](#t2fn002){ref-type="table-fn"}^ 0.135 0.181 ES 0.41 -0.22 RFD 0-200 ms\ Pre 316.2 ± 154.0 449.6 ± 236.0 2.547 0.126 (Isometric) Post 396.3 ± 126.7 429.4 ± 217.4 0.198 0.661 F^[b](#t2fn002){ref-type="table-fn"}^ 6.668 0.353 *p*^[b](#t2fn002){ref-type="table-fn"}^ 0.018 0.559 ES 0.52 -0.09 RFD 0-300 ms\ Pre 271.4 ± 107.2 364.4 ± 164.7 2.552 0.126 (Isometric) Post 328.2 ± 79.1 350.4 ± 134.2 0.232 0.635 F^[b](#t2fn002){ref-type="table-fn"}^ 7.143 0.367 *p*^[b](#t2fn002){ref-type="table-fn"}^ 0.015 0.552 ES 0.53 -0.09 RVD\ Pre 1569.6 ± 634.9 2161.2 ± 958.6 3.006 0.098 (180°/s) Post 1695.1 ± 535.3 1688.1 ± 919.6 0.001 0.982 F^[b](#t2fn002){ref-type="table-fn"}^ 0.371 4.389 *p*^[b](#t2fn002){ref-type="table-fn"}^ 0.549 0.049 ES 0.20 -0.49 --------------------------------------------------------------------------------------------------------------------------------------------------------------------- RA, recreational activities (n=15); LLHV, low-load high-velocity resistance exercises (n=15). ^a^Difference between groups. ^b^Difference between time. ###### -- Functional capacity outcomes pre and post training (Mean ± SD) -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Functional tasks Groups Pre Post F^[b](#t3fn002){ref-type="table-fn"}^ *p*^[b](#t3fn002){ref-type="table-fn"}^ ES ----------------------------------------- -------------- -------------- -------------- --------------------------------------- ----------------------------------------- ------- 8-Ft up-and-go (s) LLHV 5.5 ± 0.9 5.2 ± 0.9 2.85 0.102 -0.33 RA 5.2 ± 0.7 5.2 ± 0.7 0.079 0.780 0.00 F^[a](#t3fn001){ref-type="table-fn"}^ 0.796 0.031 *p*^[a](#t3fn001){ref-type="table-fn"}^ 0.38 0.862 30s Chair stand\ LLHV 16.3 ± 4.2 20.2 ± 5.3 15.777 0.000 0.93 (rep) RA 15.1 ± 3.6 15.8 ± 4.3 0.548 0.465 0.19 F^[a](#t3fn001){ref-type="table-fn"}^ 0.756 6.209 *p*^[a](#t3fn001){ref-type="table-fn"}^ 0.392 0.019 6min walk (m) LLHV 623.8 ± 72.9 675.0 ± 67.8 12.777 0.002 0.7 RA 584.2 ± 73.5 596.9 ± 48.2 0.849 0.366 0.17 F^[a](#t3fn001){ref-type="table-fn"}^ 1.816 10.987 *p*^[a](#t3fn001){ref-type="table-fn"}^ 0.191 0.003 -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- RA, recreational activities (n=15); LLHV, low-load high-velocity resistance exercises (n=15). ^a^Difference between groups. ^b^Difference between time. [^1]: **Author's contributions** RC and MB conceived and supervised the project. RC, MB and EC helped the elaboration of the methodology and the prototype characterization. RC, JD and FL performed the experiments and optimized the methodology. RC and FS contributed to data analysis and interpreted the results. RC wrote the manuscript and MB and EC helped in revising it.

肺癌在男性的肿瘤相关性死亡中占首位，在女性的肿瘤相关性死亡中占第二位，目前已成为世界范围内肿瘤相关性死亡的主要原因之一^\[[@b1]\]^。非小细胞肺癌（non-small cell lung cancer, NSCLC）占肺癌的85%，其中肺腺癌在NSCLC中所占的比例呈逐年增长的趋势。尽管近些年来诊断和治疗手段得到了不断的改进，但是NSCLC患者的5年生存率（约15%）却未得到明显提高。转移是NSCLC患者的主要死因之一。但是，肿瘤细胞的具体转移机制仍然不是十分明确^\[[@b2]\]^。近期研究^\[[@b3]-[@b6]\]^发现，NSCLC等恶性肿瘤的转移与上皮细胞-间叶细胞转化（epithelial to mesenchymal transition, EMT）的发生密切相关。 1. EMT的定义与鉴别 {#s1} ================== 传统的研究是从形态学上区分上皮细胞和间叶细胞^\[[@b7]\]^，因此，EMT往往被简单定义为从形态规则的上皮细胞向形态不规则的间叶细胞的转变。典型的肺上皮细胞彼此之间连接紧密，排列也十分规则^\[[@b8]\]^。上皮细胞还具有顶面和基底面之分，这种特征被定义为上皮细胞的"极性"。上皮细胞的两种不同表面与不同的基质相连。上皮细胞间的这种排列方式能够阻止上皮细胞从组织中脱离。肺的间叶细胞恰恰相反^\[[@b9]\]^。间叶细胞间的连接十分不紧密，排列方式也十分不规则。间叶细胞的外形十分瘦长，这种形态有利于间叶细胞的运动。因此，间叶细胞的运动比上皮细胞活跃，二者的运动方式十分不同。 分子生物学研究发现EMT发生的过程不仅是细胞形态的改变，还伴随着相关分子标记物的改变。例如，E-cadherin仅在上皮细胞中表达，而不在间叶细胞中表达^\[[@b10]\]^。因此，Hay^\[[@b11]\]^将EMT定义为细胞逐步丧失上皮细胞标志物，同时逐步获得间叶细胞标志物的过程。其中，上皮细胞标志物包括E-钙粘蛋白（E-cadherin）、斑珠蛋白（plakoglobin）、细胞角蛋白（cytokeratins）等，而间叶细胞标志物包括N-钙粘蛋白（N-cadherin）、波形蛋白（vimentin）、α平滑肌肌动蛋白（α smooth muscle actin, α-SMA）等。因此，应该至少从形态学和分子生物学两个方面鉴别某种肿瘤是否发生了EMT。 2. EMT的发生机制 {#s2} ================ 肿瘤细胞发生EMT需要一定的诱因，而引起EMT发生的诱导因子可分为内源性的和外源性的。其中，内源性诱因包括某些基因的突变或过表达。突变后能够导致EMT发生的基因包括*p53*等^\[[@b12]\]^；过表达后能够导致EMT发生的基因包括*K*-*ras*、*c*-*myc*和*RGC32*等^\[[@b13]-[@b15]\]^。这些基因发生异常后不但能够使细胞的形态发生改变^\[[@b13]\]^，而且还能够下调上皮细胞标志物的表达^\[[@b12]\]^，同时上调间叶细胞标志物的表达^\[[@b14],\ [@b15]\]^。外源性诱因包括吸烟、乏氧诱导因子（hypoxia inducible factor, HIF）、转化生长因子-β（transforming growth factor β, TGF-β）等多种因素^\[[@b16]-[@b18]\]^。其中，吸烟能够通过上调淋巴样增强因子1（lymphoid enhancer factor 1, LEF1）和转录因子Slug，募集组蛋白去乙酰化酶（histone deacetylase, HDAC），进而抑制E-cadherin的表达，促进EMT的发生^\[[@b16]\]^。HIF-2α能够在mRNA水平上调转录因子Snail、Zeb1以及间叶细胞标记物vimentin的表达，促进EMT的发生^\[[@b17]\]^。TGF-β能够通过Smad依赖性信号通路^\[[@b19]\]^或非Smad依赖性信号通路^\[[@b20]\]^抑制上皮细胞相关标记物E-cadherin等的表达，同时促进间叶细胞相关标记物N-cadhein等的表达。 如上所述，某些转录因子也参与了EMT的发生。与EMT相关的转录抑制因子大致可分为两类：一类能够直接抑制E-cadherin的转录和活性，如Snail1、Snail2和E47等^\[[@b21]\]^；另一类间接抑制E-cadherin的转录，如Twist等^\[[@b22]\]^。值得注意的是，Snail可以通过抑制清道夫受体A5（scavenger receptor class A member 5, SCARA5）的表达促进NSCLC发生EMT，这一机制与其他肿瘤不太相同^\[[@b23]\]^。目前，Snail是否必须受上游信号的调节才能够激活EMT尚无定论，仍然需要进一步研究。 NSCLC发生EMT不仅仅与诱导因子和转录因子有关，还与某些信号通路密切相关。Hedgehog通路是在研究果蝇胚胎时首次被发现的，与细胞的生长、侵袭密切相关，对NSCLC细胞的存活十分关键^\[[@b24],\ [@b25]\]^。这一通路中关键的效应分子包括Gli转录因子等锌指蛋白。Gli1能够迅速促进Snail的转录，进而调节E-cadherin的表达，促进NSCLC发生EMT^\[[@b25]\]^。Mizuarai等^\[[@b26]\]^研究发现核糖体蛋白S6激酶p70S6K2是Gli的下游效应分子。通过抑制p70S6K2可以抑制NSCLC细胞系中Hedgehog通路的激活。Maitah等^\[[@b27]\]^研究发现Hedgehog通路还可以与其他通路共同作用，比如通过上调配体Shh可以增强TGF-β对EMT的诱导作用。因此，Hedgehog通路既可以直接导致NSCLC发生EMT，也可以联合TGF-β等其他通路间接促进NSCLC发生EMT。并且，Hegehog通路促进NSCLC发生EMT的具体机制比既往的研究复杂得多，需要进一步深入的研究。 环氧化酶-2（cyclooxygenase 2, COX-2）是一种诱导酶，能够催化花生四烯酸和类花生酸前列腺素合成，具有抗凋亡、促增殖、促进EMT发生的作用，与NSCLC患者的预后密切相关^\[[@b28]\]^。前列腺素E2（prostaglandin E2, PGE2）是COX-2的一种代谢产物，能够促进免疫抑制、诱导肺癌中EMT的发生^\[[@b29]\]^。在COX-2/PGE2通路中，PGE2通过上调Zeb1和Snail进而促进EMT的发生^\[[@b30]\]^。基于以上理论，研究人员研发出了一些COX-2抑制剂（如阿利考西），希望借此能够治疗NSCLC^\[[@b31]\]^。但是近期的研究^\[[@b32]\]^发现，并非所有的COX-2抑制剂都能够逆转EMT。有些COX-2抑制剂，如塞来昔布（celecoxib）反而能够促进EMT的发生。因此，在研制和应用COX-2抑制剂时应当十分慎重。 与EMT相关的TGF-β通路可分为两种：一种需要转录因子Smad参与^\[[@b19]\]^，而另一种不需要Smad参与^\[[@b20]\]^。在Smad依赖性TGF-β通路中，TGF-β首先与受体结合，激活的TGF-β受体Ⅰ促使转录因子Smad2和Smad3发生磷酸化，发生磷酸化的Smad2、Smad3与Smad4结合形成三聚物^\[[@b19]\]^。这种三聚物再被转入胞核内，与其他转录因子一起调节TGF-β靶基因（如*E*-*cadherin*基因）的表达^\[[@b19]\]^。但是，Chen等^\[[@b20]\]^发现TGF-β诱导NSCLC发生EMT往往是通过PI3K/Akt通路和MEK/Erk1/2通路等不依赖Smad的通路进行的。 此外，核转录因子κB（nuclear factor-κB, NF-κB）通路、Notch通路和Wnt通路也能促进EMT的发生。但是，多项研究^\[[@b33]-[@b37]\]^表明这些通路主要促进乳腺癌等其他肿瘤发生EMT。在NF-κB通路中，NF-κB能够通过激活Twist和Snail促进EMT的发生^\[[@b34],\ [@b38]\]^。有研究^\[[@b33]\]^还发现，包括Ras在内的许多EMT相关基因的激活是通过NF-κB通路进行的。这说明NF-κB通路可能远比目前的研究结果复杂，仍需要更进一步研究。在Notch通路中，转录因子Jagged1和Notch1的过表达可以诱导转录因子Slug的表达，而Slug通过抑制E-cadherin的表达、激活β-catenin以及抵抗失巢凋亡来促进EMT的发生^\[[@b39]\]^。Sahlgren等^\[[@b35]\]^的研究发现，Notch能够诱导HIF-1α向赖氨酰氧化酶（lysyl oxidase, LOX）聚集，进而增强乏氧介导的LOX上调，而LOX具有稳定Snail（Slug）蛋白的作用。因此，他们认为Notch通路可以通过介导乏氧刺激EMT的发生^\[[@b35]\]^。在Wnt通路中，Wnt蛋白能够与Frizzled受体结合，进而在Frizzled受体、脂蛋白受体相关性蛋白（lipoprotein receptor related protein, LRP）、Dishevelled分子和轴抑制蛋白（Axis inhibition protein, Axin）之间形成一种稳定的受体复合物，使Dishevelled分子发生磷酸化^\[[@b36]\]^。磷酸化的Dishevelled分子会抑制GSK-3β活性，而GSK-3β又具有促使转录因子Snail1发生磷酸化的作用^\[[@b37]\]^。这样，Snail1就会在细胞中不断积累，最终促使EMT的发生^\[[@b37]\]^。目前仍需要大量研究证实NSCLC是否也能够通过这些通路发生EMT。 3. EMT与miRNA {#s3} ============= miRNA（或microRNA）是一种长度为20个-22个核苷酸的长链非编码RNA（long noncoding RNA），能够在转录后调节基因的表达^\[[@b40]\]^。在EMT相关的miRNA中，miR-200被研究得最多^\[[@b41]\]^。miRNA-200能够下调Zeb1 mRNA和Zeb2 mRNA的水平，上调E-cadherin水平^\[[@b42]\]^，抑制EMT的发生，进而抑制NSCLC的转移^\[[@b43]\]^。最近的研究^\[[@b44]\]^发现，MiR-134/487b/655能够通过抑制膜相关性鸟苷酸激酶的活性进而抑制肺腺癌中EMT的发生，减少肺腺癌对吉非替尼的耐药性。而miR-30a能够下调Snail的表达进而影响NSCLC细胞中E-cadherin和N-cadherin的表达水平^\[[@b45]\]^。但是，Kong等^\[[@b46]\]^发现，TGF-β在诱导小鼠乳腺上皮细胞发生EMT的同时还能够促进miR-155的表达。降低细胞中miR-155水平反而能够抑制EMT的发生、抑制细胞间紧密连接的降解，进而抑制细胞的侵袭性。反之，增加miR-155水平能够促进EMT的发生，增强细胞的侵袭性。NSCLC中是否也存在这种现象仍然不十分清楚。这说明miRNA调节EMT发生的机制不尽相同。 既然miRNA能够抑制EMT而EMT又与NSCLC的转移相关，那么miRNA能否有效地预测NSCLC的预后呢？Gao等^\[[@b47]\]^发现miR-21的高表达（HR=5.993, 95%CI: 2.518-14.264; *P*＜0.001）、miR-181a的低表达（HR=0.328, 95%CI: 0.142-0.756; *P*=0.009）与患者的总生存期密切相关，而与TNM分期和淋巴结转移情况无关。Li^\[[@b48]\]^的研究也发现miR-146的高表达与NSCLC患者的预后密切相关，而与TNM分期和淋巴结转移情况无关。可见，miRNA与NSCLC的总生存期相关，而其预后价值仍需要更深入的研究。 4. EMT与肿瘤干细胞 {#s4} ================== 肿瘤干细胞（cancer stem cells, CSC）是能够自我更新并能够分化成多种肿瘤细胞系的一类细胞。肿瘤干细胞理论最初只是一种设想，直到1997年首次通过实验证明肿瘤干细胞的存在^\[[@b49]\]^。Mani等^\[[@b50]\]^首次报道了EMT的发生能够导致肿瘤细胞成为肿瘤干细胞。他们发现经过球体培养的上皮细胞可以向干细胞样细胞转变，并且能够获得间叶细胞的某些表型^\[[@b50]\]^。Pirozzi等^\[[@b51]\]^发现，经过TGF-β1处理的NSCLC细胞系不但发生了EMT，还能够表达肿瘤干细胞的某些分子标记物，如Oct4、Nanog、Sox2、c-kit和CD133等。因此，我们推测，所谓EMT或许就是上皮细胞向肿瘤干细胞转变的一个过程。如果这一猜想成立的话，那新的问题出现了：既然肿瘤干细胞是能够自我更新并能够分化成多种肿瘤细胞系的一类细胞，那肿瘤干细胞的分化是否也能够导致EMT的发生。这些猜想尚需要大量的研究证实。 5. EMT与NSCLC预后的相关性 {#s5} ========================= 目前，有关EMT与NSCLC预后的研究多数是从相关标记物的临床意义入手的。与EMT相关的分子标志物包括上皮细胞标志物以及间叶细胞标志物、相关转录因子等^\[[@b6],\ [@b11]\]^。 E-cadherin是上皮细胞特异性标志物，表达水平在EMT的发生过程中逐渐降低^\[[@b11]\]^。Sulzer等^\[[@b4]\]^发现E-cadherin表达情况与NSCLC患者的预后密切相关。111例接受手术的患者中，高表达E-cadherin的患者的3年生存率是60%，而低表达E-cadherin的患者的3年生存率仅32%。但是，Tarin^\[[@b52]\]^通过回顾文献及自己既往的研究后认为EMT在肿瘤的进展中并不一定具有意义。Prudkin等^\[[@b53]\]^发现：E-cadherin表达的减少和N-cadherin的过表达与肺癌的预后不相关，且几乎所有的肺癌标本都存在EMT表型的变化。他们还发现，与肺癌的原发灶相比，脑转移瘤能够表达更高水平的E-cadherin^\[[@b53]\]^。Wu等^\[[@b5]\]^对13项研究中的2, 274例患者进行了*meta*分析，结果发现E-cadherin的下降确实与NSCLC患者的预后相关，但是与Ⅰ期患者的预后无关。 因此，EMT相关标志物与NSCLC患者预后的具体关系需要进一步研究及更加深入的分析，而首先需要解决的问题是筛选更多可供肺癌研究借鉴的相关标记物。例如，N-cadherin和Vimentin是间叶细胞特异性标志物，其表达水平在EMT的发生过程中逐渐增加^\[[@b11]\]^，同样可以作为检测EMT发生的特异性指标。Luo等^\[[@b54],\ [@b55]\]^发现胞核中N-cadherin和Vimentin的阳性表达率与鼻咽癌患者的临床分期相关（*P*＜0.001）。既然如此，N-cadherin和Vimentin与NSCLC的临床分期是否也存在一定的相关性呢？Hui等^\[[@b6]\]^发现Ⅲ-Ⅳ期与Ⅰ期和Ⅱ期NSCLC中N-cadherin的表达阳性率分别为39.58%、31.58%和23.53%（*P*＜0.01），而低、中、高分化NSCLC中N-cadherin表达阳性率分别为64.10%、35.71%和16.0%（*P*＜0.01）。Dauphin等^\[[@b56]\]^发现Vimentin的高表达与NSCLC的远处转移（*P*=0.024）和较晚分期（*P*=0.002, 8）相关。可见，N-cadherin和Vimentin的表达情况与NSCLC的分期同样存在一定的相关性。 转录因子在EMT的发生过程中有十分重要的调控作用，那么通过检测肿瘤细胞中转录因子的表达情况应该可以明确EMT的发生情况及其预后价值。Yin等^\[[@b57]\]^对107例骨肉瘤标本进行免疫组化染色，结果发现骨肉瘤中Twist的阳性表达率为31.8 %（34/107），并且Twist表达阳性的骨肉瘤患者在总生存期和无进展生存期方面均较差（*P*＜0.05）。类似地，Hui等^\[[@b6]\]^发现NSCLC中Twist的表达阳性率为38%，明显高于瘤旁正常组织（*P*＜0.01）。并且，Twist的表达情况与NSCLC的分期和分级相关。Ⅲ期-Ⅳ期NSCLC中Twist的表达率（56.25%）明显高于Ⅰ期（21.05%, *P*＜0.01）或Ⅱ期（32.35%, *P*＜0.01）。低分化癌与中分化和高分化癌中Twist的表达阳性率也各不相同，分别为66.67%、26.79%和20.00%（*P*＜0.01）。有淋巴结转移与无淋巴结转移的病例中Twist的表达情况也存在一定的差异（48.58% *vs* 24.0%, *P*＜0.01）。这说明，Twist对NSCLC同样具有预后意义。但是，Twist的过表达并不是NSCLC的独立预后因素，与N-cadherin的过表达呈一定的相关性（*R*=0.565, *P*＜0.001）^\[[@b6]\]^。 尽管上述的研究表明EMT相关的标记物与NSCLC患者的预后存在一定相关性，但这些研究仅仅是从分子生物学方面对EMT进行研究。既然EMT的鉴别至少需要从形态学和分子生物学两个方面进行，那么研究人员也应该将形态学因素考虑在内。 6. 问题与展望 {#s6} ============= 目前，有关EMT的研究虽然取得了一定的成果，但仍然面临许多问题。首先，多数研究表明EMT相关标志物与肿瘤的转移呈一定的相关性，但这些研究并未证明EMT与肿瘤进展是否真的存在一定的顺序关系或因果关系。Chui^\[[@b58]\]^认为，累积的遗传学和表观遗传学改变以及肿瘤微环境才是导致肿瘤获得侵袭性的重要因素，而EMT并非肿瘤发生远处转移的必要步骤。可见，EMT与肿瘤进展的关系尚存在一定的争论，仍需要深入的研究。其次，EMT相关基础研究应该不断地应用于临床。例如，Brachyury是最近发现的一种T-box转录因子，能够在多种肿瘤中表达^\[[@b59]\]^。Brachyury的高表达能够诱导上皮细胞发生EMT。针对这种转录因子，研究人员研发出了一种肿瘤疫苗，希望能够用这种疫苗治疗晚期NSCLC^\[[@b60]\]^。因此，在解决相关争论的同时，不断将EMT与NSCLC的免疫治疗相结合或许是未来的研究方向之一。

{#sp1 .472} {#sp2 .568} {#sp3 .569}

Introduction {#H1-1-ZOI200291} ============ Climate change appears to have both direct and indirect effects on human health, which may increase globally, and some populations may be affected more than others. Factors associated with health include heat-related illness and death as well as increased risk of vector-borne diseases, cardiovascular disease, and respiratory conditions from degraded air quality and threats to food and fresh water supplies.^[@zoi200291r1]^ Global health leaders, such as physicians, nurses, and public health professionals, are among the professional groups that need to understand the complex interactions of climate and health along with current and emerging health challenges.^[@zoi200291r2]^ To try and build a healthier, more secure future, health professionals should be trained to prevent, mitigate, and respond to the health consequences of climate change. However, curricula and educational programs on the topic of climate-health^[@zoi200291r3]^ (defined as the health consequences from anthropogenic climate change) are still lacking and gaps in knowledge persist. Several researchers have published climate-health education research projects and surveys. A survey conducted in 2018 and 2019 evaluated planetary health^[@zoi200291r4]^ (defined as a field focused on characterizing the human health factors associated with human-based disruptions of the earth's natural systems) teachings in all 17 Canadian medical schools; results showed variation in coverage of planetary health topics from "minimal to no teaching, to some lecture-, case-, or project-based teaching."^[@zoi200291r5]^ The researchers also identified barriers to integrating the education, such as limited time in the busy medical curricula, and provided recommendations for adding content. The Pontificia Universidad Javeriana in Colombia conducted a survey in 2017 to determine the state of climate-health education among the 59 medical schools in Colombia.^[@zoi200291r6]^ The survey revealed that, of 47 respondents, the topic of climate-health is offered at 53% of the medical schools; however, the topic is not a priority in curricula, does not have set guidelines, and is not taught by qualified professionals. Another research project searched the Curriculum Inventory database supported by the Association of American Medical Colleges for specific terms related to climate change and found that none of the medical schools in the database reported explicit inclusion of climate change education in their curriculum.^[@zoi200291r7],[@zoi200291r8]^ Other researchers have published surveys on health professionals' perceptions of climate-health topics. One study involved interviews with public health and climate change professionals to understand barriers to and opportunities for advancing work on climate-health.^[@zoi200291r9]^ Sarfaty and colleagues^[@zoi200291r10],[@zoi200291r11],[@zoi200291r12],[@zoi200291r13]^ published the findings from several surveys that were conducted to determine physicians' perceptions of, experience with, and opinions on health factors that may be associated with climate change. The surveys found that physicians observed that climate change may affect human health and supported responses to address it. The Global Consortium on Climate and Health Education (GCCHE) surveyed its members to understand the current state of climate-health curricula among health professions institutions internationally. The survey describes present practices and is intended as an initial assessment with the aim of improving this educational content, addressing gaps in knowledge on the topic, and helping to chart future GCCHE progress. The present study aimed to examine how many health professions schools and programs currently have climate-health curricula, including planetary health curricula; the existing course and programmatic offerings; plans for adding curricula; and challenges and opportunities for instituting the curricula. Methods {#H1-2-ZOI200291} ======= The GCCHE at the Columbia University Mailman School of Public Health designed and developed the survey in conjunction with the broader GCCHE Coordinating Committee, a group of 12 (7 at the time the survey was developed) researchers and professionals in the climate-health field with appointments outside Columbia University who help guide Consortium efforts. The survey was deemed exempt by the Columbia University Institutional Review Board. The Columbia GCCHE team reviewed the American Association for Public Opinion Research (AAPOR) reporting guidelines for survey studies and made efforts to devise clear questions of appropriate length that did not lead to bias; the team also characterized nonresponders to check for the association between nonresponse bias and the validity of the findings. The goal of this study was to assess the climate-health curricular offerings at health professions institutions by surveying the GCCHE membership. Member institutions are health professions institutions including schools and programs of public health, nursing, and medicine. The primary contact person from each GCCHE member institution served as survey respondent. The survey was conducted from August 3, 2017, to March 1, 2018, and was sent to the primary contact person at 160 GCCHE member institutions. Surveys were voluntary, self-administered online, and introduced by an email from GCCHE staff with brief background information on the study. It was indicated in the survey instrument that the results of the survey would be aggregated before anonymous results were reported more widely; participants did not receive financial compensation. The survey included 23 questions (eAppendix in the [Supplement](#note-ZOI200291-1-s){ref-type="supplementary-material"}). Four questions pertained to demographic characteristics of the institution and the individual completing the survey; the other 19 questions were about the climate-health educational activities at the school/program. Responses to 5 questions were required, including the respondent's name, email address, name of school, whether they offer climate-health education, and whether discussion is under way to add any climate-health offerings. Responses to the remaining 18 questions were optional, of which some were conditional (based on answers to previous questions); thus, the number of responses differs for each question. Several questions allowed respondents to select all answers that apply. Key words (ie, climate-health and planetary health) were not defined within the survey. Responses were categorized into 4 school/program types: (1) public health, health sciences, and health professions; (2) medicine; (3) nursing; and (4) other. Each response received 1 type of designation, which was selected based on the name of the school/program or the category that most closely fit the school/program. Each institutional response was given 1 designation: a school/program of public health, nursing, medicine, or other. However, a school might also include a program of another type that was not counted in this analysis. For example, a medical school might also include a program of public health; however, for the purposes of this study, the response was counted only as a medical school. To address the possibility of nonresponse bias, a sample of nonrespondents was characterized to determine whether members were more likely to respond to the survey if their institution offered education on climate-health than if their institution did not offer education on climate-health. This informal assessment was made by looking at institutions' webpages and/or online course catalogs to see if nonrespondents were less likely to offer climate-health curricula. To make this assessment, the Columbia GCCHE team first looked at whether differences existed between respondent institutions' climate-health educational offerings listed online and how these institutions responded in the survey. For a sample of 15 respondents, their institutions' webpages and/or online course catalogs were examined to determine whether information was available online to support their survey response of whether they offer climate-health curricula. Next, for a sample of 15 nonrespondents, the webpages and/or online course catalogs of these institutions were searched to determine whether these institutions appeared to offer climate-health education. These 2 samples were then compared for differences. Results {#H1-3-ZOI200291} ======= The overall response rate for the survey was 53%, with 84 of 160 institutional responses collected ([Table 1](#zoi200291t1){ref-type="table"}). Fifty-nine of the 84 responses (70%) were from schools/programs of public health, health sciences, or health professions; 15 responses (18%) were from medicine; 9 responses (11%) were from nursing; and 1 response (1%) was from another type of health profession institution. Regarding climate-health education currently offered, the institutions of 53 of 84 respondents (63%) offer climate-health education and 31 institutions (37%) do not. Thirty-seven of the 53 programs (70%) that offer climate-health education were from schools/programs of public health, health sciences, or health professions; 12 (23%) were from medicine; and 4 (8%) were from nursing. Of the 50 institutions offering climate-health education that responded, 33 institutions (66%) assess students' knowledge of climate-health and 17 institutions (34%) do not assess students' knowledge. ###### Climate-Health Educational Offerings and Evaluations[^a^](#zoi200291t1n1){ref-type="table-fn"} Survey question Institutions surveyed, No. Response, No. (%) ---------------------------------------------------------------------------------------------------------------------------------- ---------------------------- ------------------- --------- Recruitment 160 84 (53) 76 (48) Does your school offer climate-health education? 84 53 (63) 31 (37) Does your school offer planetary health modules, courses, or programs? 51 17 (33) 34 (67) Has your school received evaluations from students on their experience of and/or satisfaction with the climate-health teachings? 49 27 (55) 22 (45) Does your school assess students' climate-health knowledge? 50 33 (66) 17 (34) Climate-health refers to possible association between climate change and health. Members were asked to describe the climate-health education that their school offers ([Table 2](#zoi200291t2){ref-type="table"}). Forty-one of 54 respondents (76%) indicated that their institution provides a climate-health session as part of a required core course; 24 institutions (44%) offer a climate-health stand-alone elective course; and 21 institutions (39%) offer a climate-health session as part of a nonrequired course. Fewer institutions offer a climate-health doctoral program (4 \[7%\]), climate-health postdoctoral positions (4 \[7%\]), a climate-health stand-alone required course(1 \[2%\]), and/or a climate-health master's or certificate program (1 \[2%\]). ###### Types of Climate-Health Educational Offerings[^a^](#zoi200291t2n1){ref-type="table-fn"} Survey question Response, No. (%) ------------------------------------------------------------------------------------------------- ------------------- What climate-health education does your school offer?[^b^](#zoi200291t2n2){ref-type="table-fn"} 54 Climate-health session as part of nonrequired course 21 (39) Climate-health session as part of required core course 41 (76) Climate-health stand-alone elective course 24 (44) Climate-health stand-alone required course 1 (2) Climate-health master's or certificate program 1 (2) Climate-health doctoral program 4 (7) Climate-health postdoctoral positions 4 (7) Climate-health refers to possible association between climate change and health. Respondents were asked to select all responses that apply. The second main research question addressed how climate-health education is being taught in member institutions including this content. Of schools that offer a stand-alone course on climate-health, most courses are 3 credits (18 of 29 \[62%\] responses), ranging from 0.25 to 6 credits. Members were asked how long their climate-health educational offerings have been in place. The average course age reported by 48 respondents was approximately 5 years, ranging from less than 1 year to 17 years. Twenty-seven respondents from 49 schools (55%) reported that their school had received evaluations from students on their experience and/or satisfaction with climate-health teachings, whereas 22 institutions (45%) had not received such evaluations. Members described the main goals of their overall climate-health curriculum, and responses included the following: - Improving understanding of the science and factors responsible for climate change; - Increasing awareness of and literacy on climate-health and relevance to health professions practice; - Improving knowledge of methods or tools to analyze health risks and climate information; - Developing skills to identify vulnerable populations and strengthening understanding of climate-health--relevant policies, including mitigation of climate change and adaptation actions and health disparities; - Instigating behavior change and advocacy for sustainability in health care; and - Improving climate-health communication skills. The third main research question aimed to determine whether institutions are discussing adding climate-health content and what their experience with adding the curricula has been, including if they received a positive response to adding climate-health curricula and/or challenges. Of 82 respondents, 61 individuals (74%) reported that the addition of climate-health offerings is under discussion and 21 individuals (26%) responded that nothing is being considered ([Table 3](#zoi200291t3){ref-type="table"}). Almost all institutions have received a positive response to adding climate-health curricula. Of 58 respondents, 39 individuals (67%) have received a positive response from students, 35 (60%) from faculty, 23 (40%) from administration. Fifteen respondents (26%) selected other, which included positive responses from funders, professional organizations in the community, climate change nonprofit organizations, and departments of health as well as that it is too early to know or is unknown. ###### Challenges, Responses, and Future Plans for Climate-Health Education[^a^](#zoi200291t3n1){ref-type="table-fn"} Question Respondents, No. Response, No. (%) --------------------------------------------------------------------------------------------------------------------------------- ------------------ ------------------------------------------------------ ----------------------------------------------------------------------------- --------------------------------------------------------------- ----------------------------------------------------------------------- --------------------------------------------------------- -------------------------------------------------------------- ----------------------------------------------------------- ----------------------------------- --------------- Are any climate-health offerings under discussion to add?[^b^](#zoi200291t3n2){ref-type="table-fn"} 82 Session as part of nonrequired course, 32 (39) Session as part of required core course, 32 (39) Climate-health stand-alone elective course, 22 (27) Climate-health stand-alone required course, 6 (7) Climate-health master's or certificate program, 9 (11) Climate-health doctoral degrees, 3 (4) Climate-health postdoctoral positions, 4 (5) Nothing being considered, 21 (26) NA Have you received a positive response to adding climate-health curriculum?[^b^](#zoi200291t3n2){ref-type="table-fn"} 58 Yes, from students, 39 (67) Yes, from faculty, 35 (60) Yes, from administration, 23 (40) No, have not received a positive response, 1 (2) Other, 15 (26) NA NA NA NA Have you encountered any challenges in trying to institute climate-health curriculum?[^b^](#zoi200291t3n2){ref-type="table-fn"} 59 Yes, lack of interest or demand from students, 5 (8) Yes, administration or other skepticism about climate-health science, 4 (7) Yes, lack of funding/time to support its development, 20 (34) Yes, lack of available staff time to work on its development, 24 (41) Yes, no available space in the core curriculum, 17 (29) Yes, lack of teaching materials and staff expertise, 14 (24) Yes, competing institutional priorities/politics, 18 (31) No challenges, 17 (29) Other, 8 (14) Abbreviation: NA, not applicable. Climate-health refers to possible association between climate change and health. Respondents were asked to select all responses that apply. Members were asked if they have encountered any challenges in trying to institute climate-health curricula. Forty-two of 59 respondents (71%) encountered some challenges trying to institute the curriculum. Of 59 respondents, 24 individuals (41%) experienced a lack of available staff time to work on development of curricula, 20 individuals (34%) reported a lack of funding and/or time to support development, 18 individuals (31%) reported competing institutional priorities and/or politics, 17 individuals (29%) reported that there is no available space in the core curriculum, 14 individuals (24%) reported a lack of teaching materials and staff expertise, 5 individuals (8%) described a lack of interest or demand from students, and 4 individuals (7%) reported administrative or other skepticism about climate-health science. Seventeen respondents (29%) reported that they had no challenges and 8 individuals (14%) selected other; these responses included the following: - Issues were encountered early in the implementation, but they have successfully moved beyond them; - Virtually impossible at master of public health level owing to full course load of students; - Need updated teaching materials; and - A few students stating that the faculty member was trying to push a "liberal agenda." The fourth main research question aimed to discover the opportunities to advance climate-health education. Specifically, members were asked what they have found helpful and whether they have any partnerships on climate-health. When asked what they have found helpful in instituting or developing climate-health curricula, 47 of 59 respondents (80%) reported interest from students, 46 respondents (78%) described interest from faculty, 26 respondents (44%) reported interest from administration, 10 respondents (17%) indicated support from donors, 4 respondents (7%) reported support from institutional board members, and 8 respondents (14%) selected other ([Table 4](#zoi200291t4){ref-type="table"}). Other responses included support from community organizations, political support, being a member of groups and alliances, national and international collaboration, support from the dean, and broader university interest. ###### Opportunities in Climate-Health Education[^a^](#zoi200291t4n1){ref-type="table-fn"} Question Respondents, No. Response, No. (%) -------------------------------------------------------------------------------------------------------------------------------- ------------------ ------------------------------------------------------------- ------------------------------------------------------------- ---------------------------------------------------------------------------------------- ---------------------------------- ----------------------------- --------------- What have you found helpful in instituting or developing climate-health curriculum?[^b^](#zoi200291t4n2){ref-type="table-fn"} 59 Interest from students, 47 (80) Interest from faculty, 46 (78) Interest from administration, 26 (44) Support from board member, 4 (7) Support from donor, 10 (17) Other, 8 (14) Does your school currently have any partnerships on climate change and human health?[^b^](#zoi200291t4n2){ref-type="table-fn"} 48 Yes, with another academic institution on training, 13 (27) Yes, with another academic institution on research, 16 (33) Yes, with a nonacademic institution, 19 (40)[^c^](#zoi200291t4n3){ref-type="table-fn"} Yes, with a funder, 8 (17) No, 17 (35) NA Abbreviation: NA, not applicable. Climate-health refers to possible association between climate change and health. Respondents were asked to select all responses that apply. For example, business, government, nongovernmental organization. When we assessed the characteristics of survey respondents and nonrespondents, some evidence of bias was found, indicating that respondents were more likely to already offer climate-health curricula. Specifically, in the sample of 15 responding institutions, 9 institutions (60%) had indicated in the survey that they offer climate-health curricula. When searching these same institutions' webpages and/or online course catalogs, 12 of 15 institutions (80%) appeared to either offer (n = 8) or probably offer (n = 4) climate-health curricula. Thus, there was a 20 percentage-point difference between information online and survey responses. From the sample of 15 nonresponding institutions, 10 institutions (67%) appeared to offer (n = 5) or probably offer (n = 5) climate-health curricula. These findings identify a slight difference in rates of offering climate-health curricula between respondents and nonrespondents to our survey. Discussion {#H1-4-ZOI200291} ========== This survey provides a view of the state of climate-health education at health professions schools and programs internationally. The survey results suggest that several institutions included in the survey have successfully integrated climate-health content into their curricula. Typically, of institutions in this survey, climate-health education is offered as part of a required core course (76%), as part of a nonrequired course (39%), or as a climate-health stand-alone elective course (44%). The breadth of educational offerings, number of credits offered, years since inception, and whether members conduct evaluations and assess learning reveal considerable variation among institutions. Many schools have offered climate-health curricula for several years, and most schools assess students' climate-health knowledge ([Table 5](#zoi200291t5){ref-type="table"}). ###### Climate-Health Teaching Methods and Assessment[^a^](#zoi200291t5n1){ref-type="table-fn"} Survey question Response, No. (%) ----------------------------------------------------------------------------------------------------------------------------------------- ------------------- If your school offers a stand-alone course on climate-health, what teaching methods are used?[^b^](#zoi200291t5n2){ref-type="table-fn"} 28 Laboratories 1 (4) Lectures 24 (86) In-class exercises 20 (71) Online tutorials or MOOCs 8 (29) Internships outside the classroom 2 (7) How is climate-health knowledge assessed?[^b^](#zoi200291t5n2){ref-type="table-fn"} 34 Quizzes 19 (56) Examinations 22 (65) Papers 19 (56) Capstone 5 (15) Thesis 7 (21) Dissertation 8 (24) Abbreviation: MOOCs, massive open online courses. Climate-health refers to possible association between climate change and health. Respondents were asked to select all responses that apply. Members were asked if they are discussing adding curricula, and results showed that most are discussing a session, whether part of a nonrequired course (39%) or required core course (39%), followed by a climate-health stand-alone elective course (27%). About a quarter of respondents are not considering further content (26%). The findings suggest that although most institutions are discussing adding curricula, they are considering adding content as sessions or elective courses instead of more substantial offerings or required courses. Most respondents have encountered challenges in trying to institute climate-health curricula, and the 2 most cited issues are lack of available staff time (41%) and funding/time to support its development (34%), followed by competing institutional priorities/politics (31%). This finding suggests that challenges relate to available resources and institutional support, rather than demand for or interest in the content; 29% of respondents reported that they encountered the challenge of no available space in the core curriculum, and 24% reported lack of teaching materials and staff expertise. These data suggest that time and resource constraints as well as support for the content present challenges for instituting the curricula. There may be connections among the various challenges encountered by individual institutions. For example, the broader political context within a country might influence the priorities of an institution and present challenges for developing curricula. In addition, lack of funding could lead to an inability to hire faculty/staff with expertise in climate-health topics and insufficient time to work on the development of content, teaching materials, or training of staff. Most respondents have received a positive response to adding climate-health curricula, and the positive response was principally from students, followed by faculty; only 40% of the respondents have received a positive response from administration. In addition, most respondents reported that interest of students, followed by interest of faculty, was helpful in instituting or developing climate-health curricula, and 44% responded that interest from administration was helpful. These findings suggest that students and faculty are important groups to work with and engage when planning to institute or develop curricula, and that interest from administration also appears to be important. Most respondents have a partnership on climate-health, and most of the partnerships are with another academic institution on research or with a nonacademic institution. On the basis of these findings, providing resources to help institutions incorporate climate-health education into curricula, including information on how to integrate the curriculum in a way that synthesizes climate-health into existing course material, might help to accelerate the number of health professions schools and programs that offer this education. In addition, support for the curricula, in terms of both time and resources, and building off of the response and interest of students and faculty might help to institute curricula at individual institutions. Climate-health resources from various groups around the world are available, both on the topic generally and specifically related to curricular development. Certain groups focus on climate-health, while others cover environmental health or planetary health topics more broadly. In addition, some resources are discipline specific, such as those developed for medical professionals, while others are tailored for all health professionals or relevant across all disciplines. The GCCHE provides a set of core climate-health competencies for all health professions students and a knowledge bank of resources and recommendations to help institutions integrate content into curricula.^[@zoi200291r14]^ Curricular resources are also offered by organizations and groups, such as the National Institute of Environmental Health Sciences in the United States,^[@zoi200291r15]^ which develops lesson plans, slides, and case studies on climate-health for clinical health care and public health students; the Alliance of Nurses for Healthy Environments, which offers curricular resources to help integrate environmental health topics into nursing curricula^[@zoi200291r16]^; the NurSus project, which provides educational materials on sustainability topics for nurses in several languages^[@zoi200291r17]^; and the World Organization of Family Doctors Working Party on the Environment, which develops curricula and shares educational materials on environmental health for family physicians.^[@zoi200291r18]^ Schools and institutions are setting precedents by creating climate-health centers,^[@zoi200291r19],[@zoi200291r20],[@zoi200291r21],[@zoi200291r22]^ certificates,^[@zoi200291r23]^ curricular infusion projects,^[@zoi200291r24]^ and fellowships.^[@zoi200291r25]^ Health professions associations are taking a stance on climate change, and some have developed policies and motions on climate-health education,^[@zoi200291r26],[@zoi200291r27]^ and students are organizing and taking the lead.^[@zoi200291r28],[@zoi200291r29],[@zoi200291r30]^ Several articles have described why climate change education may be important in health professions curricula and how to integrate content without imposing a large time commitment.^[@zoi200291r8],[@zoi200291r24]^ Ready-to-use and practical resources are also available to help educate and organize health professionals,^[@zoi200291r31],[@zoi200291r32],[@zoi200291r33]^ and many organizations communicate information on climate-health in easily understood and relevant ways.^[@zoi200291r34],[@zoi200291r35]^ All of these efforts suggest the growing importance of understanding, preparing for, and helping to prevent changes in human health that may be associated with climate change. Limitations {#H2-1-ZOI200291} ----------- This study has limitations. The first is a response rate of 53%. The second is that participation was voluntary and from an overall self-selected group that does not include all health professions schools, which prevents assurance that the sample is truly representative of the overall state of climate-health education at all health professions schools internationally. A third possible limitation is the nonresponse bias identified above. Although this sample was small and did not show strong bias, potential for some bias in the overall sample is acknowledged. Conclusions {#H1-5-ZOI200291} =========== Climate change may be affecting health in a variety of ways with increasing consequences. Health professionals, including those in public health, nursing, and medical services, should be educated on how to prevent, mitigate, and respond to factors associated with climate change that may be associated with health in a negative way. We believe that the results of this survey may provide a baseline assessment of the current state of climate-health education internationally among health professions schools and programs. The survey suggests that a range of educational offerings exist on climate-health, including sessions, courses, programs, and postdoctoral positions. Some schools have offered climate-health education for several years, some are just now adding content, and others do not include any content on the subject. Although many schools are discussing adding climate-health educational offerings, considerable gaps in offerings remain at several institutions as well as challenges that extend beyond the institutional level, such as political and funding priorities that might lead to lack of staff time and materials to support the training. Opportunities also exist to facilitate the integration of curricula, such as working with students, faculty, and members of administrations who are interested in this topic. We suggest that health professions schools include this content in their curricula and that awareness as well as financial support, resources, and expertise increase to help in its uptake. To facilitate this integration, institutions can look to online resources, groups, and networks to provide guidance and information to develop curricula. ###### **eAppendix.** Survey Instrument ###### Click here for additional data file.

Chemotherapy-induced peripheral neuropathy has been increasingly recognized as a serious side effect associated with several commonly used chemotherapeutic agents, including taxanes, platinum agents, and vinca alkaloids (e.g., vincristine) during cancer treatment. Depending on the treatment regimens, chemotherapy-induced neuropathic pain can occur in 30--40% of patients and even as high as 75% under certain regimens. Common peripheral sensory symptoms include paresthesias and dysesthesias, pain, numbness and tingling, and sensitivity to touch and temperature. Motor symptoms include weakness and gait and balance disturbances[@b1]. In most cases, this kind of neuropathic pain is only partially reversible with cessation of treatment and in the worst cases damage can be permanent. To date, there is no one drug or drug class that is considered safe and effective for treatment of chemotherapy-induced neuropathic pain, making the development of alternative effective analgesics a crucial clinical need. Angelicae dahuricae radix is a perennial plant that grows naturally in broad areas of China. Angelicae dahuricae radix has a strong scent and its leaves are used to make incense. In addition, the roots of angelicae dahuricae radix (also known as Bai Zhi) are used in traditional Chinese medicine to treat harmful external influences on the skin, such as cold, heat, dampness and dryness[@b2]. Modern pharmacological studies on angelicae dahuricae radix have reported that crude extracts of angelicae dahuricae radix possesses anti-inflammatory, analgesic and antipyretic actions and acute toxicity as a guideline for clinic application[@b2]. Essential oil of angelicae dahuricae radix has analgesic effect in rat models of pain, and the antinociceptive effects have been linked to the facilitated release of endogenous opioids such as beta-endorphin[@b3]. More importantly, clinical studies have demonstrated that angelicae dahuricae radix has significant analgesic effect in humans, supporting the clinical utility of angelicae dahuricae radix as an alternative medicine for pain control[@b4]. However, the crude extract and essential oil of angelicae dahuricae radix include multiple potentially active chemical compounds and the active ingredient(s) of angelicae dahuricae radix that are responsible for its analgesic activity are currently unknown. Recent phytochemical research has purified and identified several active coumarin components of angelicae dahuricae radix[@b5], and further pharmacological studies are needed to identify the active coumarin component underlying the antinociceptive actions of angelicae dahuricae radix. In this study, we described the potent antinociceptive effects of one of the coumarin components of angelicae dahuricae radix, auraptenol (8-(2-hydroxy-3-methylbut-3-en-1-yl)-7-methoxy-2*H*-chromen-2-one, [Fig. 1](#f1){ref-type="fig"}), in a mice model of vincristine-induced neuropathic pain. We also found that a selective serotonin 5-HT~1A~ receptor antagonist, WAY100635, significantly antagonized the antinociceptive effect of auraptenol, suggesting that the observed antinociceptive effect of auraptenol was partially mediated by 5-HT~1A~ receptors. Results ======= Daily vincristine treatment (0.5 mg/kg) for 5 days led to marked mechanical hyperalgesia in mice as measured by von Frey filament ([Fig. 2](#f2){ref-type="fig"}). Paired t-test revealed that vincristine treatment produced a significant decrease in the paw withdrawal threshold (t (7) = 12.56, P \< 0.0001). In addition, repeated test every 10 min over a period of 100 min did not alter the hyperalgesic condition, which remained significantly lower than the baseline measurement prior to vincristine treatment ([Fig. 3](#f3){ref-type="fig"}). Two-way ANOVA revealed a significant main effect of vincristine treatment (F \[1, 63\] = 87.28, P \< 0.0001). Post hoc analysis found that throughout all the time points the paw withdrawal threshold was significantly lower after vincristine treatment (P \< 0.05). Auraptenol dose-dependently increased the paw withdrawal threshold in mice ([Fig. 3](#f3){ref-type="fig"}). A smaller dose of auraptenol (0.05 mg/kg) did not significantly elevate the paw withdrawal threshold. Two-way ANOVA revealed no significant main effect of auraptenol treatment (F \[1, 63\] = 0.72, P \> 0.05). A larder dose of auraptenol (0.2 mg/kg) markedly and significantly increased the paw withdrawal threshold. Two-way ANOVA revealed significant main effect of auraptenol treatment (F \[1, 63\] = 24.36, P \< 0.0001). Multiple comparison analysis found that the paw withdrawal threshold was significantly increased throughout the 20--80 min time period. When the dose of auraptenol was further increased to 0.8 mg/kg, the paw withdrawal threshold was significantly increased the pre-vincristine treatment level ([Fig. 3](#f3){ref-type="fig"}).Two-way ANOVA revealed significant main effect of auraptenol treatment (F \[1, 63\] = 87.28, P \< 0.0001). Multiple comparison analysis found that the paw withdrawal threshold was significantly increased throughout the 10--90 min time period. In order to understand the receptor mechanism underlying the anti-hyperalgesic actions of auraptenol, a dose of the selective serotonin 5-HT~1A~ receptor antagonist WAY-100635 was studied in combination with 0.8 mg/kg auraptenol ([Fig. 4](#f4){ref-type="fig"}). WAY100635 significantly attenuated the anti-hyperalgesic effects of auraptenol. Two-way ANOVA revealed that there were significant main effects of WAY100635 treatment (F \[9, 126\] = 47.52, P \< 0.0001) and time (F \[9, 126\] = 22.15, P \< 0.0001). Post hoc analysis found that the anti-hyperalgesic effect of auraptenol was significantly decreased across the 10--90 min time period. We also studied the anti-hyperalgesic actions of daily repeated auraptenol treatment ([Fig. 5](#f5){ref-type="fig"}). Daily treatment with 0.8 mg/kg auraptenol, a dose that completely reversed mechanical hyperalgesia, maintained its anti-hyperalgesic effect and no significant antinociceptive tolerance was observed. Two-way ANOVA revealed a significant main effect of auraptenol treatment (F \[1, 7\] = 464.8, P \< 0.0001), but no significant main effects of time or interaction were found. Post hoc analysis found that the paw withdrawal threshold after 0.8 mg/kg auraptenol treatment was significantly higher as compared to the daily pre-drug treatment baseline. In addition, the anti-hyperalgesic effect among the 7 daily treatments was not significantly different. The potential effect of auraptenol on the general locomotor activity in naïve mice was examined with different doses of auraptenol ([Fig. 6](#f6){ref-type="fig"}). It was found that auraptenol did not significantly alter the locomotor activity in mice across a dose range of 0.05--0.8 mg/kg. One-way ANOVA found no significant difference (F \[3, 31\] = 0.21, P \> 0.05). Discussion ========== In this study, we reported that an active component from the plant angelicae dahuricae radix, auraptenol, produced robust anti-hyperalgesic effect in a mouse model of chemotherapy-induced neuropathic pain. We also reported that the anti-hyperalgesic effect was at least partially mediated by 5-HT~1A~ receptors and the effect was not due to general behavioral impairment. Although angelicae dahuricae radix was used for the treatment of various diseases in traditional Chinese medicine, this is the first study that identified the antinociceptive active component that may explain the pain relieving effect of this plant and this important herbal medicine. In addition, these results encourage continued effort to better understand auraptenol, which may well serve as a potential novel analgesic for the control of chronic neuropathic pain. Many microtubule-targeting cancer chemotherapeutic agents including vincristine are widely recognized to cause peripheral and cranial neuropathy[@b7][@b8][@b9]. In an effort to better understand this form of neuropathy and develop novel treatment for its management, several animal models of chemotherapeutic agent-induced neuropathy was developed[@b10][@b11][@b12]. Rodents treated with chemotherapeutic agents typically develop thermal and mechanical hyperalgesia. In consistency with the literature, we found that mice treated with 0.5 mg/kg daily for 5 days developed a reliable mechanical hyperalgesia as measured by von Frey filament test. Repeated measures within a short period of time (100 min) did not significantly change the test results, which offers an opportunity to determine the duration of actions of the study drug. We found that auraptenol produced a very robust effect in decreasing mechanical hyperalgesia. This effect was both dose-dependent and time-dependent and at larger doses it completely reversed the mechanical hyperalgesia. Although angelicae dahuricae radix was used in folk medicine for centuries, and modern pharmacological studies confirmed its analgesic actions[@b3][@b4][@b13], the active components have not yet been identified. This study clearly demonstrated that one major coumarin component from angelicae dahuricae radix, auraptenol, has very robust antinociceptive effect in a mouse model of chronic neuropathic pain, marking the first effort to decipher the phytochemical substrates of angelicae dahuricae radix-induced analgesia. More importantly, repeated treatment with auraptenol did not show evidence of tolerance development. Considering the long-term therapeutic need to treat neuropathic pain, this lack of tolerance development is significant and clearly puts auraptenol in an advantageous position as a potential analgesic. Serotoninergic (5-HTergic) system is critically involved in pain modulation[@b14]. Indeed, the serotonin-norepinephrine reuptake inhibitor duloxetine has been approved to treat several chronic pain conditions including peripheral neuropathy and fibromyalgia[@b15][@b16]. In addition, 5-HT~1A~ receptor agonists demonstrate robust antinociceptive effect in animal models of chronic neuropathic pain[@b17][@b18][@b19]. This study found that a selective 5-HT~1A~ receptor antagonist, WAY-100635, significantly blocked the anti-hyperalgesic effect of auraptenol, suggesting that the anti-hyperalgesic action of auraptenol is primarily mediated by 5-HT~1A~ receptors. This dose of WAY-100635 (1 mg/kg) has been shown to significantly block 5-HT1A receptors in other studies[@b20][@b21]. In summary, this study for the first time identified an active component of angelicae dahuricae radix, auraptenol, which may be responsible for the analgesic actions of angelicae dahuricae radix. In a mouse model of chemotherapeutic agent-induced neuropathic pain, auraptenol demonstrated excellent analgesic activity with no apparent adverse effects. Although more studies are needed to examine the generality of these findings and to better understand the potential toxicology of this compound, the current data do suggest that auraptenol could be a potential novel analgesic for pain management. Methods ======= Animals ------- Male C57BL/6 mice weighing 16--22 g (Weitong Lihua, Beijing, China) were acclimated to the temperature, humidity and lighting (12 h light/dark cycle, lights on at 7:00 AM) controlled vivarium and housed in groups of four for at least one week before behavioral studies began. The animals had free access to dietary food and water except during the test sessions. All animal experimental protocols were approved by the Institutional Animal Care and Use Committee, Xinxiang Medical University. Animals were maintained in accordance with the *Guide for the Care and Use of Laboratory Animals* (8^th^ edition, Institute of Laboratory Animal Resources on Life Sciences, National Research Council, National Academy of Sciences, Washington DC). All efforts were made to minimize animal suffering and to reduce the number of animals used. Drugs ----- Vincristine sulphate injection was purchased from Haimen Pharmaceutical Co. (Zhejiang, China). Auraptenol (8-(2-hydroxy-3-methylbut-3-en-1-yl)-7-methoxy-2*H*-chromen-2-one, [Fig. 1](#f1){ref-type="fig"}) was purchased from Shanghai Lei Yun Shang Pharmaceutical Co. (\>95% purity, Shanghai, China). WAY100635 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Auraptenol was suspended in 5% DMSO. WAY100635 was dissolved in 0.9% saline. All injections were given intraperitoneally in a volume of 1 ml/100 g of body weight. Vincristine was administered at a dose of 0.5 mg/kg daily for 5 days to establish vincristine-induced neuropathy. Mechanical allodynia measurement -------------------------------- Mechanical allodynia was assessed prior to and after 5 days of vincristine treatment daily using Von Frey filaments of varying forces (0.07--4.0 g) applied to the mid-plantar surface of the right hind paw, with each application held until curved for 6 s using the up-down method[@b6]. Mice were placed in individual Plexiglas compartments atop of a wire grid floor suspended 50 cm above the laboratory bench top and acclimated to the environment for 30 min prior to each test session. For the time course studies, baseline von Frey filament measurement was immediately followed by an injection of auraptenol, and then the paw withdrawal threshold was measured every 10 min until the drug effect dissipated to a point that the paw withdrawal threshold was not significantly different from the pre-drug data. In studies that test the effect of the antagonist WAY100635, drug was administered 5 min prior to auraptenol treatment and a time course measurement was followed. For repeated treatment studies, mice were measured daily before drug treatment and 40 min after drug treatment for 7 days. Locomotor activity test ----------------------- The locomotor activity of naïve mice treated with vehicle or auraptenol was measured automatically with a Small Animal Locomotion Recording Apparatus (Shandong Academy of Medical Sciences, China), which consisted of six acrylic boxes and in each box there was one pyroelectric infrared sensor 4 cm above the floor. The sensor could detect the movements of the mice through infrared radiation. The apparatus recorded only gross movements of the mice, whereas small movements such as gnawing or grooming could not be differentiated and recorded. Data analyses ------------- For the mechanical hyperalgesia test prior to and 5 days after vincristine treatment, data were analyzed using paired t-test. For the antinociceptive studies, data were presented as paw withdrawal threshold (grams) plotted as a function of time (min or days), respectively. Data were analyzed by two-way repeated measures analysis of variance (ANOVA) (time × auraptenol treatment or time × vincristine treatment) followed by post hoc Bonferroni test. For the locomotion tests, data were analyzed with one-way ANOVA followed by post hoc Bonferroni test. Author Contributions ==================== X.Z. and P.L. designed the research; Y.W., S.C., J.T. and G.L. conducted the studies; X.Z. and P.L. analyzed the data and prepared the manuscript; all authors read and approved the manuscript. This study was supported by an intramural research grant from Xinxiang Medical University (ZD200931). {#f1} {#f2} {#f3} {#f4} {#f5} {#f6}

I. Introduction =============== In countries with advanced and modern healthcare systems, hospital pharmacies and their pharmacists play important roles in patient care \[[@B1]\]. In recent years, with the advancement of health information technology (HIT), there has been increasing attention to the adoption of the pharmacy information system (PIS) to improve the efficiency and effectiveness of services \[[@B2]\]. For example, the PIS has key roles to play in reducing medication errors and improving patient safety \[[@B3][@B4]\]. The PIS is a system that supports the distribution and management of drugs, identifying the type of intervention, determining the amount of inventory, reporting and managing of costs, and improving the accessibility of information \[[@B5]\]. Furthermore, the PIS helps clinical decision-making by alerting users about clinically important drug-drug interactions, drug allergies \[[@B6]\], and drug doses \[[@B7]\]; it also evaluates patterns of drug use \[[@B8]\] as well as other possible side effects of drugs. The PIS may operate as a separate and individual system or as part of a hospital information system (HIS), paired with the Computerized Physician Order Entry (CPOE) system \[[@B7]\]. Accordingly, to ensure efficiency and effectiveness of these systems, evaluation of the PIS is extremely important; this system could ultimately influence the safety and quality of care \[[@B9]\]. To identify and eliminate technical problems of health care systems, improve the efficiency and effectiveness of services, and minimize costs, careful evaluation of those systems is needed. In other words, the evaluation of information systems, as a key stage in the information system development life cycle \[[@B10][@B11]\] can help to assure the technical capacity of these systems, determine the effects of using the systems on users practices, and allow application of modifications as required \[[@B12][@B13]\]. Therefore, this study aimed to evaluate PISs in hospitals affiliated with three universities of medical sciences in Tehran, Iran. II. Case Description ==================== The study population included the PIS implemented in hospitals affiliated to one of the three medical universities in Tehran, Iran (Tehran, Shahid Beheshti, and Iran Universities of Medical Sciences and Health Services). In Iran, more than 80% of hospital services are provided by the public sector. Medical education is integrated with the health services, which have created universities of medical sciences and health services. Each province has at least one such university. Tehran, which is a huge province with more than 14 million inhabitants, is divided into three zones. The three abovementioned universities provide public health and treatment services to the citizens of Tehran. Since almost all of the affiliated hospitals implement a PIS from one of the well-known Iranian vendors, the sampling took place by considering two inclusion criteria: (i) there was at least one general teaching hospital for every vendor in which its PIS software was installed and (ii) hospitals were selected based on the number of hospital beds (the hospitals with the highest bed numbers). If a selected hospital was unwilling to take part in the study, the next hospital with the highest bed number was substituted. Selected hospitals provided 24 hr/7 days a week pharmacy services to in- and out-patients. The evaluation tool was a checklist created by reviewing the relevant literature; it was composed of general and specific evaluation criteria related to PIS \[[@B3][@B4][@B5][@B14][@B15][@B16][@B17]\]. There were sub-categories for both general and specific criteria. Categorizing of the criteria and the related aspects was performed according to similarity and relevance. The general evaluation criteria included those aspects that are general to all information systems, such as security, user friendliness, and integration with other systems. The specific criteria evaluated those aspects that were specifically important for PISs. These specific criteria were divided into five categories including prescriptions and medication order management, patient safety management, purchase and sale management, drug stock management, and reports management. The checklist was constructed with \'Yes\' and \'No\' options and additional space was provided for any possible applicable comments. The content validity of the checklist was examined by five experts in the field of medical informatics and by two pharmacists. Five PIS supplied by five vendors were evaluated in five general-teaching hospitals. Hospitals are called A (716 beds), B (426 beds), C (139 beds), D (111 beds), and E (379 beds). Similarly, the five software programs used in the hospitals are described as A, B, C, D, and E, respectively. The findings are presented in sections. Section A reports the findings related to the general criteria, and section B describes the findings based on the specific criteria used to evaluate the PIS. 1. General Criteria in PIS Evaluation ------------------------------------- With respect to the security aspects of the PIS, all systems had the ability to report user activities based on IP address or individual user ID. However, the capability of restricting repeated unauthorized access attempts to systems was observed only in 40% of the systems. The password strength was the other security sub-criteria; the findings indicated that the passwords of the systems were not case-sensitive and did not require a combination of letters and numbers ([Table 1](#T1){ref-type="table"}). The findings related to \'user friendliness\' showed that all systems included some user friendliness features for pharmacy end-users. In this respect, all systems had the capability of displaying patients\' drugs and demographic profiles completely and legibly; systems also had flexibility in sorting and selecting of commands from drug profiles, modifying the screen size, using multiple screens simultaneously for various activities, and using defaults to identify commands or a group of commands during order entry. Regarding the other general criteria, the findings showed that none of the pharmacy systems were connected to the national drug databank. It is notable that, in all five hospitals, the PIS was a sub-system of the integrated HIS and substantially interacted with other HIS sub-systems. 2. Specific Criteria in PIS Evaluation -------------------------------------- All of the studied systems had the ability to create reports of drugs and their dosages based on the physicians\' name and the prescription date. Furthermore, the systems had the capability to customize the list of medications and to exclude a drug from the list of current medications. However, the systems lacked functions such as showing the history of prescribed medication, prescribing drugs using different units, and renewing current medications without re-entering orders ([Table 2](#T2){ref-type="table"}). The findings related to the \'patient safety management\' showed that all five systems had the capability of identifying the drug dose and modifying medication orders. However, none of the systems had the functionality to display contraindications, drug interactions, adverse effects, or patient allergy to drugs ([Table 3](#T3){ref-type="table"}). With respect to the \'purchase and sale management\' function, as can be seen in [Table 4](#T4){ref-type="table"}, none of the systems included the ability to order drugs electronically. However, all systems had the capability of calculating the prices for drugs and medical devices. Findings related to \'drug stock management\' showed that one of the systems (system D) lacked the stock management function. However, the other four systems had the capability of controlling drug entry to, and exit from, the pharmacy. In addition, these systems had the ability to check the minimum inventory for each drug and to create inventory alerts when a drug reached a minimum stock level. Regarding \'management reports\' function of the systems, the findings indicated that all of the five PISs gave the system administrator the capability to create different reports using dynamic report builders. The reports included but were not limited to the total number of prescribed drugs for both inpatients and outpatients, daily drug distribution according to the delivery location, financial reports, and annual performance reports. III. Discussion =============== According to the findings, in terms of the security of the PIS, the users did not use appropriate passwords (a combination of capital and small letters, or a mixture of letters and numbers) to log into the system. This issue is considered a potential risk as unauthorized users could more easily gain access to confidential information stored in the system. According to studies conducted in other countries, the security of information systems will improve if a password is composed of a combination of numbers and letters \[[@B14][@B15]\]. In addition, in the current study, only two systems had the capability to restrict repeated attempts by unauthorized users to enter the system. Troiano \[[@B16]\] suggest that a powerful security system is one in which the system administrator can determine the activity limit of users on the system and their accessibility. This finding is parallel to that of the current study, in which the administrators of the systems, based on their organizational roles in all five systems, was able to define users\' access levels. In terms of integration of systems, none of the PIS were connected with the national drug data bank, though, based on the law, it is not mandatory to do so. On-line updates through the linkage with the national drug information system may result in better patient safety. For example, the database of the Federal Union of the German Association of Pharmacists, known as ABDA, and the drug database of the United States (First Databank) are two that are connected with the national drug information banks of their countries; their data are updated through these databases \[[@B17][@B18]\]. These databases include such things as drug interactions, financial information, drug prices, and drug side effects and possible adverse reactions \[[@B19]\]. Therefore, the lack of connection between the pharmacy information systems and the Iranian National Drug Information Bank may result in an increased number of errors, a reduced level of patient safety, and a lack of precise information about drug prices. This issue is considered one of the weaknesses of the existing software systems. In terms of \'prescriptions management\' and \'patient safety management\', more attention was apparently paid to prescription order entries and delivery methods than to factors such as drug dose and frequency control, cautions regarding drug usage, prevention of duplication of medication orders, evaluation of drug interactions, and reporting of adverse drug reactions and allergies. This finding indicates that in the design of these systems, the vendors did not consider prescription management or patient safety requirements. This finding is in contrast with the study conducted by Hines et al. \[[@B20]\], who reported that the PIS under study had features such as drug interaction and contraindication alerts. All drug allergies and contraindications can be added as features to the existing PISs. Furthermore, the integration of the HIS system with CPOE and clinical decision support systems can help to improve patient safety \[[@B21]\]. In fact, the CPOE system is a part of clinical information systems (CIS) that is connected with other information systems such as the PIS \[[@B19][@B22]\]. When the HIS does not provide this feature, many interconnected sub-systems such as PIS would not benefit from the integration. The majority of PISs in Iran are only designed to meet the financial aspects; the clinical aspects that can result in reduction of medication errors have been neglected. This finding is in line with the results of a study conducted by Alkelya \[[@B23]\], in which the limited capability of the PIS in supporting clinical tasks was reported as a key problem of these systems. In this study, requests to purchase medicines, medical devices, and medical consumer goods from pharmaceutical companies and their distributors in the studied hospitals were made via fax or by sale representatives when they were visiting the hospitals. In other words, none of the studied hospital pharmacies was electronically connected to the producers or their distributors. Therefore, orders of drugs, medical devices, and medical consumer goods were not recorded electronically. This is one of the weaknesses of the majority of pharmacy information systems in Iran. This finding is supported by Asadi et al. \[[@B24]\]\'s study, in which it was found that only one of the pharmaceutical companies had semi-computerized communication with the studied hospital pharmacy for marketing and sales purposes. However, in some developed countries, the pharmacy information system communicates electronically with pharmaceutical companies or their distributors, which reduces purchase and deposit process errors \[[@B25]\]. According to these findings, the evaluated systems had the capability of displaying the minimum inventory for each drug, defining different warehouses, and controlling the entry and exit of drugs from the pharmacy. This is in line with El-Mahalli et al. \[[@B26]\]\'s study, in which inventory and purchasing management were considered as core functions of PIS systems. For online checking of inventory and distribution of medicines and equipment, Mahoney et al. \[[@B27]\] state that pharmacy software systems need to be integrated with other hospital sub-systems. In the present study, one of the systems did not even have a \'purchase and sale management\' function. In the hospital using the mentioned system, a separate system was in use along with the PIS; this separate system controlled the inventory and the expiration date of drugs, and this system had no linkage with the PIS. Therefore, it was impossible to compare minimum inventories for drugs and to alert staff when order limits were reached. As these findings indicated, all of the studied hospital PISs were equipped with dynamic report builders and were capable of providing customized reports on such topics as the total number of drug deliveries, the financial earnings of the pharmacies, the pharmacies annual performance, and various reports as required by the system administrator. This finding is supported by Isfahani et al. \[[@B28]\]\'s study, which reported that the PIS understudy were capable of producing the reports required. This function plays an important role in managerial decision-making. Furthermore, insurance companies increasingly ask pharmacies for various reports, and their information needs are constantly changing. Therefore, dynamic report creation and the ability to customize reports are key requirements of pharmacy systems. **Conflict of Interest:** No potential conflict of interest relevant to this article was reported. ###### Distribution of sub-criteria related to \'security\' among the evaluated pharmacy information system  ###### Distribution of sub-criteria related to \'prescriptions and medication order management\' among the evaluated pharmacy information system  ###### Distribution of sub-criteria related to \'patient safety management\' among the studied pharmacy information system  ###### Distribution of sub-criteria related to \'purchase and sale management\' among the evaluated pharmacy information system 

Introduction {#s1} ============ Several studies have investigated the effects of the extracts of flavonoid-rich plants or flavonoid molecules as potent modulators of brain structure and function, including their neuroprotective and chemopreventive properties and their beneficial effects on memory and cognition. The effects of flavonoids have been correlated with their ability to modulate the phosphorylation state of intracellular proteins via the activation or inhibition of protein kinases and phosphatases (Gamet-Payrastre et al., [@B42]; Wang et al., [@B117]; Schroeter et al., [@B102]; Li et al., [@B65]; Hoffman et al., [@B47]; Joseph et al., [@B57]; Maher et al., [@B70]; Nakajima et al., [@B87]; Spencer, [@B108]; Vauzour et al., [@B112]; Williams et al., [@B119]; Lovera et al., [@B69]; Mansuri et al., [@B73]), to increase the level of 5-HT and its metabolites (Zhang et al., [@B124]) or to alter expression of GABA~A~ receptors (GABA~A~Rs) and/or glutamatergic N-methyl-D-aspartic acid (NMDA) receptors (NMDARs) (Wang et al., [@B115], [@B116]; Rendeiro et al., [@B92]). In addition, studies addressing the effects of specific flavonoid subgroups, including flavanols, anthocyanins, flavanones, and flavones, have shown that these constituents display potential to act as cognition-enhancing and neuroprotective agents (Vauzour et al., [@B112]; Kehr et al., [@B59]; Rendeiro et al., [@B94], [@B92]; Vauzour, [@B111]), to prevent many forms of cerebrovascular disease, or to function as anti-anxiety drugs (Hasenöhrl et al., [@B46]; Spencer, [@B107]; Zhang et al., [@B124]). Although studies *ex vivo, in vivo*, and *in vitro* have provided evidence supporting the effects of flavonoids on the central nervous system, the cellular, and molecular pathways through which these compounds modulate memory formation are not completely elucidated (Youdim et al., [@B122]; Nakajima et al., [@B87]; Spencer, [@B108]; Williams et al., [@B119]; Ballesteros et al., [@B9]; Kimura et al., [@B62]; Rendeiro et al., [@B92]). However, several studies have established that the hippocampus, which plays a central role as a substrate of fear memory and anxiety (Fendt and Fanselow, [@B40]; Sanders et al., [@B99]) and which is a component of the Behavioral Inhibition System (McNaughton and Gray, [@B76]), appears to be a target for the mnemonic effects of flavonoid metabolites (Bannerman et al., [@B10]; Wang et al., [@B118], [@B114]; Williams et al., [@B119]; Rendeiro et al., [@B93], [@B92]; Oliveira et al., [@B88]; Vauzour, [@B111]). Previous studies from our laboratory have demonstrated the role of flavonoid-rich plant extracts, such as a standardized extract of *Ginkgo biloba* L. (EGb), in the modulation of fear memory (Oliveira et al., [@B89], [@B88]) by inducing differential CREB-1, GAP-43, and GFAP gene and protein expression in the dorsal hippocampus (DH), the prefrontal cortex and the amygdaloid complex. Further, we have established that crude extracts, fractions, and flavonoid molecules isolated from the stem bark of *Erythrina falcata* (CE) improved the acquisition of conditioned fear as evaluated by single-trial, step-down inhibitory avoidance (IA) (de Oliveira et al., [@B32]). Additionally, we used an IA procedure to show for the first time that treatment with flavones produces another well-established conditioning phenomenon, spontaneous recovery (de Oliveira et al., [@B32]). These findings corroborate with the results described in the literature and expand the understanding that flavonoids act as cognition-enhancing agents. However, these results raise new questions, which are highlighted below. The first question concerns the anti-anxiety properties and cognitive effects of the flavonoid-rich fraction from CE, given the various actions of flavonoids on the central nervous system. Despite the close relationship between fear memory and anxiety, these functions are dissociable at the behavioral, pharmacological, molecular, and neuroanatomical levels (McNaughton and Corr, [@B75]; Kalueff, [@B58]; Nakajima et al., [@B87]). The conditioned emotional response (CER) is a suitable animal model for studying the behavioral, pharmacological, and molecular mechanisms underlying fear memory and anxiety. To assess these phenomena, our lab has used the conditioned suppression of the lick response, in which the conditional stimulus (CS, tone), when associated with a noxious unconditioned stimulus (US, footshock), ultimately suppresses the licking response reinforced by water; i.e., the CS leads to the suppression of the ongoing behavior (Blanchard and Blanchard, [@B15]; Bolles and Collier, [@B17]; Fanselow, [@B37]; Sotty et al., [@B106]; Sanders et al., [@B99]). Fear responses (flight/fight/freezing) increase systematically as fear memory is acquired and decrease as fear memory is extinguished (Sotty et al., [@B106]; Liu et al., [@B68]; Apergis-Schoute et al., [@B6]; Davis, [@B29]; Erlich et al., [@B36]; Furini et al., [@B41]). Conditioned fear responses are insensitive to anxiolytic drugs (McNaughton and Corr, [@B75]), but several works show that treatment with diazepam, an anxiolytic drug that is widely used in the clinic, prior to the conditioning session disrupts the initial acquisition of learned fear (Jensen et al., [@B54]; Izquierdo and Medina, [@B52]; Makkar et al., [@B71]), decreases the occurrence of freezing responses in a dose-dependent manner in rats (Fanselow and Helmstetter, [@B39]; Decker et al., [@B33]; Beck and Fibiger, [@B12]; Malkani and Rosen, [@B72]; Isoardi et al., [@B49]; Yeh et al., [@B121]) and impairs the acquisition of conditioned suppression (Oliveira et al., [@B89]). Anxiolytic compounds were effective in reducing the inhibitory response of animals to an aversive stimulus, which alleviated the suppression of the CER (McNaughton and Gray, [@B76]; Miyamoto et al., [@B81]; George et al., [@B44]). McNaughton and col. showed that anxiolytic drugs reduced theta frequency in the hippocampus (Coop et al., [@B26]; Munn and McNaughton, [@B84]). In this sense, the sensitivity of the CER to anxiolytic drugs, such as benzodiazepines and agonists of 5-HT~1A~ receptors (5-HT~1A~Rs) (Millenson and Leslie, [@B79]; Davis, [@B28]; Stanhope and Dourish, [@B109]; Mirza et al., [@B80]; George et al., [@B44]; Oliveira et al., [@B89]), substantiates the use of this model to investigate the fundamental mechanisms underlying the effects of anti-anxiety drugs in addition to their function in alleviating conditioned fear in rodents. The second question concerns the neurochemical mechanisms underlying both the acquisition and the extinction of conditioned suppression, as well as the role of the flavonoid-rich fraction from the stem bark of *Erythrina falcata* (FfB) in modulating these processes. We primarily focused on the molecular events underlying the acquisition of fear memory and the modulatory effects of FfB. Further, we were interested in determining whether treatment with flavonoids prior to conditioning can modulate the extinction process. Studies demonstrating the involvement of glutamatergic, serotoninergic and GABAergic neurotransmission in the acquisition of fear memory have been accumulating in past decades; the major targets of these neurotransmitters are NMDARs, 5-HT~1A~Rs, and GABA~A~Rs, respectively (Santini et al., [@B100]; Davis and Myers, [@B30]; Lin et al., [@B66]; Quirk and Mueller, [@B91]; Kim and Richardson, [@B60]), and the modulation of these receptors in the hippocampus is essential for the acquisition and consolidation of fear memory (Izquierdo, [@B50]; Cammarota et al., [@B20]; Alonso et al., [@B3]; Milad et al., [@B78]). Similarly, these changes are essential to consolidation of fear extinction (Myers and Davis, [@B85]). These effects are mediated by the activity of kinases and phosphatases, and ERK1/2 activation has been described to be involved in several cellular changes associated with long-term memory (LTM) (Atkins et al., [@B8]; Cammarota et al., [@B20]). Blocking NMDARs in the prefrontal cortex and the hippocampus is known to result in a deficit in the acquisition of fear extinction (Lissek and Güntürkün, [@B67]) and the retrieval of fear extinction (Lengersdorf et al., [@B63]). Evidence from *in vitro* and *in vivo* studies showed that flavones modulate GABA~A~Rs and GluN2B-NMDARs, but few studies have been conducted on the mechanisms underlying the modulatory effects of flavonoids on these processes. Therefore, in our study, we sought to elucidate the neurochemical systems involved in the acquisition of fear memory in the presence or absence of FfB treatment and to determine whether FfB treatment prior to conditioning modulates the extinction of fear memory. Further, we evaluated how these changes may control or be controlled by the activation or inhibition of specific receptors using pharmacological agonists or antagonists. Therefore, the contributions of the glutamatergic, serotoninergic, and GABAergic systems, as well as the interactions between these systems, to the effects of FfB on the acquisition and extinction of conditioned suppression were assessed for the first time by administering agonists, antagonists of receptors for GABA, glutamate (NMDA) and 5-HT or one these antagonists before FfB prior the conditioning session. Additionally, we evaluated the mRNA expression levels of the GluN2A and GluN2B subunits of the NMDAR, the receptor subunits GABA~A~R and 5-HT~1A~R and ERK1/2 in the DH of controls and treated rats subjected to acquisition and extinction of conditioned fear. This combination of molecular, behavioral and pharmacological analyses advances our understanding of the role of flavones in fear memory and anxiety. The findings regarding the molecular mechanisms of flavone action appear to be promising with respect to the development of new therapeutic strategies for the treatment of cognitive deficits or anxiety disorders. Moreover, we assessed the contribution of the hippocampus to these processes. In particular, we focused on the suppression of the licking response as a behavioral model and the hippocampus as a key component of the neural circuitry involved in the acquisition, consolidation and extinction of fear memory in animals and humans, as the hippocampus may represent a target for the action of FfB. Experimental procedures {#s2} ======================= Drugs and reagents ------------------ Methanol (HPLC grade) was obtained from Merck (Darmstadt, Germany). Formic acid, ethanol, *n*-butanol, and Tween®-80 were obtained from Synth (Diadema, Brazil). Vitexin and isovitexin standards (99.99%) were purchased from Sigma-Aldrich (São Paulo, Brazil). The 6-C-glycoside-diosmetin and vicenin-2 standards were generated in our laboratory according to the methods described by de Oliveira et al. ([@B32]). Valium® (diazepam) was purchased from Roche (São Paulo, Brazil). Sintocalmy® (standardized extract of *Passiflora incarnate* L.---extract ACH 06) was obtained from Aché (Guarulhos, Brazil). Ro25-6981, picrotoxin and (S)-WAY100135 were purchased from Tocris Biosciences (Ellisville, MO, USA). NMDA was obtained from Sigma-Aldrich (São Paulo, Brazil). Buspirone hydrochloride was obtained from LIBBS Pharmaceutical Ltd (São Paulo, Brazil). ### Standardized FfB preparation FfB was obtained by flash chromatography, as previously described by de Oliveira et al. ([@B32]). Additionally, the FfB was analyzed using high-performance liquid chromatography (HPLC) combined with electrospray ionization tandem mass spectrometry (HPLC-ESI/MS^n^) using a Thermo LCQ Fleet System mass spectrometer (Thermo Scientific, San Diego, CA, USA) equipped with an electrospray interface (ESI) and an HPLC (model Accela, Thermo Scientific). FfB separation was performed using a Luna® C18 column (250 × 4.60 mm; Phenomenex, Torrance, CA, USA) at room temperature. The mobile phase consisted of 0.1% aqueous formic acid-water (A) and methanol (B). A gradient elution method of A/B (from 64:36 to 1:1, v/v) was applied over 50 min. Ultraviolet (DAD) detection was performed at 330 nm; the flow rate was maintained at 0.8 mL/min; the sample concentrations were 1 mg.mL^−1^; and the injection volume was 10 μL. The column effluents were analyzed by ESI-MS in negative ion mode in the mass-to-charge ratio (*m/z)* range of 50--2000, with a scan time of 0.3 s in the centroid mode. The ESI conditions were as follows: nebulizer gas (nitrogen), 30 psi; drying gas, 60 L.min^−1^; drying temperature, 280°C; capillary voltage, 4000 V; collision gas, nitrogen; and collision energy,1 V. The data were acquired in the MS and MS^n^ scanning modes. The CE was dissolved in H~2~O: MeOH (1:1v/v) and was infused directly via a syringe pump (flow rate 5 μL.min^−1^) in the ESI source. The data were analyzed using Xcalibur 2.0 Software® (Thermo Scientific). The flavonoids present in the FfB were quantified by HPLC-DAD using a Luna® C18 column (Phenomenex, Torrance, CA, USA; 250 mm × 4.60 mm, 5 μm). The mobile phase consisted of 0.1% aqueous formic acid (A) and methanol (B). An isocratic elution method of A/B (64:36, v/v) was applied for 50 min. UV spectra were recorded from 200 to 400 nm, and the chromatogram was monitored at 254, 280, and 330 nm. The flow rate was maintained at 1 mL.min^−1^; the sample concentration was 1 mg.mL^−1^; and the injection volume was 20 μL. Analytical curves were obtained for vitexin, isovitexin, vicenin-2, and 6-C-glycoside-diosmetin (1 mg.mL^−1^ of each compound in 80:20 methanol/water), which peaked at concentrations ranging from 100 to 1.000 mg.mL^−1^. The sample peak areas were integrated at 254 nm. All of the procedures were performed in triplicate. Behavioral and pharmacological effects of acute treatment with FfB before conditioning on the acquisition and extinction of conditioned suppression --------------------------------------------------------------------------------------------------------------------------------------------------- ### Subjects A total of 470 adult male *Wistar* rats (±250--300 g) were obtained from the Center for the Development of Experimental Medicine and Biology (CEDEME, Federal University of Sao Paulo, SP, Brazil). The rats were housed 5 animals/cage. For 15 days, the animals had free access to food and water under a 12 h:12 h dark:light cycle (lights on at 6:00--18:00 h) at a controlled temperature (21°C ± 2°C) and relative humidity (53 ± 2%). These conditions were maintained throughout the experimental period. One minute prior to the experimental sessions, each rat was placed in an individual cage for transportation to the testing room. All of the procedures for manipulation of the animals were consistent with the Ethical Principles in Animal Research adopted by the Brazilian College for Animal Experimentation (COBEA) and were performed as suggested by the APA Guidelines for Ethical Conduct in the Care and Use of Animals. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Federal University of Sao Paulo (Permit Number: 840560). After completion of the behavioral experiments, the animals were decapitated, and the DH was extracted within 40--60 s using a magnifying glass, immediately frozen on dry ice, and maintained at −80°C until gene expression analysis. All behavioral procedures were conduced during the light phase of the dark:light cycle, and all efforts were made to minimize suffering. ### Systemic administration Diazepam and buspirone hydrochloride (a GABA~A~R and a 5-HT~1A~R agonist, respectively), Sintocalmy®(a standardized extract of *Passiflora incarnata* L.-extract ACH 06, containing 7% (21 mg) total flavonoids expressed as vitexin) and three different concentrations of FfB were dissolved in 12% Tween®-80 and administered orally via intragastric gavage (IG) 30 min before each conditioning session. The GABA~A~R, 5-HT~1A~R, and GluN2B-NMDAR antagonists (picrotoxin, S-WAY 100135 and Ro25-6981, respectively) and NMDA (an NMDAR agonist) were dissolved in saline and injected intraperitoneally (i.p.) 20 min before each conditioning session or prior to treatment with FfB. When an antagonist was administered before with FfB, the drugs were administered −50 or −30 min before conditioning, respectively. No drugs were administered before the retention test, extinction training or the extinction retention test. The drugs were administered i.p. or IG in a volume of 1 mL. The doses, administration routes and vehicles used to dissolve of the antagonists and agonists were chosen based on previous reports (Aguilar et al., [@B2]; Risbrough et al., [@B96]; Oliveira et al., [@B89]). Experimental procedure ---------------------- Rats were randomly assigned to the control group or the FfB group (*n* = 20 per subgroup) (Table [1](#T1){ref-type="table"}). The control group was subdivided into 12 subgroups as follows: (i) the paired stimulus conditioned/unconditioned stimulus (CS-US) subgroup; (ii) the unconditioned subgroup \[no footshock, i.e., only tone (CS); as such, these animals were used as controls for learning\]; (iii--iv) the negative control subgroups (12% Tween®-80 or saline); (v--xi) the positive control subgroups (4.0 mg.Kg^−1^ diazepam; 10.0 mg.Kg^−1^ NMDA; 10.0 mg.Kg^−1^ buspirone hydrochloride; 600 mg.Kg^−1^ Sintocalmy®; 0.75 mg.Kg^−1^ picrotoxin; 3.0 mg.Kg^−1^ Ro25-6981; or 0.3 mg.Kg^−1^ (S)-WAY 100135; these animals were used as controls for treatment with the respective drug together with FfB); and (xvii) a naïve subgroup (*n* = 10), which was used as a control for gene expression. The FfB groups were also divided into 12 subgroups as follows: (xiii--xiv) FfB alone (0.15 mg.Kg^−1^ FfB, 0.30 mg.Kg^−1^ FfB or 0.65 mg.Kg^−1^ FfB); (xv--xvii) picrotoxin+ FfB (Picro+0.15 mg.Kg^−1^ FfB; Picro+0.30 mg.Kg^−1^FfB; or Picro+0.65 mg.Kg^−1^ FfB); (xviii--xx) Ro25-6981+ FfB (Ro+0.15 mg.Kg^−1^ FfB; Ro+0.30 mg.Kg^−1^ FfB or Ro+0.65 mg.Kg^−1^ FfB); and (xxi--xxiii) (S)-WAY+ FfB \[(S)-WAY+ 0.15 mg.Kg^−1^ FfB; (S)-WAY+0.30 mg.Kg^−1^ FfB or (S)-WAY+0.65 mg.Kg^−1^ FfB\]. Half of the rats (*n* = 10/subgroup) were sacrificed after the retention test ended. The remaining half (*n* = 10/subgroup) were subjected to extinction training and an extinction retention test of the CER and were sacrificed 3 h after the conclusion of the extinction retention test. ###### **Experimental groups**.  ### Behavioral apparatus Rats were fear conditioned in a lick-operant chamber. Briefly, the experimental chambers consisted of an aluminum (side walls) and Plexiglas (ceiling and hinged front door) box measuring 25 × 25 × 20 cm set inside a sound-attenuation cabinet (53 × 65 × 50 cm). Three identical chambers and cabinets were used in all experiments. The floor consisted of stainless steel rods connected to grid shockers (model EP 107R, Insight, Ribeirão Petro, Brazil) set to deliver 0.4 mA, 0.5 s scrambled shocks, which were used as the US. A speaker positioned on top of the square, which produced a 2 kHz, 85 dB sound for 30 s, was used as the CS. A licking spout was slipped into the cage through a hole in the middle of the wall of the chamber; this hole protruded from the lateral wall 5.0 cm above the grid floor. Stimulus presentation and data recording were controlled using software (Refor II Software®, Insight) and a central controller box (Insight). The chambers were cleaned with 10% ethanol before each test. ### Behavioral procedure The behavioral procedure was conducted for 8 or 10 days, according to the experimental design, to assess the acquisition or extinction of a CER, respectively. All rats, except for those in the CS and naïve subgroups, were subjected to a procedure to induce acquisition of fear memory (*n* = 20/group) (Figure [1](#F1){ref-type="fig"}). Three hours after the completion of the fear acquisition test (8^th^ day), half of the rats were decapitated. Then, the DH was extracted within 40--60 s using a magnifying glass, immediately frozen on dry ice, and maintained at −80°C until gene expression analysis (acquisition analysis) (*n* = 3/subgroup). The remaining half of the animals (*n* = 10/subgroup) were subjected to extinction training (9^th^ day) and an extinction retention test (10^th^ day) performed on each of the two consecutive days following the acquisition test. Three hours after completing the extinction retention test, these rats were decapitated, and the DH was extracted as described above (*n* = 3/group). {#F1} #### Suppression of the licking response The animals were deprived of water on a daily basis for 12--16 h before all experimental sessions. For five consecutive days, the rats were placed individually in the chamber once a day for 20 min sessions with free access to the drinking spout to obtain a stable baseline of drinking behavior, but no other stimuli were presented (Figure [1A](#F1){ref-type="fig"}). After the administration of drugs or vehicle, each rat was gently placed in the experimental chamber, and after 5 min, the animal was submitted to four tone-shock (CS-US) pairings (fear conditioning, 6^th^ day; Figure [1B](#F1){ref-type="fig"}). Twenty-four hours after fear conditioning, the animals were subjected to reacquisition of the licking response sessions (7^th^ day) as performed during the acquisition of the licking response to re-establish drinking behavior after conditioning and to reduce contextual cues (Figure [1C](#F1){ref-type="fig"}). The retention test was performed 48 h after acquisition (8^th^ day) to evaluate the acquisition of fear memory as well as the effects of drug treatment. Here, each rat was placed in the experimental chamber with free access to the water spout and was subjected to the CS on 10 consecutive trials, in which the time to complete 10 licks pre-tone (no CS) and during the tone (CS) were recorded, and the suppression ratio (SR) was calculated for each trial. The tone was presented immediately after the animal completed its 90^th^ lick and was switched off after its 100^th^ lick (Figure [1D](#F1){ref-type="fig"}). The latency to complete licks 0--80 was recorded to ensure that the rats were licking when the tone was presented, but this value was not used to calculate the suppression of the lick response. The latency to complete licks 81--90 was measured as a control for time in the absence of a tone and was used to calculate the SR. Therefore, the SR was calculated as the ratio of B/(A+B) for each rat, where A is the time to complete 10 licks pre-tone (pre-CS), i.e., time to complete licks 81--90 and B is the time to complete 10 licks during the CS, i.e., time to complete licks 91--100. #### Extinction of suppression of the licking response Analysis of the effects of FfB on extinction was performed using the behavioral protocol described for acquisition. All rats were subjected to tests of adaptation (1^st^--5^th^ days), acquisition (6^th^ day), reacquisition (7^th^ day), and retention of the CER (8^th^ day). Seventy-two hours (9^th^ day) and ninety-six hours (10^th^ day) after fear conditioning, the rats were placed in the experimental chamber for extinction training and extinction test sessions, respectively (Figures [1E,F](#F1){ref-type="fig"}). In both sessions, the latencies to complete licks before the tone and during the tone for 10 consecutive CS presentations were recorded as described for the 8^th^ day. ### Data analysis The data from the first CS presentation indicated whether the association was learned. An SR approaching 1.0 indicates total suppression (high fear), whereas an SR ≤ 0.5 indicates no suppression (low fear), i.e., failure to learn the tone-shock relationship. The data are reported as the means ±SEM. A Two-way analysis of variance (ANOVA) was used to test for the presence of the effects of group and trial and the interaction between these variables; two fixed factors (group and trial), one random factor (rat), and repeated measurement of the trials were considered. *P* \< 0.05 were considered significant. Graph Pad 6.0 Software® (version 6.0; Graph Pad Inc., San Diego, CA, USA) was used for data analysis. ### Expression of *Gabra5, Htr1a, Grin2a, Grin2b*, and *Mapk1/Erk2* by quantitative PCR (qPCR) following treatments before conditioning and behavioral analysis The analysis of gene expression in the DH samples was extracted 3 h after the completion of the retention test or the extinction retention test as previously described. The candidate genes gamma-aminobutyric acid receptor subunit alpha-5 (*Gabra5*), 5-hydroxytryptamine (serotonin) receptor, subunit 1A (*Htr1a*), glutamate receptor ionotropic, NMDAR subunit GluN2A (*Grin2a*), glutamate receptor ionotropic, NMDAR subunit GluN2B (*Grin2b*), and extracellular signal-regulated kinase 2 (*Erk2*) were investigated. To this end, total RNA was isolated using Trizol reagent (Invitrogen Corp., Carlsbad, CA, USA) according to the manufacturer\'s recommendations. One microgram of total RNA was subjected to DNA-free DNase treatment (AMBION, Austin, TX, USA) and reverse-transcribed into cDNA using the SuperScript® III Reverse Transcriptase kit (Invitrogen Corp.) together with oligo~12−18~ primer and 10 units of an RNase inhibitor (Invitrogen Corp.). Reverse transcriptase-negative samples were prepared for each individual reaction and were used as controls for assay contamination. Aliquots of 1 μL of cDNA were used in 12 μL reactions containing SYBR Green Master Mix (PE Applied Biosystems, Foster City, CA) and 3pM of each primer for the target genes and the reference gene (RS8) as described previously (Cerutti et al., [@B22]). The primer sequences are displayed in (Table [S1](#SM1){ref-type="supplementary-material"}). The qPCR reactions were performed in triplicate, and the threshold for each cycle (Ct) was obtained using Applied Biosystems software (Applied Biosystems) and averaged \[standard deviation (SD) ≤ 1\]. Relative expression (RE) levels were calculated using the 2^−△△CT^ method (ddCt formula) as described previously (Cerutti et al., [@B22]). The vehicle (12% Tween®-80 or saline) was used as a control. The analyses were performed using Graph Pad 6.0 Software® (version 6.0; Graph Pad Inc., San Diego, CA, USA). For candidate gene expression analysis, normality of the data was verified using the Shapiro-Wilk normality test. One-way ANOVA followed by a *post-hoc* Bonferroni test was performed to evaluate the relationships between the expression levels of *Gabra5, Htr1a, Grin2a, Grin2b*, and *Erk2* across groups. *P* \< 0.05 were considered significant. Results {#s3} ======= Identification of flavonoids in FfB ----------------------------------- The spectroscopic and chromatographic data of the peaks (1--6) of the FfB are summarized in Figures [S1A,B](#SM10){ref-type="supplementary-material"}. The identities, fragmentation patterns and UV spectra were confirmed as follows: (1) vicenin-2:λmax = 334, 271 nm, \[M-H\]^−^ = *m/z* 593; (2) vicenin-1:λmax = 332, 271 nm, \[M-H\]^−^ = *m/z* 563; (3) vitexin:λmax = 269, 235 nm, \[M-H\]^−^ = *m/z* 431; (4) isovitexin:λmax = 335, 271 nm, \[M-H\]^−^ = *m/z* 431; (5) 6-C-glycoside-diosmetin:λmax = 342, 270 nm, \[M-H\]^−^ = *m/z* 461; and (6) apigenin:λmax = 305, 265 nm, \[M-H\]^−^ = *m/z* 269. These results were consistent with those previously reported by de Oliveira et al. ([@B32]). The identification of 6-C-glycoside-diosmetin, vicenin-2, vitexin and isovitexin was supported by the co-injection of the standards and FfB. The flavones (1, 3, 4, and 5) found in the FfB were quantified by HPLC-DAD, and the concentrations contained in the FfB were 0.15 mg/g vicenin-2, 0.20 mg/g vitexin, 0.30 mg/g isovitexin, and 0.25 mg/g 6-C-glycoside-diosmetin. Despite the evidence from our studies, few studies have examined the effects of a flavonoid fraction on fear memory. Further, previous data from our group suggest that the FfB may modulate different neurochemical systems. Behavioral, pharmacological, and molecular analysis --------------------------------------------------- The timelines illustrating the time points of drug administration and of brain removal are shown in Figures [2A](#F2){ref-type="fig"}, [3A](#F3){ref-type="fig"}, [4A](#F4){ref-type="fig"} and [5A](#F5){ref-type="fig"}. The effects of treatment with FfB and with agonists and antagonists specific to 5-HT~1A~Rs, GluN2B-NMDARs, and GABA~A~Rs or antagonists before FfB on the acquisition and extinction of the suppression of the licking response were assessed according to the mean SR for each tone, measured across 10 trials Figures [2B](#F2){ref-type="fig"}, [3B](#F3){ref-type="fig"}, [4B](#F4){ref-type="fig"} and [5B](#F5){ref-type="fig"}. Every figure shows the mean SRs for the CS on the first trial and each three-trial block from the retention test, extinction training, and extinction retention test sessions. The first trial is presented independently because it represents the first presentation of the CS after conditioning, extinction training, or retrieval of extinction; thus, the results from this trial can characterize the level of fear of the animal in each situation. In addition, the results from the first trial can show (i) the duration of fear memory expression and (ii) the occurrence of spontaneous recovery. The means (±SEM) for each first trial and block of three trials are presented in Tables [S2](#SM2){ref-type="supplementary-material"}, [S4](#SM4){ref-type="supplementary-material"}, [S6](#SM6){ref-type="supplementary-material"} and [S8](#SM8){ref-type="supplementary-material"}. {#F2} {#F3} {#F4} {#F5} To investigate the molecular mechanisms involved in modulating the suppression of the licking response by FfB, the expression levels of *Grin2a, Grin2b, Gabra5, Htr1a*, and *Erk2* in the DH were assayed by qRT-PCR. The effects of FfB, agonists and antagonists specific to the glutamatergic, serotoninergic and GABAergic systems were evaluated 3 h after the retention test session (8^th^ day; Figures [2C](#F2){ref-type="fig"}, [3C](#F3){ref-type="fig"}, [4C](#F4){ref-type="fig"} and [5C](#F5){ref-type="fig"}) and the extinction retention test session (10^th^ day; Figures [2D](#F2){ref-type="fig"}, [3D](#F3){ref-type="fig"}, [4D](#F4){ref-type="fig"} and [5D](#F5){ref-type="fig"}). The mean ± SEM values for the RE of candidate genes (ddCt) are available in Tables [S3](#SM3){ref-type="supplementary-material"}, [S5](#SM5){ref-type="supplementary-material"}, [S7](#SM7){ref-type="supplementary-material"}, [S9](#SM9){ref-type="supplementary-material"}. Further, we have made statistical comparison between control groups, which received the vehicle solutions (Saline and Tween®). To comparison of SR means during all trial of presentation of CS we used Paired *t*-test. No statistically significant difference was found between-session or intra-session (*P* = 0.1450). To comparisons of differential gene expression from samples of DH, we have used unpaired *T*-test. Comparisons between Saline and Tween® groups, after retention test (8^th^ day), were made to each gene evaluated. No significant difference was observed in the RE levels of *Htr1a* (*P* = 0.9737), *Grin2b* (*P* = 0.9691), *Gabra5* (*P* = 0.9592), *Grin2a (P* = 0.7358), or *Erk2* (*P* = 0.0962). Comparisons between Saline and Tween® groups, after extinction retention test (10^th^ day), were made to each gene evaluated, similarly to aforementioned, no significant difference between groups was observed in the RE levels of *Htr1a* (*P* = 0.5834), *Grin2b* (*P* = 0.9208), *Gabra5* (*P* = 0.9982), *Grin2a* (*P* = 0.9628), or *Erk2* (*P* = 0.1469). ### Effects of FfB on the acquisition and extinction of suppression of the licking response The effects of FfB on the acquisition and extinction of suppression of the licking response are shown in Figure [2B](#F2){ref-type="fig"} and Table [S2](#SM2){ref-type="supplementary-material"}. A Two-way ANOVA revealed a significant group × trial interaction \[*F*~(66,\ 756)~ = 2.785, *P* \< 0.0001\], a main effect of group \[*F*~(66,\ 756)~ = 24.56, *P* \< 0.0001\] and a main effect of trial \[*F*~(11,\ 756)~ = 17.27, *P* \< 0.0001\]. Comparisons of the results for the first trial in the retention test sessions between groups revealed elevated SR in the subgroups treated with FfB, CS-US or Tween and reduced SR in the subgroups treated with Sintocalmy® or CS (*P* \< 0.0001; left panel of Figure [2B](#F2){ref-type="fig"}). The analysis of SR in the first three-trial block (2^nd^--4^th^ trials) showed a significant decrease in mean SR relative to the first trial in the Tween®, CS-US, and FfB subgroups (*P* \< 0.0001); these results indicated the acquisition of extinction of fear memory within the session. An ANOVA comparing the three-trial blocks revealed no differences within sessions (*P* \> 0.05). This finding indicated a reliable decrease in suppression and a reduction of fear after each session. The middle panel of Figure [2B](#F2){ref-type="fig"} depicts the data from the extinction training session conducted 24 h after the retention test. Treatment with FfB at all doses promoted spontaneous recovery, as demonstrated by the results for the first trial in each subgroup, compared to treatment with Tween® or Sintocalmy® or to CS or CS-US alone. However, in subsequent trials, rats treated with FfB acquired fear extinction within the session (*P* \< 0.0001). The Tween® and CS-US subgroups exhibited a similar SR mean across successive exposures to the CS during the extinction training session, as observed in the retention test. The Sintocalmy® and CS subgroups showed mean SRs during all CS presentations that were similar to those measured in the retention test (*P* \> 0.05). This result indicated no conditioned fear and, as a consequence, no acquisition of fear extinction. The right panel of Figure [2B](#F2){ref-type="fig"} shows the mean SRs in the extinction retention session conducted 24 h after extinction training. The subgroups treated with FfB exhibited spontaneous recovery in the first trial, similar to the behavior observed during the extinction training session. However, all subgroups exhibited similar behavior by the end of the session. Notably, for the first trials, the mean SRs for each subgroup treated with FfB were significantly different from those for the Tween® and CS subgroups (*P* \< 0.0001). Comparisons between the first trial and the first three-trial block (2--4) showed reduced suppression of the licking response in the Tween®, CS-US and all FfB subgroups (*P* \< 0.0001). An ANOVA comparing the results for the three-trial blocks within the session demonstrated no significant differences in the mean SR between the subgroups (*P* \> 0.05). In addition, no significant differences in the mean SR were observed in the CS subgroup across all sessions (*P* \> 0.05). In summary, our data show for the first time that FfB does not impair the conditioned fear. However, rats treated with FfB showed spontaneous recovery of fear conditioning, as observed in the extinction training and extinction retention test sessions, although FfB did not prevent the acquisition within-session extinction. Furthermore, acute treatment with Sintocalmy®, a standardized extract containing 7% of the total flavonoids expressed in vitexin, impaired the conditioned fear and, consequently, resulted in no acquisition of the extinction of fear conditioning. ### FfB treatment modulates the spontaneous recovery of fear memory via *Htr1a* and *Erk2* expression within the DH FfB treatment at three different doses resulted in the overexpression of *Htr1a* \[*F*~(7,\ 16)~ = 173.0, *P* \< 0.0001\], *Grin2a* \[*F*~(7,\ 16)~ = 165.2, *P* \< 0.0001\], *Gabra5* \[*F*~(7,\ 16)~ = 40.82, *P* \< 0.0001\], and *Erk2* \[*F*~(7,\ 16)~ = 155.5, *P* \< 0.0001\] in the DH after the retention test session (8^th^ day) compared with the control treatments (Tween®, Sintocalmy®, CS-US, CS, and naïve; Figure [2C](#F2){ref-type="fig"}). The *Htr1a, Grin2b, Grin2a, Gabra5*, and *Erk2* expression levels were increased in the control subgroups (Tween® and CS-US subgroups) compared with the CS and naïve subgroups (*P* \< 0.0001). No difference was observed in *Grin2b* \[*F*~(7,\ 16)~ = 13.97\] expression after treatment with FfB compared to the treatment with FfB and CS-US groups (*P* \> 0.05; Figure [2C](#F2){ref-type="fig"} and Table [S3](#SM3){ref-type="supplementary-material"}), and *Grin2a* expression decreased after treatment with Sintocalmy®(*P* \< 0.0001). In the extinction retention test, *Htr1a* \[*F*~(7,\ 16)~ = 96.39\] and *Erk2* \[*F*~(7,\ 16)~ = 388.9; *P* \< 0.0001\] expression was significantly increased after FfB treatment in the DH compared with all other treatments (*P* \< 0.0001; Figure [2D](#F2){ref-type="fig"} and Table [S3](#SM3){ref-type="supplementary-material"}). No significant difference in the RE levels of *Grin2b* \[*F*~(7,\ 16)~ = 12.20\], *Gabra5* \[*F*~(7,\ 16)~ = 16.11\], or *Grin2a* \[*F*~(7,\ 16)~ = 181.6\] was observed in the FfB-treated subgroups compared to the CS-US or Tween® subgroups (*P* \> 0.05), although these expression levels were increased compared to the CS and naïve subgroups (*P* \< 0.05). Furthermore, *Grin2a* expression was reduced following the extinction retention test due to treatment with Sintocalmy® compared with all other treatments (Tween® and CS-US treatments; *P* \< 0.0001). In summary, the acquisition and extinction of the suppression of the licking response modulated *Htr1a, Grin2b, Grin2a, Gabra5*, and *Erk2* expression, and FfB treatment altered *Htr1a, Grin2a, Gabra5*, and *Erk2* expression after the retention test. Furthermore, the spontaneous recovery of fear memory appears to correlate with the overexpression of *Htr1a* and *Grin2a* in the DH. ### Effects of FfB on fear memory after blocking 5-HT~1A~Rs Figure [3B](#F3){ref-type="fig"} illustrates the specific effects of blocking 5-HT~1A~Rs before FfB treatment, which was administered before conditioning, on the results for the retention test, extinction training, and the extinction retention test. A Two-way ANOVA revealed a significant trial × group interaction \[*F*~(55,\ 648)~ = 1.365, *P* = 0.0453\], a main effect of group \[*F*~(5,\ 648)~ = 2.792, *P* = 0.0166\] and a main effect of trial \[*F*~(11,\ 648)~ = 9.116, *P* \< 0.0001\]. Similar mean SRs were observed between the Tween and saline groups across sessions (*P* \> 0.05). Therefore, saline was used to compare the effects of antagonists and agonists together with FfB. The left panel of Figure [3B](#F3){ref-type="fig"} shows the mean SRs in the CS, negative control (saline), positive control (0.30 mg.Kg^−1^ (S)-WAY100135 and 10.0 mg.Kg^−1^ buspirone), and treated subgroups \[0.30 mg.Kg^−1^ (S)-WAY+FfB (0.15 mg.Kg^−1^; 0.30 mg.Kg^−1^; or 0.65 mg.Kg^−1^FfB)\] for the retention test session. The analysis of mean SR for the first trial showed that treatment with (S)-WAY100135 or (S)-WAY100135 before FfB resulted in reduced suppression of the licking response compared with saline (*P* \< 0.0001) and buspirone treatment (*P* \< 0.0001). Analysis of the SR for the first three-trial block (2^nd^--4^th^ trials) showed significant differences in the mean SRs compared to the first trial in the saline and buspirone subgroups (*P* \< 0.0001). This result demonstrates acquisition within-session extinction to these subgroups. Alternatively no such differences were observed in the (S)-WAY100135 or (S)-WAY+FfB subgroups (*P* \> 0.05). An ANOVA comparing the first three-trial block (2--4) with the subsequent three-trial blocks (5--7 and 8--10) demonstrated no significant differences in mean SR on the extinction retention session between the subgroups (*P* \> 0.05; see Table [S4](#SM4){ref-type="supplementary-material"}). The data from the extinction training tests are shown in the middle panel of Figure [3B](#F3){ref-type="fig"}. Comparisons between groups showed that the groups treated with (S)-WAY+FfB, at all doses, did not demonstrate a difference in the mean SR (*P* \> 0.05). Analysis of the mean SR during the first three-trial block (2--4) showed that rats treated with (S)-WAY+FfB, saline, buspirone or (S)-WAY100135 exhibited a similar mean SR to that in the first trial (*P* \> 0.05). Moreover, similar mean SRs were observed with in all groups for the subsequent three-trial blocks (5--7 and 8--10; *P* \> 0.05; see also Table [S4](#SM4){ref-type="supplementary-material"}). The data from the extinction retention tests are shown in the right panel of Figure [3B](#F3){ref-type="fig"}. The analysis of the SR showed that the subgroups treated with (S)-WAY100135+FfB did not exhibit spontaneous recovery. Furthermore, the analysis of the mean SR showed no significant difference between the first three-trial block (2^nd^--4^th^ trial) and the first trial among the (S)-WAY100135+FfB subgroups at all doses (*P* \< 0.0001). Therefore, no significant differences in mean SRs were found among the subgroups between the first three-trial block (2--4) and the subsequent three-trial blocks (5--7 and 8--10; *P* \> 0.05; see Table [S4](#SM4){ref-type="supplementary-material"}). A Two-way ANOVA comparison between groups treated with FfB vs. (S)-WAY100135+FfB revealed a significant groups × trial interaction \[*F*~(55,\ 495)~ = 2.018, *P* \< 0.0001\] and main effects of trial \[*F*~(11,\ 99)~ = 21.25, *P* \< 0.0001\] and groups \[*F*~(5,\ 45)~ = 21.41, *P* \< 0.0001\]. Treatment with (S)-WAY100135+ 030 mg.Kg^−1^ FfB and (S)-WAY100135+ 065 mg.Kg^−1^ FfB, resulted in reduced of licking response compared with FfB group in the first trial during extinction training. No significant difference was observed among subsequent three-trial blocks. Furthermore, the analysis of the mean SR showed significant difference between (S)-WAY100135+0.65 mg.Kg^−1^ FfB and 0.65 mg.Kg^−1^ FfB to the first trial during extinction retention test (*P* \< 0.0001). No significant difference was observed among subsequent three-trial blocks. In summary, our data demonstrate that (S)-WAY+FfB, at all doses, reduces the suppression of the licking response compared with the control treatment, as demonstrated by the results from the retention test. These data suggest for the first time that the spontaneous recovery observed in the FfB subgroups is modulated by 5-HT~1A~Rs. ### (S)-WAY100135 treatment prevents the overexpression of *Htr1a* and *Erk2* within the DH caused by FfB We used treatment with (S)-WAY100135 prior to FfB administration to assess the role of the 5-HT~1A~R in the acquisition and extinction of fear memory. In addition, the roles of NMDARs, GABA~A~R~S,~and ERK2 were evaluated. Figure [3C](#F3){ref-type="fig"} shows the levels of *Htr1a, Gabra5, Grin2a, Grin2b*, and *Erk2* expression in the DH after the retention test session (8^th^ day). Consistent with the results of *Htr1a* and *Erk2* expression after FfB administration, treatment with (S)-WAY100135+FfB, at all doses, resulted in the downregulation of *Htr1a* expression \[*F*~(5,\ 12)~ = 449.9, *P* \< 0.0001\]. Treatment with (S)-WAY100135+FfB resulted in the downregulation of *Htr1a* expression when compared with saline and buspirone treatment \[*F*~(5,\ 12)~ = 40.05, *P* \< 0.0001\]. Although *Erk2* expression was similar across all groups \[*F*~(5,\ 12)~ = 3.071, *P* = 0.0516\]. A ANOVA comparison between the groups treated with (S)-WAY100135+FfB vs. FfB revealed that the overexpression of *Erk2* observed after FfB treatment was reversed by (S)-WAY100135 pretreatment, at all doses \[*F*~(5,\ 12)~ = 57.79, *P* \< 0.0001\]. Furthermore, (S)-WAY100135+FfB induced the downregulation of *Grin2a* \[*F*~(5,\ 12)~ = 124.8\] and *Grin2b* \[*F*~(5,\ 12)~ = 8.794;*P* = 0.001\] expression compared with saline and buspirone (*P* \< 0.0001). Moreover, (S)-WAY100135 treatment decreased the expression of *Grin2a*, but not *Grin2b*, and buspirone treatment reduced *Grin2b* expression compared with saline treatment (*P* \< 0.0001). No significant changes in *Gabra5* expression were observed \[*F*~(5,\ 12)~ = 2.505, *P* = 0.0894\]. These statistics are shown in Table [S5](#SM4){ref-type="supplementary-material"}. Figure [3D](#F3){ref-type="fig"} shows *Htr1a, Gabra5, Grin2a, Grin2b*, and *Erk2* expression in the DH after the extinction retention test (10^th^ day). These data show that treatment with (S)-WAY100135 before FfB administration, at three different doses, resulted in the overexpression of *Grin2a* \[*F*~(5,\ 12)~ = 278.4, *P* \< 0.0001\] compared with the control treatments \[saline, buspirone, and (S)-WAY100135\] and in the downregulation of *Gabra5* compared to treatment with 0.15 mg.Kg^−1^ FfB \[*F*~(5,\ 12)~ = 4.338, *P* = 0.0174\]. Additionally, treatment with (S)-WAY100135+FfB resulted in the downregulation of *Htr1a* \[*F*~(5,\ 12)~ = 31.18, *P* \< 0.0001\], *Erk2* \[*F*~(5,\ 12)~ = 119.9, *P* \< 0.0001\] and *Gabra5* expression \[*F*~(5,\ 12)~ = 20.48, *P* \< 0.0001\] and overexpression of *Grin2a* in the DH \[*F*~(5,\ 12)~ = 82.00, *P* \< 0.0001\]. Furthermore, treatment with buspirone resulted in the upregulation of *Htr1a* expression \[*F*~(5,\ 12)~ = 72.92; *P* \< 0.001\] compared with all other treatments. Nevertheless, no significant differences were observed in the RE of *Grin2b* \[*F*~(5,\ 12)~ = 3.039, *P* = 0.0010\] or *Erk2* \[*F*~(5,\ 12)~ = 2.94, *P* = 0.0580\]. Treatment with (S)-WAY100135 prior to FfB administration, at all three doses, prevented the upregulation of *Htr1a* expression observed after FfB treatment (Figure [2D](#F2){ref-type="fig"}; see also Table [S5](#SM5){ref-type="supplementary-material"}). In summary, our data show that treatment with (S)-WAY100135 prior to FfB administration decreases *Htr1a, Grin2b*, and *Grin2a* expression in the DH after the retention test and prevents the increase in *Htr1a* and *Erk2* expression after the extinction retention test in relation to observed after treatment with FfB alone. Conversely, *Grin2a* expression in the DH was increased after (S)-WAY100135+FfB treatment after the extinction retention test compared with FfB treatment. ### Effects of FfB on fear memory after blocking GluN2B-NMDARs Figure [4B](#F4){ref-type="fig"} shows the effects of specifically blocking GluN2B-NMDARs with Ro25-6981 before FfB treatment, which was administered before conditioning, on the results from the retention test, extinction training and the extinction retention test. A Two-way ANOVA revealed a significant trial × group interaction \[*F*~(55,\ 648)~ = 2.170, *P* \< 0.0001\], a main effect of group \[*F*~(5,\ 648)~ = 3.356, *P* \< 0.0001\] and a main effect of trial \[*F*~(11,\ 648)~ = 11.57, *P* \< 0.0001\]. The left panel of Figure [4B](#F4){ref-type="fig"} shows the mean SRs for the retention test session. Comparisons of the mean SR on the first trial revealed a difference between the Ro25-6981-treated subgroup and all other subgroups (*P* \> 0.0001); this result indicated that blockade of GluN2B impaired the acquisition of fear memory. However, treatment with Ro25-6981 before FfB administration did not affect fear memory. FfB treatment reversed the learning impairment observed in the subgroup treated with Ro25-6981 alone. Analysis of the SR for the first three-trial block (2^nd^--4^th^ trials) showed significant differences in mean SR for the saline, NMDA, Ro25-6981, andRo25-6981+FfB groups (0.15 mg.Kg^−1^;0.30 mg.Kg^−1^; or 0.65 mg.Kg^−1^FfB) compared with the first trial (*P* \< 0.0001). Furthermore, an ANOVA comparing the subsequent three-trial blocks (5--7 and 8--10) with the first three-trial block of the test revealed no differences (*P* \> 0.05). The Ro25-6981 subgroup showed similar SR values across all trials of CS presentation (statistics shown in Table [S6](#SM6){ref-type="supplementary-material"}). However, comparisons of the first trial of the extinction training test between the subgroups showed that the subgroups treated with Ro25-6981+FfB, at all doses, did not demonstrate differences in mean SR compared with the saline and NMDA subgroups (*P* \> 0.05) or the Ro25-6981 alone subgroup, which showed no acquisition of conditioned fear (middle panel of Figure [4B](#F4){ref-type="fig"}). Analysis of the SR for the first three-trial block (2^nd^--4^th^ trials) compared with the first trial showed no significant differences in the mean SR for the subgroups treated with saline, NMDA, Ro25-6981, or Ro25-6981+FfB (*P* \> 0.05). Nevertheless, no significant difference in SR was found within the groups for the first three-trial block (2--4) compared with the subsequent three-trial blocks (5--7 and 8--10; *P* \> 0.05; see also Table [S6](#SM6){ref-type="supplementary-material"}). The subgroups treated with Ro25-6981+FfB, at all doses, exhibited higher mean SRs for the first trial than the saline subgroup on the extinction retention test (*P* \> 0.05; right panel of Figure [4B](#F4){ref-type="fig"}) and remained similar throughout the trials of the extinction training test. Comparisons between the first trial and the first three-trial block (2--4) showed reduced suppression of the licking response for all groups treated with Ro25-6981+FfB (*P* \< 0.05). An ANOVA comparing the three-trial blocks demonstrated no significant differences in the mean SRs throughout the extinction retention session (*P* \> 0.05; Table [S6](#SM6){ref-type="supplementary-material"}). A Two-way ANOVA comparison between groups treated with FfB vs. Ro 25-6981+FfB revealed a significant groups × trial interaction \[*F*~(55,\ 495)~ = 2.094, *P* \< 0.0001\] and main effects of trial \[*F*~(11,\ 99)~ = 31.20, *P* \< 0.0001\] and groups \[*F*~(5,\ 45)~ = 2.873, *P* = 0.0247). Analysis of the mean SR showed significant difference between Ro 25-6981+0.65 mg.Kg^−1^ FfB vs. 0.65 mg.Kg^−1^ FfB to the first trial during extinction training (*P* \< 0.0001). No significant difference was observed among subsequent three-trial blocks (*P* \> 0.05). Furthermore, similar means SR were found to groups treated with Ro 25-6981+FfB to the first trial during extinction retention test. No significant difference was observed among subsequent three-trial blocks (*P* \> 0.05). In summary, our data show for the first time that treatment with Ro25-6981, an antagonist of the GluN2B-NMDAR, impairs the acquisition of suppression of the licking response. Conversely, treatment with after FfB after Ro25-6981 administration, at all doses, reverses the learning impairment associated with the GluN2B-NMDAR antagonist. In this sense, GluN2B is involved in the acquisition of suppression of the licking response, but the disruptive effects of Ro25-6981 appear to be offset by treatment with FfB. Additionally, we showed that the spontaneous recovery observed in the FfB subgroups may be modulated by GluN2B because rats treated with Ro25-6981 before FfB administration seems to decrease the spontaneous recovery observed during the extinction training sessions compared with the rats treated with FfB alone (see Figure [2B](#F2){ref-type="fig"}). ### Ro25-6981 treatment does not prevent the overexpression of *Grin2a* and *Erk2* caused by FfB, although it reduces *Htr1a* expression We used treatment with Ro25-6981 prior to FfB to evaluate the roles of NMDARs, 5-HT~1A~R~*S*~, GABA~A~R~S~, and ERK2. Figure [4C](#F4){ref-type="fig"} shows the *Htr1a, Grin2a, Grin2b, Gabra5*, and *Erk2* expression levels in the DH after the retention test (8^th^ day). Treatment with Ro25-6981 before FfB treatment, at all three doses, resulted in the overexpression of *Grin2a* \[*F*~(5,\ 12)~ = 107.1, *P* \< 0.0001\] and *Erk2* \[*F*~(5,\ 12)~ = 90.89, *P* \< 0.0001\] and the decreased expression of *Htr1a* \[*F*~(5,\ 12)~ = 32.67, *P* \< 0.0001\] and *Gabra5* \[*F*~(5,\ 12)~ = 12.44, *P* = 0.0002\] compared with the control treatment. No change in *Grin2b* was observed after Ro25-6981 or Ro25-6981+FfB treatment \[*F*~(5,\ 12)~ = 20.18\]. A ANOVA analysis revealed that treatment with Ro25-6981 before FfB, resulted in the overexpression of *Grin2a* \[*F*~(5,\ 12)~ = 134.8, *P* \< 0.0001\] and *Erk2* \[*F*~(5,\ 12)~ = 47.98, *P* \< 0.0001\] and the decreased expression of *Htr1a* \[*F*~(5,\ 12)~ = 361.7, *P* \< 0.0001\] and *Gabra5* \[*F*~(5,\ 12)~ = 32.57, *P* \< 0.0001\] in relation to groups treated with FfB alone. Additionally, NMDA treatment resulted in the overexpression of *Grin2b* compared with all other treatments (*P* \< 0.0001) and in the overexpression of *Erk2* compared with saline or Ro25-6981 treatment (*P* \< 0.001; see Table [S7](#SM7){ref-type="supplementary-material"}). Figure [4D](#F4){ref-type="fig"} shows that treatment with Ro25-6981+FfB increased the *Grin2a* \[*F*~(5,\ 12)~ = 14.47, *P* = 0.0001\], *Erk2* \[*F*~(5,\ 12)~ = 44.78, *P* \< 0.0001\], *Htr1a* \[*F*~(5,\ 12)~ = 158.6, *P* \< 0.0001\], and *Grin2b* \[*F*~(5,\ 12)~ = 5.37, *P* = 0.008\] expression levels in the DH after the extinction retention test (10^th^ day) compared with saline treatment. No significant differences in *Gabra5* expression were observed between the subgroups treated with Ro25-6981+FfB and the saline subgroup \[*F*~(5,\ 12)~ = 1.169, *P* = 0.3790\]. Furthermore, comparison among groups treated with Ro25-6981 before FfB treatment resulted in the overexpression of *Grin2a* \[*F*~(5,\ 12)~ = 32.68, *P* \< 0.0001\] and *Erk2* \[*F*~(5,\ 12)~ = 34.58, *P* \< 0.0001\] and *Htr1a* \[*F*~(5,\ 12)~ = 299.0, *P* \< 0.0001\]. Further, significant difference tog Grin2b was seeing to groups treated with Ro25-6981 before 0.65 mg.Kg^−1^ FfB in relation to 0.65 mg.Kg^−1^ FfB group \[*F*~(5,\ 12)~ = 4.727, *P* = 0.0128\]. In summary, treatment with Ro25-6981 reduced the expression of *Gabra5, Erk2*, and *Ht1ra* in the DH after the retention test, although treatment with FfB reduced the effects of Ro25-6981 on *Gabra5* and *Htr1a* expression and increased *Grin2a* and *Erk2* expression. Conversely, treatment with Ro25-6981+FfB increased *Htr1a* expression after the extinction retention test. Furthermore, treatment with FfB after Ro25-6981 administration increased the *Htr1a, Grin2b, Grin2a*, and *Erk2* expression levels in the DH after the extinction retention test (see Table [S7](#SM7){ref-type="supplementary-material"}). ### Effects of FfB on fear memory after blocking GABA~A~Rs The effects of specifically blocking GABA~A~Rs prior to FfB treatment before conditioning on the results of the retention test, extinction training, and the extinction retention test are shown in Figure [5B](#F5){ref-type="fig"}. A Two-way ANOVA revealed a significant trial × group interaction \[*F*~(55,\ 648)~ = 2.695, *P* \< 0.0001\], a main effect of group \[*F*~(5,\ 648)~ = 6.416, *P* \< 0.0001\] and a main effect of trial \[*F*~(11,\ 648)~ = 11.77, *P* \< 0.0001\]. Analysis of the mean SRs for the first trial of the retention test showed that treatment with picrotoxin, an antagonist of GABA~A~Rs, or picrotoxin+FfB did not prevent the acquisition of conditioned fear. These subgroups showed a similar mean SR to the saline subgroup (*P* \> 0.05). Conversely, animals treated with diazepam exhibited reduced suppression of the licking response compared with the animals treated with saline, picrotoxin or picrotoxin+FfB (*P* \< 0.001). Analysis of the SR for the first three-trial block (2^nd^--4^th^ trials) compared with the first trial showed significant differences in the mean SR for the subgroups treated with saline, picrotoxin, or picrotoxin+FfB (0.15 mg.Kg^−1^, 0.30 mg.Kg^−1^, or 0.65 mg.Kg^−1^FfB; *P* \< 0.0001). An ANOVA comparing the first three-trial block (2--4) of extinction with the other three-trial blocks (5--7 and 8--10) demonstrated no significant differences in the mean SR between the subgroups (*P* \> 0.05; see Table [S8](#SM8){ref-type="supplementary-material"}). The data from the extinction training session are shown in the middle panel of Figure [5B](#F5){ref-type="fig"}. Comparisons between the groups showed that the subgroups treated with picrotoxin+FfB, at all doses, demonstrated differences in mean SRs in the first trial compared to the saline, picrotoxin and diazepam subgroups (*P* \< 0.0001). In addition, rats treated with picrotoxin+FfB showed spontaneous recovery similar to that observed in rats treated with FfB alone (see Figure [2B](#F2){ref-type="fig"}). Furthermore, no differences in SR on the first trial were observed between the saline and picrotoxin subgroups (*P* \> 0.05). Analysis of the mean SR during the first three-trial block (2--4) showed that rats treated with picrotoxin+FfB at all doses demonstrated reduced suppression of the licking response compared with the mean SR for the first trial (*P* \< 0.0001). The saline and picrotoxin subgroups exhibited a similar mean SR across successive exposures to the CS during extinction training. Similar mean SRs were observed for all groups across the subsequent three-trial blocks (5--7 and 8--10; *P* \> 0.05; see Table [S8](#SM8){ref-type="supplementary-material"}). Similar to the previous sessions, on the extinction retention test, rats treated with picrotoxin+FfB, at all doses, showed spontaneous recovery on the first trial, as demonstrated by the higher SR means in the picrotoxin+FfB subgroups (*P* \> 0.001; Figure [5B](#F5){ref-type="fig"}, right panel). A reduced mean SR was observed in the picrotoxin+FfB subgroups on the first three-trial block compared to the first trial (*P* \< 0.0001). No significant difference in the SR was found within the groups for the first three-trial block (2--4) compared with the subsequent three-trial blocks (5--7 and 8--10; *P* \> 0.05; see also Table [S8](#SM8){ref-type="supplementary-material"}). A Two-way ANOVA comparison between groups treated with FfB vs. Picrotoxin+FfB revealed a significant groups × trial interaction \[*F*~(55,\ 495)~ = 1.091, *P* = 0.3116\] and main effects of trial \[*F*~(11,\ 99)~ = 45.11, *P* \< 0.0001\] and groups \[*F*~(5,\ 45)~ = 5.375, *P* = 0.0006\]. Analysis of the mean SR showed no significant difference between groups treated with FfB vs. Picrotoxin+FfB, at all doses, to the first trial during retention test (*P* \> 0.05), extinction training (*P* \> 0.05) and extinction test sessions (*P* \> 0.05). Significantly difference were observed among groups treated with FfB, at a dose 0.15 mg.Kg^−1^ and 0.65 mg.Kg^−1^FfB (*P* \< 0.05). Furthermore, analysis of SR means for the first trial showed significant difference among rats treated with different doses of FfB groups during extinction training and extinction retention test (*P* \< 0.001). No significant difference was observed among subsequent three-trial blocks (*P* \> 0.05). In summary, our data demonstrated that each group treated with picrotoxin prior to FfB administration acquired fear memory. Conversely, diazepam treatment impaired the acquisition of fear memory. Furthermore, we showed that treatment with picrotoxin+FfB resulted in spontaneous recovery in the first trial of extinction training and the extinction retention test, although the suppression gradually decreased over the trials. Therefore, rats treated with FfB showed within-session extinction of fear memory. These data suggest that spontaneous recovery is not modulated by GABA~A~Rs. ### Picrotoxin prevents the overexpression of *Gabra5* and *Grin2a* caused by FfB Treatment with picrotoxin prior to FfB treatment, at a dose of 0.15 or 0.65 mg.Kg^−1^ FfB, did not prevent the increase in the expression of *Htr1a* \[*F*~(5,\ 12)~ = 28.02, *P* \< 0.0001\] or *Erk2* \[*F*~(5,\ 12)~ = 84.48, *P* \< 0.0001\] in the DH after the retention test compared with saline, picrotoxin, or diazepam treatment (Figure [5C](#F5){ref-type="fig"}), as observed in the subgroups treated with FfB alone (Figure [2C](#F2){ref-type="fig"}). A ANOVA analysis revealed that treatment with picrotoxin before FfB, resulted in the downexpression of *Htr1a* \[*F*~(5,\ 12)~ = 128.7 *P* \< 0.0001\], *Grin2b* \[*F*~(5,\ 12)~ = 15.22, *P* \< 0.0001\] and *Erk2* \[*F*~(5,\ 12)~ = 22.81, *P* \< 0.0001\], at all doses, and *Gabra5* \[*F*~(5,\ 12)~ = 28.02, *P* \< 0.0001\] and *Grin2a* \[*F*~(5,\ 12)~ = 8515, *P* \< 0.00002\], at a higher doses in relation to groups treated with FfB alone. Additionally, picrotoxin+FfB treatment did reduce *Grin2b* expression \[*F*~(5,\ 12)~ = 14.42, *P* \< 0.0001\], but no change was observed in the expression of *Grin2a* \[*F*~(5,\ 12)~ = 12.24, *P* = 0.0001\] or *Gabra5* \[*F*~(5,\ 12)~ = 7.580, *P* = 0.0020\]. Furthermore, picrotoxin increased *Gabra5*, and *Erk2* expression in the DH (*P* \< 0.0001). Conversely, diazepam treatment decreased *Htr1a, Grin2a, Erk2*, and *Grin2b* expression in the DH (see Table [S9](#SM9){ref-type="supplementary-material"}). The data shown in Figure [5D](#F5){ref-type="fig"} demonstrate the upregulation of *Erk2* \[*F*~(5,\ 12)~ = 90.76, *P* \< 0.0001\] and *Grin2a* \[*F*~(5,\ 12)~ = 67.51\] expression in the DH after the extinction retention session for the subgroups treated with picrotoxin+*F*fB compared to those treated with saline, picrotoxin or diazepam (*P* \> 0.0001). Upregulated *Htr1a* \[*F*~(5,\ 12)~ = 23.98, *P* \< 0.0001\] and *Erk2* \[*F*~(5,\ 12)~ = 26.24, *P* \< 0.0001\] expression was observed in the picrotoxin+FfB in relation to FfB group. Similarly, upregulated *Htr1a* \[*F*~(5,\ 12)~ = 10.75\] expression was observed in the picrotoxin+FfB compared with the saline and diazepam subgroups (*P* \< 0.0001). Moreover, we showed that picrotoxin resulted in the overexpression of *Htr1a, Grin2b* \[*F*~(5,\ 12)~ = 31.61, *P* \< 0.0001\], and *Gabra5* \[*F*~(5,\ 12)~ = 12.13, *P* = 0.0020\] compared with saline. Downregulated *Grin2b* \[*F*~(5,\ 12)~ = 22.08, *P* \< 0.0001\], *Grin2a* \[*F*~(5,\ 12)~ = 22.04, *P* \< 0.0001\] and *Gabra5* \[*F*~(5,\ 12)~ = 14.46, *P* \< 0.0001\] expression was observed in the picrotoxin+FfB in relation to FfB group. Furthermore, diazepam increased *Htr1a* expression and decreased *Grin2a* and *Gabra5* expression (*P* \< 0.0001; see Table [S9](#SM9){ref-type="supplementary-material"}). In summary, treatment with picrotoxin before FfB administration, at all doses, increased *Htr1a* and *Erk2* expression in the DH after the acquisition and extinction of fear memory and reduced *Grin2b* expression and prevented the increase in *Grin2a* and *Gabra5* expression after the retention test. Furthermore, this treatment decreased *Grin2b* and *Gabra5* expression after the extinction retention test. Discussion {#s4} ========== The major findings of our study are as follows. (i) Rats treated with FfB acquired suppression of the licking response, and FfB upregulated the expression of *Htr1a, Grin2a, Gabra5*, and *Erk2* in the DH after the acquisition of conditioned fear, compared to rats exposed to the CS alone, naïve rats and Sintocalmy®-treated rats. (ii) Rats treated with FfB, at all doses, showed spontaneous recovery when subjected to the extinction training and extinction retention test sessions; these observations were correlated with *Htr1a* and *Erk2* overexpression in the DH. (iii) These findings were confirmed by data from treatment with (S)-WAY100135, which reduced the lick SR and inhibit spontaneous recovery. Further, data from DH samples obtained from rats treated with (S)-WAY100135 prior to FfB resulted in the downregulation of *Htr1a* expression and no modulation of *Erk2* expression after the retention test and the extinction retention test. (iv) Our data are in line with previous findings concerning the requirement of GluN2B for fear memory formation (Sotres-Bayon et al., [@B104], [@B105]). In particular, we present evidence that treatment with Ro25-6981 disrupts the acquisition of suppression of the licking response. Nevertheless, treatment with FfB after Ro25-6981 reversed the dose-dependent deficit in the acquisition of fear memory caused by Ro25-6981, which was associated with upregulation of *Grin2*a and *Erk2* expression and downregulation of *Htr1a* and *Gabra5* expression in the DH after the retention test. The occurrence of spontaneous recovery to group treated with Ro25-6981 before FfB during extinction retention test seems to be associated with increase of *Grin2b, Grin2a*, and *Erk2* expression. (v) Treatment with picrotoxin prior to FfB administration no inhibits the spontaneous recovery of fear. This observation was correlated with overexpression of *Htr1a* and *Erk2* and no modulation of *Gabra5* expression in the DH. This result suggested that spontaneous fear recovery is not modulated by inactivation of GABA~A~Rs; however, the data concerning *Gabra5* expression in the DH indicated that FfB modulated the expression of the α5-subunit, which is particularly important for mediating the process of memory formation in the hippocampus (Bannerman et al., [@B10]; Rudolph and Möhler, [@B97]; Atack, [@B7]). Additionally, treatment with diazepam and Sintocalmy® disrupt the acquisition of fear memory, in which was associated with downregulation of *Grin2a* expression in the DH. Several pharmacological studies have indicated that the administration of diazepam before training impairs LTM, as evaluated in a behavioral model such as IA (Izquierdo and Ferreira, [@B51]), contextual fear conditioning (Harris and Westbrook, [@B45]), or conditioned suppression (Oliveira et al., [@B89]). Consistent with this evidence, our results show that acute treatment with 4.0 mg.Kg^−1^ diazepam impaired fear memory acquisition and highlight the role of GABA~A~R in this process. Together with previous data, our current data further support the concept that flavonoid fractions do not prevent fear memory extinction within a session (de Oliveira et al., [@B32]). In addition, these data suggest an important role of the DH in mediating the acquisition and extinction of conditioned suppression of the lick response. The roles of the hippocampus in the acquisition, consolidation, and retrieval of fear memory (Kim and Fanselow, [@B61]; Cammarota et al., [@B21]) and in fear extinction have been extensively studied in different rodent paradigms (Izquierdo, [@B50]; Ji and Maren, [@B56]). Further, the involvement of a circuit including the hippocampus, the pre-frontal cortex and the amygdala in these processes has long been established (Vinogradova, [@B113]; Fanselow and Dong, [@B38]). However, the present data suggest an important role of the hippocampus in conditioned suppression, whereas hippocampal plasticity may represent another function of the hippocampus in addition to contextual fear memory modulation and executive and integrative functions (McNaughton and Gray, [@B76]; Anagnostaras et al., [@B5]; Vinogradova, [@B113]; Sanders et al., [@B99]). Further, many theories have attempted to explain both the neurochemical processes that occur during the acquisition and extinction of fear memory and in the mechanism by which new drug, which are designed to enhance the consolidation or facilitate the extinction of fear memories, might modulate these neurochemical systems (Ji and Maren, [@B55]; Dalton et al., [@B27]). However, much less is known about drugs that modulate the brain substrates of extinction, conditioned inhibition, and other inhibitory processes involved in the suppression of a motivated response or the basis of spontaneous recovery. In addition, very few studies have shown the effects of drug treatment prior to conditioning training on fear extinction or spontaneous recovery, i.e., the relationship between the strength of fear memory acquisition and spontaneous recovery. In contrast, the majority of the existing data show the effects of pre-extinction treatment on spontaneous recovery. Our data suggest that the role of the hippocampus in the acquisition and extinction of lick suppression is dependent on the interaction between glutamatergic, serotoninergic and GABAergic neurotransmission via the activation or inactivation of specific NMDARs, GABA~A~Rs, and 5-HT~1A~Rs, as demonstrated by the results from pharmacological manipulation and differential gene expression of *Grin2a, Grin2b, Gabra5, Htr1a*, and *Erk2*. The reappearance of a conditioned response after acquisition and training for extinction of fear memory, as shown in our subgroups treated with FfB, has been previously described (Bouton, [@B18]; Rescorla, [@B95]; Leung and Westbrook, [@B64]; Quirk and Mueller, [@B91]). Specifically, it is thought that the persistence of a fear response after extinction training is associated with anxiety-related disorders (Davis et al., [@B31]). However, we showed that FfB enabled the acquisition of extinction within a session despite the occurrence of spontaneous recovery. Although these findings may seem paradoxical, our current findings raise the hypothesis that the original memory was somewhat enhanced, i.e., better preserved; therefore, the flavones from *Erythrina falcata* may be studied as a novel pharmacotherapy for the treatment of cognitive impairment. Furthermore, we believe that the reappearance of the original memory (spontaneous recovery) observed after FfB treatment is associated with the expression of *Htr1a, Erk2*, and *Grin2a* in the DH. 5HT~1A~Rs as a potential target for the effects of FfB on spontaneous recovery ------------------------------------------------------------------------------ Drugs that modulate the serotoninergic system are important for cognitive and emotional functions, and 5-HT~1A~Rs are involved in this process. The heteromeric 5-HT~1A~R is highly expressed in the hippocampus (Barnes and Sharp, [@B11]), where it modulates GABA- and glutamate-mediated activities (Jacobs and Azmitia, [@B53]; Barnes and Sharp, [@B11]; Meneses and Perez-Garcia, [@B77]). Activation of post-synaptic 5-HT~1A~Rs (heteroreceptors) in the hippocampus is a central component of conflict resolution and anti-anxiety effects. Alternatively, reduced 5-HT~1A~R expression results in a deficit in hippocampal-dependent memory (Bert et al., [@B13], [@B14]; Altieri et al., [@B4]). However, the effect of the activation of 5-HT~1A~R on the modulation of *Erk*2 expression remains controversial and may depend on neuronal origin and maturation states. Treatment with a 5-HT~1A~R agonist increased ERK phosphorylation and activity in the hippocampal neuron-derived cell line HN2-5 and in hippocampal slices cultured from postnatal day-15 animals (Adayev et al., [@B1]). In addition to these effects, the activation of 5-HT~1A~Rs alters the dynamics of other neurotransmission systems. The serotonergic regulation of NMDAR function in the DH was described in pyramidal neurons in the prefrontal cortex (Yuen et al., [@B123]). Additionally, the activation of 5-HT~1A~Rs resulted in disruption of the transport of GluN2B subunit-containing vesicles in dendrites, and this transport is regulated by the CaMKII and ERK signaling pathways (Yuen et al., [@B123]). However, further investigations of the adaptive changes in receptor functions and their specific localization are needed to elucidate the precise role of flavonoids. Intra-hippocampal treatment with (S)-WAY100135 alone did not affect the punished response in rats (Przegalinski et al., [@B90]). Therefore, our data suggest that treatment with (S)-WAY100135 reduced lick suppression and that treatment with FfB was unable to reverse this effect. Moreover, the treatment with (S)-WAY100135 modulated *Grin2a* and *Grin2b* expression. In this sense, heteromeric 5-HT~1A~Rs in the DH appear to be related to the acquisition of conditioned fear in addition to anti-conflict functions because rats treated with (S)-WAY100135 before FfB administration did not show spontaneous recovery. The reduced *Grin2a, Grin2b*, and *Htr1a* expression in the DH in groups treated with (S)-WAY100135 or (S)-WAY+FfB may underlie the reduced lick suppression and lack of spontaneous recovery. This result suggests an interaction between neurochemical systems. Therefore, the 5-HT~1A~R represents an additional potential target for the regulation of emotion and cognition in the DH. Activation of the GluN2B-NMDARs is required for acquisition of conditioned suppression and their inactivation before FfB treatments modulates the spontaneous recovery ---------------------------------------------------------------------------------------------------------------------------------------------------------------------- Since the discovery of the involvement of NMDARs in long-term potentiation (LTP) at CA1 synapses in the hippocampus, it has become evident that NMDARs are critical for a variety of cognitive processes, such as the acquisition and extinction of fear conditioning (Morris et al., [@B82]; Bliss and Collingridge, [@B16]). GluN2A and GluN2B are the predominant subunits of NMDARs. Furthermore, both of these subunits are expressed in the adult brain, predominantly in forebrain regions such as the amygdala, the prefrontal cortex, and the hippocampus, which are involved in the signaling pathways required for aversive memory formation (Schenberg et al., [@B101]; Mathur et al., [@B74]; Sotres-Bayon et al., [@B105]; Morris, [@B83]). The hippocampal functions of NMDARs, particularly the GluN2B and GluN2A subunits, in fear memory have been reported (Zhang et al., [@B125]; Brigman et al., [@B19]). Several works have suggested that the NMDAR subunit composition could be responsible for the induction of the two forms of plasticity: LTP and long-term depression (LTD) (Shipton and Paulsen, [@B103]). The contribution of each subunit to ERK2 activation appears to be related to the localization and population of these receptors as well as the behavioral paradigm evaluated (Traynelis et al., [@B110]). NMDARs either produce weak ERK2 activation or do not activate ERK2 (Gao et al., [@B43]). Myung et al. ([@B86]) showed that the GluN2B-NMDAR is coupled to the inhibition, rather than the activation, of ERK1/2. Furthermore, differences between behavioral data and gene expression data may explain the different effects of the GluN2B-NMDAR on downstream pathways according to regional localization. Our data showed that Ro25-6981 downregulated the expression of *Erk2* in the DH, which resulted in the impairment of conditioned suppression. Alternatively, pharmacological activation of NMDARs increased *Grin2b* and *Erk2* expression but did not affect *Grin2a* expression in the DH after the acquisition of conditioned suppression. Furthermore, treatment with FfB after Ro25-6951 administration increased *Grin2a* and *Erk2* expression in the DH. Thus, *Erk2* activity is closely related to the acquisition of conditioned suppression, as well as extinction and spontaneous recovery. The increase in *Erk2* expression, in response to the acquisition of fear memory or to NMDAR stimulation, has been consistently related to memory-dependent plasticity in the hippocampus (Atkins et al., [@B8]; Cammarota et al., [@B20]). The first evidence for the involvement of MAPK in LTP and fear memory originated from studies by English and Sweatt ([@B35]) and Atkins et al. ([@B8]), which showed that ERK2 is required for the formation of LTM in a fear conditioning paradigm in the hippocampus. The levels of ERK2 are elevated following the activation of NMDARs and during the influx of calcium (Impey et al., [@B48]) but are decreased by 5-HT~1A~-receptor activation or infusion of an agonist of the serotonergic 5-HT~1A~R in the hippocampus as Erk1/2 plays an important role in neuroprotection and synaptic activity. In addition to hippocampal NMDARs and 5-HT~1A~Rs, GABA~A~Rs play an important role in synaptic plasticity and therefore contribute to the acquisition of fear memory. Accordingly, drugs that modulate GABAergic transmission have been shown to interfere with fear acquisition and extinction (Chhatwal et al., [@B23]; Delamater et al., [@B34]; Oliveira et al., [@B89]). Activation of GABA~A~Rs impairs acquisition of conditioned suppression. their inactivation, before FfB treatment, however, didn\'t prevent the spontaneous recovery ------------------------------------------------------------------------------------------------------------------------------------------------------------------- We observed that conditioned suppression was impaired in the subgroups treated with Sintocalmy® or diazepam, and this impairment appeared to be related to the downregulation of *Grin2a* expression in the DH. The pharmacological properties and behavioral actions of benzodiazepines, such as amnesic, sedative, and antianxiety effects, on GABA~A~Rs appear to be mediated by the α1 subunit, which is preferentially located in interneurons of forebrain areas (Collinson et al., [@B25]). However, evidence has demonstrated that the GABA~A~R α5 subunit is highest in the hippocampus compared with deep cortical layers and the amygdala (Rudolph and Möhler, [@B98]), where it mediates memory formation (Yee et al., [@B120]; Rudolph and Möhler, [@B97]; Atack, [@B7]) and is involved in learning and memory tasks (Harris and Westbrook, [@B45]; Collinson et al., [@B25], [@B24]). Although *Gabra5* expression was not modulated in rats subjected to fear conditioning with or without FfB treatment or to the acquisition of conditioned suppression following FfB treatment, rats treated with picrotoxin displayed upregulation of *Gabra5* expression and showed acquisition of memory. In addition to the role of the α5 subunit in the acquisition of fear memory, its modulation in the DH after extinction of fear memory is supported by data from the subgroups treated with picrotoxin+FfB; these data suggest that the α5 subunit is not correlated with spontaneous recovery. Thus, our data reveal a central role of the α5 subunit of the GABA~A~R in the acquisition of conditioned emotional suppression, as evaluated by the lick response. The memory-enhancing effects of benzodiazepine site partial inverse agonists have been shown (Yee et al., [@B120]). Conclusion {#s5} ========== The major fear memory/treatment-dependent changes observed in our study included the spontaneous recovery of fear memory, which may be related to the enhancement of consolidation of fear memory. No anti-anxiety effects were observed after treatment with FfB. Furthermore, for the first time, we showed that the spontaneous recovery of fear memory may be correlated with the combined activation of GluN2A-containing NMDARs, and 5-HT~1A~Rs in the DH, which, in turn, modulates ERK1/2 activity. Finally, the results from gene expression analysis in the DH and the results showing the modulatory effects of FfB treatment indicate that the DH appears to anatomically and functionally subserve other structures involved in the acquisition and extinction of fear memory formation, such as the amygdala and the prefrontal cortex. Together, our data provide important information concerning the molecular basis of fear-conditioned suppression and the role of the DH in these processes, and our results suggest that FfB may represent a potential therapeutic target for preventing or treating neurocognitive impairments. 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 work is dedicated to Dra Elenice Aparecida de Moraes Ferrari (*in memoriam*). This study was supported by the São Paulo State Research Foundation (FAPESP) (grant 2009/15229-3 and 2013/20378-8 to SC). DO and CZ are scholars from CAPES. JC is an investigator on the Brazilian Research Council (CNPq). Supplementary material {#s6} ====================== The Supplementary Material for this article can be found online at: <http://journal.frontiersin.org/article/10.3389/fnbeh.2015.00345> ###### **HPLC-DAD-ESI/MS^**n**^ analysis of the flavonoidic fraction (FfB) from the roots of ***Erythrina falcata*** using a C18 Luna column (A,B)**. TIC was performed in negative mode, and MS^2^ spectra of deprotonated molecules **(A)** were obtained as follows: (1) vicenin-2 \[M-H\]^−^ at *m/z* 593, (2) vicenin-1 \[M-H\]^−^ at *m/z* 563, (3) vitexin \[M-H\]^−^ at *m/z* 431, (4) isovitexin \[M-H\]^−^ at *m/z* 431, (5) 6-C-glycoside diosmetin \[M-H\]^−^ at *m/z* 461, and (6) apigenin \[M-H\]^−^ at *m/z* 269. The chromatogram was recorded at 254 nm for the UV spectra of compounds 1-6 **(A)**. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. ###### Click here for additional data file. [^1]: Edited by: Onur Gunturkun, Ruhr-Universität Bochum, Germany [^2]: Reviewed by: Carsten T. Wotjak, Max-Planck-Institute of Psychiatry, Germany; Hongjoo J. Lee, University of Texas, USA

Introduction {#Sec1} ============ Geohazards pose a severe threat to humanity, civilian properties, infrastructures, and industries, possibly leading to the loss of life and high economic values^[@CR1]^. Monitoring areas prone to geohazards is invaluable for locating their precursory signals on the surface, alerting civilians to potential disasters, mitigating the catastrophic outcomes, and facilitating the decision-making processes on the construction and operation of infrastructures and industrial facilities. The United States mid-continent has long been considered geologically stable with no large scale tectonic movements, volcanism, or seismic activities^[@CR2],[@CR3]^. Therefore, unlike California with its dense GPS networks and frequent survey (aerial, spaceborne, field) campaigns, the mid-continent has garnered less attention from scientific communities and federal/state governments. However, recent studies have revealed that some of the mid-continent, especially the Gulf Coast of the United States including Texas, Louisiana, and Mississippi, is not immune to large-scale and/or localized geohazards^[@CR4],[@CR5]^. The geohazards along the southern United States have been both naturally induced and stimulated by human activities^[@CR1],[@CR3]^. Besides the occasional, strong tropical storms and flooding in lowlands, natural geohazards include settlement due to sediment loading and glacial isostatic adjustment, which can make the coastline in the Gulf Coast vulnerable to sea-level changes^[@CR6]--[@CR8]^. However, the naturally occurring surface subsidence on the coast displays characteristics of a continuous, slow progression (millimeters per year) and a large spatial extent (\~100 km wide)^[@CR6]^. In contrast, human-induced geohazards are faster growing (up to tens of cm/yr) and encompass a varying but generally small area (up to a couple of km wide). The most prominent difference between natural and human-induced geohazards is the correlation between surface instability and anthropogenic activities (e.g., mining, groundwater extraction, hydrocarbon production)^[@CR3],[@CR9]^. Although there can be a time delay of ground deformation after human activities, depending on the geological characteristics (porosity, elasticity, compressibility, pore pressure, permeability) of soils and rocks and types of the operations, human-induced surface subsidence or uplift usually has high proximal and temporal correlation with those activities^[@CR10]--[@CR12]^. West Texas is somewhat distant from the Gulf coast, but was inundated by relatively shallow seas during the early part of the Paleozoic Era (approximately 600 to 350 million years ago). The sediments formed during this period contributed to the accumulation of sandstone, shale, and limestone. The seas constituting broad marine environments in West Texas gradually withdrew, and by the Permian Period (approximately 299 to 251 million years ago), thick evaporites (salt, gypsum) accumulated in a hot arid land encompassing shallow basins and wide tidal flats. As a consequence of geological formation in West Texas, the deposited carbonate (reef limestone) and marine evaporite sequences played an important role in the formation of oil reservoirs by helping seal the traps and preserving the hydrocarbons^[@CR13]^. This resulted in the Permian Basin of West Texas' massive hydrocarbon reservoirs that became so lucrative to the oil and gas industry^[@CR14]^. In West Texas, human activities such as groundwater exploitation, fluid injection, and hydrocarbon extraction have resulted in surface instability, leading to geohazards such as surface heave/subsidence, fault reactivation^[@CR4]^, induced seismicity^[@CR15],[@CR16]^, and sinkhole formation^[@CR17]--[@CR19]^. The vastness of West Texas challenges our ability to identify and locate the relatively small spatial scale of the deformation corresponding to human activities, particularly for fluctuations over the course of a month or a year. Without concerted focus, the small-sized signal in a short time window can go easily undetected. There have been a few studies documenting the surge of surface uplift/subsidence, sinkhole formations, and induced seismicity in oil fields^[@CR19]--[@CR22]^. However, the role of human activities on the surface and subsurface deformation has yet to be fully established, particularly regarding the identification of small-scale deformation signals over a vast region from big datasets spanning multiple years and analyzing them with supplementary information. Challenges to the effective study of the geohazards in West Texas include: identification of their locations in remote and vast regions, measurement of their long-term evolution, and characterization of the causal mechanism with accessible information. Satellite radar interferometry (InSAR) has proven capable of imaging ground surface deformation with a measurement accuracy of centimeters or better at a spatial resolution of meters or better over a large region covering tens of thousands km^[@CR2],[@CR23]^. However, satellite radar acquisitions over West Texas have previously been scarce. Here we present the analysis of the ongoing ground deformations induced by various geohazards around Pecos, Monahans, Wink, and Kermit in West Texas (Fig. [1](#Fig1){ref-type="fig"}), using multi-temporal InSAR observations based on radar imagery from the first free, open-source radar satellites Sentinel-1A/B. The objective of our study is to probe the association between the ongoing localized geohazards in West Texas and anthropogenic activities. To achieve the goal, we focus on the localized, small-sized (200 m\~2 km wide), and rapidly developing (cm/yr) geohazards in the region, which are categorized based on six possible causes: i) wastewater injection, ii) CO~2~ injection for enhanced oil recovery (EOR), iii) salt/limestone dissolution, iv) freshwater impoundment in abandoned wells, v) sinkhole formation in salt beds, and vi) hydrocarbon production. In addition, time-series measurements from two different imaging geometries are integrated to decipher the deformation phenomena. Furthermore, through comparative analysis of records of fluid injection, hydrocarbon production, and geological characteristics, we establish the relationship between the possible causes of human activities or natural perturbation and the localized observed geohazards in West Texas.Figure 1Locations of ground deformation in West Texas. 6 major sites (red stars) in West Texas display the locations influenced by human activities identified based on Sentinel-1A/B multi-temporal interferometry (background image is from Sentinel-2). To estimate 2D (east-west and vertical) deformation, the ascending (path 78; black box) and descending (path 85; white box) track Sentinel-1A/B images were integrated over the overlapped regions. West Texas' Permian Basin contains two major aquifer systems under the influence of the Pecos River, the Pecos Valley aquifer and the Edwards-Trinity aquifer. The figure has been created using open-source software Generic Mapping Tools (GMT) 5.2.2_r15292 available at <http://gmt.soest.hawaii.edu/projects/gmt/wiki/Download>. The Sentinel-1A/B data used in this study were downloaded in 2017 through the Vertex online archive <https://vertex.daac.asf.alaska.edu> provided by Alaska Satellite Facility (ASF) and the Sentinel-2 data used as a background image for this figure were obtained in 2017 through Copernicus open access hub <https://scihub.copernicus.eu> provided by the European Space Agency (ESA)'s Copernicus Programme. Results {#Sec2} ======= Here we report local geohazards occurring in West Texas, most of which have not been noticed and reported yet. Knowledge of the presence of the ongoing geohazards in West Texas is a precursor to understanding the trigger and causality of ground deformation, the revelation of which is a focal point of our study. The localized geohazards presented below may have different characteristics in spatio-temporal progression and causality (i.e. wastewater injection, CO~2~ flooding, hydraulic fracturing, freshwater impoundment), but all are happening because West Texas contains a sequence of water-soluble (limestone, evaporite) and shale formations that are highly vulnerable to human activities. Wastewater injection into formation and surface uplift {#Sec3} ------------------------------------------------------ Wastewater 'flowback fluid', a byproduct of oil and gas production^[@CR24]^, has been injected deep underground about 15 km west of Wink and Kermit, Texas (Fig. [1](#Fig1){ref-type="fig"}). The hydrocarbon production in the Bone Springs reservoir requires hydraulic fracturing, and wastewater (also called brine) containing high concentrations of total dissolved solids (TDS) is produced as a result of the operations. Two wells (API No. 49533675 and 49530150 in Fig. [2a](#Fig2){ref-type="fig"}) located near the county border between Winkler and Loving counties are classified as Class II injection wells for disposal of saltwater and non-hazardous fluids into the subsurface as a result of oil and gas production. The injection depth is from 1,590 to 1,670 m where the Bell Canyon Formation in the Delaware Basin of the larger Permian Basin lies. The upper layer (\~10 m thick) of the formation is composed of limestone that can confine the upward flow of injected fluids. Most wastewater is injected below the nearly impervious limestone units, into the Bell Canyon Formation sandstones (also called Ramsey sandstones); these sandstones have a porosity of \~20% of open pore-space for holding fluids, and a moderate-to-low permeability (a measure of how readily fluids can flow through the rock) of \~40 md (millidarcy)^[@CR25]^. Our InSAR analysis has detected the surface upheaval approximately centered on the injection well No. 49533675 (Fig. [2a](#Fig2){ref-type="fig"}). The maximum (cumulative) uplift from late 2014 to April 2017 was \~5.5 cm with the shape of a distorted ellipse, and the influential zone is within a 2 km radius of the peak deformation (white dot labeled 'point A'). Horizontal (east-west) deformation with the maximum of \~1.2 cm is also occurring on both the east and west sides of the peak uplift (inset in Fig. [2a](#Fig2){ref-type="fig"}), with the western region moving to the west (negative, blue color) and the eastern region moving to the east (positive, red color). The horizontal deformation around the injection wells represents \<\~20% of the vertical (up-down) deformation; we therefore concentrate on the vertical deformation in this study.Figure 2Ground uplift due to fluid (wastewater, CO~2~) injection. (**a**) Uplift in Winker County, TX, induced by wastewater injection in nearby wells (API No. 49533675, 49530150). Inset illustrates cumulative east-west deformation in the box outlined by a dashed rectangle. (**b**) Time-series cumulative vertical deformation in a point A (Fig. 2a) and the volume of injected wastewater (blue and gray bars) in two injection wells. (**c**) Uplift in Ward County, TX, induced by CO~2~ injection in an EOR field (triangles). (**d**) Time-series cumulative vertical deformation in a point B of Fig. 2c and the volume of injected CO~2~ (orange and gray bars) in EOR injection wells of Fig. 2c. The figures including spatial information have been created using open-source software GMT 5.2.2_r15292 available at <http://gmt.soest.hawaii.edu/projects/gmt/wiki/Download>. The National Agriculture Imagery Program (NAIP) images used as a background of the figures were downloaded in 2017 through Geospatial Data Gateway <https://datagateway.nrcs.usda.gov> provided by United States Department of Agriculture (USDA). Generally, surface uplift can be caused by the expansion of the geological formation where the fluid is injected, resulting in the upward movement of the ground surface. The injected formation experiences an increase in pore pressure as well as a decrease of the effective stress^[@CR26]^, promoting the surface uplift as we observed^[@CR27]^. At the point of maximum uplift (A in Fig. [2a](#Fig2){ref-type="fig"}), uplift was detected beginning around September 2015, with a sharp increase (at a rate of \~6 cm/yr) during the first half of 2016 (Fig. [2b](#Fig2){ref-type="fig"}), and the value after October 2016 sustained near \~5 cm cumulative deformation in spite of some monthly fluctuations. The temporal changes in uplift seem to be in concert with the changes in injection volume, suggesting that a mechanical compaction of sands by means of poroelasticity is likely the primary cause of the deformation^[@CR28]^. In addition, the small variations in the vertical deformation since mid-2016 can be depicted as the combined effects of poroelastic compaction and viscoelastic behavior of fine-grained formations surrounding an injected strata^[@CR28],[@CR29]^. We can also infer that, relatively speaking, the upper (sandy) layer responds rapidly to waste water injection, but the lower (shale/silt) formation reacts gradually to changes in overlying stress. To unravel the causality of the surface uplift, we compared our deformation observations to the sequence of wastewater injection rates in the nearby wells. Based on the H-10 forms provided by the Texas Railroad Commission (RRC)^[@CR24]^, the No. 49533675 injection well has been active since January 2016; the No. 49530150 injection well, which first became operational in 2009, experienced a period of disuse, and was then reactivated in September 2015 (Fig. [2b](#Fig2){ref-type="fig"}). The ratio of the uplift volume (i.e. the multiplication of the uplift amplitude and area extent) and the injection volume is about 0.05 m^3^/BBL (1 BBL ≈ 0.12 m^3^). Based on the onset of the uplift coinciding with the reactivation of 49530150 in September 2015, along with the increasing uplift rate aligning with the use of 49533675, it seems likely that both injection wells were affecting the surface uplift, but the effects of the two are not equivalent. It seems that the injection well No. 49533675, closest to the peak of vertical deformation, is likely situated in a geologically weak, critical formation, allowing the injection/disposal of wastewater to influence the surface deformation more dominantly. The correlation between the vertical deformation and the wastewater injection suggests that the expansion of injected formations induced a localized, relatively small-sized (\~2 km in dimension), and small-magnitude (\~5 cm) surface uplift. Although the onset of ground uplift was most likely triggered by the wastewater injection and the nearly instantaneous response of the ground surface results from the high elasticity of the underlying formations, the correlation between wastewater injection and ground surface may not be as high as we expect. The stratigraphic response to the decreased or increased pore pressure and effective stress can be a complicated process. When the injected volume is in decline, the release of relative pore pressure allows for the different response, namely immediate downward movement of coarse-grained formations and lagged upward movements of fine-grained formations. Such combined effects of the subsurface/surface processes result in retention of the intermediate correlation between the injection volume and ground uplift. The deformation has not invoked seismicity yet, but, if the injection continues, it has potential to threaten the integrity of County road 302, nearby oil/gas pipelines, and hydrocarbon production facilities. Carbon dioxide (CO~2~) injection and surface uplift {#Sec4} --------------------------------------------------- Miscible CO~2~ flooding has been applied for decades as a tool for EOR in the depleted oil and gas reservoirs of the United States^[@CR30],[@CR31]^. Unlike water and oil, supercritical fluid CO~2~ and oil mix together well, forming a single homogenous, or 'miscible', fluid (CO~2~ has different properties, depending upon its physical state; At room temperature, CO~2~ is a gas, such as what we exhale; we use liquid CO~2~ as a coolant, and we refer to the solid form of CO~2~ as "dry ice". Supercritical CO~2~, achieved under specific pressure and temperature conditions, is between gas and a liquid state, with some properties of each). CO~2~ is injected into a disposal well within a reservoir after initial hydrocarbon production rates have declined, where the CO~2~ mixes with the hydrocarbons. The CO~2~ injection causes an increase in reservoir pressure, which forces the CO~2~-oil mixture out of the pores of the rock and towards one or more producing wells, allowing more oil to be recovered from the reservoir. CO~2~ injection for EOR has been economically efficient due to its low cost, aiding the Permian Basin's EOR boom in the 1970s and 1980s and the recent years^[@CR32],[@CR33]^. The North Ward Estes Field west of Monahans, Texas and near Wickett, Texas is one of the largest cumulative oil-producing field in the Permian Basin^[@CR34]^ (Fig. [2c](#Fig2){ref-type="fig"}). The oil and gas are produced from the Yates and Queen Formations, within the Midland Basin of the larger Permian Basin. In CO~2~ (Class II) injection wells 11 km southwest of Monahans, Texas (Fig. [1](#Fig1){ref-type="fig"}), the CO~2~ is injected into both formations at depths between 750 and 810 m. Crude oil and gas can be produced from both Yates and Queen formations, but Yates consists of very-fine-grained sandstones to siltstones separated by dense dolomite beds and contains \~16% of porosity and \~37 md of permeability, providing more dominant production volume in the North Ward Estes Field^[@CR33]^. Salt water injection is also used for EOR (either 'water flooding' by itself, or in alternation with CO~2~ flooding), but its use today in this region is very limited (1% of total injected fluids) and most injection for EOR relies on the miscibility of anthropogenic CO~2~ (99% of the total injected fluids). Our multi-temporal InSAR analysis has detected the ellipse-shaped surface uplift (major and minor axis: 6 km and 4 km, respectively) in the immediate vicinity of the CO~2~ injection sites (Fig. [2c](#Fig2){ref-type="fig"}), with a cumulative uplift of \~3 cm from late 2014 to April 2017. At the point of maximum uplift (B in Fig. [2c](#Fig2){ref-type="fig"}), the cumulative uplift increased linearly (at a rate of \~3 cm/yr) until January 2016 after which the value stayed at \~3 cm cumulative uplift (Fig. [2d](#Fig2){ref-type="fig"}). Within 500 m of the maximum uplift, 11 CO~2~ injection wells (triangles in Fig. [2c](#Fig2){ref-type="fig"}) remain active as of April 2017. Although there has been variation in the injected volume (gray bars in Fig. [2d](#Fig2){ref-type="fig"}), most injections of CO~2~ occurred during 2015, with much lower injection volumes (below 5 million m^3^) since January of 2016 (Fig. [2d](#Fig2){ref-type="fig"}). The API No. 47530058 injection well (cyan triangle in Fig. [2c](#Fig2){ref-type="fig"}; orange bars in Fig. [2d](#Fig2){ref-type="fig"}) lies in approximately the same location as the maximum uplift. The mechanism of the surface uplift caused by the CO~2~ injection is almost identical to the wastewater injection-induced uplift. Injected fluids, in this case, liquid supercritical fluid CO~2~, increases the pore pressure in the rocks (sandstones in Yates Formation for the CO~2~ EOR sites) and the release of the effective stress is followed by surface uplift^[@CR26],[@CR27]^. The fluctuations in deformation after the injection was slowed down or stalled can be due to the collective effects of poroelastic compaction and viscoelastic delayed uplift in the formations surrounding the injected layer^[@CR28],[@CR29]^. The proximity between maximum uplift and the No. 47530058 injection well, implies that the CO~2~ flooding in that particular well is more influential on the movement of ground surface than other surrounding wells (black triangles in Fig. [2c](#Fig2){ref-type="fig"}). The high correlation between a large amount of uplift (\~3cm) and CO~2~ flooding during 2015 suggests that the instability of ground surface in the southwest of the North Ward Estes Field was induced by the pressurized injection of CO~2~ into the Yates formation. Contrary to the surface uplift in the vicinity of No. 47530058, no significant deformation has been detected on the other portions of the North Ward Estes Field. Differences in rock strength, porosity, compressibility, and permeability can play a role in the occurrence of deformation^[@CR28]^. CO~2~ flooding has revitalized, and continues to enhance recovery of the mature oil fields of the Permian Basin, helping to produce significant volumes of oil without CO~2~ emission^[@CR32]^. However, pressurized injection into a geologically unstable rock formation can destabilize the ground surface and risks the productivity of further oil operations^[@CR35],[@CR36]^. Dissolution of salt/limestone in Santa Rosa Spring {#Sec5} -------------------------------------------------- The Pecos County Water Improvement District No. 2 owns and operates a 2 km wide reservoir, known as the Imperial Reservoir (Fig. [3a](#Fig3){ref-type="fig"}), located about 6.4 km south of Grandfalls, Texas. Used for both irrigation of agricultural fields in Coyanosa, Texas and for recreational purposes, the Imperial Reservoir's water is pumped from the Pecos River. In addition to the pumped Pecos River water, the reservoir also receives artesian spring water from the Santa Rosa Spring, 13 km southwest of Grandfalls, Texas in Pecos County, through narrow canals and channels.Figure 3Ground subsidence in karst terrain underlain by limestone and salt. (**a**) Cumulative vertical deformation in Santa Rosa Spring. (**b**) Time-series cumulative vertical deformation at C, D, and E points of Fig. 3a. (**c**) Cumulative vertical deformation around abandoned wells in Imperial, Texas. Inset represents the averaged deformation rate in a boxed region by stacking interferograms of less than 12 days. (**d**) Time-series cumulative vertical deformation at F, G, H, I, J, and K of Fig. 3c. (**e**) Vertical deformation rate around Wink sinkholes. The figures including spatial information have been created using open-source software GMT 5.2.2_r15292 available at <http://gmt.soest.hawaii.edu/projects/gmt/wiki/Download>. The NAIP images used as a background of the figures were downloaded in 2017 through Geospatial Data Gateway <https://datagateway.nrcs.usda.gov> provided by USDA. Our InSAR analysis has detected rapid subsidence occurring in Santa Rosa Spring (Fig. [3a](#Fig3){ref-type="fig"}) from late 2014 to April 2017, with a maximum cumulative subsidence of approximately 23 cm, or at a rate of \~8.9 cm/yr (point D in Fig. [3a](#Fig3){ref-type="fig"}). The subsiding region is elliptical in shape with dimensions of \~1.4 km by \~1.0 km. Time-series deformation measurements at three points (C, D, and E in Fig. [3a](#Fig3){ref-type="fig"}) show a strong linearity (Fig. [3b](#Fig3){ref-type="fig"}), regardless of other factors of seasonal effects and irrigational uses. Around the Santa Rosa Spring, hydrogen sulfide has been produced from multiple wells. However, the hydrogen production can hardly be directly connected to the rapid subsidence because all of the wells are located outside of the deforming zone that is centered on the Santa Rosa Spring. Hence, the hydrogen production should not have apparent impact on the observed subsidence. Historically, a limestone cavern formed around Santa Rosa Spring, and the runoff water occasionally flows from and into the cavern^[@CR37]^. Stratigraphical data for the area's closest well, API No. 37137696 well (Fig. [3a](#Fig3){ref-type="fig"}), indicates Bone Springs Limestone formation is present at depth between 2,065 m and 2,911 m. However, the dissolution of this deep-seated limestone formation and the connection to the surface subsidence is not realistic as the extent of the subsidence area is less than 1.5 km (Fig. [3a](#Fig3){ref-type="fig"}). In addition, the dissolution rate of carbonate rocks like limestone is generally much smaller than that of the evaporite rocks, and a limestone cavern in a natural state forms very slowly (mm/yr)^[@CR38],[@CR39]^. Therefore, such rapid subsidence rate (8\~9 cm/yr) at Santa Rosa Spring is less likely caused by the natural dissolution of limestone. Because of the nature of the rapidity in subsidence, we believe the most likely cause of the observed subsidence is the dissolution of the Salado formation in the depth of 300\~450 m beneath Permian Basin. Investigations of groundwater conditions in Pecos County indicated that the highest salinity over the region was found at Santa Rosa Spring (7224 mg/l)^[@CR40]^. In addition, it has been documented that the salinity over the Santa Rosa Spring increased by 4,894 mg/l from the 1940s to 1987^[@CR40]^. Therefore, we interpret the rapid subsidence in the Santa Rosa Spring area to be caused by the dissolution of salt deposits. Although the subsidence has not triggered the collapse of the surface, the continuous surface subsidence in the areas can be hazardous to water management facilities and/or nearby oil/gas wells. Freshwater impoundment in abandoned wells {#Sec6} ----------------------------------------- The region near Imperial, Texas (Fig. [1](#Fig1){ref-type="fig"}), has been troubled with the growing subsidence, ground fissures, and the emergence of sinking lakes^[@CR41],[@CR42]^. Some abandoned water and oil wells were left unplugged and thus did not prevent freshwater impoundment through cracks in cement casing and/or the corroded steel pipes, and the freshwater impoundment is known to be the primary cause of rapid subsidence in the area^[@CR41]^. However, prior to this study, subsidence near many abandoned wells has gone unnoticed^[@CR42]^, and the Texas Department of Transportation is expected to spend millions of dollars to identify and plug the abandoned wells^[@CR41],[@CR42]^. Our InSAR analysis has detected rapid subsidence around 7 km southwest of Imperial, Texas (Figs [1](#Fig1){ref-type="fig"} and [3c](#Fig3){ref-type="fig"}). The region around Boehmer Lake (F and G in Fig. [3c](#Fig3){ref-type="fig"}) has sunk as much as 2\~3 cm over the course of our InSAR acquisition period (2.5 years). Boehmer Lake did not exist before 2003 and the sinking ground surface led to the formation of the lake as a result of water arising from the subsurface, thus this is continued subsidence. Farm to Market (FM) road 1053 (near H, I in Fig. [3c](#Fig3){ref-type="fig"}) is sinking so fast that we could only compare pairs of satellite data within 12 days in order to maintain coherence of the InSAR image (inset in Fig. [3c](#Fig3){ref-type="fig"}). Therefore, using InSAR pairs with small (6 or 12 days) temporal baselines, we were able to measure the round-shaped (500 m in diameter) subsidence rate (\~10 cm/yr) along FM 1053 road. Due to the safety concern, use of the road was suspended in August of 2016 and the realignment of FM 1053 was discussed by the state transportation agency^[@CR41],[@CR42]^. A third nearby area of rapid subsidence (\~10 cm/yr for 2.5 years) (near J, K in Fig. [3c](#Fig3){ref-type="fig"}) was observed near oil well API No. 37137310 (cyan triangle in Fig. [3c](#Fig3){ref-type="fig"}, near J and K). The subsidence pattern (650x350 m in dimension) is stretched NW-SE, aligning with two wells: API No. 37172505 and 37137310. Like the subsidence over the Santa Rosa Spring, vertical deformation measurements at points (F, G, H, I, J, K in Fig. [3c](#Fig3){ref-type="fig"}) show a strong linearity in time (Fig. [3d](#Fig3){ref-type="fig"}). Two points (F, G in Fig. [3c](#Fig3){ref-type="fig"}) in Boehmer Lake are experiencing 1.4 and 2.0 cm/yr subsidence. Points (H, I in Fig. [3c](#Fig3){ref-type="fig"}) near the outer edge of the deformation in FM 1053 road show the subsidence of 0.7 and 1.5 cm/yr, respectively. The areas near two oil wells are also undergoing subsidence of as much as 3.9 and 2.5 cm/yr, respectively. A few oil wells to the south of Imperial, Texas are currently active (i.e., No. 37137310 in Fig. [3c](#Fig3){ref-type="fig"}), with moderate production (less than 400 BBLs/month) for most of the time. The observed linear subsidence relatively independent of oil/gas production and seasonal effects has the characteristics of ground subsidence (subsidence sinkhole) in karst terrain^[@CR19]^. High salinity of channels along the Pecos River near Imperial, Texas and Boehmer Lake^[@CR43],[@CR44]^ suggests that the surface and underground water interact with the subsurface salt deposit. The deforming area (Fig. [3c](#Fig3){ref-type="fig"}) is located in the Central Basin Platform close to the eastern Delaware Basin of the larger Permian Basin and is underlain by the Salado Formation in the depth of 300\~500 m. Through unplugged abandoned wells, corroded pipes, or cracks in the casing, freshwater flows down and/or artisan water rises to the Salado formation, accelerating the dissolution of the evaporite, creating voids in the beds, and causing rapid subsidence on the surface^[@CR17],[@CR39]^. Indeed, all three areas of subsidence are near wells. Boehmer Lake formed over an abandoned oil well (No. 37172656), which had stopped producing decades ago, and the subsidence along FM 1053 road is occurring near an orphan well (red triangle in Fig. [3c](#Fig3){ref-type="fig"}) that was identified as an inactive, non-compliant well by Texas' petroleum regulatory agency (RRC)^[@CR45]^. The oil production in No. 37137310 or related operations may influence the large rates of subsidence. However, the downward flow of freshwater into an unplugged oil well (i.e. No. 37172505) may play a more influential role in subsidence as the subsiding areas are all underlain by salt deposits. The dissolution of salt beds (evaporite) is typically more substantial than that of the carbonate rocks (limestone), and often exceeds \~10 cm/yr subsidence^[@CR38],[@CR39]^. Expansion of the cavity and the migration of voids toward the surface can possibly result in the collapse of the surface into sinkholes. Therefore, movements around the roads and oil facilities to the southwest of Imperial, Texas should be thoroughly monitored to mitigate potential catastrophes. Dissolution of the salt bed near the Wink Sinkholes {#Sec7} --------------------------------------------------- Ground subsidence is more widely recognized near two Wink sinkholes, which collapsed in 1980 (Wink Sink \#1 in Fig. [3e](#Fig3){ref-type="fig"}) and 2002 (Wink Sink \#2 in Fig. [3e](#Fig3){ref-type="fig"})^[@CR18],[@CR19]^. The sinkholes, 4 km northeast of Wink, Texas and 9.5 km southwest of Kermit, Texas (Fig. [1](#Fig1){ref-type="fig"}), lie in the Delaware Basin part of the larger Permian Basin. The Salado Formation is near a depth of \~500 m^[@CR46]^ over this area. The oil and gas in the region are mostly produced from the Yates Formation underneath the Salado Formation^[@CR19],[@CR46]^. Both Wink sinkholes collapsed because of downward freshwater seeping through unplugged boreholes and cracked cement casing in oil and water wells. The subsidence in the immediate vicinity of the collapsed sinkholes continues at a rate of \~3--4 cm/yr (Fig. [3e](#Fig3){ref-type="fig"}). The most significant ongoing subsidence is occurring 1 km east of the Wink \#2. There are two large subsidence bowls (Fig. [3e](#Fig3){ref-type="fig"}), and the maximum subsidence in the southern bowl (380 m by 280 m in dimension) exceeds 40 cm/yr. The large gradient of subsidence in a small region cannot be observed by C-band InSAR pairs with 24-days or longer. Accordingly, only 5 InSAR pairs with 6 or 12-days temporal baselines are used to calculate the high linear deformation rate here. The peak subsidence is located at the intersection of County roads 201 and 204, and there are no existing active wells around the region. Therefore, the rapid subsidence is likely induced by the freshwater impoundments from the nearby abandoned wells. During our field trip, we observed numerous recent ground fissures around the intersection of County roads 201 and 204. These growing fissures can allow the rainwater to swiftly flow down to the Salado formation and promote the dissolution of the salt layers. Because the oil and gas production in the area has been inactive for years, the mechanism for both bowls is believed to be the same. The access to the region surrounding Wink \#1 and \#2 has been restricted out of safety concerns, but County Road 201 continues to be used to transport oil and gas products. Based on the observed rapid subsidence, the use of County Road 201 should be proactively monitored for safety. Additionally, the effect of ongoing subsidence on the pipelines in the area needs to be reviewed as well. Hydrocarbon production in Pecos and the associated seismic events {#Sec8} ----------------------------------------------------------------- The Wolfbone field 9 km south of Pecos, Texas in Reeves County (Fig. [1](#Fig1){ref-type="fig"}) has been developed for oil and gas production since 2014. Compared to other oil wells in West Texas that produced hydrocarbon for decades, the wells (API No. 38934300, 38933302, 38933668, 38934175 in Fig. [4a](#Fig4){ref-type="fig"}) in the region are recent with significant production exceeding 10,000 BBLs (1 BBL ≈ 0.12 m^3^) starting in early 2015. The drilling depth of the wells is \~4 km below the surface, and most hydrocarbons are produced from the Bone Springs and Wolfcamp formations^[@CR47]^, which lie in the depth of 2.3\~3 km and 3\~3.7 km, respectively.Figure 4Ground deformation in Pecos, Texas, induced by hydrocarbon production. (**a**) Cumulative ground deformation in a hydrocarbon production field of Pecos, Texas. (**b**) Time-series cumulative vertical deformation in points (L, M, N, O) of Fig. 4a. Yellow stars represent seismic events (along with magnitude and depth) occurring less than 15 km from the subsidence area between late 2014 and April 2017. Oil production (blue and orange bars) volumes in the surrounding wells correlate to the triangles in Fig. 4a. The figure has been created using open-source software GMT 5.2.2_r15292 available at <http://gmt.soest.hawaii.edu/projects/gmt/wiki/Download>. The NAIP images used as a background of the figures were downloaded in 2017 through Geospatial Data Gateway <https://datagateway.nrcs.usda.gov> provided by USDA. Production from the wells has been enhanced by vertical and horizontal hydraulic fracturing of the sandstone and shale formation. Approximately 4.5 cm subsidence around four producing wells in the Wolfbone field can be observed from our InSAR analysis (Fig. [4a](#Fig4){ref-type="fig"}), while the horizontal deformation is negligible. From the time-series measurements in multiple points (Fig. [4b](#Fig4){ref-type="fig"}), the subsidence rate has been at a constant, relatively slow speed (1.5 cm/yr) from 2015 to 2016. However, the subsidence was accelerated from January to March 2017 and the amount of the two-month subsidence (O in Fig. [4b](#Fig4){ref-type="fig"}) for two months reached up to 1.5 cm (at a rate of \~9 cm/yr). Following the subsidence, the surface uplifted (Fig. [4b](#Fig4){ref-type="fig"}) with a maximum magnitude of \~0.5 cm between March and April 2017. We attribute the subsidence to the hydrocarbon production, as most of the subsidence is bounded by production wells in the deep formations^[@CR28]^ and the extent of the subsidence area is consistent with a source depth of 2--4 km (Fig. [4a](#Fig4){ref-type="fig"}). Although the monthly hydrocarbon production exhibits variations, the detected ground subsidence is relatively linear in time. We can postulate that the formations in the subsiding areas behave viscously, different from other observed sites of wastewater injection and CO~2~ flooding. The removal of a huge mass of oil from the subsurface creates the stress changes in the rock/soil layers, but the ground surface gradually responds to such stress changes in the stratigraphy containing abundant viscoelastic shale formations. Although Pecos, Texas, is located in the geologically stable continental region without any seismic events before 2010s, there have been six small earthquakes in recent years (yellow stars in Fig. [4b](#Fig4){ref-type="fig"}). The magnitude of the earthquakes varies between M 1.8 and M 2.7, and all but two events occurred less than 15 km from the subsidence area. Both the timing of the April 2015 earthquake, shortly after the start of the massive increase in oil well production rates, as well as the latest changes in ground surface deformation coinciding the recent five earthquakes in 2017, suggest a close association among ground surface deformation, oil/gas production and seismic events, similar to those observed elsewhere^[@CR3],[@CR22]^. The underlying mechanism to connect oil/and gas production and surface subsidence is that the extraction of oil or gas from underground decreases the pore pressure in formations, which in turn increases the effective stress, which might favor the slip of existing faults. Hydraulic fracturing along a horizontal pipeline from a horizontal well (such as well No. 38934300) could be responsible for the lateral distribution of vertical displacement in the shale oil field. Moreover, the two-year deformation can accumulate stress on the basement faults near the deforming areas. Although the pre-existing faults in Pecos, Texas have not been documented, the surge in seismic events suggests that faults may exist in the bedrock. Ground subsidence can activate the fault(s)^[@CR3],[@CR16],[@CR48]^, and the accelerated subsidence and subsequent uplift in early 2017 can be interpreted as the co-seismic deformation and the viscoelastic relaxation during the short-term post-seismic deformation as a result of the multiple earthquakes^[@CR49]^. The focal depth of the earthquakes ranges between 3 and 5 km, just slightly below where the hydrocarbon was extracted. Unfortunately, due to the sparse number of seismic stations in Texas, the accuracy in the location of the seismicity can be on the order of 10 km^[@CR50],[@CR51]^. However, the absence of any previously reported regional earthquakes near Pecos, Texas, a shallow focal depth around the producing zone, the proximity of ground deformation to the epicenter therefore suggests a causative link between hydrocarbon production and the sequence of earthquakes (induced seismicity). Discussion {#Sec9} ========== We have compiled multiple localized geohazards in West Texas (Table [1](#Tab1){ref-type="table"}), most of which were induced by, or at least influenced by, human activities. The correlation between time-series vertical deformation and fluid injection/hydrocarbon production exhibits evident effects of human activities on the surface, but the modeling approach can also help explain the causal relationship. The inverse modeling with the observed cumulative vertical deformation (Fig. [5a](#Fig5){ref-type="fig"}) in the box outlined by a dashed rectangle (Fig. [2a](#Fig2){ref-type="fig"}) computes the best-fit model (Fig. [5b](#Fig5){ref-type="fig"}) with the least residual (Fig. [5c](#Fig5){ref-type="fig"}; root mean square (RMSE) misfit: 0.10 cm). The modeled result with a rectangular (3.5 km by 2.5 km) dislocation source at a depth of 1.63 km (known average injection depth) indicates that the peak uplift is located near the wastewater injection well (API No. 49533675). During the observation period of about 2.5 years, both wells (API No. 49533675 and 49530150 in Fig. [2a](#Fig2){ref-type="fig"}) had injected saltwater of 5,119,129 BBLs (≈610,408 m^3^) and 3,704,047 BBLs (≈442,623 m^3^), respectively (1,053,031 m^3^ in total). Our computed volume change at the source is about 790,183 ± 8,750 m^3^ and slightly lower than the total injected volume of saltwater. The difference between the two can be attributed to the diffusion of injected saltwater into the surrounding rocks without generating any measurable deformation. The comparable volume change that was calculated from our model reaffirms that the observed surface uplift was induced by disposing a massive volume of saltwater. Although modeling can shed light on revealing the causality of the ongoing ground deformation, we have to realize that models are non-unique and dependent on hydrogeological parameters of the study site. In most general cases where surface uplift and human activities are highly correlated (Fig. [2d](#Fig2){ref-type="fig"}), the comparative analysis presented in this report is sufficient for assessing the effect of human activities in West Texas.Table 1List of the observed ground deformation in West Texas.LocationLat/LonBasinPeriodMagnitudeSizeCauseSeismicityWink, TXN31.78° W103.31°Delaware Basin01/2016\~07/20165 cm (uplift)2.0 × 2.0 kmWastewater injectionNoMonahans, TXN31.51° W102.97°Midland Basin11/2014\~01/20163 cm (uplift)6 × 4 kmCO~2~ injectionNoGrandfalls, TXN31.27° W102.96°Delaware Basin11/2014\~04/201723 cm (subsidence)1.4 × 1.0 kmSalt/limestone dissolutionNoImperial, TXN31.21° W102.75°Central Basin Platform11/2014\~04/20179 cm/yr (subsidence)650 × 350 mImpounded freshwater from abandoned wellsNoWink, TXN31.78° W103.12°Delaware Basin11/2014\~04/201740 cm/yr (subsidence)380 × 280 mSalt dissolutionNoPecos, TXN31.35° W103.48°Delaware Basin01/2017\~04/20174.5 cm (subsidence)2.5 × 1.0 kmHydrocarbon productionYesEach basin represents the geologic structural basin in the deforming area. Period of the largest gradient of subsidence/uplift, magnitude of maximum cumulative deformation for 2.5 years, and the size of the largest spatial deformation in a region was presented. The cause of deformation was inferred through comparison of the observed deformation, oil/gas production, the volume of the injected fluid, and the geologic characteristics.Figure 5Modeled results of cumulative InSAR vertical deformation around wastewater injection wells. (**a**) observed cumulative vertical deformation in the box outlined by a dashed rectangle (Fig. [2a](#Fig2){ref-type="fig"}). (**b**) modeled vertical deformation. (**c**) residuals (observation -- model). The figure has been created using MATLAB R2017a licensed by Southern Methodist University. Our observations in West Texas can be separated into three groups: i) surface uplift induced by fluid injection, ii) rapid subsidence in a karst terrain due to dissolution of underlying salt deposit, and iii) ground subsidence and seismicity induced by hydrocarbon production. The first category includes two geohazards: one west of Wink, Texas and another southwest of Monahans, Texas. Although both wastewater and CO~2~ injection for EOR are economically efficient to extract oil from reservoirs, the high pressure for raising hydrocarbon production and the increased fluids in the rocks can promote the surface uplift as much as 3\~5 cm during an injection. Close correlation between the surface uplift and the injection fluid suggests a causal link between oil producing activities and ground instability. The second geohazards category includes salt (and perhaps limestone) dissolution in Santa Rosa Spring, Grandfalls and Wink with rapid subsidence in a karst terrain showing the characteristics of a strong linearity regardless of other factors (groundwater, precipitation, temperature, hydrocarbon production). Therefore, the rapid subsidence, once promoted by the freshwater impoundment and the interaction with brine water, is not slowed down by the changes of external effects. In addition, while oil and gas production does not directly impact the subsidence, poor management of the oil and gas facilities, boreholes, and pipelines allows freshwater or brine to interact with Salado salt (and possibly limestone) formations. Their subsidence rate can readily exceed 5 cm/yr, and the subsidence could lead to a collapse at the surface (collapse sinkhole^[@CR52]^). Finally, the third geohazards category includes subsidence at the recently developed hydrocarbon sites in Pecos, Texas. Subsidence of \~4 cm in 2.5 years may not be significant on the ground surface, but the continuous subsidence can exert stress on the deep-seated formations and possibly reactivate undocumented faults near the producing zone. West Texas has experienced unprecedented increases of seismicity in last 5--6 years. Earthquakes are occurring in a geologically stable region and the temporal and spatial association with hydrocarbon production suggests that these earthquakes are induced^[@CR16]^. Based on the accelerated subsidence in 2017, we can hypothesize that the increased number of seismic events is a consequence of the onset of massive hydrocarbon production and thereby ground subsidence after the increase of the effective stress. Contrary to the induced seismicity near the hydrocarbon production in Pecos, Texas, all other ground deformations identified in this study were not followed by seismic events. The ground surface undergoes significant subsidence up to 40 cm/yr (i.e. Wink sinkholes), suggesting that the basement faulting near the producing/deforming zone might not exist and the rapid subsidence can be supported by the underpinning rock formation. Another possibility is that the seismic network in West Texas is neither dense nor sensitive to the micro-earthquakes occurring around the deforming areas^[@CR3],[@CR9]^. In that case, the small-magnitude seismic events could go undetected due to the current sparse placement of seismometers in the area. Regardless of the occurrence of the induced seismicity, measuring the ground deformation from space in areas where the wastewater and CO~2~ are injected, rocks are dissolving, or the massive hydrocarbons are produced is possible using satellite radar interferometry from new free-data satellites, as we demonstrated in our research. The ground deformation in West Texas is very responsive to anthropogenic activities with little time delay after their implementations were initiated. To avoid more severe geohazards in the future, consideration of such poroelastic movements in producing formations should be carefully heeded. If we do not mitigate the possible geohazards with continuous monitoring of surface deformation, we can expect one or more possible outcomes: i) damage to infrastructures (roads, railroads, levees, dams), ii) environmental impacts (i.e. ground-water pollution), iii) risks to oil and gas pipelines (note: West Texas has one of the densest networks of oil and gas pipelines in the U.S.), iv) potential threat to residents in surrounding communities, v) economic costs in hydrocarbon productions (i.e. possible improper well managements and thereby ground deformation can lead to large spending by oil companies and governmental agencies to prevent additional damages), and vi) induced seismicity. Micro-seismicity may not result in the large drastic hazards, but the ground deformation (subsidence/uplift) itself poses more direct threat to industrial facilities, infrastructures, and residential areas. Measuring deformation can assist stakeholders as they examine the safety of the oil and gas operations and make important decisions for securing facilities and people from potential larger catastrophic events. The Texas petroleum regulators have required the submission of historical seismic events in order for an injection/disposal well permit to be approved, but the additional, continuous monitoring of the ground deformation in oil producing areas (regardless of methods including conventional oil production, water flooding, CO~2~ flooding, or hydraulic fracturing) can provide crucial, detailed information for the safe operations of oil and gas productions and the sustainable growth of the energy industry. Methods {#Sec10} ======= Sentinel-1A/B imagery {#Sec11} --------------------- Sentinel-1A/B, a constellation of two Synthetic Aperture Radar (SAR) satellites operated by the European Space Agency (ESA) within the Copernicus Program, represent the first satellite radar missions providing radar imagery freely to the public. Its radar sensor using interferometric wide swath (IW) mode as a background mode provides C-band (5.4 GHz in center frequency; 5.6 cm in wavelength) imagery with intermediate spatial resolution (20 (azimuth) x 5 (range) m) and dense temporal acquisitions with revisits of 6 days (over Europe, or 12 days outside Europe)^[@CR53],[@CR54]^. For our study, Sentinel-1A/B imagery from November 2014 to April 2017 was been processed. To estimate the deformation in two directions (vertical and east-west), it was necessary to utilize the SAR images from ascending (path 78) and descending (path 85) tracks (Fig. [1](#Fig1){ref-type="fig"}), which have the respective heading angle (clockwise from north) of 347.23° and 192.75° with an incidence angle of \~33.8° in the image center. Adaptive multi-look factors were applied to all used SAR images to maintain appropriate spatial resolution in diagnosing the small-sized deformation phenomena. Detection of deformation signal and estimation of 2D deformation with InSAR {#Sec12} --------------------------------------------------------------------------- Because Sentinel-1A/B acquires data with a swath of \~250 km, processing the large size of SAR scene for time-series measurements can be inefficient for detecting a small-sized deformation in West Texas. Moreover, our study area orientation from north to south, required merging of two or more frames. Filtering was applied to interferograms for improving InSAR coherence and also to permit retrieval of the localized deformations in an oil field feasible. To detect numerous deformation signals without losing much spatial resolution, we adopted a stepwise approach of InSAR analysis from a broad to fine scale. In the first run, all available Sentinel-1 images were coregistered based on the SAR image acquired on the first acquisition date. The precise coregistration of Sentinel-1 for avoiding the discontinuous phases between bursts and improving coherence was critical. The process of enhanced spectral diversity (ESD) was iterated until the coregistration precision of azimuth pixel became better than 0.001 pixel^[@CR55]^, and the pre-resampled SAR images closest to newly-resampled SAR image can aid rapid and more accurate coregistration. All available interferograms with maximum temporal and spatial baselines of 1 year and 200 m, respectively, were generated from the resampled SAR images and were thoroughly examined through visual inspection. Upon discovering the localized signals in our study area, we cropped the interferograms around those deformations. The adaptive multi-look filtering was then applied to each interferogram to maintain high spatial resolution (close to the original resolution of Sentinel-1A/B). We employed the multi-dimensional small baseline subset (MSBAS) method^[@CR56]--[@CR58]^, after removing topographic signatures from interferograms and completing phase unwrapping to estimate the vertical and the horizontal (east-west) deformation from Sentinel-1A/B with two different radar geometries of ascending and descending tracks^[@CR59]^. Because most SAR sensors are adopting a near-polar orbit and a single (right) look direction, the deformation in north-south direction cannot be resolved without multi-aperture interferometry, along-track interferometry, or offset tracking that is not suitable for mapping small-sized signals. The governing matrix for calculating 2D time-series deformation from multiple tracks is:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(\begin{array}{ccc}-\frac{4\pi }{\lambda }cos\,\theta \,sin\,\varphi \,A & \frac{4\pi }{\lambda }cos\,\varphi \,A & -\frac{4\pi }{\lambda }\frac{1}{R\,sin\,\varphi }{B}_{p}\\ & \beta I & \end{array})(\begin{array}{c}{V}_{E}\\ {V}_{v}\\ {\rm{\Delta }}h\end{array})=(\begin{array}{c}{\rm{\Phi }}\\ 0\end{array})$$\end{document}$$ where *R*, λ, *θ* and *ϕ* are the slant range from the satellite to the target (unit: m), the radar wavelength (\~0.056 m), the azimuth angle, and the incidence angle, respectively. When *M*~*k*~ and *N*~*k*~ are the numbers of interferograms and SAR acquisition dates from *k*^*th*^ SAR datasets (assuming that we have *K* (here *K* becomes 2 because we used ascending and descending track) SAR sets), respectively, *A* $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$({\rm{unit}}:\,{\rm{time}};\,\mathrm{dimension}:\,\sum _{k=1}^{K}{M}_{k}\times (\sum _{k=1}^{K}{N}_{k}-1))$$\end{document}$ is a matrix constructed from the time interval between consecutive SAR acquisitions, *β* is a regularization parameter, *I* (dimension: (2$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(\sum _{k=1}^{K}{N}_{k}-1)+1))\times (2(\sum _{k=1}^{K}{N}_{k}-1)+1))$$\end{document}$ is an identity matrix, *V*~*E*~ and *V*~*v*~ (each has dimensions of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(\sum _{k=1}^{K}{N}_{k}-1)\times 1)$$\end{document}$ are the east-west and vertical components (unit: m/time) of the ground deformation rate vector during each time interval, *B*~*p*~ (unit: m; dimension: $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\sum _{k=1}^{K}{M}_{k}\times 1$$\end{document}$) is the perpendicular baseline, *Δh* is the topography error (unit: m; not significant in a flat region), Φ (dimension: $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\sum _{k=1}^{K}{M}_{k}\times 1$$\end{document}$) is the observed (unwrapped) interferometric phase (unit: radian), and 0 is a zero vector with a dimension of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(2(\sum _{k=1}^{K}{N}_{k}-1)+1)\times 1$$\end{document}$^[@CR19],[@CR56]^ (thus, a left matrix, the unknown vector, and a right vector from (1) have a dimension of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(\sum _{k=1}^{K}{M}_{k}+2(\sum _{k=1}^{K}{N}_{k}-1)+1)$$\end{document}$ $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times (2(\sum _{k=1}^{K}{N}_{k}-1)+1))$$\end{document}$, ($\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2(\sum _{k=1}^{K}{N}_{k}-1)+1)\times 1$$\end{document}$, and ($\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\sum _{k=1}^{K}{M}_{k}+2(\sum _{k=1}^{K}{N}_{k}-1)+1)\times 1$$\end{document}$). The unknown parameters (*V*~*E*~, *V*~*v*~) were calculated by solving the matrix (1) via singular value decomposition (SVD) with minimum-norm constraints^[@CR56]^. All used InSAR pairs were connected to each other (full-rank matrix *A*) due to high coherence in our study area, meaning that we have more observations than unknowns (*V*~*E*~, *V*~*v*~, *Δh*). Atmospheric artifacts are not removed separately, because the multi-temporal InSAR using only less-contaminated interferograms could limit the effects of those noises and small areas with \~200--300 m in dimension are less influenced by a large variation of atmosphere. Due to the extreme summer heat in West Texas, the distribution of water vapor in the atmosphere can be still problematic particularly for July and August scenes. However, the spatio-temporal filtering applied to time-series measurements for reducing the effects of water vapor and residual noises worked nicely allowing us to successfully mitigate the influence of those noise and error sources. However, when the gradient of deformation was large, exceeding 5\~10 cm/yr, the interferograms from 24 or more temporal intervals could not maintain coherence. In that case, only the interferograms with 6 or 12-day temporal baselines were used to estimate such a high deformation rate (i.e. rapid subsidence near Wink sinkholes and Imperial, Texas). In West Texas, before mid-2016, most interferograms had 24 day intervals, and most of them were not suitable over the rapidly deforming regions. Therefore, for a large gradient deformation, the limited number of interferograms with short temporal baselines were used by applying a stacking method^[@CR60]^ that is particularly useful for reducing temporally-uncorrelated signals (atmospheric artifacts, noise) and computing the deformation rate (cm/yr). The peak subsidence near the Wink sinkholes cannot be observed by any 24-day interval Sentinel-1A/B interferograms due to the loss of coherence, but the applied stacking method with 6 or 12-day interval interferograms allows for locating and calculating the maximum subsidence rate in the intersection of County roads 201 and 204. Hydrocarbon production and injection volumes {#Sec13} -------------------------------------------- To characterize the ground deformation in West Texas, we performed comparative analysis with information from hydrocarbon production and injection volumes. Records relating to oil/gas production and wastewater injection were collected from the Texas RRC, which is the responsible regulatory authority of the petroleum industry and pipeline safety in Texas. In addition, drillinginfo^TM^ also provided additional information on geological formations in the locations of rapid subsidence. Injection wells, wastewater (generally saltwater) or carbon dioxide (CO~2~) can be injected into an oil-producing unit in the underground for boosting oil and gas production. Most wells used for hydrocarbon production can produce oil and natural gas together, but natural gas, often called casinghead gas, is regarded as a byproduct of these oil wells. Therefore, only the oil production in the wells was considered in our analysis. Modeling surface uplift due to wastewater injection {#Sec14} --------------------------------------------------- We modeled the cumulative surface uplift to estimate the volume change in the subsurface and assess the relationship between the ground deformation and human activities (here wastewater injection). We used Okada formulation^[@CR61]^ for motions in a homogeneous elastic half space, because ground deformation presented in our study shows high elastic response to the stress change. The source consists of a planar array of opening cracks at a fixed depth of the wastewater injection. First, we subsampled the cumulative vertical deformation using quadtree downsampling algorithm^[@CR62]^ for reducing the computational burden in modeling while preserving the statistically significant part of the deformation signal^[@CR63]^. The best-fitting models were searched over the grid and the best fitting parameters were obtained by minimizing the root mean square (RMS) misfit from the residuals (the observation minus the model)^[@CR64],[@CR65]^. **Publisher\'s note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. All Sentinel data were provided by the European Space Agency (ESA)'s Copernicus Programme via Alaska Satellite Facility (ASF) Distributed Active Archive Center (DAAC) and processed through high-performance computing utilizing SMU's supercomputer (ManeFrame). This research was financially supported by the NASA Earth and Surface Interior Program (NNX16AL10G) and the Shuler-Foscue Endowment at Southern Methodist University. Comments from Robert Gregory and Cathy Chickering Pace, three anonymous reviewers, and editorial board members improved the manuscript. Most geocoded images are drawn by Generic Mapping Tools (GMT) 5.2.2_r15292 available at <http://gmt.soest.hawaii.edu/projects/gmt/wiki/Download>. The National Agriculture Imagery Program (NAIP) imagery used as a background in the figures depicting ground deformation was accessed via the Geospatial Data Gateway <https://datagateway.nrcs.usda.gov> provided by United States Department of Agriculture (USDA). Z.L. and J.-W.K. designed the project and experiments, and J.-W.K. collected and processed all the SAR datasets. J.-W.K. and Z.L. validated and analyzed the results, and wrote the manuscript. J.-W.K. prepared all figures and Z.L. provided the guidance for improving the figures. Both authors discussed the results and reviewed the manuscript. Competing Interests {#FPar1} =================== The authors declare no competing interests.

Mass spectrometry (MS) is widely recognized for its high sensitivity and high selectivity in chemical analysis. In the application fields of drug discovery, clinical analysis, food safety, and environmental monitoring, mass spectrometry plays an important role in both qualitative and quantitative trace analysis. Standard analytical procedures include multiple steps of sample pretreatment and chromatographic separation prior to MS analysis, and these preliminary steps can only be performed in analytical laboratories. The miniaturization of mass spectrometry systems started more than a decade ago with the clear aim of performing chemical analysis in its entirety in the field and providing results rapidly.^[@ref1]^ Early in the miniaturization effort, significant research effort went into the miniaturization of the mass analyzer.^[@ref2]−[@ref9]^ This had direct and indirect impact on the size of the entire MS system. Later, miniature mass spectrometer systems were developed using different mass analyzers. Among these systems, the ion trap was preferred because of its modest vacuum requirements and intrinsic MS/MS capabilities. A configuration with an ion trap as mass analyzer and a pumping system composed of a Pfeiffer TPD 011 (later model HiPace 10, Pfeiffer Vacuum, Inc.) and a two-stage KNF diaphragm pump (model 1091-N84.0-8.99 or similar, KNF Neuberger, Inc.) became popular. This configuration was used in the Purdue University Mini 10,^[@ref10]^ the Griffin 600 (Griffin Analytical Technologies),^[@ref11]^ the Guardion-7 (Torion Technologies),^[@ref12]^ and the MMS-1000 (1st Detect Corp.).^[@ref13]^ Ion traps can perform MS analysis in the millitorr pressure range, significantly higher than for other mass analyzers, and the miniature pumping system provides adequate pumping speed to maintain vacuum for in-vacuum ionization, which is typically used for analysis of gas samples. The development of the discontinuous atmospheric pressure interface (DAPI) enabled the coupling of miniature mass spectrometers to atmospheric pressure ionization sources,^[@ref14]^ including electrospray ionization (ESI),^[@ref15]^ atmospheric pressure chemical ionization (APCI),^[@ref16]^ and various ambient ionization sources.^[@ref14],[@ref17]−[@ref19]^ For volatile analytes, membrane introduction systems^[@ref20]^ are useful interfaces for in situ analysis using portable mass spectrometers. However, analysis of nonvolatile analytes requires interfacing miniature mass spectrometers to external ionization sources. The ambient ionization methods were developed to bypass several steps in the analytical procedures for sample pretreatment and chromatographic separation that are traditionally required for MS analysis of complex samples.^[@ref21],[@ref22]^ They allow analytes in complex matrices to be directly sampled and ionized for MS analysis.^[@ref23],[@ref24]^ Qualitative analysis at low limits of detection (LODs) has been demonstrated with more than 30 ambient ionization methods, and the recently developed method of paper spray ionization has provided good limits of quantitation (LOQ) together with high accuracy in quantitative analysis.^[@ref25]−[@ref28]^ Development of disposable sample cartridges was also demonstrated to be feasible with paper spray.^[@ref29]^ Our recent efforts in developing miniature MS systems have resulted in backpack and benchtop MS systems that share the same in-house control electronics, but which are operated using different ambient ionization sources and have different physical configurations. The backpack MS, described in an accompanying manuscript,^[@ref30]^ typically uses a low-temperature plasma probe for screening nonvolatile compounds on surfaces. The benchtop miniature MS system described here, the Mini 12, was designed as a proof-of-concept prototype for targeted analysis. It is specifically aimed at point-of-care applications. Minimizing the size and weight of the system has been a major goal in the development of miniature MS systems, and a complete mass spectrometer has been developed that weighed only 4 kg.^[@ref31]^ The goal for the development of the Mini 12, however, was primarily to have a system that can perform direct chemical analysis without sample pretreatment and can be operated by untrained personnel. Use of ambient ionization to avoid sample preparation and chemical purification is a major convenience of such a system. Autonomous analysis, including recording of MS and MS/MS spectra, data analysis, and direct reporting, becomes possible with systems designed for specialized analysis. Specific scan functions and calibration data can be preset and loaded during operation and for data analysis, respectively. The challenge to achieving analytical performance qualified for point-of-care or other types of regulated applications is to obtain adequate sensitivity and precision in quantitation. Rectilinear ion traps (RIT)^[@ref8]^ have been used in the earlier Mini 10^[@ref10]^ and 11^([@ref31])^ and also now in the Mini 12 instrument developed at Purdue. MS/MS analysis is performed to increase the chemical specificity as well as to increase the signal-to-noise ratios for characteristic ions in the direct analysis of complex mixtures.^[@ref32]^ Quantitation at high precision of particular analytes in condensed phase samples, such as blood or food samples, has not been extensively investigated with miniature mass spectrometers, due to the technical challenges and practical difficulties in achieving highly reproducible results while maintaining the simplicity in the instrumentation and operating procedure. The use of internal standards (IS) with paper spray has been shown to improve the quantitation significantly, and several nontraditional methods of adding the standards have been reported.^[@ref33],[@ref34]^ Simple procedures for introducing IS for ambient ionization are also required for POC or similar in situ applications of miniature MS systems. Several solutions exist, including the use of IS-coated capillaries for transferring samples.^[@ref35]^ Transfer of quantitative performance to the miniaturized system involves some other technical difficulties, which will be further discussed later and favor ionization sources with stable signals. System Design and Instrumentation {#sec2} ================================= An exemplary analytical procedure intended for a miniature MS chemical analysis system is shown in Figure [1](#fig1){ref-type="fig"}a for the particular case of drugs in blood. A small volume of blood is quantitatively taken up using a capillary and transferred to a sample cartridge, which is subsequently inserted into the MS system for automatic analysis. Capillaries with internal standard coated on the inside wall are used to transfer samples of about 1 μL during which the internal standard mixes into the sample. Highly accurate quantitative data (RSD \< 3% at 10 ng/mL drug in blood) have previously been obtained using commercial triple quadrupole mass spectrometer for direct analysis using several ambient ionization methods.^[@ref35]^ To minimize the need of human intervention, an autonomous procedure for analysis and data processing was designed for the Mini 12 (Figure [1](#fig1){ref-type="fig"}b). Once the sample cartridge is inserted into the instrument, a bar code on the cartridge is read and used to search the instructions for analysis of target chemicals, which include the type of sample, target analytes, scan functions, and the calibration curve stored in the database. Using therapeutic drug monitoring as an example, two successive product ion scan functions for MS/MS analysis of the drug and the internal standard are executed; that is, the intensity of the target fragment ion generated from each precursor is recorded (Figure [1](#fig1){ref-type="fig"}c). The ratio of these ion abundances is subsequently used to calculate the concentration based on a calibration curve made and saved for the particular drug of interest in a blood sample. A report of the analytical results can then be generated automatically without requiring the user to analyze the mass spectra. For each scan, the DAPI opens for 15 ms, and the ions introduced are trapped in the RIT at elevated pressure (up to 500 mTorr). After a delay of 800 ms, an MS or MS/MS scan is performed at 1 mTorr (Figure [1](#fig1){ref-type="fig"}c). {#fig1} The integrated Mini 12 system weighs 25 kg, has dimensions of 19.6 × 22.1 × 16.5 in. (outer case measurements), consumes less than 100 W of power, and operates with 110 V AC or from a battery. The system configuration is shown in Figure [2](#fig2){ref-type="fig"}a,b, and a schematic of the instrument control is shown in Figure [2](#fig2){ref-type="fig"}d. The design of the ion source and the sample cartridges adapted to different ambient ionization methods were evaluated. NanoESI and APCI have also been used as ionization methods to characterize the instrument. As a demonstration of the possible use for POC or similar applications, the loading system shown in Figure [2](#fig2){ref-type="fig"}a was developed to accept a single paper spray sample cartridge (Figure [2](#fig2){ref-type="fig"}c) for analysis. A paper substrate with a sharp tip (∼30°) was sandwiched between top and bottom plastic covers made from polyoxymethylene (Ultraform N2320, BASF SE, Ludwigshafen, Germany) using 3D printing. Two openings on the top cover of the cartridge allow deposition of the sample and feed of the spray solvent. A stainless steel ball inserted into the top cover of the cartridge allows application of the high voltage to the paper substrate. Two solvent pumps (STH0CTCLF17, Fluid Metering, Inc., NY) with a minimum dispensing volume of 2 μL were installed, each connected to a 60 mL bottle filled with a spray solvent. This system allows a choice of spray solvent. {#fig2} A schematic of the system control is shown in Figure [2](#fig2){ref-type="fig"}d, and more details on the MS control electronics are provided in the accompanying paper.^[@ref30]^ A tablet computer (HP Slate 2) with a touch screen was integrated into the system to operate the user interface and to allow data processing. The tablet communicated with the MS control board through a USB 2.0 connection. Preoptimized scan functions and calibration functions for different types of targeted analysis were stored on the tablet computer. The scan function parameters were optimized using an expert user interface, as shown in Figure S1. A simpler and more user-friendly interface (Figure S2) was developed for end users. During operation, a user could press a button on the screen to open the cartridge tray, which was controlled through a solenoid actuator. Once the paper spray sample cartridge was placed on the tray and inserted into the system, a magnetic switch closed and triggered the analysis. A digital camera (NLV-2101, Opticon, Inc., WA) installed underneath the plastic tray read the bar code on the bottom of the paper cartridge. Identification of the needed target analysis was made reading the bar code into the program running on the tablet, and the corresponding scan functions were then automatically downloaded to the scan control board. In the particular case of analysis of a drug in blood with the use of an internal standard and paper spray ionization, a solvent was selected and dispensed on the basis of the target compound for optimal elution/ionization efficiency. Two MS/MS scan functions, each of which isolated a different precursor ion, were then executed in sequence. After the spectra data were recorded and transferred back to the tablet, the ratio of the selected fragment ions generated from the ionized drug and internal standard was calculated and used for determination of the drug concentration based on the corresponding calibration function, which was constructed using the same drug and internal standard and the same type of matrix. The vacuum manifold was fabricated with dimensions of 4.65 × 5.00 × 4.74 in. (length × width × height) and with inside dimensions of 3.95 × 4.30 × 4.04 in. It was pumped using a 2 kg turbomolecular pump (HiPace 10, Pfeiffer Vacuum, Inc., Nashua, NH) and a 500 g two-stage diaphragm pump (MPU 1091-N84.0--8.99, KNF Neuberger, Inc., Trenton, NJ). An ultimate vacuum pressure below 1 × 10^--5^ Torr was achieved with the DAPI closed. The DAPI system used a normally closed pinch valve (390NC24330, ASCO Valve, Inc., Florham Park, NJ) with conductive silicone tubing (i.d. 1/16 in., o.d. 1/8 in., and length 2 in., Simolex Rubber Corp., Plymouth, MI) connecting two stainless steel tubes (Supelco, Bellefonte, PA). The pinch valve controlled open/closed status of sampling. The stainless steel tube leading into the trap had dimensions of 0.03 in. i.d., 1/16 in. o.d., and a 10 cm length, and the other tube had dimensions of 0.02 in. i.d., 1/16 in. o.d., and a 5 cm length. Both stainless steel tubes were electrically grounded during operation. A 24 V dc pulse signal was used to open the pinch valve. An rf coil of 1 in. i.d. was used to provide a rf amplitude higher than 5.5 kV~p--p~ at 1 MHz. Resonance ejection was performed using an AC signal with the amplitudes of the AC and the main rf trapping signals ramping simultaneously to record mass spectra. A scan speed up to 10 000 mass/charge units per second was used, unless otherwise specified. The SWIFT (stored waveform inverse Fourier transform) waveform^[@ref36]^ with a notch width of 7 kHz was used for isolation of the precursor ion prior to an MS/MS scan. The detector assembly (model 397, DeTech Detector Technology, Inc., MA) with an electron multiplier and conversion dynode was used.^[@ref19]^ Experimental Section {#sec3} ==================== Whatman chromatography papers (Grade 1, ET31, and silica-coated grade SG81) were used to prepare sample substrates for paper spray (Figure S3a) and extraction spray analysis^[@ref37]^ (Figure S3b). All chemicals were commercially available and used without purification. The internal standard, amitriptyline-d6 was purchased from CDN isotopes (Pointe-Claire, Quebec, Canada). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO). Bovine whole blood stabilized with EDTAK~2~ was purchased from Innovative Research (Novi, MI). Methanol and water were purchased from Mallinckrodt (Hazelwood, MO) and used to prepare the spray solvent mixture (50/50, v/v). Tissue of a tomato was homogenized using the protocol described in a previous study^[@ref38]^ to serve as a sample for quantitative analysis. Stock solutions of analytes were directly spiked into solvents or sample matrices to make standard samples for quantitative measurements. NanoESI, APCI, paper spray, and extraction paper spray were used as ionization methods in the characterization of the instrument performance. For paper spray analysis, 2.5 μL of sample was deposited onto the paper substrate using IS-coated capillary samplers^[@ref35]^ and air-dried for up to 2 h. Methanol (40 μL) was then applied to the paper to elute compounds from the matrix, followed by the application of a high voltage of 4.3 kV to the paper substrate for ionization.^[@ref25]^ For extraction spray, the analyte prepared in blood was preloaded onto a paper strip (grade 1, length of 10 mm, width of 0.6 mm), which was inserted into a nanoESI tube made in-house from a borosilicate glass tube. After air drying (for up to 30 min), 5 μL of methanol was added into the glass tube, and a high voltage of 1.8 kV was applied to the electrode for compound elution and ionization. Further descriptions of the experimental conditions used for paper spray ionization and extraction spray ionization can be found in Supporting Information. Performance Characterization and Discussion {#sec4} =========================================== During the years the Mini 12 was in development, many features of the system were tested and improved. The discussion here focuses on the performance of the MS analysis and the quality of the system when used for quantitation of analytes in complex mixtures. The resolution and mass range of the system were characterized with nanoESI using methanol/water (50/50) solutions containing pure compounds. As shown in the mass spectrum of 1 μg/mL clenbuterol (Figure [3](#fig3){ref-type="fig"}a), isotopic peaks *m*/*z* 277, 279, and 281 were clearly separated in the mass selective instability scan performed at a scan rate of 3000 *m*/*z* per second and with a resonance ejection signal at 350 kHz (*q* = 0.80, AC amplitude scanned from 1.5 to 3.0 V~p--p~). The FWHM (full width at half-maximum) was Δ*m*/*z* = 0.6, for a resolution *m*/Δ*m* of 500. The nanoESI spectrum of a mixture containing three therapeutic drugs (Figure [3](#fig3){ref-type="fig"}b), tacrolimus (5 μg/mL, \[M + H\]^+^ at *m*/*z* 826), imatinib (1 μg/mL, \[M + H\]^+^ at *m*/*z* 494), and amitriptyline (1 μg/mL, \[M + H\]^+^ at *m*/*z* 278), was recorded at a scan rate of 10 000 *m*/*z* per second and with a resonance ejection at 300 kHz (*q* = 0.73, AC amplitude scan 3 .5--7.5 V~p--p~). A maximum *m*/*z* above 850 was obtained. {#fig3} As discussed earlier in this paper, MS/MS capabilities are essential for the deployment of a system like the Mini 12, which is aimed at direct analysis of complex samples without traditional sample pretreatment or chemical separation. Higher sensitivity can be obtained due to the elimination of chemical noise and the identification of the target analytes can be confirmed by appropriate fragmentations corresponding to structural features of the analyte. The scan function for MS/MS is shown in Figure S4. The MS/MS spectrum of 500 pg/mL thiabendazole (\[M + H\]^+^ at *m*/*z* 202, Figure [3](#fig3){ref-type="fig"}c) shows a good signal-to-noise ratio for the fragment ion *m*/*z* 175. In analysis of extremely complex mixtures, it is possible that multiple stages (\>2) of mass analysis might be necessary to confirm chemical identity or to avoid interference in quantitation.^[@ref39]^ As a demonstration of this capability, multiple stage MS analysis (up to five stages) has been performed starting with 20 μg/mL clenbuterol (\[M + H\]^+^ at *m*/*z* 277). The MS^5^ spectrum shown in Figure [3](#fig3){ref-type="fig"}d displays very good S/N ratios for fragment ions *m*/*z* 132 and *m*/*z* 140. The spectra for the intermediate stages of this experiment (viz., MS^2^, MS^3^, and MS^4^) are shown in Figure S6. The MS/MS capability of the MS 12 has also been characterized using APCI using a gas mixture sample containing vapors of dimethyl methylphosphonate (DMMP), methyl salicylate, and *N*,*N*-diethyl-meta-toluamide (DEET) (Figure S7). The ultimate goal in developing the Mini 12 MS system was to enable the direct analysis of nonvolatile analytes in samples with complex matrices. The sensitivity and the quantitative accuracy that can be achieved with ambient ionization are critical for the intended applications. Paper spray was selected as one of the main methods to be used with the Mini 12, due to the sensitivity achieved for complex mixture analysis and the potential for high-precision quantitation that has been demonstrated with lab-scale triple quadrupole mass spectrometers.^[@ref25],[@ref26]^ Another ambient ionization method used for performance development and characterization with the Mini 12 was extraction spray. Instead of spraying the solvent directly from the paper substrate, a paper strip containing the sample was inserted into a nanoESI tube, and a solvent was added to extract the analyte, which was sprayed from the tip of the nanoESI emitter (Figure S3b). Good sensitivity can be achieved with both methods. The MS/MS spectra recorded for analysis of amitriptyline at 50 ng/mL in blood using these two methods are shown in Figure [4](#fig4){ref-type="fig"}a,b. Similar S/N ratios were observed for the peaks for the fragment ions of *m*/*z* 117, 155, and 191 in both spectra. Sample amounts used for preparing the dried blood spots (DBS) on paper substrates were different, with 2.5 μL being used for paper spray but only 1 μL for extraction spray. The most significant difference in the experimental conditions is the consumption of the spray solvent and the analysis time. About 1 min spray time was obtained in paper spray using 40 μL of methanol, whereas more than 10 min spray time was available with only 5 μL of methanol in the extraction spray. A much lower spray voltage of 1.8 kV was used for extraction spray, in comparison with 4.3 kV for paper spray. The stability of the signal was also much better with extraction spray, as previously observed.^[@ref37]^ Figure S5 shows a MS/MS spectrum for analysis of 10 ng/mL verapamil in DBS prepared with 1 μL of blood sample, and the S/N ratios observed for the fragment ions *m*/*z* 165 and *m*/*z* 303 indicate that an LOD better than 10 ng/mL can be easily achieved. On the other hand, at the current stage of development, extraction spray involves more complex manipulations and is less amenable to automation and array mode operation. {#fig4} One of the advantages of using paper spray with the sample cartridge designed for the Mini 12 is the flexibility in analysis of complex samples in forms other than dried spots typically prepared from blood, homogenates, or other liquid samples. As a demonstration, a small piece (about 1 g) of the peel from an nonorganic orange was purchased from a local grocery store and dropped onto the paper spray cartridge, as shown in Figure [4](#fig4){ref-type="fig"}c. Instead of feeding the spray solvent from the other opening behind the stainless steel ball, 40 μL of methanol was directly added on top of the sample. The spray voltage of 4.3 kV was then turned on, and MS/MS analysis was performed targeting thiabendazole (precursor ion *m*/*z* 202), which is one of the most commonly used fruit fungicides. A silica-coated paper substrate was used in this case, as these have been demonstrated to be effective for paper spray due to less retention of water-soluble organic compounds. The typical fragment ions of protonated thiabendazole at *m*/*z* 131 and 175 were observed with high peak intensities (Figure [4](#fig4){ref-type="fig"}c). In many of the potential applications of the Mini 12 system, mandatory performance criteria will need to be met for regulatory purposes. The LOQ, rather than the LOD, as well as the RSD over the entire calibration range are important characteristics in the analytical performance of the system. Ambient analysis using paper spray or extraction spray with a commercial triple quadrupole instrument has been demonstrated to give LOQs in the low ng/mL level and quantitative precision (RSD) of better than 8% for drug compounds in whole blood. The use of internal standards (IS) was also found to be critical,^[@ref26]^ just as for LC-MS/MS. Although relatively good external calibration could be obtained^[@ref27]^ with tightly controlled experimental conditions, for point-of-care type applications, the use of the analyte-to-IS (A/IS) ratios could help significantly to minimize the potential errors associated with variations in operation by personnel not trained in laboratory analytical techniques. When transferring multireaction monitoring (MRM) procedures for measuring the A/IS ratios to a system like the Mini 12, some challenges were foreseen. Therefore, the A/IS ratio was measured with dual MS/MS scans in which two product ion scans were performed sequentially for the analyte and IS. The analyte-to-IS (A/IS) ratio obtained was used to calculate the analyte concentration based on the calibration curve. A delay in the signal recording exists between these two scans, which could cause a deviation in A/IS ratio especially when fluctuations in the ion current are relatively large. The delay between the scans is only about 100 ms for a commercial triple quadrupole instrument; however, it is more than 1s for Mini 12 operation with a DAPI (Figure [1](#fig1){ref-type="fig"}c), which represents a potential problem for quantitative analysis using the Mini12. To explore this issue, a set of tests was run using blood samples containing 200 ng/mL amitriptyline and 100 ng/mL amitriptyline-d6 as internal standards to characterize drug quantitation using Mini 12 with paper spray and extraction spray methods. MS/MS scans for amitriptyline and amitriptyline-d6 were run alternately, and the intensity of fragment ion *m*/*z* 233 recorded was plotted as function of scan number, as shown in Figure [5](#fig5){ref-type="fig"}. Greater fluctuations in the signal were observed for paper spray than for extraction spray. The A/IS ratios calculated for each pair of adjacent scans for amitriptyline and amitriptyline-d6 are plotted against the scan numbers (Figure [5](#fig5){ref-type="fig"}c). The comparison of the stability in the A/IS ratios certainly predicts a better opportunity for achieving high precision in quantitative analysis with extraction spray. The variance in ratio was 200% for paper spray but only 17% for extraction spray. An average of the signals recorded over multiple scans could significantly improve the accuracy in the calculation of the A/IS ratio. {#fig5} A series of DBS samples containing the IS amitriptyline-d6 at 100 ng/mL but the analyte amitriptyline at different concentrations from 7.5 to 510 ng/mL were analyzed to develop a calibration curve for quantitation. For each point in the curve, the samples were analyzed and the A/IS ratio was calculated by averaging 50 pairs of scans for each sample. The calibration curve was linear over the range from 15 ng/mL to 510 ng/mL (Figure [5](#fig5){ref-type="fig"}d), which covers the therapeutic range (80--250 ng/mL) for amitriptyline, but most importantly, RSD values of better than 10% were achieved over the entire range. As a means of monitoring the potential carryover between samples or assessing contamination problems, MS/MS analysis of blank paper substrates was performed using methanol/water (50/50, v/v) solvent between the analyses of two blood samples containing amitriptyline. No carryover problem was observed. An advantage of using paper spray or extraction spray is that the majority of the sample stays on the paper substrate and only a small portion of the analytes are eluted and ionized for analysis. Conclusion {#sec5} ========== A point-of-care miniature mass spectrometry system, the Mini 12, has been developed, built, and characterized. This system includes an RIT miniature mass spectrometer and an ambient ionization source, as well as a user-friendly interface that requires no special training in MS. This system can perform direct mass spectrometry analysis on such complex samples as whole blood, untreated food, and environmental samples, without sample preparation or chromatographic separation. In addition to fast turnaround times and in situ analysis capabilities, disposable cartridges designed for ambient ionization source are used. Further study will be continued to improve the sample handling protocol and to develop a more advanced dual miniature ion trap system to optimize time and sample usage as well as improving software for quantitative POC MS analysis. Additional information as noted in the text. This material is available free of charge via the Internet at <http://pubs.acs.org>. Supplementary Material ====================== ###### ac403766c_si_001.pdf The authors declare no competing financial interest. This work was supported by National Science Foundation (Projects CHE 0847205 and DBI 0852740), the National Center for Research Resources (5R21RR031246-03) and the National Institute of General Medical Sciences (8 R21 GM103454, 1R01GM106016) from the National Institutes of Health, and the Alfred Mann Institute at Purdue University. The authors thank John Hertig, Nick Manicke, and Ziqing Lin for helpful suggestions in development of instrumentation and analytical methods.

Disclaimer: The views expressed are those of the authors and should not be construed to represent the positions of the US Army or the Department of Defense or their employers (J.A.P., R.R., and C.S.P.). On October 28, 2014, 5 days before the annual meeting of the American Society of Tropical Medicine and Hygiene (ASTMH) in New Orleans, the Louisiana Department of Health and Hospitals (DOHH) in conjunction with the Governor\'s Office for Homeland Security and Emergency Preparedness announced to all ASTMH attendees that "individuals who traveled to and returned from the countries of Sierra Leone, Liberia or Guinea in the past 21 days, or have had contact with a known EVD \[Ebola virus disease\] patient in that time period, should NOT travel to New Orleans to attend the conference. Given that conference participants with a travel and exposure history for EVD are recommended not to participate in large group settings (such as this conference) or to utilize public transport, we see no utility in you traveling to New Orleans to simply be confined to your room." Furthermore, the letter stated that "from a medical perspective, asymptomatic individuals are not at risk of exposing others; however, the State is committed to preventing any unnecessary exposure of Ebola to the general public. In Louisiana, we love to welcome visitors, but we must balance that hospitality with the protection of Louisiana residents and other visitors."[@R1] While acknowledging recommendations of the Centers for Disease Control and Prevention (CDC) that asymptomatic individuals are not a risk to others, the statement went beyond the CDC guidelines and implied a potential threat from conference attendees, even those without exposure to EVD, based solely on travel history to countries affected by the epidemic. We believe the DOHH should appreciate the negative ramifications of unscientifically based travel bans and quarantine policies and rather, follow evidence-based guidelines to protect the public and avoid legitimizing irrational responses caused by fear. Ironically, the ASTMH is the pre-eminent professional society in tropical medicine, and the annual meeting of the society is an ideal place to share scientific advances in response to EVD, an interchange that benefits both the United States and all countries facing the current epidemic. Prospective conference attendees who are actively engaged in the EVD response were prepared to share their experiences in scientific sessions, but some could not attend. Numerous attendees from west Africa, including countries not directly affected by EVD, may have been afraid to attend because of not knowing whether they would be turned away on arrival. Moreover, the DOHH reiterated their travel ban for attendees of the annual conference of the American Public Health Association held November 15--19 in New Orleans. Ebola virus causes a deadly disease, and it typically occurs in places that have underresourced and overwhelmed health systems; whereas prior outbreaks have been small and contained, the current outbreak in west Africa is of unprecedented scale.[@R2] In September of 2014, the World Health Organization declared the current Ebola virus disease (EVD) outbreak a major threat to global health and security and requested that all global health organizations and supporting countries maximize their efforts to combat the disease at its source.[@R3] Sporadic cases in high-income countries have occurred connected to this outbreak. Because the virus is known to be transmitted by physical contact, the risk of an EVD epidemic in countries with well-equipped public health and medical systems is small. In the past, limited quarantine procedures and travel bans have been enacted for highly contagious diseases, such as Severe Acute Respiratory Syndrome (SARS). However, considering limited transmission of Ebola virus to casual contacts, there is no evidence to suggest that these strategies are needed to control EVD. On the contrary, there are detrimental consequences of inappropriately combating the outbreak in this manner. For one, health professionals who are desperately needed to combat the disease at its source are disincentivized to risk their own health.[@R4] Current fear-fueled policies issued by several states in the United States are causing significant stigma toward health workers, their families, and the organizations that respond to EVD epidemics; they also marginalize people of west African descent who live in the United States and have not had any exposure to EVD.[@R5] This would not be the first time that irrational reactions hampered scientific advancement and harmed patients---during the early Acquired Immune Deficiency Syndrome (AIDS) epidemic, at-risk populations were similarly marginalized. Unfounded policies, such as the Louisiana DOHH response, also have the potential to encourage potentially exposed individuals to travel outside of monitored routes, deny their exposure, and avoid diagnosis and isolation when symptomatic. Instead, the DOHH should adopt policies based on evidence, such as the established protocols of Médecins Sans Frontières and the CDC,[@R6] which advise monitoring returned asymptomatic health workers. These are effective and should continue to be the basis for a response to EVD in the United States. In the case of the current EVD epidemic and other public health crises, there is a need for greater advocacy on the part of health professionals and academic and professional institutions. Beyond the responsibility of providers to care for individual patients, health professionals should raise awareness about the public health implications of inappropriate responses and policies to public health crises. The medical community should unite and attack inappropriate policies to better protect our patients and their communities. Broader advocacy at the national level and within professional societies is needed to eschew fear-induced and political decisions and maintain evidence-based, neutral, and destigmatizing responses. Such actions would serve to refocus discussion on the evidence and show solidarity on the part of health professionals with the affected population as well as the heroic providers who have chosen to combat Ebola at its source. The American Society of Tropical Medicine and Hygiene (ASTMH) assisted with publication expenses. Authors\' addresses: Ramin Asgary, Departments of Population Health and Medicine, New York University School of Medicine, New York, NY, E-mail: <ramin.asgary@caa.columbia.edu>. Julie A. Pavlin, Armed Forces Surveillance Center, Silver Spring, MD, E-mail: <japavlin@gmail.com>. Jonathan Ripp, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, E-mail: <jonathan.ripp@mountsinai.org>. Richard Reithinger, Global Health Division, International Development Group, Research Triangle Institute, Washington, DC, E-mail: <rreithinger@yahoo.co.uk>. Christina S. Polyak, Military HIV Research Program, Walter Reed Army Institute of Research, Bethesda, MD, E-mail: <cpolyak@hivresearch.org>.

Introduction {#s1} ============ Precision of developmental processes is of great evolutionary importance since it conditions the accurate replication of the selected phenotype. Stabilizing selection is thus thought to favor robust developmental systems [@pgen.1002314-Landry1]. Waddington was the first to suggest that the ability to buffer variation -- referred to as developmental homeostasis -- is a fundamental property of organisms [@pgen.1002314-Waddington1]. He divided developmental homeostasis into two subcomponents: canalization that buffers genetic and environmental effects, and developmental stability that buffers developmental noise [@pgen.1002314-Debat1]. Despite numerous speculations about the evolutionary role of such buffering processes, their study has remained marginal to the mainstream of evolutionary biology until precise molecular processes were identified that might account for these properties. On the one hand, studies on the role of *Hsp90* [@pgen.1002314-Sangster1], [@pgen.1002314-Rutherford1] and more recently of other *Hsp* genes [@pgen.1002314-Takahashi1] and microRNAs [@pgen.1002314-Shomron1], [@pgen.1002314-Hornstein1], in the buffering of genetic variation, contributed to the idea that robustness is ensured by specific genes and genetic processes. On the other hand, complex genetic networks can be intrinsically robust to perturbations [@pgen.1002314-Bergman1], questioning the existence of specific robustness genes. To accommodate these contradictory results, it was suggested that some genes of particular importance for robustness might exist as hubs in complex networks [@pgen.1002314-Rutherford2]. Whether these results apply to developmental stability is not known [@pgen.1002314-Milton1]--[@pgen.1002314-Breuker1]. Developmental stability is commonly estimated by fluctuating asymmetry (FA). The two sides of bilaterally symmetrical traits are influenced by the same genes and environmental conditions and thus only differ by developmental noise. Genetics of developmental stability and FA has been controversial [@pgen.1002314-Leamy1]. The vast majority of studies report that the additive genetic variation for FA is extremely low or non significant. These low values have been interpreted either as a low signal to noise ratio, FA values being typically very small, or suggesting a non-additive genetic basis [@pgen.1002314-Leamy1]. In line with the latter hypothesis, a few QTL interacting epistatically for FA have been detected in mouse (reviewed in [@pgen.1002314-Leamy1]). The best documented case of an individual gene affecting FA is the homologue of the *Drosophila Notch* gene in the sheep blowfly (*Lucilia cuprina*) [@pgen.1002314-McKenzie1]. Its effects on FA are however limited to bristles [@pgen.1002314-Clarke1], a trait known to be controlled by *Notch*. Hence, no evidence for the existence of genes controlling developmental stability in a general way has been reported so far. Cyclins are a family of proteins primarily characterized as cell cycle regulators (for review see [@pgen.1002314-Errico1]). Nevertheless, of the 14 cyclins currently characterized, only a few (Cyclins A, B, D, and E) appear to be major actors of the cell cycle [@pgen.1002314-Satyanarayana1]. Other cyclins, such as Cyclins C, K, H or T, are involved in transcriptional processes (for review see [@pgen.1002314-Coqueret1]). We have recently shown that *Drosophila* Cyclin G is an unconventional cyclin involved, on the one hand, in transcriptional regulation *via* the Trithorax and Polycomb co-factor Corto [@pgen.1002314-Salvaing1], [@pgen.1002314-Salvaing2] and, on the other hand, in regulation of cell growth and cell cycle [@pgen.1002314-Faradji1]. *CycG* is thus suspected to play a major role in *Drosophila* development. Ubiquitous overexpression of the *Drosophila Cyclin G* (*CycG*) gene during development induces high lethality. Escaper flies exhibit various mild phenotypes: they are smaller, have a longer developmental time, reduced eyes and rotated genitalia [@pgen.1002314-Faradji1]. Most remarkably, although wings are normally structured, they exhibit a striking pattern of size asymmetry that fluctuates in the population ([Figure 1A](#pgen-1002314-g001){ref-type="fig"}). This last effect suggested that *CycG* might be a good candidate gene for the control of developmental stability in *Drosophila*. Below, we detail the effects of overexpressing or RNAi-inactivating *CycG* on FA, integrating the analyses at the macroscopic and cellular levels. {ref-type="supplementary-material"} and [S3](#pgen.1002314.s007){ref-type="supplementary-material"} for values). Only *+/+* and GOF (*da::Gal4\>RCG76*) individuals are represented here. FA10: FA index corrected for measurement error and directional asymmetry (see [Material and Methods](#s4){ref-type="sec"}) [@pgen.1002314-Palmer1]. Grey: +/+ females; black: +*/+* males; light green: GOF females; dark green: GOF males.](pgen.1002314.g001){#pgen-1002314-g001} Results {#s2} ======= The UAS/Gal4 system was used to overexpress or RNAi-inactivate *CycG* during development [@pgen.1002314-Salvaing2], [@pgen.1002314-Faradji1]. In order to minimize genetic and environmental sources of phenotypic variation, we used nearly isogenic backgrounds and conducted the experiments in a carefully standardized environment (see [Material and Methods](#s4){ref-type="sec"}). The progeny was analyzed for wing size and shape using geometric morphometrics [@pgen.1002314-Klingenberg1], [@pgen.1002314-Adams1]. Sample sizes are shown in [Table 1](#pgen-1002314-t001){ref-type="table"}. 10.1371/journal.pgen.1002314.t001 ###### Sample size. {#pgen-1002314-t001-1} Genetic background Driver Experiment Genotype females males Total -------------------- -------- ------------ --------------- --------- ------- ------- *w^1118^* *da* *GOF* *da/+* 48 46 94 *RCG76/+* 48 48 96 *da\>RCG76* 53 55 108 *+/+* 47 47 94 *yw^67c23^* *da* *GOF* *da/+* 49 48 97 *RCG76/+* 48 50 98 *da\>RCG76* 25 26 51 *+/+* 50 47 97 *da* *LOF* *da\>dsCycG2* 45 44 89 *+/+* 48 50 98 *Act* *GOF* *Act\>RCG76* 34 40 74 *+/+* 49 50 99 *sd* *GOF* *sd\>RCG76* 45 50 95 *+/+* 49 50 99 Total 638 651 1289 Overexpressing *CycG* alters wing size, wing shape, and increases fluctuating asymmetry {#s2a} --------------------------------------------------------------------------------------- We ubiquitously overexpressed *CycG* using the *daughterless* (*da*) driver. *da::Gal4/+* females were crossed with *UAS::mRFP-CycG (RCG76)/+* males as indicated in [Material and Methods](#s4){ref-type="sec"}. *da\>RCG76* flies, hereafter referred to as GOF for Gain of Function, were smaller and had smaller wings than control *+/+, da:Gal4/+* and *RCG76/+* siblings (wings around 15% smaller in both sexes; [Table S1](#pgen.1002314.s005){ref-type="supplementary-material"}). Although wing venation pattern remained normal, wing shape was clearly affected by *CycG* overexpression, with a distally rounder shape and a parallel distal shift of both cross-veins ([Figure 2](#pgen-1002314-g002){ref-type="fig"}, [Table S1](#pgen.1002314.s005){ref-type="supplementary-material"}: significant genotype effect in the MANOVAs). Similar patterns of shape change were found in both sexes. {ref-type="fig"} were analyzed (see [Table S1](#pgen.1002314.s005){ref-type="supplementary-material"} for values). Each dot represents an individual fly (wing shape averaged over the right and left sides). Canonical variates are axes that maximize variation among groups (discrimination) relative to within group variation. Percentages indicate the amount of variance explained by the axes. The first axis clearly contrasts GOF (*da::Gal4\>RCG76*) and control (+/+) flies. The difference between sexes is mostly detected on the second axis. Grey: +/+ females; black: +*/+* males; light green: GOF females; dark green: GOF males. B: Shape change along the first axis. The grey wing is the consensus wing computed from all wings (i.e. the grand mean shape); the green wing represents the shape change when moving from control to GOF flies.](pgen.1002314.g002){#pgen-1002314-g002} Wings of GOF flies exhibited a striking pattern of asymmetry detectable by sight as compared to wings of control siblings ([Figure 1A](#pgen-1002314-g001){ref-type="fig"}). This asymmetry was not related to mRFP, as no such phenotype was observed in flies overexpressing *mRFP* using the same driver (*da\>UAS::mRFP*) (data not shown). Reciprocally, flies overexpressing *CycG* without *mRFP* (i.e. *CG* transgenic lines) presented strong asymmetry ([Figure S1C](#pgen.1002314.s001){ref-type="supplementary-material"}). To further check whether the asymmetry was specific to *CycG* overexpression, we used *da::Gal4* to overexpress other genes involved in cell growth or cell cycle regulation. None of the tested genes (*dS6K*, *Myc*, *CycD Cdk4*) produced any particular asymmetric pattern (data not shown). This also demonstrated that the asymmetry was not due to an asymmetric expression of the *da::Gal4* driver that would affect tissue growth differently in the left and right sides of individuals. Taken together, these results thus suggest that the observed asymmetry is an effect of *CycG* overexpression *per se*. We therefore conducted a detailed analysis of wing size and shape asymmetry of GOF vs. control flies using replicated sets of landmark data [@pgen.1002314-Klingenberg2], [@pgen.1002314-Palmer1] ([Figure 3A](#pgen-1002314-g003){ref-type="fig"}). Conspicuous biological asymmetries are generally either directional (i.e. one side, always the same, is larger in all individuals), or antisymmetric (i.e. all individuals are strongly asymmetric but equally frequently rightwards or leftwards). These asymmetries do not reflect developmental noise and thus cannot be used to assess developmental stability [@pgen.1002314-Palmer1]. In contrast, developmental noise will randomly affect right and left sides generating the bilateral differences of random amplitude and directionality, known as fluctuating asymmetry (FA). The strong asymmetry observed in GOF flies turned out to be genuine FA of extreme amplitude (often reaching individual values of more than 10% of the trait value). The comparison of these GOF flies to control +/+ flies showed a 36.8-fold increase in wing size FA in females and a 38.9-fold increase in males ([Figure 1B](#pgen-1002314-g001){ref-type="fig"}, [Table S2](#pgen.1002314.s006){ref-type="supplementary-material"}). Wing shape asymmetry was also affected (7.6-fold increase in females and 10.2-fold increase in males as compared to +/+ controls; [Figure 1B](#pgen-1002314-g001){ref-type="fig"}, [Table S3](#pgen.1002314.s007){ref-type="supplementary-material"}). {#pgen-1002314-g003} Replication of the *CycG*-induced FA effect in another genetic background {#s2b} ------------------------------------------------------------------------- In order to investigate the potential effect of the genetic background, all transgenes, originally in a background marked by *w^1118^*, were introgressed into a new background marked by *yw^67c23^*. Microsatellite genotyping showed that the *w^1118^* background presented a low level of heterozygosity and that the *yw^67c23^* background was isogenic as far as can be detected (see [Material and Methods](#s4){ref-type="sec"}). In the new background, wings of GOF flies were again smaller than the ones of +/+ controls (18% in both sexes; [Table S1](#pgen.1002314.s005){ref-type="supplementary-material"}), and their shape was also altered ([Table S1](#pgen.1002314.s005){ref-type="supplementary-material"}), shape changes being remarkably close to those found in the *w^1118^* background (compare [Figure S1A, S1B](#pgen.1002314.s001){ref-type="supplementary-material"} to [Figure 2A, 2B](#pgen-1002314-g002){ref-type="fig"}). The GOF flies exhibited extreme wing FA (26.9-fold increase in females and 48.2-fold increase in males for wing size, 9.2-fold increase in females and 11.1-fold increase in males for wing shape as compared to +/+ controls; [Figure S2C](#pgen.1002314.s002){ref-type="supplementary-material"}; [Tables S2](#pgen.1002314.s006){ref-type="supplementary-material"}, [S3](#pgen.1002314.s007){ref-type="supplementary-material"}), clearly rejecting the hypothesis that FA increase was primarily an effect of the genetic background. *CycG* deregulation using different drivers increases FA {#s2c} -------------------------------------------------------- *CycG*-induced FA was further investigated using other drivers in the same isogenic *yw^67c23^* background. *Actin5C* (*Act*) served as an alternative ubiquitous driver. Wings of *Act\>RCG76* flies were again smaller than the ones of control +/+ siblings (15% smaller for females and 20% for males, [Table S1](#pgen.1002314.s005){ref-type="supplementary-material"}) and wing shape was altered as well ([Table S1](#pgen.1002314.s005){ref-type="supplementary-material"}). Size FA doubled in both sexes (2.3-fold increase in females, 2.4-fold increase in males as compared to +/+ controls), and shape FA increased 9.8-fold in females and 2.8-fold in males ([Tables S2](#pgen.1002314.s006){ref-type="supplementary-material"}, [S3](#pgen.1002314.s007){ref-type="supplementary-material"}). A *scalloped* (*sd*) driver, *sd^29.1^*, hereafter called *sd::Gal4*, was further used to induce a more localized overexpression of *CycG* in wing imaginal discs. This driver is an insertion of a *P-Gal4* transgene that maps in the *scalloped* gene in the first large intron after the translational start site. It has been described as a weak hypomorph allele of *scalloped* [@pgen.1002314-Shyamala1]. Whereas wings of *sd::Gal4/+* flies have a wild type phenotype, wing margin of *sd\>RCG76* flies was partially altered by notches ([Figure S1E](#pgen.1002314.s001){ref-type="supplementary-material"}), suggesting that *CycG* interacted synergistically with *sd*. Wing centroid size and wing shape could thus not be analyzed, hence only wing length was measured ([Figure 3A](#pgen-1002314-g003){ref-type="fig"}). Again, FA of *sd\>RCG76* wings increased dramatically (6.8-fold for females and up to 47.9-fold for males) as compared to control +/+ siblings ([Table S2](#pgen.1002314.s006){ref-type="supplementary-material"}). These results demonstrate that any *CycG* overexpression has a very strong effect on wing FA, although the driver can affect the intensity of the asymmetry response. qRT-PCRs showed that *CycG* overexpression driven by *Act* was weaker than the one driven by *da* ([Figure S3](#pgen.1002314.s003){ref-type="supplementary-material"}), suggesting that the strength of the FA effect depends on the level of *CycG* expression. Reducing *CycG* expression by RNAi increases shape FA {#s2d} ----------------------------------------------------- We next examined the effects of a reduction in *CycG* expression on FA. Since no *CycG* mutant has been reported so far, the *UAS::dsCycG2* transgenic line was used to silence *CycG* by RNAi in the *yw^67c23^* background [@pgen.1002314-Salvaing2]. *CycG* was ubiquitously inactivated using the *da::Gal4* driver. In *da\>dsCycG2* flies, hereafter referred to as LOF for Loss of Function, overall body size (including wing size) was not altered, in contrast to GOF flies ([Table S1](#pgen.1002314.s005){ref-type="supplementary-material"}). Interestingly, wing shape was affected ([Figure S2D, S2E](#pgen.1002314.s002){ref-type="supplementary-material"}; [Table S1](#pgen.1002314.s005){ref-type="supplementary-material"}). Shape changes were similar in both sexes and were clearly different from those induced by *CycG* overexpression (compare [Figure S2A, S2B](#pgen.1002314.s002){ref-type="supplementary-material"} to [S2D, S2E](#pgen.1002314.s002){ref-type="supplementary-material"}). Wing size FA increased significantly only in males (1.5-fold) as compared to +/+ siblings ([Figure S2F](#pgen.1002314.s002){ref-type="supplementary-material"}, [Table S2](#pgen.1002314.s006){ref-type="supplementary-material"}). However, wing shape FA increased by a factor of 3.7 in females and 2.3 in males ([Figure S2F](#pgen.1002314.s002){ref-type="supplementary-material"}; [Table S3](#pgen.1002314.s007){ref-type="supplementary-material"}), indicating that reducing *CycG* expression also impairs developmental stability. Patterns of *CycG*-induced wing shape asymmetry {#s2e} ----------------------------------------------- FA occurs at low levels in any bilateral quantitative trait. Whether an increase in asymmetry is merely an amplification of the \"normal\" asymmetry or rather involves different processes is unknown. Simple traits, like size, vary in one dimension only and are therefore not amenable to address this question. Investigating wing shape asymmetry enables us to tackle this issue. If only amplification of normal asymmetry is involved, then the patterns of shape asymmetry should be unchanged. We computed the principal components of the shape asymmetry matrices for control and experimental flies, i.e. GOF flies in both *w^1118^* and *yw^67c23^* backgrounds and LOF flies in the *yw^67c23^* background ([Figure 4](#pgen-1002314-g004){ref-type="fig"}). In contrast to +/+ siblings, shape FA matrices of GOF flies were clearly dominated by the first principal component (PC), indicating a strong structure of the FA effect along one dominant direction of shape change. This structure was less pronounced but also detected in LOF flies. Remarkably, while the wings of GOF and LOF flies differed in their mean shape, their patterns of shape FA were almost identical ([Figure 4A, 4C, 4E](#pgen-1002314-g004){ref-type="fig"}; [Table S4](#pgen.1002314.s008){ref-type="supplementary-material"}), as shown by the similarity of the FA PC1s (see [Material and Methods](#s4){ref-type="sec"}). These patterns of shape FA were also significantly correlated with those of their +/+ siblings ([Figure 4B, 4D](#pgen-1002314-g004){ref-type="fig"}; [Table S4](#pgen.1002314.s008){ref-type="supplementary-material"}) despite some differences. In the *w^1118^* background, the patterns of shape FA between +/+ and GOF female flies did not even differ more than expected from sampling error only (i.e. the angle between their respective FA PC1s was smaller than between pairs of vectors derived from a distribution obtained by resampling within a single genotype; see [Material and Methods](#s4){ref-type="sec"}; [Table S4](#pgen.1002314.s008){ref-type="supplementary-material"}). This therefore suggests that the *CycG-*induced shape asymmetry mainly consists in an amplification of the dominant pattern of \"normal\" asymmetry. {ref-type="fig"} and [Figure S3A, S3B, S3C](#pgen.1002314.s003){ref-type="supplementary-material"}, respectively) and LOF flies in the *yw^67c23^* background (same individuals as in [Figure S3D, S3E, S3F](#pgen.1002314.s003){ref-type="supplementary-material"}) were analyzed (see [Table S4](#pgen.1002314.s008){ref-type="supplementary-material"} for values). Only females are represented here. Top: Patterns of wing shape asymmetry associated with the first PC of the FA matrices. Grey shape: consensus wing computed from all wings; colored shape: shape asymmetry associated with the first principal component (PC). Bottom: Histograms of FA eigenvalues (i.e. amount of shape variance explained by each PC). Green: GOF flies (A, C); grey: control flies (B, D); orange: LOF flies (E).](pgen.1002314.g004){#pgen-1002314-g004} Effects of *CycG* overexpression on an other trait {#s2f} -------------------------------------------------- We next asked whether the observed FA in flies deregulating *CycG* was restricted to wings or affected other body parts. To avoid a bias due to developmental correlation, we focused on a structure located on a different thoracic segment i.e. the first leg. First leg femurs of GOF flies previously analyzed for wing asymmetry (*da\>RCG76* in the *yw^67c23^* background) or their +/+ siblings, were scored for length ([Figure 3B](#pgen-1002314-g003){ref-type="fig"}). As expected from the reduced size of GOF flies, their femurs were shorter than those of control siblings (-14% in females and -16% in males). Consistently with wing analyses, *CycG* overexpression induced an increase in FA in both sexes (4.2-fold for females and 2.6-fold for males as compared to +/+ siblings; [Table S5](#pgen.1002314.s009){ref-type="supplementary-material"}). Hence, the increase of asymmetry generated by deregulating *CycG* was not limited to wings but also affected other body parts. Noteworthy, we observed no individual correlation between wing FA and femur FA, i.e. the most asymmetric individuals for wings were not necessarily the most asymmetric for femurs, and *vice-versa*. Effects of *CycG* deregulation on cell size and cell number {#s2g} ----------------------------------------------------------- To identify the cellular processes mediating the observed phenotypes, cell number and cell size were estimated on wings previously measured and scored for asymmetry (GOF and LOF flies, as well as their +/+ siblings, in the *yw^67c23^* background; [Figure 3C, 3C″, 3C″](#pgen-1002314-g003){ref-type="fig"}). The overall effects of deregulating *CycG* are shown in [Figure 5](#pgen-1002314-g005){ref-type="fig"} and [Table S6](#pgen.1002314.s010){ref-type="supplementary-material"}. For cell size, overexpression and inactivation of *CycG* had opposite effects: in both sexes, overexpression reduced cell size while inactivation increased it ([Figure 5A](#pgen-1002314-g005){ref-type="fig"}; [Table S6](#pgen.1002314.s010){ref-type="supplementary-material"}, top). For cell number, the effects were sex-specific: while *CycG* overexpression and inactivation both decreased cell number in females, no significant effect was detected in males ([Figure 5B](#pgen-1002314-g005){ref-type="fig"}; [Table S6](#pgen.1002314.s010){ref-type="supplementary-material"}, bottom). {ref-type="supplementary-material"}). Each dot represents an individual fly (see [Table S4](#pgen.1002314.s008){ref-type="supplementary-material"} for values). A: Cell size (CS) vs. wing size (WS); B: Cell number (N) vs. wing size (WS). C: Cell size (CS) vs. cell number (N). Grey: *+/+* females; black: *+/+* males; light green: GOF females; dark green: GOF males; light orange: LOF females; dark orange: LOF males. Note that the tight negative relationship between cell size and cell number found in control *+/+* flies is altered in *CycG* deregulated flies. It is strongly affected in GOF female flies and lost in GOF males. While a negative correlation still holds for LOF flies, the strength of the relationship is altered (see text and [Table S7](#pgen.1002314.s011){ref-type="supplementary-material"}).](pgen.1002314.g005){#pgen-1002314-g005} Although wing size sexual dimorphism was stable across genotypes, females being typically larger than males (between 15 and 17% larger; [Table S1](#pgen.1002314.s005){ref-type="supplementary-material"}), differences were found in the cellular basis of this dimorphism ([Figure 5A, 5B](#pgen-1002314-g005){ref-type="fig"}; [Table S6](#pgen.1002314.s010){ref-type="supplementary-material"}). In control flies, cell size was similar in males and females: sexual dimorphism was mainly due to cell number, the larger female wings bearing more cells that the smaller male ones. In GOF flies, both cell size and cell number differed between sexes, males having less cells of smaller size than females. In LOF flies, sexual dimorphism was mainly due to cell size, males having as many but smaller cells as females. The smaller wing size of GOF flies was mainly explained by a decrease in cell size in both sexes as compared to +/+ siblings; cell number also decreased in females, while almost no difference was found in males ([Figure 5A, 5B](#pgen-1002314-g005){ref-type="fig"}). Interestingly, in LOF flies, although wing size was not altered, both cell size and cell number were affected. Cells were bigger than those of controls in both sexes, although the difference was stronger in females. This effect was compensated by a reduction of cell number leading to a stable wing size ([Figure 5A, 5B](#pgen-1002314-g005){ref-type="fig"}). Remarkably, the relationship between cell size and cell number observed in wild type and LOF wings was strongly affected by *CycG* deregulation ([Figure 5C](#pgen-1002314-g005){ref-type="fig"}, [Table S6](#pgen.1002314.s010){ref-type="supplementary-material"}). Cell size and cell number were indeed tightly negatively correlated in control +/+ wings (+/+ females: r = -0.78\*\*\*; +/+ males: r = -0.84\*\*\*, see [Table S6](#pgen.1002314.s010){ref-type="supplementary-material"} for statistical comparisons). This correlation sharply decreased when *CycG* was overexpressed (GOF females: r = -0.52\*; GOF males: r = -0.04, non significant, see [Table S6](#pgen.1002314.s010){ref-type="supplementary-material"} for statistical comparisons). This apparent uncoupling between the two cellular parameters was further evidenced when analyzing residuals of the regressions of cell size on cell number ([Table S6](#pgen.1002314.s010){ref-type="supplementary-material"}). Their variance was indeed found significantly higher in GOF flies compared to controls. Interestingly, while the negative correlation still held for LOF flies (LOF females: r = -0.91\*\*\*; LOF males: r = -0.77\*\*\*; [Table S7](#pgen.1002314.s011){ref-type="supplementary-material"}), the variance of the regression residuals also significantly increased relative to the controls, suggesting that the size/number relationship was altered as well when reducing *CycG* expression. FA of cell size and cell number was measured to try to account for the extreme wing size asymmetries ([Figure 6](#pgen-1002314-g006){ref-type="fig"}; [Table S8](#pgen.1002314.s012){ref-type="supplementary-material"}). In both GOF and LOF flies, FA of cell size significantly increased relative to +/+ controls whereas FA of cell number did not vary. Furthermore, only cell size FA was found positively correlated with wing size FA across individuals ([Table S8](#pgen.1002314.s012){ref-type="supplementary-material"}, bottom): flies strongly asymmetrical for wing size tended to be strongly asymmetrical for cell size (GOF females: r = 0.65\*\*; GOF males: r = 0.54\*\*) but not for cell number (GOF females: r = 0.11, non significant; GOF males: r = 0.27, non significant). Wing size asymmetry induced by *CycG* deregulation thus appeared to be predominantly generated by cell size asymmetry. {ref-type="fig"} were analyzed (see [Table S7](#pgen.1002314.s011){ref-type="supplementary-material"} for values). A: Cell size fluctuating asymmetry (FA4, see [Material and Methods](#s4){ref-type="sec"}). B: Cell number fluctuating asymmetry (FA4). Grey: +/+ females; black: +*/+* males; light green: GOF females; dark green: GOF males; light orange: LOF females; dark orange: LOF males.](pgen.1002314.g006){#pgen-1002314-g006} Discussion {#s3} ========== The *CycG* gene of *Drosophila melanogaster* encodes a cyclin involved in transcriptional regulation, cell growth and cell cycle [@pgen.1002314-Salvaing2], [@pgen.1002314-Faradji1]. We report here that upregulation of *CycG* in a context where genetic and environmental variations were minimal induces extremely high levels of fluctuating asymmetry (FA) in several traits, suggesting that Cyclin G is a major factor of developmental stability. Deregulating *CycG* alters cell growth and the compensation between cell proliferation and cell growth {#s3a} ------------------------------------------------------------------------------------------------------ Cell growth is markedly downregulated by *CycG* as *CycG* inactivation increases adult wing cell size while *CycG* overexpression reduces it. In wing imaginal discs, however, although cell size is also reduced by *CycG* overexpression, inactivation of *CycG* only induces a slight increase in cell size [@pgen.1002314-Faradji1]. This suggests that in flies where *CycG* is inactivated, extra cell growth occurs during post-larval stages. Furthermore, *CycG* impairs not only cell growth but also cell proliferation. Indeed, both inactivation and overexpression lead to a reduction in cell number in females. The fact that wings of flies where *CycG* was inactivated reach a size comparable to that of wild type flies suggests that cell growth compensates for lack of cell proliferation. A tight negative correlation between cell size and cell number is observed in control flies suggesting that wing size stability is ensured by compensation between cell proliferation and cell growth. This has also been observed in natural populations, where cell size and cell number tend to show negative covariance [@pgen.1002314-Stern1], [@pgen.1002314-McCabe1]. In addition, genetic manipulation of cell size using *cdc2* mutants [@pgen.1002314-Weigmann1], *dMyc* mutants [@pgen.1002314-Johnston1] or deregulation of cell cycle regulators [@pgen.1002314-Neufeld1] confirms that cell growth and proliferation can compensate each other to reach normal organ size. Hence, the final size of the wing seems to be determined by a compensatory mechanism between cell size and cell number. This mechanism is deeply impaired in *CycG* overexpressing flies. Remarkably, although the negative correlation is significant in LOF flies, the variance of the regression residuals presents a sharp increase relative to the controls, indicating a loosening in the relationship. Altogether these results suggest that deregulating *CycG* alters the link between cell growth and proliferation. This in turn suggests that compensation between cell growth and division is one key factor in maintaining wing size -- and thus wing developmental stability -- and that *CycG* is critical for ensuring this compensation. Deregulating *CycG* alters the cellular basis of wing sexual size dimorphism {#s3b} ---------------------------------------------------------------------------- In natural populations, differences in wing size between sexes have been suggested to involve both cell size and cell number [@pgen.1002314-McCabe1], [@pgen.1002314-Trotta1]. In our control isogenic lines though, wing sexual size dimorphism was only due to cell number, cell size being strikingly similar in both sexes. This suggests that adaptation to laboratory conditions or genetic drift might affect the cellular basis of sexual size dimorphism. Cell size is affected similarly in both sexes when manipulating *CycG* expression. In contrast, the effects on cell number are different between sexes: while no effect is detectable in males, both GOF and LOF females have fewer cells than the controls. During the pupal stage, wing cells undergo two rounds of division [@pgen.1002314-Schubiger1]. As G-type cyclins are known to be important in terminally differentiated cells [@pgen.1002314-Bennin1], it is tempting to speculate that these last divisions are differentially regulated in males and females and are controlled by Cyclin G. The last divisions in the pupal wing might be a crucial determinant of the sexual dimorphism of wing size. Deregulating *CycG* alters wing shape {#s3c} ------------------------------------- The altered wing shape in both GOF and LOF flies suggests that the ubiquitous *da* deregulation of *CycG* across the wing blade induced heterogeneous effects on cell size and cell number, possibly reflecting an interference with morphogens driving wing growth. Nevertheless, the changes in mean wing shape found in GOF flies are clearly different from those in LOF flies. As the patterns of shape change remained different in LOF and GOF flies after correcting for size -- only the GOF flies were smaller than the controls, we could rule out the hypothesis of a simple allometric effect ([Figure S4](#pgen.1002314.s004){ref-type="supplementary-material"}). A detailed mapping of the cellular effects on the wing would be needed to relate the shape changes to morphogenetic processes. These results are nevertheless consistent with QTL analyses showing that wing shape is regulated at least partly independently of wing size [@pgen.1002314-Zimmerman1]. Wing veins are important determinants of wing shape [@pgen.1002314-Birdsall1] and wing shape has notably been shown to be tightly associated with the *Egf receptor* locus that controls the amount of vein material [@pgen.1002314-Zimmerman1], [@pgen.1002314-Dworkin1], [@pgen.1002314-Palsson1]. Interestingly, *torpedo*, a mutant of the EGF Receptor, shows similar abdomen cuticle defects [@pgen.1002314-Madhavan1] than those observed in *CycG* LOF flies [@pgen.1002314-Salvaing1]. Thus, *CycG* might also interact with the *Egf receptor* in the wing imaginal disc to control vein specification and wing shape. Upregulating *CycG* increases size FA {#s3d} ------------------------------------- The amplitude of the asymmetry effect observed when overexpressing *CycG* is particularly dramatic, and such an amplitude is usually associated with directional asymmetry or antisymmetry, the two forms of conspicuous asymmetry. Similar levels of FA have -- to our knowledge -- never been reported. Comparatively, a study using deletions covering most of the *Drosophila* genome detected a maximum increase of 7-fold in size FA [@pgen.1002314-Breuker1] (Breuker, personal communication). The very low level of genetic variation and the carefully controlled environmental conditions ensured that this effect was due to developmental noise and was not confounded with genetic or environmental variation for directional asymmetry ([@pgen.1002314-Plabon1], [@pgen.1002314-Stige1]; see [Material and Methods](#s4){ref-type="sec"}). That wing size and shape as well as femurs of the first leg are affected demonstrates that, although the strength of the effect on FA may vary across body parts, this effect is not restricted to a single trait *(*i.e. the wing) or a specific segment. This result is particularly important since the only previously known cases of individual genes altering FA were trait-specific [@pgen.1002314-Clarke1]. Although wings and legs are both thoracic appendages with partly similar developmental networks, such a common FA effect suggests that the cellular processes altered by deregulation of *CycG* are likely common to many traits. It also provides some support to the hypothesis of an organism-wide source of developmental noise, and indirectly it suggests the existence of organism-wide stabilizing processes, a very contentious issue [@pgen.1002314-Polak1]. Some preliminary tests on bristle traits nevertheless suggest that bristle number FA is not affected by *CycG* deregulation. This is in agreement with previous studies suggesting that meristic and metric trait variation could be controlled *via* different processes [@pgen.1002314-Rutherford2], [@pgen.1002314-Kellermann1]. Deregulating *CycG* increases shape FA {#s3e} -------------------------------------- Whereas mean wing shape is affected differently in LOF and GOF flies, the patterns of shape variation around these different means, and specifically those of shape FA, are strikingly similar. This similarity can be interpreted in different ways. First, it might indicate that stochastic variation is constrained along a limited set of directions of shape change, consistent with the view of a wing as an integrated system [@pgen.1002314-Klingenberg3]. Alternatively, such similarity of patterns might reflect a similarity of processes. Although mean wing shape is affected differently when increasing or reducing *CycG* expression, it is conceivable that shifting *CycG* expression level away from its normal value might destabilize development in similar ways, generating these similar patterns of shape FA. Comparable - although not identical - patterns of wing shape FA were found in control flies, suggesting that similar processes are involved in generating FA in wild type and *CycG* deregulated flies. This again supports the view that *CycG* plays an important role in developmental stability. Genetics and regulation of developmental stability {#s3f} -------------------------------------------------- *CycG* thus appears as a serious candidate for the genetic control of developmental stability, and further studies should examine its role in FA amplitude differences across natural populations or samples submitted to various environmental treatments. Can we reconcile the reported lack of additive genetic variation for FA [@pgen.1002314-Leamy1] with the putative existence of (a) major gene(s) altering FA? It is likely that *CycG* is involved in a genetic network regulating cell growth, and possibly cell proliferation [@pgen.1002314-Faradji1], where it might act as a hub, as the high FA induced by overexpression suggests. Such a function, likely involving various pleiotropic effects and epistatic interactions, might be under strong selection, possibly leading to the elimination of any variation. It is also conceivable that subtle variation in *CycG* may occur with only small effects on FA. In particular, *CycG* overexpression triggered by transgenic constructs is likely of larger magnitude compared to the effects of natural variation. Investigation of natural variation in *CycG* sequence and expression across populations differing in their degree of FA would provide some insight on this question. Investigating genes interacting with *CycG* would also improve our understanding of its link with organ size stochastic variation. It was recently suggested that the ability of organs to reach a stereotypical size would depend on the competition among populations of growing cells [@pgen.1002314-NetoSilva1]. In given developmental conditions (e.g. during the last cell divisions in the pupal wing blade), Cyclin G intracellular concentration might somehow trigger cell division. Pushing this concentration away from its usual value might interfere with the process by which cells identify the appropriate stage of growth for division, potentially generating stochasticity in cell size and decoupling cell growth and division. This might in turn compromise the normal pattern of cellular competition, causing random variation in organ size. The extreme FA reported in this paper was generated by deregulating expression of a single gene. Consequently, the above hypothetical scenario focuses on the role of a single protein on the generation of random variation at the cellular level, but it does not preclude the existence of diverse processes working at various biological scales [@pgen.1002314-Hallgrimsson1]. Our results do not necessarily mean that *CycG* is a gene *for* developmental stability, but they clearly show, by the strength of its effect on cell size variation, that *CycG* normal expression is required for the formation of symmetrical flies. Materials and Methods {#s4} ===================== Fly strains {#s4a} ----------- *UAS::mRFP-CycG* and *UAS::CycG* lines (respectively referred to as *RCG* and *CG*), containing the full-length *CycG* cDNA, were established by standard transformation [@pgen.1002314-Faradji1]. The previously described *UAS::dsCycG2* line was used to downregulate *CycG* by RNAi [@pgen.1002314-Salvaing2]. *CycG* overexpression and downregulation were carried out using either ubiquitous Gal4 drivers *daughterless* (*da::Gal4*) or *Actin5C* (*Act::Gal4*) (NP3121, DGRC Kyoto), or the tissue-specific Gal4 driver *scalloped sd^29.1^ (*called *sd::Gal4)* (BL-8609). All these transgenic lines display promoter-independent *mini-white* expression. Here, we present the results for one of the *RCG* lines only (*RCG76*), but similar effects were found with other *RCG* lines and *CG* lines ([Figure S1](#pgen.1002314.s001){ref-type="supplementary-material"}). *UAS::mRFP* (BL-7118), *UAS::dS6K* [@pgen.1002314-Montagne1], *UAS::Myc* (BL-9674), *UAS::CycD* [@pgen.1002314-Datar1] and *UAS::Cdk4* [@pgen.1002314-Meyer1] were used as control lines. Strains were maintained and crossed on standard medium at 25°C. For all crosses, 5 females heterozygous for a Gal4 driver were mated with 5 males heterozygous for an UAS transgene; they were transferred in a new vial every 24 h. Genetic background {#s4b} ------------------ The first analyses were performed using a genetic background marked by *w^1118^* i.e. the original background of all the transgenes. Trangenes were then introgressed into a new background marked by *yw^67c23^* and followed by eye-color. The *yw^67c23^* line was submitted to 10 rounds of isogenization prior to transgene introgression. Males *w^1118^/Y; da::Gal4* were crossed with *yw^67c23^* isogenic females. The resulting *yw^67c23^/Y; da::Gal4/+* males were individually crossed with isogenized *yw^67c23^* females. A first round of isogenization was then performed by individually crossing *yw^67c23^; da::Gal4/+* females with isogenic *yw^67c23^/Y* males. Females *yw^67c23^; da::Gal4/+* were recovered and individually crossed with isogenic *yw^67c23^/Y* males for a second round of isogenization. *yw^67c23^Act::Gal4* females were crossed with isogenized *yw^67c23^* males. *yw^67c23^Act::Gal4/yw^67c23^* females were then individually crossed with isogenized *yw^67c23^* males for a first round of isogenization. The same scheme was adopted for *yw^67c23^sd::Gal4* females. After ten rounds of isogenization, one single isogenic line was kept for each transgene. Isogenicity of the *da::Gal4* and *RCG76* lines was assessed by analyzing 19 microsatellite markers distributed over the 4 chromosomes in 5 individual females. It revealed a single polymorphic locus for *w^1118^* females. However, the *w^1118^*; *da::Gal4* and *w^1118^*; *RCG76* females presented polymorphism at 7 and 2 additional loci, respectively. The *yw^67c23^* line was checked for the same 19 loci, revealing no polymorphism whereas *yw^67c23^; da::Gal4* and *yw^67c23^; RCG76* females each presented polymorphism at only 3 loci. Morphometrics {#s4c} ------------- ### Acquisition {#s4c1} Right and left wings of the progeny from all crosses were mounted on slides, dorsal side up, and photographed using an Imasys uEye digital camera mounted on a Leica DMRB microscope. Legs of the progeny from the cross between *da::Gal4/+* and *RCG76/+* in the *yw^67c23^* genetic background were mounted on slides and photographed using a microscope equipped with a Nikon DXM 1200 camera. 15 landmarks per wing were digitized ([Figure 3A](#pgen-1002314-g003){ref-type="fig"}). All measures were performed using Image J. ### Size {#s4c2} Log of the centroid size was used as a size variable for the wing (i.e. the square root of the sum of the squared distances from each landmark to the corresponding configuration\'s centroid [@pgen.1002314-Klingenberg1], [@pgen.1002314-Adams1]). In *sd\>RCG76* flies, wing length was measured as the distance between landmark 3 and landmark 13 ([Figure 3A](#pgen-1002314-g003){ref-type="fig"}). Length of the first leg femur was measured in arbitrary units as the distance between two landmarks as shown in [Figure 3B](#pgen-1002314-g003){ref-type="fig"}. ### Shape {#s4c3} Generalized least squares Procrustes superimposition was used to extract shape variation from the landmark data [@pgen.1002314-Klingenberg1], [@pgen.1002314-Adams1]. In order to avoid problems related to loss of dimensions due to superimposition, a principal component analysis (PCA) was applied to the Procrustes coordinates (i.e. the coordinates after superimposition) and the non-zero PC scores were used as shape variables in all subsequent shape analyses. ### Allometry {#s4c4} Allometry was investigated applying a multivariate regression of the PC scores on centroid size and using the residuals as allometry-free shape variables (e.g. [@pgen.1002314-Monteiro1]). Discriminant analyses ran before and after the regression were compared to assess the impact of allometry on wing shape changes among genotypes ([Figure S4](#pgen.1002314.s004){ref-type="supplementary-material"}). ### Cell number and cell size {#s4c5} A 256×256 pixel area, corforesponding to a 0.07 mm^2^ square, was displayed in the intervein region between vein 4 and vein 5 just behind the posterior cross-vein ([Figure 3C](#pgen-1002314-g003){ref-type="fig"}). Thresholding was performed to keep epidermal hairs of the dorsal cell layer only using Image J software ([Figure 3C′](#pgen-1002314-g003){ref-type="fig"}). Cells were automatically counted considering that each cell carries one epidermal hair. To account for potential heterogeneity in cell density in the wing, the area was then shifted twice along the proximo-distal axis and cells were counted again. As no significant difference among the three sampled regions was found, the number of cells counted over these three regions for each wing was averaged. Cell size was computed as one divided by the number of cells in the sampled area. The total number of cells in the dorsal cell layer of the wing was obtained applying the same thresholding to the whole wing. This thresholding mostly excluded cells located on the veins and the wing margin ([Figure 3C″](#pgen-1002314-g003){ref-type="fig"}). We therefore underestimated the real cell numbers, but this bias was likely stable across individuals and genotypes. The relationship between cell size and cell number was analyzed as follows. We first computed the Pearson correlation coefficient (r) for each genotype. These correlations were then compared among genotypes using Fisher r to Z transformation. To investigate more accurately the strength of the relationship between the two cellular parameters, we ran a regression model (cell size over cell number) on each genotype and computed the variance of the residuals: the stronger the association, the lower the variance. This variance was then compared across genotypes using a Levene test followed by pairwise F tests. FA analysis {#s4d} ----------- ### Antisymmetry {#s4d1} We did not detect antisymmetry in any trait investigated: no evidence for bimodality was found in right minus left distributions of size-related traits (not shown); for wing shape data, visual inspection of right-left shape vectors did not suggest occurrence of any clustering, as would be expected for antisymmetric traits. ### Measurement error {#s4d2} Measurement error (ME) is of critical importance when analyzing fluctuating asymmetry [@pgen.1002314-Palmer1]. To quantify ME, both sides were measured several times. All flies used in the overexpression experiments using *da* were digitized twice for wing size and shape, and three times for femurs. As measurement error was shown to be negligible relative to true FA (interaction MS relative to error MS in [Tables S2](#pgen.1002314.s006){ref-type="supplementary-material"}, [S3](#pgen.1002314.s007){ref-type="supplementary-material"}, [S4](#pgen.1002314.s008){ref-type="supplementary-material"}), a single measurement session was used in subsequent experiments (i.e. the ones involving *Act* and *sd*). Conventional two-way mixed model ANOVAs were applied to size data (centroid size, wing length and femur length) and Procrustes ANOVAs to wing shape data, using Individual (random), Side (fixed) and their interaction as effects. The details of this procedure can be found in [@pgen.1002314-Klingenberg2], [@pgen.1002314-Palmer1]. In addition to the estimation of ME, this ANOVA allows testing for the occurrence of directional asymmetry. For size, the side effect was never statistically significant in GOF and LOF flies (see \"side\" effect P-values on [Table S2](#pgen.1002314.s006){ref-type="supplementary-material"}) demonstrating that true FA rather than directional asymmetry is responsible for the observed asymmetry pattern. Wing shape directional asymmetry was detected in some cases (see \"side\" effect P-values on [Table S3](#pgen.1002314.s007){ref-type="supplementary-material"}). Estimators of FA corrected for ME and directional asymmetry (FA10) were then derived from these ANOVAs (FA10 following the standard terminology by Palmer and Strobeck [@pgen.1002314-Palmer1]). When only one measurement was available, variance of (R-L) was used as an FA index (FA4, [@pgen.1002314-Palmer1]). The relationship between size and asymmetry was examined to test the occurrence of allometric effect on FA. Such an effect was detected among genotypes but is mostly attributable to the fact that flies overexpressing *CycG* are smaller than the controls. No significant allometric effect was detected within genotypes. Comparisons of FA values among genotypes were done using standard F-tests. P-values were adjusted using the Holm procedure [@pgen.1002314-Benjamini1] each time the analyses involved multiple comparisons. ### Individual variation for directional asymmetry {#s4d3} Even when the average asymmetry is zero, genetic or environmental variation for directional asymmetry can occur and inflate the apparent level of fluctuating asymmetry, thereby impeding its use as an estimator of developmental stability (e.g. [@pgen.1002314-Stige1]). Genetic variation for directional asymmetry has been reported to be very low in most cases and artificial selection experiments in Drosophila have failed to generate a significant increase in asymmetry (reviewed in [@pgen.1002314-Plabon1]). Some studies have nevertheless reported significant genetic variation for directional asymmetry (see [@pgen.1002314-Rego1] for a very strong effect in Drosophila interspecific hybrids). We could rule out the hypothesis of such an individual variation for directional asymmetry as the genetic variation was reduced to a minimum by the inbreeding procedure and by raising the flies in a carefully controlled environment. ### Patterns of wing shape FA {#s4d4} Patterns of shape asymmetry were investigated following Klingenberg and McIntyre [@pgen.1002314-Klingenberg2]. For each genotype and sex, we first computed the matrix of shape FA as the covariance matrix of the individual\*side effect corrected for measurement error. We then ran a Principal Component Analysis (PCA) on this matrix. We examined the distribution of the eigenvalues \[decreasing amount of variation accounted for by the successive principal components (PCs)\]. Note that since the PCAs were ran independently, there is no one-to-one correspondence between PCs. Comparing individual eigenvalues across genotypes is thus non informative. In contrast, the shape of the whole distribution indicates whether shape FA is concentrated along one specific direction or rather is distributed over many directions. Using multivariate regression, we then displayed graphically the dominant pattern of shape FA as the shape change associated with the first FA PC. This allowed a direct visual comparison of the patterns of shape FA among genotypes. The significance of the correlation of shape FA patterns among genotypes was then tested as follows. As the angles formed by pairs of FA PCs estimate their similarity, we first compared them to a null distribution of angles formed by pairs of 26-dimensional random vectors. This allowed testing whether the recorded patterns of FA were more similar than expected from random variation. We then examined the alternative null hypothesis of identical FA vectors. To test whether the vectors differed more than expected from sampling error alone, we compared them to a null distribution of angles obtained from a bootstrap procedure resampling individual observations within genotype. ### FA of cell size and cell number {#s4d5} Fluctuating asymmetry of cell size and cell number was computed for each genotype as the variance of (R-L) values (FA4). Morphometric and statistical analyses were conducted using R version 2.6.2 [@pgen.1002314-R1] and the MorphoJ package [@pgen.1002314-Klingenberg4]. Supporting Information {#s5} ====================== ###### Superimposition of wings. In each case, photos of the two wings of the same individual were superimposed: in red the left wing and in green the right wing. A, B and C: Individuals overexpressing *CycG* ubiquitously under control of the *da::Gal4* driver using different *RCG* lines (*RCG23.3, RCG69*) or CG line (*CG2.1*). D and E: Individuals overexpressing *CycG* (*RCG76* line) under control of different drivers (*Act::Gal4* and *sd::Gal4*). F: Individual RNAi-inactivating *CycG* ubiquitously with the *da::Gal4* driver (LOF). (TIF) ###### Click here for additional data file. ###### Effects of *CycG* deregulation in a *yw^67c23^* background. A, B, and C: *CycG* overexpression; D, E and F: *CycG* inactivation. A and D: Discriminant analysis of the wing shape data. Each dot represents an individual fly (wing shape averaged over the right and left sides). GOF flies and LOF flies are completely discriminated from control flies, respectively on the first and second axes. Grey: +/+ females; black: +*/+* males; light green: GOF females; dark green: GOF males; light orange: LOF females; dark orange: LOF males; B and E: Shape change along the first axis; GOF wings (B) and LOF wings (E) (note that axes are inverted relative to the GOF experiment, due to a lesser amplitude of shape change among genotypes). The grey wing is the consensus wing computed from all wings (i.e. the grand mean shape); the colored wing represents the shape change when moving from +/+ control to GOF or LOF wings. C and F: Effect of *CycG* deregulation on wing size FA (open bars) and shape FA (dashed bars). Note that FA values reported on C and F are not directly comparable: FA10 indice was used in C, and FA4 in F (see [Material and Methods](#s4){ref-type="sec"}). (TIF) ###### Click here for additional data file. ###### Overexpression and downregulation of *CycG*. Total RNA was extracted from third instar larvae using the RNeasy kit (Qiagen). Real-time PCR was performed in triplicate using Taqman® Gene Expression Assays (Dm02151951_m1 CycG, Applied Biosystems) on a ABI prism 7700 detection system. Results were normalized against *Gapdh1* (Dm01843827_s1, Applied Biosystems) using the 2exp^-ΔΔCt^ method. (TIF) ###### Click here for additional data file. ###### Impact of allometry on mean wing shape discrimination (*yw^67c2^* background). A: Discriminant analysis applied to the shape variables (i.e. non null PC scores; grouping factor: genotype\*sex). B: Discriminant analysis applied to the residuals of a multivariate regression of size on shape variables. Grey: +/+ females; black: +*/+* males; light green: GOF females; dark green: GOF males; light orange: LOF females; dark orange: LOF males. (TIF) ###### Click here for additional data file. ###### Effects of *CycG* deregulation on mean wing size and shape. Mean centroid size values and standard deviations (Sd) are provided. Results of the ANOVAs on centroid size and MANOVAs on the PC scores (genotype and sex as fixed factors). GOF =  gain of function; LOF =  loss of function; +/+  =  controls; f =  females; m =  males. Sd =  standard deviation; Df =  degrees of freedom; SS =  sum of squares; MS =  mean squares; F =  Fisher\'s F value; Pillai =  Pillai\'s Trace; Df den =  denominator\'s degrees of freedom; Df num =  numerator\'s degrees of freedom; \* = p\<0.05; \*\* = p\<0.01; \*\*\* = p\<0.001; ns =  non significant. (DOC) ###### Click here for additional data file. ###### Wing size FA. Results of the two-way mixed model ANOVAs on centroid size (individual  =  random; side  =  fixed). The tests are presented for the three control genotypes in the GOF experiments using *da* as driver (see [Material and Methods](#s4){ref-type="sec"}). FA10 and FA4 are two FA indices (see [Material and Methods](#s4){ref-type="sec"}). FA effect refers to the ratio of the GOF or LOF FA value over the one of the corresponding *+/+* genotype. Df =  degrees of freedom; MS =  mean squares; F =  Fisher\'s F value. MS and FA10 values are multiplied by 10^5^. (DOC) ###### Click here for additional data file. ###### Wing shape FA. Results of the Procrustes ANOVAs (individual and side as main effects). MS and FA10 values are multiplied by 10^7^. Df =  degrees of freedom; MS =  mean squares; F =  Fisher\'s F value. (DOC) ###### Click here for additional data file. ###### Comparison of patterns of shape FA (females). Correlation of PC1s of FA matrices for all genotypes as measured by the angles among PCs. In brackets are the associated P-values. Top: H0 =  the null hypothesis is that the angles between FA PCs are not different from those between pairs of random vectors (10000 random 26 dimensional vectors). A significant effect means that the correlation is stronger than expected from chance only. The P-value is computed as \[1 -(number of random angles larger than the observed one)\]/10000. Bottom: H0 =  the null hypothesis is that the angles between FA PCs are not larger from those between pairs of vectors differing only by the sampling error. Statistical significance is tested against a null distribution of vectors derived from a within genotype boostrap procedure (x10000). A non significant effect means that the vectors are as strongly correlated as vectors differing only by sampling error (i.e. they are almost identical). The P-value is computed as \[(number of bootstrapped angles larger than the observed one)\]/10000. \* = p \<0.05; \*\* = p\<0.01; \*\*\* = p\<0.001, ns =  non significant. (DOC) ###### Click here for additional data file. ###### Femur length FA. Results of the two-way mixed model ANOVAs on femur length (individual  =  random; side  =  fixed). Df =  degrees of freedom; MS =  mean squares; F =  Fisher\'s F value. (DOC) ###### Click here for additional data file. ###### Effects on mean cell size and cell number. Mean wing cell size and cell number and standard deviations (Sd) are provided. Note that cell size is in arbitrary unit, being computed as one divided by the number of cells counted in the standardized area (see [Figure 3C](#pgen-1002314-g003){ref-type="fig"}′). Cell number strongly underestimate the real cell number in the wing: only one side of the wing was considered (dorsal); the wing basis was not included in the analysis; cells on - or at the direct vicinity of - veins were excluded due to the image thresholding (see [Figure 3C″](#pgen-1002314-g003){ref-type="fig"}. This underestimation is likely similar across individuals and genotypes and should thus not affect the results. ANOVAs (genotype and sex as main fixed effects) are presented with post hoc Tukey HSD test. Df =  degrees of freedom; SS =  sums of squares; MS =  mean squares; F =  Fisher\'s F-test value; Diff =  Difference in the observed means; lwr and upr  =  lower and upper limits of the interval range for each comparison; p-adj  =  adjusted P-value; \* = p \<0.05; \*\*\* = p\<0.001, ns =  non significant. (DOC) ###### Click here for additional data file. ###### Covariation between cell size and cell number. Top: Pearson \'s correlation coefficients (r) were computed for each genotypes. They were then compared statistically after applying a Fisher r to Z transformation. Bottom: Analysis of the residuals of regressions of cell size over cell number for each genotype and sex. Statistical significance of variance differences are tested using standard F tests. Z =  Fisher Z values; F =  Fisher F; p =  P-values. (DOC) ###### Click here for additional data file. ###### Effects on cell size and cell number FA. FA values for each genotype and sex is provided, as well as the statistical tests of comparison with the controls (F tests). The correlation between cell size and cell number FA with wing size FA were computed. Both Pearson parametric correlation coefficient and Spearman non parametric correlation were computed. Only Pearson\'s r values are shown with the corresponding test of statistical significance, as both tests provided very similar results. Df = degrees of freedom; MS = mean squares; F =  Fisher\'s F value; \* = p\<0.05; \*\* = p\<0.01; \*\*\*  = p\<0.001; ns =  non significant. (DOC) ###### Click here for additional data file. We thank Jean R. David, Jean-Michel Gibert, and Neel Randsholt for fruitful discussions; Jacques Montagne, Agnès Audibert, and the Bloomington stock center for fly lines; Anne Coléno-Costes for qRT-PCRs ; and Michel Baylac for granting access to a pre-release of his Rmorph library. We also thank Suzannah Rutherford, Paul Brakefield, Ary Hoffmann, and Richard Palmer for their encouraging comments on an early version of the manuscript. The authors have declared that no competing interests exist. This work was supported by grants from CNRS and UPMC to FP. FF was supported by a grant from MRT. VD was supported by a Marie Curie Reintegration grant and the ATM MNHN Formes. 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: VD SB JSD FP. Performed the experiments: VD SB FF NG PO-t VR CS JSD FP. Analyzed the data: VD NN JSD FP. Contributed reagents/materials/analysis tools: VD SB FF NG NN PO-t VR CS JSD FP. Wrote the paper: VD JSD FP.

1. Introduction {#sec1-materials-12-02190} =============== Sterilization is a fundamental step in the reuse of endodontic instruments. The sterilization procedure follows the steps of disinfection, cleaning, washing, drying, packaging, and sterilization by heat \[[@B1-materials-12-02190]\]. The disinfection and cleaning steps reduce the bacterial load and remove debris from the blades of the instruments, and then the sterilization step kills any form of microorganism, including spores \[[@B2-materials-12-02190]\]. The most widely used method described in the scientific literature for sterilization in the dental field is heat sterilization. Sterilization by heat involves the use of autoclaves that reach a temperature of 134 °C, which, with the action of steam at a pressure of 30 psi, sterilizes the instruments \[[@B3-materials-12-02190]\]. Heat sterilization can lead to changes in the physical and mechanical properties of dental instruments. These changes can affect the external surfaces with micropitting and corrosion phenomena \[[@B4-materials-12-02190]\], a reduction in cutting capacity, and/or by influencing the resistance to cyclic fatigue or torsional fatigue \[[@B5-materials-12-02190]\]. An unfavorable event that can occur during the preflaring, shaping \[[@B6-materials-12-02190]\], and probing phases is rupture of the endodontic instrument \[[@B7-materials-12-02190]\]. Ruptures can be caused by cyclic fatigue stress or torsional fatigue stress. Rupture due to torsion occurs when the instrument is blocked in the channel, losing its cutting capacity. The alloy can undergo a deformation that is first elastic and then plastic, and due to fatigue, it breaks \[[@B8-materials-12-02190]\]. Heat sterilization procedures can influence the torsional properties of endodontic instruments. Studies on resistance to torsional stress have related the number of sterilization cycles to the torsional properties, showing variations in the resistance to torsion and in the angle of deflection during torsion. In 2011, King et al. \[[@B9-materials-12-02190]\] reported a reduction in torsion resistance for GT Series X rotary instruments (Dentsply, Tulsa Dental Specialities, Tulsa, OK, USA) and an increase in resistance for Twisted Files (SybronEndo, Orange, CA, USA). Additionally, in 2011, Casper et al. \[[@B10-materials-12-02190]\] showed an increase in resistance to torsional fatigue for controlled memory (CM) wire files, whereas Hilt in 2000 reported no influence on the torsional properties of both steel and nickel-titanium alloy (NiTi) instruments \[[@B11-materials-12-02190]\]. The challenge is that sterilization cycles can either lead to deterioration, have no influence, or improve the mechanical properties of the instruments. The most recent study on the subject was conducted by Alazemi et al., in 2014, which showed a recovery effect of the instrument shape after autoclave sterilization \[[@B12-materials-12-02190]\]. The scientific literature is therefore not always in agreement about the effects of sterilization. These differences may be derived from the heterogeneity of the instruments investigated. The various endodontic instruments produced by various brands differ not only in diameter and taper at the tip, but also in the shapes of the sections and the characteristics of the alloys. The aim of this review was to try to understand these apparent controversies between the various studies with the help of systematic methodology and meta-analysis. We investigated the influences of heat sterilization on torsional properties by differentiating instruments between steel and NiTi alloys. Systematic reviews, where the relationship between torsional fatigue and sterilization procedures were investigated, were not conducted until 2019. The literature, however, includes updated revisions on sterilization procedures in the endodontic field and systematic reviews on cyclical fatigue, but they are never related to each other \[[@B1-materials-12-02190],[@B13-materials-12-02190]\]. The results of this review could therefore help endodontists follow a more accurate approach to the reuse of endodontic files, and understand the effects of autoclaving on instruments. We paid attention, using a qualitative analysis of the studies, to the variations in the torsional fatigue resistances of M wire, CM wire and Electrical Discharge Machining (EDM) alloys of endodontic instruments, following sterilization by heat. We aimed to meet the needs of modern, constantly-evolving endodontics and provide useful information on new alloys available to the clinician. 2. Materials and Methods {#sec2-materials-12-02190} ======================== The following work was performed based on the PRISMA indications \[[@B14-materials-12-02190]\]. The population, intervention, comparison, and outcome (PICO) questions of our study were endodontic instruments, modification of hot sterilization on the torsional properties of endodontic instruments, endodontic instruments not subject to hot sterilization cycles, and resistance to torsional fatigue and deflection angle for NiTi and steel instruments, respectively. The research question based on the PICO approach was how hot sterilization affects the torsional characteristics of NiTi and steel endodontic instruments compared to the same non-sterilized instruments. After a screening phase, the eligible articles were qualitatively analyzed to investigate the effects of sterilization procedures on the resistance to torsional fatigue of endodontic instruments used for probing, glide path, and shaping of the endodontic canal. 2.1. Eligibility Criteria {#sec2dot1-materials-12-02190} ------------------------- In this study, we considered literature reviews, in vitro studies, and clinical studies concerning the theme of sterilization and the influence of the sterilization on the mechanical properties of endodontic instruments, which were conducted in recent years and published with abstracts in English. We decided to choose articles from the last 40 years (1979 to 2019) because the disinfection and sterilization procedures changed radically after infectious contaminants were discovered, such as the HIV and HCV viruses and the prion of spongiform encephalopathy. The methods used to manufacture the instruments changed with the introduction of new alloys and new instruments. The articles considered potentially eligible were those that discussed the influences of sterilization and disinfection procedures on the physical and mechanical characteristics of endodontic instruments. The potentially eligible articles were finally subjected to a full-text analysis to verify the use for a qualitative and quantitative analysis. The inclusion criteria applied to the quantitative analysis included all studies that discussed the sterilization methods of endodontic instruments. The exclusion criteria included all studies that did not report data on resistance to torsional fatigue or on the angle of deflection due to torsion, of NiTi and steel endodontic instruments sterilized by heat. We also excluded the articles not in English, and those prior to 1979. 2.2. Research Methodology {#sec2dot2-materials-12-02190} ------------------------- Studies were identified through bibliographic research on electronic databases \[[@B15-materials-12-02190]\]. The literature search was conducted on the search engines PubMed and Scopus. The search was conducted between 13 January 2019 and 20 February 2019, and the last search for a partial update of the literature was conducted on 17 May 2019. The following search terms were used on PubMed and Scopus: "Endodontic sterilization" PubMed 326, Scopus 236; "endodontic autoclave" PubMed 37, Scopus 50; "cyclic fatigue" AND "sterilization" PubMed 19; and "torsional" AND "sterilization" PubMed 30 ([Table 1](#materials-12-02190-t001){ref-type="table"}). 2.3. Screening Methodology {#sec2dot3-materials-12-02190} -------------------------- The records obtained were subsequently screened by two independent reviewers (M.D. and G.I.), and a third reviewer (G.T.) acted as a decision maker in doubtful situations. The screening included analysis of the title and the abstract to eliminate records not related to the themes of the review. After the screening phase, the overlaps were removed and the full texts of the articles were analyzed, from which those eligible for qualitative analysis and inclusion in the meta-analysis for the four outcomes were identified. The outcomes sought by the two reviewers were:(1)Primary outcome: Variation in torsional fatigue resistance for NiTi endodontic instruments compared to the control not subjected to hot sterilization;(2)Secondary outcome: Variation in the flexion angle for NiTi endodontic instruments compared to the control not subjected to hot sterilization;(3)Tertiary outcome: Variation in resistance to torsional fatigue for steel endodontic instruments compared to controls not subjected to hot sterilization;(4)Quaternary outcome: Variation in the flexion angle for steel endodontic instruments with respect to the control not subjected to hot sterilization. The fourth reviewer, with supervisory duties, was L.Lo.M. The K agreement between the two screening reviewers was 0.655 ([Table 2](#materials-12-02190-t002){ref-type="table"}). The K agreement was based on the formulas in the Cochrane Handbook for Systematic Reviews \[[@B16-materials-12-02190]\]. The Newcastle-Ottawa scale for case-control studies was used to assess the risk of bias in the included studies \[[@B17-materials-12-02190]\]. The quantitative analysis was performed with the Rev Manager software 5.3 (Cochrane collaboration, Copenhagen, Denmark). 2.4. Data Analysis {#sec2dot4-materials-12-02190} ------------------ The statistical analysis of the data was performed using the Rev Manager 5.3 software (Copenhagen, 153 Denmark, The Nordic Cochrane Centre, The Nordic Cochrane Collaboration, 2014) and the results were represented by forest plots for each of the outcomes. For the primary outcome, the variation in resistance to torsional fatigue was compared following hot sterilization of NiTi instruments by measuring the torsional moment (g·cm) compared with the non-sterilized controls using a torquemeter. The comparison shows high heterogeneity of the studies, with an *I*^2^ equal to 79%. For this reason, a random effects model was used. Overall, for the primary outcome, the meta-analysis is in favor of non-sterilized NiTi instruments (control). The studies that presented data with a statistically significant difference are: Silvaggio et al. \[[@B18-materials-12-02190]\] in favor of the control and Canalda-Sahli \[[@B19-materials-12-02190]\] against the group subject to sterilization. Casper \[[@B10-materials-12-02190]\] is exactly at the center of the non-effect line, whereas the remaining three studies are unfavorable for the group subjected to heat sterilization even if their confidence intervals intercept the non-effect line ([Figure 1](#materials-12-02190-f001){ref-type="fig"}). For the secondary outcome, the variation in the angle of deflection (degrees) up to instrument rupture was compared in the NiTi instruments between the control group and the sterilization group using a torsimeter. The comparison showed high heterogeneity among the studies, with an *I*^2^ equal to 90%. For this reason, for the second outcome, a random effects model was applied so as to not minimize the roles of smaller-dimension studies. For the second outcome, the forest plot is in favor of the subject group compared to the control. The studies that reported statistically significant data in favor of the experimental group are Silvaggio et al. \[[@B17-materials-12-02190]\] and Canalda-Sahli et al. 1998 \[[@B19-materials-12-02190]\], whereas those in favor of the control group are Casper et al. 2011 \[[@B10-materials-12-02190]\] and King et al. 2011 \[[@B9-materials-12-02190]\] ([Figure 2](#materials-12-02190-f002){ref-type="fig"}). For the tertiary outcome, the variation in resistance to torsional fatigue was compared following hot sterilization of steel instruments, measuring the torsional moment (g·cm) with respect to the non-sterilized controls using a torquemeter. The comparison showed low heterogeneity between the studies, with an *I*^2^ of 4%, and a fixed effects model was applied. For the tertiary outcome, the forest plot is in a position of no effect for the two groups. None of the included studies presented statistically significant data ([Figure 3](#materials-12-02190-f003){ref-type="fig"}). For the quaternary outcome, the variation in the angle of deflection (degrees) up to instrument failure, measured using a torquemeter, in steel instruments between the control group and the sterilization group was compared. The comparison shows low heterogeneity between the studies, with an *I*^2^ of 0%, and a fixed effects model was applied. The forest plot has a slightly shifted graph in favor of the control group, but with low significance. None of the studies reported statistically significant data in favor of the control or of the group subjected to sterilization ([Figure 4](#materials-12-02190-f004){ref-type="fig"}). 3. Results {#sec3-materials-12-02190} ========== A total of 725 records were identified on the PubMed and Scopus databases ([Table 1](#materials-12-02190-t001){ref-type="table"}). After screening the articles with restriction by year of publication (1979 to 2019), there were 685 records. With the application of the eligibility criteria (all the articles pertaining to the issue of sterilization in endodontics), there were 146 articles. There were 130 articles after eliminating overlaps. There were 45 articles that discussed the influences of the sterilization procedures on the physical and mechanical characteristics of the instruments, and 12 that measured parameters on resistance to torsional fatigue. Applying the inclusion and exclusion criteria resulted in a total of eight articles for quantitative analysis. Six articles were in reference to the primary and secondary outcomes, and four to the tertiary and quaternary outcomes. The whole selection and screening procedures are described in the flow chart ([Figure 5](#materials-12-02190-f005){ref-type="fig"}). 3.1. Study Characteristics and Data Extraction {#sec3dot1-materials-12-02190} ---------------------------------------------- The included studies for quantitative analysis were:First and second outcomes: Hilt et al. 2000 \[[@B11-materials-12-02190]\], Silvaggio et al. 1997 \[[@B18-materials-12-02190]\], Canalda-Sahli et al. 1998 \[[@B19-materials-12-02190]\], King 2011 et al. \[[@B9-materials-12-02190]\], Casper et al. 2011 \[[@B10-materials-12-02190]\] and Testarelli et al. 2003 \[[@B20-materials-12-02190]\];Third and fourth outcomes: Iverson et al. 1985 \[[@B21-materials-12-02190]\], Hilt et al 2000 \[[@B11-materials-12-02190]\], Canalda-Sahli et al. 1998 \[[@B19-materials-12-02190]\] and Haikel et al. 1997 \[[@B22-materials-12-02190]\]. The extracted data included the magazine (author, data, and journal), the endodontic instrumentation object of measurement (name, taper, and diameter at tip), the method of sterilization by heat (temperature, pressure, and time), the type of instrumentation used for the measurement, and the data related to torsional fatigue (torsional moment, deflection angle, bending moment, and Knoop hardness). The data extracted for the four outcomes are shown in [Table 3](#materials-12-02190-t003){ref-type="table"} and [Table 4](#materials-12-02190-t004){ref-type="table"}. We extracted the data concerning the torsional moments and the deflection angles of the new alloys (M wire, CM, and Twisted Files) present in the market since 2010 and which hold considerable interest for the endodontist. The studies that analyzed the heat sterilization and the torsional properties of the M wire and CM alloy instruments were King et al. \[[@B9-materials-12-02190]\] and Casper et al. \[[@B10-materials-12-02190]\] ([Table 5](#materials-12-02190-t005){ref-type="table"}). 3.2. Risk of Bias {#sec3dot2-materials-12-02190} ----------------- The risk of bias was assessed through the Newcastle-Ottawa case-control scale. The results are reported in detail in [Table 6](#materials-12-02190-t006){ref-type="table"} for each category, a value of one to three was assigned (1 = low and 3 = high). The risk of bias within the individual studies was low enough that the methods of investigation adopted for the controls (not sterilized with heat) were identical to the cases (sterilized with heat) included in the meta-analysis. For Silvaggio et al. \[[@B18-materials-12-02190]\] only, we note an unclear assignment of cases and controls for all the experiments performed in the study. The risk of bias between the various studies was considered high, and partly limited the importance of the results. The heterogeneity between the studies is highlighted by three main factors: Diversity of the endodontic instruments used (only similar in taper and diameter at the tip), different apparatuses for measuring the torsional moment and the angle of deflection, and inconsistency in the method of hot sterilization. The heterogeneity of the studies is represented by funnel plots of the four outcomes in [Figure 6](#materials-12-02190-f006){ref-type="fig"}. 4. Discussion {#sec4-materials-12-02190} ============= The scientific literature provides contradictory findings about the effects of heat sterilization on the torsional properties of NiTi and steel instruments used in endodontics. The three most studied parameters in the literature for measuring torsional fatigue are: (1) The torsional moment (g-cm), which represents the force necessary to break the instrument; (2) the deflection angle (degrees), which represents the instrument's torsion angle at breaking point; and (3) the bending that represents the necessary force (g-cm) to bend an endodontic instrument at a defined angle. In the literature, the studies reporting an improvement in the resistance to torsional fatigue for endodontic instruments are:(1)Iverson \[[@B21-materials-12-02190]\], which reported a statistically significant increase in fatigue resistance only for the burn Unifile (De Trey, Bois Colombes, France) instruments after 10 sterilization cycles;(2)Silvaggio \[[@B18-materials-12-02190]\], which reported an increase in torsional fatigue resistance for profile instruments (Tulsa Dental Product Tulsa, OK), but not for all sizes;(3)Canalda-Sahli \[[@B19-materials-12-02190]\], which reported an increase in flexibility for titanium instruments and a decrease/increase for NiTi instruments;(4)King \[[@B9-materials-12-02190]\], which reported improvements in resistance to torsional fatigue for Twisted Files instruments and a reduction for the GT Series X M wire after only three sterilization cycles by autoclaving;(5)Casper et al. \[[@B10-materials-12-02190]\], which reported an increase in resistance to torsional fatigue after heat sterilization for CM wire, with a recovery effect of the shape after sterilization; and(6)Alazemi et al. \[[@B12-materials-12-02190]\], which, like Casper et al. \[[@B10-materials-12-02190]\], reported a recovery effect for HyFlex CM instruments (Coltène-Whaledent, Altstätten, Switzerland). Only one study included in the quantitative analysis reported statistically significant data for the GT Series X, whereas the remaining articles reported data with non-significant differences, showing no effect of the sterilization procedures on the torsional characteristics of endodontic instruments. The studies that showed increased resistance to torsional fatigue were mainly conducted on NiTi instruments. King et al. \[[@B9-materials-12-02190]\] reported an increase for Twisted Files. These instruments are built following a twist, and undergo thermal heating and cooling processes that stabilize the NiTi alloy. The thermal heating induced by hot sterilization make it even more stable, increasing resistance to torsional fatigue, which is also valid for M wire alloys. We obtained additional data from the meta-analysis. For the first outcome, we found an unfavorable effect of sterilization (10 cycles of autoclaving) on resistance to torsional fatigue in NiTi instruments, but with low statistical significance. The second outcome concerns the variation in the deflection angle (up to failure) following torsion. In this case, the data from the meta-analysis were significant, with a reduction in the deflection angle in the group subjected to heat sterilization. This indicates a greater stability of the austenitic and martensitic phases of the NiTi alloy following heat treatment during sterilization. For the tertiary and quaternary outcomes, with the meta-analysis we established that there is no change in the resistance to torsional fatigue, nor is there an improvement in the steel tools with hot sterilization procedures in a pejorative sense, according to Hilt et al. \[[@B11-materials-12-02190]\] and Canalda-Sahli et al. 1998 \[[@B19-materials-12-02190]\]. New Alloys: M Wire, CM, and EDM---Qualitative Analysis of the Studies {#sec4dot1-materials-12-02190} --------------------------------------------------------------------- Since 2009, the development of new alloys has led to the construction of new endodontic instruments with improved physical and mechanical characteristics, including increased resistances to cyclic fatigue and torsional fatigue. The new alloys that have been developed are M wire, CM wire, and EDM alloys \[[@B23-materials-12-02190]\]. M wire alloys were introduced in 2009, and are characterized by a more stable martensitic phase, which is reached during clinical use at higher temperatures. This depends on a thermomechanical manufacturing process that makes the martensitic phase less subject to breakage, with an intermediate phase from the austenitic phase, defined as the "R-phase". M wire alloys have a high-temperature form recovery effect, showing 6% recovery of plastic deformation induced by stress \[[@B24-materials-12-02190]\]. The endodontic instruments built using M wire alloys are: ProTaper Next (Dentsply Maillefer, Ballaigues, Switzerland) \[[@B25-materials-12-02190]\], WaveOne Gold (Dentsply Maillefer, Ballaigues, Switzerland), Twisted Files, and the Gt X and Profile Vortex series (Dentsply Tulsa Dental, Tulsa, OK, USA). CM wire alloys are always constructed with a thermomechanical process, but with shape-memory control. EDM alloys are manufactured using an electrical discharge machining technique, always with a controlled memory alloy. There are three articles related to the variations in torsional properties with the sterilization procedures, of which the data from two articles are shown in [Table 5](#materials-12-02190-t005){ref-type="table"}. King et al. \[[@B9-materials-12-02190]\] reported a statistically significant reduction for the M wire alloys, and specifically the GT Series X, after three sterilization cycles (torsional moment 85.67 ± 9.59 g·cm, angular deflector 483.11 ± 66.57 degrees) ([Table 5](#materials-12-02190-t005){ref-type="table"}). The study reports, however, a slight increase in resistance to twisting for the other M wire alloy instruments (twisted file). Casper et al. \[[@B10-materials-12-02190]\] reported no statistically significant alteration in the torsional properties in a pejorative sense for the M wire (Twisted files, ProFile Vortex) alloys and for the CM wire alloy (CM wire file). Alfoqom Alazemi et al. \[[@B12-materials-12-02190]\] reported recovery of the shape in two-thirds of the cases after hot sterilization in the HyFlex CM wire. The data reported in the literature for the new M wire, CM wire, and EDM alloys show that they have similar form recovery after heat sterilization and an increase in resistance to torsional fatigue, with some exceptions, such as the GT Series X \[[@B26-materials-12-02190]\]. The data on the increase in resistance to torsional fatigue are also supported by data on cyclic fatigue resistance. Zhao et al. \[[@B27-materials-12-02190]\] reported an increase in cyclic fatigue resistance for HyFlex CM instruments, in accordance with the data on torsional fatigue. Hilfer Pet al. \[[@B28-materials-12-02190]\] reported a reduction in the resistance to cyclic fatigue following repeated cycles of autoclaving for the GT Series X (M wire alloy), which does not affect the M wire alloy instruments. 5. Conclusions {#sec5-materials-12-02190} ============== From the literature analysis, only 12 scientific articles published in international journals that related heat sterilization procedures to the resistance to torsional fatigue in detail were identified. Of these 12 articles, only eight were included in the meta-analysis for the four outcomes. From the meta-analysis, we found that heat sterilization procedures reduce the torsional moment of NiTi instruments, with a low significance. When translated into clinical terms, this means a reduction in the resistance to torsional fatigue, with a greater risk of fracture. There is also a reduction in the angle of deflection following rupture for the group subjected to autoclaving. The clinical significance of this is not entirely clear to us, and certainly requires future investigation. For steel instruments, there is no change in the resistance to torsional fatigue, whether it is expressed in terms of torsional moment or deflection angle. The torsional fatigue data on the newer M wire, CM wire, and EDM alloys are fundamentally important for the endodontics clinician. Awareness of the better reliability of these new instruments following sterilization is beneficial for endodontists who proceed with the sterilization of the instruments before use. The data agree that there is a lower risk of fracture in the case of reuse compared to the old generation instruments. All the acknowledgements go to Lorenzo Lo Muzio, Director of the Dental Clinic and President of the Department of Clinical and Experimental Medicine of the University of Foggia, who gave fundamental technical support in the writing of this article. Conceptualization, M.D., D.S., B.R., and A.D.; methodology, K.Z.; software, R.A.; validation, G.I. and L.L.M.; formal analysis, G.I.; investigation, L.L. and V.C.A.C.; resources, L.L.M.; data curation, E.L.; writing---original draft preparation, M.D. and G.T.; writing---review and editing, M.D., G.T., and L.L.M. This research received no external funding. The authors declare no conflict of interest. {#materials-12-02190-f001} {#materials-12-02190-f002} {#materials-12-02190-f003} {#materials-12-02190-f004} {#materials-12-02190-f005} {#materials-12-02190-f006} materials-12-02190-t001_Table 1 ###### Complete overview of the search methodology. Records identified by databases: 725; records selected for qualitative analysis: 12; records selected for quantitative analysis: 8. Provider Database Key Words No. of Records Number of Records after Restriction by Year of Publication (1979 to 2019) No. of Articles Remaining after the Elimination of Records Not Related to Sterilization of Endodontic Instruments No. of Articles after Removing Duplications No. of Articles Addressing Influence of Sterilization Procedures on Mechanical Characteristics of Endodontic Instruments No. of Articles Included in The Qualitative Analysis (Influence of Hot Sterilization on Torsional Properties) No. of Articles Included in the Quantitative Analysis for the Four Outcomes ------------------- -------------------------------------- ---------------- --------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------- --------------------------------------------- -------------------------------------------------------------------------------------------------------------------------- --------------------------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------- Pub-med "Endodontic sterilization" 326 287 35 \\ \\ \\ \\ Pub-med "endodontic autoclave" 37 36 21 \\ \\ \\ \\ Pub-med "cyclic fatigue" AND "sterilization" 19 19 16 \\ \\ \\ \\ Pub-Med "torsional" AND "sterilization" 30 30 17 \\ \\ \\ \\ SCOPUS Endodontic AND Sterilization 263 263 36 \\ \\ \\ \\ Scopus "endodontic" AND" autoclave" 50 50 21 \\ \\ \\ \\ Total records 725 685 146 130 45 12 8 materials-12-02190-t002_Table 2 ###### K agreement calculation, Po = 0.86 (Proportion of agreement), Pe = 0.594 (Agreement expected), K agreement = 0.655 (\<0 no agreement, 0.0--0.20 slight agreement, 0.21--0.40 fair agreement, 0.41--0.60 moderate agreement, 0.61--0.80 substantial agreement, 0.81--1.00 almost perfect agreement). The K agreement was calculated from the 45 articles to include eight articles with the application of the inclusion and exclusion criteria. Reviewer 2 Reviewer 2 Reviewer 2 ------------ --------- ------------ ------------ ------------ ------- Include Exclude Unsure Total Reviewer 1 include 8 4 1 13 Reviewer1 exclude 0 31 0 31 Reviewer 1 unsure 1 0 0 1 total 9 35 1 45 materials-12-02190-t003_Table 3 ###### Extracted data from selected studies (primary outcome and secondary outcome). ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Autor, Date, Journal, Reference Number Endodontic Instruments, Test Instruments Sterilization Method No. of Instruments Temperature, Pressure, Exposure Time Torsional Moment (g·cm)\ Angular Deflection (degrees)\ Bending Moment\ Knoop Hardness\ Mean ± SD Mean ± SD Mean ± SD\ (kg/rnrn^2^) (g·cm) ------------------------------------------------------------------------------ ------------------------------------------------------------------------- --------------------------- -------------------- -------------------------------------- -------------------------- ------------------------------- ----------------- ----------------- Hilt. 2000 Journal of Endodontics, \[[@B11-materials-12-02190]\] NiTi file (30 size 02 taper), torsional testing apparatus 10 cycles Statim\ 10 132 °C (270 °F), 20 psi, 30 min 46.1 ± 7.0 1191 ± 190 \\ 322 ± 18 autoclave Control 10 \\ 51.1 ± 5.3 1222\. 2± 212 \\ 330 ± 32 Silvaggio, 1997, journal of endodontics, \[[@B18-materials-12-02190]\] Profile taper 4 series 5 (0.279 mm) size, Torquemeter Memocouple. Dry heat 10 cycles 10 190--204 °C Atmospheric 46 min 98.4 ± 5.1 640.1 ± 82.2 \\ \\ control 10 \\ 87.3 ± 5.0 1088.5 ± 126.9 \\ \\ Canalda-Sahli, 1998, international endodontic, \[[@B19-materials-12-02190]\] Nitiflex (30 size 02 taper), Digital Torquemeter Memocouple, Maillefer 10 cycles autoclave 10 136 °C, 2.2 bar, 10 min 48.02 ± 2.89 465 ± 35.61 17.16 ± 1.81 \\ control 10 \\ 53.61 ± 4.94 512 ± 40.55 21.57 ± 1.11 \\ King, 2011, international endodontic journal, \[[@B9-materials-12-02190]\] Twisted Files (25, size 06 taper) Torsiometer/Memocouple Autoclave statim 7 cycles 20 132 °C 2.2 bar for 6 min, 48.70 ± 4.73 702.25 ± 80.64 \\ \\ control 20 \\ 51.40 ± 6.46 586.55 ± 64.18 \\ \\ Casper, 2011, journal of endodontics, \[[@B10-materials-12-02190]\] Twisted Files (25 size 04 taper), microprocessor-controlled torsiometer 7 cycles Autoclave statim 18--18 132 °C 2.2 bar, for 6 min 28 ± 2.7 1075 ± 206.8 \\ \\ control 20--16 \\ 28 ± 4.1 948 ± 110.8 \\ \\ Testarelli, 2003 minerva stomatologica, \[[@B20-materials-12-02190]\] Hero (size 30 size 02 taper), Torsiometer/Memocouple 10 cycles autoclave 10 124 °C 2.2 bar, for 20 min 173 ± 18.2 302 ± 28.2 147 ± 12.4 \\ control 10 \\ 184 ± 17.5 311 ± 30.2 146 ± 12.9 \\ ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- materials-12-02190-t004_Table 4 ###### Extracted data from selected studies (tertiary outcome and quaternary outcome). -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Autor, Date, Journal, Reference Number Endodontic Instruments, Test Instruments Sterilization Methods No. of Instruments Temperature, Pressure, Exposure Time Torsional Moment\ Angular Deflection\ Bending Moment\ Knoop Hardness\ Mean ± SD\ Mean ± SD\ Mean ± SD\ (kg/rnrn^2^) (g·cm) (Degrees) (g·cm) ------------------------------------------------------------------------------- ------------------------------------------------------------------------------------ ----------------------- -------------------- -------------------------------------- ------------------- --------------------- ----------------- ----------------- Iverson, 1985, journal of endodontics, \[[@B21-materials-12-02190]\] K flex (35 size 04 taper), torqumeter memocouple Autoclave 10 cycles 12 127 °C, 30 psi, 30 min 97.83 ± 8.48 791.25 ± 83.63 \\ \\ Control 12 \\ 94.92 ± 12.21 807.83 ± 99.37 \\ \\ Hilt, 2000, Journal of endodontics, \[[@B11-materials-12-02190]\] stainless steel K-type files (30 size 02 taper), torsional testing apparatus 10 cycles Statim\ 10 132 °C (270 °F), 20 psi, 30 min 98.9 ± 14.7 1704 ± 325 \\ 630 ± 18 autoclave control 10 \\ 105.8 ± 4.9 1598 ± 226 \\ 616 ± 22 Canalda-Sahli, 1998, international endodontics, \[[@B19-materials-12-02190]\] stainless steel flex of file (30 size 02 taper), Torquemeter Memocouple, Maillefer 10 cycles autoclave 10 136 °C, 2.2 bar, 10 min 61.70 ± 2.40 1306 ± 167.65 64.85 ± 3.28 \\ control 10 \\ 61.55 ± 3.28 1328 ± 188.46 63.30 ± 3.37 \\ Haikel, 1997, international endodontics, \[[@B22-materials-12-02190]\] flex of file (30 size 02 taper), torsional testing apparatus 10 cycles\ 10 180 °C 2.2 bar 2 h, 58.20 ± 2.06 1420\. 6 ± 379 62.52 ± 2.19 \\ autoclave control 10 \\ 56.28 ± 5.36 1262.2 ± 293 59.60 ± 1.92 \\ -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- materials-12-02190-t005_Table 5 ###### Data extracted from studies concerning the torsional moments and the deflection angles of the instruments manufactured with the new alloys (M wire and controlled memory (CM) wire) at 0, 1, 2, 3, and 7 autoclave cycles. --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Autor, Date, Journal, Reference Number Endodontic Instruments, Test Instruments Sterilization Methods No. of Instruments Temperature, Exposure Time Torsional Moment\ Angular Deflection\ Mean ± SD\ Mean ± SD\ (g·cm) (degrees) ---------------------------------------------------------------------------- -------------------------------------------- ----------------------- -------------------- ---------------------------- ------------------- --------------------- King, 2011, international endodontic journal, \[[@B9-materials-12-02190]\] Twisted file\ control 20 \\ 51.40 ± 6.46 586.55 ± 64.18 size 25, 0.06 taper Torsiometer/Memocouple Autoclave statim 1 cycles 20 132 °C for 6 min 46.32 ± 5.84 670.63 ± 59.74 Autoclave statim 3 cycles 20 132 °C for 6 min, 43.25 ± 7.28 672.65 ± 103.16 Autoclave statim 7 cycles 20 132 °C for 6 min, 48.70 ± 4.73 702..25 ± 80.64 GT Series X M wire size 20, 0.06 taperTorsiometer/Memocouple control 20 \\ 97.95 ± 11.04 445.65 ± 62.39 Autoclave statim 1 cycles 20 132 °C for 6 min, 94.75 ± 8.84 441.80 ± 47.30 Autoclave statim 3 cycles 20 132 °C for 6 min, 85.67 ± 9.59 483.11 ± 66.57 Autoclave statim 7 cycles 20 132 °C for 6 min 78.10 ± 7.36 430.75 ± 66.49 Casper, R.B. 2011 journal of endodontic \[[@B10-materials-12-02190]\] Profile Vortex m-wire\ 0 20 \\ 76 ± 7.0 438 ± 59 microprocessor-controlled torsiometer 1 cycles Autoclave statim 20 132 °C for 6 min 80 ± 9.9 441 ± 58.0 2 cycles Autoclave statim 20 132 °C for 6 min 78 ± 8.3 448 ± 33.0 3 cycles Autoclave statim 20 132 °C for 6 min 75 ± 4.3 441 ± 35.5 7 cycles Autoclave statim 20 132 °C for 6 min 79 ± 8.2 452 ± 75.9 Twisted Files\ 0 20, 16 \\ 28 ± 4.1 948 ± 110.8 microprocessor-controlled torsiometer 1 cycles Autoclave statim 20, 19 132 °C for 6 min 29 ± 2.7 916 ± 61.0 2 cycles Autoclave statim 20, 19 132 °C for 6 min 29 ± 4.0 963 ± 147.0 3 cycles Autoclave statim 20, 16 132 °C for 6 min 29 ± 4.9 898 ± 174.6 7 cycles Autoclave statim 18, 18 132 °C for 6 min 28 ± 2.7 1075 ± 206.8 File CM Wire\ 0 19, 20 \\ 87 ± 11.0 821 ± 34.7 microprocessor-controlled torsiometer 1 cycles Autoclave statim 20 132 °C for 6 min 91 ± 11.1 829 ± 56.4 2 cycles Autoclave statim 19, 20 132 °C for 6 min 92 ± 11.2 820 ± 36.5 3 cycles Autoclave statim 20 132 °C for 6 min 90 ± 9.1 815 ± 40.5 7 cycles Autoclave statim 20, 18 132 °C for 6 min 87 ± 12.5 833 ± 89.8 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- materials-12-02190-t006_Table 6 ###### Assessment of risk of bias within the studies (Newcastle--Ottawa scale) with scores 7--12 = low quality, 13--20 = intermediate quality, and 21--24 = high quality. Selection Comparability Exposure Score ------------------------------- --- ----------- --- --- --------------- --- ---------- --- ------- \[[@B11-materials-12-02190]\] 3 3 3 3 3 3 3 0 21 \[[@B18-materials-12-02190]\] 2 2 2 2 2 2 3 0 15 \[[@B19-materials-12-02190]\] 3 3 3 3 3 3 3 0 21 \[[@B9-materials-12-02190]\] 3 3 3 3 3 3 3 0 21 \[[@B10-materials-12-02190]\] 2 2 2 2 2 3 3 0 16 \[[@B20-materials-12-02190]\] 2 2 2 2 3 3 3 0 17 \[[@B21-materials-12-02190]\] 3 3 3 3 3 3 3 0 21 \[[@B22-materials-12-02190]\] 3 3 3 3 3 3 3 0 21

1. Introduction {#s1} =============== Wood used as building material in an outdoor environment (i.e., European Standard EN 335-1:2006 use class 3--4) is frequently exposed to high relative humidity or wetting, highly increasing the risk of degradation by wood-degrading micro-organism. The resistance of wood to degradation in such a setting is mainly determined by its inherent durability (natural or artificial) and its moisture sorption properties, apart from environmental factors and design (Brischke et al., [@B10]; Meyer-Veltrup et al., [@B55]; Brischke and Alfredsen, [@B9]). Naturally durable heartwood is formed in the center of the stem of some tree species by deposition of various metabolites in the tissue, called extractives (Rowe, [@B67]). For the heartwood of many species and all of the sapwood this is not the case, and thus artificial wood protection is needed (Kutnik et al., [@B49]). In Europe, many of the old wood-preservatives were banned due to their high toxicity, and consequently, research and development focused their efforts on more environmentally benign ways to protect wood (Schultz and Nicholas, [@B73]; Singh and Singh, [@B74]). Of the new preservative compounds, many still have restricted use (ECHA, [@B21]), and thus further developments are necessary. One approach is to achieve a better understanding of the mechanisms underlying natural durability in trees, and the current study aims to contribute to this. In northern Europe, wood products from conifers are widely used for construction purposes. Most of these are susceptible to degradation by brown-rot fungi, which are known to cause a rapid loss of strength already at an early stage of degradation (Bader et al., [@B3]; Arantes and Goodell, [@B2]; Wagner et al., [@B82]). Brown-rot fungi are widely spread, cellulose-degrading fungi, currently believed to start their attacks employing non-enzymatic oxidative degradation, followed by an enzymatic stage (Bader et al., [@B3]; Arantes and Goodell, [@B2]; Zhang et al., [@B94]). Non-enzymatic oxidative degradation of the cell wall relies on secretion and diffusion of low molecular weight substances and metal ions into the cell wall, where they react to create radicals that disrupt the cell wall polymers. The success of this process depends heavily on the presence of water in and around the cell wall, but also on the amount and mechanisms of extractives present (Schultz and Nicholas, [@B73]; Jebrane et al., [@B47]). In this work, we chose to compare Kurile larch (*Larix gmelinii var. japonica*), a moderately durable conifer (Scheffer and Morrell, [@B71]; Bergstedt and Lyck, [@B5]; Metsä-Kortelainen and Viitanen, [@B53]), to non-durable Norway spruce (*Picea abies*) (Scheffer and Morrell, [@B71]; Metsä-Kortelainen and Viitanen, [@B53]) in terms of their extractive composition and their susceptibility to the brown-rot fungus *Rhodonia placenta* after various extraction procedures. Their similar xylem anatomy should provide similar geometrical conditions to the fungus, and accentuate differences arising due to their extractive composition. Extractives belong to many different chemical groups, and may be divided into more hydrophilic and more hydrophobic types (Giwa, [@B34]; Willför et al., [@B85], [@B84]). Both types are found in the tree species studied here. One role of the more hydrophobic extractives is possibly to repel water, as the amount of moisture in the wood is critical for the fungus\' successful establishment (Meyer and Brischke, [@B54]; Brischke et al., [@B11]). For instance, it has been suggested that a certain cell wall moisture content is needed to form pathways within the cell walls to allow diffusion of the fungal low molecular weight substances (Zelinka et al., [@B93]; Hunt et al., [@B46]). Hydrophobic molecules, such as those found in the oleoresin of conifers, are good candidates for such functionality by decreasing the wettability of the cell wall (Eberhardt et al., [@B20]; Harju et al., [@B40]; Nzokou and Kamdem, [@B60]; Belt et al., [@B4]; Sjökvist et al., [@B75]). Oleoresin is present throughout the xylem, but its composition varies within the stem and changes upon injury or infection (Ekman, [@B23]; Hillis, [@B43]; Bohlmann et al., [@B8]; Holmbom et al., [@B45]; Mason et al., [@B52], p. 73). Its primary components are fats and fatty acids (FAs) and various terpenoids. The non-volatile fraction is mainly composed of diterpenoids (DTs), among which resin acids (RAs) are the most abundant (50--75%) (Higuchi, [@B41]; Bohlmann et al., [@B8]; Holmbom et al., [@B45]). Apart from a role in moisture regulation, the potential of RAs as biocides against multiple insects and micro-organisms was shown in numerous *in vivo* and *in vitro* assays (Micales et al., [@B56]; Nerg et al., [@B58]; Keeling and Bohlmann, [@B48]; Mason et al., [@B52]), including white rot fungi (Eberhardt et al., [@B20]), and brown rot fungi (Micales et al., [@B56]; Nerg et al., [@B58]). Sterols (STs), which are triterpenoids, have potential as growth retardants in bacteria, and a synergistic role with RAs has been proposed (Burčová et al., [@B12]). Representative chemical structures of compounds, belonging to these families and relevant for this study, can be viewed in [Figure S1.1--7](#SM1){ref-type="supplementary-material"}. Spruce and larch heartwood also contain extractives that are more hydrophilic, mainly lignans (LI) in spruce and flavonoids (FL) in larch (see [Figure S1.9--12](#SM1){ref-type="supplementary-material"}, Willför et al., [@B85]; Gierlinger et al., [@B32]; Nisula, [@B59]). Their functions include hindering the creation of (fungal) radicals by chelation of metals needed for the latter, neutralizing occurring radicals (antioxidants) and/or directly harming the fungus (biocide) (Rice-Evans et al., [@B65]; Willför et al., [@B90]; Binbuga et al., [@B6]; Donoso-Fierro et al., [@B19]; Chen et al., [@B14]). Phenolic extractives may additionally play a role in moisture exclusion by bulking of the cell wall (Wangaard and Granados, [@B83]; Choong and Achmadi, [@B15]; Nzokou and Kamdem, [@B60]; Vahtikari et al., [@B80]). Both lignans (Rowe, [@B67]; Smith et al., [@B78]) and flavonoids (Dellus et al., [@B18]; Ostroukhova et al., [@B62]) partially form oligo- and polymers in heartwood, and so do the more hydrophobic resin components (Schaller, [@B69]; Smeds et al., [@B76], [@B77], p. 164). Larch is somewhat a special case among the conifers, because it contains up to 30% w/w of a non-structural, hemicellulose-type polysaccharide - arabinogalactan (ArGal). Its amounts increase drastically at the sapwood-heartwood boundary, and fills the lumen of tracheid cells in the heartwood, especially those closer to rays (Côté et al., [@B13]; Giwa, [@B34]; Grabner et al., [@B35],[@B36]). Experiments show that ArGal has influence on the mechanical properties of larch heartwood (Luostarinen and Heräjärvi, [@B51]), but its role in fungal degradation, if any, remains controversial (Côté et al., [@B13]; Gierlinger et al., [@B32]; Hill et al., [@B42]). Wood degrading fungi generally need a minimum water potential in the range of −4 to 0.1 MPa in order to grow (Griffin, [@B38]; Boddy, [@B7]; Griffith and Boddy, [@B39]; Schmidt, [@B72]), which corresponds to 97--99% relative humidity (RH). Above this range, liquid water accumulates in pits and cell lumina via capillary condensation (Engelund et al., [@B24]; Fredriksson and Thybring, [@B30]), while in the hygroscopic range (0 to about 98% RH) water is bound to hydroxyl groups in the cell wall. Although it has been shown that extractives alter the equilibrium moisture content (MC) in the hygroscopic range (Wangaard and Granados, [@B83]; Choong and Achmadi, [@B15]; Nzokou and Kamdem, [@B60]; Vahtikari et al., [@B80]), to the best of our knowledge, no publications exist on how extractives affect the MC in the over-hygroscopic range. The pressure plate technique allows investigation of the wood\'s MC in this range by precise regulation of the pressure applied to a water containing, tight cell (Fredriksson and Thybring, [@B30]). After a long equilibration time, the moisture content is determined gravimetrically. One aim of this study was, to explore whether extractives also influence the over-hygroscopic moisture range in Kurile larch. The best results for artificial impregnation is obtained by the combination of different functionalities (Schultz and Nicholas, [@B73]). Even more so, is it important to understand the individual roles of natural extractives, in order to be able to mimic the mechanisms at play when designing novel wood protection systems. Thus, partial removal of extractives of a certain polarity may give insight on their influence on wood durability against fungal degradation, and by assessing their effect on the MC of the wood possible interrelations between moisture and extractives content can be identified. With the aim of understanding the roles of hydrophobic and hydrophilic extractives in degradation and moisture regulation, we developed a multi-step extraction procedure that partially removes more extractives of a certain polarity from wooden sticks. The extractive composition was obtained by gas chromatography coupled to mass spectrometry and flame ionization detection to gain insights on the quantities of molecules present. Additionally, since moisture is a prerequisite for fungal attack, a specific aim of our study was to explore whether semi-selective removal of extractives with different polarity would affect the equilibrium moisture content in Kurile larch, especially in the important but under-explored over-hygroscopic range. 2. Materials and Methods {#s2} ======================== A summary of procedures and methods used in this work can be viewed in [Figure 1](#F1){ref-type="fig"}. ![**(A)** Preparation of wooden sticks, exemplified with Larch. The frozen wood disk was cut radially into slices. The mature heartwood (marked in red) was selected and one part was cut into sticks with dimensions 50 × 3--4 × 3--4 mm \[longitudinal × radial × tangential\]. The other part was milled. **(B)** Sticks and milled samples were extracted with different solvents according to the scheme. The extracts from the milled samples were quantified and identified using GC-MS/-FID. Some larch sticks were made into blocks and used for determination of the sorption isotherm. All extracted and native sticks of both species were subjected to brown-rot degradation. NAT, native; PHO, hydrophobic; PHI, hydrophilic; TOT, total; DCM, dichloromethane.](fpls-11-00855-g0001){#F1} 2.1. Wood Sampling and Preparation ---------------------------------- Two clones of spruce (*P. abies*, 48 years old) and larch (*Larix gmelinii var. japonica*, 65 years old) were harvested near Hørsholm, Denmark, in the autumn of 2017. The individual trees will be referred to as spruce 1, spruce 2, larch 1 and larch 2. [Figure 1A](#F1){ref-type="fig"} illustrates the sampling process. Disks (about 80 mm thick) were taken at 1.3 m stem height and stored at −20°C within 10 h from sampling. The disks\' diameters were 323 and 280 mm for spruce 1 and 2, respectively. For larch 1 and 2 they were 381 and 406 mm, respectively. Using a band saw, 3--4 mm thick slices were radially cut from all around the frozen stem disks. The mature heartwood (red square in [Figure 1A](#F1){ref-type="fig"}) was separated from sapwood and juvenile heartwood. For spruce, 10 growth rings from the pith were considered juvenile (Lindström, [@B50]). For larch, on average 15 year rings were similarly discarded (Gierlinger and Wimmer, [@B33]; Luostarinen and Heräjärvi, [@B51]). The preselected mature heartwood was stored at −80 °C until further processing. The samples were split into two groups: slices with 3--4 mm width were used to prepare sticks of 50 mm × 3 or 4 mm × 4 or 3 mm (L × W × B), as shown in [Figure 1A](#F1){ref-type="fig"}. The sticks were freeze dried for 30--48 h and categorized into different growth ring patterns, i.e., broad EW + broad LW, thin repeating EW+LW, EW-LW-EW, and LW-EW-LW. These were then distributed evenly into four groups of 13--17 sticks each, so as to avoid possible bias from the radial position in the heartwood, where each stick was comprised of 1--3 annual rings. The rest of the frozen material was made into 10 mm^2^ chips with garden shears. After freeze drying for 24 h, the chips were milled in a Retch ZM100 mill (1 mm sieve), freeze dried again and sieved through a 60 mesh filter, corresponding to a hole size of 250 μm. Before extraction, all samples were placed in a desiccator under vacuum with freshly dried molecular sieves for 2--18 h at room temperature. 2.2. Extraction and Further Processing -------------------------------------- Extraction of milled samples and sticks was done using the Accelerated Solvent Extractor (Dionex^TM^ ASE^TM^ 350, Thermo Electron A/S, Scientific Instrument Division, 2650 Hvidovre, Denmark). The apparatus works under N~2~ atmosphere, thereby lowering chances of oxidation artifacts possibly arising during extraction. Additionally, 1.38 MPa are applied to the extraction cell, allowing the use of volatile solvents at temperatures above their boiling points. An extraction procedure was developed, aiming at producing samples where the hydrophobic (PHO) or hydrophilic (PHI) part of extractives had been removed, as well as a total extraction, removing both portions (TOT, [Figure 1B](#F1){ref-type="fig"}). For the TOT extraction a sequence of four solvents was used in the following order: Heptane (anhydrous, 99%, Sigma-Aldrich), dichloromethane (DCM, SupraSolv®, Sigma-Aldrich), 96% ethanol (EtOH, SupraSolv®, Sigma-Aldrich), and demineralized water. For the PHO batches, only the hydrophobic solvents heptane and DCM were used. The PHI batches were treated only with hydrophilic solvents: 96% EtOH and demineralized water. For each procedure 13--17 sticks were used. To counteract losses in extraction efficiency due to the geometry of the sticks, the maximum possible number of extraction cycles was used, increasing the probability of analytes being washed out of the maze of wood cells. The extraction conditions for each solvent and batch were 9 × 5 min cycles, 90°C and 150% rinse volume, except for ethanol, for which 100°C were used. Void volume in the ASE extraction cells was filled with clean quartz sand (50--70 mesh, Sigma-Aldrich). In order to get an estimate of the extraction efficiency of the sticks, an amount corresponding to 13--17 sticks of milled material was extracted with the same procedure. For spruce 3--3.7 g were used per sample, while 5--5.6 g were needed for larch. Void volume in the ASE extraction cells was again filled with quartz sand, with a cellulose paper separating sample and sand. The ratio between the gravimetric yields of sticks and milled material (in mg/g) was used as a measure of extraction efficiency. To test for residues in the total extract, the milled samples were additionally extracted with 95:5 acetone:water (2 cycles à 5 min, 100°C). Furthermore, in order to double check the results, 4 g of milled material from each of the trees was extracted with a control sequence adapted from Willför et al. ([@B85]), employing one hydrophobic and one hydrophilic solvent. Heptane was used instead of hexane for the sake of lower toxicity and the acetone step was repeated 3 times instead of 2 times. The extracts were stored inside the pressurized extraction bottles at 4°C until further processing. The sample volume was reduced to 50 ml and 2--4 × 10 ml aliquots were used to determine the gravimetric yield. Two of the water samples were lost due to a mistake in the laboratory. For the larch water samples, the ArGal was precipitated out of the solution in triplicates, using the procedure described in Luostarinen and Heräjärvi ([@B51]). The precipitate of each sample was dried at 60°C over night in a ventilated oven and the weight determined. 2.3. Identification and Quantification of the Extracts ------------------------------------------------------ ### 2.3.1. Gas Chromatography Gas Chromatography (GC) was used for the separation of individual analytes of the respective extracts. Identification was done using the response from the Mass Spectrometrometer (MS). Quantification was done based on the signal of the Flame Ionization Detector (FID) obtained from one run per solvent fraction of each clone (*n* = 2). The extracts of all milled samples were run. Gas chromatography of the water fraction of both species did not show any peaks, likely because the majority of the material extracted by water are polysaccharides or other polymers. Thus, water-ethanol (WE) supernatant of the ArGal-free samples was pooled from 3 determinations and subsequently dried over Na~2~SO~4~ (ACS reagent, ≥ 99.0%, anhydrous, Sigma-Aldrich). After filtration and ethanol-wash of the filtrate, the now alcoholic solution was reduced to 5 ml. Only the hydrophilic extracted larch 1 WE-samples were analyzed by GC-MS, but here the extracts of the milled material and of the sticks were compared. An aliquot of 0.5 mg/ml of each extract was mixed with 200 μl internal standard (0.2 mg/ml heneicosanoic acid---HIA and betulinol---BET, both from Sigma-Aldrich, in methyl-tertiary-butyl-ether) and derivatized before subsequent separation according to the procedures found in Nisula ([@B59]), Willför et al. ([@B86]), and Zule et al. ([@B95]). Derivatization reagents were acquired from commercial sources and used as received. GC-MS and -FID experiments were performed on an Agilent 6890N/5973N-system (MS Consult, 2740 Skovlunde, Denmark), equipped with an S/SL inlet for sample introduction, using bleed and temperature optimized (BTO) high temperature septa and an Agilent Ultra Inert, split, low pressure drop liner with glass wool. The inlet was connected to the analytical column (HP-1, 25 m, 0.20 mm ID, 0.11 μm) by way of 5 m Agilent Ultimate Plus deactivated fused silica tubing (0.25 mm ID), used as a sacrificial pre-column. Eluents were split between the MSD for identification and the FID for quantification using an EPC pressure controlled CFT-splitter, sending 10% to the MSD and 90% to the FID. The original protocol for the GC-MS can be found in Örså and Holmbom ([@B61]). Before and after all runs, injections of neat derivatizing reagent were used to passivate the chromatographic system. The GC-Program was as follows: Injection volume 1 μl, split 10:1. Starting temperature 120°C, heating rate 6°C /min to 325°C, hold time 4 min. Flow rate 0.9 ml He/min, solvent delay 3 min, FID data were collected at 10 Hz. To estimate the linearity of the method over several orders of magnitude, a calibration row was run with one varying (HIA) and one constant standard (BET). The concentrations of HIA where 0, 0.005,0.025, 0.050, 0.502, 1.003, 3.010, and 5.017 mg/ml, and BET was constant at 0.55 mg/ml. The series was run once with all concentrations, and twice leaving out the 1 and 5 mg/ml samples. A linear regression based on the ratio of the two peaks was made for each of the runs (in Microsoft® Excel 2016), on a 95% confidence level. All three regressions gave an R^2^ \> 99.97%. The limits of detection (LOD) and of quantification (LOQ) were determined for each calibration row by means of the first 4 points (0--0.05 mg/g), using Equations (1) and (2), respectively. L O D = 3 S / b L O Q = 10 S / b where *S* is the standard deviation of the y-intercepts and *b* the slope of the corresponding regression. The average of the three determinations was used as the final value. The LOD was found to be 0.004 ± 0.001 mg/ml, corresponding to 0.005--0.008 mg/g dry wood (sample size 3--5 g). The LOQ was found to be 0.013 ± 0.004 mg/ml, which corresponds to 0.015--0.025 mg/g dry wood (sample size 3--5 g). 2.4. Sorption Isotherm of Kurile Larch -------------------------------------- Samples of Kurile larch were used to obtain the sorption isotherm, using two techniques: conditioning above saturated salt solutions (4 points, 64--95% RH, hygroscopic range) and the pressure plate technique (3 points, 99.64--99.99%, over-hygroscopic range). An overview of the different RH-levels and techniques used is given in [Table 1](#T1){ref-type="table"}. ###### Measurement techniques and respective conditions for the determination of the sorption isotherm of Kurile larch. **Method** **Salt used** **Relative humidity** **Pressure** **Water potential** **Calculation of** ------------------------- --------------- ----------------------- -------------- --------------------- ----------------------------------------------------------------------------------- Saturated salt solution NH~4~NO~3~ 64.1 42.01 -60364 Equation 30 in Mozurkewich ([@B57]) NaCl 75.5 26.6 -38172 Linear interpolation of RH values at 20 and 25°C in Table 2 of Greenspan ([@B37]) KCl 85.0 15.3 -21972 KNO~3~ 94.5 5.3 -7643 Pressure plate n.a. 99.67 4.44\* -44.0 Rearrangement of Equation 1 in Fredriksson and Johansson ([@B29]) n.a. 99.89 1.46\* -15.0 n.a. 99.97 0.45\* -5 *All the RH values shown for the salt solutions were calculated as detailed in the table, and the pressure as well as the water potential were determined from these. For the pressure plate method, the pressure is the pressure that was applied in the experiment (marked with \*), which was then used to calculate the corresponding RH and water potential as explained in the main text*. The TOT and NAT samples of larch 1 were used to obtain the respective absorption and desorption isotherms, in both the hygroscopic and over-hygroscopic moisture ranges, covering a total of 7 points. Thus, 7 sticks with different EW-LW patterns from the NAT and TOT groups were used. However, due to limited sample availability, sorption isotherms for the PHO and PHI samples were only determined in desorption and in the in the over-hygroscopic range. Three sticks of each treatment were used. All the sticks were labeled and cut into 5--7 equally sized pieces (≈ 0.7 × 0.3 × 0.4 mm, L × W × B). Each piece of the same stick was assigned to a different RH-level (7 for NAT and Tot, 3 for PHO and PHI). Because we suspected that the sticks were not equally well-extracted in the central part as on the borders, the pieces were distributed over the levels so that one level did not for instance contain solely center pieces. For the absorption isotherm, the samples were dried, put into individual open Eppendorf tubes in a vacuum oven at 60°C for 24 h. The oven was then allowed to cool under reduced pressure and the cooling of the samples was finalized in a vacuum desiccator. Freshly dried molecular sieves were added to each of the tubes for storage until weighing. For determination of desorption isotherms, the samples were initially water saturated. First, they were placed in round bottom flasks RH-level-wise and set under vacuum for 15 min. Degassed Milli-Q water was added to each flask using a syringe, followed by another minute of degassing with the vacuum pump. Then the samples were allowed to stand under reduced pressure for 1h, after which atmospheric pressure was re-established and they were stored in water until weighing. Due to this water saturation step, we expected that the hydrophobic extracted pieces were extracted to a certain degree---at the very least the arabinogalactan must have been affected. Weighing before conditioning: For the water-saturated samples, the surface water of the samples was removed by rolling each piece over a wet cellulose based cloth (Wettex, Vileda, Freudenberg home & cleaning, solutions, AB, Malmö). Then, the piece was quickly placed on the balance and the mass recorded with a resolution of 0.01 mg. The dry samples were weighed inside of a tared weighing glass filled with dry molecular sieves. For the hygroscopic range, saturated salt solutions according to [Table 1](#T1){ref-type="table"} were placed in small climate boxes, equipped with RH sensors. Once the RH stabilized, the samples were introduced. The relative humidities generated by the different salt solutions were determined as detailed in [Table 1](#T1){ref-type="table"}. The pressure plate technique, which gives information on the relation between the water potential and the moisture content of the material (Defo et al., [@B17]), was used to determine sorption isotherms in the over-hygroscopic range. In this study, a custom-built pressure plate system was used (Fredriksson and Thybring, [@B30]) where specimens were conditioned in the range 0.4--4.4 bar, corresponding roughly to 99--100% RH (see [Table 1](#T1){ref-type="table"}). The experimental procedure as described by Fredriksson and Thybring ([@B30]) was used. All the samples were kept in the climate boxes/pressure plate cells for a period of 2 months at 20.5 ± 0.3°C. The conversion from relative humidities to pressure (salt solutions) and vice versa (pressure plate) was done by rearrangement of Equation (1) in Fredriksson and Johansson ([@B29]). The water potential was calculated from the relative humidities by Equation (6) in Cloutier and Fortin ([@B16]). Weighing after treatment: A glove box containing an analytical balance (resolution 0.01 mg) was used. For each of the humidity levels, the RH in the glove box was adjusted accordingly using a humidity generator (2500 Humidity Generator, Thunder Scientific Corporation, Albuquerque, New Mexico, USA). For the highest levels wet cloths were additionally placed in the glove box. Finally, the desorption samples were dried and weighed as described above. The final sample size after all procedures was 4--7 replicates per group. To enable comparison between unextracted and differently extracted samples, an adjusted moisture content, *u* (g/g), was determined relative to the unextracted weight, as shown in Equation (3) below: u = ( m w / m dry ) \* ( 1 \+ \[ \% e x t r a c t i v e s \] ) where m~w~ (g) is the mass of water in the specimen and m~dry~ (g) is the dry mass of the specimen, which for the extracted material was the dry mass after extraction and \[% extractives\] corresponds to the amount of gravimetrically determined extractives in percent; i.e., for the NAT samples, this term is zero. 2.5. Fungal Degradation ----------------------- Cultures of European *R. Placenta*, European strain FPRL280, were cultivated on malt agar and stored at 4°C for 2 weeks. Mycelial flocks were taken from these cultures for the experiments below. To get a view on a more advanced state of degradation and to test for the virulence of the fungus an agar-block test was made. Six sterilized NAT sticks of spruce 2 and larch 2 were horizontally placed two by two on plastic grids in agar-filled petri dishes and incubated at 23°C and 70% RH. After 8 weeks, the samples were harvested, the mycelium removed, and the wet as well as dry weight determined. With a very simplified version of a pole in ground-contact, we chose a modified soil-block test inspired by Zhang et al. ([@B94]) to assess the impact of extractives on the initial phase of degradation. The fungus was allowed to grow bottom-up in the longitudinal direction of the wood for 2 weeks as shown in [Figure 2](#F2){ref-type="fig"}. Nine sticks from each of the different extraction treatments, and native controls were marked at 2/3 of the height. The dry weight was determined, and all sticks were equilibrated at 23°C and 70% RH for 5 weeks prior to sterilization by autoclaving and inoculation. Larch 2 was additionally autoclaved before equilibration, due to suspicion of mold. The sticks were placed vertically on pre-inoculated pine feeder strips on an autoclaved soil mixture. All glasses were incubated until the hyphal front (HF) reached the marked threshold (33.3 mm). The average height of the HF in spruce was 35.5 ± 4.5 mm and was reached within 14--18 days. For larch the average HF height was 39.8 ± 5.7 mm and was reached in 15 days. Because of the geometry of the sample, there is no guarantee the hyphal front inside of the sticks reached the same height as on the outside, and thus we consider the weight loss the appropriate measure to describe the degradation. Statistical evidence for this can be found in the [Supplementary Information](#SM1){ref-type="supplementary-material"}. ![Sample setup for directional growth of *R. placenta*, inspired by Zhang et al. ([@B94]). **(A)** Kurile larch before degradation. **(B)** Norway spruce with hyphal front after degradation.](fpls-11-00855-g0002){#F2} After harvest, the samples were weighed immediately, stored at −25°C until all the samples had been harvested, and then dried for weight loss determination. 2.6. Data Analysis ------------------ ### 2.6.1. Gravimetric Yields The gravimetric yields were analyzed in OriginPro® 2017 (OriginLab Corporation, Northamtom, MA 01060, USA, [www.OriginLab.com](https://www.OriginLab.com)). The standard deviations of sums and quotients of variables were calculated according to formulas taking into account statistical error propagation, i.e., formula (1) and (2) in Andraos ([@B1]). ### 2.6.2. Chromatograms The mass spectral data were used to identify the peaks in the chromatograms by using a mixture of the NIST database and the database created at the Laboratory of Wood and Paper Chemistry, Åbo Akademi University. The corresponding retention times (RT) were taken from the total ion current chromatograms. For quantification of all the extracts, the peak areas of the FID-chromatogram were obtained using MSD Enhanced ChemStation^©^ (Agilent Technologies Inc., CA 95051, USA, [www.agilent.com](https://www.agilent.com)) and the amounts calculated based on the area of the internal standard peaks. Fatty acids/alcohols, resin acids, other diterpenoids, flavonoids, carbohydrates, and unknown compounds were quantified using the HIA standard, while lignans and sterols where quantified using betulinol. A correction factor of 1.2 was used for lignans, as recommended elsewhere (Willför et al., [@B85]; Zule et al., [@B95]; Nisula, [@B59]; Zule et al., [@B96], a.o.). Due to the high split ratio and higher detection rate of the FID, these spectra show more resolved peaks as well as many small peaks not seen with the MS detector. The latter are quantified within the unknown (UNK) class, together with peaks that could not be identified with MS. Fatty alcohols were pooled with the fatty acids, but resin acids in different oxidation states were classified as "other diterpenoids." Due to variations in peak intensity, some peaks below the LOD were actually detected, but disregarded from analysis. Analytes detected below the LOQ, where nevertheless included in the total sums of each chemical class. ### 2.6.3. Estimation of Sticks Composition Under the assumption that similar proportions of extractives were removed in sticks as in milled material, we estimated the amount of each chemical group (*i*) left in the sticks, as compared to the milled TOT samples (*f*~i,\ sticks~, see Equation 4). This was achieved by weighing the chromatographic yields (*y*) of each milled solvent fraction *(j*) and chemical group (*i*) with the respective extraction efficiency (*EF*). Then each chemical group was summed for each procedure and divided by the sum of yields of the milled TOT samples. For the PHO samples, the chromatographic yields obtained from the respective milled TOT ethanol fractions were added. The average of 2 trees is reported. f i , s t i c k s = ∑ j = 1 N y i , j × ( 1 \- E F j ) / ∑ y i , j Where *N* is the solvents relevant for the respective treatment. We have also added the remaining ArGal present in larch, which was directly calculated from the gravimetric yields, as (1-^m~sticks~^ / ~m~milled~~). ### 2.6.4. Sorption Isotherm The data were analyzed in OriginPro® 2017. Absorption and desorption data were tested individually. First a test for normality (Shapiro-Wilk) was performed for each extraction treatment and humidity level tested. For the desorption samples, all groups were normally distributed, except the groups NAT at 94% and TOT at 74% RH, hence these were excluded from follow up analysis. Among the absorption data, only the NAT at 94% group was not normally distributed, and thus excluded from further statistical analysis. A 2-way ANOVA was performed on the rest of the desorption and absorption data, separately. The extraction treatment and the RH were used as factors, as well as the interaction terms. A power analysis was added, to test for Type II errors. ### 2.6.5. Fungal Degradation The statistical analysis of the fungal degradation was done in OriginPro® 2017. The variation in growth height and sampling days was tested using ANOVA as detailed in the [Supplementary Information](#SM1){ref-type="supplementary-material"}. It showed that WL was a suitable factor to describe the decay resistance. Normality tests showed that weight loss in both species had to be log transformed for further analysis. The equality of variance was tested before the ANOVA, showing equal variance for spruce (*p* \< 0.05, α = 0.05), and unequal variance for larch (*p* \< 0.02, α 0.05). The normalized data were subjected to 2-way-ANOVA for each species separately, testing for tree, treatment and interaction terms. Tukey\'s test for comparison of means and power analysis were used *post-hoc*. 3. Results {#s3} ========== 3.1. Milled Samples: Extract Yields and Composition --------------------------------------------------- [Figure 3](#F3){ref-type="fig"} shows the average gravimetric yields of the different extraction strategies of milled spruce and larch samples. The heptane and DCM fractions of the total extracted samples serve as the references for the milled PHO samples, which are therefore not shown separately. From total extraction ([Figure 3A](#F3){ref-type="fig"}) it is evident that the amounts of hydrophobic extracts were very similar for both species, with 7--8 mg/g dry wood for heptane and 2--4 mg/g dry wood for DCM. In spruce, the yields obtained for both hydrophilic fractions were at similar levels as the heptane extracts (≈ 10 mg/g dry wood). In larch on the other hand, ethanol yielded about 5x more material on average, and the water fraction even more with about 9x higher yields, making up about 50--60% of the total sum of all solvent fractions (see also [Table S1](#SM1){ref-type="supplementary-material"}). Substantial yield differences between the two larch trees were found, reflected by the larger standard deviations (error bars in [Figure 3](#F3){ref-type="fig"}). {#F3} When ethanol was used as the first solvent ([Figure 3B](#F3){ref-type="fig"}), the gravimetric yields where higher than when ethanol was the third solvent. The spruce yields were slightly lower than for the sum of heptane, DCM and EtOH fractions of the TOT extraction. The average ethanol yield of the two larch trees was again much higher than for spruce, and comparable to the summed yields of the first three TOT solvents. For both species, the water fractions yielded about the same amounts as for the TOT extraction. Upon the addition of cold ethanol to the water fractions of spruce, only very small amounts of material precipitated and could not be reliably quantified. In the case of larch, addition of cold ethanol resulted in large amounts of precipitate, known to be arabinogalactan (Côté et al., [@B13]; Luostarinen and Heräjärvi, [@B51]). Nearly all the yield of these water fractions was composed of the polysaccharide. For larch 1, the average yield of dry precipitate for the milled TOT and PHI extractions was 58.6 ± 2.7 mg/g. Reflecting the higher water extract yields, the ArGal precipitate of larch 2 amounted to 95.6 ± 3.8 mg/g on average. To test whether the extraction was complete after the use of four different solvents, we added a fifth extraction step for the milled TOT samples. Using an acetone:water mixture (95:5), we found that in both species, 2--3 mg/g of additional material could be extracted. In the following, the composition of the heptane and ethanol extracts of the total extraction (as assessed by GC-MS) will be described first, because the other fractions were mixtures of the latter. The composition of the additional replicates, where a control extraction procedure (heptane and acetone) was used, can be viewed in [Table S2](#SM1){ref-type="supplementary-material"}. As detailed further in the [Supplementary Information](#SM1){ref-type="supplementary-material"}, the summed yields of the individual extractive groups agreed well with the quantities obtained with the four step extraction procedure. ### 3.1.1. Hydrophobic Extracts - Heptane As shown in [Figures 4A,B](#F4){ref-type="fig"}, the hydrophobic heptane extracts of both species contained fatty acids and alcohols (FAs), resin acids (RAs), other diterpenoids (DTs), and sterols (STs), but their proportions, as well as the number of detected analytes differed. The numbers above each column in [Figure 4](#F4){ref-type="fig"} show the total chomatographic yield of the respective fraction in mg/g dry wood. The corresponding chromatograms can be viewed in the [Figures S2](#SM1){ref-type="supplementary-material"}, [S4](#SM1){ref-type="supplementary-material"}. The relative proportions of FAs and DTs were about 2 times higher in larch (20 and 22%, resp.) than in spruce (13 and 8%, resp.). On the other hand, spruce had 10 times higher proportions of STs (around 15%) and 3 times higher contribution from unidentified compounds (UNK, 30%). Only resin acids were found in similar proportions in both species, and where the most abundant group of hydrophobic analytes, amounting to around 40% of detected analytes. Please view [Table 2](#T2){ref-type="table"} for a summary of the compounds detected using four solvents. {#F4} ###### Analytes detected in each solvent fraction of Norway spruce and Kurile larch, grouped by chemical class and the class sum (*n* = 2), as compared to the literature. **Chemical group** ***P. abies*** ***L. gmelinii var. japonica*** -------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **Fatty acids /alcohols (FA), free** Total: 0.20--0.36 mg/g **Palimitic acid** (16:0), margaric acid (17:0), **pinoleic acid** (5,9,12--18:3), **linoleic acid** (9,12--18:2), **oleic acid** (9--18:1), behenyl alcohol (22:0), linoceryl alcohol (24:0) 0.6--3.4 mg/g (Willför et al., [@B85]) 0.25--1.4 mg/g depending on latitude, detected more types (Nisula, [@B59]) Total: 0.63--0.67 mg/g palmitic acid, margaric acid, **Pinoleic acid**, **linoleic acid**, **oleic acid**, sciadonic acid (5,11,14--20:3) 1.2--3.2 mg/g, found more types (Nisula, [@B59]) 0.86-1.1 mg/g in *L. decidua* (Zule et al., [@B95]) **Resin acids (RA)** Total: 0.55--1.00 mg/g levopimaric acid, pimaric acid, sandarapimaric acid, isopimaric acid, **palustric acid**, **dehydroabietic acid**, abietic acid 0.48--1.5 mg/g (Nisula, [@B59]) 0.8--2.4 mg/g (Willför et al., [@B85]) Total: 1.17--1.38 mg/g levopimaric acid, communic acid, **isopimaric acid**, **palustric acid**, dehydroabietic acid, abietic acid, neoabietic acid 0.47--1.7 mg/g (Nisula, [@B59]) 4.6--7.3 mg/g (RA+DT) in *L. decidua* (Zule et al., [@B95]) **Other diterpenoids (DT), free** Total: 0.09--0.26 mg/g Thunbergol, neoabienol, only S1: palustral^\*^, palustrol^\*^ Present (Nisula, [@B59]) 0.1--1.3 mg/g (Willför et al., [@B85])^\*^ RA precursors (Higuchi, [@B41]; Keeling and Bohlmann, [@B48]), were not reported in (Willför et al., [@B85]; Nisula, [@B59]) Total: 0.60--0.75 mg/g Thunbergol, manool (1 and 2), palustral^\*^, (iso)pimarol^\*^, neoabietol^\*^, **cedrol** Thunbergol - present, manool - 0.07--0.38 mg/g (Nisula, [@B59]) cedrol has not been reported for *Larix gmelini var. japonica* nor any other larch species^\*^ RA precursors (Higuchi, [@B41]; Keeling and Bohlmann, [@B48]) (Higuchi p 258) **Sterols (ST), free** Total: 0.26--0.33 mg/g campesterol, **sitosterol**, sitostanol 0.24--0.36 mg/g (Nisula, [@B59]) 0.2--0.3 mg/g (Willför et al., [@B85]) Total: 0.05--0.06 mg/g campesterol, **sitosterol** 0.12--0.14 mg/g (Nisula, [@B59]) 0.14--0.18 mg/g in *L. decidua* (Zule et al., [@B95]) Cycloartenol was additionally found in these references (Zule et al., [@B95]; Nisula, [@B59]) **Lignans (LI), monomeric** Total: 4.24--5.03 mg/g Dihydroxymatairesinol, 7r-todolactol, iso-lariciresionol, todolactol A, alpha-conidendric acid, lignan a, lignan b, matairesinol, hydroxymatairesinol 1, **hydroxymatairesinol 2**, mixed lignan signal, lariciresinol, iso-hydroxymatairesinol 0.87--15 mg/g (Nisula, [@B59]) 0.0--2.5 mg/g (Willför et al., [@B85]) secoisolariciresinol, pinoresinol and nortrachelogenin were not detected in our case (Willför et al., [@B85]; Nisula, [@B59]) Detected, but not confirmed: isoliovil and dihydroxymatairesinol 0.2--1 mg/g (Nisula, [@B59]) trace amounts (Zule et al., [@B96]) **Flavonoids (FL)** Detected, but not confirmed: naringenin, taxifolin Trace amounts of dihydrokaempferol, taxifolin (Willför et al., [@B88]) Total: 18.45-35.73 mg/g naringenin, dihydrokaempferol, taxifolin 1, taxifolin 2 0.49-5.4 mg/g (Nisula, [@B59]) 15-36 mg/g in *L. decidua* (Nisula, [@B59]) 10-15 mg/g (FL + LI) in *L. decidua* (Zule et al., [@B96]) *See [Table S2](#SM1){ref-type="supplementary-material"} for the quantities obtained by the 2-solvent sequence. Fatty acids are given by their common names, the numbers in brackets show (position of double bond-number of carbons:number of double bonds)*. Structures of the most abundant and/or relevant analytes are shown in [Figure S1](#SM1){ref-type="supplementary-material"}. ### 3.1.2. Hydrophilic Extracts - Ethanol Compared to the heptane extracts, the composition of the ethanol extracts was less complex in terms of the number of different chemical classes detected ([Figures 4A,B](#F4){ref-type="fig"}). The chromatograms can be found in [Figures S2](#SM1){ref-type="supplementary-material"}, [S4](#SM1){ref-type="supplementary-material"}. The spruce ethanol extracts of the TOT extraction were largely dominated by lignans (around 90%; [Figure 4A](#F4){ref-type="fig"}), while the dominant phenolics in larch were flavonoids (also around 90%; [Figure 4B](#F4){ref-type="fig"}). It may also be noteworthy that in both species the main phenolic compounds (bold in [Table 2](#T2){ref-type="table"}) were present at much greater concentrations than the other compounds, which was not the case for the hydrophobic analytes. In spruce, the difference was about 9-fold, while in larch it was much greater with over 50-fold. Note also, that the absolute amounts we found for the larch flavonoids are comparable to *L. decidua*, rather then *L. gmelinii var. japonica* as reported by Nisula ([@B59]). Additionally, larch 2 had only one largely dominating flavonoid (90% of detected flavonoids)---taxifolin (TAX), while larch 1 had dihydrokaempferol (DHK) and taxifolin at an almost 50:50 ratio, DHK being slightly higher. The total ethanol yield was also higher for larch 2 ([Figure S4](#SM1){ref-type="supplementary-material"}). In both species, low amounts of monomeric carbohydrates and lignin components were also detected (≈ 3%), and ≈ 9% of the ethanol extracts were unknown compounds. ### 3.1.3. Other Solvents and Solvent Sequences In spruce, the low-yielding DMC fraction was composed of lignans to 82--88%, while 77--80% of the corresponding larch extracts consisted of flavonoids ([Figures 4A,B](#F4){ref-type="fig"}). The rest was composed of mainly unknowns (8--12%) and residual FAs, RAs, DTs, and STs, as well as 2--4% carbohydrates and lignin monomers. The chromatograms are shown in [Figures S2](#SM1){ref-type="supplementary-material"}, [S4](#SM1){ref-type="supplementary-material"}. [Figure 4C](#F4){ref-type="fig"} shows the composition of the extracts obtained from the hydrophilic extraction procedure in spruce and larch was not specific for the hydrophilic compounds only. The PHI ethanol extracts of spruce ([Figure 4C](#F4){ref-type="fig"} and [Figure S3](#SM1){ref-type="supplementary-material"}) resulted in a mixture of the analytes found in the heptane and ethanol fractions discussed above. Thus, this extract gives an overview of the proportions of GC-detectable extractives, that is 15--20% hydrophobic analytes (FAs, RAs, DTs, and STs) and around 70--75% of lignans. The rest was small amounts of carbohydrates/lignin components and unknown compounds. Similarly, the PHI ethanol extracts of milled larch were also a mixture of the heptane and ethanol fractions ([Figure 4C](#F4){ref-type="fig"} and [Figure S5](#SM1){ref-type="supplementary-material"}). The hydrophobic analytes amounted to 4--7% of this extract, and flavonoids to 81--86% and the rest was again carbohydrates/lignin components and unknown analytes. The water fraction of spruce did not show any peaks in the chromatograms, thus, no information on their composition is available (see the chromatogram in [Figure S2](#SM1){ref-type="supplementary-material"}). In the case of larch, removal of ArGal together with a dehydration step of the remaining supernatant, enabled the now alcoholic fraction to be successfully run on the GC. For the milled specimen of larch 1, ([Figure 4C](#F4){ref-type="fig"}), flavonoids accounted for 55% of the yield, and carbohydrates for about 30%. ### 3.1.4. Differences in Gravimetric and Chromatographic Yields We compared the gravimetric and chromatographic yields of all milled samples in [Figure 5](#F5){ref-type="fig"} and found that the GC-detectable fraction varies between solvents and species. Only about 30% of the less polar spruce extracts were reliably detected with GC-FID (including unknown peaks), while up to 38% of the ethanol fractions (TOT and PHI) were detected as compared to the gravimetric yields. In case of larch, 40--50% of the heptane and DCM fractions were detected, and around 65% of the ethanol fractions (TOT and PHI). {#F5} 3.2. Composition of the Sticks ------------------------------ The gravimetric yields obtained for the sticks are listed in [Table S1](#SM1){ref-type="supplementary-material"}. The resulting extraction efficiency of the sticks compared to the milled material is shown in [Table 3](#T3){ref-type="table"}. Note that the composition of the milled TOT heptane and DCM fractions were used for the calculation of the extraction efficiency of the PHO sticks, because we considered them representative for both groups. We found that the extraction efficiency was similar in spruce and larch. With some exceptions, the sticks yielded 50--90% for the heptane, DCM and water fractions relative to the milled material. With ethanol maximally 30--60% could be extracted from the sticks. ###### Average extraction efficiency of the sticks that were used in the sorption isotherm and fungal degradation experiments. **Tree ID** **Stick/milled** **Heptane** **DCM** **EtOH** **H~2~O** ------------- ------------------ ------------- --------- ---------- ----------- S1 TOT-s/TOT-m 0.4 0.3 0.4 n.a. PHO-s/TOT-m 0.5 0.5 − − PHI-s/PHI-m − − 0.4 0.9 S2 TOT-s/TOT-m 0.8 0.5 0.6 1.1 PHO-s/TOT-m 0.6 0.6 − − PHI-s/PHI-m − − 0.4 1.2 L1 TOT-s/TOT-m 0.6 0.6 0.3 0.5 PHO-s/TOT-m 0.5 1.2 − − PHI-s/PHI-m − − 0.3 0.6 L2 TOT-s/TOT-m 0.8 1.0 0.2 n.a. PHO-s/TOT-m 0.6 0.5 − − PHI-s/PHI-m − − 0.2 0.5 *The extraction efficiency was calculated as the ratio between the average gravimetric yield of sticks and associated milled sample. The yields of heptane and DCM fraction of the TOT extraction were considered representative for the respective fractions of the PHO procedure, and the calculations therefore based on the TOT yields. DCM, dichloromethane; EtOH, ethanol; S, spruce; L, larch; 1/2, tree 1 or 2; s, stick; m, milled; TOT, total extracted; PHO, hydrophobic extracted; PHI, hydrophilic extracted*. Precipitation of ArGal from the larch water fractions of TOT and PHI sticks yielded about 40% of what the corresponding milled specimen yielded (not shown in [Table 3](#T3){ref-type="table"}). The chromatograms of the remaining dehydrated water extract of larch PHI sticks revealed that the composition was not the same as for the milled counterpart, as shown in [Figure 4C](#F4){ref-type="fig"}. Flavonoids and carbohydrates were identified in both samples, but in the sticks\' extract the amount of flavonoids was much higher than for milled specimen---in absolute and relative amounts---making up 80% of the yield (≈ 5 mg/g). Despite uncertainties, we tentatively estimated the residual amounts of different extractive groups in the sticks after the different extraction procedures ([Figure 6](#F6){ref-type="fig"}), using Equation (4). The milled TOT samples were used as a reference, because we consider it the most complete extraction. Residual ArGal was estimated based on the gravimetric yields of the precipitates. {#F6} The levels of retained hydrophobic extractives was slightly higher for the PHO extractions than for the TOT extraction in both species, even though a contribution from ethanol in the TOT procedure can be excluded, as no hydrophobic analytes were detected in this fraction. Differences may arise from experimental variations, such as the moisture content of the material upon starting the extraction. On the other hand, total extraction removed lignans (LI, SL) more efficiently in spruce (approx. 50% left), than flavonoids in larch (approx. 70% left). One reason could be that DCM extracted about 20% of the total monomeric lignans in spruce, while flavonoids in larch seem to be more polar, because DCM removed only about 1--4 % of total flavonoids. The ethanol step of the PHI extraction removed a significant portion of the rather hydrophobic extractives (FA, RA, DT, ST), although lower amounts as compared the other extraction procedures in both species. The extraction efficiency of ethanol as the first solvent (PHI) was lower in larch than in spruce. A contributing factor here could be the presence of ArGal in larch, which is highly insoluble in alcohol, and while occupying the lumina of many tracheid cells, may reduce the flux of solvent during extraction. More than half of the polysaccharide remained in the TOT and the PHI groups ([Figure 6B](#F6){ref-type="fig"}), although it is likely that the water extraction step relocated parts of it. As water was not used in the PHO group, we consider the relocation issue to be smaller for these samples. Nevertheless, solvent flux during extraction might be different in spruce than in larch. The metabolic profiles described above can be summarized as follows: While the TOT samples contain the lowest amount of extractives in all categories, the PHO samples contain the highest amounts of hydrophilic extractives, including most unidentified compounds and large amounts of ArGal in larch. The PHI extraction resulted in samples having a higher proportion of hydrophobic material compared to the TOT group, but also higher amounts of hydrophilic compounds. It should thus be considered the least efficient extraction, especially in larch. A likely consequence of the incomplete extraction of the sticks is a relocation of extractives to areas where they might not be present in the native wood, which might influence their mode of action and/or efficacy. 3.3. Sorption Isotherm of *Larix gmelinii var. japonica* -------------------------------------------------------- The sorption isotherm for Kurile larch is shown in [Figure 7A](#F7){ref-type="fig"} for native and total extracted wood in the range of 64--100% RH. The MC of the TOT samples was generally slightly higher than for NAT in both absorption and desorption. The adjusted MC of the TOT samples deviated more from the NAT samples in the range of 94.5--99.89% RH in desorption mode and up to 99.97% in absorption mode. Above and below that, the curves coincide ([Figure 7A](#F7){ref-type="fig"}). Nevertheless, 2-way ANOVA showed that this difference was only significant in absorption mode (*p* = 0.028, α = 0.05, power = 0.59) in the RH range of 64--99.97%. Also, as expected, the differences in MC at different RH-levels was significant (*p* = 0, α = 0.05). {#F7} [Figure 7B](#F7){ref-type="fig"} shows the desorption isotherm in the over-hygroscopic range for native, totally extracted, hydrophobic and hydrophilic extracted larch. Although NAT had the lowest MC compared to the extracted samples, none of these differences were significant (*p* \> 0.05, α = 0.05). Again, the difference in MC was different between the RH-levels as expected (*p* = 0, α = 0.05). 3.4. Fungal Degradation ----------------------- The 8 week degradation test of native Norway spruce 2 and native Kurile larch 2 with *R. placenta* resulted in an average weight loss of 30.0 ± 12% for spruce (*n* = 5), while it was about half for larch, with 14.8 ± 6 % (*n* = 6). This shows that, as expected, Kurile larch was more resistant to degradation by this brown-rot fungus. A similar pattern was found for the 2 weeks degradation test, after which a small weight loss could already be observed. For every treatment group, spruce lost close to twice the amount of mass compared to larch, except for the hydrophilic extraction, where the weight loss was almost identical for both species. The average weight loss percentage of each species and treatment is shown in [Table 4](#T4){ref-type="table"}. ###### Average weight loss percentage and associated standard deviations of sticks of two clones of Norway spruce and Kurile Larch after different extraction treatments. **Treatment** **Spruce 1^A^** **Spruce 2^A^** **Average spruce** **Larch 1^D^** **Larch 2^E^** **Average larch** --------------- ------------------ ------------------ -------------------- ------------------ ------------------ ------------------- Native 3.7 ± 1.2^a,\ b^ 3.3 ± 0.8^a^ 3.5 ± 1.0^a^ 2.5 ± 1.5^d^ 1.2 ± 0.3^d^ 1.9 ± 0.9^d^ Total 5.3 ± 1.5^a,\ c^ 5.1 ± 1.3^b^ 5.2 ± 1.4^b^ 3.2 ± 1.0^d,\ f^ 2.8 ± 0.8^e^ 3.0 ± 0.9^e^ Hydrophobic 6.0 ± 1.9^c^ 4.2 ± 0.5^a,\ b^ 5.1 ± 1.2^b,\ c^ 3.8 ± 1.0^e,\ f^ 1.8 ± 0.4^d,\ e^ 2.8 ± 0.7^e^ Hydrophilic 3.6 ± 1.1^a,\ b^ 4.6 ± 1.7^a,b^ 4.1 ± 1.4^a,\ c^ 4.3 ± 1.2^e,\ f^ 2.6 ± 0.4^e^ 3.4 ± 0.8^e^ *For each treatment n = 18, except native of trees with index 1, where n=17. Statistically significant differences on a 95% confidence level are denoted with different letters pairs, all at p ≪ 0.001. Uppercase letters are used for the average trees (column-wise), lowercase for the treatments (row-wise); Letters "a-c" were used for spruce, "d-f" for larch*. The average weight loss of spruce was between 3.5 and 5% over all groups. The 2-way ANOVA performed on the spruce data showed that the trees were not significantly different from each other (*p* \> 0.05, α = 0.05, power = 0.15), but that the treatments were different (*p* ≪ 0.001, α = 0.05, power = 0.99), but also that the trees were differently affected by the treatments (interaction terms, *p* = 0.046, α = 0.05, power = 0.94). Tukey\'s test showed that the TOT and the PHO groups were more affected than the NAT group, and that the TOT group was also different from the PHI group. For the interaction terms, the same was found for spruce 1, but in the case of spruce 2 only the TOT group was different from the NAT group. For Kurile larch, the range of weight loss was between 1.8 and 3% over all groups. The ANOVA showed that the trees were significantly different from each other (*p* ≪ 0.001, α = 0.05, power = 0.999), which can be seen from the individual weight loss of the trees, were larch 2 lost about half the weight compared to larch 1 in all treatment groups ([Table 4](#T4){ref-type="table"}). In fact, the weight loss recorded for larch 1 was much more comparable to the weight loss of the spruce specimen, despite having much higher phenolics content. The treatments affected the weight loss of larch differently (*p* ≪ 0.001, α = 0.05, power = 0.999) and also the trees were affected differently by different treatments (interaction terms, *p* = 0.03, α = 0.05, power = 0.96). Tukey\'s test revealed that the WL after all extraction procedures were different from the native group, but not from each other. For the interactions, we found that in both trees the WL of the PHI group was different from the NAT group. However, in larch 1, only the weight loss of the PHO group was higher than the NAT group, while in larch 2 the weight loss of the PHI group was higher than for NAT. We also looked at the moisture content, calculated relative to non-degraded mass of the samples. After 8 weeks of degradation the reduced moisture content was the same for native spruce (47 ± 13%) and native larch (43 ± 9%). A similar relation is found for samples after 2 weeks of exposure to *R. placenta*, although the MC was lower. The reduced MC of 35% was again the same for all four trees, irrespective of the extraction procedure or weight loss. 4. Discussion {#s4} ============= 4.1. Extraction and Analysis ---------------------------- Before any further conclusions are drawn from the collected data, a few methodological issues should be acknowledged. The performance of a living tree is yet different than what we can observe here, as we removed the majority of volatile monoterpenes during drying, which are known to play an important role in defense (Schaller, [@B69], p. 164). Furthermore, despite having increased the number of extraction cycles to a maximum, the yields of the sticks were much lower than for the milled counterparts. It is questionable whether an increase in cycle duration would have significantly increased the yields further. We also found differences in the composition of the water extracts of larch 1 sticks and milled specimen. Similarly, Ekeberg et al. ([@B22]) compared the yields and content of acetone-extracted powders and blocks of *Pinus sylvestris L*. and found that i.e., fatty acids were extracted more efficiently from blocks than resin acids or stilbenes. As we did not run the sticks\' extracts on the GC, we can base our discussion only on the estimated composition of the sticks ([Figure 6](#F6){ref-type="fig"}). ### 4.1.1. Gas Chromatography and Polymers We obtained a detailed view on the monomeric composition of our extracts, but noticed that, depending on the solvent, up to 70% of the gravimetrically determined mass, was not detected with the GC-MS/FID method used in this work. There can be various (cumulative) reasons for this, which will be shortly outlined below: Low-molecular-weight compounds, especially many of the acidic extractives, are known to form esters with different alcohols, such as glycerol, sterols and other diterpenyl and aliphatic alcohols, as well as with various carbohydrates (Fengel and Wegener, [@B28]; Rowe, [@B67]; Otto and Wilde, [@B63]; Willför et al., [@B85]). As a consequence, some compounds do not have sufficient volatility to reach the detector with the column used in this work (Zule et al., [@B95]). The most significant contribution is certainly the presence of polymers, the molecular weight of which is too high for regular gas chromatography. Polymerization reactions may also occur during extraction and work-up (Holmbom, [@B44], i.e., p.128), and thus introduce artifacts. Our extracts were analyzed fresh, but we saw some precipitates in the acetone extracts of both species as the samples aged. Detailed information, as well as respective countermeasures can be found in the literature (i.e., Willför et al., [@B91]). Nevertheless, most of the mass not detected with the GC-MS method used int his work is expected to be from natural polymers. The rather hydrophilic lignans and flavonoids (Willför et al., [@B89], [@B91]; Fedorova et al., [@B27]), and more recently, also the rather hydrophobic resin acids and fatty acids (Smeds et al., [@B76], [@B77]) have been shown to be present as oligomers and polymers. In *Picea obovata Ledeb*. from Siberia, oligomeric lignans were found to constitute up to 40% of ethyl acetate extracts (Fedorova et al., [@B27]). Several studies on spruce knotwood found that up to 70% of the ethanolic extracts were polymeric lignans (Willför et al., [@B85], [@B89]), but also polymerized fatty acids, resin acids and other diterpenoids (Smeds et al., [@B76]). This agrees surprisingly well with our findings, since only 30% of the spruce extracts could be quantified by GC-FID. Therefore we suggest, that there may be similar proportions of polymeric material in the stemwood as in the knotwood of spruce, although with much higher abundance in knots (Willför et al., [@B85]). Similarly in larch, Ostroukhova et al. ([@B62]) mention a publication where the resin of Siberian larch should contain 30% of a polymer, consisting of taxifolin subunits. This again corresponds rather well with the amount of substance we were able to quantify from the ethanol/acetone extracts using gas chromatography. Investigations of pine knotwood determined large amounts (\> 50%) of polymerized fatty acids, resin acids and other diterpenoids (Smeds et al., [@B77]). Although, to our knowledge, such studies have not been conducted for larch, and only in knotwood of pine (Smeds et al., [@B77]) and spruce (Smeds et al., [@B76]), it may very well be that it is a property of resin in general to form polymers of its constituents. Thus, it could explain the difference in gravimetric and chromatographic yields of the heptane fractions of spruce, as well as larch. There are many techniques that allow identification and quantification of the polymeric fractions (Holmbom, [@B44]; Willför et al., [@B85], [@B91]; Fang et al., [@B26]; Smeds et al., [@B76]; Zule et al., [@B96]). However, none of these options were used in the present study. ### 4.1.2. Extractives in Spruce and Larch Our Norway spruce specimens showed fewer analytes in some of the extractive groups than reported in literature, and slight differences between the two clones were seen. Our data from the 2-solvent sequence confirm the quantities ([Table S2](#SM1){ref-type="supplementary-material"}), and these also corresponded well with literature references ([Table 2](#T2){ref-type="table"}). We found relatively low amounts of fatty acids and fewer types than reported in the references (Nisula, [@B59]); also levopimaric acid (a RA) was not found. This might be due to natural variation with regard to genetics or the environment, or because these analytes were below the LOD of our setup, or even due to artifacts from extraction and/or work-up. Alternatively, some infection might have happened already in the live tree, which has been shown to decrease the amount of free FAs, but also deplete the amount of levopimaric acid (Ekman, [@B23]). The fatty alcohols lignoceryl alcohol and behenyl alcohol were also detected and are known to be the most common alcohols found in *P. abies* (Fengel and Wegener, [@B28], p. 194). Due to the detection method used, we have no information about the amounts and types of esterified FAs, but they are known to be less abundant in spruce heartwood than in sapwood (Willför et al., [@B85]). Among the diterpenoids, neoabienol had a higher concentration in spruce 1, explaining the different proportions of DTs among the two trees ([Figure 4](#F4){ref-type="fig"}). The relative amounts of sterols in our spruce specimen were also different, but the absolute amounts were the same, the difference likely arising from the difference in unknown compounds. Also the lignans detected in our specimen were fewer than described in the literature (Willför et al., [@B85]; Nisula, [@B59]), but again, some of these might have been below our LOD. The precipitate obtained from the water fraction of spruce suggests the presence of hemicelluloses, possibly of arabinogalactan type (Willför and Holmbom, [@B87]). Similarly, in Kurile larch a few analytes were not detected---or detected and not found in the literature. Apart from this, the extractive profiles corresponded well with previous literature for the same (Nisula, [@B59]) and other species of larch (Zule et al., [@B95], [@B96]; Nisula, [@B59]). As opposed to spruce, the heptane fraction of Kurile larch did not contain fatty alcohols, but triglycerides are also expected to be present (Zule et al., [@B95]). We found relatively large amounts of cedrol in our specimens, but could not find evidence for this in the literature for any species of larch. We do not know if this is an artifact or not. As mentioned, there was a rather important difference in the flavonoid profiles of the two larch clones. Firstly, larch 2 contained almost twice the amount of flavonoids of larch 1. Secondly, and as reported by several sources, Taxifolin 1 (TAX) was the dominating flavonoid of larch 2 (Venäläinen et al., [@B81]; Zule et al., [@B96]; Nisula, [@B59]), whereas in larch 1, DHK and TAX were present at a 50:50 ratio. According to literature, DHK is one of the direct precursor molecules of TAX (Winkel-Shirley, [@B92]), indicating that larch 1 had a problem with the conversion to TAX. The large difference in yields of the water fractions resulted in a 30 mg/g difference of precipitated arabinogalactan, being higher in larch 2. The amounts detected correspond to 6--10% w/w. These quantities are in agreement with findings from other authors and species of larch, where the amounts range from 5 to 20% (Côté et al., [@B13]; Luostarinen and Heräjärvi, [@B51]). The ArGal yields for the sticks were approximately 40--50% of the milled counterpart. After removal of the hemicellulose, the water fractions were successfully run on the GC and revealed that the water fraction was composed of carbohydrates and flavonoids, but the relative composition between sticks and milled specimens was not the same. Not surprisingly, a larger contribution of carbohydrates was found for the milled samples, but in absolute numbers this difference was not so drastic. The relatively higher release of flavonoids in the sticks was probably due to the limited extraction efficiency as compared to the milled material. Because the extraction efficiency of the ethanol fraction in spruce was higher on average (50%) than for larch (30%), we also suggest that the presence of ArGal in the lumen of tracheids might hinder the flow of solvent and extractives during extraction with solvents other than water. Summarizing, we should keep in mind that (1) we do not know the composition of the fraction not detected by GC, (2) that we cannot say for sure whether the extracts of the sticks contain the same proportions of molecules as do their milled counterparts and (3) that extraction was incomplete for the sticks, possibly causing migration of analytes within the wooden tissue. 4.2. Extractives in Relation to Moisture and Fungal Degradation --------------------------------------------------------------- ### 4.2.1. Moisture Content at Isothermal Equilibrium and After Fungal Degradation Investigating the moisture content of the Kurile larch samples, we found that the TOT samples had a higher MC than the NAT samples in absorption, especially in the range between 94.5 and 99.89% RH. This difference, however, was not statistically significant in desorption, although we observed that at saturation (100% RH), the NAT samples had the lowest MC (about 140%) and the TOT samples the highest with 150% MC. Previous studies, performed in the hygroscopic range, indicate that there is a difference in both absorption and desorption modes and that it is dependent on the amount of extractives (Wangaard and Granados, [@B83]; Choong and Achmadi, [@B15]; Nzokou and Kamdem, [@B60]; Vahtikari et al., [@B80]). In the hygroscopic moisture range, this observation can be attributed to a bulking effect of (polymeric) extractives, occupying space in cell walls. However, changes seen in the over-hygroscopic range where water is present also outside cell walls should rather be related to presence of extractives in pits and macro voids. It seems that also the proportions of hydrophilic and hydrophobic extractives influence the MC in the hygroscopic range, which can be seen from the data of Nzokou and Kamdem ([@B60]) where the resin-rich pine had a lower equilibrium MC as compared to cherry and oak at comparable extractives content. No such trend was seen from our desorption data in the over-hygroscopic range, obtained for all types of extractions of larch. Another factor may be the radial position the sample was taken from, as the MC of earlywood and latewood may differ from year to year (Hill et al., [@B42]). The differences in MC reported in the above mentioned references are all in the same range with the standard deviations of our findings. Therefore, it might not be so straightforward to interpret the cause of these observations and studies should be conducted to assess whether these effects can actually be distinguished from each other. Different fungi have different moisture requirements for successful infestation of wood (Meyer and Brischke, [@B54]; Brischke et al., [@B11]). Yet, our data suggest that the extractives do not play a key role in the moisture regulation of our larch specimen, as the MC was not different enough between the extracted and native samples to explain the differences we found in degradation. This is supported by the fact that we found the same moisture content for all samples after degradation by the brown-rot, no matter what kind of extractives had been removed, and also independent of the species. It would be necessary to repeat this experiment with a non-degraded control sample, incubated under the same conditions, to see how much of that water comes from the humidity conditions inside the petri-dish and the soil-block jar. This would allow the estimation of how much water comes from fungal respiration and/or active water transport by the fungus (Thybring, [@B79]). Similar to our study, it was also concluded for Scots pine (Jebrane et al., [@B47]) and several species of larch (Venäläinen et al., [@B81]; Jebrane et al., [@B47]) that the natural durability of wood is not necessarily determined only by its moisture content, but also by chemical properties of extractives, as will be further discussed below. ### 4.2.2. Defense Strategy of Spruce The weight loss of the Norway spruce samples was in the same range with Finnish spruce (Metsä-Kortelainen and Viitanen, [@B53]), but higher than found for Austrian spruce, which was more in range with larch specimen of the present study (Fackler et al., [@B25]). Despite its overall low durability, we found that spruce hydrophobic extractives play a relevant role in defense of the cell wall material against fungal attack, supported by the action of lignans. Very indicative was that the average weight loss we found for the PHO group of spruce was statistically the same as the TOT group, which both had a higher weight loss than the native samples. This suggests that the removal of the hydrophobic extractives alone had a more significant impact on the brown-rot attack, than did the additional removal of hydrophilic lignans, which are removed and/or relocated to higher extends by the PHI and TOT extractions. We found that the major contributors to the heptane extracts were fatty acids, resin acids, some diterpenoids and sterols, out of which resin acids were dominant. These compound classes are all part of the typical oleoresin found in rays and resin channel of conifers (Higuchi, [@B41]), which is also used for the defense in active tissue (i.e., sapwood). Micales et al. ([@B56]) tested pure and mixed RAs *in vitro* and detected fungitoxic effects at concentrations of 0.02% on pine wood. They noted that, generally, the abietan type resin acids (i.e., dehydro-/abietic acid, palustric acid, [Figure S1.3,4](#SM1){ref-type="supplementary-material"}) were more fungitoxic than the pimarane-types (i.e., iso-pimaric acid, sandaracopimaric acid, [Figure S1.5](#SM1){ref-type="supplementary-material"}). In our specimens the total resin acid concentration was higher than that (around 0.04%), and the most abundant analyte, dehydroabietic acid, was present at about 0.02%. The more hydrophilic lignans in the ethanol fraction exhibit relatively strong antioxidant behavior when used in higher concentrations (i.e., 2%) (Willför et al., [@B90]). The total concentration of monomeric lignans of our specimens was only about 0.4%, with HMR as the main analyte. This molecule was found to be a good antioxidant *in vitro* (Rice-Evans et al., [@B64]; Saarinen et al., [@B68]; Willför et al., [@B90]). In our study, resin acids were found at concentrations levels found to be fungitoxic, and lignans found at much lower concentrations than their proven effect levels. This could explain why the extraction of the hydrophobic analytes affected the spruce wood proportionally more than did the removal of lignans. Note also that spruce 1 was more severely affected, which might be due to the fact that it had a higher amount of hydrophobic substances, but a lower amount of hydrophilic ones than spruce 2. The fewer lignans in that tree could not back-up the loss of the RAs, while in spruce 2, which had a higher monolignan content, they could protect the cell wall material to a higher degree. We saw that the TOT extraction affected the sticks samples significantly more than the PHI extraction (at least in spruce 1), but the slightly higher yield of the total extraction might not suffice to explain this difference alone. A possible additional effect could be the migration of hydrophobic molecules upon extraction, as mentioned in Nzokou and Kamdem ([@B60]). Following this thought, and assuming more hydrophobic extractives are infiltrated in the cell wall, the extraction with heptane and DCM might have caused migration of hydrophobic substances to the cell wall surface, which were subsequently removed by ethanol extraction. In the case of the PHI samples, with ethanol as the first extraction step, the same migration may have happened, but the subsequent water extraction could not remove them efficiently, thus leaving a fungitoxic and hydrophobic layer on the cell wall surface. It was suggested that the absorption rate could be additionally reduced by such an effect and thus, potentially, early phase of fungal growth slowed down (Vahtikari et al., [@B80]; Sjökvist et al., [@B75]). Likely we were not able to see this effect because our samples were pre-equilibrated for several weeks prior to inocculation. ### 4.2.3. Defense Strategy of Larch For the 8 weeks degradation test, we found the same weight loss of Kurile larch grown in Denmark, as reported for European and Siberian larch of several provenances, degraded by *R. placenta* (Jebrane et al., [@B47]). The hydrophilic flavonoids, especially taxifolin, seemed to be the crucial ingredient to the defense of the wood via their antioxidative potential, but being supported by hydrophobic extractives. We saw that the two larch clones performed very differently in all tested groups, except in the TOT group, where the WL was almost the same. In this group the difference to the control was only significant for larch 2, indicating that the extractives it contained were more relevant to its durability than for larch 1. In contrast, larch 1 was more affected by the hydrophobic treatment, as compared to larch 2 and the controls, thus the hydrophobic extractives played a bigger role in this tree. The phenolics content of larch 2 was about twice as high as for larch 1, while the hydrophobic extractives were present in similar amounts. This supports findings from previous studies that used the total phenolics or water-soluble extractives as a way to predict or explain decay resistance in larch trees (Scheffer and Cowling, [@B70]; Gierlinger et al., [@B31]; Jebrane et al., [@B47]; Nisula, [@B59]). Notably, the difference between the two trees lay in their respective taxifolin content, being much larger in larch 2. Venäläinen et al. ([@B81]) also found a good correlation between the taxifolin content of several clones of Siberian larch to their weight loss. The major phenolic compounds in larch 1 were DHK and TAX at almost equal concentration, but this did not give nearly as good a protection as did TAX alone at a higher concentration in larch 2 (see [Table 4](#T4){ref-type="table"}). Interestingly, the only structural difference between DHK and TAX is an additional hydroxyl group at the 3\' position of the B-ring ([Figure S1.8](#SM1){ref-type="supplementary-material"}) of TAX, as seen in [Figure S1.9,10](#SM1){ref-type="supplementary-material"}. It was shown that this greatly enhances the antioxidant potential in aqueous systems (Rice-Evans et al., [@B65]; Binbuga et al., [@B6]). One of the reasons is a better stabilization of the flavonoid radical when both of these positions are hydroxylated. The same configuration also increases their metal chelating abilities (Rice-Evans et al., [@B65]), which is important when considering that brown-rot fungi need iron to generate radicals (Binbuga et al., [@B6]; Ringman et al., [@B66]). The difference in durability of these two larch trees may thus be explained not only by the quantity, but also the increased antioxidative power, as well as metal chelating abilities of TAX over DHK. The fact that larch 1 was more affected by the hydrophobic extraction points at the possibility that, similarly to spruce, the hydrophobic extracts are an important back-up to the more abundant hydrophilic compounds, especially when these are not so efficient. That is, larch 2 was less affected by the PHO extraction, because the high taxifolin content protected the wood well, which the mixture of TAX and DHK could not do for larch 1. Finally, the case of larch 1 gives a good example of why the taxifolin content may be a more reliable indicator for larch durability, rather than total phenolics or flavonoid content, because it was found to have a similar WL as our spruce specimen, even though the phenolics content was much higher. ### 4.2.4. Degradation - Spruce vs. Larch The total phenolics content is often used as a parameter in durability studies (Scheffer and Cowling, [@B70]; Gierlinger et al., [@B31]; Jebrane et al., [@B47]; Nisula, [@B59]). In the case of these two species the 4× higher phenolics content of larch, only lead to about half the weight loss within the first 2 and 8 weeks as compared to spruce. This suggests that lignans in spruce at higher concentrations could contribute to a similar or greater durability than larch flavonoids do, presumably through their antioxdiative potentials. The work of Willför et al. ([@B90]) supports this suggestion, as they found that the performances of spruce and larch extracts, as well as isolated compounds thereof, were similar, but not equally good at scavenging different types of oxygen radicals, or inhibiting lipid peroxidation. We suggest that the antioxidative function of poly(phenolics) in heartwood could be especially relevant in the early-phase of degradation, where brown-rot fungi disrupt cell wall polymers by employing small radicals. Although there is no quantitative information on how efficient the cellulolytic enzymes of brow-rots could be without prior oxidative disruption of the cell wall, we find it unlikely that the fungus invests so much energy into a non-enzymatic oxidation phase, if it was not necessary. Apart from the moisture content of the wood, the amount and efficacy of antioxidants may play a significant role in the on-setting brown-rot attack, because they could potentially prolong the early-phase, as is the case with larch, showing a lower weight loss as compared to spruce in equal time. Ideally, it would even starve the fungus, because it cannot access the cell wall with its enzymes. We found that the weight loss after 2 weeks was nearly the same for the PHI groups of both species, where spruce was not significantly affected compared to their native control, but both larch trees were less durable compared to their controls. This could be related to the presence of oligolignans in spruce, that may be harder to extract and have been shown to be good radical scavengers (Willför et al., [@B90]). On the other hand, the larch specimen is also expected to contain polymeric material with similar activity. A more likely reason is that the removal of hydrophilic components did not affect our spruce samples as much, because the quantities of lignans were low, and are generally low in spruce heartwood. Therefor, spruce may simply have to rely on the fungicidal oleoresin more heavily, than larch does. In this scenario, the composition of the oleoresin is crucial, and known to differ among more and less susceptible trees (Holmbom et al., [@B45]; Mason et al., [@B52]). We tentatively conclude, that hydrophobic extractives play a more important role in spruce than in larch, and vice versa. Note though, that we cannot exclude the possibility that the observed effects were caused by a relocalization of certain substances by the extraction, to places where they may not be as effective as they would have been in their native surroundings. 5. Conclusion {#s5} ============= In order to investigate the influence of hydrophobic and hydrophilic extractives on water sorption and brown-rot degradation, we prepared milled samples and sticks of Norway spruce and Kurile larch extracted with only hydrophobic or only hydrophilic solvents, as well as extracted with all four solvents (total extraction). The extraction efficiency was lowest for the ethanol fraction, indicating that ethanol-soluble analytes are harder to remove from the wood, which might be connected to their localization. For both species we found that most of the extracted material must be either polymeric or esterified in some way, because we only detected a fraction of the extracts with gas chromatography. This implies that the conclusions we draw could not consider much of the effect of the polymers in the extract, although it is known that they contribute to bulking of the cell wall. However, this effect was overall small as seen from the sorption isotherm we obtained for Kurile larch after different extractions, because of the incomplete extraction. We conclude that the changes observed are likely not responsible for the differences in degradation among the differently extracted samples. The degradation test with European *R. placenta* resulted in measurable weight loss already after 2 weeks of incubation. We suggest that the more fungitoxic, hydrophobic substances found in spruce may play a bigger role in its protection, while on the other hand, larch durability is additionally boosted by its antioxidative flavonoid content, especially its taxifolin content. In this scenario, the hydrophilic lignans in spruce and hydrophobic extracts in larch can complement or back-up the function of the respective other part, especially in case of metabolic disturbances as likely seen with larch 1. Regarding non-toxic wood protection systems, this study indicates that large amounts of antioxidant material can provide some protection against the brown-rot *R. placenta*, but systems with multiple mechanisms of action are still preferrable. Data Availability Statement {#s6} =========================== The datasets generated for this study are available on request to the corresponding author. Author Contributions {#s7} ==================== The research was initiated and designed by SF and LT. TB-N and SF did the GC-MS/GC-FID instrument setup and measurements. AS contributed significantly to the analysis of the GC-MS data and the manuscript revisions. MF and SF did the design and execution of the sorption isotherm experiments, interpretation of data together with LT. AP helped design and discuss the fungal degradation experiments and with manuscript revisions. SF did the extractions, data analysis and interpretation of all experiments. SF, TB-N, MF, and LT co-wrote the paper. All authors read and approved the final manuscript. Conflict of Interest {#s8} ==================== 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 would like to thank Stefan Willför from the Åbo Akademi University for kindly providing his facilities for the identification of extractives. Many thanks also go to Morten Alban Knudsen and Sofie Wikkelsø Jensen (UCPH, Denmark), Flemming Grauslund (DTI, Denmark), and Anja Vieler (TU Munich, Germany) for their valuable help with experimental practicalities. All the chemical structures were drawn with MarvinSketch^©^ 17.20.0, an application by ChemAxon Ltd. **Funding.** This project was funded by the VILLUM FONDEN (grant number 12404). The short-term scientific mission to Åbo Akademi University was funded by the Northern European Network for Wood Science and Engineering (WSE). Supplementary Material {#s9} ====================== The Supplementary Material for this article can be found online at: <https://www.frontiersin.org/articles/10.3389/fpls.2020.00855/full#supplementary-material> ###### Click here for additional data file. [^1]: Edited by: Lauri Rautkari, Aalto University, Finland [^2]: Reviewed by: Tiina Belt, Natural Resources Institute Finland (Luke), Finland; Miha Humar, University of Ljubljana, Slovenia [^3]: This article was submitted to Technical Advances in Plant Science, a section of the journal Frontiers in Plant Science

Dear Editor Myasthenia gravis (MG), an autoimmune disease, is characterized by physical exhaustion and weakness caused by autoantibody-induced dysfunction of the neuromuscular junction. MG is frequently accompanied with thymoma, and approximately 25% of patients with thymoma-associated MG exhibit at least one non-motor symptom (taste disorder, myocarditis, neuromyotonia, limbic encephalitis, pure red cell aplasia, immunodeficiency, or alopecia areata) \[[@bb0005]\]. Further, approximately 74%--88% of patients with MG have anti-acetylcholine receptor (AChR) antibodies that bind to postsynaptic membrane and induce complement-mediated injury \[[@bb0010]\]. Moreover, anti-striational antibodies are often detected in patients with thymoma-associated refractory MG \[[@bb0015], [@bb0020]\], which putatively induce complement activation \[[@bb0025]\]. Herein, we report the case of refractory thymoma-associated generalized MG with anti-AChR and anti-striational antibodies. The motor symptoms remarkably improved by the administration of eculizumab, a humanized monoclonal antibody that targets the terminal complement protein C5 \[[@bb0030]\]. Moreover, taste disorder was alleviated, suggesting the involvement of complement activation in the onset of taste disorder in patients with MG. 1. Case report {#s0005} ============== A 34-year-old male presented to our hospital after 3 months of the onset of limb weakness, bilateral ptosis, diplopia, and dysphagia. A diagnosis of thymoma-associated generalized MG was established based on positive serum anti-AChR antibodies (51 nmol/L) and detection of thymoma by computed tomography. Among anti-striational antibodies, anti-titin and anti-Kv1.4 antibodies were positive, whereas serum anti-RyR antibody was negative. The patient had no arrhythmia or heart failure. Serum IgG was 1873 mg/dL, and the patient had no past history of immunodeficiency. Although treatment with oral prednisolone was initiated prior to performing extended total thymectomy, the patient exhibited rapid aggravation of dysphagia and dyspnea. Eventually, mechanical ventilation was required due to myasthenic crisis. Following treatment with intravenous high-dose immunoglobulin (IVIg, 400 mg/kg bodyweight for 5 days) and intravenous methylprednisolone (IVMP, 1000 mg/day for 3 days) followed by oral prednisolone, thymectomy was performed \[[@bb0035]\]. Postoperatively, the patient was treated with oral prednisolone (60 mg/day) with tacrolimus. Although prednisolone dose tapering was attempted, the patient experienced relapse of MG every 3--6 months; the prednisolone dose could not be reduced below 20 mg/day. Approximately 10 months following the onset, the patient gradually developed impairment of taste sensation and diffuse alopecia areata. Each relapse of MG was repeatedly treated with IVIg, IVMP, intravenous cyclophosphamide, and plasmapheresis. However, long-term remission of motor symptoms could not be achieved with prednisolone and tacrolimus therapy, and there was a gradual worsening of taste disorder and alopecia areata. Eighteen months following the onset, the patient developed pulmonary infection with *Mycobacterium avium complex*, ostensibly owing to the long-term immunosuppressant therapy. To taper predonisolone dose, eculizumab was introduced as a maintenance therapy for refractory MG. Eculizumab administration was initiated after antibiotics against the *M. avium complex* had been administered for at least 1 year and chest X-ray showed signs of remission. Just before eculizumab administration was initiated, the patient was receiving predonisolone (20 mg/day) and tacrolimus (2.5 mg/day) and exhibited severe physical exhaustion and bilateral ptosis. The quantitative myasthenia gravis (QMG) score was 13 and Myasthenia Gravis Activities of Daily Living (MG-ADL) score was 18. The induction dosing schedule of eculizumab was 900 mg on day 1 and weeks 1, 2, and 3; the maintenance dose was 1200 mg every second week after week 4. Eculizumab substantially improved the motor symptoms, and prednisolone could be safely tapered; the QMG score reduced to 4 and MG-ADL score to 5 at week 12 with 2.5 mg/day tacrolimus and 12.5 mg/day prednisolone. Remarkably, the taste disorder and motor symptoms together showed improvement, whereas alopecia areata did not improve. Following the introduction of eculizumab and tapering of steroids, there was no deterioration in the *M. avium complex* infection. At week 26, the QMG score was 3 and MG-ADL score was 2 with 2.5 mg/day tacrolimus and 8 mg/day prednisolone. After week 34, the patient exhibited minimal manifestations with 3 mg/day tacrolimus and 5 mg/day prednisolone. 2. Discussion {#s0010} ============= Eculizumab blocks complement-mediated injury to postsynaptic AChR \[[@bb0030]\] and has been used for patients with anti-AChR antibody-associated refractory generalized MG \[[@bb0010]\]. Although in the REGAIN study, the change in the MG-ADL score from baseline to week 26 did not significantly differ between the eculizumab and placebo arms, additional prespecified sensitivity and secondary analyses suggested the efficacy of eculizumab \[[@bb0010]\]. However, patients with a history of thymoma or thymic neoplasms were excluded from the REGAIN study, and the patients were not investigated for anti-striational antibodies. The present case suggests that eculizumab is effective in patients with thymoma-associated generalized MG who have anti-striational antibodies. Anti-striational antibodies can recognize the epitopes on skeletal and cardiac muscle tissues. Among these, antibodies against titin, RyR, and Kv1.4 are frequently observed in patients with anti-AChR antibody-positive thymoma-associated MG \[[@bb0020]\]. Presence of these antibodies is associated with severe manifestations of MG including complications of myositis and cardiomyositis \[[@bb0040]\]. Anti-striational antibodies have been implicated in complement activation and the subsequent muscle dysfunction \[[@bb0020],[@bb0025]\]. In the present case, eculizumab possibly inhibited complement activation mediated by anti-striational antibodies, which resulted in a favorable clinical course. In addition, this case is unique in that taste disorder improved following eculizumab administration, although no improvement in alopecia areata was observed. Among the non-motor symptoms of MG, taste disorders are putatively autoantibody-mediated disorders and typically respond to immunotherapy in parallel with the disease activity of MG \[[@bb0005],[@bb0045]\]. Conversely, alopecia areata is considered to be due to CD8 T-cell cytotoxicity and independently develops of the clinical course of MG \[[@bb0005]\]. Although further studies are needed, the present case suggests that complement activation is involved in the causation of taste disorder, but not of alopecia areata, in patients with MG. We cannot eliminate the possibility that the long-term immunotherapy administered prior to the use of eculizumab contributed to the remission; however, MG did not relapse after the induction of eculizumab for more than 12 months and prednisolone was successfully tapered. We hypothesize that eculizumab inhibited complement-mediated damage triggered by anti-AChR antibodies and anti-striational antibodies and that it is effective in patients with anti-striational antibodies associated MG. Funding {#s0015} ======= This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declarations of interest {#s0020} ======================== None. The authors would like to thank Enago ([www.enago.jp](http://www.enago.jp/){#ir0005}) for the English language review.

Introduction {#S1} ============ High-throughput sequencing has facilitated major advances in our understanding of microbial ecology and is now widespread in biotechnological applications from personalized medicine^[@R1]^ to bioenergy^[@R2]^. Markers such as the 16S ribosomal RNA gene (16S) of bacteria and archaea are frequently used to characterize the taxonomic composition and phylogenetic diversity of environmental samples. Because marker gene studies focus on one or a few universal genes, they cannot directly identify metabolic or other functional capabilities of the microorganisms under study. Conversely, metagenomic sequencing aims to sample all genes from a community and can produce detailed metabolic and functional profiles. Although relatively little sequencing is needed to characterize the diversity of a sample^[@R3],\ [@R4]^, deep, and therefore costly, metagenomic sequencing is required to access rare organisms and genes^[@R5]^. Thus, marker gene profiling of large sample collections is now routine, but deep metagenomic sequencing across many samples is prohibitively expensive. Although marker gene and shotgun sequencing strategies differ in the type of information produced, phylogeny and biomolecular function are strongly, if imperfectly, correlated. Phylogenetic trees based on 16S closely resemble clusters obtained based on shared gene content^[@R6]-[@R9]^, and researchers often infer properties of uncultured organisms from cultured relatives. For example, the genome of a *Bacteroides* spp. might reasonably be inferred to contain many genes encoding glycoside hydrolase activity, based on the commonality of these activities in sequenced *Bacteroides* isolates^[@R10]^. This association is in turn closely related to the pan- and core-genomes of each phylogenetic subtree^[@R11]^, in that larger and more strongly conserved core genomes result in more confident linkages of genes with clades. Conversely, a clade's core genome consists of genes its genomes can be expected to carry with high probability. The correlation between phylogeny and functional attributes depends on factors including the complexity of the trait^[@R12]^, but the overall degree of correlation suggests that it may be fruitful to predict the functions encoded in an organism's genome on the basis of functions encoded in closely related genomes. Recently, some 16S studies have extended these intuitions to infer the functional contribution of particular community members by mapping a subset of abundant 16S sequences to their nearest sequenced reference genome^[@R13]-[@R15]^. The accuracy of such approaches has not been characterized, but the correlation between gene content and phylogeny^[@R8],\ [@R9],\ [@R16]^ (excepting special cases such as laterally transferred elements and intracellular endosymbionts with reduced genomes) suggests that it may be possible to approximately predict the functional potential of microbial communities from phylogeny. Widespread and reproducible application of such a strategy requires an automated method that formalizes the relationship between evolutionary distance and functional potential across the entire metagenome, accounts for variation in marker gene copy number^[@R17]^, and accurately recaptures insights from shotgun metagenomic sequencing. Here we describe PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States), a technique that uses evolutionary modeling to predict metagenomes from 16S data and a reference genome database. We investigated the accuracy of this approach as a function of the phylogenetic proximity of reference genomes to sampled environmental strains and the rate of decay of the phylogeny-function correlation owing to a variety of factors including gene duplication, loss, and lateral gene transfer. Lateral gene transfer is particularly relevant because it allows distantly related genomes to share functions that are missing from closer relatives and appears to be particularly widespread in microbes sharing a common environment, including constituents of the human microbiome^[@R18],\ [@R19]^ as well as extreme and contaminated environments^[@R20],\ [@R21]^ Quantitative predictions also depend on accurate modeling of community member abundance, which can be affected by 16S copy-number variation^[@R17]^ ([Supplementary Results](#SD2){ref-type="supplementary-material"}). Although these caveats could theoretically limit the accuracy of any inference of microbial function from 16S sequence data, their quantitative effects on this relationship have not previously been explored in detail. Our results using published data show that PICRUSt recaptures key findings from the Human Microbiome Project and predicts metagenomes across a broad range of host-associated and environmental samples. We applied PICRUSt to a range of datasets from humans^[@R22]^, soils^[@R23]^, other mammalian guts^[@R24]^ and the hyper-diverse and underexplored Guerrero Negro microbial mat^[@R23],\ [@R24]^, which allowed us to model how the accuracy of PICRUSt varies based on the availability of reference genomes for organisms in each environment. In the best cases, correlations between inferred and metagenomically measured gene content approached 0.9 and averaged approximately 0.8. PICRUSt recaptured most of the variation in gene content obtained by metagenomic sequencing using only a few hundred 16S sequences and in some cases outperforms the metagenomes measured at particularly shallow sampling depths Additionally, we quantified the effects of several other factors on PICRUSt's accuracy, including reference database coverage, phylogenetic error, gene functional category (a potential surrogate for the effects of lateral gene transfer), ancestral state reconstruction method, microbial taxonomy and 16S sequencing depth. Finally, we applied PICRUSt to several 16S-only datasets to identify previously undescribed patterns in gene content in oral, vaginal and coral mucus samples. Our implementation of these techniques, associated documentation and example datasets are made freely available via the PICRUSt software package at <http://picrust.github.com>. Results {#S2} ======= The PICRUSt algorithm {#S3} --------------------- We developed PICRUSt to predict the functional composition of a microbial community's metagenome from its 16S profile. This is a two-step process. In the initial 'gene content inference' step, gene content is precomputed for each organism in a reference phylogenetic tree. This reconstructs a table of predicted gene family abundances for each organism (tip) in the 16S-based phylogeny. Because this step is independent of any particular microbial community sample, it is pre-calculated only once. The subsequent 'metagenome inference' step combines the resulting gene content predictions for all microbial taxa with the relative abundance of 16S rRNA genes in one or more microbial community samples, corrected for expected 16S rRNA gene copy number, to generate the expected abundances of gene families in the entire community ([Fig. 1](#F1){ref-type="fig"}). In the genome prediction step, PICRUSt predicts genes present in organisms that have not yet been sequenced based on the genes observed in their sequenced evolutionary relatives. To do this, PICRUSt uses existing annotations of gene content and 16S copy number from reference bacterial and archaeal genomes in the IMG database^[@R25]^. Any functional classification scheme can be used with PICRUSt; here, we demonstrate the use of the popular KEGG Orthology (KOs)^[@R26]^ and Clusters of Orthologs Groups (COGs)^[@R27]^ classification schemes. PICRUSt uses ancestral state reconstruction, along with a weighting method we developed for this work, to make predictions of gene content (with estimates of uncertainty)for all organisms represented in the Greengenes phylogenetic tree of 16S sequences^[@R28]^. Prediction of a microbe's gene content starts by inferring the content of the organism's last common ancestor with one or more sequenced genomes. Inference of the genes in each ancestor (and uncertainty in that estimate) is handled by existing methods for ancestral state reconstruction (ASR). ASR algorithms infer the traits of ancestral organisms by fitting evolutionary models to the distribution of traits observed in living organisms using criteria such as maximum likelihood or Bayesian posterior probability. PICRUSt extends existing ASR methods to predict the traits of extant (in addition to ancestral) organisms. This allows the contents of the genomes of environmental strains to be inferred, with uncertainty in that inference quantified based on each gene family's rate of change. This approach accounts both for gene families' propensities for lateral transfer and for the degree to which each gene family is part of a 'core'conserved within particular microbial clades. The gene contents of each reference genome and inferred ancestral genomes are then used to predict the gene contents of all microorganisms present in the reference phylogenetic tree. This initial genome prediction step is computationally intensive, but it is independent of any specific experiment and needs to be performed only once, allowing a single reference to be pre-computed 'off-line' and provided to users. The metagenome inference step relies on a user-provided table of operational taxonomic units (OTUs) for each sample with associated Greengenes identifiers. Such tables are typically produced as one of the main data products in a 16S rRNA gene sequencing assay by analysis systems such as QIIME^[@R29]^. Because 16S rRNA copy number varies greatly among different bacteria and archaea, the user's table of OTUs is normalized by dividing the abundance of each organism by its predicted 16S copy number. The 16S rRNA copy numbers for each organism are themselves inferred as a quantitative trait by ASR during the genome prediction step. Normalized OTU abundances are then multiplied by the set of gene family abundances pre-calculated for each taxon during the gene content inference step The final output from metagenome prediction is thus an annotated table of predicted gene family counts for each sample, where gene families can be orthologous groups or other identifiers such as KOs, COGs, or Pfams. The resulting tables are directly comparable to those generated by metagenome annotation pipelines such as HUMAnN^[@R30]^ or MG-RAST^[@R31]^. As with metagenome sequence data, the table of gene family counts can optionally be further summarized as pathway-level categories. However, in addition to estimating the aggregate metagenome for a community, PICRUSt also estimates the contribution of each OTU to a given gene function, which is not as easily obtained from shotgun metagenome sequencing^[@R32]^. PICRUSt recapitulates Human Microbiome Project metagenomes {#S4} ---------------------------------------------------------- The value of PICRUSt depends on the accuracy of its predicted metagenomes from marker gene samples and the corresponding ability to recapitulate findings from metagenomic studies. The performance of PICRUSt was first evaluated using the set of 530 HMP samples that were analyzed using both 16S rRNA gene and shotgun metagenome sequencing^[@R22]^. Although a shotgun metagenome is itself only a subset of the underlying biological metagenome, accurate prediction of its composition constitutes a critical test for PICRUSt. Human-associated microbes have been the subject of intensive research for decades, and the HMP alone produced \>700 draft and finished reference genomes, suggesting that the human microbiome would be a worthwhile benchmark for testing the accuracy of PICRUSt's metagenome predictions. We tested the accuracy of PICRUSt by treating HMP metagenomic samples as a reference and calculating the correlation of PICRUSt predictions from paired 16S samples across 6,885 resulting KO groups. PICRUSt predictions had high agreement with metagenome sample abundances across all body sites (Spearman r=0.82, p\<0.001, [Fig. 2a](#F2){ref-type="fig"}, [Supplemental Fig. 1](#SD2){ref-type="supplementary-material"}). Using two synthetic communities from the HMP constructed from a set of known microorganisms^[@R33]^, we used PICRUSt to make predictions that were even more accurate for both communities (Spearman r=0.9, p\<0.001, [Supplemental Fig. 2](#SD2){ref-type="supplementary-material"}). As a targeted example, we also tested PICRUSt's accuracy in specifically predicting the abundance of glycosaminoglycan (GAG) degradation functions, which are more abundant in the gut than elsewhere in the body^[@R30]^. Using the same differential enrichment analysis on both PICRUSt and metagenomic data yielded identical rankings across body sites and very similar quantitative results ([Fig. 2b](#F2){ref-type="fig"}), suggesting that PICRUSt predictions can be used to infer biologically meaningful differences in functional abundance from 16S surveys even in the absence of comprehensive metagenomic sequencing. Inferring host-associated and environmental metagenomes {#S5} ------------------------------------------------------- Next, we then evaluated the prediction accuracy of PICRUSt in metagenomic samples from a broader range of habitats including mammalian guts^[@R24]^, soils from diverse geographic locations^[@R34]^ and a phylogenetically complex hypersaline mat community^[@R23],\ [@R24]^. These habitats represent more challenging validations than the human microbiome, as they have not generally been targeted for intensive reference genome sequencing. Because PICRUSt benefits from reference genomes that are phylogenetically similar to those represented in a community, this evaluation allowed us to quantify the impact of increasing dissimilarity between reference genomes and the metagenome. To characterize this effect, we developed the Nearest Sequenced Taxon Index (NSTI) to quantify the availability of nearby genome representatives for each microbiome sample (Methods). NSTI is the sum of phylogenetic distances for each organism in the OTU table to its nearest sequenced reference genome, measured in terms of substitutions per site in the 16S rRNA gene and weighted by the frequency of that organism in the OTU table. As expected, NSTI values were greatest for the phylogenetically diverse hypersaline mat microbiome (mean NSTI=0.23 +/- 0.07 s.d.), least for the well-covered HMP samples (mean NSTI=0.03 +/- 0.02 s.d.), mid-range for the soils (mean NSTI=0.17 +/- 0.02 s.d.) and varied for the mammals (mean NSTI=0.14 +/- 0.06 s.d.) ([Fig. 3](#F3){ref-type="fig"}). Also as expected, the accuracy of PICRUSt in general decreased with increasing NSTI across all samples (Spearman r=-0.4, p\< 0.001) and within each microbiome type (Spearman r=-0.25 to -0.82, p\<0.05). For a subset of mammal gut samples (NSTI\<0.05) and all of the soil samples that we tested, PICRUSt produced accurate metagenome predictions (Spearman r=0.72 and 0.81, respectively, both p\<0.001). It should be noted that both the mammal and hypersaline metagenomes were shallowly sequenced at a depth expected to be insufficient to fully sample the underlying community's genomic composition, thus likely causing the accuracy of PICRUSt to appear artificially lower for these communities (see below). Although the lower accuracy on the hypersaline microbial mats community (Spearman r =0.25, p\<0.001) confirms that PICRUSt must be applied with caution to the most novel and diverse communities, the ability to calculate NSTI values within PICRUSt from 16S data allows users to determine whether their samples are tractable for PICRUSt prediction prior to running an analysis. Moreover, the evaluation results verify that PICRUSt provides useful functional predictions for a broad range of environments beyond the well-studied human microbiome. PICRUSt outperforms shallow metagenomic sequencing {#S6} -------------------------------------------------- These validations showed that other factors in addition to NSTI also influence PICRUSt accuracy. Because sequenced metagenomes were used as a proxy for the true metagenome in our control experiments, metagenome sequencing depth was an additional contributing factor to the (apparent) accuracy of PICRUSt. This is because sequenced metagenomes themselves are incomplete surveys of total underlying functional diversity. Indeed, we found that metagenome sequencing depth for each sample correlated with PICRUSt accuracy (Spearman r=0.4, p\<0.001), suggesting that samples with particularly low sequencing depth may be poor proxies for the community's true metagenome and may lead to conservative estimates of PICRUSt accuracy ([Supplemental Fig. 3](#SD2){ref-type="supplementary-material"}). Similarly, we found a weak correlation between 16S rRNA gene sequencing depth and PICRUSt accuracy (Spearman r=0.2, p\<0.001), also suggesting a statistically significant but numerically smaller impact on PICRUSt predictions ([Supplemental Fig. 4](#SD2){ref-type="supplementary-material"}). This is likely because proportionally more sequencing is needed to profile functional diversity than phylogenetic diversity. To test the relationship between sequencing depth and accuracy, we used rarefaction analysis of the soil dataset to assess the effects of subsampling either the 16S rRNA genes (for PICRUSt predictions) or the shotgun metagenomic data ([Fig. 4](#F4){ref-type="fig"}). We found that PICRUSt predictions converged rapidly with increasing sequencing depth and reached a maximum accuracy with only 105 16S sequences assigned to OTUs per sample (final Spearman r=0.82, p\<0.001). This suggests that PICRUSt predictions could be performed on 16S data even from shallow sequencing (including many clone library/Sanger datasets) with little loss of accuracy. At this sequencing depth, subsamples from the full metagenome were very poor (though still significant) predictors of overall metagenome content (Spearman r=0.18, p \< 0.001). Approximately 15,000 annotated metagenomic sequences per sample were required before being able to provide the same accuracy as PICRUSt with 105 assigned 16S reads. Accounting for the percent of genes surviving annotation (17.3% of metagenomic reads) or closed-reference OTU-picking (68.9% of post-QC 16S rRNA reads), this analysis indicates that PICRUSt may actually outperform metagenomic sequencing for read depths below \~72,000 total sequences per sample. Although most metagenomes exceed this threshold, it is worth noting that 16.7% (411/2,462) of bacterial and archaeal WGS samples in MG-RAST as of November 2012 are reported as containing fewer than 72,000 sequences. Our results clearly demonstrate the value of deep metagenomic sequencing, but also show that the number of sequences recovered per sample in a typical 16S survey (including those using Sanger sequencing) is more than sufficient to generate high-quality predictions from PICRUSt. Functional and phylogenetic determinants of PICRUSt accuracy {#S7} ------------------------------------------------------------ We further tested and optimized the genome prediction step of PICRUSt using additional information from sequenced reference genomes ([Supplemental Online Results, Supplemental Figs. 5-9](#SD2){ref-type="supplementary-material"}). The prediction accuracy of PICRUSt was largely consistent across diverse taxa throughout the phylogenetic tree of archaea and bacteria ([Fig. 5](#F5){ref-type="fig"}). Notably, PICRUSt predictions were as accurate for archaeal (mean=0.94 +/- 0.04 s.d., n=103) as for bacterial genomes (mean=0.95 +/- 0.05 s.d., n=2,487). Most of the variation seen across groups was due to differences in their representation by sequenced genomes. For example, of the 40 taxonomic families that had an associated accuracy less than 0.80, each of these families had at most six sequenced members, whereas the 53 families with a predicted accuracy greater than 0.95 had on average 30 sequenced representatives. This coincides with our findings that the accuracy of PICRUSt at both the genome and metagenome level depends on having closely sequenced relatives with accurate annotations. Analysis of PICRUSt predictions across functional groups ([Fig. 6](#F6){ref-type="fig"}; [Supplemental Fig. 10](#SD2){ref-type="supplementary-material"}) revealed that, as a positive control, core or 'housekeeping' functions, such as genetic information processing, were best predicted (mean accuracy=0.99 +/- 0.03 s.d.). Conversely, gene families that are variable across genomes and more likely to be laterally transferred, such as those in environmental information processing, had slightly lower accuracy (mean accuracy=0.95 +/- 0.04 s.d.). The subcategories of this group predicted least well were membrane-associated and therefore expected to change rapidly in abundance in response to environmental conditions^[@R35]^. Such functional categories also typically show large differences in relative abundance between similar communities (e.g. metal cation efflux^[@R36]^ and nickel/peptide transporters^[@R19]^) and are enriched for lateral gene transfer^[@R21],\ [@R37]^. However, even these more challenging functional groups were well predicted by PICRUSt (min. accuracy=0.82), suggesting that our inference of gene abundance across various types of functions is reliable. Biological insights from the application of PICRUSt {#S8} --------------------------------------------------- As a final illustration of PICRUSt's computational efficiency and ability to generate biological insights, we applied PICRUSt to three large 16S rRNA datasets. In the first example, all 6,431 16S samples from the HMP were analyzed to predict metagenomes using PICRUSt, requiring \<10 minutes of runtime on a standard desktop computer. One of the many potential applications of such data is in functionally explaining shifts in microbial phylogenetic distributions between distinct habitats. Previous culture-based studies had detected higher frequencies of aerobic bacteria in the supragingival plaque relative to subgingival plaque^[@R38]^, and an analysis of HMP 16S rRNA sequences detected taxonomic differences between these two sites^[@R39]^. Analysis of the PICRUSt-predicted HMP metagenomes revealed an enrichment in the metabolic citrate cycle (M00009) genes in supragingival plaque samples in comparison to subgingival plaque (p\<1e-10; Welch's t-test with Bonferroni correction), supporting previous claims that aerobic respiration is more prevalent in the supragingival regions^[@R38]^. In the second example, we applied PICRUSt to generate functional predictions for ecologically-critical microbial communities associated with reef-building corals. The system under study is subject to an experimental intervention simulating varying levels of eutrophication and overfishing^[@R40]^. One hypothesis to explain the role of algae in the global decline of coral populations posits that eutrophication favors algal growth, which in turn increases dissolved organic carbon (DOC) loads. DOC favors overgrowth of fast-growing opportunist microbes on the surface of coral, outcompeting more-typical commensal microbes, depleting O~2~^15^ and ultimately causing coral disease or death. This is known as the dissolved organic carbon, disease, algae and microbes model^[@R41]^ (although direct algal toxicity through secreted allelochemicals also appears to play a role^[@R42]^). To shed light on this hypothesis using PICRUSt, we predicted metagenomes for 335 coral mucus samples collected *in situ* from corals in experimental plots with varying levels of algal cover ([Supplemental Fig. 11](#SD2){ref-type="supplementary-material"}). Consistent with algae-driven increases in opportunistic pathogen loads, genes in the secretion system were perfectly correlated with relative algal cover (Spearman r=1.0, p=0.0), with 46% enrichment in corals from high- vs. low- algal cover plots. Algal cover also produced significant variation in ribosomal biogenesis genes (ANOVA raw p=1.6 × 10-4; Bonferroni-corrected: 0.049; FDR q= 0.0047), indicating an effect on generally faster-growing organisms. This variation was strongly correlated with relative algal cover across plots and timepoints (Spearman r=0.90, p=0.037) and represented a 25% increase in this gene category between corals in plots with the highest versus lowest algal cover. Further evidence that supported a decrease in typical consumers of coral mucus carbohydrates in favor of fast-growing opportunists was provided by significant depletion of two categories of carbohydrate metabolism genes (Spearman r=-1.0; p=0.0 "Galactose metabolism"; Spearman r=-0.90, p=0.037 "Ascorbate and alderate metabolism"). As the weighted NSTI in this case was 0.12 (+/- 0.02 s.d.), these results suggest that PICRUSt may provide biologically actionable hypotheses even in challenging environments with fewer available reference genomes. Finally, we assessed 993 samples from time courses covering \~16 weeks each from the vaginal microbiomes of 34 individual subjects^[@R43]^. These samples have been previously analyzed only in the context of longitudinal changes in microbial taxonomic composition over time; PICRUSt provided insights into what additional putative microbial pathway changes might explain or accompany this compositional variation. The first observation this enabled was a comparison of community beta-diversity within subjects over time, contrasting the degree of similarity of microbial composition over time with the similarity of the accompanying inferred metagenomes. In all cases, the mean Bray-Curtis diversity using KOs predicted by PICRUSt was more stable over time than when using OTU composition ([Supplemental Fig. 12](#SD2){ref-type="supplementary-material"}). To our knowledge, this provides the first longitudinal results mirroring the functional stability in metagenomes that has been observed cross-sectionally^[@R22],\ [@R44]^. Second, we identified seven KEGG modules that had significant differences in mean abundances in samples taken during menses ([Supplemental Fig. 13](#SD2){ref-type="supplementary-material"}). The KEGG module with the largest significant increase in mean proportion during menses was "M00240: Iron complex transport system", suggesting a shift in the microbiome that might be explained by pathways that utilize the iron-rich environment provided during menstruation. Discussion {#S9} ========== The application of PICRUSt to diverse metagenomic data sets shows that the phylogenetic information contained in 16S marker gene sequences is sufficiently well correlated with genomic content to yield accurate predictions when related reference genomes are available. Our validation results support widespread application of PICRUSt to 16S datasets containing as few as a few hundred sequences per sample, provided that NSTI or a similar measure is used to quantify the expected prediction accuracy. Although PICRUSt's predictive approach neither precludes nor outperforms deep metagenomic sequencing, it can predict and compare probable functions across many samples from a wide range of habitats at a small fraction of the cost of such sequencing. This approach thus opens up new avenues for tiered, more cost-effective study designs and provides functional insights into the tens of thousands of existing samples for which only 16S data is available. To best leverage the strengths both of (meta)genomic sequencing and of PICRUSt, we recommend its incorporation into marker gene studies using a deliberate, tiered approach. Because phylogenetic dissimilarity among environmental organisms and sequenced genomes (as captured by NSTI) affects PICRUSt accuracy, NSTI values can be calculated from preliminary 16S rRNA data to assess whether reference genome coverage is sufficiently dense to allow for accurate PICRUSt prediction. If adequate reference genomes are not available, additional genome sequences can be collected to fill in phylogenetic "gaps" in the reference database and allow for accurate prediction. This can be performed either through traditional culture-based techniques, single-cell genomic approaches or deep metagenomic sequencing of samples targeted based on 16S data. If NSTI appears sufficient but additional controls are desired, a preliminary set of paired 16S rRNA and shotgun metagenomic samples can be compared using PICRUSt's built-in tools to empirically test prediction accuracy on the sample types of interest. On the basis of such validations from select samples, PICRUSt can then be used to extend approximate functional information from a few costly metagenomes to much larger accompanying 16S rRNA gene sequence collections. However, the limitations of this approach must be considered in interpreting PICRUSt predictions. For example, only 16S marker gene sequences corresponding to bacterial and archaeal genomes are currently included; thus this version of the system does not infer viral or eukaryotic components of a metagenome. PICRUSt's ability to detect patterns also depends on the input data used: the software cannot distinguish variation at the strain level if the marker gene sequence used is identical among strains, and it cannot detect genes families (or summarize them into pathways) if those genes are not included in the input genomic data used, or if pathway annotations are currently poor (e.g. for acetogenesis genes). However, because PICRUSt can accept trees produced by alternative marker genes or gene/pathway annotations, users have the flexibility to customize the tool to meet the needs of their system. Although high overall accuracy was obtained despite microbial lateral gene transfer and other processes of gene gain and loss, gene families or pathways (e.g. methane oxidation) with highly variable distribution throughout the tree of life can still lead to incorrect predictions in individual cases. PICRUSt thus provides confidence intervals for each functional abundance prediction that reflect the degree of variation in that function among sequenced phylogenetic neighbors of predicted (meta)genomes, with wide confidence intervals indicating a high degree of uncertainty ([Supplemental Fig. 7](#SD2){ref-type="supplementary-material"}). If individual gene abundances (rather than aggregate patterns) are of interest, users can choose to either discard predictions with low confidence, or confirm them experimentally. We anticipate several experimental and computational improvements that will further refine the predictive accuracy of PICRUSt. In addition to extending genome coverage and metagenome calibration as above, PICRUSt predictions could also likely be improved by including habitat information in a predictive model. This may provide additional predictive power in that some genes might correlate strongly with environmental parameters as well as phylogenetic similarity to reference organisms^[@R9],\ [@R16]^. Modification of prediction methods that incorporate information from partial genome sequences could expand the sensitivity of predictions in under-studied environments by including additional reference gene content information. Finally, as reference genome sequence databases continue to expand and incorporate isolates from ever more diverse environments, the prediction accuracy of PICRUSt will improve by default over time. Predictive metagenomics thus holds the promise of uniting completed genome sequences, 16S rRNA gene studies and shotgun metagenomes into a single quantitative approach for assessing community function. Methods {#S10} ======= Reference genomes and 16S data used by PICRUSt {#S11} ---------------------------------------------- PICRUSt requires a phylogenetic tree of marker genes that includes both tips with known data (e.g. complete reference genomes) and unknown tips (e.g. environmental sequences). Although any type of marker gene tree could be used with PICRUSt, the 16S 'tax2tree' version of Greengenes^[@R45]^ was downloaded and used for all presented research. Similarly, PICRUSt can make inferences about any type of continuous trait, but for this research we used the popular KEGG^[@R26]^ and COG^[@R27]^ databases for annotations. Specifically, we obtained all KEGG Ortholog (KO) and COG annotations from v3.5 of IMG^[@R25]^ to produce a table of 6,885 KO and 4715 COG abundances for 2,590 genomes that had identifiers in the Greengenes reference tree. The number of copies of the 16S gene in each of these genomes was also obtained from IMG. The PICRUSt algorithm {#S12} --------------------- PICRUSt begins by formatting the marker phylogenetic tree and functional annotation file in preparation for ancestral state reconstruction. This includes creation of internal node labels in the tree, matching tree tips with reference genomes to the annotation file and creating a pruned version of the tree that contains only tips with corresponding reference genomes. An ancestral state reconstruction method is then applied to the pruned tree. This provides predicted values for each of the KOs (and the additional 16S copy number trait) for all internal nodes in the pruned tree. Four different ancestral state reconstruction methods were tested including Wagner Parsimony from the COUNT package (v11.0502)^[@R46]^ and ACE ML, ACE REML and ACE PIC of the APE R library (v2.8)^[@R47]^. The next step makes predictions for all tips in the reference tree that do not have corresponding genomes using the inferences for the internal nodes from the ancestral state reconstruction step. A prediction of gene content is generated using an average of the contents of extant and inferred ancestral genomes, weighted exponentially by the reciprocal of phylogenetic distance. This causes very closely related existing or ancestral genomes to be counted much more heavily than more distant relatives, and it is also consistent with previous research suggesting an exponential relationship between 16S phylogenetic distance and gene content conservation^[@R9]^. (Confidence intervals on this prediction are also optionally calculated when using any of the ACE methods ([Supplemental Fig. 7](#SD2){ref-type="supplementary-material"}).) It is important to note that the prediction of gene content for tips in the trees without reference genomes is an estimate only, and that although our method does model gene gain and loss, some instances of gain or loss or laterally transferred genes will be poorly predicted (with broad confidence intervals as a result). This is rare in practice, however, as validated at the genome and metagenome level by comparing our predictions with the known gene contents from actual sequencing (see below). This genome prediction step only needs to be precomputed once, resulting in a pre-calculated file that is provided with the PICRUSt package containing predicted genome contents for all tips in the marker reference tree. For metagenome prediction, PICRUSt takes an input OTU table that contains identifiers that match tips from the marker gene (e.g. greengenes identifiers) with corresponding abundances for each of those OTUs across one or more samples. First, PICRUSt normalizes the OTU table by the 16S copy number predictions so that OTU abundances more accurately reflect the true abundances of the underlying organisms. The metagenome is then predicted by looking up the pre-calculated genome content for each OTU, multiplying the normalized OTU abundance by each KO abundance in the genome and summing these KO abundances together per sample. The prediction yields a table of KO abundances for each metagenome sample in the OTU table. For optional organism-specific predictions, the per-organism abundances are retained and annotated for each KO. Paired 16S and metagenome validations and metagenome predictions from 16S data {#S13} ------------------------------------------------------------------------------ Several microbiome studies that included both 16S sequencing and WGS metagenome sequencing for the same samples were used to test the accuracy of PICRUSt. These included 530 paired human microbiome samples^[@R22]^, 39 paired mammal gut samples^[@R24]^, 14 paired soil samples^[@R34]^, 10 paired hypersaline microbial mats^[@R23],\ [@R24]^ and two even/staggered synthetic mock communities from the HMP^[@R33]^. We additionally used PICRUSt to make predictions on three 16S-only microbiome studies, specifically 6,431 HMP samples (<http://hmpdacc.org/HMQCP>), 993 vaginal time course samples^[@R43]^ and 335 coral mucus samples(<http://www.microbio.me/qiime/>; Study ID 1854). For 16S data, PICRUSt-compatible OTU tables were constructed using the closed-reference OTU picking protocol in QIIME 1.5.0-dev (pick_reference_otus_through_otu_table.py) against Greengenes+IMG using 'uclust'^[@R48]^. For paired metagenomes, WGS reads were annotated to KOs using v0.98 of HUMAnN^[@R30]^. Expected KO counts for the HMP mock communities were obtained by multiplying the mixing proportions of community members by the annotated KO counts of their respective reference genomes in IMG. PICRUSt was used to predict the metagenomes using the 16S-based OTU tables, and predictions were compared to the annotated WGS metagenome across all KOs using Spearman rank correlation. In addition, KOs were mapped to KEGG Module abundances, following the conjugative normal form as implemented in HUMAnN script "pathab.py" for the HMP and vaginal datasets to compare modules and pathways. Bray-Curtis distances (for Beta-diversity comparison between OTU or PICRUSt KO abundances across samples) were calculated using as implemented in the QIIME "beta_diversity.py" script. The PCA plot and identification of KEGG modules with significant mean proportion differences for both the HMP and vaginal datasets was created using STAMP v2.0^[@R36]^. The Nearest Sequenced Taxon Index (NSTI) was developed as an evaluation measure describing the novelty of organisms within an OTU table with respect to previously sequenced genomes. For every OTU in a sample, the sum of branch lengths between that OTU in the Greengenes tree to the nearest tip in the tree with a sequenced genome is weighted by the relative abundance of that OTU. All OTU scores are then summed to give a single NSTI value per microbial community sample. PICRUSt calculates NSTI values for every sample in the given OTU table, and we compared NSTI scores and PICRUSt accuracies for all of the metagenome validation datasets. In the metagenome rarefaction analysis ([Fig. 4](#F4){ref-type="fig"}), a given number of counts were randomly selected from either the collection of microbial OTUs for each sample (i.e. the 16S rRNA OTU table) or the collection of sequenced genes in that sample using the multiple_rarefactions.py script in QIIME 1.5.0-dev^[@R29]^. To estimate the number of raw reads at which PICRUSt outperforms metagenomic sequencing the annotated shotgun reads were transformed to total sequenced reads by dividing by the mean annotation rates from the original manuscript (17.3%), while 16S rRNA reads were transformed using the success rate for closed-reference OTU picking at a 97% 16S rRNA identity threshold (68.9%). Both the subsampled metagenome and the PICRUSt predictions from the subsampled OTU table were compared for accuracy using Spearman rank correlation versus the non-subsampled metagenome. Single-genome, phylogenetic and pathway-specific validations {#S14} ------------------------------------------------------------ The accuracy of metagenomic prediction depends on accurate prediction of the gene families (e.g. KOs) present in unsequenced organisms. The accuracy of this gene content prediction step was assessed by using fully sequenced genomes (in which gene content is known) as controls. A test dataset was generated for each sequenced genome in IMG in which that genome was excluded from the reference gene by genome table. PICRUSt was then used to infer the content of the excluded genome. Subsequently, this predicted gene content was compared against the actual gene content, i.e. the sequenced genome annotations. The results were compared using Spearman rank correlation for the actual versus estimated number of gene copies in each gene family or using accuracy and/or balanced accuracy for presence/absence evaluations. These results are presented as the 'genome holdout' dataset. In addition to using this dataset to calculate the accuracy of each genome, it was also used to calculate the accuracy per functional gene category. This was done by first mapping KOs to KEGG Modules (described above) for each genome (for both real and PICRUSt predictions) and then comparing each module across all genomes. For visualization, the accuracy of each module was mapped into more general functional categories using the BRITE hierarchy^[@R26]^. The accuracy of PICRUSt across different taxonomic groups in the phylogenetic tree of bacteria and archaea was visualized using GraPhlAn v0.9 (<http://huttenhower.sph.harvard.edu/graphlan>). The phylogenetic tree for display was constructed by pruning the Greengenes tree down to tips with corresponding genomes as above, with taxonomic labels at the phylum and genus level obtained for each genome from NCBI Taxonomy^[@R49]^. We expected that the accuracy of PICRUSt's predictions would decrease when large phylogenetic distances separated the organism of interest and the nearest sequenced reference genome(s). To test this expectation, 'distance holdout' datasets were constructed. These datasets were constructed in the same manner as 'genome holdout' datasets described above, except that all genomes within a particular phylogenetic distance (on the 16S tree) of the test organism were excluded from the reference dataset. For example, when predicting *Escherichia coli* MG1655, a distance holdout of 0.03 substitutions/site would exclude not only that genome, but also all other *E. coli* strains. These tests were conducted at phylogenetic distances ranging from 0.0 to 0.50 substitutions/site in the full-length 16S rRNA gene, in increments of 0.03 substitutions/site. Finally, we tested the effects of local inaccuracy in tree construction on PICRUSt's performance. These 'tree randomization holdouts' were constructed the same as the 'genome holdout' dataset (above), except that in addition to excluding one genome, the labels of all organisms within a specified phylogenetic distance of the test organism were randomized on the 16S tree. For example, our 'tree randomization holdout' targeting *E.coli* with a distance of 0.03 scrambled the phylogeny of all reference *E.coli* strains around the tip to be predicted, while leaving the rest of the tree intact. These tests were conducted at phylogenetic distances ranging from 0.0 to 0.50 substitutions/site in the 16S rRNA gene, in increments of 0.03 substitutions/site. Supplementary Material {#S15} ====================== We would like to thank Adam Robbins-Pianka and Nicola Segata, along with all members of the Knight, Beiko, Vega Thurber, Caporaso and Huttenhower labs, for their assistance during PICRUSt conception and development. This work was supported in part by the Canadian Institutes of Health Research (MGIL,RGB), the Canada Research Chairs program (RGB), NSF OCE \#1130786 (RVT,DB), the Howard Hughes Medical Institute (RK), NIH P01DK078669, U01HG004866, R01HG004872 (RK), the Crohn's and Colitis Foundation of America (RK), the Sloan Foundation (RK), NIH 1R01HG005969 (CH), NSF CAREER DBI-1053486 (CH), and ARO W911NF-11-1-0473 (CH). ![The PICRUSt workflow. PICRUSt is composed of two high-level workflows: *gene content inference* (top box) and *metagenome inference* (bottom box). Beginning with a reference OTU tree and a gene content table (i.e., counts of genes for reference OTUs with known gene content), the *gene content inference* workflow predicts gene content for each OTU with unknown gene content, including predictions of marker gene copy number. This information is precomputed for 16S based on Greengenes^[@R28]^ and IMG^[@R25]^, but all functionality is accessible in PICRUSt for use with other marker genes and reference genomes. The *metagenome inference* workflow takes an OTU table (i.e., counts of OTUs on a per sample basis), where OTU identifiers correspond to tips in the reference OTU tree, as well as the copy number of the marker gene in each OTU and the gene content of each OTU (as generated by the *gene content inference* workflow) and outputs a metagenome table (i.e. counts of gene families on a per-sample basis).](nihms510850f1){#F1} {#F2} {#F3} {#F4} {#F5} {#F6} [^1]: These authors contributed equally.

Introduction ============ Rheumatoid arthritis (RA) is an autoimmune disease that is mainly mediated by cytokines and is characterized by the abnormal proliferation of synovial cells, the massive infiltration of inflammatory cells and the progressive destruction of joints ([@b1-etm-0-0-8557]). T helper cells (T~h~) are divided into T~h~1, T~h~2 and T~h~17 subgroups according to the different expression profiles of secretory cytokines ([@b2-etm-0-0-8557]). It has become clear in the last few years that T cell-derived cytokines expressed preferentially by T~h~1 cells contribute to joint destruction and inflammatory response in RA, whereas T~h~2 cell-associated cytokines may be protective ([@b2-etm-0-0-8557],[@b3-etm-0-0-8557]). However, recent research on T~h~17 cells and regulatory T cells (Treg) has demonstrated that an imbalance between T~h~1/T~h~2/T~h~17/Treg cells is important to the pathogenesis of RA ([@b4-etm-0-0-8557]). Although the origin of RA is unclear, it has been determined that lymphocytes accumulate around the terminal blood vessels of the subsynovial layer prior to the inflammatory response ([@b7-etm-0-0-8557]). The synovium is composed of two layers, namely the synovial lining layer (lining cells) and the lower synovial lining layer (or supporting layer) ([@b8-etm-0-0-8557]). Another major feature of RA is the abnormal proliferation of synovial cells in the lining layer and the expression of transformed cells, which erode the surrounding bone and cartilage ([@b7-etm-0-0-8557]). Increasing evidence has confirmed that the activation state of RA synovial fibroblasts (RASFs) is critically dependent on TLR expression, which in turn is known to serve an essential role in T cell differentiation and function ([@b10-etm-0-0-8557]). TLR2 and TLR4 have been revealed to serve important functions in pathogenesis of RA and they has been determined that the expression of TLR-2 and TLR-4 is increased and regulated by proinflammatory cytokines that are present in the synovial compartment ([@b10-etm-0-0-8557]). Activation of these RASF-expressed TLRs exacerbates inflammatory T~h~1 and T~h~17 cell expansion in cell-cell contact-dependent and inflammatory cytokine-dependent pathways, inducing the increased accumulation of interferon (IFN)-γ and interleukin (IL)-17([@b11-etm-0-0-8557]). Targeting TLRs may therefore modulate inflammation in RA and provide novel therapeutic strategies for overcoming this persistent disease ([@b12-etm-0-0-8557]). Xixin is isolated from the dried roots and rhizomes of *Asarum heterotropoides* f. *mandshuricum* (Maximowicz) Kitagawa, *A. sieboldii* Miq. var. *seoulense* Nakai and *A. sieboldii* Miq. (Aristolochiaceae), and is a commonly used herbal medicine in China ([@b13-etm-0-0-8557]). Xixin is used to treat colds, fever, chills, headaches, acute toothaches, sinusitis, coughs and RA in traditional Chinese medicine ([@b13-etm-0-0-8557]). Asarinin (molecular weight, 354.35) is one of the main active chemical components isolated from Xixin ([@b13-etm-0-0-8557]). Many pharmacological effects have been identified in asarinin, including anti-inflammatory effects, antipyretic properties and immune inhibition ([@b13-etm-0-0-8557]). A previous study indicated that asarinin significantly inhibited the macroscopic score and cartilage destruction of collagen-induced arthritis ([@b17-etm-0-0-8557]). However, little is known about the effect of asarinin on RA synovial fibroblasts. The present study aimed to determine the pharmacological profile of asarinin and its potential effect on RA ([@b18-etm-0-0-8557]). The present study used RA synovial fibroblasts to examine the effect of asarinin and the possible roles of T~h~17, T~h~1 and TLRs in the pathogenesis of arthritis. Materials and methods ===================== ### Reagents Recombinant Human tumor necrosis factor (TNF)-α and IL-1β were obtained from PeproTech China. Fetal bovine serum and high-glucose DMEM were purchased from HyClone; GE Healthcare Life Sciences. Trypsin-EDTA solution and the Trypan Blue Staining Cell Viability Assay kit were purchased from Beyotime Institute of Biotechnology. The RNA PCR kit (EX TAQ R-PCR Version 2.1) was obtained from Takara Biotechnology Co., Ltd., and primers and probes were purchased from Sangon Biotech Co., Ltd. TRIzol^®^ reagent was purchased from Invitrogen. Asarinin was purchased from Chengdu Must Bio-Technology Co., Ltd., and ELISA kits for TNF-α (cat. no. 555212), IL-6 (cat. no. 555220) and IFN-γ (cat. no. 555142) from BD Biosciences. The ELISA kit for IL-17A (cat. no. D1700) was obtained from R&D Systems, Inc. Primary antibodies for TNF-α (cat. no. TA808184), IL-17A (cat. no. TA337063), IL-6 (cat. no. TA500067) and IFN-γ (cat. no. TA353236) were from OriGene Technologies, Inc. Peptidoglycan (PGN) and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich. ### Preparation of the asarinin-medicated serum Asarinin and olive oil were mixed in a ratio of 5:1 to form a suspension for the asarinin-medicated serum. The asarinin-medicated serum was generated according to previous studies ([@b19-etm-0-0-8557],[@b20-etm-0-0-8557]). A total of 20 female Sprague-Dawley (age range, 6-8 weeks; weight 200-250 g) were supplied by the Experimental Animals Department of the Second Affiliated Hospital of Harbin Medical University. Animals were housed in an airconditioned room (temperature, 22±2˚C; humidity, 55±5%) with a 12 h light-dark cycle and free access to a standard diet with free access to tap water. Sprague-Dawley (SD) rats were randomly divided into an asarinin group and a blank serum group. Asarinin (30 mg/kg) was administered orally to SD rats twice daily for 5 days. Rats in the blank serum group received saline twice daily for 5 days. The same dose was administered orally to the blank serum group and the asarinin group. Then, 1 h after the last administration, rats were anesthetized via intraperitoneal injection of pentobarbital and blood samples (10 ml) were collected from the abdominal aorta for subsequent experiments. The Ethics Committee for Experimental Animals of Harbin Medical University reviewed and approved the current study and all animals were treated according to the guidelines of the animal ethics committee. ### Rat plasma sample preparation for high-performance liquid chromatography (HPLC) analysis Rat medicated sera and blank sera were subjected to HPLC analysis using a Thermo Fisher Ultimate 3000 UHPL (Thermo Fisher Scientific, Inc.) ([@b21-etm-0-0-8557],[@b22-etm-0-0-8557]). Chromatographic conditions: The C18 column used was supplied by Thermo Fisher Scientific, Inc. (4.6x150 mm; 5 µm). The mobile phase consisted of acetonitrile-water (50:50) and the running time was 32 min. Additionally, the column temperature was 30˚C and the flow rate 1 ml/min. The detection wavelengths were set at 287 nm with a sample size of 10 µl. Asarinin serum (1 ml) was obtained and dissolved by ultrasonography with methanol added to a final volume of 5 ml. Sera were filtered through 0.45 microporous membranes for analysis. ### Patients, tissue specimens and ethics statement Synovial tissue specimens used for the culture of OA synovial fibroblasts (OASFs; n=3) and RASFs (n=4) were obtained from the knees of patients following joint replacement surgery, which was a procedure performed in patients who had severe, long-term disease and who had received numerous therapies over many years. All patients fulfilled the American College of Rheumatology 2010 criteria for RA and the 1995 criteria for osteoarthritis (OA) and provided their written consent to participate in the current study ([@b23-etm-0-0-8557],[@b24-etm-0-0-8557]). Synovial tissue specimens from 4 patients with RA \[2 females (mean age, 67.9±4.8 years) and 2 males (age, 70.1±3.5 years)\] and 3 patients with OA \[3 females (age, 68.2±5.3 years)\]. Patients were recruited from September 2017 to September 2018.The study protocols, consent forms and consent procedure were approved by the Institutional Medical Ethics Review Board of the Second Affiliated Hospital of Harbin Medical University. ### Synovial fibroblast (SF) isolation, culture and treatment Human SFs were isolated from synovial tissue obtained at the time of knee replacement surgery from 4 patients with RA and 3 patients with OA. Synoviocytes were trypsinized and suspended in DMEM containing 10-20% fetal bovine serum, inoculated in a culture flask and cultured in a cell incubator at 37˚C and 5% CO~2~. The following day, the medium was changed, and the unattached cells were discarded. The cells that remained adherent were considered to be synovial cells. The medium was subsequently changed once every 3-4 days and the culture solution was discarded once the synovial cells reached 80-90% confluence in the flask. Cells were then rinsed twice with PBS. Digestion was terminated when the majority of cells changed shape from diamond to round and were bright in appearance. Cells were generally passaged at a ratio of 1:2 or 1:3. After 3 generations, it was revealed that 95% of cells were fibroid synovial cells, which were used in the present study. OASFs and RASFs were pretreated with IL-1β (2 ng/ml) and TNF-α (10 ng/ml). OASFs (5x10^6^ cells) and RASFs (5x10^6^ cells) per well were stimulated with medicated serum of asarinin, toll-like receptor (TLR)2 ligand PGN (10 ng/ml) and TLR4 ligand LPS (100 ng/ml). ### Cell viability determination OASFs (5x10^6^ cells) and RASFs (5x10^6^ cells) were seeded into 96-well plates and treated with medicated serum of asarinin at a series of concentrations (5, 10, 15, 20, 25 or 30%) for 6 h or medicated serum of asarinin (15%) for 6, 12, 18 or 24 h. Asarinin at a dosage of 15% was selected as cell death did not affect subsequent experiments. Cell suspensions in PBS (10 µl) were mixed with trypan blue (40 µl) for 5 min at room temperature and the number of stained (dead) and unstained (alive) cells were counted using a hemocytometer ([@b25-etm-0-0-8557],[@b26-etm-0-0-8557]). ### ELISA OASFs (5x10^6^ cells) and RASFs (5x10^6^ cells) per well were stimulated with medicated serum of asarinin (15%) for 6 h, after which the culture supernatants were collected and stored at -20˚C for subsequent ELISA. Levels of TNF-α, IFN-γ, IL-17A and IL-6 in cell culture supernatants were measured using ELISA kits, in accordance with the manufacturer\'s protocol. ### RNA preparation and reverse transcription-quantitative (RT-q) PCR OASFs (5x10^6^ cells) and RASFs (5x10^6^ cells) per well were stimulated with medicated serum of asarinin (15%) for 6 h and the cells were harvested and stored at -80˚C until analysis. Total mRNA was isolated from synovial fibroblasts using TRIzol^®^ and cDNA was synthesized from 1 µg total RNA using oligo-dT primers and AMV reverse transcriptase according to the manufacturer\'s protocol. The conditions of reverse transcription were as follows: 30˚C for 10 min, 42˚C for 30 min, 99˚C for 5 min and 5˚C for 5 min. Samples were stored at -20˚C until further use. The following primer sequences were used for RT-qPCR. β-actin forward, 5\'-AGCGGTTCCGATGCCCT-3\' and reverse, 5\'-AGAGGTCTTTACGGATGTCAACG-3\'; IFN-γ forward, 5\'-TGAACGCTACACACTGCATCTTGG and reverse, 5\'-CGACTCCTTTTCCGCTTCCTG G-3\'; IL-17A forward, 5\'-AGTGAAGGCAGCAGCGATCAT-3\' and reverse, 5\'-CGCCAAGGGAGTTAAAG-3\'; TNF-α forward, 5\'-TCTCATCAGTTCTATGGCCC-3\' and reverse, 5\'-GGGAGTAGACAAGGTACAAC-3\'; IL-6 forward, 5\'-TCCAGTTGCCTTCTTGGGAC-3\' and reverse, 5\'-GTGTAATTAAGCCTCCGACTTG-3\'; TLR2 forward, 5\'-ACCAAGTGAAGGTACCTGTGGGGC-3\' and reverse, 5\'-GCACCAGAGCCTGGAGGTTCAC-3\'; TLR4 forward, 5\'-CCCCGACAACCTCCCCTTCTCA-3\' and reverse, 5\'-TCCAGAAAAGGCTCCCAGGGCT-3\'. RT-qPCR was performed at a final concentration of 0.15 µl primer and 0.25 µl of Ex Taq HS polymerase (Takara Biotechnology Co., Ltd.) in standard PCR buffer. Transcripts were quantified using EX TAQ R-PCR. PCR was performed with a reaction mix comprising 0.5 µl forward and reverse primers, 2 µl cDNA and 1 µl TaqMan probe template to a total volume of 25 µl. PCR was initiated for 2 min at 95˚C, continued with 40 cycles of 10 sec at 95˚C, 40 sec at 60˚C and final extension for 5 min at 72˚C. The fold changes in the expression of each gene were calculated using the 2^-ΔΔ^Cq method ([@b27-etm-0-0-8557]), with the housekeeping gene β-actin mRNA as an internal control. ### Western blot analysis RASFs (5x10^6^ cells) and OASFs (5x10^6^ cells) were pretreated with IL-1β (2 ng/ml) and TNF-α (10 ng/ml), and incubated with medicated serum of asarinin at 37˚C for 6 h. The cells were harvested and stored at -80˚C until analysis. Ice-cold lysis buffer (40 ml) was used to lyse cells. Protein concentration was measured using the BCA method. Samples (10 µl) were separated using SDS-PAGE in a 10% polyacrylamide gel and transferred electronically to a PVDF membranes. The membranes were then blocked with Tris-buffered saline containing 0.1% Tween-20 and 5% nonfat milk at 37˚C for 1 h. Samples were then incubated with the following primary antibodies at 4˚C overnight: IFN-γ, IL-6, TNF-α and IL-17A (1:500). After three washes with PBST (0.5% Tween 20 in PBS), membranes were incubated with horseradish peroxidase-conjugated IgG (cat. no. PV6002; OriGene Technologies, Inc.) secondary antibodies for 1 h at 37˚C. Samples were visualized via chemiluminescence using an ECL Plus Detection kit (Beyotime Institute of Biotechnology). The protein expression levels were then determined by analyzing the signals captured on the PVDF membranes using a ChampGel 5500 (Beijing Sage Creation Science Co, Ltd.). ### Statistical analysis Samples were run in triplicate and the data were presented as the mean ± standard deviation. Statistically significant differences were analyzed via one-way analysis of variance followed by and SNK test using SPSS software 13.0 for Windows (SPSS, Inc.). P\<0.05 was considered to indicate a statistically significant difference. Results ======= ### HPLC of medicated serum of asarinin The chemical structures of asarinin are provided in [Fig. 1A](#f1-etm-0-0-8557){ref-type="fig"}. No peak for asarinin was detected in the blank serum group ([Fig. 1B](#f1-etm-0-0-8557){ref-type="fig"}) but was present for the control asarinin ([Fig. 1C](#f1-etm-0-0-8557){ref-type="fig"}) and in rat medicated serum ([Fig. 1D](#f1-etm-0-0-8557){ref-type="fig"}). The extra peak (\~26.635 min) may represent a secondary metabolite of asarinin ([Fig. 1D](#f1-etm-0-0-8557){ref-type="fig"}). ### Medicated serum of asarinin inhibits human RASF proliferation To examine the effect of medicated serum of asarinin on the proliferation of RASFs, trypan blue staining was performed to detect the viability of RASFs. The results demonstrated that medicated serum of asarinin decreased the viability of RASFs and OASFs in a dose- and time-dependent manner ([Fig. 2A-C](#f2-etm-0-0-8557){ref-type="fig"}). A significant decrease was observed in the cell number of RASFs compared with that of OASFs ([Fig. 2D](#f2-etm-0-0-8557){ref-type="fig"}). ### Regulation of inflammatory cytokines on rheumatoid synoviocytes by medicated serum of asarinin RA and OA samples were collected from the discarded tissue of patients following knee joint replacement surgery. Third generation rheumatoid synoviocytes and osteoarthritic synoviocytes were used in the current study. The results revealed that third generation rheumatoid synoviocytes and osteoarthritic synoviocytes exhibited fibroblast-like synovial cell morphology ([Fig. 3A](#f3-etm-0-0-8557){ref-type="fig"}). TNF-α, IFN-γ, IL-6 and IL-17A are important inflammatory mediators in RA ([@b2-etm-0-0-8557]). RT-qPCR analysis for IL-17A, TNF-α, IFN-γ and IL-6 expression revealed a decrease following asarinin treatment compared with control and treated cells ([Fig. 3B](#f3-etm-0-0-8557){ref-type="fig"}). Western blot analyses for TNF-α, IFN-γ, IL-6 and IL-17A expression also revealed a decrease compared with the controls ([Fig. 3C](#f3-etm-0-0-8557){ref-type="fig"}). The effect of asarinin on IL-17A, TNF-α, IFN-γ and IL-6 in RASFs was also measured by ELISA, revealing similar results ([Fig. 3D](#f3-etm-0-0-8557){ref-type="fig"}). ### Inhibition of TLR2 and TLR4 on rheumatoid synoviocytes by medicated serum of asarinin RT-qPCR analysis for TLR2 and TLR4 expression revealed a decrease following asarinin treatment ([Fig. 4](#f4-etm-0-0-8557){ref-type="fig"}). Discussion ========== Xixin is a commonly used herbal medicine in China and other Asian countries. Asarinin is one of the main active chemical components isolated from Xixin ([@b13-etm-0-0-8557]). Despite a previous study revealing that asarinin significantly inhibited the macroscopic score and cartilage destruction of collagen-induced arthritis ([@b17-etm-0-0-8557]), little is known about the effect of asarinin on RA synovial fibroblasts. The present study therefore aimed to assess the effects of asarinin on RASFs. RA is an autoimmune disease characterized by chronic inflammation that leads to joint destruction ([@b28-etm-0-0-8557]). Although the pathogenesis of RA, mediated by T cells, is not completely clear, certain T~h~1-type cytokines, including IFN-γ, TNF-α and IL-1, T~h~2-type cytokines, including IL-4 and IL-10, T~h~17 cytokines, including IL-17 and IL-22, and Treg cells interact to activate synovial macrophages, fibroblasts and osteoclasts to affect chronic inflammation and joint destruction ([@b29-etm-0-0-8557]). RASFs are leading cells in joint erosion and contribute actively to inflammation. Synovial cells are the most important cells in the synovial layer. The primary target of RA immune activity is located in the synovium. There are two types of cells in the synovial layer: Type A (macrophage-like synovial cells) and type B (fibroblast-like synoviocytes; FLSs) ([@b33-etm-0-0-8557]). Synovial cells, particularly FLSs of the synovial layer, participate in the function of motor joints, producing joint lubrication fluids and maintaining the compliance and integrity of the synovial fluid, cartilage nutrition and the synovial layer ([@b33-etm-0-0-8557],[@b34-etm-0-0-8557]). Rheumatoid arthritis synovial fibroblasts are leading cells in joint erosion and contribute actively to inflammation, and enhance cell proliferation and invasiveness under the action of various cytokines and growth factors ([@b28-etm-0-0-8557]). During the disease progression of RA, the fibroid synovial cells function as passive responders and active invaders after transformation ([@b35-etm-0-0-8557]). Therefore, the present study used third generation rheumatoid synoviocytes and osteoarthritic synoviocytes. Since certain compounds of asarinin may change at room temperature after being dissolved in solvents, the current study used medicated serum of asarinin on cells. The results demonstrated that medicated serum of asarinin decreased the viability of RASFs and OASFs in a time-dependent manner within 24 h. Furthermore, when the concentration and duration of drug-containing serum were significantly increased, the number of RASFs was significantly reduced and the number of OASFs was correspondingly reduced. However, the reduction of the latter was less compared with that of the former. When OASFs and RASFs were treated with blank serum, no significant decrease in cell viability was observed. FLSs of RA exhibit tumor-like growth characteristics, and the excessive abnormal proliferation and apoptosis of these cells serve an important role in synovial inflammation and bone destruction in RA ([@b36-etm-0-0-8557]). The effect of asarinin on FLS may be associated with the immunosuppressive effect of asarinin. However, whether the medicated serum of asarinin causes RASF necrosis or apoptosis requires elucidation. To clarify the mechanism of action of asarinin on RA, OASFs were used as control cells to detect the cytokines associated with RA pathogenesis. The results demonstrated that asarinin downregulated the expression of TNF-α, IFN-γ, IL-6 and IL-17A in RA synovial cells. The expression of T~h~2 cytokines (IL-4 and IL-5) were too low to be detected (data not shown). TNF-α, IFN-γ, IL-6 and IL-17A were also expressed in OA synovial cells, but TNF-α, IFN-γ, IL-6 and IL-17A expression was low and no significant differences were observed before and after asarinin treatment. IFN-γ, IL-6, TNF-α and IL-17A are inflammatory cytokines and IFN-γ and TNF-α are T~h~1-type cytokines ([@b6-etm-0-0-8557]). IL-6 is also the main inflammatory factor in RA joint inflammation, but its pathogenic effect is different from that of IL-1 and TNF-α, which mainly induces the production of immunoglobulins and the formation of acute phase proteins ([@b37-etm-0-0-8557]). Although T~h~2 cells secrete IL-6, IL-6 still serves a role as an inflammatory cytokine. Additionally, T~h~17 cells secrete IL-17, which is generally considered an important inflammatory mediator that modulates local infiltration and tissue injury of inflammatory cells by inducing the expression of other inflammatory cytokines, such as IL-6 and TNF-α, and chemokines, such as monocyte chemotactic protein 1 and macrophage inflammatory protein-2 ([@b1-etm-0-0-8557],[@b6-etm-0-0-8557]). Mice deficient in IL-17 exhibit reduced severity of arthritis, and mice with increased IL-17 levels exhibit exacerbated levels of the disease ([@b41-etm-0-0-8557],[@b42-etm-0-0-8557]). Inflammatory cytokines are selectively recruited to the synovial cavity in RA and various relevant cytokines in the synovial tissue were detected in the present study ([@b13-etm-0-0-8557],[@b43-etm-0-0-8557]). IL-1 and TNF-α stimulate the proliferation of FLS to secrete cytokines and prostaglandins, inducing FLS proliferation ([@b44-etm-0-0-8557]). FLS produce large numbers of cytokines (including IL-1, TNF-α and IL-6) in spontaneous and stimulating conditions ([@b45-etm-0-0-8557]). The cytokine production profiles by synovial cells in patients with RA and OA are similar regarding the types of cytokines produced ([@b44-etm-0-0-8557],[@b48-etm-0-0-8557]). However, the quantity of inflammatory cytokines secreted in patients with RA is significantly higher compared with that in patients with OA, indicating that synovial cells may be excessively activated in patients with RA ([@b49-etm-0-0-8557]). Streptococcal cell wall induced arthritis is markedly reduced in TLR2^-/-^ mice and TLR4 deficiency results in impaired osteoclast formation as well as a reduction in Th-17 inducing cytokines such as IL-1, IL-6 and IL-23 in the same animals ([@b50-etm-0-0-8557]). TLR4 and TLR2 serve crucial roles in the production of inflammatory cytokines and TLR activation exacerbates RASF-mediated inflammatory T~h~1 and T~h~17 responses ([@b11-etm-0-0-8557]). In conclusion, the inhibitory effect of asarinin drug serum on human RASFs may be achieved by inhibiting T~h~1 and T~h~17 cytokines via the suppression of TLR2 and TLR4. Not applicable. Funding ======= The present study was supported by the Fund of Heilongjiang Science and Technical Office (grant no. QC2011C059), the Harbin Science and Technology Bureau of Heilongjiang Province (grant no. 2012RFQXS020), the Postdoctoral Foundation of China and Heilongjiang Province (grant nos. 2013M531081, LBH-Z11004 and LBHQ15138) and the Excellent Innovative Talents Support Program of Heilongjiang University of Traditional Chinese Medicine (Excellent Young Academic Leader; grant no. 2018). Availability of data and materials ================================== The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Authors\' contributions ======================= QD, YL and JL conceived and designed the current study, and wrote and revised the manuscript. QD, MW and YZL conducted the experiments and analyzed and interpreted the data. All authors read and approved the final manuscript. Ethics approval and consent to participate ========================================== The animal experiments were performed in accordance with the guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Harbin Medical University. The study protocols, consent forms and consent procedure were approved by the Institutional Medical Ethics Review Board of the Second Affiliated Hospital of Harbin Medical University. Patient consent for publication =============================== Not applicable. Competing interests =================== The authors declare that they have no competing interests. {#f1-etm-0-0-8557} {#f2-etm-0-0-8557} {#f3-etm-0-0-8557} {#f4-etm-0-0-8557}

CASE DESCRIPTION ================ A 56-year-old male with history of alcohol abuse was admitted to our trauma service following a motor vehicle collision, where he was a passenger. The patient was found unresponsive with a Glasgow Coma Score of 3 (E1, V1, M1). Initial brain imaging demonstrated small subdural hematoma along the falx cerebri as well as the frontal convexities. A cervical spine magnetic resonance imaging (MRI) demonstrated severe post-traumatic changes at C5 -- C6 level without evidence of cord compression. The patient regained consciousness 36 hours after admission. His neurological examination one week later was remarkable for an awake patient able to blink on command. Pupils were equal and reactive and extraocular movements had restricted bilateral abduction. Remaining bilateral vertical eye movements were intact. He had bilateral facial, lingual, and palatal weakness that impaired articulation, swallowing, and respiratory ability. He had flaccid quadriplegia with hyper-reflexia and bilateral Hoffman signs were present, and he required full ventilator support through tracheostomy. His neurological examination was consistent with "Locked-in Syndrome",[@b1-12-2-56] suggestive of a lesion at the level of the pons. MRI of the brain demonstrated symmetric areas of subtle restricted diffusion at pons and medulla involving corticospinal and corticobulbar fibers, suggestive of brainstem traumatic axonal injury ([Figure 1](#f1-12-2-56){ref-type="fig"}). DISCUSSION ========== Locked-in Syndrome is described clinically as quadriplegia and anarthria with preserved consciousness.[@b2-12-2-56] Disruption of the corticospinal, corticopontine, and corticobulbar tracts in the brainstem lead to these clinical features. There are multiple etiologies to Locked-in Syndrome, most commonly due to ischemic or hemorrhagic stroke, pontine hemorrhage related to hypertension of vascular malformations, or trauma.[@b3-12-2-56] We described a patient with Locked-in Syndrome due to traumatic axonal injury (TAI) or diffuse axonal injury (DAI), a common finding in patients with severe traumatic brain injury.[@b4-12-2-56] Angular and rotational acceleration and deceleration forces to the head lead to stretching of brain tissue and subsequent damage to white matter. After traumatic brain injury, axonal degeneration from DAI is considered progressive. Disruptive axonal transport results in axonal swelling followed by secondary disconnection and anterograde degeneration (more common) or even orthograde degeneration (less common).[@b5-12-2-56] MRI can characterize these lesions typically located in the white matter of cerebral hemispheres, corpus callosum, and brainstem.[@b6-12-2-56] {#f1-12-2-56}

Background ========== We are witnessing a dramatic shift in the design of personal computer systems, where speedups are achieved by porting the parallel traits of supercomputers into the world of personal computing. Modern computers are heterogeneous platforms with many different types of computational units, including central processing units (CPUs), graphics processing units (GPUs), digital signal processors (DSPs), coprocessors and custom acceleration logic. Today's CPUs contain from two to twelve cores, each capable of executing multiple instructions per clock cycle. Assisting the CPU, graphics processing units usually render 3D graphics, but can also provide a general-purpose computing platform. Current GPUs are designed as massively parallel processors offering substantially more computing power than CPUs. GPUs are the most powerful computational hardware available at an affordable price \[[@B1],[@B2]\]. The availability of general-purpose GPUs with computing abilities in commodity laptop and desktop computers has generated a wide interest, including applications in bioinformatics \[[@B3]-[@B9]\]. The newest addition to the commodity computer parallel processing hardware is the Intel Xeon Phi family of coprocessors \[[@B10]\] designed for computationally intensive applications. Xeon Phi implements Intel's Many Integrated Core (MIC) architecture and offers a theoretical performance similar to that of modern GPUs, but promises easier porting of existing software to the new architecture. Tianhe-2, currently the world's fastest supercomputer has 48 000 Xeon Phi coprocessors \[[@B11]\]. Many computational problems in bioinformatics require substantial computational resources \[[@B12]\]. Problems that can be computed with a high degree of parallel and independent processing are most suited for heterogeneous massively parallel hardware. Our aim was to investigate how these modern architectures cope with problems that are typical for bioinformatics, such as the problem of SNP-SNP interaction detection. As a proof-of-concept, we focused on a parallel implementation of computational core for the web-application SNPsyn \[[@B13]\] by exploiting heterogeneous processing resources, multi-core CPUs, GPUs, and the new MIC coprocessors. SNPsyn \[[@B13]\] (Figure [1](#F1){ref-type="fig"}) was developed as an interactive software tool for efficient exploration and discovery of interactions among single nucleotide polymorphisms (SNPs) in case-control genome-wide association study (GWAS) data. It uses an information-theoretic approach to evaluate SNP-SNP interactions \[[@B14]\]. Information gain is computed for every individual SNP, which allows the user to identify SNPs that are most associated with the disease under study. When searching for interesting pairs of SNPs, SNPsyn estimates the synergy between a pair of SNPs by computing the interaction gain. Information gain can identify SNP pairs with non-additive effects. Results are presented in an interactive graphical user interface that allows the user to select the most synergistic pairs, perform Gene Ontology enrichment analysis and visualize the synergy network among the selected SNP-SNP pairs. {#F1} SNPsyn computes the information gain exhaustively across all SNP pairs to avoid missing any pair where SNPs on their own provide no information about the phenotype under study. Because the number of pairs is quadratic to the number of SNPs, the exhaustive search quickly becomes computationally intractable for commodity computer systems. The information-theoretic-based detection of SNP-SNP interactions has a high degree of data parallelism and requires much more processing power than memory storage. This makes it a perfect candidate for processing on modern massively parallel architectures. Implementation ============== Below we describe the SNP-SNP interaction scoring approach we use in SNPsyn and discuss its implementation on CPU, CUDA and MIC architectures. Our particular concern is to evaluate Intel's new MIC architecture and compare its advantages against currently prevailing CUDA architecture. SNP-SNP interaction scoring --------------------------- The SNP-SNP interaction scoring scheduler, written in Python, partitions and distributes the computational tasks to all available, user-specified resources: CPUs, GPUs, and Xeon Phi coprocessors (Figure [2](#F2){ref-type="fig"}). It then merges the results from individual units into a final result file. Each thread (CPU, GPU or Xeon Phi) takes one pair of SNPs and performs all the calculations needed to compute the synergy score of the pair. The synergy of a pair of SNPs *X* and *Y* with respect to phenotype *P* is obtained by subtracting the information gains of individual SNPs from the information gain of the combined pair \[[@B13]\]: $$G\left( X,Y \right) = I\left( X,Y;P \right) - I\left( X;P \right) - I\left( Y;P \right).$$ {#F2} Given the two SNPs and the phenotype as random variables *X*, *Y* and *P*, respectively, the information gains required in Equation 1 are calculated as \[[@B14]\]: $$\begin{matrix} {I\left( X;P \right)} & = & {\underset{x \in X,p \in P}{\sum}q\left( x,p \right)\log\limits_{2}\frac{q\left( x,p \right)}{q\left( x \right)q\left( p \right)},} \\ \end{matrix}$$ $$\begin{matrix} {I\left( Y;P \right)} & = & {\underset{y \in Y,p \in P}{\sum}q\left( y,p \right)\log\limits_{2}\frac{q\left( y,p \right)}{q\left( y \right)q\left( p \right)},} \\ \end{matrix}$$ $$\begin{matrix} {I\left( X,Y;P \right)} & = & {\underset{x \in X,y \in Y,p \in P}{\sum}q\left( x,y,p \right)\log\limits_{2}\frac{q\left( x,y,p \right)}{q\left( x,y \right)q\left( p \right)}.} \\ \end{matrix}$$ Computation of marginal probabilities *q*(*x*), *q*(*y*), *q*(*p*) and joint probability distributions *q*(*x*,*p*), *q*(*y*,*p*), *q*(*x*,*y*), *q*(*x*,*y*,*p*) requires a single scan through case and control samples. The number of joint probability distributions *q*(*x*,*y*) and *q*(*x*,*y*,*p*) that need to be determined grows quadratically with the number of SNPs. This ensures enough computational load to compensate for the memory transfer costs and makes it efficient for an implementation on parallel hardware. Permutation analysis is used to evaluate the significance of results on true data. Data is randomly shuffled thirty times. Each time, information gain and synergy for all pairs are calculated to obtain the null distribution, which is used to determine the significance of results on true data. Details on permutation analysis are described in Curk *et al.*\[[@B13]\]. Parallel implementations of interaction scoring ----------------------------------------------- Calculations are performed in parallel for as many pairs of SNPs as allowed by the hardware. We took special care to efficiently use the GPU and Xeon Phi hardware. We minimized memory transfers between the main CPU and the coprocessors to avoid bottlenecks and vectorized the code wherever possible. We optimized the number of threads running on the GPU to maximize throughput. To cope with the memory limitation of the GPU, SNPsyn includes optional heuristics to quickly estimate the importance of SNPs and reduce the data set prior to analysis. In the following sections we present the implementation details regarding both architectures. GPU and CUDA ------------ GPUs gain their computational power from the numerous processing cores packed into one chip. For example, the modern Nvidia Tesla K20 GPU has 13 streaming multiprocessors, each containing 192 computational units called CUDA cores. These cores lack sophisticated control units and are thus likely to work best when executing the same instruction on many data elements in parallel with no divergent program paths in the algorithm. A programmer sees the GPU as a parallel coprocessor and can use it to speedup computationally intensive parts of the algorithm. Of course, there must be enough data parallelism in the code to make it worthwhile. Different tools are available for programming GPUs. Nvidia offers the CUDA toolkit \[[@B15]\] for programming its own products. It includes a proprietary compiler and a set of libraries that extend the C++ syntax with parallel programming constructs. Another popular option is the OpenCL framework \[[@B16]\]. It supports hardware from different vendors but usually lags slightly in terms of performance when compared to specialized development kits such as CUDA. Regardless of the development tool used, the programmer must follow certain rules to obtain maximum performance \[[@B17]\]. The most important one is to partition the algorithm in blocks small enough to simultaneously start a sufficient number of threads to utilize all available resources. For example, consider the code snippet in Figure [3](#F3){ref-type="fig"}, a simplified version of a code that scores pairs of SNPs. Function computeIGain calculates the information gain of a SNP pair using Equation 1. The details of the calculation are omitted to emphasize the architecture specific parts of code. The snippet includes all the peculiarities of programming for GPUs. The program has to implement the GPU-specific part separately from the CPU code and explicitly transfer data from the host to the GPU. Special functions called kernels (marked with the keyword \_\_global\_\_) must be written to be executed on the GPU. Memory transfer and allocation functions must be called to supply the necessary data to the GPU and collect the results afterwards. Usually, the programmer performs measurements to determine which thread configuration is most suitable for a particular problem size and the appropriate number of threads to launch. {#F3} Xeon Phi and MIC ---------------- Intel designed the Xeon Phi family of coprocessors around the new MIC architecture \[[@B18]\] to compete with GPUs specialized in general-purpose computing. The design follows a different approach in comparison to GPUs. Coprocessors consists of many simple, but fully functional processor cores derived from the Intel Pentium architecture. Intel improved the original design by adding a 512-bit wide vector unit and Hyper-Threading Technology. This enables Xeon Phi to achieve similar theoretical performance as modern GPUs. The model 5510P, which we used in this study, includes sixty cores interconnected with a bidirectional ring bus. Each core is capable of running four threads in parallel. The cores fetch data from the 8 GB of on-board RAM and communicate with the host CPU through the PCIe bus. In comparison to GPUs, each core on a Xeon Phi can efficiently execute the code even if threads do not follow the same program path. This makes it suitable for a wider range of problems, including multiplications of sparse matrices \[[@B19]\], and operations on trees and graphs \[[@B20]\]. Intel provides a C++ compiler suite and all the tools needed to exploit the hardware \[[@B21]\]. The code can be parallelized using OpenMP directives or the MPI library and compiled for the MIC architecture. Resulting applications can then run only on the Xeon Phi coprocessors. Another, more general way to specify parallel execution is to use offload constructs along with OpenMP to mark the data and the code to be transferred and executed on the Xeon Phi. All other parts of the program will run normally on the host computer CPU. A third possibility is to use OpenCL framework in the same manner as with GPUs.MIC development tools facilitate data management through compiler directives. The example in Figure [4](#F4){ref-type="fig"} demonstrates this programming paradigm. It performs the same operation as the snippet from Figure [3](#F3){ref-type="fig"}. The programmer marks the data and the code that is needed on the coprocessor. All memory allocations and transfers are done implicitly. To obtain best performance, the programmer must tailor the algorithms to fully utilize the vector unit. The Intel compiler automatically vectorizes sections of code where possible. {#F4} If a computer lacks Xeon Phi, the MIC code can be executed by the main CPU, which is not the case with CUDA-specific implementation. The MIC code looks much cleaner and easier to handle than CUDA code. The current drawbacks of using Xeon Phi are the shortage of supporting Linux distributions (officially only RedHat and SuSE) and the pricey development environment for the Windows operating system. The main aspects (relevant to the developer) of each of the architectures are shown in Table [1](#T1){ref-type="table"}. ###### Comparison of parallel computer architecture platforms with key aspects from the viewpoint of software development   **x86/x64 single CPU** **Nvidia GPU** **Intel Xeon Phi** --------------------------------- ------------------------ ------------------------------------- -------------------------------------- **Tools** Arbitrary compiler CUDA Toolkit or OpenCL framework Intel compiler suite **OS support** Many Windows, Linux, Mac OSX Linux (RedHat and SuSE), Windows **Required programming skills** Low High Medium **Lines of code\*** 260 460 360 **Programming remarks** None Architecture specific optimizations Recommended optimizations using     are crucial vector unit **Platform maturity** Mature Extensive documentation, many Bugs in drivers, documentation needs     programming examples to improve Lines of code (\*) reports on the approximate length of the code that implements the computationally intensive tasks of SNPsyn. Results ======= We benchmarked SNPsyn on a workstation with two six-core Intel Xeon E5-2620 2.00 GHz CPUs capable of running up to twenty-four threads in parallel, 64 GB of RAM, two Nvidia Tesla K20 general-purpose computing cards with 5 GB of RAM each and one Intel Xeon Phi 5110P coprocessor with 8 GB of RAM. The operating system was CentOS 6.4. We evaluated the performance on a series of representative WGAS data sets constructed from the Infinium_20060727fs1_gt_MS_GCf data set found in the WTCCC study \[[@B22]\]. Our goal was to observe the effect of the number of SNPs and WGAS study subjects to the execution time on different configurations. We sampled with replacement the original data on 994 subjects and 15 436 SNPs to obtain data sets with the desired number of subjects and SNPS. We performed the analysis on data with 1 000, 6 000, and 20 000 subjects and 10 000, 100 000, and 660 000 SNPs. The study considered only the data sets that could fit into the GPU memory. Xeon Phi is clearly in advantage when compared to K20 regarding the amount of RAM (8 GB versus 5 GB). We tested six hardware configurations including one CPU core running a single thread, twelve CPU cores running twelve threads, twelve CPU cores running twenty-four threads, one GPU core, both GPU cores, and Xeon Phi.Figure [5](#F5){ref-type="fig"} reports on execution times of the exhaustive SNP-SNP interaction analysis and the speedups achieved using various hardware configurations. For easier comparison, execution times are plotted on a logarithmic scale. As expected, execution times increase proportionally with the number of subjects and are quadratic with the number of SNPs included in the analysis. {#F5} The single thread CPU configuration takes more than 30 days to analyze the data on 660 000 SNPs and 1 000 subjects. Running twelve threads in parallel, one on each of the CPU cores, speeds up the computation by a factor of 10 and reduces the execution time to approximately 3 days. Increasing the number of threads to twenty-four reduces the time to perform the analysis to around 2 days with the speedup peaking at 12.8 compared to a one thread configuration. Memory bottleneck is the main factor for the poor speedup, which is far below the theoretical value of 24. Interestingly, similar speedups are achieved on all (smaller) data sets, meaning that there is enough data parallelism to keep the CPU busy. Nvidia K20 provides for considerable reduction in execution times, with the analysis of the largest data set taking only around 17 hours, demonstrating a speedup of 42 in comparison to a single CPU thread. Sharing the work between both GPU cards doubles the speedup and reduces the execution time to 8 hours. Increasing the number of subjects leads to a noticeable decrease in speedup, as more data is being transferred between the main memory and the GPU. On the other hand, increasing the number of SNPs introduces more data parallelism into the computations, reflecting in an improved speedup. Xeon Phi is positioned somewhere in-between K20 and CPU-only implementation. It achieves a speedup of nearly 20 on the largest data set, making the analysis run a day and a half, which is double the time needed on a K20. The speedup behaves similarly for Xeon Phi as for K20 -- it increases with the number of SNPs and decreases with the number of subjects. This confirms that the drop is caused by transferring larger amounts of data without introducing additional parallelism. Using only CPUs to analyze the data is unfeasible except for small data sets since the computations can take days to complete even on multiple cores. Xeon Phi provides a considerable performance boost with a maximum speed-up of nearly 20 and lots of on-board memory to store the data. Nvidia K20 clearly outperforms every other configuration in terms of speed and is the perfect choice when one wants to cut on the execution times as much as possible. This comes at a price of cumbersome programming and less on-board memory, which limits the size of data. Technical specifications presented in Table [2](#T2){ref-type="table"} show similar trends: Nvidia K20 offers the highest theoretical performance in terms of TFLOPS and has the most complex design. Xeon Phi has considerably less computing power, but interestingly draws the same amount of power as K20 at maximum load. The Xeon E5-2620 CPU is the least efficient of all and lacks the performance to remain competitive at computationally intensive tasks. ###### Technical specification of hardware platforms   **Intel Xeon E5-2620** **Nvidia Tesla K20** **Intel Xeon Phi 5110P** ------------------------------------------------- ------------------------ ---------------------- -------------------------- **Number of transistors** 2.3 billion 7.1 billion 5 billion **Peak power consumption** 95 W 225 W 225 W **Single precision floating point performance** 96 GFLOPS 3.5 TFLOPS 2.0 TFLOPS **Main memory** 64 GB can be expanded 5 GB 8 GB Conclusion ========== We investigated how modern heterogeneous architectures cope with a selected computational problem typical for bioinformatics. The proof-of-concept implementation of SNPsyn on heterogeneous systems greatly reduces the (wall-clock) time needed for analysis of large GWAS data sets. GPUs proved to be a mature platform that offers a large amount of computing power to address inherently parallel problems, but is demanding for the programmer. A user who is only interested in using SNPsyn to analyze their data will profit the most by having multiple GPUs in their system. The new MIC architecture greatly alleviates programming but lacks in performance. Its ease of programming combined with good performance has a lot to offer to developers who don't want to spend too much time optimizing their algorithms. Nevertheless, MIC is a general platform capable of tackling a wider range of more complex problems. This makes it very promising to excel in more complex analysis of SNP-SNP interactions such as adjustment for covariates \[[@B23]\]. Availability and requirements ============================= **Project name:** SNPsyn **Project home page:**<http://snpsyn.biolab.si> **Operating systems:** Linux, Windows, Mac OS **Programming language:** C++ **Other requirements:** CUDA 2.0 or higher, Intel Composer XE 2013 or newer, make **License:** GNU GPLv3 **Restrictions to use by non-academics:** none Competing interests =================== The authors declare that they have no competing interests. Authors' contributions ====================== UL, DS, TC, and BZ designed the study. DS implemented the CUDA and MIC software and measured the performance of the software. TC implemented the CPU and Python part of the software. DS wrote the first draft of the manuscript. All authors have written, read and approved the final manuscript. Acknowledgements ================ BZ and TC were supported by the Slovenian Research Agency (ARRS, P2-0209). UL and DS were supported by the Slovenian Research Agency (ARRS, P2-0241).

{#sp1 .781} {#sp2 .782}

T[he]{.smallcaps} D- and E-type cyclins and the cyclin-dependent kinases (cdks)^1^ they activate, are now recognized to be key regulators of G1 to S phase progression in mammalian cells ([@B52]; [@B64]). Overexpression of cyclin D1 or E in fibroblasts shortens their G1 interval, decreases cell size, and renders the cell less dependent on mitogens for S phase entry ([@B49]; [@B53]), indicating that both G1 cyclins can be rate-limiting factors for S phase initiation. However, their functions are likely to be quite different (Resnitzki and Reed, 1995). Microinjection of antisense plasmids or neutralizing antibodies to cyclin D1 in serum-stimulated fibroblasts ([@B2]; [@B32]), or an antibody to cyclin D2 in lymphocytes ([@B34]), during early to mid G1 phase prevents cellular DNA synthesis, suggesting that cyclin D--dependent kinases execute critical functions during middle to late G1 but are not required thereafter ([@B65]). Until now, no such G1 phase requirement has been demonstrated for cyclin D3, which is more distantly related to the other two cyclins D ([@B64]). Cyclins D bind to and activate cdk4 ([@B40]) or the closely related kinase cdk6 ([@B42]; [@B70]). The major activity of cyclin D1--cdk4/6 is to phosphorylate and inactivate proteins of the Rb family ([@B74]; [@B6]) that in turn releases active transcription factors, including those of the E2F family (Bejiersbergen et al., 1996; [@B36]). In cells lacking functional Rb proteins, the microinjections of a cyclin D1--blocking antibody ([@B33]), or of the cdk4 inhibitor p16^ink4A^ ([@B41]; [@B35]), fail to block DNA synthesis. By contrast, the lack of functional Rb protein does not obviate the requirement of cyclin E for DNA synthesis initiation ([@B45]). A fundamental role of D-type cyclins is thus to integrate extracellular signals with the cell cycle machinery by neutralizing the Rb inhibitory pathway ([@B74]; [@B6]; [@B66]). By contrast cyclin E is crucial for initiation of DNA synthesis ([@B45]; [@B38]). Cyclin E transcription is regulated in part by Rb via E2F proteins, which provides a mechanism for cyclin E induction downstream to cdk4 activation ([@B44]; [@B22]). The three D-type cyclins (D1, D2, and D3) are combinatorially expressed in a cell lineage--specific manner in proliferating mammalian cells. It is unclear whether they carry out redundant functions in response to different signals or whether their roles are distinct. In addition to activating cdk4 and cdk6 ([@B65]), they might play distinct roles in cell differentiation (Kato et al., 1993; [@B26]). Moreover, whereas cyclin D2 and cyclin D3 can also activate cdk2, cyclin D1--cdk2 complexes are inactive ([@B23]). D-type cyclins are synthesized as long as growth factor stimulation persists and exhibit only moderate oscillations during the cell cycle, in contrast to other cyclins, with peak levels achieved near G1-S ([@B2]; [@B63]; [@B34]). However they are rapidly degraded when mitogens are withdrawn, regardless of the position of the cell in the cycle ([@B65]). How cAMP, probably through PKA, can impinge on the cell cycle machinery to either inhibit or induce cell proliferation in different systems is still poorly understood ([@B10]; [@B19], [@B20]; [@B61]). In some cell types, cAMP has been found to inhibit mitogenic stimulations in mid-G1 or at the restriction point in late G1 as in rat hepatocytes ([@B71]) and murine macrophages, which involves the inhibition of Rb protein phosphorylation ([@B12]; [@B25]). In this case, early mitogenic responses may not be affected by cAMP ([@B56]), but the G1 block by cAMP was ascribed to result from a suppression of the expression of cyclin D1 ([@B14]; [@B63]; [@B30]), cyclin D3 ([@B73]), and/or an increased expression of the cdk inhibitor p27^kip1^ ([@B25]; [@B73]; [@B30]). In sharp contrast with these systems, cAMP as the main second messenger for thyrotropin (TSH) and thyroid-stimulating antibodies elicits in thyroid gland in vivo a pathway leading to hyperplasia (goiter) and tumorigenesis ([@B19], [@B20]; [@B39]; [@B31]; [@B46]; [@B15]). Dog thyroid epithelial cells in primary culture reproduce these proliferative effects and constitute a model of positive control of DNA synthesis initiation and G0-S prereplicative phase progression by TSH and cAMP ([@B57], [@B61], [@B62]; [@B20]). In this system, cAMP positively controls a late G1 restriction point ([@B58]; [@B3]). In contrast to what is observed in the signaling cascades elicited by growth factors and tumor promoters in this system, the cAMP-dependent mitogenic stimulation does not involve the phosphorylation and nuclear translocation of MAP kinases ([@B28]), it poorly induces c-Fos protein accumulation ([@B4]), it downregulates *c-jun* mRNA ([@B54]) and, after a short initial induction, *c-myc* mRNA and protein ([@B47]). Despite this early divergence, the cAMP- dependent and -independent mitogenic pathways of dog thyrocytes converge on the phosphorylation of Rb family proteins ([@B16]), late common changes of subcellular localization and phosphorylation of cdk2 and cdc2, and common induction of cyclin A and cdc2 ([@B5]). The passage of normal cells through the restriction point has been suggested to require both the induction of D-type cyclins and a reduction of p27^kip1^ concentration in response to growth factors ([@B66]). Recently, we have observed in dog thyroid cells that, paradoxically, p27^kip1^ expression is increased during the cAMP-dependent mitogenic stimulation but not in response to EGF+serum stimulation ([@B17]). Here we compare, using this system, the expression, subcellular localization, activation, and requirement of cyclins D and cdk4 in the cAMP-dependent and cAMP-independent cell cycles, and demonstrate a major involvement of cyclin D3 in the positive regulation by cAMP of the cell cycle at the restriction point. Materials and Methods {#MaterialsMethods} ===================== Primary Cultures of Dog Thyroid Follicular Cells ------------------------------------------------ Dog thyrocytes, seeded as follicles (2 × 10^4^ cells/cm^2^) were cultured in monolayer in the following mixture that constitutes the control medium ([@B59]): DME + Ham\'s F12 medium + MCDB104 medium (2:1:1, by volume; GIBCO BRL, Paisley, Scotland), supplemented with ascorbic acid (40 μg/ml), insulin (5 μg/ml; Sigma Chemical Co., St. Louis, MO), and antibiotics. The medium was changed every other day. At day 4, the cells were quiescent and were treated with the following stimulants: bovine TSH (Sigma Chemical Co.), murine EGF (Collaborative Research, Waltham, MA), recombinant human hepatocyte growth factor (HGF; a kind gift of T. Nakamura, Osaka University Medical School), forskolin (Calbiochem-Novabiochem Corp., La Jolla, CA), 12-*O*-tetradecanoylphorbol 13-acetate (TPA; Sigma Chemical Co), and fetal bovine serum (Sera-Lab, Sussex, UK). Antibodies ---------- Mouse monoclonal antibodies to cyclin D1 (DCS-6; [@B32]), cyclin D2 (DCS-3 and -5; [@B34]), and cyclin D3 (DCS-22 and -29; [@B7]) were characterized previously. DCS-28, -20, -24, and -27 are new mouse monoclonal antibodies generated upon immunization of BALB/c mice with bacterially produced human cyclin D3, using techniques described in [@B32]. The mouse monoclonal antibodies DCS-31, -32, and -35 against recombinant cdk4 were generated using similar techniques. Epitope mapping of monoclonal antibodies against cyclin D3 was performed using an ELISA technique based on a library of biotinylated 20-mer peptides (with 5--amino acid overlap) covering the complete sequence of cyclin D3 (Bartkova, J., unpublished data). Rabbit polyclonal antibodies against cdk4 (C22) and Rb (C15) were from Santa Cruz Biotechnology (Santa Cruz, CA). Gel Electrophoresis and Immunodetection of Proteins --------------------------------------------------- Cell proteins were separated by PAGE and immunodetected after Western blotting as previously described ([@B4]). ^125^I-Labeled anti--mouse antibody from sheep (ICN Pharmaceuticals Inc., Irvine, CA) and ^125^I-protein A (Amersham International, Little Chalfont, UK) were used as secondary reagents to detect monoclonal and polyclonal antibodies, respectively. Autoradiographs were quantitated by densitometry (Ultroscan; Bio-Rad Laboratories, Hercules, CA). Indirect Immunofluorescence --------------------------- Cells in petri dishes (2 × 10^4^ cells/cm^2^) were fixed with 2% paraformaldehyde for 90 s at 4°C and then with methanol for 10 min at −20°C, permeabilized with 0.1% Triton X-100 in PBS, pH 7.5, at room temperature and blocked for 30 min with normal sheep serum (5% in PBS containing 0.3% bovine serum albumin \[PBS/BSA\]; [@B3], [@B5]). For staining of cyclin D3, D2, D1, or cdk4 cells were then incubated overnight at 4°C with the respective mouse monoclonal antibodies (culture supernatant: 1/3; ascite liquid: 1/200). After washing, cells were successively incubated for 2 h at room temperature with biotinylated sheep anti--mouse immunoglobulins (1/50, RPN1001; Amersham International) and for 1 h with fluorescein-streptavidin (1/30, RPN1232; Amersham International). For double staining of proliferating cell nuclear antigen PCNA used as a cell cycle marker ([@B3], [@B5]) and cyclin D3 using two mouse monoclonal antibodies, fixed cells were incubated overnight at 4°C with DCS-22 and then for 2 h at room temperature with biotinylated anti-mouse immunoglobulins and for 1 h with Texas red streptavidin. Washed cells were then successively incubated for 30 min with normal mouse serum (1/100), for 2 h with unconjugated anti-mouse immunoglobulin G Fab fragment (50 g/ml; Jackson Immuno-Research Laboratories, West Grove, PA), for 1 h with PC10 (1/400; DAKOPATTS, Copenhagen, Denmark), and for 1 h with fluorescein-conjugated anti-mouse immunoglobulin (1/50; Amersham International). This "sandwich" procedure is designed to avoid binding of PC10 (the PCNA monoclonal antibody) to the first secondary antibody and binding of the second secondary antibody to the cyclin D antibody. To unmask the DCS-22 epitope, cells were fixed and permeabilized as above, and then incubated for 10 min at room temperature with a solution of 0.01% trypsin (ICN Pharmaceuticals Inc.) in MCDB104 Hepes-buffered culture medium. This dilution of trypsin was established from a range of different dilutions in preliminary experiments. The trypsin digestion was stopped by rapidly rinsing cells with PBS/BSA and incubating them with normal sheep serum as above. Cells were then normally processed for immunofluorescent detection of cyclin D3 using DCS-22. Photometry ---------- The nuclear immunofluorescence of cyclin D3 was quantitated using a photometric head attached to the Zeiss Axiovert 135 microscope (Carl Zeiss Inc., Thornwood, NY) and a 100× oil immersion lens exactly as described previously ([@B4]). Fluorescence was measured from ∼100 nuclei selected at random in each dish. All the conditions were assayed in duplicate with an excellent reproducibility. DNA Synthesis ------------- Cells in 3-cm Petri dishes were incubated either for 1 or 24 h before fixation in the presence of 10^−4^ M 8-bromo-deoxyuridine (BrdU) and 2 10^−6^ M fluorodeoxycytidine. Cells were fixed and the incorporation of BrdU into nuclei was revealed by immunofluorescence as described ([@B60]). The percentage of BrdU-labeled nuclei was evaluated by counting 1,000 nuclei per dish. Immunoprecipitation ------------------- Subconfluent cultures of thyrocytes that contain the same number of cells 20 h after stimulation by TSH, EGF+serum, or none were washed with calcium/magnesium-free PBS and lysed in 1 ml lysis buffer ([@B75]) containing 150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.5% NP-40, 50 mM NaF, 1 mM sodium orthovanadate, DTT, and protease inhibitors (pefablock, leupeptin). 1 ml of precleared cellular lysate was incubated with 2 g of antibody at 4°C for 2 h (monoclonal antibody against cyclin D3 \[DCS-28\] or against cdk4 \[DCS-35\]). A rabbit anti--mouse immunoglobulin antibody was added in the last 30 min. The immune complexes were recovered by incubation with protein A/G Agarose (Santa Cruz Biotechnology) for 1 h. The protein A/G Agarose immune complexes were suspended in SDS lysis buffer, boiled for 4 min, and analyzed on 10% SDS-- polyacrylamide gels. The proteins were immunodetected as described above using either the DCS-22 cyclin D3 antibody or the DCS-35 cdk4 antibody. Cyclin D3 and cdk4 were not precipitated by a control mouse immunoglobulin G or irrelevant mouse monoclonal antibodies in the present conditions. Rb Kinase Assay --------------- Cells were suspended in IP buffer (50 mM *N*-2-hydroxyethylpiperazine- *N*′-2-ethanesulfonic acid \[Hepes, pH 7.5\] 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 1 mM DTT, and 0.1% Tween 20) containing 10% glycerol, 0.1 mM phenylmethylsulfonyl fluoride, 10 μg of leupeptin per ml, 20 U of aprotinin per ml, 10 mM β-glycerophosphate, 1 mM NaF, and 0.1 mM sodium orthovanadate, and sonicated at 4°C (Virsonic 475, full microtip power two times for 10 s each time; [@B40]). Lysates were clarified by centrifugation at 10,000 *g* for 5 min, and the supernatants were precipitated for 2--6 h at 4°C with protein A/G--Agarose beads precoated with saturating amounts of the indicating antibodies. Protein A/G--Agarose was pretreated with rabbit anti--mouse to provide a suitable affinity matrix. Immunoprecipitated proteins on beads were washed four times with 1 ml of IP buffer and twice with the kinase buffer (50 mM Hepes, pH 7.5, 10 mM MgCl~2~, 1 mM dithiothreitol). The beads were suspended in 30 μl of kinase buffer containing substrate (1 μg of soluble glutathione *S*-transferase-Rb COOH terminus fusion protein \[GST-Rbc\] prepared from *Escherichia coli* transformed with pGEX-Rb \[773-928\] as previously described \[[@B42]\]) and 2.5 mM EGTA, 10 mM β-glycerophosphate, 0.1 mM sodium orthovanadate, 1 mM NaF, 50 μM ATP, and 5 μCi of γ-\[^32^P\]ATP. After incubation for 30 min at 30°C with occasional mixing, the samples were boiled in polyacrylamide gel sample buffer containing SDS and separated by electrophoresis. Phosphorylated proteins were visualized by autoradiography or phosphorimaging (PhosphorImager; Molecular Dynamics, Inc., Sunnyvale, CA) of the dried slab gels. Microinjection of Dog Thyroid Cells ----------------------------------- Thyrocytes were microinjected at day 4 of the culture as described ([@B18]) with affinity-purified monoclonal antibodies against cyclin D3 (DCS-29) or cyclin D1 (DCS-6). At this time, cells were quiescent and well spread. As the capacity to proliferate can differ for each follicle-derived cell cluster, but is generally homogeneous within each, one half of each cell cluster was microinjected, the other half being used as a control. The microinjected and nonmicroinjected cells were stimulated by various mitogenic factors just after the microinjection. BrdU (10^-4^M) and fluorodeoxycytidine (2 × 10^−6^ M) were added 16 h later and cells were fixed 40 h after the stimulation with methanol for 10 min at −20°C. Injected cells were identified by biotinylated anti-mouse antibody followed by Texas red--coupled streptavidin (incubations with the antibodies for 1 h). BrdU incorporation was then codetected as described ([@B60]), using a FITC-coupled anti-BrdU antibody (overnight incubation; Becton Dickinson, Mountain View, CA) and all the nuclei were counterstained with Hoechst 33342 dye. The fraction of cells entering into DNA synthesis was estimated by the percentage of BrdU-labeled nuclei. All the countings were realized independently by two persons. All the experiments were performed three to five times with similar results. Results {#Results} ======= After 4 d without mitogenic agents but in the presence of insulin, dog thyrocytes were spread and quiescent. Cells were then stimulated to proliferate using either TSH (maximum cAMP-dependent mitogenic stimulation \[[@B57]\]), or EGF+serum (maximum cAMP-independent mitogenic stimulation), or these factors in combination. As previously shown ([@B59]; [@B3]) the first S-phase cells appeared 18--20 h after stimulation by TSH or EGF+serum (Fig. [1](#F1){ref-type="fig"}). S-phase began earlier (14 h) in response to the combination of EGF+serum+TSH. This illustrates the fact that TSH (cAMP) cooperates with cAMP-independent mitogens by stimulating steps that are rate limiting for DNA synthesis initiation ([@B58]; [@B3], [@B5]). The combination of TSH and EGF+serum also resulted in an increase of the fraction of cells that enter S phase ([@B59]; Fig. [1](#F1){ref-type="fig"}). Differential Expression of D-type Cyclins during Distinct Mitogenic Stimulations -------------------------------------------------------------------------------- The various mouse cyclin D1, D2, and D3 monoclonal antibodies used throughout this study were monospecific in Western immunoblotting analyses. In immunoblotting experiments, as illustrated in Fig. [2](#F2){ref-type="fig"}, very different exposure times were necessary to obtain comparable autoradiography signals for the three cyclins D: 1--3 d for the detection of cyclin D3, ∼10 d for cyclin D1, and ∼3 wk for cyclin D2 (Fig. [2](#F2){ref-type="fig"} *A*). In some experiments cyclin D2 was even undetectable. Similar data, i.e., strongest signals for the detection of cyclin D3 as compared with cyclin D1 and even more to cyclin D2, were obtained using several different monoclonal antibodies used at similar concentrations (compare the detections with similar exposure times of cyclin D1 \[Fig. [2](#F2){ref-type="fig"} *A*\] and cyclin D3 \[Fig. [2](#F2){ref-type="fig"} *B*\]; and data not shown). This indicates that cyclin D3 is the most abundant one in dog thyrocytes, unlike in most cell types used until now for proliferation studies. In response to EGF+serum, cyclins D1 and D2 were markedly induced. This effect was detectable 12 h after stimulation, and maximum at 16--20 h, but at 26 h the expression of cyclins D1 and D2 decreased (Fig. [2](#F2){ref-type="fig"}, *A* and *C*), consistently with a maximum accumulation in mid to late G1 as in other systems ([@B2]; [@B32], [@B34]). At all the time points, TSH did not stimulate cyclin D1 and D2 expression and even slightly inhibited it in the presence of EGF+serum (Fig. [2](#F2){ref-type="fig"}, *A* and *C*). At variance with cyclins D1 and D2, cyclin D3 was already abundant in quiescent control cells, its expression was less affected by mitogens, and it did not disappear at late cell cycle stages (Fig. [2](#F2){ref-type="fig"}, *A*, *B*, and *D*). EGF+serum and EGF+serum+TSH stimulated cyclin D3 accumulation by ∼50%, as first detected 12 h after stimulation. On the contrary, TSH inhibited by ∼50% the basal expression of cyclin D3 during the whole prereplicative phase (until 24 h). At late time points of TSH stimulation (26--32 h), after many cells had already reached S phase, cyclin D3 levels increased (Fig. [2](#F2){ref-type="fig"}, *A* and *D*). This effect could result from the delayed induction by TSH of insulin receptors ([@B11]) and the moderate stimulation of cyclin D3 accumulation by insulin (unpublished observations). HGF and TPA qualitatively mimicked the EGF+serum action on the expression of cyclins D (data not shown). Thus, TSH markedly differs from all the cAMP-independent mitogens studied so far, as it triggers DNA synthesis and cell cycle progression without stimulating the accumulation of cyclins D during G1 phase. TSH Enhances the Immunofluorescence Detection of Cyclin D3 ---------------------------------------------------------- Using indirect immunofluorescence, the presence and subcellular location of D-type cyclins were studied at various time points (8, 12, 16, 20, 26, 32 h) after stimulation. Using DCS-6 (cyclin D1), DCS-3 or -5 (cyclin D2), and DCS-22 (cyclin D3), the three cyclins D were detected as mostly nuclear proteins (Fig. [3](#F3){ref-type="fig"}). EGF+serum treatment increased their nuclear staining in 30--50% of cells, starting at 12 h of stimulation (Fig. [3](#F3){ref-type="fig"}), in agreement with immunoblotting data on the accumulation of these proteins (Fig. [2](#F2){ref-type="fig"}). Also consistent with the immunoblotting analyses, TSH did not induce cyclin D1 and D2 nuclear stainings at any time points during the prereplicative phase (Fig. [3](#F3){ref-type="fig"} illustrating the 20 h time point and data not shown). However, in sharp contrast with the inhibition of cyclin D3 expression detected on immunoblots of TSH-treated cells (Fig. [2](#F2){ref-type="fig"}), we observed with the same antibody (DCS-22) that TSH like EGF+serum markedly enhanced the immunofluorescent labeling of cyclin D3 in the nuclei of a majority of cells (first seen 16 h after TSH stimulation; gradually increased afterwards; Fig. [3](#F3){ref-type="fig"}). This effect of TSH was mimicked by the adenylyl cyclase activator forskolin (not shown). The nuclear DCS-22 immunofluorescent staining of cyclin D3 was measured by photometry from 200 individual cells per condition 20 h after the addition of various mitogens. The individual responses of cells were heterogeneous. Compared with the average fluorescence measured from the nuclei of control cells, TSH in the presence of insulin enhanced the DCS-22 detection of cyclin D3 by a factor of 2--8 in 51% of the cell population. When comparing the different cAMP-dependent and cAMP-independent mitogenic treatments, a good correlation (*r* = 0.929) was found between the average intensity of the nuclear labeling of cyclin D3 and the subsequent accumulation of BrdU-labeled cells during a further 28 h incubation (Fig. [4](#F4){ref-type="fig"}). Thus, the increase of the nuclear immunoreactivity of cyclin D3, in response to various treatments was a better predictive factor of their mitogenic potency than the accumulation of this protein. The enhanced nuclear immunoreactivity of cyclin D3 might therefore reflect an important event for cell cycle progression. The correlation between the enhanced immunoreactivity of cyclin D3 and cell cycle progression was further assessed by the analysis of the double immunofluorescence labeling of cyclin D3 (DCS-22) and PCNA, which has been previously demonstrated in dog thyroid cells as a marker of the different phases of the cell cycle ([@B3], [@B5]). In response to either TSH or EGF+serum, the nuclear immunoreactivity of cyclin D3 was strongly increased in all the cells in late G1 and S (Fig. [5](#F5){ref-type="fig"}). There were also some cells with a low PCNA immunoreactivity but an intense DCS-22 staining (Fig. [5](#F5){ref-type="fig"}). Whether such cells were at a G1 stage before PCNA appearance and would eventually enter S phase is not known. Epitope Unmasking and Nuclear Translocation of Cyclin D3 -------------------------------------------------------- The TSH-induced increase of cyclin D3 immunoreactivity was reproduced using several cyclin D3 monoclonal antibodies (DCS-20, 22, 24, 27, and 29, which recognize epitopes located between amino acids 241 and 260 of cyclin D3 sequence close to the "cyclin box"; data not shown). The comparison of immunoblotting and immunofluorescence data thus suggested that these epitopes were inaccessible to these related cyclin D3 antibodies in fixed quiescent cells and unmasked in the nuclei of cells stimulated by TSH (and by EGF+serum). A different situation was revealed using another cyclin D3 monoclonal antibody (DCS-28). This antibody recognizes an epitope localized within the COOH-terminal 20--amino acid residues of cyclin D3; it binds cyclin D3 in Western blots and is the most efficient for precipitating the cyclin D3-associated kinase activity (our unpublished data). Using DCS-28, control quiescent cells were more heavily stained with an overall cytoplasmic and nuclear distribution of the labeling (Fig. [6](#F6){ref-type="fig"}). In response to TSH, this labeling was not further enhanced, but in many cells it moved to a more nuclear localization that suggests a nuclear translocation (Fig. [6](#F6){ref-type="fig"}). Thus, the comparison of DCS-22 and DCS-28 immunofluorescent patterns also suggests that DCS-22 epitopes of cyclin D3 were masked in quiescent cells. To substantiate this inaccessibility of the DCS-22 epitope, we gently digested fixed cells with a diluted trypsin solution, a classical method for retrieval of masked antigens. The mild trypsin digestion enhanced the detection of cyclin D3 by DCS-22 in quiescent cells but not in TSH-treated cells, confirming the overall cytoplasmic and nuclear localization of cyclin D3 before stimulation and its nuclear translocation in response to TSH, as revealed using DCS-28 (Fig. [6](#F6){ref-type="fig"}). Nuclear Translocation of cdk4 ----------------------------- Unlike some other normal cell types ([@B21]; [@B40]), quiescent dog thyrocytes expressed cdk4 before mitogenic stimulation, as shown by Western blotting analysis (Fig. [7](#F7){ref-type="fig"}). In response to TSH or TSH+EGF+serum, but not in all the experiments in response to EGF+serum, the abundance of cdk4 moderately increased (Fig. [7](#F7){ref-type="fig"}). In various experiments this effect was mostly observed at 20--40 h after stimulation, i.e., when most responding cells were already progressing into S and G2 phases. The immunofluorescent detection of cdk4 using the DCS-31 and -32 monoclonal antibodies, or the polyclonal antibody from Santa Cruz Biotechnology, showed that cdk4 was distributed in both the cytoplasm and nucleus of quiescent control cells. As first observed 12 h after EGF+serum or 16--20 h after TSH addition, many cells displayed a strong increase of the nuclear staining of cdk4 at the expense of the cytoplasmic labeling (Fig. [8](#F8){ref-type="fig"}). Since cdk4 contents, as measured by Western blottings, varied little at these times (Fig. [7](#F7){ref-type="fig"}), this suggests a nuclear translocation of cdk4. The double immunofluorescent labeling of cdk4 and PCNA showed that the nuclear staining of cdk4 remained elevated until mitosis onset in TSH-treated cells. By contrast, in EGF+serum--treated cells, the cdk4 nuclear staining was increased in many cells well before the appearance of PCNA and it decreased at 20--26 h in S-phase cells. In response to the combination of both stimulations (TSH+EGF+serum), cdk4 nuclear staining increased early, at 8--12 h, but remained elevated at later time points like in TSH treatment (data not shown). Assembly and Activity of Cyclin D3--cdk4 Complexes -------------------------------------------------- The above immunofluorescence results have suggested that TSH and EGF+serum could induce the formation of nuclear cyclin D3--cdk4 complexes. To test this hypothesis, we precipitated cdk4 using the DCS-35 monoclonal antibody and analyzed by Western blotting the coimmunoprecipitation of cyclin D3. Conversely, cyclin D3 was precipitated using DCS-28 and precipitates were examined for the presence of cdk4 (Fig. [9](#F9){ref-type="fig"} *A*). Cells were used 20 h after stimulation, i.e., when a maximum of cells are in mid to late G1 phase. In the experiment shown, the amount of total DCS-35--precipitable cdk4 slightly increased in response to TSH (+37% as evaluated by densitometry) and EGF+serum (+33%). The amount of total DCS-28--precipitable cyclin D3 also moderately increased in response to EGF+serum (+60%) and weakly in response to TSH (+20%), which in this last case contrasted with the moderate reduction of cyclin D3 content consistently observed in whole lysates of TSH-stimulated cells (Fig. [2](#F2){ref-type="fig"}). Thus, TSH treatment might somewhat affect the efficiency of cyclin D3 precipitation by DCS-28 in the present nondenaturing conditions. In contrast to these modest variations of cdk4 and cyclin D3 concentrations and/or immunoreactivity, the amount of cyclin D3 complexed with cdk4 strongly increased in TSH- or EGF+serum--stimulated cells (Fig. [9](#F9){ref-type="fig"} *A*). Both TSH and EGF+serum provoked a 10-fold elevation of the amount of cdk4 coprecipitated with cyclin D3. As grossly evaluated by the comparison of the amounts of cdk4 precipitated by cyclin D3 or cdk4 antibodies, ∼50% of total cdk4 was associated with cyclin D3 in TSH- or EGF+serum--stimulated cells, compared with 5% in control cells (Fig. [9](#F9){ref-type="fig"} *A*). The Rb-kinase activity in cyclin D3 immunoprecipitates was analyzed using a recombinant COOH-terminal fragment of Rb as substrate ([@B40]). In these precipitates, the phosphorylation of GST-Rb was stimulated by both TSH and EGF+serum and paralleled the presence of cdk4 (Fig. [9](#F9){ref-type="fig"} *B*). TSH and EGF+serum also induced the phosphorylation of Rb demonstrated by its electrophoretic shift in Western blots of total cell lysates (Fig. [9](#F9){ref-type="fig"} *C*; [@B16]). This demonstrates that both TSH and EGF+serum mitogenic stimulations converge on the assembly of active cyclin D3--cdk4 complexes and Rb phosphorylation. Requirement for Cyclin D3 in cAMP-dependent G1 Phase Progression ---------------------------------------------------------------- To assess whether cyclin D3 is required for cell cycle progression, we microinjected a cyclin D3--specific antibody (DCS-29) into quiescent dog thyrocytes and examined its effect upon S-phase entry induced by different mitogenic factors. DCS-29 ([@B7]) like DCS-22, very efficiently and quite specifically recognized dog cyclin D3 in immunoblotting and immunofluorescence experiments (Fig. [2](#F2){ref-type="fig"} *B*; data not shown). DCS-29 like DCS-22 recognizes an epitope very close to the "cyclin box" domain required for cdk4 activation, and it does not precipitate cyclin D3--associated kinase activity (Bartkova, J., unpublished data). As shown in Fig. [10](#F10){ref-type="fig"} and summarized in Fig. [11](#F11){ref-type="fig"}, cells microinjected with DCS-29 and stimulated by TSH or the direct adenylyl cyclase activator forskolin were prevented from entering DNA synthesis (70--80% of inhibition), whereas microinjection of a control immunoglobulin did not result in any significant inhibition. By contrast the DCS-6 cyclin D1 antibody did not prevent DNA synthesis induced by TSH (Fig. [10](#F10){ref-type="fig"}), whereas in parallel microinjection experiments performed under identical conditions the same batch of purified DCS-6 blocked G1 phase progression in the human MCF7 cell line (Fig. [2](#F2){ref-type="fig"} in [@B37]). It is worth mentioning that the DCS-6 epitope is conserved among species and lies within the "cyclin box" domain, which explains that this antibody prevents both the association of cyclin D1 with cdks and DNA synthesis in a variety of human and murine cells that predominantly express cyclin D1 ([@B32], 1995*a,b*; [@B45]). Since DCS-6 did recognize dog cyclin D1 (Figs. [2](#F2){ref-type="fig"} *A* and [3](#F3){ref-type="fig"}), it had very likely the capacity to neutralize dog cyclin D1 in the present microinjection experiments. The results (Figs. [10](#F10){ref-type="fig"} and [11](#F11){ref-type="fig"}) thus suggest that cyclin D3, but not cyclin D1, is required for the cAMP-dependent G1 phase progression of dog thyrocytes. On the other hand, in the same experiments the microinjection of DCS-29 had no or a weak effect on the capacity of dog thyrocytes to enter DNA synthesis after stimulations that induce the accumulation of cyclins D1 and D2, including HGF, EGF+serum, and EGF (Figs. [10](#F10){ref-type="fig"} and [11](#F11){ref-type="fig"}). Whereas the microinjection of 2 mg/ml DCS-29 was sufficient to block DNA synthesis in TSH-treated cells, the microinjection of a higher DCS-29 concentration (6 mg/ml) did not inhibit the entry of cells after the stimulation by HGF and only slightly affected the weaker stimulation by EGF. These data demonstrate that the cell cycle inhibitory effect of DCS-29 in TSH-treated cells is specific, rather than attributable to some nonspecific toxic component in the antibody preparations. The absence of DCS-29 effect in growth factor--stimulated dog thyrocytes did not suggest a lack of cdk4 requirement, since the microinjection of the p16^INK4A^ cdk4 inhibitor completely inhibited the stimulations of DNA synthesis by both TSH and EGF ([@B37]). Rather, the present observation is consistent with the view that different cyclins D might play overlapping roles in cdk4 activation and G1 phase progression in thyrocytes stimulated by cAMP-independent growth factors. Discussion {#Discussion} ========== The dog thyrocyte culture system constitutes an interesting model of the diversity of the mechanisms of cell cycle control by distinct intracellular signaling cascades. In these cells, we have previously proposed that the potent MAP kinase--independent mitogenic pathway elicited by TSH via cAMP and the rapidly converging cAMP-independent ones of growth factors and tumor promoters could remain partly separated during most of the G1 phase, and in parallel lead to the commitment for DNA replication ([@B59]; [@B19], [@B20]; [@B28]; [@B61], [@B62]). This study strongly supports this proposal. In dog thyrocytes as in a variety of cell types, cAMP-independent mitogens including EGF+serum, HGF, and TPA induced the accumulation of cyclins D1 and D2 and slightly enhanced the high basal expression of cyclin D3. In sharp contrast, TSH via cAMP did not induce the accumulation of cyclin D1 and D2 and even partially inhibited the basal expression of cyclin D3 at least during the G0-S prereplicative phase. Moreover, the synergy between TSH and EGF+serum on DNA synthesis initiation was not associated with a similar synergy at the level of cyclin D expression, and TSH even slightly reduced the cyclin D1 accumulation induced by EGF+serum. To our knowledge, there is no precedent for such an observation in the case of a mitogenic stimulation of a normal cell. Mitogenic pathways stimulated by agents as diverse as growth factors through tyrosine kinase receptors ([@B63]), activators of protein kinases C ([@B1]), transforming growth factor β (Rao and Kohtz., 1995), or steroid hormones ([@B43]), have all been found to involve the induction of at least one of the three cyclins D, as a prerequisite for the activation of cdk4 and the inactivation of Rb-like cell cycle suppressors. Our results also contrast with the induction of cyclin D2 mRNA by FSH through cAMP recently demonstrated in rat ovarian granulosa cells ([@B68]), and the reported increase of cyclin D1 accumulation in TSH-stimulated rat FRTL5 thyroid cell line ([@B76]). In different cell systems, cAMP may thus use different strategies, not only to inhibit cell cycle progression ([@B61]), but also to directly stimulate it. Strikingly, in dog thyrocytes the effects of cAMP on the accumulation of cyclins D and p27^kip1^ ([@B17]; a cdk inhibitor whose cellular concentration, as generally considered \[[@B25]; [@B13]\], should decrease as a prerequisite for the passage through the restriction point in response to growth factors) are thus similar to those observed in cells blocked in G1 by cAMP ([@B14]; [@B63]; [@B25]; [@B73]; [@B30]). Paradoxically, in the dog thyrocyte system they are associated with a late G1 cAMP-dependent commitment to DNA synthesis ([@B58]) and mitosis ([@B3]), which involves, as after a cAMP-independent mitogenic stimulation, the phosphorylation of Rb and related p107 and p130^Rb2^ ([@B16]), the nuclear translocation and phosphorylation of cdk2 and the accumulation of cyclin A and cdc2 ([@B5]). This has raised the question of the mechanism of Rb phosphorylation in this cAMP-dependent pathway. Here we demonstrate that the TSH and EGF+serum mitogenic pathways do converge on the assembly of precipitable complexes of cdk4 and cyclin D3, and the stimulation of the Rb kinase activity associated with cyclin D3. The activation of cdk4 is indeed an essential step both in the cAMP-dependent and growth factor mitogenic pathways, since the microinjection of the p16^INK4A^ inhibitor of cdk4 blocked the stimulation of DNA synthesis by TSH and EGF in dog thyrocytes ([@B37]), exactly as in growth factor--stimulated fibroblasts with a normal Rb function ([@B35]). These results support the concept that the inactivation of Rb family proteins through phosphorylation by cyclin D--cdk4 is indeed the ultimate integrator of quite diverse mitogenic stimulations at the restriction point ([@B74]; [@B6]; [@B66]; [@B16]). In TSH-stimulated dog thyrocytes cyclin D3 appears to be the main D-type cyclin involved in cdk4 activation and DNA synthesis induction: (*a*) cyclins D1 and D2 were not induced (cyclin D2 was often undetectable); (*b*) cyclin D3 appears to be the most abundant cyclin D in quiescent cultured dog thyrocytes and in the thyroid gland of mice in vivo (Coppée, F., F. Depoortere, J. Bartek, C. Ledent, M. Parmentier, J.E. Dumont, manuscript submitted for publication), like in some T lymphocytes ([@B70]) but at variance with most cell types used for cell cycle studies. Even after a 50% reduction of its concentration, cyclin D3 remains significantly expressed since it was evaluated to recruit ∼50% of cdk4 molecules in TSH-stimulated dog thyrocytes; (*c*) the microinjection of the DCS-6--neutralizing antibody to cyclin D1 failed to inhibit TSH-stimulated DNA synthesis, at variance with a wide variety of cell types, but like other cells that weakly express cyclin D1 ([@B32]); (*d*) the microinjection of a cyclin D3 antibody (DCS-29) directly showed that cyclin D3 is required for DNA synthesis in cells stimulated by TSH or forskolin, but very poorly or not at all in cells stimulated by growth factors (HGF, EGF, and the combination of EGF+serum) that induce cyclin D1 and D2. This specific requirement of cyclin D3 is a novel observation. Cyclin D1 or D2 knockouts by gene targeting have only impaired the development of tissues that predominantly express these cyclins, and have suggested partly overlapping functions for the three cyclins D in G1 progression (Sicinsky et al., 1995, 1996; [@B29]). In dog thyrocytes, cyclins D1 and/or D2 induced by growth factors but not by TSH might thus compensate for the neutralization of cyclin D3. It remains to be seen whether cyclin D3 knockout in mice could specifically impair the development and/or TSH-dependent hyperplasia of the thyroid gland, but we surmise that in other cell types the requirement for cyclin D3 also might be cryptic in the presence of other cyclins D. Other mechanisms of the differential requirement of cyclins D have not been excluded, such as a critical influence of different cdk inhibitor levels in the parallel mitogenic pathways. Whatever the mechanism(s), the TSH (cAMP)-dependent cell cycle of dog thyrocytes is unique as it mainly depends on cyclin D3 for cdk4 activation and DNA synthesis, which allows this first direct demonstration of a G1 phase requirement for cyclin D3. The assembly and activation of cyclin D3--cdk4 complexes in TSH-treated cells that were paradoxically associated with a partial inhibition of cyclin D3 accumulation should thus involve a new mechanism. This mechanism might also play a nonessential role (as argued above) in growth factor--dependent G1 phase progression, since the formation of cyclin D3--cdk4 complexes in EGF+serum-- stimulated dog thyrocytes is also poorly explained by only a 50% increase of cyclin D3 levels. At variance with some other systems ([@B21]; [@B40]), in dog thyrocytes the stimulation of the activity of cdk4 is not associated with important variations of its concentration. In quiescent thyrocytes, the simultaneous expression of cyclin D3 and cdk4 does not suffice for cdk4 activation. This is consistent with observations from rodent fibroblasts that despite constitutive engineered overexpression of cyclin D3 together with cdk4, remain dependent on mitogenic stimulation for the assembly of active cyclin D3--cdk4 complexes ([@B40]). This study provides some new insights about the poorly understood assembly of cyclin D--cdk4 complexes: (*a*) As detailed in Results, some but not all the epitopes of cyclin D3 were inaccessible to antibodies in fixed quiescent thyrocytes, but specifically unmasked in cycling cells in response to both cAMP-dependent and cAMP-independent mitogenic stimulations. An attractive speculation would be that DCS-22 and related antibodies are preferentially recognizing the active cyclin D3 molecules in the present cell fixation conditions. Interestingly, the epitope made accessible to these antibodies in stimulated cells is very close to the "cyclin box" domain of interaction with cdk4. The cAMP-dependent activation step in G1 (through modifications of protein--protein interactions or conformational changes) could thus allow both the access of DCS-22 to its antigen and the assembly of cyclin D3--cdk4 complexes. (*b*) Both in TSH- and in EGF+serum--treated dog thyrocytes, the formation of cyclin D3--cdk4 complexes was associated with nuclear translocations of cyclin D3 and cdk4 that initially were equally distributed in the cytoplasm and nucleus of quiescent cells. To our knowledge, such nuclear translocations of cyclin D3 and cdk4 have not been previously reported in other systems. They could be crucial for cdk4 activation, as recently suggested by studies of cells engineered to overexpress a cyclin D and cdk4 with various cdk inhibitors ([@B27]; [@B55]). The cdk4 translocation occurred later during the prereplicative phase of TSH-treated thyrocytes than in EGF+serum--treated cells, and thus might well correspond to the "X" event we have postulated previously as being especially rate limiting for the initiation of DNA synthesis in TSH-stimulated cells ([@B3]). Previously we have evidenced the nuclear translocation of cdk2 and cdc2 at G1/S and G2/M transitions, respectively ([@B5]). Subcellular localization might thus be a general determinant of the activation of cyclin-dependent kinases. (*c*) Cyclin D3 and cdk4 are not known to contain a nuclear localization signal. Their nuclear accumulation might thus depend on their interaction with other nuclear proteins. After completion of the present experiments, [@B55] and La Baer et al. (1997) have demonstrated using cotransfected cell lines that p27^kip1^ can target cdk4 and D-type cyclins to the nucleus. p27^kip1^ and other cdk inhibitors of the p21 family might also promote the association of cdk4 with cyclins D ([@B27]). Unlike initial claims ([@B25]), the presence of p27^kip1^ in cyclin D-cdk4 complexes does not necessarily prevent their activity ([@B69]; [@B9]). The TSH (cAMP)-induced accumulation of p27^kip1^ we have observed including in the nuclei of G1 and S phase thyrocytes ([@B17]) might thus contribute to the cAMP-dependent nuclear translocation and/or assembly of cyclin D3 and cdk4. The unique characteristics of the TSH-controlled cAMP-mediated growth pathway might explain how it can be compatible with the cAMP-dependent induction of differentiation expression that is inhibited by cAMP-independent mitogens ([@B48]). In addition to their role as cdk4 activators, the different D-type cyclins have distinct influences on differentiation in a variety of cell types ([@B24]; [@B51]; [@B72]). Cyclin D1 expression inhibits the terminal differentiation of myoblasts that is associated with a strong induction of cyclin D3 ([@B26]; [@B50]). Thyroid growth accompanies chronic stimulation of function by TSH. The limited number of cAMP-dependent mitotic cycles of fully differentiated thyrocytes that this process involves ([@B20]), must obey specific constrains ([@B61]). The cAMP-dependent mitogenic pathway could thus be viewed, in thyrocytes and possibly other cells, as a differentiation characteristic, adjunctive to the more general mechanisms operated by growth factors ([@B60], [@B61]). This could help to understand the various odd characteristics of this pathway, such as the lack of cyclins D induction and the specific cyclin D3 requirement. In summary, this study has precisely identified the earliest observed convergence point of the cAMP-dependent and cAMP-independent mitogenic pathways of dog thyrocytes on cyclin D3 "activation," cdk4 nuclear translocation, and assembly of active cyclin D3--cdk4 complexes. However, these pathways lately differ in essential steps of the cdk4 activation mechanism: the expression of the various cyclins D and p27^kip1^ and the requirement for cyclin D3. This study was supported by the Belgium Program on University Poles of Attraction initiated by the Belgian State, Prime Minister\'s Office, Science Policy Programming, and by grants from the National Fund for Scientific Research (FNRS, Belgium), the Caisse Générale d\'Epargne et de Retraite, the Danish Cancer Society, and the Nordic Cancer Union. F. Depoortere and A. Van Keymeulen are fellows of the Fonds pour la Formation a la Recherche dans l\'Industrie et l\'Agriculture (FRIA), S. Dremier is a fellow of the "Televie", and P.P. Roger is a Research Associate of the FNRS. Address all correspondence to Pierre Roger, IRIBHN, Campus Erasme, 808 Route de Lennik, B-1070 Brussels, Belgium. Tel.: 322 555 41 53. Fax: 322 555 46 55. The first two authors made equal contributions to this work. BrdU : 8-bromo-deoxyuridine cdk : cyclin-dependent kinase HGF : hepatocyte growth factor TPA : 12-*O*-tetradecanoylphorbol 13-acetate TSH : thyrotropin {#F1} ###### Western blotting analyses of the accumulation of the three cyclins D in dog thyrocytes after stimulation by different mitogenic treatments. Cells remained quiescent in control conditions (*C*) or were stimulated by TSH (1 mU/ml), EGF (25 ng/ ml)+serum (10%; *ES*), or the combination of these factors (*ES*+*TSH*) for 8 to 32 h. (*A*) Immunoblot autoradiograph. 30 μg of cellular proteins were loaded per lane. The exposure durations were different for the autoradiographical detection of the different cyclins D: cyclin D1 (DCS-6), 8 d; cyclin D2 (DCS-3), 16 d; cyclin D3 (DCS-22), 2 d. *ND*, not done or lost. (*B*) Detection of cyclin D3 with DCS-29 from control cells (*C*) and cells stimulated by TSH (*T*) or ES for 20 h. This autoradiograph was exposed for 8 d like the cyclin D1 immunoblot shown in *A*, in order to illustrate the relative abundances of cyclin D3 and cyclin D1. It also shows the high specificity of the DCS-29 antibody used in the microinjection experiments of Figs. [10](#F10){ref-type="fig"} and [11](#F11){ref-type="fig"}. (*C*) Densitometry quantitation of cyclin D1 from the autoradiograph details in *A*. (*D*) Densitometry quantitation of cyclin D3 (shown with a more precise kinetics from another dog thyrocyte primary culture in order to illustrate the qualitative reproducibility compared with the results in *A*). In *C* and *D*, the relative amount of cyclin D1 or cyclin D3 after cell stimulation is expressed compared with the average (fixed as 100) of values obtained at different times from unstimulated (control) cells in the same experiments.     {#F10} {ref-type="fig"}, after stimulation for 40 h as indicated, with TSH (1 mU/ml), forskolin (*Fo*; 10^−5^M), HGF (50 ng/ml), EGF (25 ng/ml)+serum (*S*), or EGF alone. Results are expressed (mean+SD) relative to the percentages of BrdU-labeled nuclei in neighboring noninjected cells. Average percentage of BrdU-labeled nuclei in noninjected cells were 37% (*TSH*), 50% (*Fo*), 46% (*HGF*), 69% (*EGF*+*S*), 35% (*EGF*), and 3% in control unstimulated cells. The number of separate experiments and the total number of injected cells analyzed are indicated for each condition. In each separate experiment, the stimulation by TSH (or Fo) was compared with one of the cAMP-independent stimulations (*HGF*, *EGF*+*S*, or *EGF*).](JCB29387.f11){#F11} {#F3} {#F4} ![Double immunofluorescence labeling of cyclin D3 (detected using DCS-22), and PCNA used as a cell cycle marker in the same cells. Before fixation, 4-d-old dog thyrocytes were stimulated for 26 h with TSH (1 mU/ml), EGF (25 ng/ml)+serum (10%; *ES*), or remained quiescent in control medium (*C*) for this time. Arrowheads and large arrows show cells identified, respectively, in late G1 and S phase as described previously ([@B3], [@B5]). Small arrows show G0/G1 cells with a low PCNA staining.](JCB29387.f5){#F5} {ref-type="fig"}, or using DCS-22 after a retrieval treatment of masked antigen by trypsin. All the DCS-22 labelings were photographed with the same exposure time. Note the more intense staining of control cells with DCS-28 or with DCS-22 after the trypsin preincubation, and in these two last conditions, the increase of the nuclear staining of cyclin D3 at the expense of its cytoplasmic staining in many TSH-stimulated cells, which suggests a nuclear translocation.](JCB29387.f6){#F6} {#F7} {#F8} ![Convergence of cAMP-dependent and -independent mitogenic treatments on cyclin D3--cdk4 complex formation and Rb phosphorylation. 4-d-old dog thyrocytes were stimulated for 20 h with either TSH (1 mU/ml; *T*), EGF (25 ng/ml)+serum (10%; *ES*), or remained quiescent in control medium (*C*) for this time. (*A*) Assembly of cyclin D3--cdk4 complexes. The complexes were immunoprecipitated as indicated from equal amounts of cell extract using either DCS-28 (*cyclin D3*) or DCS-35 (*cdk4*) monoclonal antibodies, and the presence of cyclin D3 and cdk4 was detected by Western blot in these immunoprecipitates. A same autoradiograph exposure is provided throughout in order to allow the direct comparison of amounts of total versus complexed cdk4 and cyclin D3. (*B*) Cyclin D3--associated Rb kinase activity using GST-Rb COOH-terminal protein (GST-Rbc) and γ-\[^32^P\]ATP as substrates. (*C*) Phosphorylation of Rb detected by its electrophoretic shift in Western blot of whole cell lysates.](JCB29387.f9){#F9}

**Introduction** {#Sec1} ================ A stochastic fractional integro-differential equation (SFIDE), where order of derivative is non integer, is a generalization of the fractional Folkker-Plank equation which describes the random walk of a particle \[[@CR2]\]. This model has the following form$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \begin{aligned} D^{\alpha }u(t)&= f(t)+\int _{0}^{t}u(s)k_{1}(s,t)ds+\int _{0}^{t}u(s)k_{2}(s,t)dW(s),~t\in [0,T),\\ u(0)&= u_{0}, \end{aligned} \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$D^{\alpha },$$\end{document}$ $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ 0< \alpha < 1,$$\end{document}$ denotes the Caputo fractional derivative, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$W(s),s \in [0,T)$$\end{document}$ is the standard Wiener process and the integral with respect to it is the It$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\hat{o}$$\end{document}$ integral. Presence of the It$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\hat{o}$$\end{document}$ integral in Equation ([1](#Equ1){ref-type=""}) causes randomness in the solution and hence it becomes non deterministic. In this paper, we develop a novel approach to quantify this uncertain behavior in the numerical solution. In recent decade, the need to obtain the numerical solution of SFIDE has increased significantly. However, in literature, only a handful of papers are available that actually discuss about the numerical solution of SFIDE. In \[[@CR8]\], Maleknejad *et al.* provided an operational matrix method based on block pulse functions to solve stochastic Volterra integral equations. In \[[@CR12]\], Taheri *et al.* formulated the spectral collocation method based on shifted Legendre polynomials to solve SFIDE. In \[[@CR10]\], Mirzaee and Samadyar constructed an efficient scheme to solve SFIDE based on Bernstein polynomials. In \[[@CR11]\], Mirzaee and Samadyar provide a meshless discrete collocation method based on radial basis functions to solve SFIDE. In this paper, a new scheme is derived based on Legendre wavelet collocation method and block pulse function involving the operational matrix for solving SFIDE ([1](#Equ1){ref-type=""}). In Sect. [2](#Sec2){ref-type="sec"}, we give basic definition of fractional calculus and construction of Legendre wavelet based on Multi-resolution analysis. Then in Sect. [3](#Sec5){ref-type="sec"}, operational matrix of fractional order integration and integration operational matrix are derived. The proposed scheme for the SFIDE is discussed in Sect. [4](#Sec9){ref-type="sec"}, while Sect. [5](#Sec10){ref-type="sec"} provides numerical experiments performed to showcase the effectiveness of the approach. In Sect. [6](#Sec11){ref-type="sec"}, we present various applications of SFIDE. Finally, Sect. [7](#Sec12){ref-type="sec"} gives the brief conclusion. Preliminaries {#Sec2} ============= In this section, we discuss the mathematical preliminaries of fractional calculus and construction of wavelet which are required for subsequent development. Definition 1 {#FPar1} ------------ \[[@CR5]\] The left Riemann-Liouville fractional integral of order $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha \ge 0$$\end{document}$ of a function $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f(t),~t\in (a,b)$$\end{document}$ is defined as follows$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \begin{aligned} _{a}I_{t}^{\alpha }f(t)&= \frac{1}{\varGamma (\alpha )}\int _{a}^{t}(t-s)^{\alpha -1}f(s)ds, \\ _{a}I_{t}^{0}f(t)&= f(t). \end{aligned} \end{aligned}$$\end{document}$$Similar to integer order integration, the left Riemann-Liouville fractional integral operator is a linear operator$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} _{a}I_{t}^{\alpha }(\lambda f(t)+\mu g(t)) = \lambda _{a}I_{t}^{\alpha }f(t)+\mu _{a}I_{t}^{\alpha }g(t), \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\lambda $$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mu $$\end{document}$ are constants. Definition 2 {#FPar2} ------------ \[[@CR5]\] The left Caputo derivative with order $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha > 0$$\end{document}$ of the given function $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f(t),~t\in (a,b)$$\end{document}$ is defined as$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} D_{a,t}^{\alpha }f(t) = \frac{1}{\varGamma (m-\alpha )}\int _{a}^{t}(t-s)^{m-\alpha -1}f^{(m)}(s)ds, \end{aligned}$$\end{document}$$where *m* is a positive integer satisfying $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m-1 < \alpha \le m.$$\end{document}$ Multi-resolution Analysis (MRA) {#Sec3} ------------------------------- An MRA is an increasing family of closed subspace $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V^{j} \subset L^{2}(\mathbb {R})$$\end{document}$ which satisfies the following axioms \[[@CR9]\] : $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V^{j} \subset V^{j+1}$$\end{document}$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\overline{\cup _{j \in \mathbb {Z}}V^{j}} = L^{2}(\mathbb {R})$$\end{document}$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\{\phi (x-k) : k \in \mathbb {Z}\}$$\end{document}$ is an orthonormal basis of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V^{0}$$\end{document}$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f(\cdot ) \in V^{j}$$\end{document}$ if and only if $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f(2(\cdot )) \in V^{j+1}$$\end{document}$ for all $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$j \in \mathbb {Z}$$\end{document}$. For given nested sequence subspace $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V^{j}$$\end{document}$, define the space $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$W^{j}$$\end{document}$ as the orthogonal complement of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V^{j}$$\end{document}$ in $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V^{j+1}$$\end{document}$, i.e., $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V^{j} \perp W^{j}$$\end{document}$ and$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} V^{j+1} = V^{j}\oplus W^{j}, \end{aligned}$$\end{document}$$applying recursively, we get$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} V^{j} = V^{j_{0}}\oplus \bigoplus _{k=j_{0}}^{j-1}W^{k},~~j > j_{0}. \end{aligned}$$\end{document}$$Now, based on the above analysis, to construct a wavelet define a space $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V_{M}^{J}$$\end{document}$ of piecewise polynomial functions as follows :$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \begin{aligned} V_{M}^{J} := \{\phi ~: \text { the restriction of } \phi \text { to the interval } [2^{-J+1}(k-1),2^{-J+1}k) \\ \text {is a polynomial of degree less than M for } k = 1,2,\cdots ,2^{(J-1)},\\ \text { and } \phi \text { vanishes elsewhere} \}. \end{aligned} \end{aligned}$$\end{document}$$The space $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V_{M}^{J}$$\end{document}$ has dimension $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2^{(J-1)}M$$\end{document}$ and$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V_{M}^{1} \subset V_{M}^{2} \subset \cdots V_{M}^{J} \subset \cdots \subset L^{2}([0,1)). $$\end{document}$$Next, consider the $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2^{(J-1)}M$$\end{document}$-dimensional space $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$W_{M}^{J}$$\end{document}$ which is an orthogonal complement of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V_{M}^{J}$$\end{document}$ in $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V_{M}^{J+1}$$\end{document}$, i.e.,$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V_{M}^{J+1} = V_{M}^{J}\oplus W_{M}^{J+1}.$$\end{document}$$Inductively, one can obtain$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} V_{M}^{J} = V_{M}^{1}\oplus \bigoplus _{j=1}^{J-1}W_{M}^{j}. \end{aligned}$$\end{document}$$Unlike Haar, the element of the space $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$W_{M}^{J}$$\end{document}$ do not have a general form. To construct the elements of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$W_{M}^{J}$$\end{document}$ one can refer \[[@CR1]\]. Further, if$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ V_{M}^{J} = \text {span}\{\phi _{k,m}^{J},~m=0,1,\cdots ,M-1,~k = 1,2,\cdots ,2^{(J-1)}\},$$\end{document}$$then we define the projection operator $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P_{V_{M}^{J}} : L^{2}[0,1] \rightarrow V_{M}^{J}$$\end{document}$ as$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} P_{V_{M}^{J}}(f(x)) := \sum _{k=1}^{2^{J-1}}\sum _{m=0}^{M-1}c_{k,m}^{J}\phi _{k,m}^{J}(x), \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{k,m}^{J} = \int _{\frac{(k-1)}{2^{(J-1)}}}^{\frac{k}{2^{(J-1)}}}f(x)\phi _{k,m}^{J}(x)dx.$$\end{document}$ Set$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$W_{M}^{J} = \text {span}\{\psi _{k,m}^{J}(x),~m=0,1,\cdots ,M-1,~k = 1,2,\cdots ,2^{(J-1)}\},$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\psi _{k,m}^{J}(x) = 2^{\frac{(J-1)}{2}}\psi _{m}(2^{(J-1)}x-k+1)$$\end{document}$. The support of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\psi _{k,m}^{J}$$\end{document}$ is $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[\frac{k-1}{2^{J-1}},\frac{k}{2^{J-1}})$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\psi _{m}(x)$$\end{document}$ satisfies the following property (vanishing moment property)$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \int _{0}^{1}x^{i}\psi _{m}(x)dx = 0,~i = 0,1,\cdots ,M-1. \end{aligned}$$\end{document}$$Now, using $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V_{M}^{J}$$\end{document}$, we introduced the subspace $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V_{M}^{J,2}$$\end{document}$ of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$L^{2}([0,)\times [0,1))$$\end{document}$ defined by$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ V_{M}^{J,2} := \{\phi ~|~ \phi = \phi _{1}\phi _{2}~\text {where}~\phi _{1},\phi _{2} \in V_{M}^{J}\}.$$\end{document}$$Moreover,$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V_{M}^{J,2} = \text {span}\{\phi _{k,m,k',m'}^{J} = \phi _{k,m}^{J}\phi _{k',m'}^{J}~:~k,k' = 1,\cdots ,2^{J-1}~\text {and}~m,m' = 0,\cdots ,M-1 \}.$$\end{document}$$ Then, define the projection operator $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P_{V_{M}^{J,2}}:L^{2}([0,1]\times [0,1]) \rightarrow V_{M}^{J,2}$$\end{document}$ as$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} P_{V_{M}^{J,2}}f(s,t) = \sum _{k=1}^{2^{J-1}}\sum _{m=0}^{M-1}\sum _{k'=1}^{2^{J-1}}\sum _{m'=0}^{M-1}c_{k,m,k',m'}^{J}\phi _{k,m,k',m'}^{J}(s,t), \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{k,m,k',m'}^{J} = \int _{\frac{(k-1)}{2^{(J-1)}}}^{\frac{k}{2^{J-1}}}\int _{\frac{(k'-1)}{2^{(J-1)}}}^{\frac{k'}{2^{J-1}}}f(s,t)\phi _{k,m,k',m'}^{J}(s,t)dsdt$$\end{document}$. Next, introduce the space $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$W_{M}^{J,2}$$\end{document}$ which is defined by$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$W_{M}^{J,2} = \{\psi ~|~ \psi = \psi _{1}\psi _{2}~\text {where}~\psi _{1},\psi _{2} \in W_{M}^{J} \},$$\end{document}$$and$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$W_{M}^{J,2} = \text {span}\{\psi _{k,m,k',m'}^{J} = \psi _{k,m}^{J}\psi _{k',m'}^{J}~:~ k,k'=1,\cdots ,2^{J-1}~\text {and}~m,m'=0,\cdots ,M-1 \},$$\end{document}$$ where$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \psi _{k,m,k',m'}^{J}(s,t)&= 2^{J-1}\psi _{m,m'}(2^{J-1}s-k+1,2^{J-1}t-k'+1)\\ {}&:= 2^{J-1}\psi _{m}(2^{J-1}s-k+1)\psi _{m'}(2^{J-1}t-k'+1) \end{aligned}$$\end{document}$$and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\psi _{m,m'}(s,t)$$\end{document}$ satisfies the following property$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \int _{0}^{1}\int _{0}^{1}s^{i}t^{j}\psi _{m,m'}(s,t)dsdt = 0,~i,j=0,\cdots ,M-1. \end{aligned}$$\end{document}$$The subspace $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$W_{M}^{J,2}$$\end{document}$ is orthogonal complement of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V_{M}^{J,2}$$\end{document}$ in $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V_{M}^{J+1,2}$$\end{document}$. Therefore, one can write$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$V_{M}^{J+1,2} = V_{M}^{J,2}\oplus W_{M}^{J,2},$$\end{document}$$and hence$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} V_{M}^{J,2} = V_{M}^{1,2}\oplus \bigoplus _{j=1}^{J-1}W_{M}^{j-1,2}. \end{aligned}$$\end{document}$$Now, if we choose $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\phi _{k,m}^{J}$$\end{document}$ as in \[[@CR13]\], i.e., for $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m = 0,1,2,\cdots ,M-1$$\end{document}$, and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\hat{k} = 2k-1$$\end{document}$ with $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k=1,2,\cdots ,2^{(J-1)}$$\end{document}$$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \phi _{k,m}^{J}(t) = {\left\{ \begin{array}{ll} \sqrt{m+\frac{1}{2}}2^{J/2}P_{m}(2^{J}t-\hat{k}) &{} \text {for}~~ \frac{\hat{k}-1}{2^{J}} \le t < \frac{\hat{k}+1}{2^{J}}\\ 0 &{} \text {otherwise}, \end{array}\right. } \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P_{m}(t)$$\end{document}$ is a Legendre polynomials of order *m* are defined in the interval $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$[-1,1]$$\end{document}$ and given by the following recurrence formulas$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P_{0}(t) = 1,~P_{1}(t) = t,$$\end{document}$$ $$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P_{m+1}(t) = \Big (\frac{2m+1}{m+1}\Big )tP_{m}(t)-\Big (\frac{m}{m+1}\Big )P_{m-1}(t),~m=1,2,3,\cdots .$$\end{document}$$The wavelet constructed above using Legendre polynomials are called as Legendre wavelet \[[@CR7]\]. Function Approximation {#Sec4} ---------------------- A function *f*(*t*) defined over $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$L^{2}[0,1)$$\end{document}$ can be expanded with Legendre scaling functions $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\phi _{k,m}^{J}(t)$$\end{document}$ as$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} f(t) = \sum _{k=1}^{2^{J-1}}\sum _{m=0}^{\infty }c_{k,m}^{J}\phi _{k,m}^{J}(t), \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$c_{k,m}^{J} = \int _{0}^{1}f(t)\phi _{k,m}^{J}(t)dt$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$J\rightarrow \infty $$\end{document}$. If the infinite series in ([14](#Equ14){ref-type=""}) is truncated, then ([14](#Equ14){ref-type=""}) can be written as$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} f(t) \approx P_{V_{M}^{J}}(f(t)) = \sum _{k=1}^{2^{J-1}}\sum _{m=0}^{M-1}c_{k,m}^{J}\phi _{k,m}^{J}(t) = C^{T}\varPhi (t), \end{aligned}$$\end{document}$$where *C* and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\varPhi (t)$$\end{document}$ are $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2^{J-1}M\times 1$$\end{document}$ matrices given by$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} C =&[c_{1,0}^{J},c_{1,1}^{J},\cdots ,c_{1,M-1}^{J},c_{2,0}^{J},\cdots ,c_{2,M-1}^{J},\cdots ,c_{2^{J-1},0}^{J},\cdots ,c_{2^{J-1},M-1}^{J}]^{T},\\ \varPhi _{2^{J-1}M}(t) =&[\phi _{1,0}^{J}(t),\phi _{1,1}^{J}(t),\cdots ,\phi _{1,M-1}^{J}(t),\cdots ,\phi _{2^{J-1},M-1}^{J}(t)]^{T} \\ =&[\phi _{1}^{J}(t),\dots ,\phi _{2^{J-1}M}^{J}(t)]^{T}. \end{aligned}$$\end{document}$$In similar way, a bivariate function $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f(s,t) \in L^{2}[[0,1)\times [0,1)]$$\end{document}$ can be expanded with Legendre wavelets as$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} f(s,t) \approx \sum _{i=1}^{2^{J-1}M}\sum _{j=1}^{2^{J-1}M}\phi _{i}^{J}(s)f_{ij}\phi _{j}^{J}(t) = \varPhi ^{T}(s)F\varPhi (t), \end{aligned}$$\end{document}$$where$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f_{ij} = \int _{0}^{1}\int _{0}^{1}f(s,t)\phi _{i}^{J}(s)\phi _{j}^{J}(t)dsdt.$$\end{document}$$ Legendre Wavelet Matrix and Block Pulse Operational Matrix {#Sec5} ========================================================== Let the collocation points be$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} t_{i} = \frac{2i-1}{2^{J}M},~ i = 1,2,\dots ,2^{J-1}M. \end{aligned}$$\end{document}$$We denote the Legendre wavelet matrix as $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\phi _{2^{J-1}M\times 2^{J-1}M}$$\end{document}$ and define it as the combination of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\phi _{k,m}^{J}(t_{i})$$\end{document}$ at the collocation points $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$(t_{i})$$\end{document}$ as$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \phi _{2^{J-1}M\times 2^{J-1}M} = \begin{pmatrix} \phi _{1,0}^{J}(t_{1}) &{} \phi _{1,1}^{J}(t_{2}) &{} \dots &{} \phi _{2^{J-1},M-1}^{J}(t_{2^{J-1}M}) \\ \phi _{1,0}^{J}(t_{1}) &{} \phi _{1,1}^{J}(t_{2}) &{} \dots &{} \phi _{2^{J-1},M-1}^{J}(t_{2^{J-1}M}) \\ \vdots &{} \vdots &{} \ddots &{} \vdots \\ \phi _{1,0}^{J}(t_{1}) &{} \phi _{1,1}^{J}(t_{2}) &{} \dots &{} \phi _{2^{J-1},M-1}^{J}(t_{2^{J-1}M}) \end{pmatrix}. $$\end{document}$$ Legendre Wavelet Operational Matrix of Fractional Order Integration {#Sec6} ------------------------------------------------------------------- If *f*(*t*) is expanded as in Eq. ([14](#Equ14){ref-type=""}), then the Riemann-Liouville fractional order integration is given by$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} _{0}I_{t}^{\alpha }f(t) \approx \frac{1}{\varGamma (\alpha )}\int _{0}^{t}(t-\tau )^{\alpha -1}C^{T}\varPhi (\tau )d\tau = C^{T}(_{0}I_{t}^{\alpha }\varPhi _{2^{J-1}M}(t)). \end{aligned}$$\end{document}$$The $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2^{J-1}M-$$\end{document}$set of block pulse functions (BPFs) are also defined as$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} b_{i}(t) = {\left\{ \begin{array}{ll} 1 &{} \frac{(i-1)}{2^{J-1}M} \le t < \frac{i}{2^{J-1}M} \\ 0 &{} \text {otherwise}, \end{array}\right. } \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$i = 1,2,\dots ,2^{J-1}M$$\end{document}$. The function $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$b_{i}(t)$$\end{document}$ has the following disjoint and orthogonal properties$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$b_{i}(t)b_{j}(t) = \delta _{ij}b_{i}(t)$$\end{document}$,$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\int _{0}^{1}b_{i}(t)b_{j}(t) = \frac{\delta _{ij}}{2^{J-1}M}$$\end{document}$,where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta _{ij}$$\end{document}$ is the Kronecker delta. The Legendre wavelet can be expanded into $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2^{J-1}M$$\end{document}$ - term block pulse function as$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \varPhi _{2^{J-1}M}(t) = \phi _{2^{J-1}M\times 2^{J-1}M}{} \mathbf{b} _{2^{J-1}M}(t), \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathbf{b} _{2^{J-1}M}(t) = [b_{1}(t),\dots ,b_{2^{J-1}M}(t)]^{T}.$$\end{document}$ The block pulse operational matrix of fractional-order integration $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$G^{\alpha }$$\end{document}$ is given in \[[@CR6]\] as follows$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} _{0}I_{t}^{\alpha }{} \mathbf{b} _{2^{J-1}M}(t) \approx G^{\alpha }{} \mathbf{b} _{2^{J-1}M}(t), \end{aligned}$$\end{document}$$where$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ G^{\alpha } = \frac{1}{(2^{J-1}M)^{\alpha }}\frac{1}{\varGamma (\alpha +2)} \begin{pmatrix} 1 &{} \xi _{1} &{} \xi _{2} &{} \xi _{3} &{} \cdots &{} \xi _{2^{J-1}M-1}\\ 0 &{} 1 &{} \xi _{1} &{} \xi _{2} &{} \cdots &{} \xi _{2^{J-1}M-2} \\ 0 &{} 0 &{} 1 &{} \xi _{1} &{} \cdots &{} \xi _{2^{J-1}M-3} \\ \vdots &{} \vdots &{} \vdots &{} \vdots &{} \ddots &{} \vdots \\ 0 &{} 0 &{} 0 &{} 0 &{} \cdots &{} 1 \end{pmatrix}, $$\end{document}$$with $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\xi _{i} = (i+1)^{\alpha +1}-2i^{\alpha +1}+(i-1)^{\alpha +1}$$\end{document}$. Let$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} _{0}I_{t}^{\alpha }\varPhi _{2^{J-1}M}(t) = P_{2^{J-1}M\times 2^{J-1}M}^{\alpha }\varPhi _{2^{J-1}M}(t), \end{aligned}$$\end{document}$$where the matrix $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P_{2^{J-1}M\times 2^{J-1}M}^{\alpha }$$\end{document}$ is called the Legendre wavelet operational matrix of fractional order integration. Using Eqs. ([18](#Equ18){ref-type=""}) and ([19](#Equ19){ref-type=""}) in ([20](#Equ20){ref-type=""}), we get$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} P_{2^{J-1}M\times 2^{J-1}M}^{\alpha } \approx (\phi _{2^{J-1}M\times 2^{J-1}M})G^{\alpha }(\phi _{2^{J-1}M\times 2^{J-1}M})^{-1}. \end{aligned}$$\end{document}$$ Deterministic Integration Operational Matrix {#Sec7} -------------------------------------------- Let $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m = 2^{J-1}M$$\end{document}$ and compute $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\int _{0}^{t}b_{i}(s)ds$$\end{document}$ as follows$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \int _{0}^{t}b_{i}(s)ds = {\left\{ \begin{array}{ll} 0 &{} 0 \le t< \frac{i-1}{m}\\ t-\frac{i-1}{m} &{} \frac{i-1}{m} \le t< \frac{i}{m} \\ \frac{1}{m} &{} \frac{i}{m} \le t < 1. \end{array}\right. } \end{aligned}$$\end{document}$$We approximate $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$t-\frac{i-1}{m}$$\end{document}$, for $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{i-1}{m} \le t < \frac{i}{m},$$\end{document}$ by $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{1}{2m}$$\end{document}$ and express $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\int _{0}^{t}b_{i}(s)ds$$\end{document}$ in terms of BPFs as follows$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \int _{0}^{t}b_{i}(s)ds \approx \big (0,\dots ,0,\frac{1}{2m},\frac{1}{m},\dots ,\frac{1}{m}\big )\mathbf{b} _{m}(t), \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{1}{2m}$$\end{document}$ is the *i*th component of vector. Therefore$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \int _{0}^{t}{} \mathbf{b} _{m}(s)ds \approx P\mathbf{b} _{m}(t), \end{aligned}$$\end{document}$$where the operational matrix of integration is given by$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ P = \frac{1}{2m} \begin{pmatrix} 1 &{} 2 &{} 2 &{} \cdots &{} 2 \\ 0 &{} 1 &{} 2 &{} \cdots &{} 2 \\ 0 &{} 0 &{} 1 &{} \cdots &{} 2 \\ \vdots &{} \vdots &{} \vdots &{} \ddots &{} \vdots \\ 0 &{} 0 &{} 0 &{} \cdots &{} 1 \end{pmatrix}_{m\times m} $$\end{document}$$ Stochastic Integration Operational Matrix {#Sec8} ----------------------------------------- The It$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\hat{o}$$\end{document}$ integral of each single BPFs $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$b_{i}(t)$$\end{document}$ can be computed as follows$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \int _{0}^{t}b_{i}(s)dW(s) = {\left\{ \begin{array}{ll} 0 &{} 0 \le t< \frac{i-1}{m}\\ W(t)-W(\frac{i-1}{m}) &{} \frac{i-1}{m} \le t< \frac{i}{m} \\ W(\frac{i}{m})-W(\frac{i-1}{m}) &{} \frac{i}{m} \le t < 1. \end{array}\right. } \end{aligned}$$\end{document}$$We can approximate $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$W(t)-W(\frac{i-1}{m})$$\end{document}$, for $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{i-1}{m} \le t < \frac{i}{m}$$\end{document}$, by $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$W(\frac{i-0.5}{m})-W(\frac{i-1}{m})$$\end{document}$ and express $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\int _{0}^{t}b_{i}(s)dW(s)$$\end{document}$, in terms of BPFs as follows$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \begin{aligned} \int _{0}^{t}b_{i}(s)dW(s) \approx&\bigg (0,\dots ,0,W(\frac{i-0.5}{m})-W(\frac{i-1}{m}),W(\frac{i}{m})-W(\frac{i-1}{m}),\\ {}&\dots , W(\frac{i}{m})-W(\frac{i-1}{m})\bigg )b_{m}(t), \end{aligned} \end{aligned}$$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$W(\frac{i-0.5}{m})-W(\frac{i-1}{m})$$\end{document}$ is the *i*th component of vector. Therefore, we obtain the following expression (for details, see \[[@CR8]\])$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \int _{0}^{t}{} \mathbf{b} _{m}(s)dW(s) \approx P_{s}{} \mathbf{b} _{m}(t), \end{aligned}$$\end{document}$$where stochastic operational matrix of integration is given by$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ P_{s} = \begin{pmatrix} W(\frac{1}{2m}) &{} W(\frac{1}{m}) &{} W(\frac{1}{m}) &{} \cdots &{} W(\frac{1}{m}) \\ 0 &{} W(\frac{3}{2m})-W(\frac{1}{m}) &{} W(\frac{2}{m})-W(\frac{1}{m}) &{} \cdots &{} W(\frac{2}{m})-W(\frac{1}{m}) \\ 0 &{} 0 &{} W(\frac{5}{2m})-W(\frac{2}{m}) &{} \cdots &{} W(\frac{3}{m})-W(\frac{2}{m}) \\ \vdots &{} \vdots &{} \vdots &{} \ddots &{} \vdots \\ 0 &{} 0 &{} 0 &{} \cdots &{} W(\frac{2m-1}{2m})-W(\frac{m-1}{m}) \end{pmatrix}_{m\times m} $$\end{document}$$ Description of Numerical Method {#Sec9} =============================== Here we present the wavelet collocation method based on the Legendre wavelets for solving SFIDE ([1](#Equ1){ref-type=""}). We use the relation between the fractional derivative and integral to obtain the solution *u*(*t*) derived as followsLet $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$D^{\alpha }u(t) \approx C^{T}\varPhi (t)$$\end{document}$, this implies that $$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} u(t) \approx {C^{T}} {_{0}I^{\alpha }}\varPhi (t)+u_{0}. \end{aligned}$$\end{document}$$Let $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k_{1}(s,t) \in L^{2}([0,1)\times [0,1))$$\end{document}$. It can be expanded with respect to Legendre wavelet as $$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} k_{1}(s,t) \approx \varPhi ^{T}(s)K_{1}\varPhi (t) = \varPhi ^{T}(t)K_{1}^{T}\varPhi (s), \end{aligned}$$\end{document}$$ where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$K_{1} = (k_{1})_{ij},~i=1,2,\dots ,m,~j=1,2,\dots ,m$$\end{document}$ is the $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m\times m$$\end{document}$ Legendre wavelets coefficient matrix with $$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} (k_{1})_{ij} = \int _{0}^{1}\int _{0}^{1}k_{1}(s,t)\phi _{i}(s)\phi _{j}(t)dsdt. \end{aligned}$$\end{document}$$ Similarly $$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} k_{2}(s,t) \approx \varPhi ^{T}(s)K_{2}\varPhi (t) = \varPhi ^{T}(t)K_{2}^{T}\varPhi (s), \end{aligned}$$\end{document}$$ where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$K_{2} = (k_{2})_{ij},~i=1,2,\dots ,m,~j=1,2,\dots ,m$$\end{document}$ is the $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m\times m$$\end{document}$ Legendre wavelets coefficient matrix with $$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} (k_{2})_{ij} = \int _{0}^{1}\int _{0}^{1}k_{2}(s,t)\phi _{i}(s)\phi _{j}(t)dsdt. \end{aligned}$$\end{document}$$ Substituting the above approximation in ([1](#Equ1){ref-type=""}), we get$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} C^{T}\varPhi (t)&= f(t) +\int _{0}^{t}C^{T}(_{0}I^{\alpha }\varPhi (s))\varPhi ^{T}(s)K_{1}\varPhi (t)ds + u_{0}\int _{0}^{t}\varPhi ^{T}(s)K_{1}\varPhi (t)ds\\&+\int _{0}^{t}C^{T}(_{0}I^{\alpha }\varPhi (s))\varPhi ^{T}(s)K_{2}\varPhi (t)dW(s) + u_{0}\int _{0}^{t}\varPhi ^{T}(s)K_{2}\varPhi (t)dW(s) \\&= f(t)+C^{T}P^{\alpha }\big (\int _{0}^{t}\varPhi (s)\varPhi ^{T}(s)ds\big )K_{1}\varPhi (t)+u_{0}\big (\int _{0}^{t}\varPhi ^{T}(s)ds\big )K_{1}\varPhi (t) \\&+C^{T}P^{\alpha }\big (\int _{0}^{t}\varPhi (s)\varPhi ^{T}(s)dW(s)\big )K_{2}\varPhi (t)+u_{0}\big (\int _{0}^{t}\varPhi ^{T}(s)dW(s)\big )K_{2}\varPhi (t) \\&= f(t)+C^{T}P^{\alpha }\phi \big (\int _{0}^{t}{} \mathbf{b} (s)\mathbf{b} ^{T}(s)ds\big )\phi ^{T}K_{1}\varPhi (t)+u_{0}\big (\int _{0}^{t}{} \mathbf{b} ^{T}(s)ds\big )\phi ^{T} K_{1}\varPhi (t) \\&+C^{T}P^{\alpha }\phi \big (\int _{0}^{t}{} \mathbf{b} (s)\mathbf{b} ^{T}(s)dW(s)\big )\phi ^{T}K_{2}\varPhi (t)+u_{0}\big (\int _{0}^{t}{} \mathbf{b} ^{T}(s)dW(s)\big )\phi ^{T} K_{2}\varPhi (t). \end{aligned}$$\end{document}$$ Let $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P^{\alpha }\phi = Q_{1},~\phi ^{T}K_{1} = Q_{2},~\phi ^{T}K_{2} = Q_{3}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$Q_{2}^{i}$$\end{document}$ be the *i*th row of constant matrix $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$Q_{2}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$Q_{3}^{i}$$\end{document}$ be the *i*th row of constant matrix $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$Q_{3}$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$R^{i}$$\end{document}$ be the *i*th row of the integration operational matrix *P* and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$R_{s}^{i}$$\end{document}$ be the *i*th row of the stochastic operational matrix $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$P_{s}$$\end{document}$. We haveThen$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} C^{T}(I-Q_{1}B_{1}-Q_{1}B_{2})\varPhi (t) \approx f(t)+u_{0}(\mathbf{b} ^{T}(t)P^{T}Q_{2}+\mathbf{b} ^{T}(t)P_{s}^{T}Q_{3})\varPhi (t) \end{aligned}$$\end{document}$$So, by setting$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ A = \begin{pmatrix} \varPhi ^{T}(t_{1})(I-Q_{1}B_{1}-Q_{1}B_{2})^{T} \\ \vdots \\ \varPhi ^{T}(t_{m})(I-Q_{1}B_{1}-Q_{1}B_{2})^{T} \end{pmatrix}$$\end{document}$$and$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ F = \begin{pmatrix} f(t_{1})+u_{0}(\mathbf{b} ^{T}(t_{1})P^{T}Q_{2}+\mathbf{b} ^{T}(t_{1})P_{s}^{T}Q_{3})\varPhi (t_{1}) \\ \vdots \\ f(t_{m})+u_{0}(\mathbf{b} ^{T}(t_{m})P^{T}Q_{2}+\mathbf{b} ^{T}(t_{m})P_{s}^{T}Q_{3})\varPhi (t_{m}) \end{pmatrix}, $$\end{document}$$where $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$t_{i}$$\end{document}$ are the collocation points. We have$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} AC = F, \end{aligned}$$\end{document}$$which is a linear system of equations that gives the Legendre wavelets coefficient. Numerical Experiments {#Sec10} ===================== To illustrate the proposed method discussed in Sect. [4](#Sec9){ref-type="sec"}, we consider the following examples. Example 51 {#FPar3} ---------- Consider the SFIDE ([1](#Equ1){ref-type=""}) with $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f(t) = \frac{t^{2}}{2}+\frac{\varGamma (2)}{\varGamma (2-\alpha )}t^{1-\alpha },~k_{1}(s,t) = 1,~k_{2}(s,t) = 0,$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$u_{0} = 0$$\end{document}$. For $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha = 0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$u(t) = -(2+t)+2e^{t}$$\end{document}$ is exact solution of ([1](#Equ1){ref-type=""}). Example 52 {#FPar4} ---------- Consider the SFIDE ([1](#Equ1){ref-type=""}) with $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f(t) = -\frac{t^{5}e^{t}}{5}+\frac{6t^{2.25}}{\varGamma (3.25)},~k_{1}(s,t) = e^{t}s,~k_{2}(s,t) = 0,$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$u_{0} = 0$$\end{document}$. For $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha = 0.75$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$u(t) = t^{3}$$\end{document}$ is exact solution of ([1](#Equ1){ref-type=""}). Example 53 {#FPar5} ---------- Consider the SFIDE ([1](#Equ1){ref-type=""}) with $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f(t) = 0,~k_{1}(s,t) = s^{2},~k_{2}(s,t) = s^{3},$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$u_{0} = 1$$\end{document}$. For $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha = 0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$u(t) = e^{\frac{t^{3}}{3}+\int _{0}^{t}s^{3}dW(s)}$$\end{document}$ is exact solution of ([1](#Equ1){ref-type=""}). Let $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$u_{num}(t_{i},l)$$\end{document}$ denotes the approximate solution of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$l^{th}$$\end{document}$ simulation at $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$t_{i}$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$u_{exact}(t_{i},l)$$\end{document}$ denotes the exact solution of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$l^{th}$$\end{document}$ simulation at $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$t_{i}$$\end{document}$. The efficiency of the proposed method, for Examples [51](#FPar3){ref-type="sec"}, [52](#FPar4){ref-type="sec"} and [53](#FPar5){ref-type="sec"} are highlighted in Tables [1](#Tab1){ref-type="table"}, [2](#Tab2){ref-type="table"} and [3](#Tab3){ref-type="table"}, respectively, which showcase the values of maximum absolute error and root mean square (RMS) error that are defined as$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \mathbb {E}\Vert e\Vert _{\infty } = \frac{1}{N}\sum _{l=1}^{N}\max _{1\le i \le m}|u_{exact}(t_{i},l)-u_{num}(t_{i},l)|, \end{aligned}$$\end{document}$$ $$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} \mathbb {E}\Vert e\Vert _{2,m} = \frac{1}{N}\sum _{l=1}^{N}\sqrt{\frac{1}{m}\sum _{i=1}^{m}|u_{exact}(t_{i},l)-u_{num}(t_{i},l)|^{2}} \end{aligned}$$\end{document}$$respectively, where *N* is total number of simulation and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathbb {E}$$\end{document}$ is mathematical expectation. For deterministic function $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathbb {E}\Vert .\Vert _{\infty } = \Vert .\Vert _{\infty }$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathbb {E}\Vert .\Vert _{2,m} = \Vert .\Vert _{2,m}$$\end{document}$. Table [1](#Tab1){ref-type="table"} shows the maximum absolute errors obtained for Example [51](#FPar3){ref-type="sec"} via the proposed method discussed in Sect. [4](#Sec9){ref-type="sec"} for $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha = 0$$\end{document}$ and different values of *J*,  *M* and *m*. Table [2](#Tab2){ref-type="table"} shows the comparison of our method with Gaussian radial basis function (GA RBF) and thin plate splines radial basis function (TBS RBF) in terms of the absolute maximum error and RMS-error obtained for Example [52](#FPar4){ref-type="sec"} with $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha =0.75$$\end{document}$ and different values of *m*. These methods (GA RBF and TBS RBF \[[@CR11]\]) need smaller value of shape parameter for higher accuracy which increases the condition number of coefficient matrix and as a result the methods become unstable. However, the method proposed in Sect. [4](#Sec9){ref-type="sec"} has no such behavior. Finally, Table [3](#Tab3){ref-type="table"} shows the calculation of the mean and standard deviation which are denoted by $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathbb {E}\Vert e\Vert _{\infty }$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$S_{e}$$\end{document}$, respectively, of the maximum absolute error for Example [53](#FPar5){ref-type="sec"} with $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha = 0$$\end{document}$ and different number of simulation trajectories (*N*). For different values of *N*, the upper and lower limit of $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$95\%$$\end{document}$ confidence interval (C.I.) are also listed in Table [3](#Tab3){ref-type="table"}. In Fig. [1](#Fig1){ref-type="fig"}, we plot the mean approximate solution and mean exact solution of Example [53](#FPar5){ref-type="sec"} along with $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$95\%$$\end{document}$ confidence interval region for $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha = 0$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m =32$$\end{document}$ with different values of *N*.Table 1.Maximum absolute error in *u*(*t*) corresponding to $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha =0$$\end{document}$ with different value of *J* and *M*.*J*222333444*M*234234234$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m = 2^{J-1}M$$\end{document}$46881216162432$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Vert e\Vert _{\infty }$$\end{document}$0.04120.01940.01130.01130.00520.00300.00300.00130.0008 Table 2.Results of Example [52](#FPar4){ref-type="sec"} for different values of *m* and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha = 0.75$$\end{document}$*m*GA RBF \[[@CR11]\]TBS RBF \[[@CR11]\]Present method$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Vert e\Vert _{\infty }$$\end{document}$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Vert e\Vert _{2,m}$$\end{document}$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Vert e\Vert _{\infty }$$\end{document}$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Vert e\Vert _{2,m}$$\end{document}$*JM*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Vert e\Vert _{\infty }$$\end{document}$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Vert e\Vert _{2,m}$$\end{document}$4--------224.39$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!2$$\end{document}$2.53$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!2$$\end{document}$108.60$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!2$$\end{document}$7.62$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!2$$\end{document}$3.70$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!2$$\end{document}$4.63$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!2$$\end{document}$258.30$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!3$$\end{document}$4.10$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!3$$\end{document}$204.11$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!3$$\end{document}$7.30$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!3$$\end{document}$5.60$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!3$$\end{document}$4.89$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!3$$\end{document}$352.20$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!3$$\end{document}$1.0$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!3$$\end{document}$32--------529.0052$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!4$$\end{document}$4.0463$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!4$$\end{document}$403.5988$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!4$$\end{document}$8.6523$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!3$$\end{document}$4.3341$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!4$$\end{document}$2.3569$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!4$$\end{document}$453.7848$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!4$$\end{document}$1.5841$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$e\!-\!4$$\end{document}$ Fig. 1.The trajectory of the approximate solution and exact solution of Example [53](#FPar5){ref-type="sec"} along with 95$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\%$$\end{document}$ confidence interval (C.I) for $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$M = 2,~J = 5,~m = 32$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha = 0$$\end{document}$. Table 3.Mean, Standard deviation and mean confidence interval for maximum absolute error in Example ([53](#FPar5){ref-type="sec"}) with $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$m = 32,~J = 5,~ M = 2$$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha =0$$\end{document}$.*N*$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\mathbb {E}\Vert e\Vert _{\infty }$$\end{document}$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$S_{e}$$\end{document}$95$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\%$$\end{document}$ confidence interval for $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\chi _{e}$$\end{document}$LowerUpper300.29050.22460.21010.37091000.28360.20740.24290.32432000.27120.18980.24490.29755000.26580.16870.25100.2806 Applications {#Sec11} ============ In this section, we present some special cases of the proposed model and their applications in real life examples. SFIDE ([1](#Equ1){ref-type=""}) have many practical applications in scientific field such as physics, finance and biology etc. When $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\alpha =0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$f(t)=0$$\end{document}$, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k_1(s,t)=\mu $$\end{document}$ and $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$k_2(s,t)=\sigma $$\end{document}$, the proposed model reduces in the following form$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} u(t) = u_{0}+\int _{0}^{t}\mu u(s)ds+\int _{0}^{t}\sigma u(s)dW(s), \end{aligned}$$\end{document}$$or,$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{aligned} du(t) = \mu u(t)+\sigma u(t)dW(t). \end{aligned}$$\end{document}$$The above stochastic differential equation is called geometric Brownian motion model which is used for modeling the stock prices in the finance \[[@CR3]\]. Also in physics, ([35](#Equ35){ref-type=""}) is the Langevin equation with multiplicative noise \[[@CR4]\]. The Langevin equation is very important tool in physics for describing many physical process. Suppose *u*(*t*) is any physical process described by the ([35](#Equ35){ref-type=""}) then we always get probability distribution $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$p(u,t|u_{0})$$\end{document}$ corresponding to ([35](#Equ35){ref-type=""}) which satisfies the Fokker-Plank equation. Conclusion {#Sec12} ========== In this paper, we develop a wavelet collocation method based on the Legendre wavelets to solve SFIDE ([1](#Equ1){ref-type=""}). For this purpose, we compute the deterministic and stochastic operational matrices based on block pulse function. The SFIDE ([1](#Equ1){ref-type=""}) is then converted to a system of linear equations by employing the collocation method and making use of operational matrices. The solution of SFIDE is obtained using our proposed method. In Sect. [5](#Sec10){ref-type="sec"}, we solve several examples to indicate the accuracy and efficiency of the proposed method as discussed in Sect. [4](#Sec9){ref-type="sec"}. Supported by University Grants Commission, New Delhi-110002, India.

Introduction {#Sec1} ============ Cushing syndrome (CS) is characterized by chronic exposure to excessive glucocorticoid, and thus classified into adrenocorticotropic hormone (ACTH)-dependent and ACTH-independent CS. ACTH-independent CS accounts for approximately 15--20% of CS, primarily due to the unilateral adrenal tumor such as adrenocortical adenoma (ADA) and adrenal carcinoma that accounts for 10 and 5%, respectively. The rare causes of ACTH-independent CS include primary bilateral macronodular adrenal hyperplasia (BMAH), primary pigmented nodular adrenal disease (PPNAD), and McCune--Albright syndrome \[[@CR1]\]. PPNAD is rarely encountered and accounts for 0.6--1.9% of CS \[[@CR2]\]. It can occur isolated or as a component of the Carney complex (CNC). CNC is a hereditary multiple neoplasia syndrome characterized by skin lentigines, myxomas, and endocrine tumors, first described by J. Aidan Carney in 1985 \[[@CR3]\]. According to Stratakis et al., 95% patients with PPNAD fulfilled the diagnostic criteria of CNC \[[@CR4]\]; however, other reports on the incidence rate of CNC in patients with PPNAD are limited. The pathological feature of PPNAD constitutes of multiple pigmented cortical nodules and atrophy of the internodular cortex. The typical radiological features of PPNAD may present as multiple nodules of bilateral adrenal glands; however, in most cases, the appearance of adrenal glands with PPNAD might be normal or presented with macronodules \[[@CR5]\] that are indistinguishable from BMAH, which is characterized by large adrenal nodules, \>5 cm. Thus, occasionally, the preoperative diagnosis through imaging is misleading and rather challenging \[[@CR5], [@CR6]\]. Therefore, a new method with high sensitivity and specificity that can discriminate PPNAD from other adrenal diseases is an urgent prerequisite. Several reports have mentioned an anomalous increase in the glucocorticoid level in patients with PPNAD \[[@CR7]--[@CR9]\]. Stratakis et al. reported a paradoxical increase in urinary free cortisol (UFC) in response to dexamethasone during the Liddle test. Moreover, an increase of more than 50% in 24 h UFC on day 6 could distinguish PPNAD patients from those with other primary adrenal disorders causing CS with a 69.2% sensitivity and 80.0% specificity \[[@CR4]\]. Dexamethasone suppression tests (DST) are widely used in the diagnosis of CS. Low-dose dexamethasone suppression test (LDDST) serves as the definitive test in the confirmation of CS, while the high-dose dexamethasone suppression test (HDDST) differentiates between pituitary-dependent and non-pituitary-dependent forms of CS. Nevertheless, the usefulness of DST in the diagnosis of PPNAD is yet to be elucidated. In addition, since the level of cortisol differs among races, whether an increase in \>50% of 24 h UFC in the Liddle test could also be applied to Asian people is yet to be investigated. Herein, we analyzed data from 25 patients with PPNAD and control groups consisting of 27 patients with BMAH and 84 with ADA were admitted to the Peking Union Medical College Hospital (PUMCH) from 2001 to 2016. To our knowledge, this was the largest single-center study of PPNAD in Asia. LDDST and HDDST were conducted, and comparisons were made among the three groups. We used the ratio of 24 h UFC post-dexamethasone to prior-dexamethasone, for the first time, to determine the cut-off value for the evaluation of PPNAD. Materials and methods {#Sec2} ===================== Patients {#Sec3} -------- The present study was approved by the Ethics Committee of PUMCH. We retrospectively analyzed the records of 136 patients with ACTH-independent CS who were admitted to PUMCH from 2001 to 2016. All of the patients underwent unilateral epinephrectomy for the preserving of the adrenal gland function. Twenty-five patients were histologically confirmed for PPNAD, 27 patients had BMAH, and 84 were diagnosed with ADA. Methods {#Sec4} ------- All the clinical information was extracted from the medical records of PUMCH. The CNC was diagnosed based on the published criteria in 2001 \[[@CR3]\]. According to the criteria, the CNC could be diagnosed if patients fulfilled two major criteria or one major and one supplementary criterion. The screening tests, including echocardiogram, thyroid ultrasonography, breast ultrasonography, pituitary MRI, testicular ultrasonography in males, and transabdominal pelvic ultrasonography in females, were conducted for the confirmation of CNC in all participants. A total of 109 patients received LDDST, and 27 patients were administered 1 mg overnight dexamethasone test. All patients presented abnormal screening test results; moreover, they undertook HDDST immediately after LDDST. LDDST: After 2 days of baseline measurement of 24 h UFC, dexamethasone was administered orally at the dosage of 0.5 mg every 6 h for 2 days at 8:00, 14:00, 20:00, and 8:00. The 24 h UFC was measured on the second day of dexamethasone administration. The ratio of 24 h UFC post-LDDST to before LDDST was denoted as 24 h UFC (Post-L-Dex)/(Pre-L-Dex). Similar to LDDST, dexamethasone was administered at the dosage of 2 mg during HDDST. The time point of 24 h UFC was identical to that of LDDST. The ratio of 24 h UFC after HDDST to before HDDST was denoted as 24 h UFC (Post-H-Dex)/(Pre-H-Dex). Serum cortisol, serum ACTH, and 24 h UFC were measured with the chemiluminescence method using the commercial kits (DPC Biotechnology and Medical Products Cooperation, Tianjin, China). The data of arterial blood pressure, serum sodium and potassium, fasting plasma glucose, plasma glucose after the 75 g oral glucose tolerance test, and HbA1c levels were collected. Computed tomography (CT) of adrenal glands was performed for each patient, and bone mineral density (BMD) was evaluated using Dual-Energy X-ray absorption assay method to assess the influence of hypercortisolism to bone metabolism. Statistical analysis {#Sec5} -------------------- Statistical analyses were performed using the SPSS statistical package version 22.0. Kolmogorov--Smirnov test was utilized for normal distribution and homogeneity test for a variance. Normal distribution data were expressed as mean ± standard deviation. Skewed distribution data were expressed as median (P25, P75). Paired *t*-test compared the difference between 24 h UFC at the basal level and after DST. Chi-square test was used to assess the fundamental ratio differences among the groups. Kruskal--Wallis test was used in skewed distribution data to compare the differences among multiple groups of measurement data. The level of significance was corrected in the case of pairwise comparisons and *P*-value \< 0.017 was considered statistically significant. The receiver-operating characteristic (ROC) curve was constructed to assess the usefulness of both LDDST and HDDST in the diagnosis of PPNAD. 24 h UFC (Post-L-Dex)/(Pre-L-Dex) and 24 h UFC (Post-H-Dex)/(Pre-H-Dex) were employed as test variables independently. Results {#Sec6} ======= Baseline characteristics and clinical features of the three groups {#Sec7} ------------------------------------------------------------------ The baseline characteristics are presented in Table [1](#Tab1){ref-type="table"}. A total of 25 patients with PPNAD comprised of 8 males (32%) and 17 females (68%). The BMAH group consisted of 14 females (51.9%) while the ADA group comprised of 92.9% females (78/84), suggesting that the female constituent ratio was higher in the ADA group as compared to the other two groups (*P*-value \< 0.017). The mean age of the patients with PPNAD, BMAH, and ADA was 23 ± 11, 50 ± 10, and 34 ± 9 years, respectively. Although no significant difference was observed between the groups, patients with PPNAD seemed to have an earlier onset age.Table 1Baseline characteristics and clinical presentations of different groupsPPNADBMAHADA*P*-value*N*252784Male8 **\$**13 **\#**60.000Female17 **\$**14 **\#**780.000Age of diagnosis (years)23 ± 1150 ± 1034 ± 90.58Hypertension19 of 25 (76.0%)**\***27 of 27 (100%) **\#**49 of 61 (80.3%)0.03Osteoporosis or osteopenia18 of 23 (78.3%)20 of 27 (74.1%)24 of 50 (48.0%) **\$**0.015Hypokalemia3 of 25 (12.0%)**\***14 of 27 (51.9%)28 of 84 (33.3%)0.009IGT or DM14 of 25 (56.0%)18 of 27 (66.7%)39 of 80 (48.8%)0.263*PPNAD* primary pigmented nodular adrenocortical disease, *BMAH* bilateral macronodular adrenal hyperplasia, *ADA* adrenocortical adenoma, *IGT* impaired glucose tolerance, *DM* diabetes mellitus**\*** PPNAD vs. BMAH, *P* \< 0.017; **\#** BMAH vs. ADA, *P* \< 0.017; **\$** PPNAD vs. ADA *P* \< 0.017 As shown in Table [1](#Tab1){ref-type="table"}, a majority of the patients exhibited typical CS manifestations such as hypertension, weight gain, and abnormal glucose metabolism, and decreased BMD. In the present study, patients in the PPNAD group seemed to have a higher incidence rate of developing decreased BMD (osteopenia or osteoporosis) than those with ADA (78.3 vs. 48.0%, *P* \< 0.017), while no differences were found between PPNAD and BMAH groups. In addition, patients with PPNAD had a lower incidence rate of hypokalemia as compared to BMAH (12.0 vs. 51.9%, *P* \< 0.017). In the BMAH group, all the 27 patients developed hypertension, indicating a significant difference between BMAH and other groups (100 vs. 76.0%, *P* \< 0.017; 100 vs. 80.3%, *P* \< 0.017). With respect to glucose metabolism, no statistically significant differences were observed among the three groups (*P* = 0.263). Laboratory examinations of the three groups {#Sec8} ------------------------------------------- In all participants, the serum ACTH levels were \<10 pg/mL, providing support for the diagnosis of ACTH-independent CS accordingly. Table [2](#Tab2){ref-type="table"} displayed the 24 h UFC levels before and after administration of dexamethasone. The baseline of 24 h UFC in patients with PPNAD was higher than that of the patients with ADA (383.50 vs. 224.42, normal range: 12.3--103.5 µg/24 h, *P* \< 0.017, Fig. [1a](#Fig1){ref-type="fig"}). No statistical difference was observed between the other groups. In the ADA group, the majority of the patients showed an increased UFC level, while 9.4% of the patients exhibited a 24 h UFC level within the normal range. After LDDST and HDDST, the 24 h UFC elevated in patients with PPNAD (paired *t*-test, *P* = 0.007 and *P* = 0.001); among these, 12 patients (48%) presented an elevation of 24 h UFC \> 50% after HDDST. On the other hand, 24 h UFC remained unaltered in the BMAH group (paired *t*-test, *P* = 0.471 and *P* = 0.414) and decreased in the ADA group (paired *t*-test, *P* = 0.002 and *P* = 0.004, Fig. [1b, c](#Fig1){ref-type="fig"}). The 24 h UFC level after LDDST was higher in the PPNAD and BMAH groups as compared to ADA (*P* \< 0.017), while no significant difference was observed between PPNAD and BMAH. After administration of HDDST, 24 h UFC was higher in patients with PPNAD as compared to that of ADA and BMAH (*P* \< 0.017).Table 2Levels of 24 h UFC before and after dexamethasoneGroupPPNADBMAHADA*P*-valueBaseline (µg/d)383.50 (271.61, 452.97) **\#**314.70 (151.04, 557.16)224.42 (144.50, 334.88)0.002LDDST (µg/d)497.35 (389.86, 717.70) **\#**325.60 (124.28, 557.03)181.66 (108.00, 252.00) **\$**0.000HDDST (µg/d)495.30 (402.97, 876.55) **\#**339.84 (175.42, 526.13) **\***184.50 (116.20, 309.61)0.000Post/Pre (L)1.29 (0.91, 1.71) **\#**0.93 (0.82, 1.23)0.78 (0.51, 1.07)0.000Post/Pre (H)1.48 (1.14, 2.09) **\#**0.94 (0.76, 1.59) **\***0.92 (0.71, 1.07)0.000*PPNAD* pigmented nodular adrenocortical disease, *BMAH* bilateral macronodular adrenal hyperplasia, *ADA* adrenocortical adenoma, *LDDST* low-dose dexamethasone suppression test, *HDDST* high-dose dexamethasone suppression test, *Post/Pre* (*L*) the ratio of 24 h UFC posterior to LDDST and prior to LDDST, *Post/Pre* (*H*) the ratio of 24 h UFC posterior to HDDST and prior to HDDST**\*** PPNAD vs. BMAH, *P* \< 0.017; **\#** PPNAD vs. ADA, *P* \< 0.017; \$ BMAH vs. ADA, *P* \< 0.017 Fig. 124 h UFC levels before and after administration of dexamethasone. The basal level of 24 h UFC in the PPNAD group was higher than that of ADA (Fig. 1a, *P* \< 0.017), after low-dose and high-dose dexamethasone. 24 h UFC in patients with PPNAD increased, while in patients with ADA and BMAH the 24 h UFC decreased or remained unaltered. The 24 h UFC level after LDDST was higher in the PPNAD group and BMAH as compared to ADA (*P* \< 0.017), while no significant difference between PPNAD and BMAH was observed (Fig. 1b). After administration of HDDST, 24 h UFC was higher in patients with PPNAD as compared to that of ADA and BMAH (Fig. 1c, *P* \< 0.017). The ratio of 24 h UFC posterior to DST to before DST (Fig. 1d, e) showed a significant difference between PPNAD and the other two groups In order to clarify the level of 24 h UFC variation, the ratio of 24 h UFC post-DST to 24 h UFC pre-DST was calculated and denoted as 24 h UFC (Post-L-Dex)/(Pre-L-Dex) and 24 h UFC (Post-H-Dex)/(Pre-H-Dex) after LDDST and HDDST, respectively. The ratio \> 1 suggested that 24 h UFC elevated after DST. Furthermore, after LDDST, the 24 h UFC (Post-L-Dex)/(Pre-L-Dex) in the PPNAD group was significantly higher than that of the ADA group (1.29 vs. 0.78, *P* \< 0.017), whereas the ratio between PPNAD and BMAH showed no difference (Fig. [1d](#Fig1){ref-type="fig"}). After administration of high-dose dexamethasone, 24 h UFC (Post-H-Dex)/(Pre-H-Dex) in PPNAD, BMAH, and ADA was 1.48 (1.14, 2.09), 0.94 (0.76, 1.59), and 0.92 (0.71, 1.07), respectively, and the ratio in the PPNAD group was distinctively higher than that of BMAH and ADA (*P* \< 0.017, Fig. [1e](#Fig1){ref-type="fig"}). Clinical features and laboratory examinations between isolated PPNAD and CNC {#Sec9} ---------------------------------------------------------------------------- In 9/25 patients (36.0%), PPNAD occurred as a component of CNC; the clinical features were summarized in Table [3](#Tab3){ref-type="table"}. The cohort comprised of two males and seven females, with an average age of 19.6 (range, 14--28) years. Spotty hyperpigmentation was the most frequent manifestation and could be observed in all the patients. In addition to spotty hyperpigmentation, a 19-year-old male also demonstrated a cyst in the head of the epididymis. Multiple solid hypoechoic nodules of the thyroid gland were found in three females, whereas two had nodules in the breast. Two females had solid nodules in the unilateral or bilateral breast. No cardiac myxoma and tumors of other endocrine glands were found in patients with CNC. Furthermore, comparisons between patients with isolated PPNAD and patients with CNC (Table [4](#Tab4){ref-type="table"}) did not reveal any significant differences in the clinical features such as age, sex, constituent ratio, body mass index (BMI), the incidence rate of hypertension, disturbance of carbohydrate metabolism, dyslipidemia, and decreased BMD. The 24 h UFC levels before and after DST did not show any difference between the patients with isolated PPNAD and those with CNC (*P* \> 0.05).Table 3Clinical manifestations of the patients with CNCNumberGenderAge (years)PPNADOther manifestations of CNC1M14YesSpotty hyperpigmentation in lower eyelid and lips2F23YesSpotty hyperpigmentation in face and bulbar conjunctiva3F17YesSpotty hyperpigmentation in face and hands, with similar family history4F28YesSpotty hyperpigmentation in lips and buccal mucosa, pancreatic cyst, nodule in unilateral breast5F19YesSpotty hyperpigmentation in face and lips, solid nodules in bilateral breast6F14YesMultiple hypoechoic nodules of the thyroid gland7M19YesSpotty hyperpigmentation in face, cyst in the head of epididymis8F25YesSpotty hyperpigmentation in lips and buccal mucosa, hypoechoic nodules in thyroid gland and breast, skeletal lesions9F17YesSpotty hyperpigmentation in lips and buccal mucosa, hypoechoic nodules in thyroid gland and breast*PPNAD* pigmented nodular adrenocortical disease Table 4Clinical features and laboratory examinations of patients with isolated PPNAD and patients with CNCIsolated PPNADCarney syndrome*P*-valueAge (years)22.13 ± 9.9119.56 ± 4.570.318Number1690.667  Female107  Male62BMI (kg/m^2^)24.8526.630.327Hypertension1180.380  Grade I32  Grade II23  Grade III63IGT or DM951.000  IGT84  DM11TC (mmol/L)6.015.250.213TG (mmol/L)1.811.460.491LDL-C (mmol/L)3.813.030.122HDL-C (mmol/L)1.651.610.869Potassium (mmol/L)4.003.960.873Osteopenia or osteoporosis1261.000  Osteopenia31 Osteoporosis95  Severe osteoporosis21Fatty liver531.000Adrenal gland CT4 with normal adrenal glands2 with normal adrenal glands5 with unilateral nodules1 with unilateral nodules8 with bilateral nodules6 with bilateral nodules24 h UFC  Baseline375.05 (282.18, 411.40)423.10 (216.88, 622.96)0.301  LDDST482.20 (344.96, 770.60)556.92 (389.86, 778.85)0.558   HDDST485.42 (339.17, 745.01)862.90 (391.21, 948.76)0.229Post/Pre (L)1.31 (1.04, 1, 75)1.50 (0.79, 2.15)0.978Post/Pre (H)1.49 (1.12, 2.08)1.59 (1.05, 2.18)0.846*PPNAD* pigmented nodular adrenocortical disease, *BMI* body mass index, *IGT* impaired glucose tolerance, *DM* diabetes mellitus, *TC* total cholesterol, *TG* triglyceride, *LDL-C* low-density lipoprotein cholesterol, *HDL-C* high-density lipoprotein cholesterol, *24 h UFC* 24 h urinary free cortisol, *Post/Pre* (*L*) the ratio of 24 h UFC posterior to LDDST and prior to LDDST, *Post/Pre* (*H*) the ratio of 24 h UFC posterior to HDDST and prior to HDDS ROC and cut-off values in the diagnosis of PPNAD {#Sec10} ------------------------------------------------ 24 h UFC (Post-L-Dex)/(Pre-L-Dex) and 24 h UFC (Post-H-Dex)/(Pre-H-Dex) were used as the test variables. The ROC curve was constructed with PPNAD that was served as the state variable, and its value was 1. Taking the leftmost point of the curve as the cut-off point, the area under the ROC curve (AUC) was 0.733 after LDDST, and the cut-off value of 24 h UFC (Post-L-Dex)/(Pre-L-Dex) was 1.16 with 64.0% sensitivity and 77.9% specificity. Similarly, the AUC was 0.802 after HDDST, and the cut-off value of 24 h UFC (Post-H-Dex)/(Pre-H-Dex) was 1.08 with 84.0% sensitivity and 75.6% specificity (Fig. [2](#Fig2){ref-type="fig"}).Fig. 2ROC curve was constructed with the ratio of 24 h UFC Post/Pre (L) and Post/Pre (H) as the detection variable. The AUC was 0.733 and 0.802 after low-dose and high-dose dexamethasone, respectively Discussion {#Sec11} ========== PPNAD occurs as a rare cause of CS, accounting for 0.6--1.9% of all such patients \[[@CR2]\]. CNC is an autosomal dominant inherited and multiple neoplasia syndrome characterized by skin tumors and pigmented lesions, cardiac myxomas, schwannomas, breast adenomas, bone lesions and various endocrine disorders caused by tumors of the pituitary and thyroid glands, pancreas, and/or gonads \[[@CR10], [@CR11]\]. A total of 26--60% of the CNC patients exhibited PPNAD, and it is the most common endocrine tumor associated with CNC \[[@CR3], [@CR10], [@CR12]\]. In our study, 36.0% PPNAD patients had the components of CNC, and all of them presented spotty pigmentation in the skin or the mucosa, five females demonstrated the hypoechoic nodules of the thyroid gland and/or breast, and a male developed a cyst in the head of the epididymis. Nevertheless, the commonly reported manifestation, cardiac myxoma \[[@CR13]\], was not found in our patients. Stratakis et al. reported that 20/21 patients (95%) showed PPNAD that occurred as a component of CNC \[[@CR4]\]. Such an enormous difference in the incidence rate of CNC in patients with PPNAD between the current study (36%) and that by Stratakis et al. (95%) might be attributed to the ethnic differences. Additionally, the sample size of both studies was not sufficiently large, which leads to inevitable bias to the results. Further investigations with expanded sample size need to be undertaken in order to clarify the genetic distinctions among various races. DST is commonly used in the diagnosis and differential diagnosis of ACTH-dependent CS. LDDST or 1 mg overnight DST is used for screening the presence of CS in the patient, while HDDST is used to distinguish CS from other ACTH-dependent CS; \>50% suppression of cortisol concentration indicates CS. However, limited studies are available concerning the administration of dexamethasone in ACTH-independent CS since the baseline of serum ACTH level is relatively low. Several reports have mentioned a paradoxical increase in the glucocorticoid level after administration of dexamethasone in patients with PPNAD \[[@CR14]--[@CR17]\]. Silverman et al. reported a patient with CS secondary to bilateral nodular adrenocortical hyperplasia, in whom urinary 17-OHCS could not be suppressed by a large dose of dexamethasone, and the adrenal pathology demonstrated cortical hyperplasia with multiple small nodules \[[@CR14]\]. In 1999 Stratakis et al. found that UFC levels on day 6 of the Liddle test had the highest accuracy in the diagnosis of PPNAD, and an increase of \>50% UFC levels could discriminate 9/13 patients with PPNAD from other primary adrenocortical disorders \[[@CR4]\]. In the current study, we also found that after DST, the 24 h UFC levels in patients with PPNAD increased, while in the other two groups the 24 h UFC levels remained unchanged or even decreased, which suggested the usefulness of DST in the diagnosis of PPNAD. In patients with ADA, the 24 h UFC levels were slightly decreased after DST, which might be partially attributed to the suppressibility of those patients whose basal 24 h UFC values were within the normal range. Herein, we used the ratio of 24 h UFC post-dexamethasone to prior-dexamethasone, for the first time, in the diagnosis of PPNAD. After LDDST, the ratio of 24 h UFC Post/Pre (L) in the PPNAD group was significantly higher than that in the ADA group, while it could not differentiate PPNAD from BMAH. After administration of high-dose dexamethasone, a statistically significant difference between PPNAD and BMAH was noted, indicating the diagnostic value of DST, especially the HDDST could identify PPNAD from both BMAH and ADA. Our study revealed the ratio of 24 h UFC after LDDST to the baseline value \>1.16 with 64.0% sensitivity and 77.9% specificity. Similarly, when the value of 24 h UFC (Post-H-Dex)/UFC (Pre-H-Dex) is \>1.08, the sensitivity and specificity increased to 84.0 and 75.6%, respectively. Compared to the previous study using 24 h UFC of \>50% increase on day 6 of the Liddle test, the degree of UFC level was slightly lower in our study. This phenomenon might be attributed to the theory that the proportion of patients with isolated PPNAD was different between our study and that by Stratakis et al. The molecular mechanism underlying the paradoxical response to dexamethasone in patients with PPNAD was due to an increased expression of glucocorticoid receptor in PPNAD nodules \[[@CR17]\]. The CNC is primarily caused by germline mutations in the protein kinase A regulatory subunit 1A (*PRKAR1A*) gene \[[@CR18]--[@CR22]\]. Nevertheless, several studies demonstrated that patients with isolated PPNAD exhibited specific molecular genetic abnormalities with mutations in phosphodiesterase *PDE11A*, *PDE8B*, and the PKA catalytic subunit *PRKACA* gene \[[@CR23]--[@CR25]\]. The mutations in various gene loci may differentially influence the cAMP/PKA pathway in patients with isolated PPNAD and CNC, leading to variable levels of UFC. Nevertheless, the present study exhibited some limitations. Since we mainly focused on the clinical study regarding the usefulness of DST in the diagnosis of PPNAD, the genetic mutations were not assessed in the participants, which made it impossible to compare the genetic abnormities between patients with isolated PPNAD and those with CNC. Furthermore, due to the retrospective nature of the current study, some clinical data were missing, which could lead to a potential bias in our study. In conclusion, DST, especially HDDST, is greatly beneficial in the early diagnosis of PPNAD. When the radiological examination could not provide valuable information, DST could detect the patients with subclinical, atypical PPNAD-associated manifestations and distinguish PPNAD from other adrenal diseases. With respect to the Chinese population, the cut-off value of 24 h UFC (Post-H-Dex)/(Pre-H-Dex) was 1.08 with 84.0% sensitivity and 7.6% specificity. However, the cut-off values of 24 h UFC levels between different races need to be investigated further. We thank Yimin Qu for the valuable suggestions for statistical analysis. Conflict of interest {#FPar1} ==================== The authors declare that they have no competing interests.

Background {#Sec1} ========== The contemporary response to vector-borne infectious diseases still mainly relies on low-technology interventions, with a major emphasis on vector control through the use of insecticides. As vector-borne diseases continue to present significant threats to human, animal and plant health, there is an urgent need to develop control efforts that remain effective over time. Genetically modified disease vectors that are rendered resistant (refractory) to pathogen transmission can provide unique tools for developing new or complementing existing control strategies (reviewed by \[[@CR1]\]). Paratransgenesis is one such approach that aims to reduce vector competence by genetically modifying symbionts of disease vectors and has been demonstrated for *Rhodnius prolixus*, the triatomine vector of Chagas disease \[[@CR2]\] and *Anopheles gambiae* \[[@CR3],[@CR4]\], the principal malaria vector in Africa. A paratransgenic approach in tsetse flies, the sole vector of major African trypanosome parasites (*T. brucei* sp. and *T. congolense*), is of high interest since tsetse flies are not amenable to germ-line transformation due to their viviparous reproductive biology (intrauterine development and parturition of live offspring \[[@CR5]\]). The tsetse fly harbors a natural commensal bacterium i.e. *Sodalis glossinidius*, which is ideally suited as a paratransgenic platform organism since it i) resides in different tsetse tissues that are in close proximity to pathogenic trypanosomes (e.g. midgut) \[[@CR6]\]; ii) can be cultured and genetically modified *in vitro* \[[@CR7],[@CR8]\]; iii) is maternally transmitted to the offspring \[[@CR9]\] and iv) is restricted to the tsetse host niche ensuring that this symbiont is a safe candidate for use in the paratransgenic strategy \[[@CR10]\]. The characteristics of the selected effector molecule will largely determine the efficacy and specificity of this paratransgenesis approach. Low specificity will generally result in bystander effects that could have a direct or indirect fitness cost for the arthropod. The use of highly specific compounds from the adaptive immune system of vertebrates such as antibody derived fragments is likely to enable highly specific effects without conferring a selective disadvantage to the (para)transgenic arthropods \[[@CR11]\]. Nanobodies® (Nbs), representing the smallest known intact antigen-binding fragments derived from camelid heavy-chain only antibodies (HCAbs) \[[@CR12]\], are therefore considered as excellent candidates to increase the immune competence of tsetse. Nanobodies targeting distinct epitopes of the variant-specific surface glycoprotein (VSG), abundantly present on the surface of bloodstream trypanosomes have already been identified, some of which were shown to exert direct *in vitro* and *in vivo* trypanolytic activity by interfering with the complex endocytotic machinery that is organized in the flagellar pocket of this parasite \[[@CR13]\]. Recently, we developed recombinant *Sodalis* (rec*Sodalis*) strains expressing functional anti-trypanosome nanobodies. These strains were shown to release considerable amounts of functional anti-trypanosome Nbs to the extracellular culture environment and to have no negative effects on the bacterium in an *in vitro* context \[[@CR14]\]. Another prerequisite in developing paratransgenic tsetse flies is the development of a methodology that allows the stable repopulation of tsetse flies with rec*Sodalis* strains expressing trypanosome-interfering proteins in insect tissues where trypanosome parasites reside. Previously, rec*Sodalis* expressing green fluorescent protein (GFP) has been successfully introduced into tsetse through thoracic microinjection \[[@CR6]\]. Here, GFP-expressing *Sodalis* was found to be present in the haemolymph and gut tissues of injected females and their progeny. However, to date no studies have focused on the densities whereby genetically modified *Sodalis*, expressing heterologous genes, are maintained within the fly and the efficiency of their transmission to the offspring. In this study, a rec*Sodalis* strain was tested for its ability to deliver functional anti-trypanosome Nbs in the tsetse fly *Glossina morsitans morsitans*. The *in vivo* long-term stability of the recombinant strain and transmission to the progeny was measured using a quantitative PCR (qPCR) analysis. We show that rec*Sodalis* expressing a potent trypanolytic nanobody, i.e. Nb_An46, was stably maintained *in vivo* only when the WT *Sodalis* population was significantly reduced (\>95% of the normal population) prior to rec*Sodalis* introduction. Furthermore, we demonstrated that significant levels of functional Nb_An46 accumulated in different fly tissues, including the midgut where an important developmental stage of the trypanosome parasite occurs. Results {#Sec2} ======= *In vitro* culture characteristics of recombinant *Sodalis* expressing Nb_An46 {#Sec3} ------------------------------------------------------------------------------ Prior to the introduction of rec*Sodalis* into experimental flies, the Nb expression profile, *in vitro* growth rate and plasmid stability of rec*Sodalis* expressing a FliCpelBNb_An46 fusion protein (*Sod*:FliCpelBNb46*fliC*) was established. Extracellular Nb_An46 expression was confirmed by Western blot analysis of supernatant from cultures grown to the beginning of stationary phase (OD~600~ 0.5-0.6) (Figure [1](#Fig1){ref-type="fig"}). Nb_An46 expression and release was quantified at different time points during bacterial growth over a 10-day period by measuring the concentration of active Nb in the whole cell lysate and culture medium using a VSG-binding ELISA assay. Functional Nb_An46 was expressed from day 2 onwards and accumulated in the culture medium to a concentration of 88 ng/ml by day 10 (Figure [2](#Fig2){ref-type="fig"}B). RecS*odalis* showed normal growth kinetics (Figure [2](#Fig2){ref-type="fig"}A) with cell population doubling times comparable to a WT *Sodalis* strain i.e., 15.0 hrs and 14.8 hrs respectively. The number of plasmid copies per cell was estimated to be approximately 20 during the lag and exponential phases of *Sod*:FliCpelBNb46*fliC* grown in the presence of antibiotic selection. The stability of the FliCpelBNb46*fliC* plasmid in rec*Sodalis* was measured by maintaining the recombinant bacteria in log phase growth for 27 generations in liquid MM medium in the absence of antibiotic selection. Colony counts on antibiotic-selective plates showed that 94% of the *Sodalis* population remained antibiotic resistant after 27 generations (corresponding to a 2-month test period) (Table [1](#Tab1){ref-type="table"}).Figure 1**Qualitative analysis of extracellular Nb_An46 expressed by** ***Sod*** **\_pFliCpelBNb46** ***fliC in vitro*** **.** The extracellular expression of Nb_An46 was analyzed by immunoblotting of the medium supernatant using an anti-His antibody (1:1000 Serotec) for detection. Presented data are representative for at least three independent experiments. The PageRuler 10--170 kDa prestained protein ladder (Fermentas) was used as a molecular size marker (MM).Figure 2***In vitro*** **characteristics of rec** ***Sodalis*** **expressing Nb_An46. A)** Growth curve analysis of WT *Sodalis* and *Sod*\_pFliCpelBNb46*fliC*. The error bars show the ± SD of two biological replicates. Samples were taken every 24 h. **B)** ELISA-based Nb_An46 quantitation (bar-chart) of the intra- and extracellular nanobody concentration produced by *Sod*\_pFliCpelBNb46*fliC* at selected time points in relation to bacterial cell density (OD~600nm~, solid line) using a 6 × His tag specific detection antibody. Values are presented as ng recombinant protein per ml culture medium.Table 1**Stability of pFliCpelBNb46** ***FliC*** **in** ***Sodalis*** **under nonselective growth conditionsGenerations (n)Plasmid-containing CFU (%)**10**100**15**77**20**91**27**94**Plasmid stability was expressed as the ratio between the number of colonies formed on MM blood agar plates with (50 μg/ml) and without kanamycin. Prior reduction of WT *Sodalis* is a prerequisite for an efficient host colonization with *recSodalis* {#Sec4} ------------------------------------------------------------------------------------------------------ We explored the capability of rec*Sodalis* to colonize the tsetse fly after introduction through intrathoracic injection. We first evaluated the necessity of the prior reduction of the WT *Sodalis* population in tsetse for rec*Sodalis* to establish and colonize its host. In female flies that received 3 streptozotocin supplemented blood meals, the WT *Sodalis* population was reduced by 95% and 88% in abdomen and thorax tissues respectively, compared to flies fed on normal blood. This treatment did not affect the obligatory *Wigglesworthia* symbiont population that mainly resides in the tsetse fly abdomen (Additional file [1](#MOESM1){ref-type="media"}: Figure S1). Next, the *in vivo* persistence and growth of the recombinant bacteria in streptozotocin-treated and non-treated flies injected intrathoracically with 5×10^4^ CFU *Sod*\_FliCpelBNb46*fliC* was evaluated using qPCR (Figure [3](#Fig3){ref-type="fig"}). In streptozotocin-treated flies, rec*Sodalis* was able to proliferate inside its host, reaching densities of 10- and 20-fold the initial injected dose in respectively abdomen and thorax, whereas in non-treated flies the injected rec*Sodalis* population was not able to expand and remained present at its initial density.Figure 3**Number of rec** ***Sodalis*** **CFU (DNA equivalent) in abdomen and thorax of streptozotocin-treated and non-treated male flies injected with 5x10** ^**4**^ **CFU of** ***Sod*** **\_pFliCpelBNb46** ***fliC*** **.** Prior to injection, treated flies were given three blood meals supplemented with 20 μg/ml streptozotocin during the first week after emergence, while non-treated flies received normal blood meals. qPCR on the pFliCpelBNb46*FliC* plasmid was used as a means to estimate the number of rec*Sodalis*. The bars represent the mean rec*Sodalis* CFU (DNA equivalent) ± SD present in abdomen and thorax tissues of at least 5 individual flies from each treatment group at the time of sampling. The number of CFU (DNA equivalent) is represented in log scale on the y-axis. P values were calculated using the Mann--Whitney U test (\*p \<0.05, \*\*p \<0.01). The effect of differential doses of rec*Sodalis* inoculum on tsetse fly colonization and viability {#Sec5} -------------------------------------------------------------------------------------------------- We determined the optimal dose for rec*Sodalis* injection in terms of host colonization and its viability. For this, streptozotocin-treated male flies were microinjected with either 5 × 10^4^, 5 × 10^5^, 10^7^ and 5 × 10^7^ CFU *Sod*\_FliCpelBNb46*fliC* and rec*Sodalis* densities in abdomen and thorax tissues were determined 7 and 14 days post-injection (dpi) using qPCR (Figure [4](#Fig4){ref-type="fig"}). Injection of 5 × 10^7^ CFU resulted in an increased fly mortality (up to 59% mortality 14 dpi), whereas limited mortality (≤25% 14 dpi) was observed within the other injection groups. In these groups, rec*Sodalis* was able to repopulate the abdomen and thorax to densities comparable to natural *Sodalis* levels present in WT flies (on average respectively 2.5 × 10^6^ and 1.5 × 10^6^ CFU), demonstrating that a wide range of rec*Sodalis* doses (i.e. 5 × 10^4^ to 1 × 10^7^) are suitable to initiate colonization without affecting fly viability.Figure 4**Number of rec** ***Sodalis*** **CFU (DNA equivalent) present in abdomen (A) and thorax (T) tissues of flies injected with respectively 5×10** ^**4**^ **, 5×10** ^**5**^ **, 1×10** ^**7**^ **and 5×10** ^**7**^ **CFU of** ***Sod*** **\_pFliCpelBNb46** ***fliC*** **. qPCR on the pFliCpelBNb46** ***FliC*** **plasmid was used as a means to estimate the number of rec** ***Sodalis*** **.** The bars represent the mean rec*Sodalis* CFU (DNA equivalent) ± SD present in abdomen and thorax tissues of at least 5 individual flies from each treatment group at the time of sampling. The number of CFU (DNA equivalent) is represented in log scale on the y-axis. P values were calculated using the Kruskal--Wallis test followed by Dunn\'s test for multiple comparison (\*p \<0.05, \*\*p \<0.01). *recSodalis* persists in the tsetse fly and is vertical transmitted to the offspring but at a low extent {#Sec6} -------------------------------------------------------------------------------------------------------- The *in vivo* persistence of *Sod*\_FliCpelBNb46*fliC* was evaluated by qPCR based estimation of the amount of rec*Sodalis* CFU in abdomen and thorax tissues of streptozotocin-treated male flies injected with 1 × 10^7^ recombinant CFU over a 28 day period (Figure [5](#Fig5){ref-type="fig"}A). *Sod*\_FliCpelBNb46*fliC* was able to remain present at high densities in abdomen and thorax tissues of experimental flies throughout the course of the 28-day observation period. In flies injected with *Sod*\_FliCpelBNb46*fliC*, the entire *Sodalis* population in abdomen and thorax remained recombinant. Next, we evaluated rec*Sodalis* densities in the haemolymph and midgut tissues of flies injected with 1 × 10^7^ recombinant CFU (Figure [5](#Fig5){ref-type="fig"}B). *Sod*\_FliCpelBNb46*fliC* was able to reach the fly midgut where it persisted at densities between 5 × 10^4^ and 1 × 10^5^ CFU (DNA equivalent) throughout the 21-day observation period. In these flies the obligatory *Wigglesworthia* symbiont population was not affected by the presence of *Sod*\_FliCpelBNb46*fliC* (Additional file [1](#MOESM1){ref-type="media"}: Figure S2) nor did we observe any effect upon the fecundity of female rec*Sodalis* colonized flies. Transmission dynamics of the recombinant bacteria to the F~1~ progeny was evaluated by qPCR. *Sod*\_FliCpelBNb46*fliC* was transmitted to the F~1~ generation, although non-plasmid containing *Sodalis* were dominant in these flies. Indeed, *Sod*\_FliCpelBNb46*fliC* constituted only 7 and 5% of the entire *Sodalis* population in respectively abdomen and thorax (Figure [6](#Fig6){ref-type="fig"}).Figure 5**Evaluating the host colonization properties of rec** ***Sodalis*** **. A)** Number of rec*Sodalis* CFU (DNA equivalent) present in abdomen (solid red line) and thorax (solid black line) of Streptozotocin-treated flies injected with 1x10^7^ CFU of *Sod*\_pFliCpelBNb46*fliC* versus the total number of *Sodalis* CFU DNA equivalent (WT + recombinant *Sodalis*) present in abdomen (dashed red line) and thorax tissues (dashed black line). **B)** The number of recombinant and total *Sodalis* CFU (DNA equivalent) present in haemolymph (solid/dashed blue line) and midgut (solid/dashed grey line) were estimated as described above. Data points represent the mean rec*Sodalis* and total *Sodalis* CFU (DNA equivalent) ± SD present in the respective tissues of at least 5 individual flies at the time of sampling. The number of CFU (DNA equivalent) is represented in log scale on the y-axis.Figure 6**Transmission of rec** ***Sodalis*** **to the F** ~**1**~ **generation was evaluated.** Recombinant and total *Sodalis* numbers were estimated as described above in abdomen and thorax of the F~1~ progeny (freshly emerged) produced by streptozotocin-treated female flies injected with 5 × 10^4^ CFU of *Sod*\_pFliCpelBNb46*fliC*. Data points represent the mean rec*Sodalis* and total *Sodalis* CFU (DNA equivalent) ± SD present in the respective tissues of at least 5 individual flies at the time of sampling. The number of CFU (DNA equivalent) is represented in log scale on the y-axis. Functional Nb_An46 is expressed in different tsetse fly tissues {#Sec7} --------------------------------------------------------------- Nb_An46 expression in flies injected with 1 × 10^7^*Sod*\_FliCpelBNb46*fliC* CFU was quantified using a VSG-binding ELISA. Nanobody concentrations were determined at different time points post-injection in whole abdomen and thorax extracts, haemolymph and midgut (Figure [7](#Fig7){ref-type="fig"}). Functional Nb_An46 accumulated in haemolymph and thorax samples of injected flies over time, indicating a continuous transgene expression by rec*Sodalis* in these tissues. In the thorax the Nb_An46 concentration increased from 22 ng on day 14 post-injection to 35 ng on day 21. Although significant quantities of active Nb_An46 were detected in abdomen and midgut using a VSG-binding ELISA, the accuracy of this quantitation could have been hampered by the abundant presence of proteolytic enzymes in the tsetse fly gut, probably resulting in fast degradation of the nanobodies in the tissue homogenates and possibly resulting in the underestimation of the active Nb content of the non-digestive part of the tsetse fly gut.Figure 7**ELISA based quantitation of the amount of Nb_An46 present in haemolymph, abdomen, thorax and midgut of streptozotocin-treated flies injected with 1×10** ^**7**^ **CFU of** ***Sod*** **\_pFliCpelBNb46** ***fliC*** **at selected time points (○:7 dpi, ▽: 14 dpi, □: 7 dpi) using a 6 × His tag specific detection antibody.** Values are presented as ng nanobody per whole tissue extract or 1 μl haemolymph. Discussion {#Sec8} ========== The aim of this study was to develop a functional *Sodalis*-based shuttle system that is able to deliver and express anti-trypanosome nanobodies within the tsetse fly. In this study a recombinant *Sodalis* strain was used expressing a potent trypanolytic nanobody Nb_An46 through a plasmid-based expression system. The functionality of the system was assessed by monitoring if rec*Sodalis* bacteria can (1) establish a stable population in the tsetse fly over time, (2) express significant levels of active anti-trypanosome nanobodies in the tsetse fly and (3) be efficiently transferred to the offspring. Although the growth characteristics of Nb-expressing rec*Sodalis* were shown to be similar with those of the WT *Sodalis* in *in vitro* culture conditions it is plausible that in the tsetse fly *in vivo* environment the endogenous WT bacteria could have a competitive advantage over the introduced recombinant bacteria. Our results clearly suggest that rec*Sodalis* is competitive with WT *Sodalis* in *in vivo* conditions, however the maximal total *Sodalis* density in the inner tsetse fly environment seems to be limited to approximately 5×10^6^ CFU. Indeed, only when the existing WT *Sodalis* population is priorly reduced (\>95%) by a selective antibiotic treatment, the introduced rec*Sodalis* population is able to maintain and proliferate to comparable density levels observed for wildtype *Sodalis* in normal tsetse flies. This rec*Sodalis* population is then able to outgrow the WT population which remains present at low density. In contrast, in non-treated flies rec*Sodalis* is not able to displace the WT *Sodalis* population but remains present at low density, confirming the importance of creating a WT *Sodalis* deprived niche that allows rec*Sodalis* to proliferate inside its host. An important factor when using episomally located plasmids in a paratransgenic system is the persistence of these recombinant strains in the tsetse fly in the absence of antibiotic selection as premature loss of expression due to plasmid instability would not be desirable. Therefore, we assessed the stability of the recombinant strains during *in vitro* culture maintenance and after re-introduction in the tsetse fly. Results from the bacterial plate assay showed that in the absence of selection pressure in the culture medium the FliCpelBNb46*fliC* plasmid proved to be stably maintained with 94% of the *Sodalis* population retaining the plasmid after 27 generations. This strong long-term plasmid retention in rec*Sodalis* was not always observed as for another strain (containing the FliCpelBNb33*fliC* plasmid) a complete plasmid loss in the total *Sodalis* population after 20 generations in culture. We postulate that the strong persistence of the FliCpelBNb46*fliC* plasmid in the rec*Sodalis* culture in the absence of a selection pressure is related to the high plasmid copy number that is observed in this strain and which was 20 fold higher than in the unstable *Sod*\_FliCpelBNb33*fliC* strain. *Sod*\_FliCpelBNb46*fliC* remained present at high densities in the fly throughout the 28 day observation period. Furthermore, rec*Sodalis* was able to disseminate into the digestive tract reaching densities comparable to those in flies harboring WT symbionts. Furthermore, the majority of the total *Sodalis* population remained recombinant for the duration of the experiment indicating that the high plasmid stability that was observed in the *in vitro* culture is also present in the *in vivo* tsetse fly environment. Although transmission to the F~1~ generation was observed, this seemed to be highly inefficient as only a small percentage of the total *Sodalis* population was found to be recombinant. This could be due to plasmid loss by rec*Sodalis* during the 30-day pupal stage or the inability of rec*Sodalis* to efficiently colonize the milk glands upon injection in the adult female fly which is a prerequisite for rec*Sodalis* transmission to the intra-uterine larvae through the milk secretion. Indeed analysis of transcripts encoding *Sodalis* motility genes, *fliC* and *motA*, and cell invasion genes, *invA1* and *invA2*, are up regulated in the larval and early pupal stages, and not in adult tsetse flies \[[@CR15]\]. These results indicate that the Type-III secretion system and flagellum may be important for the transmission and establishment of symbiont infections in the intra-uterine progeny. Our results clearly indicate that for a successful use of *Sodalis* as paratransgenic vehicle in the tsetse fly several methodologies should still be improved allowing i) the generation of more stable rec*Sodalis* i.e. through an efficient methodology to insert exogenous DNA directly into the bacterial genome and ii) a more efficient transfer of the rec*Sodalis* to the next generations. Rec*Sodalis* was found to continuously express functional nanobody in the tsetse fly as indicated by the Nb_An46 accumulation in the haemolymph over time. Abdomen and midgut extracts from the majority of insects that carried the recombinant nanobody-producing bacteria were positive in the ELISA, indicating the presence of functional Nb in these tissues. However the abundant presence of proteolytic enzymes in the tsetse fly midgut probably interfered with detection, prohibiting accurate quantification of the Nb expression in this tissue. These results suggest the benefit of lowering the susceptibility of potential effector proteins to proteolytic degradation especially when they have to be functional in a digestive tissue such as the tsetse fly midgut. Nbs have the advantage that they can be mutagenized and selected for increased proteolytic stability \[[@CR16]\]. From our experimental data, the expected levels of *in vivo* Nb expression are in the lower ng range. However, these levels of Nb expression should be sufficient to interfere with trypanosome development given that an infective blood meal of a tsetse fly in nature is estimated to contain around 10^3^ parasites as the average parasitaemia in *T. brucei* infected cattle fluctuates from 1.5 × 10^5^ parasites per ml during the acute phase of infection to 5 × 10^4^ parasites per ml during the chronic phase \[[@CR17]\] and *in vitro* studies have shown that nanomolar concentrations of trypanolytic Nbs are sufficient to saturate \>95% of the surface VSG molecules and to provoke efficient trypanosome lysis \[[@CR13],[@CR18]\]. Indeed, since a VSG:Nb molar ratio of 1:20 is sufficient to cause efficient lysis, 5 ng of Nb_An46 should be adequate to efficiently lyse 10^3^ parasites. Conclusion {#Sec9} ========== These data are the first to show the potential of *Sodalis* as a delivery system for anti-trypanosome effector molecules in tsetse fly tissues relevant for trypanosome development. Given the ability of recombinant *S. glossinidius* to efficiently establish in different tsetse fly tissues at high densities and their capacity to release significant levels of functional anti-trypanosome Nbs in tissues were trypanosomes reside, the foundation has been laid for further exploration of the inhibitory effect on trypanosome development in the tsetse fly. Moreover, a paratransgenic approach using *Sodalis* to deliver Nbs that target the trypanosome-tsetse fly crosstalk could open a new avenue to unravel the molecular determinants of this specific parasite-vector association. Material and methods {#Sec10} ==================== Insects, bacterial strains and culture conditions {#Sec11} ------------------------------------------------- *G. morsitans morsitans* (Westwood) from the colony at the Institute of Tropical Medicine Antwerp (ITM), originated from pupae collected in Kariba (Zimbabwe) and Handeni (Tanzania), were used in all experiments. Flies, maintained at 26°C and 65% relative humidity, were fed 3 days per week with defibrinated bovine blood using an artificial membrane system. *Sodalis glossinidius* strains used in this study were isolated from the haemolymph of surface-sterilized tsetse flies from the colony maintained at ITM. Cultures were maintained *in vitro* at 27°C in liquid Mitsuhashi-Maramorosch (MM) insect medium (PromoCell) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS). Where appropriate, selection antibiotics were added to the media at the following concentrations: 100 μg/ml of ampicillin or 50 μg/ml of kanamycin. Flies used in this study were maintained at 26°C and 65% relative humidity, and fed 3 days per week with defibrinated bovine blood using an artificial membrane system. Plasmid constructs {#Sec12} ------------------ In this study the pFliCpelBNb46*fliC* plasmid was used in which a 6 × His tagged *Nb*\_*An46* gene was fused to two secretion signals (FliC and pelB) and cloned into the multiple cloning site of the pCM66 expression vector. The pFliCpelBNb46*fliC* plasmid was derived from pFliCpelBNb33*fliC* \[[@CR12]\] by replacement of *pelBNb*\_*An33* between the *Xba*I and *Eco*RI sites by *pelBNb*\_*An46* amplified as a *Xba*I-*EcoR*I fragment (465 bp) by PCR from the pHen6C plasmid containing the *pelBNb*\_*An46* gene using the following primer set: Nb46_FW, 5'-*TCTAGA*ATGAAATACCTATTGCCTACGG-3' and Nb46_Rev, 5'-*GAATTC*TTAGTGATGGTGATGGTGGTGGCGGCCGCGTGAGGAGAC-3' (*Xba*I-*Eco*RI restriction sites are underlined). Transformation of *Sodalis glossinidius* {#Sec13} ---------------------------------------- *Sodalis* transformation with the pFliCpelBNb46*fliC* plasmid was conducted using a heat-shock based method as described in \[[@CR14]\]. Following transformation, the cells were allowed to recover overnight at 27°C in liquid MM medium prior to plating. Growth curve measurements {#Sec14} ------------------------- Logarithmically growing cultures were used to inoculate 25 ml of MM-medium to an optical density at 600 nm (OD~600~) of 0.05. *Sodalis* cultures harboring the FliCpelBNb46*fliC* plasmid were allowed to grow without shaking for the first 24 h, after which they were transferred to a shaking incubator. Samples were taken every 24 h for optical density measurements and Nb protein quantification in culture supernatants and whole cell extracts. The cell population doubling time was calculated from the growth rate during exponential growing phase using the following equation: doubling time (in hours) = h\*ln(2)/ln(c2/c1) where c1 is the initial concentration and c2 is the concentration when cultures reached maximum densities. Plasmid copy number assay {#Sec15} ------------------------- The plasmid copy number per cell was calculated by dividing the absolute copy number by the number of *Sodalis* CFU present in the culture. The absolute number of plasmid and *Sodalis* CFU present in the *Sod*\_FlicpelBNb46*fliC* culture grown in the presence of antibiotic selection (kanamycin 50 μg/ml) was estimated by quantitative real-time PCR (qPCR) on DNA extracted from culture samples taken during the lag and exponential growth phases using the QIAGEN DNeasy extraction kit (QIAGEN). PCR reactions were carried out on a LightCycler™ (Roche Diagnostics, Mannheim, Germany). To estimate the number of *Sodalis* CFU present in the culture, a standard curve was generated using DNA extracts from a serial dilution series (1:10) ranging from 10^7^*Sod*\_FlicpelBNb46*fliC* CFU/ml to 10^2^*Sod*\_FlicpelBNb46*fliC* CFU/ml. For pFliCpelbNb46*fliC* plasmid number determination a dilution series (1:10) ranging from 10-10^10^ plasmid copies μl^−1^ was prepared to establish a linear standard curve for real-time PCR assays. For *Sodalis*, primers that target a 120-bp region of the single-copy *S. glossinidius exochitinase* gene were used: Qchi_Fw, 5'- TGGGGACAGTACGATGGCAGAGC −3 ; Qchi_Rev, 5'- TCATAGGCGGTCGGGGATAATTGCG -3'. For the plasmid number determination, a 433-bp common region present on both pFliCpelBNb33*fliC* and pFliCpelBNb46*fliC* plasmids was amplified using the following primer set: pCM66_Fw, 5'- CTTGGCCCTCACTGACAG-3'; pCM66_rev, 5'- GCAGCCCTGGTTAAAAAC-3'. qPCR was performed in a 20-μl reaction mixture volume containing 10 μl of 2 × iQ™ SYBR green supermix, 0.3 μM of each primer, template (DNA ) and RNase-free sterile water to a final volume of 20 μl. Measuring *in vitro* plasmid stability {#Sec16} -------------------------------------- The *in vitro* stability of the pFliCpelBNb46*fliC* plasmid in *Sodalis* was measured by maintaining recombinant bacteria in log phase growth for 27 generations in MM without kanamycin selection. Samples were taken every two to four generations and plated on MM blood agar with and without kanamycin. The ratio of the number of colony forming units (CFU) grown on the selective blood agar plate to those grown on the non-selective blood agar was used to determine the percentage of plasmid-carrying cells. This ratio allows to monitor the plasmid stability during subsequent generations in the cultured rec*Sodalis*. Western blot analysis {#Sec17} --------------------- Cells were pelleted from bacterial cultures by centrifugation (15 min, 10000 × g) and the supernatant was clarified from residual bacterial cells by a second centrifugation step. Proteins in the growth medium were precipitated with 10% trichloroacetic acid (TCA) for 1 h on ice. For SDS-PAGE, samples were heat denatured at 95°C in the presence of SDS-PAGE loading buffer containing β-mercaptoethanol and analyzed on a 12% (w/v) polyacrylamide gel (Biorad). Proteins were transferred onto a Hybond C nitrocellulose membrane (Whattman). After overnight blocking with 1% (w/v) bovine serum albumin, the membrane was incubated sequentially with a mouse anti-6 × His-tag IgG1 antibody (1:1000) (Serotec) and a rabbit anti-mouse-IgG antibody (1:1000) (Serotec) conjugated to horseradish peroxidase. In between these successive 2 h incubations, the membrane was washed with PBS-0.1% Tween 20. Thirty minutes after adding the substrate (TMB 1-Component Membrane Peroxidase Substrate, KPL) the reaction was stopped by washing the membrane with water. *RecSodalis* introduction into tsetse flies {#Sec18} ------------------------------------------- To evaluate the effect of reducing the WT *Sodalis* population in tsetse on rec*Sodalis* growth inside its host, newly emerged male *G. morsitans morsitans* flies were divided into two experimental groups. The treated group was given three blood meals supplemented with 20 μg/ml streptozotocin while the non-treated group received normal blood meals. Next, treated and non-treated flies were briefly anaesthetized by cold shock and microinjected intrathoracically with a suspension of 5 × 10^4^*Sod*\_pFliCpelBNb46*fliC* CFU, using a 5 μl Hamilton 75RN microsyringe with gauge 33 removable electrotapered needles. After injection, males were kept in separate cages and received an antibiotics-free blood meal every 48 h throughout the course of the experiment. The optimal rec*Sodalis* injection dose was evaluated by injecting streptozotocin treated adult male flies with respectively 5 × 10^4^, 5 × 10^5^, 10^7^ and 5 × 10^7^ CFU *Sod*\_pFliCpelBNb46fliC followed by determination of the number of rec*Sodalis* CFU in abdomen and thorax 7 and 14 days post-injection. To evaluate the *in vivo* persistence of *Sod*\_pFliCpelBNb46*fliC*, streptozotocin treated adult male flies were injected intrathoracically with 10^7^*Sod*\_pFliCpelBNb46*fliC* CFU. At different time points post-injection i.e. day 1 (teneral), 7, 14, 21 and 28, the number of WT and rec*Sodalis* CFU present in abdomen, thorax, haemolymph and midgut was measured using qPCR. To evaluate the transmission of rec*Sodalis* to the F~1~ progeny, streptozotocin-treated female flies were injected intrathoracically with 5 × 10^4^*Sod*:pFliCpelBNb46*fliC* CFU. Females were allowed to mate with WT males and their offspring (teneral stage) was evaluated for the presence of rec*Sodalis*. Measuring *in vivo Sodalis* and *Wigglesworthia* density {#Sec19} -------------------------------------------------------- At various time points post-injection flies were sacrificed for genomic DNA extraction using the QIAGEN DNeasy extraction kit (QIAGEN). We used a quantitative PCR (qPCR) method for the estimation of the number of WT *Sodalis* and rec*Sodalis* cells in tsetse fly tissues (abdomen, thorax, midgut and haemolymph). For this, triplicate cultures of WT and rec*Sodalis* were serially diluted (10-fold) in PBS to yield a *Sodalis* density ranging from 10^7^ CFU/ml to 10^2^ CFU/ml. DNA was extracted from each *Sodalis* dilution followed by qPCR using primers that amplify a region of the pCM66 plasmid backbone present in rec*Sodalis*: (pCM66_Fw, 5'-CTTGGCCCTCACTGACAG-3' and pCM66_Rev, 5'- GCAGCCCTGGTTAAAAAC-3') and primers that target a 120-bp region of the single-copy *Sodalis glossinidius exochitinase* (Qchi) gene to determine the corresponding Ct values. Standard curves were generated by plotting these C~t~ values against the corresponding log of *Sodalis* CFU/ml. This qPCR approach in combination with the *Sodalis* CFU standard curves allowed us to estimate the CFU (DNA equivalent) values present in the tsetse fly tissues of the different experimental fly series. An internal control was included to evaluate the DNA extraction efficiency in all the tissue samples. For this, samples were spiked with 0.4 ng of plasmid DNA (pBAD24_GFP) prior to extraction. qPCR with *gfp*-specific primers (pBAD24_GFP: GFP_Fw, 5'-TGGCCAACACTTGTCACTAC-3' and GFP_Rev, 5'-AGAAGGACCATGTGGTC-3') revealed a C~t~ value of approximately 24.5 in all the DNA extracts corresponding to the C~t~ value of the plasmid dilution used to spike to internal control alone demonstrating a uniform highly efficient extraction of the DNA in all the samples. *Wigglesworthia* quantification was done by amplifying the *thiamine* locus with the following primer set: QthiC_F, 5'-AAGTTATGATAGAAGGACCAGGAC-3' and QthiC_R, 5'-CCCGGAGCAATATCAGTAGTTAG-3'. All Ct values were normalized to the *G. morsitans morsitans* reference gene β-actin for each sample using the following primer set: β-actin_F, 5'-GGCTTCTGGTCGTACTACT-3' and β-actin_R, 5'-CCGGACATCACAATGTTGG-3'. For each sample the obtained Ct value of the thiamine gene was normalized to the Ct value of the reference gene (β-actin) by subtracting the β-actin Ct value from the thiamine Ct value to yield a ΔCt value for each sample. ΔCt values were log-transformed and compared using the Mann--Whitney U test to establish differences among treated and control groups. A value of p \<0.05 was considered significant in all analyses. qPCR was performed in a 20-μl reaction mixture volume containing 10 μl of 2 × iQ™ SYBR green supermix, 0.3 μM of each primer, template (genomic DNA, standard) and RNase-free sterile water to a final volume of 20 μl. The amount of fluorescence generated was measured during each amplification cycle using the following program: (i) initial denaturing at 95°C for 3 min; (ii) 40 cycles, with 1 cycle consisting of denaturation at 95°C for 10 s, annealing at 60°C for 10 s, and extension at 72°C for 30 s. All assays were carried out on a LightCycler™ (Roche Diagnostics, Mannheim, Germany) in 96-well format plates in duplicate and were averaged for each sample. For each PCR run, a negative (no-template) control was used to test for false-positive results or contamination. Analysis of *in vitro* and *in vivo* Nanobody concentrations using ELISA {#Sec20} ------------------------------------------------------------------------ i.***In vitro***. The amount of active Nb_An46 present in cytoplasmic extracts and growth medium was quantified using an optimized nanobody-detection ELISA \[[@CR19]\]. For this purpose, Maxisorb 96-well plates (Nunc) were coated overnight (4°C) with 200 ng purified soluble AnTat 1.1 VSG per well in 0.1 M NaHCO~3~, pH 8.2. Residual protein binding sites were blocked for two hours at room temperature with 0.5% bovine serum albumin (BSA) in PBS. Standards and samples were added for 1 h at room temperature. Detection of antigen-bound nanobodies was performed with a mouse anti-6 × His IgG antibody (Serotec) directly conjugated to horseradish peroxidase. Thirty minutes after adding peroxidase substrate, the reaction was stopped with 0.33 M H~2~SO~4~ and the optical density was measured at 450 nm (690 nm was used as reference filter). Protein concentrations were calculated from a standard curve fitted to a four parameter logistic equation using the Ascent software (Labsystems). Samples from the *in vitro Sodalis* culture were taken at the same time points indicated for the growth curve measurements. For each sample, 1 ml of culture media was centrifuged two times (8000 × g) to obtain the extracellular and whole cell fractions. The whole cell extracts were prepared by resuspending the cell pellets in 0.2 ml PBS supplemented with complete protease inhibitor (Roche) followed by sonication at an amplitude of 10 microns for 5 seconds (3 cycles on ice). For quantification of Nb_An46 present in cytoplasmic extracts and growth medium, a standard serial dilution series (1:2) starting from 2500 to 5 ng/ml of purified Nb_An46 was prepared in PBS and MM-medium respectively. MM medium and PBS alone were included as blanks.ii.***In vivo***. Quantification of active Nb_An46 present in different tsetse fly tissues samples (abdomen, thorax, midgut and haemolymph) was performed at different time points post-injection. The preparation of the whole tissue extracts by sonication and the Nb-quantification by ELISA were performed as described above. To mimic the sample complexity of the abdomen, thorax, midgut and haemolymph extracts, nanobody standards were prepared in the corresponding tissue extracts from non-injected wildtype flies. The respective tissues from flies harboring WT *Sodalis* were included as blanks. Additional file {#Sec21} =============== Additional file 1: Figure S1.The presence of *Wigglesworthia* in abdomen tissues from streptozotocin treated and non-treated male flies. **Figure S2.** The presence of *Wigglesworthi*a in abdomen of male flies injected with 107 CFU of *Sod\_*pFliCpelBNb46*fliC* at day 7, 14 and 21 post injection and control flies. **Competing interests** The authors declare that they have no competing interests. **Authors' contributions** Conceived and designed the experiments: LDV, GC, JVDA. Performed the experiments: LDV, GC, KDR. Wrote the paper: LDV, GC, JVDA. All authors read and approved the final manuscript. This research was supported by an ITM SOFI grant, the InterUniversity Attraction Pole programme (IAP), FWO and the ERC-Starting Grant 'NANOSYM'. This work is also performed in the frame of a FAO/IAEA Coordinated Research Project on "Improving SIT for tsetse flies through research on their symbionts and pathogens". We wish to acknowledge the technical assistance of Jos Van Hees.

Introduction {#Sec1} ============ Anemia is common in patients with chronic kidney disease (CKD) and prevalence increases with CKD severity \[[@CR1]\]. Renal erythropoietin-producing cells sense low tissue oxygen tension and respond by the production of erythropoietin, a hormone that stimulates red blood cell production \[[@CR2]\]. CKD leads to a disruption of this process, erythropoietin deficiency and subsequent anemia, characterized by lower than normal number of circulating red blood cells or decreased levels of hemoglobin (Hgb) \[[@CR3]\]. Other possible causes of anemia include blood loss, iron deficiency, inflammation and accumulation of uremic toxins \[[@CR4]\]. Prevalent anemia in CKD has been associated with cognitive impairment, sleep disturbances, CKD progression, cardiovascular disease and higher mortality in mostly older studies and in selected populations \[[@CR5]--[@CR13]\], whereas contemporary unselected population-based data on clinical outcomes associated with anemia are scarce. Treatment options for anemia include iron (oral and intravenous), erythropoietin stimulating agents (ESAs) and red blood cell transfusion to restore hemoglobin levels. Concerns have been raised regarding the cardiovascular safety of treating anemia to higher Hgb levels, in particular when using ESAs to target Hgb levels \> 12 g/dl \[[@CR14]--[@CR16]\]. This has resulted in a change of anemia management practices since 2011, with generally less intensive therapy and lower Hgb treatment targets \[[@CR17], [@CR18]\]. Following this change, high-quality longitudinal real-world data on the current impact of anemia on clinical outcomes are scarce. Furthermore, the exact risk of cardiovascular and other adverse outcomes in patients following progression to more severe anemia remains unknown. The overall aim of our study was to identify major clinical consequences of anemia in dialysis dependent (DD) and non-dialysis dependent (NDD) patients with severe CKD. Specifically, we examined the association between anemia and time to first dialysis (in NDD patients) and major cardiovascular events (MACE), acute hospitalizations and all-cause death (in NDD and DD patients). Methods {#Sec2} ======= We linked Danish population-based healthcare and administrative databases including all laboratory plasma-creatinine tests from primary and hospital care from The Laboratory Information Systems Database (LABKA) \[[@CR19]\], all hospital contacts and diagnoses from The Danish National Patient Registry (DNPR), and all drug prescriptions from The Aarhus University Prescription Database \[[@CR20], [@CR21]\] for the entire population in Northern Denmark from 2000 through 2016 where residence, migration and vital status was obtained from The Civil Registration System (CRS). The cumulative source population comprised of \~ 2.2 million persons from which we included a study population with prevalent severe CKD at study start on Jan 1, 2009 (identified from 2000 to 2008), or with incident severe CKD between 2009 and 2016. Study population {#Sec3} ---------------- Patients with severe CKD were defined as individuals with two plasma-creatinine tests at least 3 months (90 days) apart showing an estimated glomerular filtration rate (eGFR) \< 30 mL/min/1.73 m^2^ in the period 2000--2016. eGFR was computed by the simplified Modification of Diet in Renal Disease (MDRD) equation based on creatinine measurements, taking sex and age and into account \[[@CR22]\]. During 2009--2016, we assigned CKD patients (who had either prevalent severe CKD on Jan 1, 2009 or incident severe CKD between 2009 and 2016) to different anemia grade cohorts. We excluded patients with any of the following at any time prior to the index date: any cancer (except non-melanoma skin cancer), hereditary hematologic disease, chronic inflammatory disease, gastrointestinal bleeding, or organ transplants. Anemia {#Sec4} ------ Anemia grades were classified as no anemia (Hgb level of ≥ 12/≥ 13 g/dl in women/men); grade 1 (Hgb level of 10 to \< 12/\< 13 g/dl in women/men); grade 2 (Hgb level of 8 to \< 10 g/dl) and grade 3+ (Hgb level of \< 8 g/dl) \[[@CR23]\]. For prevalent severe CKD patients, anemia grade was determined based on the lowest Hgb recording during a 1 year lookback period from Jan 1st 2009. If Hgb was not measured in this period, the follow up started following the first recorded Hgb measurement after Jan 1st 2009. For incident severe CKD patients, follow up began on the date of the second measurement showing an eGFR \< 30 mL/min/1.73 m^2^ after Jan 1st 2009 if Hgb measurements were recorded within 1 year before that date. For these patients, anemia grade was determined based on the lowest Hgb recording during that year. If no prior Hgb measurements were available, follow up started following the first available Hgb measurement after the patient's second eGFR measurement below 30 mL/min/1.73 m^2^. A schematic presentation of inclusion of prevalent severe CKD patients is shown in Fig. [1](#Fig1){ref-type="fig"}a and similarly for incident severe CKD patients in Fig. [1](#Fig1){ref-type="fig"}b. Patients could be included in more than one anemia grade cohort if they "moved up" in anemia grade; i.e., one person was allowed to contribute risk time in consecutively more severe anemia grade cohorts. Since our aim was to predict the subsequent outcome risk for a given CKD patient from the date of first reaching a specific anemia grade, patients were not censored from the cohort at time following inclusion into a more severe anemia grade cohort and were not re-entered into a lower anemia grade cohort once they were included in a more severe grade cohort irrespective of the results of subsequent Hgb measurements.Fig. 1Overview of the inclusion based on eGFR and anemia grade of prevalent (**a**) and incident (**b**) severe CKD patients for follow up showing examples of how included patients may move to a more severe anemia grade cohort Outcomes {#Sec5} -------- Study outcomes were: (1) incident dialysis (among NDD patients only) defined as a first ever record of dialysis (acute or chronic) during follow-up; (2) all-cause acute hospitalization, defined as an acute inpatient record in DNPR during follow-up; (3) all-cause death, defined as death before the end of follow-up; (4) MACE (composite of tenth revision of the International Classification of Diseases and Related Health Problems (ICD-10) codes for myocardial infarction \[I21--I23\], unstable angina pectoris \[I200\], stroke \[I61, I63 and I64\], or heart failure \[I50\]); and (5) fatal and non-fatal cardiovascular events (CVE) separately (composite and individual). Patients who were already hospitalized at the index date were not considered at risk of an acute hospitalization and thus excluded from this analyses. Data analysis {#Sec6} ------------- For the incident severe CKD patients, we generated cumulative incidence function curves for incident dialysis among NDD patients and for CVE and all-cause death stratified by dialysis status at time of entry in each anemia grade cohort. For both prevalent and incident severe CKD patients, we computed incidence rates per 100 person years and estimated adjusted hazard ratios (HRs) using Cox regression. To account for the dependency between observations, we used the robust sandwich estimator to compute 95% CIs \[[@CR24]\]. HRs were simultaneously adjusted for the following covariates assessed at the start of follow-up in each anemia grade cohort: age, gender, marital status, severe CKD duration (time since 2nd eGFR \< 30 mL/min/1.73 m^2^), last measured eGFR level, Charlson Comorbidity Index \[[@CR25]\] (based on complete hospital contact history, excluding CVE and renal disease categories), alcohol dependency related disorders, previous history of CVE and number of acute hospitalizations 1 year before anemia. Since the proportional hazard assumption was not fulfilled for incident dialysis, angina and acute hospitalization in the NDD population and for all outcomes in the DD population, supplementary analyses were performed dividing the analyses into 0--2 years and 2--8 years of follow-up. Since the average age of the included study population was relatively high we performed a stratified analyses by ages \< 65 and 65 + years, and as diabetes is associated with increased baseline risk of the included outcome events we also performed stratified analyses by diabetes status. Results {#Sec7} ======= In total, we included data from 16,972 unique patients with severe CKD, who contributed to one or more of the anemia grade cohorts over time, resulting in a total of 28,510 patient profiles across all anemia cohorts. These included 24,916 (87%) NDD and 3594 (13%) DD patients, with different anemia grades. Characteristics are shown for all patients in Table [1](#Tab1){ref-type="table"}, for NDD patient profiles in Supplementary Table 1, and for DD patient profiles in Supplementary Table 2.Table 1Characteristics of the 28,510 severe CKD patient profiles included in the study by first observation of anemia grade 0, 1, 2 or 3+ following severe CKD^a^No anemia = grade 0 (Hgb ≥ 13/≥ 12 g/dL)^b^Anemia grade 1 (Hgb 10 to \< 13/\< 12 g/dL)^b^Anemia grade 2 (Hgb 8 to \< 10 g/dL)Anemia grade 3+ (Hgb \< 8 g/dL)TotalOverall, N410510,0339632474028,510Hgb g/dl, median (IQR)13.5 (12.9--14.5)11.8 (11.3--12.1)9.7 (9.3--10.0)7.6 (7.3--7.9)10.0 (9.3--11.9)Age, median (IQR)76 (13.8)77 (13.7)77 (13.8)74 (14.5)76 (13.9)Male sex, N (%)1485 (36.2)4700 (46.8)4380 (45.5)2251 (47.5)12,816 (45.0)Marrital status Divorced, N (%)431 (10.5)1033 (10.3)1038 (10.8)601 (12.7)3103 (10.9) Married, N (%)1566 (38.1)3954 (39.4)3610 (37.5)1833 (38.7)10,963 (38.5) Unmarried, N (%)406 (9.9)1088 (10.8)1118 (11.6)676 (14.3)3288 (11.5) Widowed, N (%)1702 (41.5)3958 (39.4)3866 (40.1)1630 (34.4)11,156 (39.1)Severe CKD duration (months), median (IQR)0 (0--26)0 (0--29)9 (0--42)15 (0--50)4 (0--37)Dialysis dependent, N (%)239 (5.8)725 (7.2)1326 (13.8)1304 (27.5)3594 (12.6)Latest eGFR ml/min/1.73 m^2^ level before cohort entry 60 + , N (%)154 (3.8)222 (2.2)253 (2.6)167 (3.5)796 (2.8) 45 ≤ 60, N (%)213 (5.2)433 (4.3)534 (5.5)256 (5.4)1436 (5.0) 30 ≤ 45, N (%)670 (16.3)1656 (16.5)1673 (17.4)754 (15.9)4753 (16.7) 15 ≤ 30, N (%)2941 (71.6)6831 (68.1)5209 (54.1)2228 (47.0)17,209 (60.4)  \< 15, N (%)127 (3.1)891 (8.9)1963 (20.4)1335 (28.2)4316 (15.1)eGFR, Median (IQR)28.2 (25.1--30.1)27.1 (22.3--29.8)25.3 (17.0--30.3)23.0 (13.6--30.0)26.5 (19.8--29.9)Charlson comorbidity index^c^ 0, N (%)1935 (47.1)4023 (40.1)3325 (34.5)1380 (29.1)10,663 (37.4) 1--2, N (%)1794 (43.7)4674 (46.6)4465 (46.4)2250 (47.5)13,183 (46.2) 3+ , N (%)376 (9.2)1336 (13.3)1842 (19.1)1110 (23.4)4664 (16.4)Diabetes, N (%)1163 (28.3)3349 (33.4)3451 (35.8)1843 (38.9)9806 (34.4)History of heart failure, N (%)826 (20.1)2310 (23.0)2378 (24.7)1138 (24.0)6652 (23.3)History of CVD, N (%)1816 (44.2)4905 (48.9)4985 (51.8)2419 (51.0)14,125 (49.5)Number of acute hospitalizations one year back, median (IQR)0 (0--1)0 (0--1)1 (1, 2)2 (1--3)1 (0--2)Alcoholism related disorders, N (%)290 (7.1)787 (7.8)924 (9.6)612 (12.9)2613 (9.2)Antihypertensive/antilipid-/antiplatelet therapy, N (%)3868 (94.2)9560 (95.3)9197 (95.5)4525 (95.5)27,150 (95.2)^a^Severe CKD was defined based on the 2nd of two eGFR calculations at least 3 months apart showing a eGFR \< 30. At the date of first observation in each anemia grade, eGFR may be higher than 30 as shown in the table^b^Hgb ≥ 13 g/dL for men and ≥ 12 g/dL for women^c^Charlson comorbidity score was calculated based on complete hospital contact history, excluding cardiovascular disease and renal disease categories Overall we identified 4105 (14%) patient profiles with no anemia, 10,033 (35%) with anemia grade 1, 9631 (34%) with anemia grade 2 and 4740 (17%) with anemia grade 3+. The duration of severe CKD, the number of acute hospitalizations (within the last year), the proportion of patients on dialysis, with low eGFR (\< 15 ml/min/1.73 m^2^) and with a high comorbidity score all increased with increasing anemia grade (Table [1](#Tab1){ref-type="table"}). The incidence rates of incident dialysis among NDD patients and of acute hospitalization, all-cause deaths and MACE among all patients (Table [2](#Tab2){ref-type="table"}) increased markedly with increasing anemia grades both when analyzing crude incidence rates per 100 person years (IR) and hazard ratios for clinical outcomes by anemia grade, and when these were adjusted for baseline differences in potential confounders (Table [2](#Tab2){ref-type="table"}). The adjusted estimates were attenuated compared to the crude estimates. Among the NDD patients, the IR for newly initiated dialysis at anemia grade 3+ was 8.6 per 100, 95% CI 7.8--9.5 and the adjusted HR was markedly elevated (HR 1.91, 95% CI 1.61--2.26) at anemia grade 3+ when compared to no anemia. Anemia was strongly associated with acute hospitalization for any cause (IR at anemia grade 3 + 103.9, 95% CI 96.4--111.8 and HR 1.74, 95% CI 1.57--1.93) and with all-cause death (IR at anemia grade 3+ 39.4, 95% CI 37.8--41.1 and HR 1.82, 95% CI 1.70--1.94). The HR for MACE at anemia grade 3+ was only modestly elevated, although statistically significant (IR at anemia grade 3+ 15.4, 95% CI 14.3--16.6 and HR 1.14, 95% CI 1.02--1.26). Among the individual MACE endpoints, heart failure revealed the strongest association with anemia (IR at anemia grade 3+ 10.7, 95% CI 9.8--11.6 and HR 1.24, 95% CI 1.09--1.41).Table 2Crude Incidence rate as well as crude and adjusted^a^ hazard ratios for incident dialysis, any and specific cardiovascular events, acute hospitalization and all-cause death associated with different anemia grades among non-dialysis dependent patients with severe CKDNNo. of eventsFollow-up time, yearsCrude incidence rate per 100 person-years (95% CI)Crude hazard ratio (95% CI)Adjusted hazard ratio (95% CI)^a^Incident dialysis No anemia386628312,3802.3 (2.0--2.6)(ref)(ref) Anemia grade 1930897724,0664.1 (3.8--4.3)1.71 (1.55--1.90)1.27 (1.14--1.42) Anemia grade 28306106015,6186.8 (6.4--7.2)2.70 (2.41--3.03)1.69 (1.49--1.93) Anemia grade 3+343643550438.6 (7.8--9.5)3.26 (2.83--3.76)1.91 (1.61--2.26)Acute hospitalization No anemia31112187620335.3 (33.8--36.8)(ref)(ref) Anemia grade 163704609993746.4 (45.1--47.7)1.24 (1.19--1.30)1.12 (1.07--1.17) Anemia grade 237092795381673.2 (70.5--76.0)1.79 (1.69--1.89)1.41 (1.33--1.50) Anemia grade 3+943713686103.9 (96.4--111.8)2.41 (2.19--2.65)1.74 (1.57--1.93)All-cause death No anemia3866192112,93914.8 (14.2--15.5)(ref)(ref) Anemia grade 19308521026,32619.8 (19.3--20.3)1.29 (1.24--1.35)1.15 (1.10--1.19) Anemia grade 28306533117,76030.0 (29.2--30.8)1.86 (1.78--1.95)1.47 (1.40--1.55) Anemia grade 3+34362275577039.4 (37.8--41.1)2.32 (2.19--2.47)1.82 (1.70--1.94)Cardiovascular events No anemia386689811,5417.8 (7.3--8.3)(ref)(ref) Anemia grade 19308230422,86710.1 (9.7--10.5)1.23 (1.15--1.31)1.07 (1.00--1.14) Anemia grade 28306194315,19912.8 (12.2--13.4)1.44 (1.34--1.55)1.10 (1.02--1.19) Anemia grade 3+3436749485515.4 (14.3--16.6)1.62 (1.47--1.78)1.14 (1.02--1.26)Myocardial infarction No anemia386620412,6201.6 (1.4--1.9)(ref)(ref) Anemia grade 1930853425,5232.1 (1.9--2.3)1.25 (1.09--1.43)1.10 (0.96--1.26) Anemia grade 2830645117,1462.6 (2.4--2.9)1.48 (1.27--1.73)1.20 (1.01--1.42) Anemia grade 3+343614855932.6 (2.2--3.1)1.41 (1.15--1.75)1.11 (0.88--1.39)Stroke No anemia386625512,6022.0 (1.8--2.3)(ref)(ref) Anemia grade 1930855025,5812.2 (2.0--2.3)1.03 (0.92--1.16)0.95 (0.84--1.07) Anemia grade 2830644017,2142.6 (2.3--2.8)1.17 (1.01--1.35)0.98 (0.84--1.15) Anemia grade 3+343615755742.8 (2.4--3.3)1.23 (1.01--1.49)1.01 (0.81--1.25)Heart failure No anemia386656112,1094.6 (4.3--5.0)(ref)(ref) Anemia grade 19308154324,0946.4 (6.1--6.7)1.31 (1.21--1.41)1.11 (1.03--1.20) Anemia grade 28306133716,0678.3 (7.9--8.8)1.55 (1.42--1.70)1.15 (1.04--1.27) Anemia grade 3+3436547512510.7 (9.8--11.6)1.84 (1.64--2.06)1.24 (1.09--1.41)Unstable angina pectoris No anemia38664012,8260.3 (0.2--0.4)(ref)(ref) Anemia grade 193088426,1360.3 (0.3--0.4)1.00 (0.75--1.35)0.88 (0.65--1.20) Anemia grade 283065517,6570.3 (0.2--0.4)0.93 (0.63--1.37)0.74 (0.48--1.15) Anemia grade 3+34362057310.3 (0.2--0.5)1.00 (0.58--1.70)0.71 (0.38--1.32)^a^Adjusted for age, gender, marital status, history of CVD, alcohol dependency, other comorbidities, recent acute hospitalizations, eGFR level and CKD duration For the DD population (Table [3](#Tab3){ref-type="table"}), similar risk estimates as in the NDD population were observed for acute hospitalization (IR 121.9, 95% CI 109.8--135.0 and HR 1.51, 95% CI 1.20--1.90) all-cause death (IR 23.4, 95% CI 21.6--25.0 and HR 1.91, 95% CI 1.50--2.43) and MACE (IR 10.4, 95% CI 9.3--11.7 and HR 1.16, 95% CI 0.84--1.61) although the estimates were less precise, probably due to lower number of subjects included in the analyses.Table 3Crude Incidence rate as well as crude and adjusted^a^ and hazard ratios for any and specific cardiovascular events, acute hospitalization and all-cause death associated with different anemia grades among dialysis dependent patientsNNo. of eventsFollow-up time, yearsCrude incidence rate per 100 person-years (95% CI)Crude hazard ratio (95% CI)Adjusted hazard ratio (95% CI)^a^Acute hospitalization No anemia19915555128.1 (23.9--32.9)(ref)(ref) Anemia grade 158451792456.0 (51.3--61.0)1.68 (1.44--1.95)1.09 (0.92--1.29) Anemia grade 272066273590.0 (83.3--97.2)2.37 (2.02--2.78)1.21 (0.99--1.47) Anemia grade 3+434371304121.9 (109.8--135.0)2.99 (2.47--3.61)1.51 (1.20--1.90)All-cause death No anemia2398613006.6 (5.3--8.2)(ref)(ref) Anemia grade 1725390329311.8 (10.7--13.1)1.73 (1.42--2.11)1.29 (1.05--1.58) Anemia grade 21326770447617.2 (16.0--18.5)2.36 (1.92--2.91)1.52 (1.22--1.91) Anemia grade 3+1304712306023.3 (21.6--25.0)2.95 (2.37--3.68)1.91 (1.50--2.43)Cardiovascular events No anemia2394912174.0 (3.0--5.3)(ref)(ref) Anemia grade 172518329286.2 (5.4--7.2)1.43 (1.09--1.88)1.03 (0.78--1.37) Anemia grade 2132636138739.3 (8.4--10.3)1.92 (1.44--2.54)1.16 (0.86--1.57) Anemia grade 3+1304280268210.4 (9.3--11.7)1.92 (1.43--2.59)1.16 (0.84--1.61)Myocardial infarction No anemia2391112830.9 (0.4--1.5)(ref)(ref) Anemia grade 17256431962.0 (1.5--2.6)2.22 (1.24--3.96)1.15 (0.62--2.16) Anemia grade 2132610243292.4 (1.9--2.9)2.40 (1.30--4.41)1.03 (0.52--2.03) Anemia grade 3+13046729702.3 (1.7--2.9)2.06 (1.08--3.95)0.91 (0.44--1.90)Stroke No anemia2391912641.5 (0.9--2.3)(ref)(ref) Anemia grade 17256331532.0 (1.5--2.6)1.26 (0.82--1.95)0.90 (0.59--1.37) Anemia grade 2132612242612.9 (2.4--3.4)1.68 (1.08--2.61)1.06 (0.68--1.65) Anemia grade 3+13048829353.0 (2.4--3.7)1.62 (1.01--2.59)1.04 (0.63--1.71)Heart failure No anemia2392712622.1 (1.4--3.1)(ref)(ref) Anemia grade 17259831183.1 (2.6--3.8)1.37 (0.95--1.97)1.17 (0.80--1.71) Anemia grade 2132620841755.0 (4.3--5.7)1.97 (1.34--2.89)1.45 (0.96--2.19) Anemia grade 3+130416728795.8 (5.0--6.7)2.05 (1.37--3.07)1.53 (0.98--2.40)Unstable angina pectoris No anemia^b^239112990.1 (0.0--0.4)---- Anemia grade 17251632420.5 (0.3--0.8)(ref)(ref) Anemia grade 213262743960.6 (0.4--0.9)1.11 (0.73--1.68)0.87 (0.55--1.38) Anemia grade 3+13041730130.6 (0.3--0.9)0.91 (0.50--1.66)0.69 (0.35--1.36)^a^Adjusted for age, gender, marital status, history of CVD, alcohol dependency, other comorbidities, recent acute hospitalizations, eGFR level and CKD duration^b^Due to low number of events in the no anemia group the Anemia grade 1 group is used as reference Fatal MACE were more strongly associated with anemia (HR 1.42, 95% CI 1.19--1.68) than non-fatal events (Supplementary Tables 3 and 4). In particular, fatal events of stroke, myocardial infarction and unstable angina were associated with anemia, while similar non-fatal events revealed HRs close to one. Both fatal and non-fatal heart failure events were clearly associated with anemia in both the NDD and the DD patients. In supplementary analyses, outcomes were evaluated based on follow up time dividing this into 0--2 years and 2--8 years. Among the NDD patients, the HRs for incident dialysis, acute hospitalization and all-cause death associated with anemia grade 3+ compared to no anemia were markedly higher during the first 2 years of follow up when compared to 2--8 years (Supplementary Table 5). Similarly, in DD patients higher HRs for acute hospitalization and all-cause death associated with anemia were observed during the first 2 years when compared to 2--8 years (Supplementary Table 6). Stratification by age \< 65 years and 65+ years and by presence or absence of diabetes revealed similar results to the comparator, indicating that these were not key effect modifiers (data not shown). NDD patients with a known hospital history of heart failure at baseline had lower HRs than those without previously known heart failure for death (HR 1.55, 95% CI 1.38--1.74 vs HR 1.97, 95% CI 1.82--2.13), incident heart failure hospitalization (HR 1.11, 95% CI 0.93--1.32 vs HR 1.55, 95% CI 1.28--1.88) and MACE (HR 1.04, 95% CI 0.89--1.23 vs HR 1.29, 95% CI 1.12--1.48). For the DD population similar but less precise estimates were obtained (data not shown). Cumulative incidence function curves for study outcomes in NDD and DD patients are shown in Fig. [2](#Fig2){ref-type="fig"}a--g. The risk of dialysis initiation in NDD patients as well as the risks for acute hospitalization and all-cause death among both NDD and DD increases successively with increasing anemia grades. The risk of MACE increased modestly with higher anemia grades among NDD patients, while no clear association was observed among DD patients (Fig. [2](#Fig2){ref-type="fig"}a--e).Fig. 2Cumulative incidence curves for patients with incident severe CKD. **a** incident dialysis for non-dialysis dependent patients; **b** acute hospitalization for non-dialysis dependent patients; **c** acute hospitalization for dialysis dependent patients; **d** all-cause mortality for non-dialysis dependent patients; **e** all-cause mortality for dialysis dependent patients; **f** cardiovascular events for non-dialysis dependent patients; **g** cardiovascular events for dialysis dependent patients Discussion {#Sec8} ========== In the current setting of less frequent treatment of NDD anemia and treatment to lower Hgb targets among patients with severe CKD and anemia, the present study provides contemporary data showing that increasing grades of anemia in severe CKD patients are associated with increased absolute and relative risks of a number of adverse health outcomes, including dialysis initiation, MACE, acute hospitalization and all-cause death. In general, similar results were seen among DD and NDD patients. Compared with previous cross-sectional studies of anemia prevalence in CKD, we observed higher proportions of severe CKD patients experiencing grade 2 or 3 anemia at any given point of time, likely due to the longitudinal nature of our study enabling us to identify incident anemia events. Our results corroborate earlier findings on the association between anemia and clinical outcomes in smaller and selected populations from the United States or Japan \[[@CR5], [@CR11]--[@CR13]\]. The observed and markedly increased HR of incident dialysis, acute hospitalization, all-cause death and MACE during the first 2 years after anemia may indicate that CKD four to five patients surviving the first 2 years after anemia have an only slightly elevated, subsequent risk of adverse events when compared to non-anemic patients. This interpretation, however, is limited by the fact that only survivors were followed in this study. In addition, in this study anemia grade was defined by the lowest Hgb level observed during 1 year before inclusion. This Hgb level may not have been constant during the follow-up period. Since our analyses did not differentiate between patients with sustained and chronic anemia and patients with acute anemia or anemia that was corrected during follow up, our risk estimates may have underestimated the effects of chronic anemia on long term adverse outcomes. Future studies should look into the causes and mechanisms through which increased anemia associates with acute hospitalizations and death, other than CVE. This study was a population-based study using extensive registry health data. This is a major strength of the study since the universal health care system in Denmark implies that all individuals with contact to the hospital system are included in the registers thereby providing an unselected real-world study population. Also, compared to previous studies we were able to include a large study population of 16,930 patients with severe CKD, with ascertainment of progressing anemia over time. We could follow these patients longitudinally for up to 8 years after anemia and adjust our outcome estimates for a large set of relevant confounders. In general, the registration of exposures and outcomes in the included databases is accurate and the validity of registration of major diagnoses including cardiovascular diseases in the Danish National Patient Registry is high \[[@CR26], [@CR27]\]. Several limitations also apply to the study. The inclusion of severe CKD patients relied on routine plasma-creatinine measurements and some misclassification of CKD patients may occur based on missing or infrequent plasma creatinine measurements to determine CKD stage. Although the definition of CKD was based on registered eGFR measurements and the diagnosis was not verified by a nephrologist, we do not expect this to be associated to major misclassification of CKD, since we used the common definition of CKD as specified above. The estimation of GFR was based on the MDRD equation, which was the predominant equation used in clinical practice throughout the period of data collection. Currently, the CKD-EPI equation is more often used. The divergence between the equations occurs mainly at eGFR values \> 30 mL/min/1.73 m^2^ \[[@CR28]\], and thus, this most likely has limited impact on the results of the present study. Sicker patients with more severe CKD may have had a higher likelihood of any anemia being detected due to higher frequency of Hgb testing, leading to potential overestimation of anemia and possibly also its clinical consequences. However, our patients all had severe CKD and are all likely to receive regular blood test monitoring including Hgb in a universally covering healthcare system. We did not have complete treatment data for ESAs, iron, and transfusions recorded in our prescription registries. Therefore, the effects of anemia alone from the potential adverse effects of anemia treatment could not be differentiated in our study. It is likely that patients with severe anemia and late stage CKD did receive ESAs, IV iron or RBC transfusions occasionally, as is suggested by international guidelines \[[@CR29], [@CR30]\]. Overall, our results suggest that higher Hgb levels are associated with better outcomes; however, the potential positive or negative effects of anemia treatment cannot be assessed from this study. Finally, we did not account for differences in blood pressure and proteinuria, and we cannot exclude unmeasured confounding from e.g. smoking, socioeconomic status or lifestyle factors on which did not have reliable data, or from other, unknown confounding factors. Conclusions {#Sec9} =========== The presence and increasing severity of anemia were associated with a substantially increased risk for progression to dialysis in non-dialysis patients with CKD stage 4--5, and with an increased risk for acute hospitalization and all-cause death both in non-dialysis and dialysis patients. The presence and increasing severity of anemia was also associated with a moderately elevated risk of MACE which was more pronounced during the first 2 years after entering a lower anemia grade and with particularly increased risks of heart failure and fatal CVE. In the current setting of generally less intensive anemia therapy, the present large study emphasizes the need for awareness of the potential risk of adverse clinical events in patients with CKD and anemia, and for further exploration of the interaction between anemia and progressive disease in CKD. Electronic supplementary material ================================= {#Sec10} Below is the link to the electronic supplementary material. Supplementary material 1 (DOCX 52 kb) **Publisher\'s Note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. The present study, received funding in part from AstraZeneca. HvH, GJ and KH are employees and stockholders of AstraZeneca. GT, UHJ, HB, RWT and CFC do not report any personal conflicts of interest relevant to this study. The Department of Clinical Epidemiology is, however, involved in studies with funding from various companies as research grants to (and administered by) Aarhus University. This includes the present study, which received funding in part from AstraZeneca. The study was approved by the Danish Data Protection Agency. No approval from an ethics committee or informed consent from patients is required for registry studies in Denmark. All procedures performed were in accordance with the ethical standards of the Danish National Committee on Health Research Ethics and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Children\'s low intake of vegetables and fruits is a nutritional challenge in many European countries ([@CIT0001], [@CIT0002]). Healthy dietary patterns in childhood, specifically diets rich in vegetables and fruits, have been demonstrated to be associated with a lower risk of cardiovascular diseases in adulthood ([@CIT0003]). Kindergartens in Finland are served meals daily according to national dietary recommendations, and a variety of vegetables and fruits are served every day ([@CIT0004]). However, the total intake of vegetables, fruits, and berries is low among Finnish children ([@CIT0005]). Nordic wild berries are part of Finnish food culture, but their bitter, sour, and astringent taste may limit their consumption. Sensory properties, such as taste and flavour, have been shown to be critical factors in the preferences for vegetables and berries among children ([@CIT0006], [@CIT0007]). Food neophobia is also common among preschool-aged children and may be associated with lower consumption of vegetables and fruits ([@CIT0008]). New and innovative approaches exploiting sensory practices have been introduced in kindergarten and school settings to promote healthy eating ([@CIT0009]). Sensory-based food education is a training concept based on sensory perception and experiences and their impact in learning processes related to food. Sensory education offers activities for the learning process via our senses by smelling, touching, hearing, watching, and tasting ([@CIT0010], [@CIT0011]). French 'Glasses du Goût' (Sapere taste education), developed for school-aged children, is a well-known sensory education method ([@CIT0012]). In Finland, project funding has been available for training day care personnel and various sensory-based activities have been implemented in many kindergartens ([@CIT0013]). The practical experiences have been encouraging, but there is a lack of documented scientific evidence on the effectiveness of these activities among kindergarten children. The challenge in assessing food preferences and dietary intake among children is that most methods rely on parental reports that may be prone to parental subjective impressions and misreporting ([@CIT0014], [@CIT0015]). Parents rely on their opinion of food habits at home, and they may also have misunderstandings of how their children behave in meal situations at day care. Thus, objective child-centred methods to measure food acceptance, preferences, and consumption are important to develop and evaluate. Young children\'s cognitive and verbal skills need to be considered when developing new testing protocols ([@CIT0016]). The choice, preparation, and presentation of test samples for children requires further consideration, and the testing location and research personnel may also have an impact on the test situation ([@CIT0017]). In the present study, sensory-based food education activities previously used in Finland were implemented in a new kindergarten with no previous experience with these activities. The main aim of the study was to evaluate the effectiveness of sensory-based food education in a kindergarten setting among children aged 3--6 years. To measure the programme impact, a child-orientated test protocol was developed to evaluate children\'s willingness to eat the test samples of vegetables and berries (primary objective of the present study). Furthermore, parent\'s opinions on their children\'s food preferences were compared to the children\'s willingness to eat the samples in the actual test situation (secondary objective). Methods {#S0002} ======= Study population and protocol {#S0002-S20001} ----------------------------- This study was conducted in Hanko, located in southern Finland, with a total population of 9,100. Two kindergartens were chosen to participate in this study in collaboration with the Director of Early Childhood Education in Hanko. In one kindergarten, sensory-based food education activities were performed with the children (intervention), as described in detail in this article, while the other acted as a control with no activities. The same project researcher was responsible for the food education activities and the measurements with the children. The study was targeted at children aged 3--6 years. In the intervention kindergarten, there were 44 children eligible for the study, and in the control kindergarten, 42 children were eligible. All of the parents of these children were sent a notice explaining the study and consent forms, and all of the parents of the 44 (intervention) and 24 (control) children provided written consent for their children to participate. Children could also refuse to participate or discontinue the activities and/or the test. The ethical committee of the University of Turku approved the study procedures. Questionnaire for the parents {#S0002-S20002} ----------------------------- At baseline, the parents completed a questionnaire that contained background data about the parents (year of birth, education, employment situation, and smoking status). Parents also completed a questionnaire including the child food neophobia scale (the Finnish version focusing on children) ([@CIT0018], [@CIT0019]). Parents were also asked to assess how much their child liked certain vegetables and berries (the same that were tested in practice) on a scale of seven different options (from fully agree to fully disagree) and complemented with the option 'not served at home'. For data processing, responses that took on values 1--3 (1=fully disagreed, 2=rather disagreed, 3=slightly disagreed) were regrouped as disliked, while values 5--7 (5=slightly agreed, 6=rather agreed, 7=fully agreed) were grouped as liked. Value 4 'not known' referred to responses that were not in agreement or disagreement. Measurements with the children {#S0002-S20003} ------------------------------ Measurements with the children were performed at baseline and after the intervention (5 weeks) in both kindergartens. The samples chosen for tasting contained a selection of vegetables (carrot, cabbage, swede, rucola, and romaine lettuce) and Finnish berries (bilberry, lingonberry, and sea buckthorn). The vegetable samples were cut in pieces (total sample weight 10 g in a plastic, transparent cup), and leafy vegetables were cut in strips (4--5 pieces in the cup). Frozen, whole bilberries and lingonberries (10 g sample) were placed in a cup and served frosty. The sea buckthorn juice sample was of 2 ml. The sea buckthorn was sampled as a juice due to its availability and because the juice is a common preparation of this berry in Finland. All of the samples were placed on a tray and the samples were stored in the refrigerator prior to the test. Good hygienic practices according to national food regulations were followed in the preparation and storage of the food samples. The children\'s food allergies were discussed and taken into account in all phases. The measurements with the children were performed with the project researcher in a peaceful and isolated place in the kindergarten. All children attended the measurement individually, and the session lasted approximately 15--20 min. The instructions were explained to the children and the tray with the samples in plastic cups was placed in front of them. The vegetable samples were served first, followed by the berry samples. Children were allowed to freely explore the samples by seeing, smelling, and touching before tasting. They could pick the samples by hand, but a spoon was also offered. They were allowed to decide the order in which the samples were to be tasted. The results of the tasting were recorded immediately on the study form by the researcher. First, the researcher recorded if the child refused to taste the sample. If the child tasted the sample, the amount eaten was inspected and further categorised as the proportion of the sample eaten (categorisation: not tasted, tasted a bit, ate half of the sample, or ate the whole sample). This four-category variable is the main outcome measure of the present study and is defined by a single term, 'willingness to eat'. Description of the sensory-based food education activities {#S0002-S20004} ---------------------------------------------------------- In the intervention kindergarten, five different food education sessions were implemented, once per week for 5 weeks. Every child attended five food education sessions. There were usually three to four children (with a maximum of 7 children) in these groups. Activities were scheduled between 9.00 and 11.30 am, and the sessions lasted for 20--30 min. Activities were planned according to the sensory-based activities implemented previously in Finnish kindergartens ([@CIT0013]). The contents of the sessions are described briefly in [Table 1](#T0001){ref-type="table"}. They activated all five senses: taste, smell, sight, hearing, and the sense of touch. For example, smell was activated by odour bottles containing food-related odours. Pure taste samples (sweet, salty, sour, and bitter) were tasted in dilute water solutions. The sense of touch was activated by sensing different food samples in opaque fabric bags. ###### Goals and tools of the sensory-based food education programme Activating session Goal of a session Tools -------------------------- --------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------ 1st 'Familiarisation' Explore and get familiar with own senses (sense of smell, touch, taste, hearing, and sight). Learning by playing evocative activating games Sensory cards, puppet, cloth bags for touching, food samples ([@CIT0009]), magnifying glass, odour bottles ([@CIT0007]) 2nd 'Applying to senses' Observing food samples and surrounding with smelling, touching, hearing, looking, and tasting Sensory cards, puppet, cloth bags with food samples ([@CIT0004]), variety of food samples, magnifying glass, kitchen utensils, play shop, food cards 3rd 'Sweet-sour' Learning sweet and sour in food focusing on vegetables, fruit and berries with five senses Sensory cards, food cards, puppets, activating fairy tale, variety of food samples, magnifying glass, kitchen utensils, play shop 4th 'Bitter-salty' Learning bitter and salty focusing on cooking with five senses Sensory cards, variety of food samples, kitchen utensils, stories, salad materials 5th 'Umami' Learning umami and food talk focusing on food exploring with five senses Sensory cards, variety of food samples, kitchen utensils, stories, variety of food samples, play shop, food posters The sessions included the tasting and exploration of real food samples, concentrating on vegetables and berries. Of the samples tested in the test protocol, carrots, cabbage, swede, rucola, romaine lettuce, and lingonberries were included in the activities. In addition to tasting, food samples were investigated with a magnifying glass, they were smelled and touched, and the sounds of chewing were heard. Additionally, pictures of the foods were used and children could play with the food cards and identify different vegetables and berries. All activities, including tasting the foods, were voluntary, but children were generally highly curious, and most of them were willing to taste the foods in these sessions. Statistical analyses {#S0002-S20005} -------------------- Background characteristics of the groups were compared with chi-square test (categorical variables) and *t*-test (continuous variables). The non-parametric related-samples Wilcoxon signed rank test was used to analyse the median of differences for willingness to eat comparing the baseline measurement (m1) and the post-intervention measurement (m2) within the control and intervention groups independently. Non-parametric related-samples Friedman\'s two-way analysis of variance was used to analyse the willingness to eat (comparing m1 and m2) in the control and intervention groups independently. All food samples, both together and separately (*n*=8), were analysed to study the effect of the sensory-based activities. Statistical analyses were performed using IBM SPSS Statistics 21.0 (IBM Corporation, Armonk, NY, USA). Results {#S0003} ======= The characteristics of the intervention and control children are presented in [Table 2](#T0002){ref-type="table"}. The differences in the background variables between the groups were not statistically significant. The results of the test protocol implemented in kindergartens at baseline and after the intervention are presented in [Table 3](#T0003){ref-type="table"}. When the willingness to eat (total amount of all samples) was compared, there was a significant difference both in the distribution (*p*\<0.001, Friedman\'s) and the median of differences (*p\<*0.001, Wilcoxon) in intervention kindergarten between measurements at baseline and after the intervention. There was no significant change within the control group between measurements. Specifically, the proportion of samples completely eaten increased significantly (*p\<*0.001) in the intervention group. The willingness to eat all test samples together is also illustrated in [Fig. 1](#F0001){ref-type="fig"}. {#F0001} ###### Background characteristics of the intervention and control children and their mothers Intervention Control *p* [\*](#TF0001){ref-type="table-fn"} ----------------------------------------------- -------------- ------------- ---------------------------------------- N consents 44 24 Girls (*n*, %) 22 (50%) 14 (58%) 0.40 Age of the child, years (mean, SD) 5.1 (0.8) 4.7 (0.9) 0.054 *N* responded to questionnaire 39 22 Children\'s neophobia score, mean (SD) 34.0 (10.0) 39.8 (14.8) 0.069 Age of the mother, years (mean, SD) 34.1 (5.7) 33.0 (7.0) 0.52 Maternal education, university or polytechnic 23% 32% 0.46 Maternal employment, working 74% 77% 0.80 Maternal smoking 26% 14% 0.31 Categorical variables compared with chi-square test and continuous variables with *t*-test. ###### Willingness to eat (%) test samples in the intervention (INT, *n*=37) and control groups (CNTR, *n*=19) at baseline (m1) and after the intervention (m2) Phase Not tasted Tasted a bit Half of the sample eaten Whole sample eaten Wilcoxon, *p* Friedman\'s, *p* ----------------- ------- ------------ -------------- -------------------------- -------------------- --------------- ------------------ Carrot  INT m1 10.8 18.9 21.6 48.6 0.001 \<0.001 m2 5.4 8.1 5.4 81.1  CNTR m1 52.6 5.3 0.0 42.1 0.25 0.48 m2 31.6 15.8 5.3 47.4 Swede  INT m1 18.9 32.4 5.4 43.2 0.022 0.004 m2 10.8 10.8 13.5 64.9  CNTR m1 52.6 31.6 0.0 15.8 1.0 1.0 m2 63.2 10.5 10.5 15.8 Cabbage  INT m1 21.6 37.8 0.0 40.5 0.039 0.074 m2 21.6 13.5 5.4 59.5  CNTR m1 57.9 21.1 0.0 21.1 0.39 0.41 m2 68.4 15.8 0.0 15.8 Romaine lettuce  INT m1 27.0 35.1 0.0 37.8 0.17 0.061 m2 21.6 21.6 5.4 51.4  CNTR m1 78.9 5.3 0.0 15.8 0.033 0.034 m2 47.4 26.3 0.0 26.3 Rucola  INT m1 27.0 54.1 2.7 16.2 0.048 0.108 m2 29.7 21.6 16.2 32.4  CNTR m1 68.4 21.1 0.0 10.5 0.59 0.41 m2 78.9 10.5 0.0 10.5 Bilberry  INT m1 18.9 27.0 0.0 54.1 0.001 0.001 m2 5.4 8.1 5.4 81.1  CNTR m1 52.6 10.5 0.0 36.8 0.49 0.70 m2 42.1 21.1 0.0 36.8 Lingonberry  INT m1 27.0 54.1 2.7 16.2 0.012 0.074 m2 24.3 27.0 13.5 35.1  CNTR m1 52.6 42.1 0.0 5.3 0.17 0.41 m2 52.6 26.3 0.0 21.1 Sea buckthorn  INT m1 40.5 59.5 0.0 0.0 0.22 0.47 m2 37.8 51.4 2.7 8.1  CNTR m1 47.4 47.4 5.3 0.0 0.11 0.41 m2 42.1 36.8 0.0 21.1 All samples  INT m1 24.0 39.9 4.1 32.1 \<0.001 0.001 *n*=8×37 = 296 m2 19.6 20.3 8.4 51.7  CNTR m1 57.9 23.0 0.7 18.4 0.25 0.11 *n*=8×19 = 152 m2 53.3 20.4 2.0 24.3 Statistical analyses (Wilcoxon and Friedman) were made within the group (INT and CNTR) by comparing the results of m1 and m2. When the samples were inspected separately ([Table 3](#T0003){ref-type="table"}), it was found that the willingness to eat carrots (*p*\<0.001), swede (*p*=0.004), and bilberries (*p\<*0.001) increased statistically (Friedman\'s) in the intervention group. There was also a significant difference in the median intake of carrots, cabbage, swede, rucola, bilberries, and lingonberries (Wilcoxon). In the control group ([Table 3](#T0003){ref-type="table"}), only the willingness to eat romaine lettuce increased between measurements (*p=*0.033; Wilcoxon, *p=*0.034; Friedman). [Figure 2](#F0002){ref-type="fig"}, which depicts comparison of parent opinion and child behaviour, illustrates how parents reported their children\'s preferences for certain vegetables and how the children tasted or ate the samples in the test situation. The figure highlights that in many cases, the parental answers and children\'s behaviour did not interrelate with each other. For example, in the case of swede, cabbage, or lingonberries, many parents reported that their child disliked these foods; however, many children were willing to taste or even eat the sample in the test situation. {#F0002} Discussion {#S0004} ========== In the present study, sensory-based food education activities concentrating on vegetables and berries were employed in a kindergarten. These activities were shown to increase the willingness to eat test samples of vegetables and berries. The arrangement of sensory tests for small children is often laborious and time demanding, but the developed test procedure was found to be practical and easy to use in kindergarten. It was challenging to motivate the control group subjects in particular to participate in the study, and although the number of children was quite low, the results are promising. The period with activities was quite short (5 short sessions over 5 weeks) and the measurements were carried out shortly after the activity sessions were finished (within the next week). Thus, an evaluation of the long-term impact requires further studies with larger study populations and with longer intervention and follow-up periods. There are very few scientific reports on the effectiveness of sensory-based activities, including all five senses, among children. Previous studies have concentrated in school-aged children and have found positive effects in promoting the willingness to taste novel foods among children aged 8--10 years ([@CIT0011]), and in reducing neophobia among children aged 8--11 years ([@CIT0019]). Sensory education also improved the children\'s skills in describing the sensory properties of foods ([@CIT0020]). Among Dutch schoolchildren aged 9--12 years, the taste lessons were shown to result in an increased number of foods known and tasted in the intervention group, as reported by the children themselves in the questionnaire after 4 weeks of the intervention ([@CIT0021]). Dazeley and Houston-Price ([@CIT0010]) reported the results of a non-taste sensory activity programme among children aged 12--36 months in the UK. They found that in a meal-time taste test, children touched and tasted more of the vegetables that they had been familiarised with in activities. Additionally, some other programmes promoting healthy eating for preschool-aged children, such as *Color Me Healthy*, have included some sensory components, but the effects of sensory activities in multicomponent interventions are difficult to assess ([@CIT0022]). It is well-known that repeated exposure to vegetables may increase intake ([@CIT0023], [@CIT0024]), and frequent exposure during sensory activities might thus promote the tasting and eating of new vegetables. Additionally, visual exposure alone, such as looking at picture books about fruits and vegetables, may influence the willingness to taste these foods ([@CIT0025]). Recently, Wagner et al. ([@CIT0026]) reported that toddlers' food preferences were correlated with their liking of the odour of the food. Sensory characteristics, including texture, have been shown to be important to children, and therefore different preparation methods of vegetables may be related to children\'s preferences ([@CIT0027]). Thus, more information on the combination of all senses in children\'s food perceptions and further sensory-based food education activities are required. Kindergarten offers a good operational environment for sensory-based education. The pedagogical skills of the personnel help to develop activities appropriate for different age groups. The activities implemented in this study are easy and cost little to incorporate into daily pedagogical activities. Peer modelling in kindergarten may promote children\'s acceptance of new foods ([@CIT0028]). Food services at day care may also be involved in food education, and daily lunch and snack times offer constant possibilities for sensory experiences. The day care practices and meal services at day care differ between countries, and thus new food education activities need to be developed for local conditions. Involving children in meal preparation ([@CIT0029]) and gardening ([@CIT0030]) may offer possibilities for motivational activities to influence children\'s attitudes, knowledge, and behaviours related to vegetables and fruit. Thus, sensory-based food education could be part of the normal daily activities in kindergartens and could promote healthy eating in the long run. We also observed that there were clear discrepancies between the parental report on their child\'s liking and the child\'s actual eating of the test samples. Parents rely on their opinion of the children\'s eating habits and meal situations at home, and although the test situation differs from this, the differences are still notable. This result is consistent with findings in previous studies, for example, Liem et al. ([@CIT0031]) found that only 39% of mothers were able to correctly predict their children\'s most preferred flavour in a preference ranking task of five ice-cream flavours. They also found that older children (5--10 years) reported more stable flavour preferences than younger children (3--5 years) ([@CIT0031]). Vereecken et al. ([@CIT0032]) compared children\'s and parents' responses on young children\'s fruit and vegetables preferences and they found that the agreement between parents and their children was rather low to moderate. Thus, relying only on parental reports may distort the reality of children\'s preferences and consumption of foods. Previous studies aiming to increase and measure children\'s preferences and consumption of vegetables have used various tasting protocols. The choice of test samples and measuring methods depends on the aims of the study ([@CIT0033]). The arrangement of the test situation also depends on local practices in kindergartens and the place (in separate room or in the lunch room), time of day (in the morning or during snack time), and personnel (familiar kindergarten personnel or study personnel), as well as whether the measurements are performed individually or in group need to be considered ([@CIT0017]). Thus, careful planning and pilot testing of methods suitable for the study question, age group, food culture, and other factors are required of future studies. Clear indicators and outcomes should be defined for evaluating interventions ([@CIT0034]). The method developed and tested here seemed to be motivational for children, practical, precise enough and easy to use and thus suitable for testing Finnish children in a day care setting. The strengths of the present study are also the presence of a control group and the measurements with the same test protocol both at baseline and after the intervention. Because children\'s preferences and behaviours will change as they grow older, it is important to have a control group when evaluating the effectiveness of sensory-based education, especially for long-term interventions. The limitation of the present study is the low number of participants, but for practical reasons we were able to include only two kindergartens in this small-scale study. As the kindergartens are located in different areas, the background variables of families may also differ at baseline. This a common challenge in all real-life interventions carried out in kindergarten settings. The two kindergartens participating in the study represented typical day care settings in small Finnish towns, but the generalisability of the results on a larger scale needs to be further studied. Interventions in kindergarten may increase children\'s willingness to eat vegetables served in day care. However, the total consumption of vegetables cannot be markedly improved if the supply is not increased at home. Parents are responsible for the foods offered at home and the availability of vegetables and fruits at home ([@CIT0035]), as well as serving as parental role models ([@CIT0036]), and have been demonstrated to be important determinants of children\'s food intake. Therefore, parental involvement and motivation to increase vegetable consumption should have a role in further interventions. Additionally, Finnish adults consume fewer vegetables and fruits than recommended and many adults may not enjoy the taste of vegetables, such as their bitterness, thereby limiting their intake ([@CIT0037]). Food neophobia may also reduce vegetable intake of adults ([@CIT0038]). Therefore, various sensory-based activities could also be developed for the whole family to encourage both adults and children to diversify and increase their vegetable intake. In conclusion, food education activities in kindergarten may promote children\'s willingness to eat vegetables and berries. Further research on effective methods to implement sensory-based food education and scientific research examining the impact of these activities on children\'s dietary choices are warranted. We thank the parents and children for participating in the study and the director and personnel of the day care in Hanko. Conflict of interest and funding {#S0005} ================================ The authors have no conflict of interest. This study was supported by the Academy of Finland (grant for M. Sandell \#252005, \#283070, \#263747).

INTRODUCTION {#s1} ============ Lung cancer and chronic obstructive pulmonary disease (COPD) combined, present a major cause of morbidity and mortality globally that has persisted for several decades \[[@R1]--[@R3]\]. These two complex diseases are closely linked to each other as shared environmental and genetic risk factors are implicated in both \[[@R4], [@R5]\]. COPD coexists in 40-70 % of patients with lung cancer and is increasingly recognized as a predecessor of lung cancer development, independent of tobacco exposure \[[@R6]\] and most published genetic studies focused on either lung cancer or COPD \[[@R5], [@R7]--[@R9]\]. Chronic inflammation plays a central role and key inflammatory cells such as macrophages, neutrophils, and T lymphocytes are involved in COPD \[[@R10]--[@R14]\]. Macrophages from COPD patients release higher levels of pro-inflammatory cytokines (TNF-α and IL-6) compared to nonsmoking control subjects \[[@R15], [@R16]\]. Inflammation and COPD together are strongly associated with lung cancer but the molecular basis for inflammation and cancer has not been established yet. Loss of function mutations in the gene *PARK2* encoding Parkin, an E3 ubiquitin ligase, leads to death of dopaminergic neurons and causes Autosomal Recessive Juvenile Parkinsonism (AR-JP) \[[@R17], [@R18]\], the neuroprotective role of *PARK2* is linked to mitophagy \[[@R19]\]. Recently, *PARK2* has been suggested as a haplo-insufficient tumor suppressor gene, based on the frequent deletion or mutation in human cancer \[[@R20], [@R21]\]. Consistent with this notion, overexpression of *PARK2* in hepatocarcinoma, glioblastoma, lung cancer, breast cancer and colon cancer cell lines repressed cell growth \[[@R20]--[@R24]\]. Furthermore, *PARK2*-null mice displayed increased vulnerability to inflammation-induced degeneration when exposed to chronic systemic lipopolysaccharide (LPS) \[[@R25]\]. Cancer cell lines that harbor a deleted *PARK2* gene show resistance to TNF-α induced cell death \[[@R16]\]. These observations suggest a potential function of *PARK2* as a regulator of immune response. However, how *PARK2* functions in immune response and its relationship to tumorigenesis remains unclear. In this study, we investigated a potential role of *PARK2* in inflammation, COPD and lung cancer. RESULTS {#s2} ======= Loss of *PARK2* maintains higher expression of cytokines for inflammation {#s2_1} ------------------------------------------------------------------------- To assess the role of PARK2 in inflammation, we generated *PARK2* knockout (KO) in C57BL/6 mice and assessed IL-6 levels in mouse serum. Notably, IL-6 levels in *PARK2* KO mice were significantly higher than those in wild type (WT) mice (Figure [1A](#F1){ref-type="fig"}). Similarly, mouse primary bronchus epithelial cells and mouse embryonic fibroblasts (MEFs) from *PARK2* KO mice produced more IL-6 than controls (Figure [1B and 1C](#F1){ref-type="fig"}). Furthermore, the expression of other inflammation markers, IL-1β and TNF-α also increased in *PARK2* deleted cells (Figure [1B and 1C](#F1){ref-type="fig"}). In addition, the increases caused by *PARK2* KO were reversed by exogenous expression of WT *PARK2* (Figure [1D](#F1){ref-type="fig"}). These results suggest that PARK2 has anti-inflammation functions. We also performed immunohistochemistry (IHC) in *PARK2* wild type (WT) and knockout (KO) mouse at 10 months (Figure [1E](#F1){ref-type="fig"}). *PARK2* KO mice showed increased inflammation phenotypes, as evinced by densely packed plasma cells around lung bronchia. This data suggested that *PARK2* deficiency leads to increased inflammation. {#F1} Overexpression of *PARK2* inhibits localization of the nuclear NF-κB for inflammation {#s2_2} ------------------------------------------------------------------------------------- Nuclear factor kappa B (NF-κB) is a broadly expressed transcription factor that induces cytokines and immunoglobulin (Ig) gene expression in COPD-related inflammation \[[@R26]\]. Although NF-κB is present in its inactive state in the cytoplasm, its p50--p65 heterodimer translocates to the nucleus and binds the DNA (at the promoter region) when NF-κB is activated by carcinogens, tumor promoters, inflammatory cytokines, and other chemotherapeutic agents \[[@R27]\]. To understand PARK2′s role in inflammation, we monitored NF-κB localization in *PARK2*-depleted primary human bronchial epithelial BEAS-2B cells using fluorescence microscopy (Figure [2A and 2B](#F2){ref-type="fig"}) and immunoblot (Figure [2C](#F2){ref-type="fig"}). The depletion of *PARK2* led to increased nuclear NF-κB localization in the absence of stimuli (Figure [2A, 2B and 2C](#F2){ref-type="fig"}). In addition, TNF-α treatment-induced nuclear NF-κB translocation was significantly blocked by the expression of WT *PARK2* (Figure [2D and 2E](#F2){ref-type="fig"}). Interestingly, expression of mutant *PARK2* that inactivates its E3 ligase has a partial effect in blocking NF-κB translocation. These results demonstrate that *PARK2* suppresses NF-kB activation in an E3 ligase-dependent and -independent manner. {#F2} We next explored how PARK2 suppresses NF-kB activation. Several papers reported involvement of PARK2 in the antioxidant defense \[[@R28], [@R29]\]. We confirmed increased Reactive oxygen species (ROS) levels in *PARK2* KO MEFs (Figure [2F](#F2){ref-type="fig"}). ROS has been linked to increased Akt activation, and NF-kB activation \[[@R30]--[@R33]\]. Consistent with this, we found increased Akt S473 phosphorylation in *PARK2* KO MEFs (Figure [2G](#F2){ref-type="fig"}). Treatment of *PARK2* KO MEFs with NAC, ROS scavenger or MK 2206, Akt inhibitor (Figure [2H](#F2){ref-type="fig"}) decreased NF-kB translocation, suggesting that PARK2 suppresses NF-kB signaling through ROS/Akt regulation \[[@R30], [@R32], [@R33]\]. Deletion of *PARK2* induced chromosome stability and tumor initiation {#s2_3} --------------------------------------------------------------------- In addition to increased inflammation, *PARK2* deficiency also resulted in genomic instability \[[@R15], [@R21], [@R34]\]. We found that loss of *PARK2* induced centrosome duplication. Using an antibody to γ-tubulin, we compared the number of centrosome in *PARK2* KO and wild-type MEFs at Passage 5. Our data showed that 11 % of *PARK2 KO* cells contained three or more centrosomes, whereas very few *WT* MEFs have abnormal centrosomes (Figure [3A](#F3){ref-type="fig"}). The reconstitution of *PARK2* KO cells with *PARK2* corrected the centrosome abnormities (Figure [3B](#F3){ref-type="fig"}). Similar results were obtained using IMR-90 lung fibroblasts (PDL=33) or MEFs supporting the idea that *PARK2* deficiency results in genomic instability (Figure [3C, 3D and 3E](#F3){ref-type="fig"}). {#F3} Our results suggest that *PARK2* deficiency results in increased inflammation and genomic instability, both of which are potential drivers of tumorigenesis \[[@R20], [@R21], [@R23], [@R25]\]. To confirm whether *PARK2* contributes to tumorigenesis in ex-vivo models, we seeded *PARK2* WT or KO MEFs in 3-D organoid culture system. Culture of cells as three-dimensional (3-D) aggregates can enhance *in vitro* tests for basic biological research as well as mimics tissue organization condition *in vivo* \[[@R35], [@R36]\]. We found that *PARK2* KO MEFs, but not WT MEFs, formed colonies in 3-D culture (Figure [4A](#F4){ref-type="fig"}). Furthermore, we found that *PARK2* knockout MEFs became transformed and promoted cancer cell growth by foci and colony formation assay, where the frequency of foci number and colony number were increased (Figure [4B and 4C](#F4){ref-type="fig"}), suggesting a role of *PARK2* loss in tumor initiation. Treatment of *PARK2*-deficient MEFs with N-acetyl-l-cysteine (NAC), ROS scavenger, significantly decreased colony formation, suggesting that ROS is a contributing factor in cellular formation induced by *PARK2* loss. We next examined the potential role of *PARK2* on the growth of lung cancer cells. After the *PARK2* gene was expressed in seven lung cancer cell lines (H1437, H522, H1650, A549, H460, H1299 and H196), the growth of tumor cell lines was significantly inhibited compared to controls (Figure [4D, 4E and 4F](#F4){ref-type="fig"}), which suggests that *PARK2* overexpression plays a critical role in the inhibition of lung cancer cell proliferation \[[@R28]\]. Multiple *PARK2* mutations have also been identified in human lung cancer ([Supplementary Table S1](#SD2){ref-type="supplementary-material"}). These results suggest that *PARK2* deficiency might contribute to lung cancer development. {#F4} *PARK2* as a potential target to reduce the risk of COPD and lung cancer {#s2_4} ------------------------------------------------------------------------ As *PARK2* deficiency results in both increased inflammation and genomic instability \[[@R37]--[@R39]\], we assessed a potential role of *PARK2* in lung cancer and COPD through a comprehensive validation study of 114 informative single nucleotide polymorphism (SNP) variants of *PARK2* ([Supplementary Table S2](#SD3){ref-type="supplementary-material"}) in 2,484 cases and controls with well-defined lung cancer (LC) and COPD phenotypes (Table [1](#T1){ref-type="table"}). For specific comparisons, we assigned the four groups into the six models as follows: Model 1: LC+COPD+ vs. LC-COPD- (cases with both compared to controls with neither); Model 2: LC+COPD- vs. LC-COPD- (cases with LC compared to controls with neither); Model 3: LC-COPD+ vs. LC-COPD- (cases with COPD compared to controls with neither); Model 4: LC+COPD+ vs. LC-COPD+ (cases with both compared to controls with COPD); Model 5: LC+ vs. LC- (LC cases compared to LC-free controls, adjusting for COPD and other covariates including age, sex, and smoking history); and Model 6: COPD+ vs. COPD- (COPD cases compared to COPD-free controls, adjusting for LC and other covariates including age, sex, and smoking history). After using two methods of minimizing false positive results and a penalized logistic regression analysis \[[@R40]--[@R42]\], rs577876, rs6455728 and rs9346917 (*p\<0.01*) were confirmed to be associated with lung cancer development in people with COPD (in Model 4 only, Table [2](#T2){ref-type="table"}). Figure [5](#F5){ref-type="fig"} shows the LD haplotype blocks of five SNPs that were tested significant in Model 4. Noted is rs34424986 \[[@R24], [@R43]\], which lies between rs2223468 and rs9346917. ###### Distribution of lung cancer (LC) and chronic obstructive pulmonary diseases (COPD) in 2484 cases and controls COPD + COPD - Total ------- ------------- ------------- ------- LC + n +/+ (573) n +/− (612) 1185 LC − n −/+ (537) n −/− (762) 1299 Total 1110 1374 2484 ###### Significant results of Testing 114 Informative PARK2 SNPs *PARK2 Gene (Chromosome 6) SNP rs-ID* Alleles(major: minor) MAF controls[^\*^](#tfn_001){ref-type="table-fn"} MAF cases[^\*^](#tfn_001){ref-type="table-fn"} Penalized regression --------------------------------------- ----------------------- --------------------------------------------------- ------------------------------------------------ ---------------------- ------ ------------- *PARK2* rs577876 A:G 0.41 0.35 0.73 5.49×10^−4^ *PARK2* rs6455728 G:T 0.29 0.24 0.70 3.28×10^−3^ *PARK2* rs9346917 C:T 0.35 0.30 0.68 1.66×10^−3^ MAF is Minor Allele Frequency calculated from the controls (LC+COPD-) and cases (LC+COPD+). ![Linkage disequilibrium (LD) plot of 6 SNPs in *PARK2* gene\ Among the six SNPs, five (SNPs 1-4 and 6) are tested significant in our Model 4 that comparing cases with lung cancer and chronic obstructive pulmonary diseases (COPD) to controls with COPD only. One SNP rs34424986, which was recently reported from an independent study \[[@R24]\] but not included on GWAS panels, is shown relative position among the five tested significant SNPs. Numbers shown in blocks are D\' values (i.e., D\'x100; 99 means D\' = 0.99), which measures the strength of LD between two neighboring SNPs. The higher of D\' value, the more statistically associated between two SNPs. Because the high LD between rs9456676, rs577876 and rs2223768, final results of penalized regression model removed two SNPs (rs9456676 and 2223768). LOD, means logarithm of odds score, is a statistical test used in genetic linkage analysis. **Legends**: White - D\'\<1, LOD \<2; Pink shades - D\'\<1, LOD ≥ 2; Red - D\' =1, LOD ≥ 2;None: SNPs not correlated.](oncotarget-07-44211-g005){#F5} This is the first study that presents lung cancer with COPD as a separate disease entity from the lung cancer or COPD phenotype \[[@R44]\]. We found the *PARK2* gene associates with the phenotype of lung cancer with COPD, compared to COPD patients. Notably, altered PARK2 expression induced chromosomal instability and increased inflammation, similar to that found in the COPD phenotype. We demonstrated that *PARK2* may be a bona fide tumor suppressor gene that may be responsible for the development of progression lung cancer with COPD, suggesting *PARK2* as a potential target to reduce the risk of COPD and lung cancer. Our results evinced the biological plausibility as well as the common and unique mechanisms in lung cancer with COPD development \[[@R45]\]. DISCUSSION {#s3} ========== Our analysis of *PARK2*, located at 6q25.2-q27, revealed four significant yet independent SNPs to be associated with lung cancer with COPD. Although the function of *PARK2* in lung cancer remains unknown, we suggest that PARK2-deficiency induced inflammation and genomic instability are possible factors contributing to lung cancer. *PARK2* mutations are known to cause Parkinson\'s disease and have also been identified in human malignancies, including lung cancer, and are associated with the potential to inhibit cancer cell growth *in vitro* \[[@R21]\], suggesting that *PARK2* may function as a tumor suppressor gene. Additionally, a SNP in *PARK2* was associated with a decline in forced expiratory volume in 1 second (FEV1) and a recurrent *PARK2* mutation was recently reported \[[@R24], [@R46]\]. Our study demonstrated that *PARK2* expression significantly inhibited cell growth in lung cancer cells lines compared to controls, while abnormal inflammation and cell division was induced by *PARK2* deficiency. This result could be interpreted as a role in cancer initiation, progression or both. As inflammation and chromosomal instability are a driving forces for tumor initiation, it is possible that *PARK2* mutation/loss may also involved in lung cancer progression and prognosis ([Supplementary Figure S1](#SD1){ref-type="supplementary-material"}). In conclusion, our multi-level validation study suggests a critical role of *PARK2* in the development of lung cancer with COPD. We showed that *PARK2* inhibiting role in inflammation, lung cancer cell growth, and the chromosomal instability that might contribute to tumor initiation when *PARK2* expression is altered. We used strict inclusion criteria in the study. Lung cancer diagnoses were re-confirmed by pulmonary pathologists and COPD was confirmed by lung function testing. The definition of COPD phenotype based on lung function tests minimizes the phenotypic heterogeneity caused by the use of clinical records or patient reported symptoms. As a result, the reliability and validity of our study is sound. These findings have advanced the current understanding of the development of lung cancer with COPD, and the progression from COPD to lung cancer, potentiating the discovery of new targets for prevention, early diagnosis, and treatment. MATERIALS AND METHODS {#s4} ===================== Mouse strains and MEFs {#s4_1} ---------------------- All animal procedures were approved by the Institutional Animal Care and Use Committee which also approved experimental procedures. Mouse strains were described previously \[[@R34], [@R47]\]. *PARK2* KO C57BL/6 (6--8 weeks old, female) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and mated. Mouse embryonic fibroblasts were isolated from individual day 11.5-13.5 (E11.5 - E13.5) embryos by uterine dissection. Each embryo was washed gently with 1x PBS (pH 7.2), followed by removal of the mouse embryo\'s head and liver. The embryo body was suspended in 0.5 ml of 0.25 % Trypsin-EDTA, and then forced through a 1 ml syringe with an 18-gauge needle. The tissue homogenate was incubated for 30 min at 37°C, triturated by drawing the suspension through a pipette, and then evenly divided into two 10 cm tissue culture dishes in Dulbecco\'s modified Eagle\'s medium (DMEM) with 15 % fetal bovine serum (FBS). Early-passage MEFs (passage 1--5) were used for all experiments, and at least three lines were examined for all studies. Animals were housed in a pathogen-free barrier environment throughout the study. Cells and cell lines and reagent {#s4_2} -------------------------------- All cell lines were sourced from the American Type Culture Collection (ATCC, Manassas, VA). These included seven lung cancer cells, H1437, H522, H1650, A549, H460, H1299 and 196, and one normal human lung fibroblast cell line, IMR-90. c. For the human lung fibroblast IMR-90 cells ranging from 29 to 34 in population doubling level (PDL) were used. Cell lines were maintained in Eagle\'s minimal essential media (EMEM, Gibco-Invitrogen, Grand Island, NY) and contained 10 % (15 % for IMR-90 cells) heat-inactivated FBS (Gibco-Invitrogen), sodium bicarbonate (2 mg/ml; Sigma-Aldrich, St Louis, MO), penicillin (100 units/ml), and streptomycin (100 μg/ml; Gibco-Invitrogen). An ELISA kit (R&D Systems) was used to assay IL-6 production, according to the manufacturer\'s instructions. The concentrations of IL-6 were normalized to the cell numbers in the cell culture. 2′,7′- dichlorofluorescein diacetate (DCF-DA) was obtained from Molecular Probes. MK 2206 was obtained from selleckchem. N-acetylcystein (NAC) was obtained from Gibco-Invitrogen. Immunoblotting and antibodies {#s4_3} ----------------------------- For immunoblotting, extraction of proteins from cultured cells with a modified buffer was followed by immunoblotting with corresponding antibodies. Briefly, protein samples were fractionated on 10 - 15 % SDS polyacrylamide gels and electroblotted onto Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech) using a semidry transfer apparatus (Bio-Rad). Rabbit polyclonal antibodies recognizing PARK2 (ab15954), were obtained from Abcam. Mouse monoclonal antibody recognizing PARK2 (sc-32282) and Lamin B1 (sc-20682) were purchased from Santa Cruz Biotechnology. Rabbit polyclonal antibody recognizing NF-κB (sc-372) was obtained from Santa Cruz Biotechnology. Anti-β-tubulin and HA mouse antibodies were purchased from Sigma. Immunofluorescence {#s4_4} ------------------ For immunofluorescence staining, MEF or IMR90, Beas-2B cells were plated on glass coverslips and transfected with the indicated constructs. Cells were then fixed in 3.7 % Paraformaldehyde for 10 min at room temperature and stained using standard protocols. Immunofluorescence images were taken using fluorescent microscopy (Nikon Microscope, Melville, New York). Plasmid and gene silencing by lentiviral shRNAs {#s4_5} ----------------------------------------------- The pMX retroviral vector containing the human cDNAs for HA-PARK2 was obtained from Addgene (plasmid 38248, Cambridge, MA). *PARK2* vectors were obtained from Sigma-Aldrich and Open Biosystems. Company Species Clone Set ID Names Target sequence (5′- -3′) -------------- --------- -------------- ------- ------------------------------- Open Bio. Human NM_013988 84517 5′-GAGAGAGTTCTCACATTTAAT-3′ Open Bio. Human NM_013988 84518 5′-ACTCACTAGAATATTCCTTAT-3′ Open Bio. Human NM_013988 84520 5′-GAACGTTTAGAAATGATTTCAAA-3′ Sigma (TRC1) Human NM_013988 2399 5′-CGTGAACATAACTGAGGGCAT-3′ Sigma (TRC1) Human NM_013988 341 5′-CGCAACAAATAGTCGGAACAT-3′ Sigma (TRC1) Human NM_013988 425 5′-CGTGATTTGCTTAGACTGTTT-3′ Sigma (TRC1) Human NM_013988 434 5′-CTTAGACTGTTTCCACTTATA-3′ Sigma (TRC1) Human NM_013988 872 5′-CTCCAAAGAAACCATCAAGAA-3′ Colony formation or foci assay {#s4_6} ------------------------------ For colony formation or foci assay, early-passage MEFs (passage 5) cells were plated at low density into 60-mm cell culture plates. When sufficient colonies were visible, typically after 2-3 weeks, cells were washed twice in PBS before fixing in ice-cold 70 % methanol for 30 min, stained by 0.2 % Crystal violet for 2-3 h. The following day cells were rinsed in PBS and air-dried. Cell fractionation assay {#s4_7} ------------------------ Cell preparations were described previously. \[[@R31]\] The Nuclear extract kit (California, USA) was used to perform cellular fractionation in accordance with the manufacturer\'s instructions. The purity of the extract was confirmed by western blot analysis against anti-cytosol-specific-tubulin-actin (Sigma-Aldrich) or anti-nuclear-specific-Lamin B1. 3-dimensional (3-D) organoid culture Assay {#s4_8} ------------------------------------------ For 3-dimensional (3-D) assays, *PARK2* WT or KO MEFs were cultured on NanoCulture plates (SCIVAX Corp.). After seeding cells, cell were cultured in same condition with 2-D culture and observe cell images and recover sample 0, 3, 5, 7 days after seeding. Cells were lysed in Spheroid Lysis Buffer (Scivax). Phenotype definitions {#s4_9} --------------------- Lung cancer cases were pathologically diagnosed as defined by the World Health Organization \[[@R48]\]. COPD diagnosis was based on the criteria of the Global Initiative for Chronic Obstructive Lung Disease (GOLD) \[[@R49]\], i.e., subjects with a FEV1/FVC \< 70 % by lung function test (PFT) as COPD+ regardless of sub-groups (emphysema and/or chronic bronchitis). All subjects provided written informed consent, and the study protocol was approved by the Mayo Clinic Institutional Review Board. Experimental design for PARK2 SNPs validation {#s4_10} --------------------------------------------- ### Subject selection and detailed phenotyping {#s4_11} A total of 2,484 well-defined subjects were included in the current study following a 4-step sampling strategy in order to minimize the confounding factors and maximize the uncommon groups: \[[@R1]\] enriching never-smoking cases; \[[@R2]\] balancing three phenotype groups, i.e., COPD only (COPD+), lung cancer only (LC+), both COPD and lung cancer (LC+COPD+) and controls (LC-COPD-); \[[@R3]\] matching on current vs. former smoking history with gender and age; and \[[@R4]\] for former smokers, matching on quitting years. To minimize the heterogeneity due to population stratification, we restricted our study to subjects of European descent \[[@R9], [@R44], [@R50]--[@R52]\]. In total, the study consisted of 612 with LC+, 537 COPD+, 573 LC+COPD+, and 762 controls; yielding 1,110 COPD cases and 1,185 lung cancer cases. Genome wide analysis and candidate SNPs validation {#s4_12} -------------------------------------------------- Using GWAS Data from two Illumina platforms (OmniExpress, 610k and 370k Human BeadChips; Illumina, San Diego, California, USA) we performed genome-wide association analyzes using the models described above Concordance between platforms was \> 99.5 %. We removed samples with genotyping call rates \< 95 % from structure analysis. SNP quality control removed SNPs with a call rate \< 95 %, minor allele frequencies (MAF) \< 0.01, Hardy-Weinberg equilibrium test *p* \< 0.0001. A quantile-quantile plot was generated using logistic regression for additive genetic model, and 4 sets of above-defined group comparisons of genomic inflation factors (λ~GC~) were 1.006, 1.011, 0.98122, 0.98185, respectively. A total of 114 candidate SNPs for the *PARK2* gene were matched in our genotyping platforms, after removing the redundant SNPs using linkage disequilibrium (LD) test (r^2^≥0.8) \[[@R53]\]. Real time PCR {#s4_13} ------------- RNA preparation, cDNA, and qRT-PCR were described previously \[[@R31]\]. The following primers were used: Primers Used for Real-Time PCR IL-6 Primer sequence: Forward 5′-CCTGCGTTTAAATAACATCAGCTTTAGCTT-3′, Reverse 5′-GCACAATGTGACGTCGTTTAGCATCGAA-3′, IL-1β Primer sequence: Forward 5′-CACAGCAGCA CATCAACAAG-3′, Reverse 5′-GTGCTCATGTCCT CATCCTG-3′, TNF-α Primer sequence: Forward 5′-CCAGCCAGCAGAAGCTCCCTCAGCGAG-3′, Reverse 5′-GCGGATCATGCTTTCTGTGCTCATGGTGTC-3′ Statistical analysis {#s4_14} -------------------- Each assay was performed in triplicate and independently repeated at least three times. The results are presented as mean ± standard error of mean (SEM). Statistical analyses were performed using GraphPad Prism software (version 4.02; GraphPad Software, San Diego, CA). One-way analysis of variance (ANOVA) followed by T-test was used to compare the results. A difference was considered significant if *P*\<0.05. Statistical significance was defined as *p*\<0.05 (^\*^), *p*\<0.01(^\*\*^), and *p*\<0.001(^\*\*\*^). SUPPLEMENTARY TABLES AND FIGURES {#s5} ================================ The study was supported by the National Heart, Lung, and Blood Institute (NHLBI) grant, R01-HL107612 (Yang and Wendt), and National Cancer Institute grants, R01-CA80127 (Yang), R01-CA84354 (Yang), R01-CA130996 (Lou), R01-CA108961 (Lou) and R01-CA148940 (Lou), and Mayo Clinic Foundation Funds (Yang and Lou), Mayo Clinic Cancer Center grant CA15083. We thank Dr. Thomas C. Smyrk (Mayo clinic) for IHC analysis. Jun She was supported by the National Natural Science Foundation for Young Scholars of China, No. 81200051 and Scientific Project for Fudan University, No. 20520133474. Bo Deng was supported by National Natural Science Foundation of China, No. 81101782 and Third Military Medical University MiaoPu project. Ola Larsson was supported by the Swedish Research Council and the Wallenbberg Academy Fellows Programme. Andrew Limper was supported by funds from the Annenberg Foundation. Biospeciens accessioning and processing, genotyping and RNA microarray analysis were supported partly by the Mayo Clinic Cancer Center grant CA15083. **CONFLICTS OF INTEREST** The authors declare no conflict of interest.

Related literature {#sec1} ================== For applications of Schiff base compounds, see: Kahwa *et al.* (1986[@bb4]); Santos *et al.* (2001[@bb5]). Experimental {#sec2} ============ {#sec2.1} ### Crystal data {#sec2.1.1} C~10~H~13~N~3~O~2~*M* *~r~* = 207.23Monoclinic,*a* = 7.3079 (11) Å*b* = 10.2150 (17) Å*c* = 14.763 (2) Åβ = 100.058 (9)°*V* = 1085.1 (3) Å^3^*Z* = 4Mo *K*α radiationμ = 0.09 mm^−1^*T* = 296 K0.28 × 0.21 × 0.11 mm ### Data collection {#sec2.1.2} Bruker SMART CCD area-detector diffractometerAbsorption correction: none7304 measured reflections2116 independent reflections1099 reflections with *I* \> 2σ(*I*)*R* ~int~ = 0.024 ### Refinement {#sec2.1.3} *R*\[*F* ^2^ \> 2σ(*F* ^2^)\] = 0.047*wR*(*F* ^2^) = 0.159*S* = 0.932116 reflections141 parameters1 restraintH atoms treated by a mixture of independent and constrained refinementΔρ~max~ = 0.17 e Å^−3^Δρ~min~ = −0.15 e Å^−3^ {#d5e367} Data collection: *SMART* (Bruker, 1998[@bb1]); cell refinement: *SAINT* (Bruker, 1998[@bb1]); data reduction: *SAINT*; program(s) used to solve structure: *SHELXS97* (Sheldrick, 2008[@bb6]); program(s) used to refine structure: *SHELXL97* (Sheldrick, 2008[@bb6]); molecular graphics: *ORTEP-3 for Windows* (Farrugia, 1997[@bb2]); software used to prepare material for publication: *WinGX* (Farrugia, 1999[@bb3]). Supplementary Material ====================== Crystal structure: contains datablocks global, I. DOI: [10.1107/S1600536809031420/xu2581sup1.cif](http://dx.doi.org/10.1107/S1600536809031420/xu2581sup1.cif) Structure factors: contains datablocks I. DOI: [10.1107/S1600536809031420/xu2581Isup2.hkl](http://dx.doi.org/10.1107/S1600536809031420/xu2581Isup2.hkl) Additional supplementary materials: [crystallographic information](http://scripts.iucr.org/cgi-bin/sendsupfiles?xu2581&file=xu2581sup0.html&mime=text/html); [3D view](http://scripts.iucr.org/cgi-bin/sendcif?xu2581sup1&Qmime=cif); [checkCIF report](http://scripts.iucr.org/cgi-bin/paper?xu2581&checkcif=yes) Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: [XU2581](http://scripts.iucr.org/cgi-bin/sendsup?xu2581)). The authors would like to express their deep appreciation to the start-up Fund for PhDs of the Natural Scientific Research of Zhengzhou University of Light Industry (No. 2005001) and the Fund for Natural Scientific Research of Zhengzhou University of Light Industry, China (000455). Comment ======= The chemistry of Schiff base has attracted a great deal of interest in recent years. These compounds play an important role in the development of various proteins and enzymes (Kahwa *et al.*, 1986; Santos *et al.*, 2001). As part of our in the study of the coordination chemistry of Schiff bases, we synthesized the title compound and determined its crystal structure. The molecular structure of (I) is shown in Fig. 1. The molecules is roughly planar, with the largest deviations from the mean plane defined by all non-H atoms, except the nitro group, being -0.120 (2) for atom O2. Intramolecular N---H···O hydrogen bond is observed in compound (I), and this helps to stabilize the configuration of the molecule. Experimental {#experimental} ============ 2-Nitrophenylhydrazine (1 mmol, 0.153 g) was dissolved in anhydrous ethanol (15 ml). The mixture was stirred for several min at 351 K, then butan-2-one (1 mmol, 0.72 g) in ethanol (8 ml) was added dropwise and the mixture was stirred at refluxing temperature for 2 h. The product was isolated and recrystallized from methanol, red single crystals were obtained after 3 d. Refinement {#refinement} ========== Imino H atom was located in a difference Fourier map and positional parameters were refined with a fixed isotropic thermal parameter of 0.08 Å^2^. Other H atoms were positioned geometrically and refined as riding with C---H = 0.93 (aromatic), 0.97 (methylene) and 0.96 Å (methyl), with U~iso~(H) = 1.5U~eq~(C) for methyl H atoms and 1.2U~eq~(C) for the others. Figures ======= {#Fap1} Crystal data {#tablewrapcrystaldatalong} ============ ------------------------- --------------------------------------- C~10~H~13~N~3~O~2~ *F*(000) = 440 *M~r~* = 207.23 *D*~x~ = 1.268 Mg m^−3^ Monoclinic, *P*2~1~/*c* Mo *K*α radiation, λ = 0.71073 Å Hall symbol: -P 2ybc Cell parameters from 1572 reflections *a* = 7.3079 (11) Å θ = 2.4--26.0° *b* = 10.2150 (17) Å µ = 0.09 mm^−1^ *c* = 14.763 (2) Å *T* = 296 K β = 100.058 (9)° Block, red *V* = 1085.1 (3) Å^3^ 0.28 × 0.21 × 0.11 mm *Z* = 4 ------------------------- --------------------------------------- Data collection {#tablewrapdatacollectionlong} =============== ----------------------------------------------- -------------------------------------- Bruker SMART CCD area-detector diffractometer 1099 reflections with *I* \> 2σ(*I*) Radiation source: fine-focus sealed tube *R*~int~ = 0.024 graphite θ~max~ = 26.0°, θ~min~ = 2.4° ω scans *h* = −9→8 7304 measured reflections *k* = −11→12 2116 independent reflections *l* = −13→18 ----------------------------------------------- -------------------------------------- Refinement {#tablewraprefinementdatalong} ========== ------------------------------------- ------------------------------------------------------------------------------------- Refinement on *F*^2^ Primary atom site location: structure-invariant direct methods Least-squares matrix: full Secondary atom site location: difference Fourier map *R*\[*F*^2^ \> 2σ(*F*^2^)\] = 0.047 Hydrogen site location: inferred from neighbouring sites *wR*(*F*^2^) = 0.159 H atoms treated by a mixture of independent and constrained refinement *S* = 0.93 *w* = 1/\[σ^2^(*F*~o~^2^) + (0.0917*P*)^2^\] where *P* = (*F*~o~^2^ + 2*F*~c~^2^)/3 2116 reflections (Δ/σ)~max~ = 0.010 141 parameters Δρ~max~ = 0.17 e Å^−3^ 1 restraint Δρ~min~ = −0.14 e Å^−3^ ------------------------------------- ------------------------------------------------------------------------------------- Special details {#specialdetails} =============== ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Geometry. All e.s.d.\'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.\'s are taken into account individually in the estimation of e.s.d.\'s in distances, angles and torsion angles; correlations between e.s.d.\'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.\'s is used for estimating e.s.d.\'s involving l.s. planes. Refinement. Refinement of *F*^2^ against ALL reflections. The weighted *R*-factor *wR* and goodness of fit *S* are based on *F*^2^, conventional *R*-factors *R* are based on *F*, with *F* set to zero for negative *F*^2^. The threshold expression of *F*^2^ \> σ(*F*^2^) is used only for calculating *R*-factors(gt) *etc*. and is not relevant to the choice of reflections for refinement. *R*-factors based on *F*^2^ are statistically about twice as large as those based on *F*, and *R*- factors based on ALL data will be even larger. ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å^2^) {#tablewrapcoords} ================================================================================================== ------ ------------ -------------- --------------- -------------------- -- *x* *y* *z* *U*~iso~\*/*U*~eq~ N1 0.3024 (2) 1.04656 (17) 0.09960 (11) 0.0674 (5) C1 0.2088 (2) 0.89169 (19) −0.01835 (13) 0.0551 (5) N2 0.2496 (2) 1.01728 (17) 0.00803 (12) 0.0678 (5) C2 0.2183 (3) 0.79113 (19) 0.04780 (12) 0.0621 (6) H2 0.2495 0.8122 0.1099 0.075\* C6 0.1570 (2) 0.85236 (19) −0.11104 (12) 0.0587 (5) C5 0.1221 (3) 0.7221 (2) −0.13432 (14) 0.0690 (6) H5 0.0889 0.6985 −0.1959 0.083\* N3 0.1382 (3) 0.9459 (2) −0.18488 (14) 0.0817 (6) C3 0.1830 (3) 0.66460 (19) 0.02310 (14) 0.0698 (6) H3 0.1907 0.6007 0.0685 0.084\* C4 0.1358 (3) 0.6288 (2) −0.06817 (15) 0.0732 (6) H4 0.1136 0.5415 −0.0841 0.088\* O2 0.1590 (3) 1.0630 (2) −0.16759 (12) 0.1089 (7) C7 0.3445 (3) 1.1663 (2) 0.11902 (16) 0.0713 (6) O1 0.1019 (3) 0.90665 (19) −0.26427 (11) 0.1204 (7) C8 0.4002 (3) 1.2013 (2) 0.21782 (18) 0.0938 (8) H8A 0.5240 1.2387 0.2266 0.113\* H8B 0.3166 1.2687 0.2325 0.113\* C10 0.3419 (3) 1.2749 (2) 0.05076 (18) 0.0974 (8) H10A 0.4223 1.2531 0.0080 0.146\* H10B 0.3843 1.3543 0.0824 0.146\* H10C 0.2175 1.2869 0.0180 0.146\* C9 0.4004 (4) 1.0931 (3) 0.28360 (19) 0.1200 (10) H9A 0.2803 1.0523 0.2740 0.180\* H9B 0.4286 1.1266 0.3452 0.180\* H9C 0.4926 1.0298 0.2745 0.180\* H2A 0.240 (3) 1.0806 (15) −0.0338 (11) 0.080\* ------ ------------ -------------- --------------- -------------------- -- Atomic displacement parameters (Å^2^) {#tablewrapadps} ===================================== ----- ------------- ------------- ------------- -------------- ------------- -------------- *U*^11^ *U*^22^ *U*^33^ *U*^12^ *U*^13^ *U*^23^ N1 0.0685 (11) 0.0696 (12) 0.0636 (12) −0.0003 (9) 0.0106 (8) −0.0109 (9) C1 0.0509 (11) 0.0590 (12) 0.0556 (12) 0.0053 (9) 0.0102 (8) 0.0030 (10) N2 0.0764 (12) 0.0608 (12) 0.0652 (13) 0.0025 (9) 0.0095 (9) 0.0037 (8) C2 0.0672 (13) 0.0693 (14) 0.0485 (11) 0.0010 (10) 0.0062 (9) 0.0024 (10) C6 0.0557 (11) 0.0734 (14) 0.0468 (11) 0.0076 (10) 0.0081 (8) 0.0083 (10) C5 0.0601 (13) 0.0917 (17) 0.0549 (12) −0.0007 (11) 0.0087 (10) −0.0156 (12) N3 0.0826 (13) 0.1009 (16) 0.0613 (13) 0.0115 (11) 0.0116 (9) 0.0134 (12) C3 0.0758 (15) 0.0658 (14) 0.0680 (14) −0.0028 (11) 0.0126 (11) 0.0065 (11) C4 0.0764 (15) 0.0667 (14) 0.0777 (16) −0.0052 (11) 0.0169 (12) −0.0077 (12) O2 0.1446 (17) 0.0932 (13) 0.0870 (13) 0.0062 (12) 0.0148 (11) 0.0327 (10) C7 0.0523 (12) 0.0700 (15) 0.0934 (17) 0.0034 (11) 0.0173 (11) −0.0162 (13) O1 0.1572 (18) 0.1516 (17) 0.0490 (11) 0.0180 (13) 0.0086 (10) 0.0100 (10) C8 0.0826 (16) 0.0953 (18) 0.106 (2) −0.0056 (13) 0.0236 (14) −0.0338 (16) C10 0.0861 (18) 0.0704 (16) 0.136 (2) 0.0024 (13) 0.0191 (15) 0.0039 (15) C9 0.135 (2) 0.144 (3) 0.0799 (17) −0.036 (2) 0.0144 (16) −0.0224 (18) ----- ------------- ------------- ------------- -------------- ------------- -------------- Geometric parameters (Å, °) {#tablewrapgeomlong} =========================== --------------- ------------- ------------------- ----------- N1---C7 1.282 (2) C3---C4 1.380 (3) N1---N2 1.372 (2) C3---H3 0.9300 C1---N2 1.359 (2) C4---H4 0.9300 C1---C6 1.413 (3) C7---C10 1.496 (3) C1---C2 1.411 (2) C7---C8 1.488 (3) N2---H2A 0.888 (9) C8---C9 1.471 (3) C2---C3 1.355 (2) C8---H8A 0.9700 C2---H2 0.9300 C8---H8B 0.9700 C6---C5 1.387 (3) C10---H10A 0.9600 C6---N3 1.438 (2) C10---H10B 0.9600 C5---C4 1.356 (3) C10---H10C 0.9600 C5---H5 0.9300 C9---H9A 0.9600 N3---O1 1.223 (2) C9---H9B 0.9600 N3---O2 1.227 (2) C9---H9C 0.9600 C7---N1---N2 116.33 (18) C5---C4---H4 120.3 N2---C1---C6 123.67 (17) C3---C4---H4 120.3 N2---C1---C2 120.49 (18) N1---C7---C10 125.6 (2) C6---C1---C2 115.84 (18) N1---C7---C8 117.5 (2) C1---N2---N1 119.87 (16) C10---C7---C8 116.8 (2) C1---N2---H2A 120.1 (14) C9---C8---C7 115.8 (2) N1---N2---H2A 120.0 (14) C9---C8---H8A 108.3 C3---C2---C1 121.61 (18) C7---C8---H8A 108.3 C3---C2---H2 119.2 C9---C8---H8B 108.3 C1---C2---H2 119.2 C7---C8---H8B 108.3 C5---C6---C1 121.30 (17) H8A---C8---H8B 107.4 C5---C6---N3 117.42 (19) C7---C10---H10A 109.5 C1---C6---N3 121.28 (19) C7---C10---H10B 109.5 C4---C5---C6 120.57 (19) H10A---C10---H10B 109.5 C4---C5---H5 119.7 C7---C10---H10C 109.5 C6---C5---H5 119.7 H10A---C10---H10C 109.5 O1---N3---O2 121.1 (2) H10B---C10---H10C 109.5 O1---N3---C6 119.0 (2) C8---C9---H9A 109.5 O2---N3---C6 119.9 (2) C8---C9---H9B 109.5 C2---C3---C4 121.22 (18) H9A---C9---H9B 109.5 C2---C3---H3 119.4 C8---C9---H9C 109.5 C4---C3---H3 119.4 H9A---C9---H9C 109.5 C5---C4---C3 119.5 (2) H9B---C9---H9C 109.5 --------------- ------------- ------------------- ----------- Hydrogen-bond geometry (Å, °) {#tablewraphbondslong} ============================= --------------- ---------- ---------- ----------- --------------- *D*---H···*A* *D*---H H···*A* *D*···*A* *D*---H···*A* N2---H2A···O2 0.89 (2) 1.97 (2) 2.604 (3) 127 (1) --------------- ---------- ---------- ----------- --------------- ###### Hydrogen-bond geometry (Å, °) *D*---H⋯*A* *D*---H H⋯*A* *D*⋯*A* *D*---H⋯*A* --------------- ------------ ------------ ----------- ------------- N2---H2*A*⋯O2 0.889 (16) 1.969 (16) 2.604 (3) 127.2 (14)

Systems biology presents an extreme challenge to the traditional human-based scientific method ([@r1], [@r2]). The fundamental difficulty is the high degree of complexity of biological systems, where even simple "model" systems such as *Escherichia coli* and *Saccharomyces cerevisiae* have thousands of genes, proteins, and small molecules all interacting together in complicated spatial-temporal ways. This biological complexity implies a need for a similar complexity, probably beyond human intuitive understanding, in the corresponding systems biology models. In the development of systems biology models, biological knowledge is integrated to form a model, experiments are planned and executed to test the model, the experimental results are used to refine the model, new biological knowledge is generated, and the cycle repeated ([@r1]). To radically improve existing system biology models, it will be necessary to execute hundreds/thousands of such cycles of model improvement. However, little current research completes even a single cycle. We therefore argue that greater automation is required, which will in turn require the combination and integration of multiple systems biology software tools into closed-loop cycles with laboratory robotics. To evaluate the integration of software tools and laboratory robotics for systems biology we selected as a test case the diauxic shift of the yeast *S. cerevisiae.* This is the standard model system for understanding eukaryotic cellular transformation, and it is relevant to understanding cancer (Warburg effect), the immune system, and aging. In *S. cerevisiae* growing in batch culture on glucose with aeration a diauxic shift is commonly observed: During the first growth phase, yeast metabolizes glucose using the fermentative Embden--Meyerhof pathway to produce ethanol ([@r3]); when the glucose is exhausted, it switches to a fully respiratory metabolism utilizing the tricarboxylic acid cycle and oxidative phosphorylation in the mitochondria ([@r3]). This transition requires the large-scale remodeling of the metabolic apparatus ([@r4]). However, despite being one of the most studied of all eukaryotic cellular transformations, the diauxic shift is still very poorly understood, and existing systems biology models of this transformation could be greatly improved. We combined system biology software for data analysis, model formation, experiment generation, experiment execution, model refinement, systems biology modeling, bioinformatics, laboratory robotic control, and semantic web techniques to execute three cycles of diauxic shift model improvement ([Fig. 1*A*](#fig01){ref-type="fig"}). The wide range of software and tools required to achieve this are shown in [Fig. 2](#fig02){ref-type="fig"}. (CoRegNet and CoRegFlux are available in bioconductor. All of the other software is available on request at LIPN GitLab). {#fig01} {#fig02} Results {#s1} ======= Modeling of the yeast diauxic shift is especially challenging because of the complexity of the biology involved, and the need to include subsystems operating at different time scales, and serving different purposes ([@r5]). The modeling requires integration of (1) a model of control of metabolism (cell signaling), and (2) a genome scale model of metabolism ([@r1]). The key difference between gene regulatory/signaling and metabolic networks is that the former carry signal flows, whereas metabolic pathways generate mass flows. We modeled the metabolic network as a biochemical (mechanistic) network based on the stoichiometry and reversibility of the reactions involved. Specifically, we chose the iMM904 model ([@r6]), ([*SI Appendix 1*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900548116/-/DCSupplemental)). This model is widely used, its structure is suitable for integration with signaling, and it is the most accurate model available for predicting growth phenotypes ([@r7]). For cell-signaling modeling we used a two-time slice dynamic Bayesian network (DBN) with conditional linear Gaussian parameters ([Fig. 3*A*](#fig03){ref-type="fig"}). We selected this form of model because: it belongs to a well-studied family of continuous models, is easily interpretable in terms of activation and repression effects, and they enable the inference of gene states from known states in a versatile way. Each node in the model corresponds to either a regulatory protein or an enzyme, the former being the only type of node allowed to have children in the network, ([*SI Appendix 2* and *3*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900548116/-/DCSupplemental)). The starting point for our cell-signaling model was the model of Geistlinger et al. ([@r8]), which was assembled by compiling the findings of hundreds of scientific articles. We extracted the regulatory part of the model (Mz) and integrated this with iMM904m ([Fig. 3*A*](#fig03){ref-type="fig"}). Mz is of high quality in terms of dependencies recall (small number of false positive links), but it is relatively incomplete (missing links). Mz is also optimistic in that it predicts the occurrence of diauxic shifts for almost all strains with gene/protein deletions in the model. ![(*A*) The form of the integrated diauxic shift models. The regulatory model (blue box) is a DBN with linear Gaussian conditionals, overregulatory (parents and children) + metabolic (only parents) genes/proteins. The metabolic model (orange box) is composed of a stoichiometric matrix, and a set of enzymatic relations between metabolic genes and reactions. Simulation for *n* time steps consists of *n* repeats of: (1) DBN inference; (2) metabolic inference with dynamic flux balance analysis (DFBA); (3) regularization of gene states for the next time step using two results, and diauxic shift metabolite to gene rules. (*B*) The ensemble network inference procedure ELSA for learning DBNs. Simple models ("components") are combined to form a consensual "composite" model. Each component is built by computing the Edmonds directed maximal spanning arborescence over a graph obtained by double sampling. The final composite model is built by aggregating all components by edge frequency to produce a ranking and postfiltering this using information from the Brauer dataset ([@r4]) (S5).](pnas.1900548116fig03){#fig03} In the first cycle of model improvement, the initial step was application of the bioinformatic program CoRegNet ([@r9], [@r10]) to identify genes potentially involved in control of the diauxic shift. CoRegNet integrates information from microarray experiments, regulatory interactions from the YEASTRACT database, and the *S. cerevisiae* Kinase and Phosphatase Interactome resource. CoRegNet uses a cooperative network based on shared transcription factor targets to identify coregulatory relationships from gene expression data ([*SI Appendix 4*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900548116/-/DCSupplemental)). We then applied a two-step model refinement process to its output ([Fig. 3*B*](#fig03){ref-type="fig"}): (*i*) We applied the ensemble network inference algorithm ELSA (Ensemble Learning of Spanning Arborescences) ([@r11]) to the Brauer microarray dataset ([@r4]), with Mz as a learning prior on the model space composed of the union of the Mz regulatory genes, the top 40 transcription factors identified by CoRegNet, and the top 40 kinases identified by CoRegNet ([*SI Appendix 5*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900548116/-/DCSupplemental)); (*ii*) We then applied a forward selection step to add to Mz edges that improve gene state predictions on the Brauer microarray dataset, using leave 1 out cross-validation. This generated model M1 ([Fig. 1*B*](#fig01){ref-type="fig"}). At the start of the second cycle, we used tools to design experiments to provide the maximum amount of information to optimize the improvement of Mz to form M1, see [Fig. 4*A*](#fig04){ref-type="fig"}. We developed two tools for this task. The first tool is AdactiveFB (active learning based), which compares estimated protein/gene states (forward simulation) with the most likely protein/gene states consistent with the observed growth and metabolite state (backward simulation) ([*SI Appendix 6*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900548116/-/DCSupplemental)). In forward simulation a standard simulation from genes to phenotypes is performed, using both regulatory and metabolic simulators ([*SI Appendix 3*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900548116/-/DCSupplemental)). This produces an estimated time series of states for each gene in the DBN---as Gaussian distributions means and SDs. These forward simulations are compared with backward ones, i.e., simulations using phenotypes evidence to infer gene states ([*SI Appendix 6*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900548116/-/DCSupplemental)). Due to the unavailability of inferred states for several genes, the method used for backward simulation is designed to deal with partial evidence. The result of backward simulation is a set of backward time series for all of the genes in the regulatory model---also as Gaussian distributed means and SDs. Kullback--Leibler divergence is then calculated between the forward and backward Gaussian distributions (using their means and SDs) for each gene and each time point. This generates a divergence value for each (gene, time) pair. The genes selected for knockout experiment are those with the highest node divergence values. The strength of the AdactiveFB approach is that it focuses directly on optimizing the model rather than using a proxy. Its current main weakness is that the observed growth curve is the only phenotype used to inform backward simulation. Growth curve experiments are relatively robust ([@r12]), but they are not highly informative. In the future we plan to include many more phenotypic experiments. {#fig04} The second tool, CoRegMine, initially uses CoRegNet ([@r10]) to infer a graph in which the vertices are coregulators labeled according to their influence profile ([@r13]), and the edges relate predicted coregulators. This graph is then processed by the graph mining tool MinerLC\* ([@r14]) to extract subgraphs, each consisting of coregulators with similar influence profiles. Subgraph pairs were selected with a) antagonistic influence profiles, and b) edges relating coregulators from the two subgraphs, suggesting differential regulation of their common targets during the diauxic shift. The coregulators identified in this manner were then selected for use in experiments ([*SI Appendix 7*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900548116/-/DCSupplemental)). The strength of this approach is its use of background knowledge. Its weakness is that it does not directly focus on improving the model. We used the cloud laboratory robotics system Eve ([@r15]) to execute the experiments selected by AdactiveFB and CoRegMine. Eve executed two complementary types of automated experiment on selected yeast deletion strains: determinations of growth curves ([Fig. 5*A*](#fig05){ref-type="fig"}) and glucose consumption curves ([Fig. 5*B*](#fig05){ref-type="fig"}). The observed growth curves were preprocessed, normalized, and descriptive parameters calculated ([@r12]) ([*SI Appendix 8*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900548116/-/DCSupplemental)). Periodic colorimetric resorafin-based assays were used to track glucose levels in the culture medium. {#fig05} The next step in the cycle of model improvement is to refine the model based on the obtained new experimental results. We developed the tool Adarev for this task. As Eve's experiments do not directly observe the time-series of protein/gene states, these need to be inferred from observations of growth and limited metabolite states. The approach used by Adarev is built on the identification of a local curve error reduction improvements to an input model based on simulation vs. real growth curves. Computational model simulation is very costly in terms of computer time. A prior scoring step was therefore included to predict local changes to the model likely to be interesting. Model refinement was restricted to selecting the edges to be removed from the cell-signaling submodels, although the addition of edges is also possible with the algorithm. The main steps in the model refinement algorithm are shown in [Fig. 4*B*](#fig04){ref-type="fig"}. ([*SI Appendix 9* and *10*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900548116/-/DCSupplemental)). In total, three closed-loop system biology cycles were executed. In the first cycle, the model Mz was semiautomatically improved using bioinformatic data to form M1. To assess the utility of cycle 1, we compared Mz and M1's predictions with the empirical growth curves observed by Eve using a set of yeast gene deletant strains not used to form M1 ([*SI Appendix 9*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900548116/-/DCSupplemental)). The 192 strains selected for the experiments were taken from genes identified by CoRegNet as potentially involved in the diauxic shift ([@r10]) and randomly selected regulatory genes (kinases and transcription factors) ([*SI Appendix 12*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900548116/-/DCSupplemental)). The experimental results demonstrate that M1 is significantly better than Mz ([Fig. 6](#fig06){ref-type="fig"}). {#fig06} The second and third closed-loop cycles differed from the first in including new planned experiments ([Fig. 1*A*](#fig01){ref-type="fig"}). In the second closed loop, inference tools were run to generate experiments, the experiments were executed, and the models were refined. Two sets of experiments were generated to improve M1: set (a) of 80 hypothesis-led experiments designed with our tools (AdactiveFB and CoRegMine), and set (b), consisting of 80 randomly selected experiments. Eve executed both sets of experiments. Model M1-smart was refined from M1 based on the results of hypothesis-led experiments, and model M1-random was refined from M1 based from the random experiments ([Fig. 1 *A* and *B*](#fig01){ref-type="fig"}). The motivation for generating two separate sets of experiments was to test the belief that hypothesis-led experiments (experiments designed to improve/test models) are more efficient in systems biology model development than random/high-throughput experiments ([@r16]). The M1-smart model has 298 nodes and 1,760 edges ([Fig. 1*B*](#fig01){ref-type="fig"}). We compared M1-smart and M1 using their predictions for 281 test strains. The results show that M1-smart is significantly more accurate at prediction than M1 ([Fig. 6](#fig06){ref-type="fig"}). We ensured the maximal improvement of M1-random and made the comparison between M1-random and M1-smart as rigorous as possible by selecting the 80 randomly selected genes from known yeast regulators (kinases and transcription factors) ([*SI Appendix 13*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900548116/-/DCSupplemental)). The M1-random model has 298 nodes and 1,778 edges ([Fig. 1*B*](#fig01){ref-type="fig"}). To compare M1 and M1-random, we applied their predictions on the same 281 test strains. M1-random was significantly better at prediction than M1 ([Fig. 6](#fig06){ref-type="fig"}). In the third cycle, the M1-smart and M1-random models were compared by generation and execution of experiments. To generate the "crucial" experiments used to compare M1-smart and M1-random we applied the tool Adana to select 81 deletant strains with the largest predicted postshift growth rate disagreement between M1-smart and M1-random. We found that M1-smart was significantly better than M1-random ([Fig. 6](#fig06){ref-type="fig"}). We therefore concluded, as expected, that hypothesis-led experiments are more efficient at improving systems biology models than high-throughput/random experiments. An essential part of systems biology is the analysis of new models to provide biological insight ([@r1]). Our most accurate model, M1-smart adds a substantial amount of knowledge about the yeast diauxic shift: 92 extra genes (+45%) and 1,048 interactions (+147%). We used the Adana tool to rank the genes in terms of relative importance in the M1-smart model. To evaluate the biological insight possible from these additions, and to illustrate the biological utility of the knowledge generated by the system, we selected two genes highly ranked in M1-smart, but absent from Mz: MRK1 and TIS11 ([*SI Appendix 14*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900548116/-/DCSupplemental)). MRK1 (YDL079C) is homologous to human protein kinase glycogen synthase kinase-3 (GSK-3). [Fig. 7*A*](#fig07){ref-type="fig"} shows the fragment of M1-smart incorporating MRK1. GSK-3 genes are highly conserved and ubiquitous in eukaryotes and involved in differentiation, cell fate determination, and spatial patterning ([@r17]). These two highly homologous isoforms have been implicated in type II diabetes (Diabetes mellitus type 2), Alzheimer's disease, inflammation, cancer, and bipolar disorder ([@r17]). {#fig07} TIS11 is a member of the 12-O-tetradecanoylphorbol-13-acetate inducible sequence 11 family. TIS11 genes are involved in posttranscriptional gene regulation by micro-RNA (miRNA) and short interfering RNA (siRNA) ([@r18], [@r19]). Note that RNA processing is not explicitly included in M1-smart, and TIS11 was automatically incorporated as a putative transcription factor based on its zinc finger motif. This illustrates a strength of automating systems biology modeling: a human biologist would have excluded TIS11, yet its inclusion proved interesting, highlighting a possibly important role for RNA processing in the diauxic shift. [Fig. 7*B*](#fig07){ref-type="fig"} shows the fragment of M1-smart incorporating TIS11. In humans, changes in *TIS11* expression have been associated with both the suppression and promotion of cancer, and with autoimmune diseases ([@r18]). Formal languages promote the reproducibility and reusability of results, and the exchange of information between human scientists and computer systems. We developed a suite of complementary ontologies to support the application of systems biology tools and their integration: (1) AdaLab-meta, an ontology for the description of metadata about datasets; (2) AdaLab, a domain ontology to represent relevant biological entities in systems biology; and (3) Eve-CV, a controlled vocabulary that defines typical Eve experiments and experimental conditions ([*SI Appendix 15*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900548116/-/DCSupplemental)). When combined these ontologies consist of ∼20,000 RDF (Resource Description Framework) triples. We collected and formalized in RDF all of the bioinformatic data used for this study to form a knowledge base of 1,301,017 RDF triples grouped in five separate RDF graphs: imported genes, genes annotations, genes expressions, Eve strains, and relevant metadata. The data are accessible via the linked data web interface ([*SI Appendix 15*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900548116/-/DCSupplemental)). We developed a dedicated communication mechanism SciCom (Scientific Communication) to communicate information about experiments to Eve. The requests for experiments and experimental results are stored in an RDF triple store in Manchester that consists of 10,187,417 RDF triples combined in two graphs. Discussion {#s2} ========== The fundamental motivation for studying the diauxic shift in yeast (*S. cerevisiae*) is that it serves as a model for transformation in human cellular systems. It is therefore important to consider how well the methods can be scaled up for use in mammalian systems. This scaling up entails two main challenges: ensuring the same experimental reproducibility as is achievable in yeast, and scaling up the computational methods. We consider experimental reproducibility to be the most difficult of these challenges ([@r20]). For the scaling up of computational methods, the different parts of the software pipeline have different sensitivities to an increase in input network size ([*SI Appendix 16*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900548116/-/DCSupplemental)), but all of the methods scale polynomially, implying that the increase in size and complexity associated with the move to mammalian systems should be tractable with our approach. We have successfully combined multiple systems biology software tools and laboratory robotics to execute three cycles of improvement for a model of the yeast diauxic shift. The cycles were not fully automated, as in the Robot Scientists Adam ([@r12]) and Eve ([@r15]), as the automation of systems biology is very much more complicated. However, full automation will be necessary to execute the hundreds or thousands of model improvement cycles required. The achievement of this full automation will require the software tools to be more robust and more modularly designed. Many of the software tools we have used are based on techniques originating in artificial intelligence (AI), especially machine learning: CoRegNet, CoRegMine, ELSA, ActiveFB, MinerLC\*, Adarev, and Adana ([Fig. 2](#fig02){ref-type="fig"}). However, more advanced ideas from AI will be required to improve performance ([@r21]). For example, the tools have no high-level understanding of what they are doing, just as chess programs do not known that they are playing chess. One approach to providing them with such an understanding would be to give the system high-level goals to achieve, along with a higher-level planning ability. Another fundamental enhancement would be to give the AI tools the ability to communicate goals and intentions to human scientists. In conclusion, we foresee a future in which combinations of software tools, laboratory automation, and human scientists will work together to create systems biology models that fully reflect and predict the underlying biology. Supplementary Material ====================== We received support from the CHIST-ERA AdaLab project: The Engineering and Physical Sciences Research Council (EPSRC), UK (EP/M015661/1), ANR-14-CHR2-0001-01. The authors declare no conflict of interest. This article is a PNAS Direct Submission. This article contains supporting information online at [www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900548116/-/DCSupplemental](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900548116/-/DCSupplemental). [^1]: Edited by Michael S. Waterman, University of Southern California, Los Angeles, CA, and approved July 23, 2019 (received for review January 15, 2019) [^2]: Author contributions: A.C., K.R., D.T.B., M.E., J.R., C.R., L.N.S., and R.D.K. designed research; A.C., K.R., D.T.B., M.C., J.G., and M.E. performed research; A.C., D.B., G.S., H.S., M.E., C.R., L.N.S., and R.D.K. contributed new reagents/analytic tools; A.C., K.R., M.E., J.R., C.R., L.N.S., and R.D.K. analyzed data; and A.C., K.R., M.E., C.R., L.N.S., and R.D.K. wrote the paper. [^3]: ^1^A.C., K.R., and D.T.B. contributed equally to this work.

Mechanical trauma (MT), such as that caused during motor vehicle crashes, athletic competition, and war, currently represents a primary economic and medical burden. Trauma has become the most common cause of death in people under the age of 45 years[@b1][@b2]. Several studies have indicated that MT can cause direct heart damage, such as coronary artery dissection[@b3][@b4]. As a result of advanced prehospital care and regional trauma system development, fewer seriously injured patients are dying at the scene of an accident. However, several recently published clinical reports have suggested that nonfatal MT can induce secondary cardiac dysfunction even in the absence of direct cardiomyocyte injury during the first 24 h[@b5]^,^[@b6]^,^[@b7]^,^[@b8]. Secondary cardiac dysfunction can present as many severe cardiac diseases, such as myocardial infarction[@b9], severe congestive heart failure[@b8], traumatic ventricular septal defect (VSD)[@b10], and even multiple organ dysfunction syndrome (MODS), which has detrimental effects on human health and living standards[@b11]. Secondary cardiac dysfunction is difficult to diagnose because the clinical presentation, course and severity are variable[@b12]. In addition, the primary traumatic injury may obscure the typical chest pain associated with cardiac ischaemia[@b13] and the mechanism has never been elucidated. Thus, the mechanism of secondary cardiac dysfunction needs to be investigated. Our previous study[@b14] demonstrated that trauma-induced overproduction of TNF-α is the main reason for cardiomyocyte apoptosis, which contributes to posttraumatic cardiac dysfunction. Moreover, we have provided clear evidence that TNF-α initiates cardiomyocyte apoptosis by the overproduction of cytotoxic reactive oxygen/nitrogen species in cardiomyocytes. These results support the hypothesis that the overproduction of TNF-α and ROS may be the primary cause of secondary cardiomyocyte apoptosis, which eventually leads to secondary cardiac dysfunction after MT. Therefore, developing drugs with anti-inflammatory and anti-oxidative properties may have profound significance for patients who have experienced MT. Quercetin is a natural flavonoid compound that is widely distributed in fruits and vegetables. It exerts numerous beneficial effects on neuroprotective, anti-inflammatory, anti-ischaemic, antimutagenic, antiviral and cardiovascular protection processes[@b15][@b16][@b17][@b18][@b19][@b20]. Many studies have shown that quercetin acts as a novel protectant by mitigating the increased levels of TNF-α and ROS[@b21][@b22][@b23][@b24]. Given its protective properties and therapeutic potential, we speculated that quercetin may have a protective effect against MT-induced cardiomyocyte apoptosis. To test our hypothesis and explore its mechanism, *in vivo* and *in vitro* experiments were undertaken. Changes in cardiac function, cell viability, and TNF-α, ROS and \[Ca^2+^\]~i~ levels with or without quercetin treatment were observed and analysed. Results ======= MT induced cardiomyocyte apoptosis ---------------------------------- To determine the most appropriate trauma intensity in the nonlethal MT model, the cardiomyocyte apoptosis states of rats subjected to 0 r, 100 r, 200 r, and 400 r were assessed using TUNEL and DAPI staining 12 h after trauma. [Figure 1A](#f1){ref-type="fig"} shows the results of DAPI (a, b, c, d) and TUNEL (e, f, g, h) staining, which indicated the density of cardiomyocytes (a, b, c, d are of the same intensity) and the degree of apoptosis. There were nearly no TUNEL-positive nuclei detected in the samples isolated from rats subjected to sham trauma. However, the cardiomyocytes of rats subjected to 100 r, 200 r and 400 r showed significant apoptosis ([Fig. 1B](#f1){ref-type="fig"}). As the mortality rate of the rats in the 400 r group (30%) was significantly higher than that of the rats in the 200 r (0%) and 100 r (0%) groups, we chose 200 r as the standard number of revolutions in the nonlethal MT model to reduce mortality while obtaining the greatest apoptosis effect after trauma. Quercetin decreased MT-induced cardiomyocyte apoptosis ------------------------------------------------------ To test the protective effect of quercetin on MT-induced cell apoptosis *in vivo*, rats with MT (200 r) were pretreated with quercetin or vehicle (20 mg/kg i.p.; 0.5 h before trauma). The dose for quercetin (20 mg/kg) was based on the dose used in previously published articles[@b25][@b26]. TUNEL and DAPI staining were performed as above ([Fig. 2A](#f2){ref-type="fig"}). The apoptotic cardiomyocytes of posttraumatic rats increased significantly, whereas quercetin significantly reduced the number of apoptotic cells. However, the changes in the degree of apoptosis caused by pretreatment with vehicle were not statistically significant ([Fig. 2B](#f2){ref-type="fig"}). Taken together, these data indicated that quercetin attenuated MT-induced cardiomyocyte apoptosis. Quercetin improved cardiac dysfunction -------------------------------------- Evaluation of myocardial function is the basis for managing heart disease. Left ventricular developed pressure (LVDP, [Fig. 3A](#f3){ref-type="fig"}), peak rates of rise in the first derivative of the left ventricular pressure (+*dp*/*dtmax*, [Fig. 3B](#f3){ref-type="fig"}), and peak rates of fall in the first derivative of the left ventricular pressure (−*dp*/*dtmax*, [Fig. 3C](#f3){ref-type="fig"}) were continuously monitored on a recorder to evaluate cardiac function. [Figure 3A--C](#f3){ref-type="fig"} showed cardiac dysfunction with a lower LVDP, reduced +*dp*/*dtmax* and lessened −*dp*/*dtmax* compared with each control group, respectively. While quercetin attenuated this downtrend significantly, vehicle treatment did not. These results demonstrated that quercetin effectively improved MT-induced cardiac dysfunction. Appropriate concentration of quercetin for H9c2 cells ----------------------------------------------------- To determine the most appropriate quercetin concentration, 10 μM, 20 μM, 40 μM, or 80 μM quercetin was added to H9c2 cells, and the cells were analysed using the MTT assay. As shown in [Fig. 4](#f4){ref-type="fig"}, there was no statistically significant difference in cell viability when the concentrations of quercetin were 10 μM or 20 μM compared with the control group (with vehicle) and blank group (without vehicle), indicating that quercetin had no toxicity at low concentrations. Cell viability decreased when the concentration of quercetin was 40 μM and significantly decreased at 80 μM compared with the control group, indicating that quercetin had toxicity at these two concentrations. Thus, concentrations under 20 μM may be safe for H9c2 cells. Quercetin protected TP-induced H9c2 cells ----------------------------------------- TP (50% V/V)-induced cells *in vitro* is a simple model of organ MT, at least partly reflecting the pathophysiology *in vivo*. To simulate *in vivo* nonlethal mechanical trauma, H9c2 cells were exposed to TP. To determine whether quercetin has a protective effect on TP-induced H9c2 cells, quercetin (2.5 μM, 5 μM, 10 μM, or 20 μM) was added 24 h in advance. Cell viability was detected using an MTT assay. The cell viability of H9c2 cells induced by TP notably decreased compared with that induced by normal plasma (NP), while previous treatment with quercetin significantly inhibited the decrease. As [Fig. 5](#f5){ref-type="fig"} shows, 10 μM quercetin was the most effective at inhibiting TP-induced viability decreases. Accordingly, the NP + 10 μM group was added to the study to rule out the toxicity of quercetin at the concentration of 10 μM in NP. Quercetin reduced circulating TNF-α level ----------------------------------------- In our previous study[@b27], cardiomyocytes were cultivated with cytomix (a mixture of IFN-γ, IL-1β, and TNF-α) and with IFN-γ, IL-1β, or TNF-α alone. The caspase-3 activation of cardiomyocytes incubated with cytomix and TNF-α was much higher than that with normal plasma. In addition, the exposure of normal cardiomyocytes to TP revealed apparent cell apoptosis; this phenomenon was virtually abolished through the incubation of TP with anti-TNF-α or TP isolated from TNF-α−/− mice, indicating that TNF-α plays a critical role in cardiac injury after MT. As the amount of TNF-α in the circulation system of posttraumatic rats peaked at 1.5 h after trauma (2 h ahead of significant cardiomyocyte apoptosis), ELISA kits were used to determine the amount of TNF-α in the circulatory system 1.5 h after MT in the four groups (sham group, trauma group, trauma + quercetin group and trauma + vehicle group). As shown in [Fig. 6](#f6){ref-type="fig"}, with quercetin pretreatment, overproduction of TNF-α was significantly inhibited, whereas there was no apparent change in TNF-α in the trauma + vehicle group compared with the trauma group. Quercetin suppressed ROS overproduction in H9c2 cells ----------------------------------------------------- Oxidative stress was proven in our previous study to play an important role in MT-induced cardiomyocyte apoptosis. Therefore, we examined the protective effect of quercetin on TP-induced oxidative imbalance in H9c2 cells. As shown in [Fig. 7](#f7){ref-type="fig"}, the intracellular ROS level of TP-treated cells showed a remarkable increase, while quercetin scavenged excess ROS to protect cells at the concentration of 10 μM in the presence of TP. Interestingly, pretreatment with quercetin in NP significantly decreased intracellular ROS in H9c2 cells, indicating that quercetin could eliminate ROS both in NP and TP. Quercetin inhibited Ca^2+^ in H9c2 cells ---------------------------------------- Given the important role of cellular Ca^2+^ in apoptosis and necrosis[@b28], MT-initiated Ca^2+^ overload in H9c2 cells was measured. As shown in [Fig. 8](#f8){ref-type="fig"}, TP significantly enhanced the level of \[Ca^2+^\]~i~, and quercetin pretreatment for 2 min suppressed this change, providing evidence that MT-initiated \[Ca^2+^\]~i~ overload may contribute to posttraumatic cell apoptosis that can be relieved by quercetin. Discussion ========== In the present study, we have demonstrated the protective effects of quercetin on posttraumatic cardiac injury, the mechanism of which is mediated by reducing cell apoptosis through the inhibition of inflammation and oxidative stress and attenuating intracellular Ca^2+^ overproduction in cardiomyocytes. Many previous studies have investigated the effect of flavonoids in other post-traumatic injury models[@b25][@b26]. Xu *et al*. found that luteolin protected the mouse brain from traumatic brain injury by inhibiting the inflammatory response and that luteolin-induced autophagy may play a pivotal role in neuroprotection. Itoh *et al*. demonstrated the neuroprotective effect of (−)-epigallocatechin-3-gallate in rats when administered pre- or post-traumatic brain injury. However, the post-traumatic injury models used in those studies were different from those used in our study in terms of the regions involved and the methods used to produce trauma. To the best of our knowledge, this study is the first to examine the protective effect of quercetin on MT-induced cardiac injury. Mechanical trauma, even nonlethal mechanical trauma not causing death at the moment of an accident, can lead to secondary organ dysfunction, which is characterized by a lack of circulatory shock and no direct cardiac injury[@b11]. To detect the protective effect of quercetin on secondary cardiac dysfunction, a well-accepted nonlethal traumatic model was created *in vivo*. As shown in [Figs 1](#f1){ref-type="fig"}, [2](#f2){ref-type="fig"}, [3](#f3){ref-type="fig"}, rats subjected to trauma (200 r) exhibited significant cardiomyocyte apoptosis and decreased cardiac function; these trends were reversed by pretreatment with quercetin (20 mg/kg i.p.; 0.5 h before trauma), strongly supporting the hypothesis that quercetin can effectively protect against MT-induced cardiomyocyte apoptosis and alleviate secondary cardiac dysfunction. The dose for quercetin (20 mg/kg) was established based on available articles[@b25][@b26]. The levels of quercetin in plasma following intraperitoneal administration (50 mg/kg) in Sprague--Dawley rats were detected by Mauro Piantelli *et al*.[@b29] in a published article. As described in their study, the highest plasma concentrations were found at the earliest sampling time, approximately 15 min after dosing. The maximal concentration is (0.081 + 0.34) μg/ml, which is much less than the minimum toxic dose detected in our study (40 μM/L approximately equalling 12 μg/ml). Thus, it is safe to use quercetin at the concentration of 20 mg/kg (\<50 mg/kg). As shown in [Fig. 4](#f4){ref-type="fig"}, to determine the most appropriate concentration of quercetin *in vitro*, 10 μM, 20 μM, 40 μM, or 80 μM quercetin was added to H9c2 cells; the cells were analysed using an MTT assay, which proved that quercetin decreased cell viability at the concentrations of 40 μM and 80 μM but not at 10 μM and 20 μM. The reason for this cytotoxic effect has never been elucidated. It has been proven that, owing to its specific planar chemical structure, quercetin readily forms chelates with metal ions and that these complexes of bioactive compounds and metal ions, such as lanthanum, often show cytotoxic properties in human cervical carcinoma cells and induce dose-dependent pro-oxidative effects[@b30]. Another study demonstrated that quercetin may act as a cytotoxic pro-oxidant after its metabolic activation to semiquinone and quinoidal products[@b31]. Our previous study showed that some antioxidants, such as procyanidin and curcumin, exhibit toxicity when cultured with H9c2 cells individually, resulting in excessive clearance of ROS in H9c2 cells. These results are in accordance with the idea that ROS are useful for the existence and proliferation of cells[@b32]. *In vitro*, H9c2 cells were cultivated with TP to reflect the pathophysiology of trauma *in vivo*. It is shown in [Figs 4](#f4){ref-type="fig"} and [5](#f5){ref-type="fig"} that 10 μM quercetin had no cytotoxicity with or without NP and could reverse TP-induced cell viability decreases to the maximum extent compared with 2.5, 5, or 20 μM, indicating that 10 μM quercetin can be used as a protective dose. We have previously proven that TNF-α produced by injured peripheral tissues is the main reason for the overproduction of the intracellular ROS that initiate cardiomyocyte apoptosis following non-lethal mechanical trauma[@b14]. Given our previous study and the anti-inflammatory and anti-oxidative properties of quercetin, TNF-α in blood and ROS in H9c2 cells were detected with or without quercetin. As shown in [Figs 6](#f6){ref-type="fig"} and [7](#f7){ref-type="fig"}, TNF-α in blood and ROS in H9c2 cells were enhanced by TP and were decreased by pretreatment with quercetin. Several studies have demonstrated that quercetin inhibits lipid peroxidation effectively by scavenging free radicals and/or chelating transition metal ions[@b33], results similar to those of our study, indicating that the anti-oxidative effect of quercetin may contribute to the protective effect on MT-induced cardiomyocyte apoptosis. It has been reported that intracellular free Ca^2+^ is essentially involved in the mechanism of apoptosis[@b28] and that Ca^2+^ and ROS release are two cross-talk events that are important in TNF-α-mediated apoptosis[@b34]. As shown in [Fig. 8](#f8){ref-type="fig"}, \[Ca^2+^\]~i~ increased after TP treatment and decreased after pretreatment with quercetin, indicating that quercetin may attenuate the overproduction of \[Ca^2+^\]~i~ to inhibit cardiomyocyte apoptosis. In the experiments with calcium overload in H9c2 cells, quercetin was added 2 min prior to their exposition to TP. In our previous study, quercetin added 30 min, 15 min, 5 min, and 2 min in advance was tested and showed a similar effect on calcium overload. Thus, we chose the shortest time as the standard. In conclusion, our study demonstrated for the first time that quercetin can attenuate cardiomyocyte apoptosis and improve cardiac dysfunction induced by MT. Its anti-inflammatory, anti-oxidative and Ca^2+^ scavenging properties may contribute to this protective effect. Given its extensive distribution and the high incidence of nonlethal mechanical trauma, quercetin may become a promising agent for preventing posttraumatic cardiac injury in the future. Methods ======= Materials --------- Quercetin (purity \>98%) was purchased from Melone Pharmaceutical Co., Ltd. (Dalian, China). Dulbecco's modified Eagle medium (DMEM) was purchased from Gibco BRL Co., Ltd. (Grand Island, NY, USA). H9c2 cells were obtained from American Type Culture Collection (ATCC,Manassas, VA, USA; CRL-1446). The DCFH-DA ROS Detection Kit was purchased from Beyotime Institute of Biotechnology (Nanjing, China). The TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) apoptosis detection kit was purchased from Roche (Shanghai, China). 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), RPMI-1640 media, 10% heat-inactivated foetal bovine serum (FBS), Fluo-4/Am and all other chemicals were purchased from Sigma (USA). The TNF-α assay kit was purchased from Sangon (Shanghai, China). The modified Noble-Collip drum was obtained from the Department of Physiology, Dalian Medical University. The BL-420 biological and functional experimental system and pressure sensors were purchased from Taimeng Sciences and Technology Limited (Chengdu, China). The BX51 fluorescence microscope was purchased from Olympus Co. (Japan). The microplate reader was purchased from BioTek (VT, USA). The SP8 laser confocal microscope was purchased from Leica Co. (USA). Ethics statement ---------------- This study conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health and the Guide for the Care and Use of Laboratory Animals' protocol published by the Ministry of the People's Republic of China (issued 3 June, 2004) and was approved by the Institutional Animal Care and Use Committee of Dalian Medical University. The methods were carried out in accordance with the approved guidelines. Animals and groups ------------------ A total of 120 healthy and clean adult male Sprague Dawley (SD) rats (body weight: 210 ± 20 g, from Dalian Medical University) were fed a standard laboratory diet and water ad lib. and were maintained at 22 °C under a constant 12-hour light-dark cycle. The animals were acclimated to their surroundings for 3 days before experimentation and were divided into four groups equally according to the random indicator method (30 in each): sham group, trauma group, trauma + quercetin group (quercetin and DMSO at the ratio of 10 mg to 1 ml, 20 mg/kg ip; 0.5 h before MT), and trauma + vehicle group (DMSO, the same volume as above ip; 0.5 h before MT). Nonlethal traumatic rat model ----------------------------- Noble-Collip drum exposure is a well-accepted traumatic model that results in whole-body nonpenetrative mechanical trauma. Briefly, after anesthetization with 10% chloral hydrate (300 mg/kg i.p.), the rats were placed in a Noble-Collip drum and were subjected to 5-min rotations (200 rotations at a rate of 40 r/min). The traumatic rats (for trauma, trauma + quercetin, trauma + vehicle groups) were injured when the wheel was rotated, while the sham trauma rats (for sham group) were taped on the inner wall of the drum to avoid traumatic injury. The nonlethal MT rat models used in this Noble-Collip drum experiment were characterized by the lack of circulatory shock, no direct cardiac injury, and a 100% 24 h survival rate. Determination of cardiomyocyte apoptosis by TUNEL staining ---------------------------------------------------------- The hearts of traumatized rats were isolated 12 h after trauma for each group (5--7 in each). To determine cardiomyocyte apoptosis in a quantitative manner, the hearts were perfused first with 0.9% NaCl for 5 min and then with 4% paraformaldehyde in PBS (pH 7.4) for 20 min. Four longitudinal sections from the free wall of the left ventricle were cut and further fixed in 4% paraformaldehyde in PBS for 24 h at room temperature. Fixed tissues were embedded in a paraffin block, and two slides at 4- to 5-μm thickness were cut from each tissue block. Immunohistochemical procedures for detecting apoptotic cardiomyocytes were performed using an apoptosis detection kit according to the manufacturer's instructions. After rinsing with PBS, the slides were coverslipped with mounting medium containing DAPI to permit total nuclei counting. Using a ×20 objective, the tissue slide was digitally photographed using a QICAM-Fast Digital Camera mounted onto an Olympus BX51 fluorescence microscope. The total nuclei (blue) and TUNEL-positive nuclei (green) in each field were counted in five randomly chosen fields. The index of apoptosis (number of TUNEL-positive nuclei/total number of nuclei ×100) was calculated and exported to Microsoft Excel for further analysis. Assessments of cardiac function *in vivo* ----------------------------------------- At 12 h after trauma, a thin cannula was inserted into the left ventricle of the rats (anesthetized by pentobarbital sodium, 40 mg/kg) through the right common carotid and was then connected to a pressure transducer for the measurement of left ventricular pressure (LVP). With the pressure transducer connected to a biological mechanic experiment system (BL-420), left ventricular peak systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP = LVSP -- LVEDP), peak rates of rise in the first derivative of the left ventricular pressure (+*dp*/*dtmax*), and peak rates of fall in the first derivative of the left ventricular pressure (−*dp*/*dtmax*) were continuously monitored on a recorder. Preparation of plasma --------------------- Blood was harvested from rats 1.5 h after trauma. The rats were deeply anesthetized with chloral hydrate throughout the process. Cardiac puncture into the apex cordis was used to collect blood into Eppendorf tubes containing 1% heparin. The tubes were then centrifuged (3000 r, 20 min, 4 °C), and the supernatant (TP) was collected. NP was obtained from the rats without trauma in the same way as above. TP and NP were both stored at −80 °C. Cultivation of H9c2 cells ------------------------- H9c2 cells were cultured in high-glucose DMEM supplemented with 10% foetal bovine serum, 4 mM L-glutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycin at 37 °C in a humidified chamber of 95% air and 5% CO~2~ atmosphere. Cell culture media were changed every 2--3 days, and the cells were sub-cultured once they reached 70--80% confluence. Cells between passages 7 and 13 were used in the experiments. Most appropriate concentration of quercetin detected by MTT ----------------------------------------------------------- A 100-μl H9c2 cell suspension was loaded into each well of a 96-well plate and was cultured in the logarithmic growth phase until the cell concentration reached 5 × 10^7^/L. The cells were then treated with quercetin at a series of diluted concentrations (80, 40, 20, or 10 μM, respectively), except the cells in the blank group (without vehicle) and control group (with vehicle) for 24 h. Before harvesting, the cells were incubated with 20 μl of MTT in medium for 4 h at 37 °C. The viable cells converted the MTT to a blue-purple colour after dissolving in 150 μl of dimethyl sulfoxide (DMSO). The absorbance at 570 nm was measured using a microplate reader. All experiments were carried out in triplicate. Cell viability (%) = \[(absorbance of cells treated with quercetin)/absorbance of control cells\] ×100%. Protective effect of quercetin on H9c2 cells as detected by MTT --------------------------------------------------------------- H9c2 cells were prepared in the same way until the concentration reached 5 × 10^7^/L. The cells were divided into a control group, NP group (pretreatment with NP, 50% V/V), TP group (pretreatment with TP, 50% V/V), TP + 2.5 μM quercetin group, TP + 5 μM quercetin group, TP + 10 μM quercetin group and TP + 20 μM quercetin group (pretreatment with quercetin, 2.5/5/10/20 μM; TP added 24 h later). We added the NP + 10 μM quercetin group after 10 μM was proven to be the best concentration for protecting cells from TP-induced viability decrease. After processing as described, all of the cells were cultured for 3 h before the MTT assay was performed as above. Determination of circulating TNF-α level ---------------------------------------- To determine the circulating TNF-α level of the sham group, trauma group, trauma + quercetin group and trauma + vehicle group quantitatively, we used prepared animal serum and the TNF-α assay kit for rats. The blank, standard and specimen samples were set in triplicate. After adding the samples following the instruction manual, the plates were closed with the membrane and incubated for 60 min at 37 °C. Next, the plates were washed 5 times, the chromogenic agent was administered, and then the plates were incubated for another 5--15 min. Finally, after adding stop solution, we acquired the absorbance at 450 nm to calculate the results. Intracellular ROS measurement ----------------------------- H9c2 cells (5 × 10^7^/L, in logarithmic growth phase) were divided into five groups: a control group, NP group, TP group, NP + quercetin group (pretreatment with quercetin, 10 μM; NP added 24 h later), TP + quercetin group (pretreatment with quercetin, 10 μM; TP added 24 h later) and TP + vehicle group (pretreatment with DMSO, the same volume; TP added 24 h later). Then, all of the cells were incubated for 3 h. The culture fluids were subsequently removed, and intracellular ROS generation was measured as previously described[@b35]. The intracellular ROS accumulation of H9c2 cells was measured by 2′,7′-dichlorofluorescin diacetate (DCFH-DA)[@b36] using an intracellular ROS assay kit. Fluorescence intensity was measured by a laser scanning confocal microscope using an excitation of 488 nm and an emission of 525 nm (at a detection spectrum of 488 nm). Measurement of intracellular calcium concentration (\[Ca^2+^\]~i~) ------------------------------------------------------------------ H9c2 cells were washed three times and were incubated for 1 h with Ca^2+^ solution. Fluo-4 acetoxymethyl ester (Fluo-4/AM, 2 μl)[@b37] was added to the cells 30 min before the Ca^2+^ solution was removed. The cells were then cultivated in a Ca^2+^ solution containing BSA for 30 min. Thereafter, quercetin (10 μM) was added to the NP + quercetin group and TP + quercetin group, DMSO was added to the TP + vehicle group, and PBS was added to the NP group and TP group at the same volume. All cells were cultured for 30 min at 37 °C in a 5% CO~2~ atmosphere in the dark. Fluorescence intensity (images analysed with Image-Pro Plus 6.0 software, recorded as F~0~) was measured by laser scanning confocal microscopy using an excitation of 488 nm and an emission of 525 nm. Two minutes later, NP and TP (50% V/V) were added respectively and the fluorescence intensity (recorded as F) was measured. The final fluorescence intensity was recorded as F/F~0~. Statistical analysis -------------------- Data are presented as means ± standard deviations (means ± SDs). Statistical comparisons were performed using one-way analysis of variance (ANOVA, GraphPad Software, USA). Probabilities of 0.05 or less were considered to be statistically significant. Additional Information ====================== **How to cite this article**: Jing, Z. *et al*. Protective Effect of Quercetin on Posttraumatic Cardiac Injury. *Sci. Rep.* **6**, 30812; doi: 10.1038/srep30812 (2016). The authors would like to thank Mr. Shuo Ma, Dalian Medical University, for his assistance with the trauma model preparation. **Author Contributions** Z.J. performed the modelling and comparisons and helped write the manuscript; Z.W. performed the *in vivo* measurements and quantification of the *in vivo* data and helped write the manuscript; X.L. performed the reprogramming of the origin analyses and helped write the manuscript; X.L. prepared the data and helped write the manuscript; T.C. performed the *in vitro* measurements; Y.B. performed the *in vitro* measurements; J.Z. generated data and helped write the manuscript; X.C. advised on model building and helped write the manuscript; D.Y. devised comparisons and helped write the manuscript; L.Z. devised the experiments, performed the *in vivo* measurements, and helped write the manuscript; S.L. was responsible for the overall study coordination, devised the experiments, and wrote the manuscript. {#f1} {#f2} {#f3} {#f4} {#f5} {#f6} {#f7} {#f8} [^1]: These authors contributed equally to this work.

INTRODUCTION {#sec1-1} ============ Oocyte retrieval is a key step in assisted reproduction, the transvaginal route being the current preferred method. The quality of the oocyte so collected is an important determinant of the overall embryo quality and post embryo-transfer outcomes. Apart from the inherent characteristics of the oocyte, its quality is affected by the actual process of oocyte retrieval; including the expertise of the aspirating doctor, the type of equipment and the techniques used. The aspirating pressure may affect the integrity of the oocytes aspirated. It is generally postulated that increasing the vacuum aspiration pressure might decrease the quality of oocytes retrieved.\[[@ref1]\] In cases of anticipated difficult oocyte retrievals the practice is to flush and aspirate 2--4 times maintaining the standard pressure.\[[@ref1][@ref2]\] This can causes loss of ovarian granulosa cells that may result in subsequent corpus luteal insufficiency. The Cochrane review in 2010, followed by the meta-analysis in 2012\[[@ref3][@ref4]\] concluded that follicular flushing in unselected women undergoing *in-vitro* fertilization (IVF) did not affect the oocyte yield or pregnancy outcomes. It, in fact, increased the operating time marginally and increased the analgesic requirement. More recently, a small study\[[@ref5]\] showed that even in poor responders the method of follicular flushing does not improve the oocyte yield. There is a lack of studies looking at the effect of aspiration pressure on oocyte quality and pregnancy outcomes. A few studies have quoted using aspirating pressures ranging between 150 mmHg and 200 mmHg\[[@ref6]\] or occasionally, as low as 80 mmHg.\[[@ref5]\] We routinely used aspirating pressures of 120 mmHg but like many others in this field found that oocyte yield was less in women with low antral follicle count (AFC) (≤10). Hence, we decided to try oocyte retrieval at a slightly higher aspiration pressure of 140-mmHg in women with low follicular counts. We analyzed 3-year data from our center to compare the outcomes following the three methods of oocyte retrieval--aspirating at the standard negative pressure of 120-mmHg, higher negative aspiration pressure of 140-mmHg, and flushing and aspiration at 120-mmHg. Aim(s) and objectives {#sec2-1} --------------------- To compare oocyte retrieval outcomes using three methods of aspiration: Direct aspiration pressure of 120-mmHg in those with normal AFCDirect aspiration pressure at 140-mmHg in those with low AFCAspiration pressure of 120-mmHg with flushing in those with low AFC. The AFC in both the ovaries together of ≥10 was considered normal and anything less was low. MATERIALS AND METHODS {#sec1-2} ===================== This retrospective study was conducted at the assisted reproduction center in our Tertiary Care Hospital. We included data from IVF records of 172 women who underwent oocyte retrieval and IVF/intracytoplasmic sperm injection in our center over a period of 3 years from May 2010 to June 2013. The data were divided into three groups as described previously. Group A consisted of 96 women with normal AFC who underwent oocyte retrieval with negative aspiration pressure of 120-mmHg, Group B consisted of 41 women with low AFC who had oocyte retrieval using negative pressure of 140-mmHg, and Group C consisted of 35 women with low AFC in whom oocyte retrieval was done after flushing if initial aspiration at 120-mmHg did not yield oocytes. Women who had oocyte retrieval with 120-mmHg from one ovary and 140-mmHg from the other ovary were excluded from the study. The groups were comparable with respect to semen parameters and other co-morbidities like endometriosis. They underwent stimulation with antagonist protocol. The number of oocytes retrieved, the oocyte yield (the number of oocytes retrieved divided by follicles aspirated), and the fertilization rate (the number of embryos developed divided by number of eggs retrieved) for each patient was calculated.\[[@ref7]\] The oocyte and embryo quality were graded according to the Istanbul consensus criteria \[[Table 1](#T1){ref-type="table"}\].\[[@ref8]\] Serum beta-human chorionic gonadotropin (bHCG) level was done 2 weeks after embryo transfer. Those who had a bHCG level ≥30 mIU/mL but showed no evidence of pregnancy on ultrasound scan were termed biochemical pregnancies. The rest were clinical pregnancies. Pregnancy losses at \<24 weeks were termed miscarriages. Those that delivered live after 24 weeks were termed live births. Ethical Committee Clearance was obtained prior to the study. ###### Consensus scoring system for cleavage-stage embryos\[[@ref8]\]  Analysis {#sec2-2} -------- Statistical analysis was done by Statistical package for the social sciences version 16 (SPSS, Chicago, USA). Data was analyzed by applying Chi-square tests, and *P* \< 0.05 was considered significant. RESULTS {#sec1-3} ======= As shown in [Table 2](#T2){ref-type="table"}, a higher mean number of oocytes and embryos were noted in Group A, compared to Groups B or C (*P* \< 0.001). As Group A had a higher AFC to begin with, we analyzed the oocyte yield (number of oocytes divided by number of follicles tapped in each patient) and the embryo yield (number of embryos formed divided by the number of oocytes retrieved per patient) to enable comparison between the three groups \[[Table 3](#T3){ref-type="table"}\]. No statistically significant difference was noted between the oocyte yield in Groups A and B (*P* = 0.404) while that in Group C was comparatively less. The mean embryo yield in all three groups was comparable, but the maximum yield was in Group B. ###### Mean parameters from each group  ###### Oocyte and embryo yield  The pregnancy rates and outcomes are depicted in [Table 4](#T4){ref-type="table"} and [Figure 1](#F1){ref-type="fig"}. The number of biochemical pregnancies in all the three groups was small and did not differ much. The clinical pregnancies in Group A (35.4%) and Group B (39%) were significantly higher than the 17.1% in Group C. Of these clinical pregnancies, 76% in Group A and 69% in Group B resulted in live births as against 50% in Group C. Considering the original number of women in each group, the live birth rates were comparable in Group A (27%) and B (26.8%) but significantly less in Group C (8.57%). The miscarriages in Group A and B were 21 and 31%, respectively as against the 50% in Group C. ###### Pregnancy rates  {#F1} DISCUSSION {#sec1-4} ========== Aspiration of oocyte cumulus complex is the defining step in assisted reproduction.\[[@ref5]\] Transvaginal oocyte recovery is the least invasive, least painful, most accurate, and simple method to collect oocytes.\[[@ref9][@ref10]\] Apart from the inherent characteristics of the oocyte, its quality is affected by the techniques used for oocyte retrieval. The aspirating pressure used for oocyte retrieval can affect the integrity of the oocytes.\[[@ref11]\] Initially, manual syringe aspiration was used for oocyte pick up. Maintaining a steady aspiration pressure below 120-mmHg was difficult with this technique. Manual syringing has consequently been replaced by electronic aspiration pumps, which can maintain a steady aspiration pressure.\[[@ref10]\] Morphologically abnormal oocytes have been seen more at higher aspiration pressures particularly around 180-mmHg, which were frequently used during laparoscopic oocyte retrieval.\[[@ref12][@ref13][@ref14][@ref15][@ref16]\] Aspiration pressures between 90 mmHg and 120 mmHg have been associated with good oocyte yield and minimal damage.\[[@ref1][@ref2][@ref8]\] Few published studies that have assessed the effect of higher aspiration pressure for oocyte retrieval in humans.\[[@ref10][@ref11][@ref17][@ref18]\] Higher than standard pressures have been used for aspiration of immature human oocytes for *in-vitro* maturation. A study done in this field noted that aspiration pressures of more than 180-mmHg cause immature oocyte damage and poor embryogenesis.\[[@ref10]\] However, there is no literature citing the effects of slightly higher aspiration pressure of 140-mmHg on oocytes. Moreover, many recent studies have shown that most morphologic abnormalities especially the fractured zona pellucida that is attributed to higher than standard aspiration pressure does not significantly affect favorable assisted reproduction technology (ART) outcomes.\[[@ref19][@ref20]\] A review published in 2011 also stated that the predictive potential of oocyte morphology in IVF outcome is not clear at present.\[[@ref19]\] In women with low ovarian reserve (AFC \< 10), the standard aspirating pressure of 120-mmHg often yields very less oocytes.\[[@ref1][@ref2]\] In these women flushing the follicles with tubal fluid and aspiration was believed to improve the oocyte yield and thereby the ART outcome.\[[@ref21][@ref22]\] In fact, a study published in 2005 found that flushing up to four times was optimal in maximizing the oocyte recovery rate per follicle tapped.\[[@ref22]\] However, the 2010 Cochrane review on flushing, found no added benefit in using flushing compared to direct aspiration. Flushing and aspiration lengthened the oocyte retrieval time.\[[@ref4]\] Flushing was also believed to increase loss of granulosa cells causing luteal phase defects and miscarriages.\[[@ref23][@ref24]\] This study showed that the oocyte yield, quality of oocytes retrieved, as well as pregnancy rate (39%) following direct oocyte aspiration at 140-mmHg, was comparable to that with the standard aspiration pressure of 120-mmHg (34.4%); whereas flushing and oocyte retrieval resulted in only 17.1% clinical pregnancies, significantly less compared to the other groups. Oocyte quality was determined based on nuclear maturation. Morphological differences in oocytes retrieved were not seen among the groups on analysis by our embryologists. Cleavage stage embryo assessment was done either on day 2 or 3. These were done as per the Istanbul consensus criteria as mentioned earlier.\[[@ref8]\] No significant difference in the morphology of oocytes retrieved was noted between the three groups. Nevertheless, the oocyte and embryo yields were more in the direct aspiration groups compared to the flushing group. The clinical pregnancy rate in the flushing group was a dismal 17.1% compared to 39% in the 140 mmHg group \[[Table 1](#T1){ref-type="table"} and [Figure 1](#F1){ref-type="fig"}\]. The flushing group also had a high miscarriage rate (50%). On the other hand, pregnancy outcomes in groups undergoing oocyte aspiration at 120 and 140-mmHg were comparable, even though, the latter group included women with low AFC. The abortion rate of 31% was higher in the 140-mmHg compared to the 21% in the 120-mmHg group, but the difference was not statistically significant \[[Figure 1](#F1){ref-type="fig"}\]. Loss of granulosa cells results in poor ovarian hormonal support in the luteal phase, as well as the first trimester of pregnancy, which might explain the poor pregnancy rate and increased abortions in the flushing group.\[[@ref25]\] The present study showed that increasing oocyte pickup pressure to 140-mmHg did not adversely affect the oocyte yield, embryo yield, and quality or pregnancy outcome compared to the standard aspiration pressure of 120-mmHg, even though it was used in women with low AFC. This increased pickup pressure seemed logical since it was practically feasible and gave hope to a segment of patients where the oocyte retrieval was difficult with the stipulated standard pressure of 120-mmHg. Moreover, oocyte aspiration pressure of 140-mmHg gave promising retrieval rates and pregnancy outcomes in women with poor AFC, where the alternative practice of flushing and aspiration showed dismal outcome. Assisted reproduction technology outcome in women with low AFCs was expected to be poorer compared to those with the good follicular count. However, in our study the ART outcome using direct aspiration at 140-mmHg in these women was comparable to that in women with normal AFC. Thus direct oocyte aspiration at negative pressure of 140-mmHg was found to be a good alternative to flushing and aspiration in women with poor AFC as flushing and aspiration was associated with significantly lesser pregnancy rates and more early miscarriages. Our study is the only study to our knowledge comparing two specific aspiration pressures and comparing them with follicular flushing. The results open newer horizons for safe and productive oocyte aspiration for women with low AFC. The limitation of our study is that, it is retrospective and confined to one center only. Nevertheless, the study model may be used to conduct large multicentric prospective trials to arrive at a definitive recommendation creating a new trend for ART in women with low AFC. CONCLUSION {#sec1-5} ========== Direct oocyte retrieval using higher aspiration pressure of 140-mmHg resulted in better oocyte yield and pregnancy outcomes compared to flushing and aspiration in women with poor AFCs. This innovation also showed ART outcomes comparable to those in women with normal ovarian reserve. Hence, it offers a safe and promising alternative to flushing in women with low AFCs. We would like to acknowledge Professors Dr. Lavanya Rai and Dr. Muralidhar V Pai Additional Professor Dr. Jyothi Shetty and Associate Professor Dr. Sapna Vinit Amin, Department of OBG, Kasturba Medical College, Manipal University, Manipal for providing generous academic insight for our study. **Source of Support:** Nil **Conflict of Interest:** The authors of this publication are affiliated with Department of OBG, Kasturba Medical College, Manipal University, Manipal. The terms of this arrangement have been reviewed and approved by the Manipal University, Manipal, in accordance with its policy on objectivity in research.

Cancer Sci 107 (2016) 155--161 **Funding Information** Ministry of Education, Culture, Sports, Science and Technology of Japan; Japan Society for the Promotion of Science; Japan Agency for Medical Research and Development; Princess Takamatsu Cancer Research Fund. Lung cancer has long been the leading cause of cancer death and lung adenocarcinoma is the most prevalent among the four major histologic types. We previously reported that *NKX2‐1/TTF‐1*, an indispensable transcription factor for peripheral lung development and physiological functions, plays a role as a lineage‐survival oncogene in lung adenocarcinoma.[1](#cas12858-bib-0001){ref-type="ref"} In addition, we have shown that neuroendocrine lung cancer cells, such as small‐cell lung cancer, have dependence on lineage‐survival signaling conferred by continued expression of ASH1, a transcription factor indispensable for pulmonary neuroendocrine cell development.[2](#cas12858-bib-0002){ref-type="ref"}, [3](#cas12858-bib-0003){ref-type="ref"}, [4](#cas12858-bib-0004){ref-type="ref"} Together, those findings suggested that a "lineage‐specific addiction" to signaling programmed for development of normal progenitor cells[5](#cas12858-bib-0005){ref-type="ref"} is involved in the molecular pathogenesis of lung cancer.[6](#cas12858-bib-0006){ref-type="ref"} The importance of *NKX2‐1/TTF‐1* in lung adenocarcinoma development was further substantiated by subsequent identification of the NKX2‐1/TTF‐1 locus as a genomic region with frequent focal amplification.[7](#cas12858-bib-0007){ref-type="ref"}, [8](#cas12858-bib-0008){ref-type="ref"}, [9](#cas12858-bib-0009){ref-type="ref"} Notably, we identified the receptor tyrosine kinase‐like orphan receptor 1 (ROR1) as a direct transcriptional target of NKX2‐1/TTF‐1, which is crucial for maintaining a favorable balance between pro‐survival phosphatidylinositol 3‐kinase--protein kinase B (PI3K‐AKT) and pro‐apoptotic apoptosis signal‐regulating kinase 1 (ASK1)‐p38MAPK signaling for lung adenocarcinoma survival.[10](#cas12858-bib-0010){ref-type="ref"} Mechanistic insight into how ROR1 sustains epidermal growth factor receptor (EGFR)‐mediated PI3K‐AKT signaling in both kinase‐dependent and ‐independent manners has been well presented. In contrast, how ROR1 inhibits pro‐apoptotic signaling remains elusive. A member of the MAPKKK family, ASK1 is activated by various stimuli such as oxidative and endoplasmic reticulum stress, as well as by tumor necrosis factor‐ and Fas‐elicited signals. In turn, ASK1 activates the MAPKK 4 (MKK4)‐JNK and MKK3/6‐p38 pathways, leading to induction of cellular apoptosis.[11](#cas12858-bib-0011){ref-type="ref"} ASK1 knockout mice were shown to be resistant to oxidative stress‐induced cell death,[12](#cas12858-bib-0012){ref-type="ref"} suggesting the functional importance of ASK1 for coping with cellular stress induced by reactive oxygen species (ROS). To date, some proteins have been identified as negatively regulating ASK1 activity through their interactions, including TRX,[13](#cas12858-bib-0013){ref-type="ref"} 14‐3‐3,[14](#cas12858-bib-0014){ref-type="ref"} GSTM1‐1,[15](#cas12858-bib-0015){ref-type="ref"} Raf1,[16](#cas12858-bib-0016){ref-type="ref"} CIIA,[17](#cas12858-bib-0017){ref-type="ref"} and CIB1.[18](#cas12858-bib-0018){ref-type="ref"} In this study, we attempted to gain insight into the mechanism of ROR1 repression of pro‐apoptotic signaling to the ASK1--p38 axis. Our findings showed that ROR1 inhibits ASK1 activity in an ROR1‐kinase dependent manner through physical interaction at the C‐terminal serine threonine‐rich domain of ROR1. Materials and Methods {#cas12858-sec-0002} ===================== Cell lines {#cas12858-sec-0003} ---------- NCI‐H1975 and NCI‐H441 cells were purchased from ATCC (Manassas, VA, USA), and PC‐9 cells from RIKEN Cell Bank (Tsukuba, Japan). The SK‐LC‐5 cell line was a generous gift from the late Lloyd J. Old (Memorial Sloan‐Kettering Cancer Center, New York, NY, USA). All cell lines were authenticated by short tandem repeat DNA profiling and confirmed to be free from mycoplasma contamination. A summary of NKX2‐1/TTF‐1 and ROR1 expression is provided in Table S1. Generations of wild‐type or kinase‐dead ROR1‐expressing MSTO‐211H cells were produced using a previously described technique.[10](#cas12858-bib-0010){ref-type="ref"} All cells were maintained in RPMI‐1640 with 10% FBS. Antibodies {#cas12858-sec-0004} ---------- Recombinant MKK6 was obtained from Millipore (Watford, UK). Anti‐ROR1, anti‐ASK1, anti‐phospho‐ASK1 (T845), anti‐phospho‐ASK1 (S83), anti‐MKK3/6, anti‐phospho‐MKK3/6 (S189/S207), anti‐p38, anti‐phospho‐p38 (T180/Y182), anti‐mouse IgG, and anti‐rabbit IgG were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti‐c‐myc came from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti‐GST and anti‐maltose‐binding protein (MBP) from MBL (Nagoya, Japan), anti‐ROR1 and anti‐goat IgG from R&D Systems (Minneapolis, MN, USA), anti‐JNK and anti‐phospho‐JNK (T183/Y185) from BD Biosciences (Bedford, MA, USA), and anti‐α‐tubulin from Sigma (St. Louis, MO, USA). RNA interference {#cas12858-sec-0005} ---------------- The following RNA oligomers were obtained from Qiagen (Hilden, Germany) and Sigma‐Aldrich (Dorset, UK), and used for RNA interference: siROR1 \#1, 5′‐CAGCAAUGGAUGGAAUUUCAA‐3′; siASK1 \#1, 5′‐GGUAUACAUGAGUGGAAUU‐3′; and siASK1 \#2, 5′‐CACUAUUGGAUGUUCUCUA‐3′. AllStars Negative Control siRNA (siControl) was also obtained from Qiagen. Transfection of siRNAs (each at 20 nM) was carried out using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA), according to the manufacturer\'s instructions. Cells were harvested at 72 h after transfection and subjected to Western blotting (WB). Cell proliferation was determined 5 days after transfection by colorimetric assays with a Cell‐Counting kit‐8 (Dojindo Laboratories, Kumamoto, Japan). Constructs {#cas12858-sec-0006} ---------- Construction of expression vectors of full‐length human ROR1 cDNA (pCMVpuro‐ROR1) as well as its various derivatives was carried out using a previously described method.[10](#cas12858-bib-0010){ref-type="ref"} Full‐length human ASK1 cDNA was purchased from RIKEN and inserted into a pCMV‐puro expression vector. The entire ASK1 ORF of the resultant construct (pCMVpuro‐ASK1) was thoroughly sequenced. In addition, a myc‐tagged derivative of pCMVpuro‐ASK1 (pCMVpuro‐ASK1‐myc) was also constructed by *in vitro* mutagenesis using KOD‐plus‐DNA polymerase (Toyobo, Osaka, Japan) and the following oligonucleotides: ASK1‐myc‐F, 5′‐TCCGAAGAGGACCTGTGACTGTTGCTCAATCTAATCTTC‐3′; and ASK1‐myc‐R, 5′‐GATCAGCTTCTGCTCAGTCTGTTTGTTTCGAAAGTCAAT‐3′. Preparation of recombinant proteins {#cas12858-sec-0007} ----------------------------------- The GST‐tagged ROR1 (intracellular domain) was expressed in Sf9 insect cells using a Gateway system (Invitrogen) and purified by glutathione‐affinity chromatography, as previously described.[10](#cas12858-bib-0010){ref-type="ref"} An *Escherichia coli*‐expressing protein of MBP‐tagged kinase‐dead MKK6 was purchased from Millipore. Western blotting and immunoprecipitation--WB analyses {#cas12858-sec-0008} ----------------------------------------------------- Western blotting and immunoprecipitation (IP)‐WB analyses were carried out according to standard procedures using Immobilon‐P filters (Millipore) and an Amersham ECL Western Blotting Detection Reagent (GE Healthcare, Amersham, UK). For analysis of physical interactions between ROR1 and ASK1, pCMVpuro‐ASK1 was transfected with various ROR1 expression constructs, including wild‐type, ∆tyrosine kinase domain 1 (∆TK1), ∆TK2, ∆TK3, ∆ST1, ∆P, and ∆ST2. Cells were harvested 24 h after transfection with NP‐40 lysis buffer containing 20 mM Tris--HCl (pH 8.0), 137 mM NaCl, 2 mM EDTA, 1% NP‐40, 10% glycerol, and 1 mM NA~3~VO~4~. Analysis of responses to ROS‐inducing conditions {#cas12858-sec-0009} ------------------------------------------------ Wild‐type and kinase‐dead ROR1‐expressing MSTO‐211H cells were plated into 6‐well plates. After 24 h, the cells were harvested for WB analysis after treatment with or without 10 mM hydrogen peroxide (H~2~O~2~) for 15 min. Colorimetric assays were also carried out to determine the influence on cell proliferation using a Cell‐Counting kit‐8 (Dojindo), following prolonged incubation in the presence of 100 μM H~2~O~2~ for 24 h. Oxidative stress‐generating ROS was also produced by treating cells with 100 μM tert‐butyl hydroperoxide (TBHP; Molecular Probes, Paisley, UK) for 120 min, after which the cells were harvested for WB analysis. *In vitro* kinase assays {#cas12858-sec-0010} ------------------------ The 293T cells were transiently transfected with pCMVpuro‐ASK1‐myc, then myc‐tagged ASK1 was purified using a c‐Myc tagged Protein Mild Purification Kit (version 2; MBL). ASK1‐myc and MBP‐MKK6 cells were co‐incubated at 37°C for 1 h with GST or GST‐ROR1‐WT in phosphorylation buffer (25 mM Tris--HCl \[pH 7.5\], 5 mM MgCl~2~, 0.1 mM ATP), and subjected to SDS‐PAGE, followed by WB analysis with a specific antibody. Flow cytometric analysis {#cas12858-sec-0011} ------------------------ Cell apoptosis was measured by flow cytometry after propidium iodide staining. Briefly, cells were treated with 100 μM H~2~O~2~ for 24 h, then harvested by trypsinization. All cells were collected and fixed with 70% ethanol and 30% PBS at −20°C, followed by treatment with RNase and propidium iodide (Sigma‐Aldrich), essentially as previously described.[4](#cas12858-bib-0004){ref-type="ref"} Using a FACSCalibur system, 1 × 10^4^ cells were analyzed and sub‐G~1~ cells were quantitated with CellQuest Pro software (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Results {#cas12858-sec-0012} ======= Knockdown of ASK1 alleviates siROR1‐elicited growth inhibition {#cas12858-sec-0013} -------------------------------------------------------------- We examined the effects of co‐treatment with siASK1 and siROR1 in the lung adenocarcinoma cell lines PC‐9 and NCI‐H1975, which readily express both ROR1 and ASK1. Co‐knockdown of ASK1 significantly repressed siROR1‐induced phosphorylation of both MKK3/6 and p38 but not JNK in PC‐9 and NCI‐H1975 cells (Fig. [1](#cas12858-fig-0001){ref-type="fig"}). Co‐knockdown of ASK1 also significantly alleviated siROR1‐induced growth inhibition in PC‐9 cells (Fig. [1](#cas12858-fig-0001){ref-type="fig"}a), while similar but more modest effects were also observed in NCI‐H1975 cells (Fig. [1](#cas12858-fig-0001){ref-type="fig"}b, as well as Fig. S1a,b with siASK1 \#2). These findings suggested that ASK1‐mediated signaling to the MKK3/6--p38 axis is involved, at least in part, in siROR1‐elicited growth inhibition in lung adenocarcinoma cells. {#cas12858-fig-0001} Repression of ROS‐induced ASK1 phosphorylation requires ROR1 {#cas12858-sec-0014} ------------------------------------------------------------ In order to investigate whether ROR1 is involved in regulation of the ASK1‐p38 MAPK signaling pathway, we first compared steady state levels of phosphorylation of ASK1 and p38 in MSTO‐211H cells, which had been stably transfected with either a wt‐ROR1 expression construct or an empty vector. Significant decreases in autophosphorylation of ASK1 at Thr845 and p38 phosphorylation at T180 and Y182 were observed in ROR1‐transfected MSTO‐211H cells, suggesting ROR1‐mediated negative regulation of the ASK1--p38 axis (Fig. [2](#cas12858-fig-0002){ref-type="fig"}a). Phosphorylation of ASK1 and p38 was also analyzed by WB analysis in the presence or absence of H~2~O~2~. Consequently, we observed marked repression of oxidative stress‐induced, activation‐reflecting phosphorylation of ASK1 (T845) and p38 (F180/Y182) in MSTO‐211H cells overexpressing ROR1 (Fig. [2](#cas12858-fig-0002){ref-type="fig"}b). It was also noted that ROR1 overexpression significantly maintained ASK1 phosphorylation at serine 83, which is known to be phosphorylated by AKT and inhibits ASK1 function,[19](#cas12858-bib-0019){ref-type="ref"} even in the presence of hydrogen peroxide. Similar results were obtained with another oxidative stress inducer, TBHP (Fig. [2](#cas12858-fig-0002){ref-type="fig"}c). An *in vitro* kinase assay was also carried out using cellular ASK1 prepared from ASK1‐transfected 293T cells as well as recombinant MBP‐tagged kinase‐deficient MKK6. While readily detectable autophosphorylation of ASK1 was found along with phosphorylation of MKK6, which is a substrate of ASK1 and directly upstream of p38, co‐incubation of ASK1 with recombinant GST‐tagged ROR1 clearly diminished ASK1 autophosphorylation and MKK6 phosphorylation (Fig. [3](#cas12858-fig-0003){ref-type="fig"}). Taken together, these findings indicated that ROR1 negatively regulates pro‐apoptotic signaling of the ASK1--MKK6--p38 axis. {#cas12858-fig-0002} {#cas12858-fig-0003} Physical interaction between ROR1 and ASK1 {#cas12858-sec-0015} ------------------------------------------ We next investigated whether ROR1 physically interacts with ASK1. 293T cells were co‐transfected with ROR1 and ASK1, then subjected to IP‐WB analysis. ASK1 was co‐immunoprecipitated with an anti‐ROR1 antibody, while an anti‐ASK1 antibody was conversely co‐immunoprecipitated with ROR1 (Fig. [4](#cas12858-fig-0004){ref-type="fig"}a). An association of transfected ROR1 and endogenous ASK1 was also observed in MSTO‐211H cells transfected with ROR1 (Fig. [4](#cas12858-fig-0004){ref-type="fig"}b). Interactions between endogenous ROR1 and ASK1 proteins were further analyzed by IP‐WB analysis using four different lung adenocarcinoma cell lines, which clearly demonstrated their physical associations (Fig. [4](#cas12858-fig-0004){ref-type="fig"}c for PC‐9 and NCI‐H1975; Fig. S2 for NCI‐H441 and SK‐LC‐5). We also found a consistent association between ASK1 and ROR1 regardless of the presence of H~2~O~2~ (Fig. [4](#cas12858-fig-0004){ref-type="fig"}d). {#cas12858-fig-0004} We then carried out detailed mapping of the ASK1 binding site of the ROR1 protein. Various deletion mutants of ROR1 were co‐transfected with ASK1 into COS‐7 cells (Fig. [5](#cas12858-fig-0005){ref-type="fig"}a), followed by IP‐WB analysis of their interactions. We found that the interaction between ROR1 and ASK1 required the presence of the C‐terminal serine/threonine‐rich domain of ROR1 (Fig. [5](#cas12858-fig-0005){ref-type="fig"}b). These results showed that ROR1 interacts with ASK1 at the C‐terminus in both steady state and ROS‐inducing conditions. {#cas12858-fig-0005} Kinase activity of ROR1 is required for inhibition of ASK1 phosphorylation {#cas12858-sec-0016} -------------------------------------------------------------------------- Next, we examined whether the kinase activity of ROR1 is required for inhibition of ASK1 activity. MSTO‐211H cells, which had been stably transfected with either a wild‐type ROR1 (ROR1‐WT) or a kinase‐dead (ROR1‐KD) mutant, were treated with H~2~O~2~ and subjected to WB analysis. In contrast to ROR1‐WT, ROR1‐KD failed to significantly repress H~2~O~2~‐induced ASK1 phosphorylation as well as consequential p38 phosphorylation (Fig. [6](#cas12858-fig-0006){ref-type="fig"}a). In addition, we noted that H~2~O~2~‐induced cell growth inhibition was partly rescued by overexpression of ROR1‐WT but not by that of ROR1‐KD in MSTO‐211H cells (Fig. [6](#cas12858-fig-0006){ref-type="fig"}b). Consistent with these findings, flow cytometric analysis revealed a significant reduction in sub‐G~1~ cells among MSTO‐211H cells overexpressing ROR1‐WT but not ROR1‐KD (Fig. [6](#cas12858-fig-0006){ref-type="fig"}c). Together, the present findings showed that ROR1 physically interacts with ASK1 and that ROR1 kinase activity is required to reduce that activation of ASK1, leading to repression of pro‐apoptotic ASK1‐p38 signaling in lung adenocarcinoma cells. {#cas12858-fig-0006} Discussion {#cas12858-sec-0017} ========== We previously reported that ROR1 sustains a favorable balance between pro‐survival and pro‐apoptotic signaling, thus playing a role as a key downstream molecule of the *NKX2‐1/TTF‐1* lineage‐survival oncogene.[6](#cas12858-bib-0006){ref-type="ref"} In contrast to significant advances in understanding of the molecular mechanisms related to ROR1 pro‐survival signaling, how ROR1 represses pro‐apoptotic signaling remains elusive. The present findings clearly show that ROR1 is involved in negative regulation of the pro‐apoptotic ASK1--p38 axis through its physical association with ASK1. Overexpression of ROR1 inhibited activation of ASK1 in response to oxidative stress in an ROR1 kinase‐dependent manner. Intriguingly, ASK1 phosphorylation was also repressed in a steady state conditions without exogenous ROS‐inducing stress, suggesting the existence of ROR1‐mediated repression of ASK1 activation in response to intrinsic cellular stresses. Thus, our results suggest that ROR1 may be part of an important cellular machinery for repression of irrelevant ASK1--p38 activation. Activity of ASK1 is known to be tightly regulated by phosphorylation, as it has been observed at a representative Thr845 residue within the activation loop of the kinase domain,[20](#cas12858-bib-0020){ref-type="ref"} while there are additional residues phosphorylated by various kinases, including Ser83,[21](#cas12858-bib-0021){ref-type="ref"} Ser967,[22](#cas12858-bib-0022){ref-type="ref"} Ser1034,[23](#cas12858-bib-0023){ref-type="ref"} and Tyr718.[24](#cas12858-bib-0024){ref-type="ref"} Nevertheless, it remains to be elucidated which amino acid(s) of ASK1 among its more than 40 possible phosphorylation sites is affected either directly or indirectly by ROR1. As ASK1 is also known to be both positively[25](#cas12858-bib-0025){ref-type="ref"}, [26](#cas12858-bib-0026){ref-type="ref"}, [27](#cas12858-bib-0027){ref-type="ref"} and negatively[13](#cas12858-bib-0013){ref-type="ref"}, [14](#cas12858-bib-0014){ref-type="ref"}, [15](#cas12858-bib-0015){ref-type="ref"}, [16](#cas12858-bib-0016){ref-type="ref"}, [17](#cas12858-bib-0017){ref-type="ref"}, [18](#cas12858-bib-0018){ref-type="ref"} regulated through interaction with various other proteins, it is also possible that ASK1 activation may be inhibited by ROR1 by its effects on such ASK1‐interacting proteins, or a yet unidentified one. The *NKX2‐1/TTF‐1* oncogene is crucially involved in maintenance of physiological functions in normal lungs, thus it cannot be considered as a therapeutic target, even though its genetic aberrations are among the most frequent in lung adenocarcinoma.[6](#cas12858-bib-0006){ref-type="ref"} ROR1 is a crucial mediator of lineage‐survival signaling of NKX2‐1/TTF‐1 and ROR1 inhibition represses lung cancer cells, which even include EGFR tyrosine kinase inhibitor‐resistant ones due to the presence of T790M EGFR, MET amplification, and/or hepatocyte growth factor overexpression.[10](#cas12858-bib-0010){ref-type="ref"} Thus, ROR1 appears to be a promising molecular target for a future novel therapeutic method to treat this devastating cancer. As the present study indicates that ROR1 counteracts oxidative stress‐induced ASK1 activation and apoptosis, enhanced treatment efficacy of a ROS‐inducing chemotherapeutic agent when combined with ROR1 inhibition is anticipated. In addition to lung adenocarcinoma development, ROR1 has been suggested to be involved in the pathogenesis of various other human cancers, such as ovarian, colon, lung, skin, pancreatic, testicular, bladder, uterus, prostate, gastric cancers, melanoma, and lymphomas.[28](#cas12858-bib-0028){ref-type="ref"}, [29](#cas12858-bib-0029){ref-type="ref"}, [30](#cas12858-bib-0030){ref-type="ref"}, [31](#cas12858-bib-0031){ref-type="ref"}, [32](#cas12858-bib-0032){ref-type="ref"}, [33](#cas12858-bib-0033){ref-type="ref"}, [34](#cas12858-bib-0034){ref-type="ref"} In addition, onco‐fetal expression of ROR1 is another attractive feature.[10](#cas12858-bib-0010){ref-type="ref"}, [28](#cas12858-bib-0028){ref-type="ref"}, [29](#cas12858-bib-0029){ref-type="ref"} In conclusion, ROR1 directly interacts with ASK1, repressing ASK1 phosphorylation and resultant ASK1‐p38 pro‐apoptotic signaling. A future study is awaited for development of a therapeutic means to inhibit this attractive molecular target. It would also be interesting to investigate whether ROR1 has a role as a mediator of outside‐in signaling to the ASK1--p38 axis. Disclosure Statement {#cas12858-sec-0019} ==================== The authors have no conflict of interest. Supporting information ====================== ###### **Fig. S1.** Receptor tyrosine kinase‐like orphan receptor 1 (ROR1) interacts with apoptosis signal‐regulating kinase 1 (ASK1). Effects of co‐treatment with siASK1 and siROR1 in (a) PC‐9 and (b) NCI‐H1975 cells using siASK1 \#2. Colorimetric analyses were carried out at 5 days after co‐transfection. Data are shown as the mean ± SEM (*n* = 3). \**P* \< 0.05. ###### Click here for additional data file. ###### **Fig. S2.** Receptor tyrosine kinase‐like orphan receptor 1 (ROR1) interacts with apoptosis signal‐regulating kinase 1 (ASK1). Immunoprecipitation--Western blot analysis of endogenous ROR1 and ASK1 using lysates of NCI‐H441 and SK‐LC‐5 cells. ###### Click here for additional data file. ###### **Table S1.** Endogenous expression of NKX2‐1/TTF‐1 and receptor tyrosine kinase‐like orphan receptor 1 (ROR1) in cell lines used in this study. ###### Click here for additional data file. This work was supported in part by a Grant‐in‐Aid for Scientific Research on Innovative Areas from the Ministry of Education, Culture, Sports, Science and Technology of Japan; Grants‐in‐Aid for Scientific Research (A) and (C) from the Japan Society for the Promotion of Science; the Program for Development of Innovative Research on Cancer Therapeutics (P‐Direct) from the Japan Agency for Medical Research and Development; and a grant from the Princess Takamatsu Cancer Research Fund.

Introduction {#Sec1} ============ Multiple sclerosis (MS) is an autoimmune inflammatory demyelinating disease, pathologically characterized by perivascular CD4^+^ T cells and monocytes inflammation, resulting in axonal demyelination and transection^[@CR1]--[@CR3]^. Current therapy of MS such as fingolimod, glatiramer acetate mainly depends on non-specific suppression of the immune system to delay the progression^[@CR4]^ and reduce the frequency and severity of disease relapse periods. However, they cannot protect myelin from future damage^[@CR5],[@CR6]^. Thus, more effective pharmacological strategies for MS to reduce inflammation and diminish myelin damage in injured CNS is urgently needed. In recent years, it has become clear that macrophages play a very important role in the pathogenesis of MS^[@CR7]^. The function of macrophages in experimental autoimmune encephalomyelitis (EAE), a widely used animal model of MS^[@CR8]^, is often opposite^[@CR9]^. M1 cells are classically activated macrophages, generally exhibit proinflammatory activity, whereas M2 cells are alternatively activated macrophages, predominantly inhibit immune responses^[@CR10]^. M1 and M2 macrophages co-exist in vivo and present plastic properties regulated by members of the STAT family in the peripheral lymph system and CNS during the disease progress. STAT1/NF-κB axis promotes IFNs and Toll-like receptor (TLR) to skew macrophage function toward the M1 phenotype, whereas STAT3/STAT6 signaling pathway activated by IL4, IL10, or IL13 induces M2 macrophages phenotype^[@CR11]^. The distribution of M1 macrophages in CNS lesion causes oligodendrocyte precursor cells death^[@CR12]^ while M2 macrophages induce pro-repair molecules such as brain-derived neurotropic factor (BDNF), IL10, and ferritin increasing the maturation of oligodendrocyte precursor cells^[@CR13],[@CR14]^. Moreover, M2 macrophages remove inhibitory debris or toxic products enabling subsequent remyelination^[@CR15]^. Macrophages interactions with CD4^+^ T cells in demyelinating lesions have been suggested to play a critical role in modifying the pathobiology of MS^[@CR16]^. As professional antigen-presenting cells (APCs), M1 macrophages prime naive CD4^+^ T cells and initiate auto-reactive immune response in CNS^[@CR17]^, releasing many inflammatory mediators such as interleukin-1 (IL-1)^[@CR18]^, tumor necrosis factor-alpha (TNF-α)^[@CR19]^, and NO^[@CR20]^, that are critical for EAE development^[@CR21]^. However, M2 macrophages suppress CD4^+^ T cells activity in demyelinated CNS. Indeed, transferring of microglia- or monocyte-derived M2 macrophages could reduce the severity of clinical signs of EAE by inhibiting T cell proliferation^[@CR22]^. The transition from M1 to M2 macrophages has been reported to be crucial for preserving myelin integrity^[@CR12]^. Thus, the modulations of molecules associated with macrophage plasticity and polarized activation are critical to modulate CNS inflammation and tissue repair. Here, we identified that lenalidomide, an oral drug approved by FDA for the treatment of myelodysplastic syndromes and multiple myeloma^[@CR23],[@CR24]^, markedly promoted the M2 polarization of macrophages alone thus mediated immunosuppressive and neuroprotective effect in EAE. Moreover, lenalidomide induced M2 macrophages polarization mainly through expression and autocrine secretion of IL10 and subsequent activation of STAT3. Results {#Sec2} ======= Lenalidomide alone directly induces M2 phenotype in macrophages {#Sec3} --------------------------------------------------------------- Recent studies have revealed that the balance between activation and polarization of M1 and M2 macrophages is important for the progression of EAE. Polarized macrophages can be modulated by pharmacological intervention. Therefore, we employed qRT-PCR experiment screening a variety of compounds/drugs, including anti-metabolites (pemetrexed and gemcitabine), anti-histamine drugs (loratadine and promethazine), 5-HT receptor antagonists (azasetron and palonosetron), immunomodulators (lenalidomide and pomalidomide), and novel compounds (referred to as C1--C2) in bone marrow-derived macrophages (BMDMs) in order to find a compound/drug, which selectively induces M2 phenotype or inhibits M1 phenotype. We found that the upregulation of *Arg1* and *Mrc1* mRNA level induced by lenalidomide, about 27.3 and 6.7 times, is closer to that induced by IL13, while pomalidomide upregulated the mRNA level of *Arg1* and *Mrc1* only by 18.6 and 3.9 times, respectively (Supplementary Figure [S1a, b](#MOESM1){ref-type="media"}). Those compounds/drugs could not markedly inhibit the mRNA level of *Inos* and *Cxcl10* induced by LPS stimulation (Supplementary Figure [S1c, d](#MOESM1){ref-type="media"}). The further qRT-PCR analysis demonstrated lenalidomide significantly increased the mRNA level of M2 macrophages-related genes of *Ym*1, Ar*g1* and *Mrc1* in RAW264.7 (monocyte-derived macrophage cell line), as well as in BMDMs (Fig. [1a, b](#Fig1){ref-type="fig"} and Supplementary Figure [S2a](#MOESM1){ref-type="media"}). And western blotting analysis showed that lenalidomide enhanced the expression of Arg1 and Ym1 in macrophages (Fig. [1c, d](#Fig1){ref-type="fig"}). Moreover, flow cytometry analysis indicated that lenalidomide treatment significantly increased CD206^+^ M2 cells (Fig. [1e, f](#Fig1){ref-type="fig"}). Nevertheless, lenalidomide did not inhibit LPS-induced M1 polarization in BMDMs (Fig. [1g, h](#Fig1){ref-type="fig"} and Supplementary Figure [S2b](#MOESM1){ref-type="media"}). Taken together, our results clearly demonstrate that lenalidomide alone could obviously increase M2 macrophages polarization.Fig. 1Lenalidomide promotes M2 macrophages polarization.**a**,**b** qRT-PCR analysis for expression of macrophage M2 phenotype genes in RAW264.7 (**a**) and BMDMs (**b**) with lenalidomide treatment for 4 h, IL13 (10 ng/ml) was used as positive control (*n* = 3). **c**, **d** Western blotting analysis for expression of Arg1 and Ym1 in RAW264.7 (**c**) and BMDMs (**d**) with lenalidomide treatment for 4 h. GAPDH or β-actin was the loading control (*n* = 3). **e** Flow cytometry analysis of the percentage of M2 phenotype (CD206^+^ cells) in RAW264.7 cells with lenalidomide (25 nM) treatment for 4 h. IL13 (10 ng/ml) was used as positive control (*n* = 3). **f** Flow cytometry analysis of the percentage of M2 phenotype (CD206^+^F4/80^+^ cells) in BMDMs with lenalidomide (25 nM) treatment for 4 h (*n* = 3). Cells were gated at F4/80^+^ cells. **g**, **h** BMDMs were pretreated with LPS (50 ng/ml) for 24 h and then administrated with lenalidomide (25 nM) for additional 4 h. qRT-PCR was carried out to analyze mRNA level of macrophage M1 phenotype genes (*n* = 3) (**g**). Flow cytometry analysis was carried out to characterize the percentage of M1 phenotype (CD86^+^F4/80^+^ cells), cells were gated at F4/80^+^ cells (*n* = 3) (**h**). Data are presented as means ± SEM; *\*P* \< 0.05, *\*\*P* \< 0.01, *\*\*\*P* \< 0.001 versus untreated control Lenalidomide ameliorates EAE symptom and reduces demyelination {#Sec4} -------------------------------------------------------------- Given that M2 macrophages have effect on inflammation resolution and tissue repair in EAE^[@CR25]^, we tested whether lenalidomide might be able to ameliorate the development of autoimmune diseases. Therefore, C57BL/6 mice were immunized with MOG~35--55~ peptide and treated daily with lenalidomide after onset of clinical symptoms. Expectedly, lenalidomide ameliorated the disease severity from the early stage and lasted until the end of experiment, whereas vehicle-treated mice showed continued disease progression (Fig. [2a, b](#Fig2){ref-type="fig"}). Moreover, inflammatory foci areas in spinal cord of lenalidomide-treated mice were reduced compared with vehicle-treated mice (Fig. [2c](#Fig2){ref-type="fig"}, left). The inflammatory cells will attack the myelin to cause demyelination^[@CR26]^, we next detect the myelin-forming cells and myelin integrity. Immunofluorescence analysis showed that the number of Olig2^+^ cells and Olig2^+^CC1^+^ cells in the spinal cords white matter of lenalidomide-treated mice compared with EAE control mice were significantly increased (Fig. [2d--f](#Fig2){ref-type="fig"}). Consistently, LFB analysis also indicated a decreased demyelination in the spinal cord of lenalidomide recipients in contrast to vehicle-treated EAE mice (Fig. [2c](#Fig2){ref-type="fig"}, right). Moreover, vehicle-treated EAE mice showed extensive demyelination compared to lenalidomide-treated EAE mice, with unraveling of the myelin sheaths and loss of axon (Fig. [2g](#Fig2){ref-type="fig"}). g-ratios (the ratio between the diameter of the axon to myelinated axon diameter) was used to quantify myelin thickness. Lenalidomide markedly decreased the g-ratios from 0.82 to 0.73 (Fig. [2h](#Fig2){ref-type="fig"}). These results indicate that lenalidomide could alleviate the symptoms of EAE mainly through decreased inflammatory cells infiltration in CNS and strong attenuating typical demyelination.Fig. 2Lenalidomide ameliorates EAE progression.**a**, **b** Wild-type (WT) mice were immunized with MOG~35--55~ peptide and treated with lenalidomide (30 mg/kg, i.g.), dexamethasone (10 mg/kg, i.g.), or vehicle (0.9% CMC-Na, i.g.) at indicated time points (arrows). Mean clinical score (**a**) and loss of body weight (**b**) are shown (*n* = 15 per group). **c** H&E and Luxolfast blue (LFB) staining of mouse spinal cords from vehicle- and lenalidomide-treated WT EAE mice at day 16. Arrowheads indicate leukocyte infiltration area in H&E staining. Outlines indicate demyelination in LFB staining. Scale bars: 100 μm. **d** Immunostaining of mouse spinal cords from vehicle- and lenalidomide-treated WT EAE mice at day 16 using antibodies against Olig2 (red) and CC1 (green). Scale bars: 50 μm. **e** Quantification of Olig2^+^ cells density in spinal cords in **d**. **f** Quantification of the percentage of Olig2^+^CC1^+^ cells among Olig2^+^ cells from spinal cords in **d**. **g** Representative electron micrographs of spinal cords from vehicle- and lenalidomide-treated WT EAE mice at day 16. Scale bars: 2 μm. **h** g-ratios of axons in spinal cords from vehicle- and lenalidomide-treated WT EAE mice at day 16 (*n* \> 100). Data are presented as means ± SEM; *\*P* \< 0.05, *\*\*P* \< 0.01, *\*\*\*P* \< 0.001 versus vehicle-treated controls Lenalidomide suppresses proinflammatory Th1 and Th17 cells responses {#Sec5} -------------------------------------------------------------------- T cells and B cells contribute to autoimmune CNS inflammation and demyelination^[@CR27]^, we therefore performed flow cytometry analysis to evaluated possible regulatory immune responses by lenalidomide in vivo. The experiments data demonstrated that lenalidomide treatment dramatically reduced IFN-γ^+^ and IL17^+^ CD4^+^ T cells in draining lymph node (DLN) and spleen (Fig. [3a, b](#Fig3){ref-type="fig"}), but had no effect on Foxp3^+^CD4^+^ T and B220^+^ B cells (Supplementary Figure [S3a--d](#MOESM1){ref-type="media"}), suggesting that lenalidomide injection inhibited pathogenic Th1 and Th17 cells activity in vivo. To verify the modulated effect of lenalidomide on immune cells infiltration into CNS, we separated and quantified mononuclear cells (MNCs) from the whole spinal cord and brain at the peak phase of disease. In contrast to vehicle-treated EAE mice, the number of CNS-infiltrating MNCs in lenalidomide-treated EAE mice were reduced by 2.5 times (Fig. [3c](#Fig3){ref-type="fig"}). Similarly, flow cytometry analysis observed that Th1 and Th17 cells penetrated into CNS were threefold reduction in lenalidomide-injected mice (Fig. [3d](#Fig3){ref-type="fig"}). Taken together, we speculate that lenalidomide inhibits effector T cells polarization in spleen and DLN, which induces the reduction of inflammatory Th1 and Th17 cells penetrating into the CNS.Fig. 3Lenalidomide suppresses autoimmune responses in EAE.**a**, **b** Flow cytometry analysis of Th1 and Th17 cells in spleen (**a**) and DLN (**b**) from vehicle- and lenalidomide-treated WT EAE mice at day 17. Representative fluorescence activated cell sorting (FACS) plots (left) and statistics from six mice per group (right) are shown; cells are gated for CD4^+^ T cells. **c** The total numbers of MNCs in whole spinal cord and brain were isolated from vehicle- and lenalidomide-treated mice on day 17 (*n* = 6). **d** Flow cytometry analysis of Th1 and Th17 cells in CNS-infiltrating MNCs from vehicle- and lenalidomide-treated WT EAE mice at day 17. Representative FACS plots (left) and statistics from six mice per group (right) are shown; cells are gated for CD4^+^ T cells. Data are presented as means ± SEM; *\*P* \< 0.05, *\*\*P* \< 0.01, *\*\*\*P* \< 0.001 versus vehicle-treated controls Lenalidomide-attenuated EAE is dependent on polarized M2 macrophages {#Sec6} -------------------------------------------------------------------- To analyze whether macrophages related to the beneficial effect of lenalidomide in EAE, macrophages in spleen and spinal cord were selectively depleted using the "suicide technique" by application of clodronate liposomes (c-lipo). After administrating c-lipo, EAE mice could not be protected from disease progression upon lenalidomide treatment, particularly obvious at day 15 post immunization, clearly indicating that lenalidomide ameliorates CNS autoimmunity depending on the effect of macrophages (Fig. [4a](#Fig4){ref-type="fig"}). This hypothesis was further strengthened by the observation of macrophages subtypes in EAE mice. Reduction of disease severity in lenalidomide-treated mice was in paralleled with increased numbers of F4/80^+^CD206^+^ M2 cells both in spleen and DLN compared to vehicle-treated mice, however, the percentage of F4/80^+^CD86^+^ M1 cells remained unchanged (Fig. [4b, c](#Fig4){ref-type="fig"}). In addition, the frequency of activated CD11b^+^CD45^hi^CD206^+^ M2 macrophages in spinal cords was increased from 3.94 to 8.36% after treatment with lenalidomide (Fig. [4d](#Fig4){ref-type="fig"}). Based on our findings so far, we next transferred lenalidomide-pretreated BMDMs (Supplementary Figure S[4a, b](#MOESM1){ref-type="media"}) into EAE mice every 5 days from the day of the appearance of clinical symptoms (Fig. [4e](#Fig4){ref-type="fig"}). After injection, adoptive transferred mice with M2 macrophages had lower clinical scores (Fig. [4f](#Fig4){ref-type="fig"}). The previous data showed that lenalidomide had effect on both CD4^+^ T cells and M2 macrophages in EAE mice, it is still unsure whether lenalidomide directly affects the function of myelin-specific CD4^+^ T cells or indirectly by regulating the function of macrophages. Indeed, we observed that CD4^+^ T cells were insensitivity to lenalidomide at a concentration of M2 cells polarization. However, higher concentration of lenalidomide (2 μM) augmented IFN-γ^+^CD4^+^ T cells (Supplementary Figure [S5a, b](#MOESM1){ref-type="media"}), which is consistent with the previous studies^[@CR28]^. Moreover, MOG-induced reaction of CD4^+^ T cells in vitro was not inhibited by lenalidomide (Supplementary Figure [S5c](#MOESM1){ref-type="media"}), indicating that the therapeutic effect of lenalidomide on EAE mice was dependent on M2 macrophages polarization rather than direct inhibition of CD4^+^ T cells. Thus, we addressed whether lenalidomide-induced M2 macrophages have the capacity to inhibit myelin-specific CD4^+^ T cells, the result showed that MOG peptide-induced proliferation of CD4^+^ T cells was significantly inhibited when presented macrophages stimulated with lenalidomide (Fig. [4g](#Fig4){ref-type="fig"}). These results demonstrate that lenalidomide indirectly altering the proinflammatory activity of myelin-specific CD4^+^ T cells mainly through promoting the polarization of M2 macrophages.Fig. 4The protective effect of lenalidomide on EAE is dependent on M2 phenotype.**a** WT mice were immunized with MOG~35--55~ and treated with lenalidomide (30 mg/kg, i.g.) or vehicle (0.9% CMC-Na, i.g.) combined with empty or clodronate liposome (50 mg/kg, i.v.) at indicated time points (bottom arrows represent lenalidomide or vehicle treatment, upper arrows represent liposome treatment). Mean clinical score is shown (*n* = 15 per group). **b**, **c** Flow cytometry analysis of M2 and M1 macrophages in spleen (**b**) and DLN (**c**) from vehicle- and lenalidomide-treated WT EAE mice at day 17. Representative FACS plots (left) and statistics from six mice per group (right) are shown; cells are gated for F4/80^+^ cells. **d** Flow cytometry analysis of M2 macrophages in whole spinal cord and brain. CNS-infiltrating MNCs isolated from vehicle- and lenalidomide-treated WT EAE mice at day 17 stained for antibodies, including CD11b, CD206, and CD45. CD11b^+^CD45^hi^CD206^+^ cells were M2 macrophages. Representative FACS plots (left) and statistics from six mice per group (right) are shown. **e** WT mice were immunized with MOG~35--55~ on day 0 and 3 × 10^6^ BMDMs treated with or without 25 nM lenalidomide for 4 h were injected intravenously into these mice on day 9, 14, and 19 (*n* = 5 per group). **f** The mean clinical score of mice in **e**. **g** BMDMs were treated with or without 25 nM lenalidomide for 4 h. Splenic CD4^+^ T cells were isolated from MOG-treated WT mice, labeled with1 μM CFSE and subsequently cocultured with BMDMs at a ratio of 1:4 supplemented with MOG~35--55~ peptide (20 μg/ml) for 72 h. The proliferation of CD4^+^ T cells was confirmed by flow cytometry analysis based on CFSE dilution. Left panel represents flowcytometric dot plot of CFSE-labeled CD4^+^ T cells, right panel shows the percentage of CD4^+^ T cells that have proliferated based on CFSE dilution (*n* = 3 per group). Data are presented as means ± SEM; \**P* \< 0.05, \*\**P* \< 0.01, \*\*\**P* \< 0.001 Macrophages-derived IL10 is essential for the therapeutic effect of lenalidomide on EAE {#Sec7} --------------------------------------------------------------------------------------- M2 macrophages secrete anti-inflammation cytokines including IL4, IL10, IL13, and TGF-β^[@CR10]^. Based on this, we then tested whether lenalidomide was able to upregulate these factors. Expectedly, BMDMs stimulated with lenalidomide, confirmed by qRT-PCR analysis, led to a significant increase of *Il4*, *Il10*, *Il13*, and *Tgf-β* mRNA level, especially, *Il10 mRNA* increased about 25 times (Fig. [5a](#Fig5){ref-type="fig"}). Consistently, lenalidomide markedly increased IL10 production in serum, spleen, and spinal cord from EAE mice harvested at day 17 (Fig. [5b, c](#Fig5){ref-type="fig"}). IL10 can be produced in response to proinflammatory signals by virtually all immune cells, including T cells, B cells, and macrophages^[@CR29]--[@CR31]^. We conducted IHC and flow cytometry analysis to validate the source of IL10 in the presence of lenalidomide, the data revealed that the majority of IL10^+^ cells are also CD68^+^ macrophages (Fig. [5d--g](#Fig5){ref-type="fig"}), but not CD4^+^ T cells or CD8^+^ T cells (Supplementary Figure [S6a--d](#MOESM1){ref-type="media"}), suggesting that IL10 was mainly produced by M2 macrophages. We next detected IL10's role in lenalidomide-induced macrophages M2 polarization. In contrast to higher protein levels of Ym1 in IL10-sufficient BMDMs induced by lenalidomide, IL10-deficient BMDMs showed remarkably decreased Ym1 protein (Fig. [5h](#Fig5){ref-type="fig"}). To clearly distinguish whether the protective effect of lenalidomide was indeed mediated by IL10 in vivo, WT and IL10-deficient (IL10^−/−^) mice were immunized and treated with lenalidomide. Expectedly, IL10^−/−^ EAE mice treated with lenalidomide had higher mean clinical scores and more severe demyelination compared to lenalidomide-treated WT mice (Fig. [5i, j](#Fig5){ref-type="fig"}). Then, we used adoptive transfer experiment injecting lenalidomide-treated WT or IL10^−/−^ BMDMs to EAE mice on day 12. Consistent with in vivo data, IL10^−/−^ BMDMs were not able to inhibit the process of disease, while WT-BMDMs transferred mice displayed lower clinical scores (Fig. [5k](#Fig5){ref-type="fig"}). These results confirm that lenalidomide-ameliorated EAE is, at least partially, dependent on IL10-producing M2 macrophages.Fig. 5IL10-producing M2 macrophages play a critical role in lenalidomide treatment.**a** qRT-PCR analysis for expression of *Il4*, *Il10*, *Il13*, and *Tgf-β* in WT BMDMs with 25 nM lenalidomide treatment for 4 h (*n* = 3). **b** Concentration of IL10 in serum from vehicle or lenalidomide-treated WT EAE mice at day 17 (*n* = 6). **c** Concentration of IL10 in spleen and spinal cord from vehicle- or lenalidomide-treated WT EAE mice at day 17 (*n* = 6). **d** Immunostaining of mouse spinal cords from vehicle- and lenalidomide-treated WT EAE mice at day 16 using antibodies against CD68 (green) and IL10 (red). Scale bars: 150 μm (left), 50 μm (right). **e** Quantification of the percentage of CD68^+^ IL10^+^ cells among CD68^+^ cells from spinal cords in **d**. **f**, **g** Flow cytometry analysis of IL10^+^CD206^+^ cells in spleen and DLN from vehicle or lenalidomide-treated WT EAE mice at day 17. Representative FACS plots (**f**) and statistics from six mice per group (**g**) are shown. Cells are gated for CD206^+^ cells. **h** Western blotting analysis for expression of Ym1 in WT or IL10^−/−^ BMDMs with 25 nM lenalidomide treatment for 4 h. GAPDH was the loading control. **i** WT or IL10^−/−^ mice were immunized with MOG~35--55~ and treated with lenalidomide (30 mg/kg, i.g.) or vehicle (0.9% CMC-Na, i.g.) at indicated time points (arrows). Mean clinical score is shown (*n* = 15 per group). **j** MBP and LFB staining of spinal cord from lenalidomide-treated WT or IL10^−/−^ EAE mice in **i** at day 15. Scale bars: 200 μm (upper), 100 μm (bottom). **k** WT mice were immunized with MOG~35--55~ on day 0 and 3 × 10^6^ WT or IL10^−/−^ BMDMs treated with or without 25 nM lenalidomide for 4 h were injected intravenously into these mice at day 12. Mean clinical scores are shown (*n* = 8 per group). Data are presented as means ± SEM; \**P* \< 0.05, \*\**P* \< 0.01, \*\*\**P* \< 0.001 Lenalidomide promotes M2 macrophages polarization via IL10-STAT3-IL10 positive feedback loop {#Sec8} -------------------------------------------------------------------------------------------- Subsequently, we investigated how lenalidomide regulates M2 macrophages polarization. It has been reported that activated STAT6, as well as AKT and STAT3 can shift macrophages from the destructive M1 phenotype toward the beneficial M2 phenotype^[@CR11],[@CR32]^. The western blotting analysis was performed in lenalidomide-treated RAW264.7 and BMDMs. The data showed that lenalidomide did not activate AKT and STAT6 in RAW264.7. However, lenalidomide upregulated STAT3 phosphorylation in a concentration-dependent fashion in both RAW264.7 cells and BMDMs (Fig. [6a](#Fig6){ref-type="fig"}). Moreover, IL10 protein level was upregulated then subsequently activated STAT3 in macrophages after lenalidomide treatment (Fig. [6b](#Fig6){ref-type="fig"}). We also detected that IL10 cytokine secretion began to increase after 4 h lenalidomide treatment (Fig. [6c](#Fig6){ref-type="fig"}). Moreover, pretreatment with recombinant anti-IL10 mAb suppressed Tyr-705 phosphorylation-mediated Ym1 expression. In addition, pretreatment with STAT3 inhibitor Cucurbitacin (CuCu) could partially reverse lenalidomide-stimulated macrophages M2 polarization (Fig. [6d, e](#Fig6){ref-type="fig"}). In vivo study confirmed that lenalidomide obviously increased Arg1^+^ p-STAT3^+^ cells in spleen of EAE mice (Fig. [6f](#Fig6){ref-type="fig"}). Accordingly, lenalidomide significantly increases expression and autocrine secretion of IL10, subsequently activates STAT3-mediated expression of Ym1.Fig. 6Lenalidomide-induced M2 macrophage polarization is dependent on STAT3.**a** Western blotting analysis for expression of STAT3, p-STAT3(Y705), AKT, p-AKT(Y473), STAT6, and p-STAT6 (Y641) in RAW264.7 (upper panel) and BMDMs (lower panel) treated with lenalidomide in different concentration for 4 h (*n* = 3). **b** Western blotting analysis for expression of IL10, p-STAT3 (Y705) in RAW264.7 (upper panel), and BMDMs (lower panel) with 25 nM lenalidomide treatment for a gradient time (*n* = 3). **c** RAW264.7 (upper panel) and BMDMs (lower panel) were cultured in serum-free medium and treated with lenalidomide for different times. Supernanant was cultured at indicated time to test the concentration of IL10 (*n* = 3). **d** RAW264.7 (upper panel) and BMDMs (lower panel) were cultured in serum-free medium, added anti-IL10 antibodies for 12 h and treated with lenalidomide for an additional 4 h. Western blotting analysis for expression of p-STAT3 (Y705) and Ym1. **e** RAW264.7 (upper panel) and BMDMs (lower panel) were pretreated with CuCu for 2 h and then treated with lenalidomide for an additional 4 h. Western blotting analysis for p-STAT3 (Y705) and Ym1. **f** Immunostaining of spleen from vehicle- and lenalidomide-treated EAE mice at day 16 using antibodies against p-STAT3 (Y705) (red) and Arg1 (green). Scale bars: 150 μm. GAPDH and β-actin were the loading control Discussion {#Sec9} ========== Here, we demonstrated that lenalidomide efficiently inhibited prototypical inflammatory demyelinating disease of CNS. Lenalidomide alone significantly activated M2 macrophages and inhibited proinflammatory Th1 and Th17 cells both in the peripheral lymph system and CNS, thus resulting in reduced inflammation and demyelination in injured CNS tissues. At the cellular level, lenalidomide efficiently promoted the expression and autocrine of IL10, subsequently phosphorylated STAT3 Tyr-705, and finally led to the induction of M2 macrophages (Fig. [7](#Fig7){ref-type="fig"}).Fig. 7Schematic representation of lenalidomide-mediated M2 polarization and neuroprotective effect.Lenalidomide is uptaken by macrophages and directly promotes macrophages M2 polarization by IL10-STAT3 signaling pathway. Specifically, lenalidomide effectively increases IL10 expression and subsequently phosphorylates STAT3 Tyr-705. Phosphorylated STAT3 translocated into the nucleus encoding M2 macrophages-related genes such as Arg1, Ym1, and CD206. Anti-inflammatory cytokines secreted by M2 macrophages can reduce the infiltration of peripherally generated autoreactive Th1 and Th17 cells into CNS, inhibit demyelination, and therefore prevent of EAE It has been reported that lenalidomide administration is neuroprotective in a mouse model of amyotrophic lateral sclerosis at symptom onset stage^[@CR33],[@CR34]^, as well as in a transgenic model of Parkinson's disease^[@CR35]^ by decreasing the expression of the proinflammatory cytokines. Here, we found that administration of lenalidomide by gavage prevented EAE from the early stage and lasted until the end of experiment by reducing inflammation and demyelination. However, this observation was inconsistent with previous studies. The paper indicated that intraperitoneal administration of lenalidomide delayed symptom onset of EAE, but did not prevent demyelination^[@CR36]^. We speculated that lenalidomide could not be fully dissolved in 1% carboxymethyl cellulose and 0.9% saline solution according to our formulation preparation (data not shown), lenalidomide by intraperitoneal administration was unable to fully absorbed in the abdominal cavity. Furthermore, the doses of lenalidomide was lower than we used in this paper. Although many cytokines, including IL4, IL13, and IL10, efficiently direct M2 macrophages polarization^[@CR10]^, they are not suitable for clinical use^[@CR37]^. The previous trials that treated EAE mice with systemic IL10 did not show any promising effect and was found, at least in some studies, to aggravate disease^[@CR38]^. A small quantity of compounds, such as pidotimod and azithromycin, could induce the polarization of M2 macrophages in an IL4- or IL13-depedent manner^[@CR39],[@CR40]^; however, azithromycin had no therapeutic effect on EAE mice^[@CR41]^. Here, we found that lenalidomide alone obviously induced M2 phenotype and did not affect M1 phenotype both in RAW264.7 and primary macrophages, however, whether lenalidomide modulates M2 macrophages polarization in human macrophages still need to be studied. Moreover, we observed that depletion of macrophages with c-lipo completely abolished the beneficial effects of lenalidomide on EAE while adoptive transferring of lenalidomide-induced macrophages ameliorated EAE, demonstrating that lenalidomide-alleviated inflammation and demyelination is mainly through promoting M2 macrophages polarization. Macrophages and CD4^+^ T cells both play important roles in the neuropathology of MS, and the interactions between CD4^+^ T helper cell subsets and M1 and M2 macrophages often result in particular functions and outcomes^[@CR16]^. Autoreactive CD4^+^ T cells from peripherally infiltrate into the CNS, where they are restimulated by M1 macrophages, leading to local expansion of pathogenic Th1 and Th17 cells, and subsequently damage to oligodendrocytes^[@CR42]^. Indeed, injection of M2 macrophages into mice with EAE has been shown to reduce the frequency and severity of disease relapse periods by suppressing T cell activity^[@CR22],[@CR43]^. Exposing of T cells to lenalidomide leads to increased IFN-γ and IL-2 secretion^[@CR44]^, however, we found that it did not directly inhibit MOG-loaded CD4^+^ T cells. Moreover, we here provided evidence that lenalidomide-induced M2 macrophages diminished the proliferation ability of MOG-specific CD4^+^ T cells ex vivo. IL10 is an anti-inflammatory cytokine and its primary function is to limit inflammatory responses, which has been well studied in the pathogenesis of several autoimmune diseases including EAE^[@CR45]^. Lenalidomide could reduce the survival support of NLCs for chronic lymphocytic leukemia (CLL) cells in vitro by increasing secretion of IL10^[@CR46]^. Consistently, we showed here that lenalidomide promoted IL10 expression both in vivo and ex vivo in macrophages. IL10 played important roles in lenalidomide therapy as IL10^−/−^ mice could not be protected from actively induced EAE upon lenalidomide treatment. IL10 can be produced in response to proinflammatory signals by virtually all immune cells, including T cells, B cells, and macrophages^[@CR29]--[@CR31]^. Plasmablasts in the DLN serve as IL10 producers to limit autoimmune inflammation^[@CR47]^ and CD4^+^ T cells-induced IL10 was associated with the beneficial effects of Bowman--Birk inhibitor (BBI) on EAE^[@CR48]^. Here, we show that the effect of lenalidomide strictly depended on M2 macrophages-secreted IL10, as adoptive transferring of lenalidomide-activated IL10-sufficient macrophages improved EAE progress, however, IL10^−/−^ macrophages could not rescue EAE clinical scores. These findings emphasize the importance of M2 macrophages as IL10 producer for control of CNS autoimmunity. Up to now, the role of IL10 in macrophages has been clearly characterized and two important signaling axes have been reported to be activated through IL10 receptor ligation in macrophages, the JAK1/STAT3 and PI3K/Akt/GSK3β pathways^[@CR49],[@CR50]^. We found here that lenalidomide-induced M2 macrophages depended on STAT3 activation. IL10/STAT3 axis is successfully studied in M2 macrophages, IL10 binding to IL10R^[@CR51],[@CR52]^ activates the IL10/JAK1/STAT3 cascade, then phosphorylates STAT3 homodimers translocate to the nucleus within seconds to activate the expression of M2 macrophages-related genes^[@CR53]^. STAT3 has pathological implications for MS due to its critical roles in myeloid cell activation, T cell polarization, and cytokine/chemokine induction^[@CR54],[@CR55]^. However, whether hyperactivation of STAT3 in macrophages renders mice resistant to EAE disease need to be further studied. Here, we found that therapeutic administration of lenalidomide significantly controls disease severity, and improves behavior in mice with EAE, suggesting that lenalidomide could be used for the treatment of inflammation-associated degenerative CNS diseases, such as relapsing-remitting MS. Lenalidomide is therefore a promising therapeutic drug candidate for attenuating inflammatory responses and reducing neuronal demyelination in CNS in MS. Materials and methods {#Sec10} ===================== Animals and EAE mice induction {#Sec11} ------------------------------ C57BL/6 mice, 6--8-week old, female, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. IL10^−/−^ mice, 6--8-week old, female, were purchased from Model Animal Research Center of Nanjing University. Those mice were immunized subcutaneously with 400 μg MOG~35--55~ peptide (Sangom Biotech) emulsified in CFA (Sigma) containing 4 mg/ml heat-killed Mycobacterium tuberculosis H37Ra (BD Biosciences) on day 0. In addition, 200 ng pertussis toxin (List Biological Laboratories) in 0.1 ml PBS was administered intravenously on day 0 and day 1 post immunization per mice. Animals were assessed daily for weight loss and clinical signs with a 0--5 point scoring system as follows: 0, healthy; 0.5, tip of tail is partial paralysis; 1, limp tail; 1.5, inhibition of hindlimb; 2, weakness of hindlimb; 2.5 dragging of hindlimb; 3, one hindlimb paralysis; 3.5, complete hindlimb paralysis; 4, one forelimb paralysis; 4.5, complete forelimb paralysis; and 5, severe paralysis or death. Cell culture {#Sec12} ------------ RAW264.7 cells were purchased from Cell Bank of Chinese Academy of Sciences, cultured in DMEM (GIBCO) containing 10% (vol/vol) FBS (GIBCO). BMDMs: bone marrow cells were isolated from femurs and tibias of C57BL/6 mice and cultured in DMEM containing 10% (vol/vol) FBS and 50 ng/ml of M-CSF (PEPROTECH) for 7 days. Purity was determined by flow cytometry. Drugs/compounds screening {#Sec13} ------------------------- Drug/compounds screening for promoting M2 macrophages polarization: BMDMs were seeded in 24-well plate presented with 100 nM drugs/compounds for 24 h. IL13 (10 ng/ml, PEPROTECH) was used as positive control for macrophages M2 polarization. Following qRT-PCR analysis for expression of *Arg1* and *Mrc1*. Drug/compounds screening for inhibiting macrophages M1 polarization: BMDMs were seeded in 24-well plate presented with LPS (50 ng/ml) for 24 h to induce M1 phenotype and then treated with drugs/compounds (100 nM) for additional 24 h. Following qRT-PCR analysis for expression of *Inos* and *Cxcl10*. Compounds and pharmacological treatment {#Sec14} --------------------------------------- For RAW264.7 and BMDMs, lenalidomide was dissolved in DMSO at proper concentration. For EAE mice, vehicle (0.9% CMC-Na, i.g.), dexamethasone (Tianjin Kingyork Group Co., Ltd, 10 mg/kg, i.p.) and lenalidomide (Chia Tai Tianqing Pharmaceutical Group Co., Ltd, 30 mg/kg in 0.9% CMC-Na, i.g.) were daily administrated after the appearance of clinical symptom (average clinical score ≥0.5). To selectively deplete macrophages, clodronate liposome (Encapsula, 50 mg/kg, i.v.) was administrated on day 7, 8, 11, 12, 15, and 16 after immunization. Suppression assays {#Sec15} ------------------ BMDMs were generated as described above and on day 7 of differentiation, cells were treated with or without 25 nM lenalidomide. After 4 h, splenic CD4^+^ T cells were isolated from MOG-treated WT mice via magnetic separation (EasySep Mouse CD4 Isolation Kit, Stem Cell Technologies), labeled with1 μM CFSE (Biolegend) and subsequently cocultured with BMDMs at a ratio of 1:4 in RPMI 1640 (GIBCO) media supplemented with MOG~35--55~ peptide (20 μg/ml) for 72 h. The proliferation of CD4^+^ T cells was assessed by flow cytometry based on CFSE dilution. Adoptive transfer of macrophages {#Sec16} -------------------------------- BMDMs from WT or IL10^−/−^ mice were cultured for 7 days and then treated with DMSO or lenalidomide for 4 h. Cells were collected and resuspended in DMEM without serum. In total, 3 × 10^6^ BMDMs were injected intravenously into EAE mice at specific days depending on the severity of disease. Immunohistology {#Sec17} --------------- Spinal cords and spleen from lenalidomide- or vehicle-treated EAE mice were embedded in OCT (SAKURA Finetek) and spinal cords were cut into 12 μm sections while spleen were cut into 8 μm sections. Immunofluorescence staining was performed and incubated with the appropriate dilutions of primary antibodies against Olig2 (Millipore), CC1 (Oncogene Research), CD68, CD4, CD8 (all purchased from Santa Cruz), IL10 (Abcam), Arg1, p-STAT3(705) (all purchased from Cell Signaling Technology). Subsequently, slides were incubated with Alexa Fluor (AF) 568- or AF488-coupled secondary antibodies (Life Technologies). Nuclei were counterstained with DAPI (Dojindo). For LFB staining, slides were stained with LFB solution (Sigma-Aldrich) overnight, rinsed with distilled water, and differentiated with 0.05% lithium carbonate solution followed by 70% ethanol. Differentiation was stopped by rinsing in distilled water. For HE staining, spinal cords from lenalidomide- or vehicle-treated EAE mice were embedded in paraffin and cut into 3 μm sections, deparaffinized and stained with hemotaxylin and eosin. For MBP IHC, slides were incubated with the appropriate dilutions of primary antibodies against MBP (Covance) and incubated with secondary antibody (Beijing Zhongshan Biotechnology) for 1 h before subjected to DAB staining. Images were taken by Zeiss LSM510 meta fluorescence confocal microscope or Leica DM2500 microscope. Electron microscope {#Sec18} ------------------- Spinal cords from lenalidomide- or vehicle-treated EAE mice were fixed with 2.5% glutaraldehyde for 3 days. The tissues were then PBS washed, fixed in 1% osmium tetroxide for 1 h, and stained with 2% uranyl acetate for 30 min, subsequently dehydrated in graded ethanol series, and embedded in Epon. g-ratio defined as the ratio of the diameter of a given axon and the myelinated fiber diameter. Axon and axon plus myelin units were measured for more than 100 times for each group using ImageJ. Western blotting and ELISA {#Sec19} -------------------------- Protein extracts were subjected to SDS-PAGE (8--12% gels) and blotted onto PVDF membranes. After blocking with 5% fat-free milk, the membranes were incubated with the following antibodies: anti-AKT, anti-p-AKT, anti-Arg-1, anti-GAPDH, anti-β-actin (all purchased from Santa Cruz Biotechnology), anti-STAT6, anti-p-STAT6, anti-STAT3, anti-p-STAT3(705) (all purchased from Cell Signaling Technology), anti-Ym1, anti-IL10 (all purchased from Abcam) at 4 °C overnight. The bound antibodies were detected using horseradish peroxidase (HRP)-conjugated IgG (MULTI Sciences) and visualized with enhanced chemiluminescence (ECL, PerkinElmer) detection reagents (Thermo scientific, USA). β-actin or GAPDH was used as a loading control. IL-10 concentration in plasma, tissues, and cell culture supernatants were determined by ELISA kit (DAKEWE). The tissue ELISA measurements were normalized to the protein content of the homogenates. RNA isolation and qRT-PCR {#Sec20} ------------------------- Total RNA was isolated from cells with Trizol reagent (Invitrogen), cDNA was transcribed using TransScript kit (TransGen Biotech). qRT-PCR analysis was performed using the SYBR Green (Bio-Rad) method on the ABI Fast 7500 real-time PCR instrument (Perkin-Elmer Applied Biosystems). The gene expression was normalized to the expression of the gene encoding GAPDH. Monocytes isolation and flow cytometry analysis {#Sec21} ----------------------------------------------- Single-cell suspensions from spleen and draining lymph nodes (DLN) were obtained by mechanical disruption. For spleen cells, using NH~4~Cl lysis buffer to remove red blood cells. CNS-infiltrating monocytes were isolated from spinal cord and brains as follows. CNS tissues were cut into small pieces and digested in Hank's balanced salt solution (HBSS) containing 20 mM Hepes (Sigma), 0.25% collagenase D (GIBCO), 0.025 U/ml DNaseI (Sigma) at 37 °C for 35 min with brief vortex mixing every 15 min. At the end of digestion, the solution was mixed thoroughly and the cells were washed twice in PBS and suspended in 30% Percoll, overlaid on 70% Percoll and centrifuged for 40 min at 1300 × *g* at 25 °C. Cells at the interface were CNS monocytes. Single-cell suspensions from DLN, spleen, and CNS were incubated for 30 min at 4 °C with fluorochrome-conjugated anti-CD4, anti-CD8, anti-CD45, anti-B220 (all purchased from BD Biosciences), anti-F4/80, anti-CD11b, anti-CD86, and anti-CD206 (all purchased from Biolegend) for staining of surface markers. For intracellular staining of cytokines, the cells were stained with anti-CD4, followed by staining with anti-IFN-γ, anti-IL17A (all purchased from eBioScience) and anti-IL10 (Biolegend) using Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer's protocol. Intracellular staining with anti-Foxp3 (eBioscience) was performed using an Fixation/Permeabilization kit (eBioscience) according to the manufacturer's protocol. Data and statistical analysis {#Sec22} ----------------------------- All data from at least three different experiments. All values were presented as means ± SEM, and statistically significant differences were assessed by one-way ANOVA. A value of *P* \< 0.05 was considered statistically significant. Electronic supplementary material ================================= {#Sec23} supplementary material These authors contributed equally: Qinjie Weng, Jiaying Wang, Jiajia Wang. Edited by T Brunner **Electronic supplementary material** **Supplementary Information** accompanies this paper at (10.1038/s41419-018-0290-x). **Publisher\'s note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. We thank Dr. Richard Lu and Dr. Lie Wang for comments and suggestions. This study was supported by the National Natural Science Foundation of China (No. 81625024), the International Science & Technology Cooperation Program of China (2014DFE30050), the Zhejiang Provincial Natural Science Foundation of China (LGF18H310001 and LY16H310008), the General Medical Research Program of Zhejiang Province (2014KYB020). Conflict of interest {#FPar1} ==================== The authors declare that they have no conflict of interest.

Introduction ============ MicroRNA (miRNA) regulation of protein expression adds a subtle layer of complex fine-tuning on top of numerous modes of protein production and destruction. miRNAs typically bind to specific sites within the 3′ untranslated regions (3′-UTRs) of their target mRNAs, to impart post-transcriptional silencing, either through translational repression and/or mRNA decay.[@b1-ebo-10-2014-107],[@b2-ebo-10-2014-107] In animals, miRNAs typically exhibit partial complementary base pairing to their mRNA targets, with the "seed region" of six to eight nucleotides toward the 5′end of the miRNA being important for target specificity.[@b3-ebo-10-2014-107] miRNA target efficacy is predicted to increase with the number of Watson-Crick matches to the seed region and is also dependent on base composition at nucleotide position 1.[@b2-ebo-10-2014-107],[@b4-ebo-10-2014-107] For example, a perfect match to nucleotides 2--8, referred to as a "7Mer-m8" target site, has a higher hierarchy rating compared to a "7Mer-A1" target site, which only confers Watson-Crick base pairing to nucleotides 2--7.[@b3-ebo-10-2014-107] Noticeably, the latter has an A at nucleotide position 1, which again rates it higher than a "6Mer" with the same 2--7 seed match, but a different number 1 nucleotide (C, G, or T).[@b4-ebo-10-2014-107] However, other factors such as AU-rich neighbor nucleotides and accessibility may also influence site-efficacy.[@b5-ebo-10-2014-107],[@b6-ebo-10-2014-107] Hence, miRNAs target specific genes for post-transcriptional regulation, fine-tuning protein expression levels during critical processes such as cell proliferation, differentiation, and maturation.[@b7-ebo-10-2014-107] The functional roles of microRNA-7 (miR-7) have been extensively studied, as it is highly conserved, being detected in 81 species (October 2013: <http://www.mirbase.org>),[@b8-ebo-10-2014-107]--[@b10-ebo-10-2014-107] and controls multiple cell signaling networks, including epidermal growth factor receptor,[@b11-ebo-10-2014-107] insulin-like growth factor,[@b12-ebo-10-2014-107] Hedgehog,[@b13-ebo-10-2014-107] and the mammalian target of rapamycin (mTOR) signaling pathways.[@b14-ebo-10-2014-107] miR-7 is also a critical regulator of multiple regulatory genes, including paired box gene 6 (*PAX6*) in mice.[@b8-ebo-10-2014-107],[@b9-ebo-10-2014-107] *PAX6* encodes a neurogenic transcription factor, which is highly conserved, both structurally and functionally, and is critical for neural tube polarization, brain regionalization, and eye formation.[@b15-ebo-10-2014-107]--[@b17-ebo-10-2014-107] *PAX6* is widely co-expressed with miR-7 in several tissues including the forebrain, pancreas, and retina where it is involved in regulating cellular differentiation.[@b8-ebo-10-2014-107]--[@b10-ebo-10-2014-107] Given the functional importance of the miR-7/PAX6 regulatory apparatus in controlling cell fate in mouse, we sought to evaluate whether this mechanism was conserved in humans. Herein we use bioinformatics to predict three miR-7 target sites in the human *PAX6* 3′-UTR and reporter gene assays to confirm the functional capacity of two of these predicted target sites. Further, we compare the human *PAX6* 3′-UTR region across species and identify a conserved miR-7 3′-UTR target site present in many species, as well as a second functional miR-7 target site in the human *PAX6* 3′-UTR that is specific to primates. Materials and Methods ===================== Prediction of miR-7 target sites -------------------------------- 3′-UTRs were identified using the "Spidey" freeware, which is part of the NCBI toolkit (<http://www.ncbi.nlm.nih.gov/spidey/>). Target sites for hsa-miR-7 within human *PAX6* 3′-UTR (GenBank accession number: NM_001127612) were predicted using TargetScan software (release 6.2)[@b4-ebo-10-2014-107] with the following search criteria: species: human, human Entrez gene symbol: *PAX6*, and miRNA name: hsa-miR-7. Target sites for miR-7 in 3′-UTRs of other species (GenBank accession numbers given in [Supplementary Table 1](#SD1-ebo-10-2014-107){ref-type="supplementary-material"}) were predicted using a "Find in This Sequence" function available at GenBank to search for the miR-7 seed region (5′-GUCUUCC-3′). Notably, only species that encode miR-7 (mirbase.org) are included in the analysis. Nucleotide position numbers for 3′-UTRs are listed in [Supplementary Table 1](#SD1-ebo-10-2014-107){ref-type="supplementary-material"}. Luciferase reporter constructs ------------------------------ Luciferase reporter plasmids were generated by ligating annealed DNA oligo nucleotides containing each of the three predicted *PAX6* 3′-UTR hsa-miR-7 7mer targets sites (NM_001127612.1; m8\#1: nt. 2389--2441, m8\#2: nt. 3613--3664, and A1\#3: nt. 4170--4216) to the pMiR-REPORT luciferase plasmid (Ambion) via *Hind*III and *Spe*I restriction sites. Mutated (Mut) miR-7 seed target sites with three nucleotide substitutions were generated following the same procedure (for sequences refer to [Supplementary Table 2](#SD2-ebo-10-2014-107){ref-type="supplementary-material"}). A perfect hsa-miR-7 target site (forward: 5′-CAACAAAATCACTAGTCTTCCA-3′ and reverse: 5′-TGGAAGACTAGTGATTTTGTTG-3′) inserted in the pGL3-control (Promega) firefly luciferase reporter vector was used as a control as previously described.[@b11-ebo-10-2014-107],[@b18-ebo-10-2014-107] Sequences of all plasmids were confirmed by Sanger DNA sequencing. Cell culture ------------ HeLa and HEK293 cells were obtained from the American Type Culture Collection (ATCC) and maintained at 37 °C and 5% CO~2~ in Dulbecco's modified Eagle's medium (DMEM)/F-12-GlutaMAX (Life Technologies) supplemented with 5% fetal bovine serum (Serana) and 100 units/mL of Penicillin/Streptomycin (Life Technologies). Luciferase assays ----------------- HEK293 cells in 24-well plates were co-transfected with 1) firefly luciferase reporter plasmid DNA (100 ng) containing either *PAX6* 3′-UTR WT or Mut miR-7 target sites (7mer-m8\#1, 7mer-m8\#2, and 7mer-A1\#3), 2) a control pRL-CMV *Renilla* luciferase reporter plasmid (5 ng), and 3) precursor miRNAs (30 nM) purchased from Ambion, corresponding to human miR-7 (ID: PM10047) or a miRNA negative control (miR-NC) (ID: AM17110) using Lipofectamine 2000 (Invitrogen). Cells were harvested 24 hours post-transfection and assayed using a Dual-Luciferase Reporter Assay System (Promega) and an EnSpire Multimode Plate Reader (PerkinElmer). For data analysis, firefly luciferase activity was firstly normalized to *Renilla* (transfection control) and then hsa-miR-7 was normalized to miR-NC transfected cells. Transfection ------------ HeLa cells were seeded 24 hours before transfection with miR-7 or miR-NC miRNA precursor molecules (30 nM) using RNAi/MAX transfection reagent (Invitrogen). Cells were harvested at 24 hours (for RNA) or 72 hours (for protein) post-transfection. Western blotting ---------------- HeLa cells were lysed by sonication (4 × 5 seconds) in ice-cold lysis buffer (0.1% SDS, 10 mM Tris--HCl pH 7.5) containing 1× complete inhibitor cocktail tablet (Roche) and protein concentrations determined by Bradford assay (Bio-Rad). Samples (70 μg) were electrophoretically separated on a 12.5% polyacrylamide gel, transferred to a Hybond-P membrane (Amersham Biosciences), and probed with anti-PAX6 (1:1,000; Abcam Clone AD2.38) or anti-β-actin (1:10,000; Sigma-Aldrich Clone AC-15) mouse monoclonal antibodies before detection with the commercially available Qdot 625 system (Molecular Probes) and UV light using Universal Hood II (Bio-Rad) for visualization of a representative image. A colored ladder was used to determine protein size (Marker Precision Plus Protein Kaleidoscope standard; Bio-Rad). RT-qPCR ------- Total RNA was extracted (SV Total RNA Isolation System; Promega), quality assessed, and quantified using a NanoDrop 1000 Spectrophotometer (Thermo Scientific) with threshold cutoff for DNA contamination (260/280) at 2.0. cDNA was synthesized (SuperScript III; Invitrogen) using 250 ng RNA and random hexamers according to the manufacturer's instructions. qPCR assays were performed using a Rotor-Gene Q (Qiagen) with SYBR-Green chemistry (GoTaq qPCR Master Mix; Promega) and previously published gene-specific primers: *PAX6*[@b19-ebo-10-2014-107]--[@b21-ebo-10-2014-107] forward: 5′-TCTTTGCTTGGGAAATCCG-3′ and reverse: 5′-CTGCCCGTTCAACATCCTTAG-3′; glyceraldehyde 3-phosphate dehydrogenase (*GAPDH*)[@b22-ebo-10-2014-107]--[@b25-ebo-10-2014-107] forward: 5′-GAAGGTGAAGGTCGGAGTC-3′ and reverse: 5′-GAAGATGGTGATGGGATTTC-3′. Primer-specificity was tested using gel electrophoresis (to verify single band and amplicon size), DNA sequencing, and melting profile. A no-reverse transcription control was included in each run for detection of potential PCR contamination. Normalized *PAX6* expression relative to *GAPDH* was calculated using the 2^−ΔΔ*Ct*^ method.[@b26-ebo-10-2014-107] Statistical analysis -------------------- Data are presented as mean of three independent experiments (*n* = 3) + standard deviation (SD). Significance (*P* \< 0.05) was assessed using a paired *t*-test (RT-qPCR) and ANOVA Tukey's post-hoc analysis (luciferase assay) in R (version 3.0.2).[@b27-ebo-10-2014-107] Results and Discussion ====================== The human *PAX6* 3′-UTR contains two functional miR-7 target sites ------------------------------------------------------------------ There are seven verified human *PAX6* transcripts in the NCBI database, which result from alternative splicing and alternative initiation of transcription. However, it is noteworthy that they all share identical 3′-UTRs accounting for approximately 75% of the total mRNA sequence, which allows for the suggestion that *PAX6* expression is possibly regulated by miRNAs. Using TargetScan software,[@b4-ebo-10-2014-107] we identified three miR-7 target sites: one of which (m8\#1) has previously been shown to regulate *PAX6* expression in mouse[@b9-ebo-10-2014-107] and two additional miR-7 target sites (m8\#2 and A1\#3) were predicted when poorly conserved target sites were considered ([Fig. 1A](#f1-ebo-10-2014-107){ref-type="fig"}). Two of the three predicted miR-7 target sites within the *PAX6* 3′-UTR contained 7mer-m8 seed matches (m8\#1 and m8\#2), whereas the third target site contained a lower hierarchy 7mer-A1 seed match (A1\#3)[@b4-ebo-10-2014-107] ([Fig. 1B](#f1-ebo-10-2014-107){ref-type="fig"}). Noticeably, the A1\#3 target site has a mismatch at position 8 in the miR-7 sequence ([Fig. 1B](#f1-ebo-10-2014-107){ref-type="fig"}), which has been shown to be essential for functional miR-7-target binding.[@b28-ebo-10-2014-107] To examine the functional significance of the three predicted human miR-7 target sites within the *PAX6* 3′-UTR, we generated firefly luciferase reporter constructs containing each of these individual sites, as well as Mut constructs of each. We then co-transfected HEK293 cells with the firefly and *Renilla* (transfection control) luciferase constructs and either hsa-miR-7 or miR-NC (a negative control) precursor miRNAs to assess target specificity. To confirm the reliability of our assay, we first measured luciferase activity of a construct containing a perfect miR-7 target site (5′-CAACAAAATCACTAGTCTTCCA-3′), which resulted in a 99% reduction in luciferase activity (*Renilla* normalized) compared to cells transfected with miR-NC precursors ([Fig. 1C](#f1-ebo-10-2014-107){ref-type="fig"}). Assessment of cells transfected with *PAX6* 3′-UTR target site firefly luciferase reporter plasmids revealed that two of the three miR-7 target sites (m8\#1 and m8\#2) were functionally active, each yielding a significant reduction in luciferase activity of approximately 45% with miR-7 relative to their Mut target site reporters ([Fig. 1C](#f1-ebo-10-2014-107){ref-type="fig"}). Both of these active sites contained 7mer-m8-predicted miR-7 target sites, whereas the third site (7mer-A1\#3) did not show any regulation by miR-7. Our study therefore confirms previous findings, showing that Watson-Crick base pairing to nucleotide position 8 is essential for functional miR-7-target binding.[@b28-ebo-10-2014-107] Taken together, these data indicate that the *PAX6* 3′-UTR contains two 7 mer-m8 miR-7 seed sites. A third predicted 7 mer-A1 miR-7 target site that did not comply with Watson-Crick pairing at nucleotide 8 failed to show functional activity in luciferase reporter assays. PAX6 protein expression is regulated by miR-7 in human cells ------------------------------------------------------------ To assess whether miR-7 regulates PAX6 protein expression in human cells, we transiently transfected HeLa, a human cervical cancer cell line, with miR-7 precursor molecules and measured PAX6 levels by Western blotting. Compared with a negative miRNA precursor molecule (miR-NC), miR-7 reduced expression of PAX6 in HeLa cells ([Fig. 2A](#f2-ebo-10-2014-107){ref-type="fig"}). To determine whether miR-7 reduced PAX6 protein levels through interfering with the translational pathway or mediating mRNA decay, we assessed endogenous *PAX6* mRNA levels by RT-qPCR; however, we saw no significant difference in *PAX6* mRNA expression between HeLa cells transfected with hsa-miR-7 or miR-NC miRNA precursor molecules ([Fig. 2B](#f2-ebo-10-2014-107){ref-type="fig"}). Taken together, these data indicate that miR-7 regulates PAX6 protein levels in human cells, but has no significant effect on mRNA levels, which suggests that miR-7 interferes with the translational pathway rather than mediating *PAX6* mRNA decay. Diversification of *PAX6* 3′-UTR miR-7 target sites across species ------------------------------------------------------------------ Recent experiments in mice have demonstrated that miR-7 directly represses the expression of *PAX6* through a single miR-7 target site in the mouse *PAX6* 3′-UTR.[@b8-ebo-10-2014-107],[@b9-ebo-10-2014-107] Given our identification of a second functional miR-7 target site (7mer-m8) in human *PAX6* 3′-UTR compared to mouse, we examined the evolutionary conservation of *PAX6* 3′-UTRs and their miR-7 target sites across species, and found that the length of *PAX6* 3′-UTRs varied considerably and that this length seemed to be associated with the number of predicted miR-7 target sites ([Fig. 3A](#f3-ebo-10-2014-107){ref-type="fig"}). For example, in primates, human (*Homo sapiens*) and rhesus monkey (*Macaca mulatta*), *PAX6* has relatively long 3′-UTRs (accounting for more than 70% of the total mRNA sequences), and we demonstrated the existence of two functional 7mer-m8 miR-7 target sites within these regions. In comparison, the 3′-UTRs of pig (*Sus scrofa*), rodents (*Mus musculus* and *Rattus norvegicus*), frog (*Xenopus tropicalis*) and fish (*Danio rerio*) account for between 30 and 39% of total *PAX6* mRNA length and contain only one predicted miR-7 target site ([Fig. 3A](#f3-ebo-10-2014-107){ref-type="fig"}). In other species such as chicken (*Gallus gallus*) and fruit fly (*Drosophila melanogaster*), where the 3′-UTR accounts for less than 20% of the total *PAX6* mRNA sequence, no miR-7 target sites were predicted ([Fig. 3A](#f3-ebo-10-2014-107){ref-type="fig"}). Notably, in zebrafish, there are two duplicated *PAX6* gene variants, *PAX6a* and *PAX6b*, with distinct 3′-UTR lengths, accounting for 34%--39% of the total mRNA lengths, respectively ([Fig. 3A](#f3-ebo-10-2014-107){ref-type="fig"}). *PAX6a* contains a single predicted miR-7 target site, whereas no site was predicted in the shorter 3′-UTR of the *PAX6b* variant, suggesting that perhaps only *PAX6a* is post-transcription-ally regulated by miR-7. Also, the fruit fly has multiple *PAX6* gene loci, from which six different *PAX6* 3′-UTRs are transcribed; none have a predicted miR-7 target site in the 3′-UTR despite one transcript having a 3′-UTR that accounts for more than 45% of the total mRNA sequence ([Fig. 3A](#f3-ebo-10-2014-107){ref-type="fig"}), suggesting that miR-7 might not regulate PAX6 expression levels in fruit flies. These results are in agreement with previous work that suggested the expansion of 3′-UTR sequences increases with organism complexity throughout evolution.[@b29-ebo-10-2014-107] In species with a predicted miR-7 target site in the *PAX6* 3′-UTR, sequence alignment revealed that 20 of 23 nucleotides were conserved and that all predicted miR-7 target sites shared identical 7mer-m8 seed regions ([Fig. 3B](#f3-ebo-10-2014-107){ref-type="fig"}). Thus, the first functional human 7mer-m8 *PAX6* miR-7 target site (more 5′-located; m8\#1 in [Figure 1](#f1-ebo-10-2014-107){ref-type="fig"}) appears to be conserved, whereas the second functional human 7mer-m8 miR-7 target site (more 3′-located; m8\#2 in [Figure 1](#f1-ebo-10-2014-107){ref-type="fig"}) does not. The identification of a miR-7 target site positioned outside the conserved region suggests that this site may have resulted from the expansion of the *PAX6* 3′-UTR. The appearance of multiple functional miR-7 binding sites within the human *PAX6* 3′-UTR suggests an added level of complexity by allowing the combinatorial regulation of *PAX6* expression by miR-7.[@b7-ebo-10-2014-107] In summary, given our finding that miR-7 can regulate PAX6 expression via two miR-7 target sites in a human cell line, future experiments are needed to examine the physiological importance of this regulatory system, in particular, examining whether there is a combinatory effect between the two functional human miR-7 target sites (m8\#1 and m8\#2) or if a dysfunctional miR-7/PAX6 regulatory system promotes tumorigenesis, as both factors are highly associated with cancer.[@b30-ebo-10-2014-107]--[@b34-ebo-10-2014-107] Conclusions =========== In conclusion, we provide the first demonstration that miR-7 regulates the expression of PAX6 protein in human cells, and show that this regulation occurs via two target sites within the *PAX6* 3′-UTR: the first site is highly conserved across species, whereas the second functional miR-7 site is conserved only in primates. This finding leads to the intriguing possibility that the expanded 3′-UTR found in human *PAX6* may allow for a tighter control of *PAX6* expression by miR-7 than that already documented in mouse. Supplementary Data ================== ###### *PAX6* 3′untranslated regions. SPECIES VARIANTS ACCESSION NUMBERS 3′UTR (START-STOP) --------------------------- ------------------ ---------------- ------------------- -------------------- *Homo Sapiens* N/A NM_001127612.1 1739--6883 *Macaca mulatta* N/A NM_001266257.1 1711--5908 *Sus scrofa* N/A NM_001244172.1 1728--2643 *Mus musculus* N/A NM_001244198.1 1597--2619 *Rattus norvegicus* N/A NM_013001.2 1430--2191 *Gallus gallus* N/A NM_205066.1 1641--1961 *Xenopus tropicalis* N/A NM_001006762.1 1651--2361 *Danio rerio* *PAX6a* NM_131304.1 1845--2809 *PAX6b* NM_131641.1 1575--1733 *Drosophila Melanogaster* *Eyeless* (*Ey*) NM_166789.2 2621--2851 *Twin of eyeless* (*Toy*) Variant a NM_079899.4 2160--2287 Variant c NM_00127215.1 2109--3870 *Eyegone* (*Eyg*) NM_001014582.1 2350--2586 *Twin of eyegone* (*Toe*) Variant a NM_07 9 317. 3 2279--2400 Variant b NM_001274833.1 2030--2303 **Notes:** List of *PAX6* GenBank accession numbers, N/A = "not applicable." Nucleotide position numbers for the 5′end (start) and 3′end (stop) of the 3′-UTRs are listed. ###### Oligonucleotides used for generating firefly luciferase constructs. TARGET SITES OLIGONUCLEOTIDES (5′→3′) -------------- -------------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------------- ----------------------------------------------------------------- m8\#1 WT SE *AGCT*TTCTGAGGATTTCT**AGGGAAGACAAATACTTACATTTT**GACATAAAACAAATT AS *CTAG*AATTTGTTTTATGTC**AAAATGTAAGTATTTGTCTTCCCT**AGAAATCCTCAGAA Mut SE *AGCT*TTCTGAGGATTTCT**AGG[A]{.ul}A[G]{.ul}G[G]{.ul}CAAATACTTACATTTT**GACATAAAACAAATT AS *CTAG*AATTTGTTTTATGTC**AAAATGTAAGTATTTG[C]{.ul}C[C]{.ul}T[T]{.ul}CCT**AGAAATCCTCAGAA m8\#2 WT SE *AGCT*AGTGAAGTTTGCAG**AGGAAGACTTATCTGTATTGACT**TATATGTTGCACAGA AS *CTAG*TCTGTGCAACATATA**AGTCAATACAGATAAGTCTTCCT**CTGCAAACTTCACT Mut SE *AGCT*AGTGAAGTTTGCAG**AG**[A]{.ul}**A**[G]{.ul}**G**[G]{.ul}**CTTATCTGTATTGACT**TATATGTTGCACAGA AS *CTAG*TCTGTGCAACATATA**AGTCAATACAGATAAG**[C]{.ul}**C**[C]{.ul}**T**[T]{.ul}**CT**CTGCAAACTTCACT A1\# 3 WT SE *AGCT*CAAATAATCTCCATCC**TGGAAGATGGTGTCAAAACATC**CCTGCAGATACCCCA AS *CTAG*TGGGGTATCTGCAGG**GATGTTTTGACACCATCTTCCA**GGATGGAGATTATTTG Mut SE *AGCT*CAAATAATCTCCATCC**TG**[A]{.ul}**A**[G]{.ul}**G**[G]{.ul}**TGGTGTCAAAACAT**CCCTGCAGATACCCCA AS *CTAG*TGGGGTATCTGCAGG**GATGTTTTGACACCA**[C]{.ul}**C**[C]{.ul}**T**[T]{.ul}**CA**GGATGGAGATTATTTG **Notes:** List of sense (SE) and antisense (AS) oligonucleotides used for generating firefly luciferase constructs containing WT and Mut miR-7 7mer target sites (m8\#1, m8\#2, and A1\#3). Italic, bold, and underlined nucleotides represent restriction recognition-, miR-7 target- and Mut sites, respectively. **Author Contributions** MN, RBW, KMG, SAD, and MGT conceived and designed the experiments. MN, RBW, KMG, and MGT analyzed the data. MN wrote the first draft of the manuscript. MN, RBW, KMG, SAD, and MGT contributed to the writing of the manuscript. MN, RBW, KMG, SAD, and MGT agreed with manuscript results and conclusions. MN, RBW, KMG, SAD, and MGT jointly developed the structure and arguments for the paper. MN, RBW, KMG, SAD, and MGT made critical revisions and approved the final version. All authors reviewed and approved the final manuscript. **DISCLOSURES AND ETHICS** As a requirement of publication the authors have provided signed confirmation of their compliance with ethical and legal obligations including but not limited to compliance with ICMJE authorship and competing interests guidelines, that the article is neither under consideration for publication nor published elsewhere, of their compliance with legal and ethical guidelines concerning human and animal research participants (if applicable), and that permission has been obtained for reproduction of any copyrighted material. This article was subject to blind, independent, expert peer review. The reviewers reported no competing interests. **ACADEMIC EDITOR:** Jike Cui, Associate Editor **FUNDING:** MGT was funded by Edith Cowan University's Office of Research and Innovation, the Faculty of Computing, Health and Science, and the Vario Health Institution. RBW was funded by a Raine Medical Research Foundation priming grant and an Early Career grant from the Cancer Council of Western Australia. SAD was funded by an NHMRC Principal Research's fellowship (1002347), MN received a postgraduate research scholarship from Edith Cowan University, and KG was the recipient of a Royal Perth Hospital Medical Research Foundation's fellowship. **COMPETING INTERESTS:** Authors disclose no potential conflicts of interest. {#f1-ebo-10-2014-107} {#f2-ebo-10-2014-107} {#f3-ebo-10-2014-107}

{#sp1 .115} {#sp2 .116}

All relevant data are within the paper and its Supporting Information files. Introduction {#sec001} ============ Type 1 diabetes is an autoimmune inflammatory disease that is caused by immune cell mediated destruction of the insulin-producing beta cells in the pancreas. T cells are assumed to play a considerable role in the pathogenesis of this disease which comes from the demonstration that its strongest genetic risk is conferred by the HLA locus and other loci affecting the biology of these cells \[[@pone.0146516.ref001], [@pone.0146516.ref002]\]. Islet specific CD4^+^ and CD8^+^ cells mediate diabetogenesis in NOD mice \[[@pone.0146516.ref003]\] and have been identified in human type 1 diabetes patients \[[@pone.0146516.ref004]--[@pone.0146516.ref006]\]. Other cell types including B cells, dendritic cells and macrophages are also necessary for the initiation of the anti-islet immune responses (reviewed in \[[@pone.0146516.ref007]\]). Once the beta cell pool is destroyed by the immune system, insufficient amounts of insulin are produced to sustain glucose uptake into insulin dependent cells, notably muscle cells, causing wasting due to protein breakdown, even though high levels of glucose are present in the blood. The disease was fatal until Banting and Best discovered insulin in 1922 \[[@pone.0146516.ref008]\]. Although much has been learned about type 1 diabetes since then, no protocols for sustained cure or prevention have been discovered. Strategies attempting to abrogate immune responses have been successful in mouse models \[[@pone.0146516.ref007]\], and some have also resulted in delay of beta cell destruction in patients, such as the anti-CD3 treatment clinical trials \[[@pone.0146516.ref009], [@pone.0146516.ref010]\] and CTLA-4 Ig \[[@pone.0146516.ref011]\]. CTLA-4 Ig prevents the activation of T cells by inhibiting interaction with the co-stimulatory molecule CD28. However, CD28 blockade alone is unlikely to be sufficient to prevent autoimmunity due to the fact that memory and CD8^+^ T cells are not as dependent as naïve CD4^+^ T cells on CD28 costimulation. As none of the clinical trials have resulted in long term reversal of diabetes, it has been suggested that a combination of treatments targeting various stages of immune activation may be more successful \[[@pone.0146516.ref012]\] A widely explored approach is the inhibition of key signalling enzymes involved in the activation and metabolism of immune cells. PI3Ks constitute a family of enzymes involved in cellular functions such as cell growth, proliferation, differentiation, motility, survival and intracellular trafficking \[[@pone.0146516.ref013]\]. The class I PI3Ks catalyse the final step to PIP3 by phosphorylating PI(4,5)P2 to become PI(3,4,5)P3. PIP3 activates Akt, which in turn leads to activation of mTOR and inhibition of Foxo. Of the four class I PI3K subunits, PI3K p110δ is uniquely expressed in immune cells \[[@pone.0146516.ref014]\]. Specific inhibition of this subunit therefore primarily affects cells of the immune system, leaving other cell types unchanged. p110δ inhibitors such as IC87114 have been used to inhibit p110δ activity *in vitro* and *in vivo*. It has been shown that IC87114 inhibits TCR-induced cytokine production by both naïve and effector T cells \[[@pone.0146516.ref015]\]. In addition to its effects on T cells, p110δ inhibitors also target other immune cells such as B cells, NK and myeloid cells (reviewed in \[[@pone.0146516.ref014], [@pone.0146516.ref016]\] The typical outcome of PI3Kδ inactivation is a reduction but not a complete abrogation of leukocyte functions, probably due to the overlapping functions with other PI3K members that are also present in leukocytes. IC87114 reduced disease severity in preclinical rodent models of rheumatoid arthritis, asthma, and allergy \[[@pone.0146516.ref017]--[@pone.0146516.ref019]\]. The PI3Kδ inhibitor idelalisib was approved in 2014 for the treatment of chronic lymphocytic leukaemia. CD28 and p110δ act synergistically to provide full T-cell stimulation \[[@pone.0146516.ref020]\], and we hypothesised that dual inhibition of CD28 and p110δ may achieve more potent alleviation of pathologic immune responses than can be achieved with either inhibitor alone. To test this hypothesis, we used IC and CTLA-4 Ig, either alone or in combination, to inhibit signalling via p110δ, CD28 or both in naïve and activated T cells from non obese diabetic (NOD) mice and TCR transgenic mice on a NOD background, and monitored proliferation and cytokine production. In addition, we have investigated whether such a combination could limit the development of autoimmune diabetes in the NOD mouse model after the transfer of either naïve or activated islet reactive BDC2.5 CD4^+^ T cells. Materials and Methods {#sec002} ===================== Mice {#sec003} ---- Female NOD, NOD-*scid*, BDC2.5 NOD mice \[[@pone.0146516.ref021]\] NOD-CD2-GFP mice \[[@pone.0146516.ref022]\], G9C8 TCR transgenic NOD mice \[[@pone.0146516.ref023], [@pone.0146516.ref024]\] and NOD-Foxp3-GFP \[[@pone.0146516.ref025]\] were bred in the Department of Pathology, University of Cambridge and maintained under specific pathogen---free conditions. The mice are housed in individually ventilated cages with free access to standard chow and water. The facility is kept on a 12 hour light, 12 hour dark cycle. The humane endpoints for these experiments specify that any mouse that loses more than 15% of its body weight (compared to healthy littermates), or in other ways looks unwell and likely to exceed the Home Office standard of moderate severity must be culled. However, no mice used in this study required early culling. At the end the experiments, mice were culled using a CO2-chamber followed by dislocation of the neck. In cases where we were harvesting islets for transplantation it was important to maintain an intact bile duct, and death was instead confirmed through palpation of the chest to assess the absence of a heart beat. Ethics statement {#sec004} ---------------- This study was carried out in strict accordance with U.K. Home Office project licence regulations (Project Licence number 80/2442) after approval by the Ethical Review Committee of the University of Cambridge. PI3K-δ inhibitor {#sec005} ---------------- The PI3K p110δ inhibitor IC87114 was synthesized by Jonathan Clark (Babraham Institute) as described (D030 from patent WO 01/81346) \[[@pone.0146516.ref015]\]. For *in vivo* administration, IC87114 was dissolved in methyl cellulose 400 cps (Sigma) using a sonicator (Heat Systems Ultrasonics), and administered through oral gavage twice daily in 100μl at a dose of 30mg/kg body weight. This dose was chosen based on previous reports of its efficacy in vivo \[[@pone.0146516.ref017]\]. In our hands, a 30 mg/kg by gavage achieves \~2 μM 90 min post-administration and the drug is cleared from the blood 4--7 hours post administration. IC87114 is selective for p110δ at plasma concentrations of 5 μM \[[@pone.0146516.ref017]\]. CTLA4-Ig {#sec006} -------- CTLA4-Ig (Abatacept) was provided by Bristol Myers Squibb (BMS). CTLA4-Ig was administered by intraperitoneal (ip) injection starting on day 0 with 500 μg, then 250μg every other day \[[@pone.0146516.ref026]\]. For *in vitro* assays, CLTA4-Ig was added to cultures at 100 ng/ml. Th1 differentiation for *in vitro* studies and adoptive transfer {#sec007} ---------------------------------------------------------------- CD4^+^CD25^-^ T cells (for *in vitro* studies) or CD4^+^CD62L^hi^ CD25^-^B220^-^ T cells (for adoptive transfer) were isolated by cell sorter from 5-week-old BDC2.5 TCR transgenic NOD mice and differentiated into Th1 cells by culturing them with plate bound anti-CD3 (2μg/mL), soluble anti-CD28 (10μg/mL), IL-2 (100u/ml), IL-12 (10ng/ml) and IFN-γ (100u/ml) for 4 days at 37°C with 5% CO~2~. Afterwards, the production of IFN-γ was checked by specific ELISA (R&D). T cell activation for functional assays {#sec008} --------------------------------------- Cells were isolated from spleen and lymph nodes and cultured in IMDM with 10% fetal calf serum, 1% penicillin-streptomycin, and β-mercaptoethanol. 2x10^5^ total lymphocytes were stimulated as appropriate (see below) for 3 days in the presence or absence of rising concentrations of IC (0.6, 1.25, 2.5, 5 and 10μg/mL) at 37°C with 5% CO2. NOD mouse cells were stimulated with plate bound anti-CD3 (2μg/mL) and soluble anti-CD28 (10μg/mL), whereas cells isolated from BDC2.5 or G9C8TCR transgenic NOD mice were stimulated with BDC2.5 mimotope or insulin peptide insB 15--23, respectively. In other experiments, 2.5x10^5^ Th1 cells were cultured with 1x10^4^ APCs and BDC2.5 mimotope (0.5μg/mL) with or without increasing concentrations of IC as previously described for 72 hours. Cells cultured in the presence of the proliferative stimulus but without IC87114 were positive controls, whereas non-stimulated cells were negative controls. Proliferation and cytokine analysis {#sec009} ----------------------------------- In all experiments proliferation was assessed by CFSE staining (5μM). After gating on CD4^+^ and/or CD8^+^ T cells, the percentage of proliferating cells in each population was determined. For cytokine analysis, supernatants were taken at the end of the time cultures and IFN-γ production or IL-10 was assessed by specific ELISA (R&D Systems). Levels of other cytokines were detected using a cytometric bead array (eBioscience). For intracellular cytokine staining, cells were washed and stimulated with PMA (50 ng/mL) and ionomycin (2000 ng/mL) for 5 hours. BFA (5μg/mL) was added for the last 3 hours. Afterwards, the staining of cell surface markers was performed. Cells were washed, fixed, permeabilized (intracellular cytokine staining kit, eBioscience), and stained for detection of IFN-γ. *In vitro* assessment of regulatory T cells {#sec010} ------------------------------------------- CD4^+^ CD25^+^ GFP^+^ cells from spleens and lymph nodes of 5-week-old Foxp3/GFP^+^ BDC2.5 TCR transgenic NOD mice were isolated by cell sorter (MoFlo, BD). Tregs were cultured with anti-CD3 (5 μg/mL), anti-CD28 (20μg/mL) and IL-2 (1000 u/ml) with or without IC87114 (5 and 10μM) for 72 hours. Proliferation was assessed by dilution of CFSE staining (5μM, Invitrogen) after gating on CD4^+^CD25^+^ cells. Supernatants were assessed for IL-10 by specific ELISA (R&D). Positive control cells were stimulated with anti-CD3/28 antibodies and IL-2 whereas negative control cells were not stimulated at all. Islet transplantation {#sec011} --------------------- Diabetes was induced in recipient mice (normal (WT), CD28-/-, p110δ^D910A/D910A^ and CD28-/-; p110δ^D910A/D910A^ double knockouts (DKOs)) by streptozotocin injection. All strains were backcrossed on the C57BL/6 background. Islets were prepared from MHC-mismatched donors (Cba1-C57BL/6 F1). Pancreatic islets were isolated through inflation of the pancreas via the bile duct \[[@pone.0146516.ref027]\], and islet transplantation was performed according to standard protocols \[[@pone.0146516.ref028]\]. Confirmed diabetic recipient mice received between 300 and 500 islets, giving approximately 15 islets per gram of body weight. We anaesthetised the recipient mice with isoflurane inhalation anaesthesia, and gave them sc temgesic for post-surgery analgesia. Islets were injected beneath the kidney capsule of female recipients that have been confirmed to be diabetic after the streptozotocin injection (Blood glucose level \> 20 mM at two consecutive readings). Blood glucose was assessed three times per week for the period of graft survival, and daily at the onset of graft rejection---for a maximum of three days. Some mice which retained their grafts for longer than 100 days had the engrafted kidney removed at the end of the experiment to ascertain that the graft was responsible for the restoration of euglycemia. Adoptive transfer models {#sec012} ------------------------ CD4^+^ T cells were isolated by cell sorting from spleen and lymph nodes of BDC2.5 TCR transgenic mice. 1x10^6^ CD4^+^ T cells were transferred by the ip route to 5-week old NOD-*scid* mice. Mice received IC87114 treatment (30mg/Kg) by oral gavage twice per day and/or CTLA-4 Ig administrated by the ip route at a concentration of 500μg/mL (the first time) and then 250μg/mL every other day from day 0 to 10. Positive control mice did not receive any treatment. After day 7, mice were monitored for the development of diabetes by measuring blood glucose or urine glucose levels with Diastix strips (Bayer) every day. In other experiments, 5x10^5^ Th1 cells (see above) were transferred by the ip route to 5-week old NOD mice. Mice received the same treatment as described above, but from day 0 to 5. After day 3, mice were monitored for the development of diabetes as previously described. Statistical analysis {#sec013} -------------------- Differences between groups were tested using the student t-test, significant p-values are indicated with \* (p≤0.05), \*\* (p≤0.001) or \*\*\* (p≤0.0001). Differences between animals regarding diabetes incidence or recurrence after islet transplant were tested using the Log rank survival test, with actual p-values displayed within the relevant figure or legend. All analysis was performed using GraphPad Prism software. Results {#sec014} ======= IC87114 inhibition of the PI3K p110δ subunit does not diminish T cell proliferation but has profound effects on cytokine production {#sec015} ----------------------------------------------------------------------------------------------------------------------------------- We assessed the effects of increasing levels of IC87114 on T cells from diabetes-prone NOD mice in *in vitro* culture, looking at anti-CD3 and anti-CD28 induced proliferation of CD4^+^ T cells ([Fig 1A](#pone.0146516.g001){ref-type="fig"}, left hand panel) and CD8^+^ T cells ([Fig 1A](#pone.0146516.g001){ref-type="fig"}, right hand panel) and production of IFN-γ ([Fig 1B](#pone.0146516.g001){ref-type="fig"}). We found that the presence of IC87114 did not suppress proliferation of either CD4^+^ or CD8^+^ T cells ([Fig 1A](#pone.0146516.g001){ref-type="fig"}) but that the production of IFN-γ was severely impaired even at the lowest concentration of IC87114 tested ([Fig 1A](#pone.0146516.g001){ref-type="fig"}). Unaffected proliferation (as measured by % of cells divided ≥1 time) and suppressed IFN-γ production were seen both in islet specific TCR transgenic BDC2.5 CD4^+^ T cells \[[@pone.0146516.ref021]\] ([Fig 1C and 1D](#pone.0146516.g001){ref-type="fig"}), which recognise a posttranslationally modified peptide of chromogranin A \[[@pone.0146516.ref029]\], and in TCR transgenic G9C8 CD8^+^ T cells which recognise an insulin peptide \[[@pone.0146516.ref023], [@pone.0146516.ref024]\] ([Fig 1E and 1F](#pone.0146516.g001){ref-type="fig"}). We performed an extended analysis of the effects on cytokine production by IC87114, and found that increasing concentration of IC87114 suppressed all the pro-inflammatory cytokines assessed in supernatants from peptide-stimulated BDC2.5 CD4^+^ T cells, including IFN-γ, IL-2, GM-CSF, and IL-6, while there was a trend towards increased production of anti-inflammatory cytokine IL-10 and Th2 associated cytokines IL-4 and IL-5 ([S1 Fig](#pone.0146516.s001){ref-type="supplementary-material"}). Production of the cytokines that could be detected in supernatants from insulin peptide stimulated G9C8 CD8^+^ T cells was also decreased, with lower levels of IFN-γ, IL-2, TNF, IL-17, GM-CSF, IL-10 and IL-6 recorded even at the lowest concentrations of added IC87114 ([S2 Fig](#pone.0146516.s002){ref-type="supplementary-material"}). An interesting finding was that T cell proliferation was if anything increased in the presence of higher concentrations of IC87114, with more cells dividing several times ([S3A Fig](#pone.0146516.s003){ref-type="supplementary-material"}) and with BDC2.5 CD4^+^ T cells upregulating CD25 in response to increasing concentration of IC87114 ([S3B Fig](#pone.0146516.s003){ref-type="supplementary-material"}). {#pone.0146516.g001} Administration of IC87114 via oral gavage does not delay onset of diabetes after BDC2.5 cell transfer into NOD-*scid* recipients {#sec016} -------------------------------------------------------------------------------------------------------------------------------- As production of proinflammatory cytokines is central to the process whereby islet-specific T cells cause type 1 diabetes through incapacitating and killing beta cells \[[@pone.0146516.ref030]\], we wanted to test whether oral administration of IC87114 could prevent or delay onset of diabetes in NOD*scid* mice after transfer of potentially diabetogenic islet-specific BDC2.5 CD4^+^ T cells. We found that oral administration of 30mg/kg body weight twice per day ([Fig 2A](#pone.0146516.g002){ref-type="fig"}) had no effect of the development of diabetes after cell transfer, as all mice in both groups had developed diabetes by day 13 after transfer ([Fig 2B](#pone.0146516.g002){ref-type="fig"}). {#pone.0146516.g002} IC87114 suppresses IFN-γ production from Th1-differentiated islet specific effector T cells but does not stop them from causing diabetes after transfer into wt NOD recipients {#sec017} ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ As IC87114 did not have an effect on development of diabetes after transfer of BDC2.5 cells we wished to elucidate whether it could decrease IFN-γ production from already Th1 differentiated effector CD4^+^ T cells. We found that, just as seen in *in vitro* cultures with naïve cells, the Th1 differentiated effector cells proliferated equally well in the presence of IC87114 ([Fig 3A](#pone.0146516.g003){ref-type="fig"}), but that their IFN-γ production, although more robust than from naïve T cells, was decreased by IC87114 in a dose-dependent way ([Fig 3B](#pone.0146516.g003){ref-type="fig"}). Th1 differentiated effector BDC2.5 CD4^+^ T cells cause diabetes after transfer into wildtype NOD mice in a process heavily dependent on production of IFN-γ ([Fig 4A](#pone.0146516.g004){ref-type="fig"}) \[[@pone.0146516.ref030]\], and we hypothesised that administration of IC87114 via oral gavage which can stop IFN-γ production from these cells in vitro, would inhibit or delay onset of diabetes in the IFN-γ dependent model of disease. However, oral administration of 30mg/kg body weight twice per day from the day of cell transfer did not affect disease development, and both the IC87114-treated and the vehicle treated group developed diabetes on day 5 after adoptive transfer ([Fig 4B](#pone.0146516.g004){ref-type="fig"}). Oral administration did not appear to affect IFN-γ production *in vivo* as cells isolated from pancreas ([Fig 4C](#pone.0146516.g004){ref-type="fig"}) or pancreatic lymph nodes ([Fig 4D](#pone.0146516.g004){ref-type="fig"}) of IC87114 or vehicle-treated mice produced equal amounts of IFN-γ. {#pone.0146516.g003} {#pone.0146516.g004} Combination with CTLA4-Ig does not alter or potentiate the *in vitro* effects of IC87114 {#sec018} ---------------------------------------------------------------------------------------- As IC87114 showed no effect on *in vivo* diabetes development despite its dramatic effects on cytokine production *in vitro*, we hypothesised that a combination with another agent that targets a different pathway leading to the activation of T cells might synergise with the IC87114. Indeed, in work leading up to the current study, we found that p110δ-CD28 double knockout mice are immune suppressed and failed to reject allogeneic islet grafts (\[[@pone.0146516.ref020]\] and [S4 Fig](#pone.0146516.s004){ref-type="supplementary-material"}). Since CTLA-4 Ig inhibits the ability of T cells to receive signals via CD28, we assessed the effects of combining increasing levels of IC87114 with CTLA4-Ig on T cells from NOD mice in *in vitro* culture, looking at anti-CD3 induced proliferation of CD4^+^ T cells ([Fig 1A](#pone.0146516.g001){ref-type="fig"}, left hand panel) and CD8^+^ T cells ([Fig 1A](#pone.0146516.g001){ref-type="fig"}, right hand panel) and production of IFN-γ ([Fig 1B](#pone.0146516.g001){ref-type="fig"}). We found that the combination of CTLA4-Ig with IC87114 did not suppress proliferation of either CD4^+^ or CD8^+^ T cells any differently than IC87114 on its own ([Fig 5A](#pone.0146516.g005){ref-type="fig"}) and that the production of IFN-γ was similarly impaired in the presence of CTLA4-Ig ([Fig 5B](#pone.0146516.g005){ref-type="fig"}) compared with IC87114 alone ([Fig 1B](#pone.0146516.g001){ref-type="fig"}). The presence of CTLA4-Ig modestly impaired proliferation of BDC2.5 CD4^+^ T cells, but this was not potentiated by increasing concentrations of IC87114 ([Fig 5C](#pone.0146516.g005){ref-type="fig"}). However, CTLA4-Ig did have a significant effect on the production of IFN-γ from the BDC2.5 CD4^+^ T cells, and combination with IC87114 decreased IFN-γ production even further ([Fig 5D](#pone.0146516.g005){ref-type="fig"}). The proliferation and IFN-γ production of G9C8 CD8^+^ T cells was not affected by the addition of CTLA4-Ig ([Fig 5D and 5E](#pone.0146516.g005){ref-type="fig"}), demonstrating the same reduction of IFN-γ production in response to IC87114 as when no CTLA4-Ig was present ([Fig 1F](#pone.0146516.g001){ref-type="fig"}). We performed an extended analysis of the effects on cytokine production by combining CTLA4-Ig and IC87114, and found that addition of CTLA4-Ig on its own decreased the production of IL-2 in supernatants from peptide-stimulated BDC2.5 CD4^+^ T cells, but that other cytokines assessed demonstrated no additional downregulation in response to the additional presence of CLTA4-Ig to the increasing concentrations of IC87114 in the culture ([S5 Fig](#pone.0146516.s005){ref-type="supplementary-material"}). We did not detect an effect of CTLA-4 Ig with the bead array, which could be due to the fact that the high concentration of IFN-γ in the samples exceeding the top limit of the assay ([S5 Fig](#pone.0146516.s005){ref-type="supplementary-material"}). {#pone.0146516.g005} Combination treatment with CTLA4-Ig does not increase protection from diabetes by IC87114 {#sec019} ----------------------------------------------------------------------------------------- As production of IFN-γ was further decreased in BDC2.5 CD4^+^ T cells *in vitro* when CTLA4-Ig was added to IC87114 in culture, we tested whether oral administration of IC87114 in combination with CTLA4-Ig could prevent or delay onset of diabetes in NOD *scid* mice after transfer of BDC2.5 CD4^+^ T cells ([Fig 6A](#pone.0146516.g006){ref-type="fig"}). We found that intraperitoneal injections of CTLA4-Ig every other day for the first 10 days after transfer significantly delayed onset of diabetes in recipient mice ([Fig 6B](#pone.0146516.g006){ref-type="fig"}), but that combination with twice daily administration of IC87114 did not enhance the protective effect, but rather appeared to attenuate it ([Fig 6B](#pone.0146516.g006){ref-type="fig"}). {#pone.0146516.g006} Combination with CTLA4-Ig does not alter or potentiate the *in vitro* or *in vivo* effects of IC87114 on Th1 differentiated effector T cells {#sec020} -------------------------------------------------------------------------------------------------------------------------------------------- We also assessed whether addition of CTLA4-Ig had an additional inhibitory affect on IFN-γ production from already Th1 differentiated effector CD4^+^ T cells. We found that, just as seen in [Fig 3](#pone.0146516.g003){ref-type="fig"}, that the Th1 differentiated effector cells proliferated equally well in the presence of IC87114 ([Fig 3A](#pone.0146516.g003){ref-type="fig"}), but that their IFN-γ production, although more robust than from naïve T cells, was decreased by IC87114 in a dose dependent way. Addition of CTLA4-Ig to the culture had no effect on proliferation ([Fig 7A](#pone.0146516.g007){ref-type="fig"}), and had some effect on IFN-γ production, not appearing to result in synergistic suppression ([Fig 7B](#pone.0146516.g007){ref-type="fig"}). Neither treatment with CTLA4-Ig on its own nor in combination with IC87114 affected development of diabetes after transfer of Th1 differentiated effector BDC2.5 CD4^+^ T cells into wt NOD mice ([Fig 7C](#pone.0146516.g007){ref-type="fig"}), as all mice regardless of treatment developed diabetes 5 to 6 days after injection of the diabetogenic cells. {#pone.0146516.g007} IC87114 does not alter the percentage of regulatory T cells, but reduces their production of IL-10 {#sec021} -------------------------------------------------------------------------------------------------- Treatment with IC87114 appeared to break the temporary tolerance afforded by CTLA4-Ig treatment after islet specific T cell transfer ([Fig 6B](#pone.0146516.g006){ref-type="fig"}). As the effects of IC87114 on the cytokine production of these cells were so striking, we wondered if effects of IC87114 on the regulatory T cell pool could be even more important, and thus cancel out and even override any anti-inflammatory effects. We found that oral administration of IC87114 had no effect on the percentages of Treg present in the spleen, inguinal or pancreatic lymph nodes of treated mice ([Fig 8A](#pone.0146516.g008){ref-type="fig"}) or on their absolute numbers (data not shown), and that presence of IC87114 in culture medium did not affect proliferation of sorted Treg from Foxp3-GFP reporter NOD mice *in vitro* ([Fig 8B](#pone.0146516.g008){ref-type="fig"}). However, we found that cytokine production was strongly affected by IC87114, demonstrating a \~65% drop in IL-10 production in the presence of 5mM IC87114 ([Fig 8C](#pone.0146516.g008){ref-type="fig"}). {#pone.0146516.g008} Discussion {#sec022} ========== IC87114 has a profound suppressive effect on the production of proinflammatory cytokines such as IFN-γ and IL-17 as well as IL-2 from islet-specific cells activated *in vitro*, while increasing the production of anti-inflammatory cytokines such as IL-4 and IL-10. As the development of diabetes in NOD mice depends on the production of IFN-γ from CD4^+^ T cells \[[@pone.0146516.ref030]\], and IFN-γ production and IL-17 production is elevated in islet-reactive CD4^+^ T cells in recent onset type 1 diabetes patients \[[@pone.0146516.ref005], [@pone.0146516.ref031]\], we hypothesised that administration of IC87114 could have beneficial effects on anti-islet inflammation and be a feasible option of type 1 diabetes therapy. A previous study has demonstrated modest positive effects of IC87114 on the development of diabetes in NOD mice \[[@pone.0146516.ref032]\], and we hypothesised that combination of this agent which has such dramatic effects on production of pro-inflammatory cytokines even from already activated cells \[[@pone.0146516.ref015]\] with another well tolerated treatment targeting the activation of T cells \[[@pone.0146516.ref011], [@pone.0146516.ref026]\] would have a chance of preventing disease completely. Indeed, preliminary studies showed that genetic inactivation of PI3Kδ and CD28 resulted in indefinite acceptance of islet allografts. However, we found that, despite seeing considerable inhibition of pro-inflammatory cytokine production even at the lowest concentrations of IC87114 tested, treatment with IC87114 had no effect on the development of diabetes either after adoptive transfer of naïve BDC2.5 CD4^+^ T cells into NOD *scid* mice or after transfer of Th1 differentiated effector BDC2.5 CD4^+^ T cells into wild type NOD mice. The group sizes we used for *in vivo* experiments were small, and it cannot be excluded that differences between groups could have been detected had the sample size been larger. However, as no positive effects of IC87114 were detected in the preliminary *in vivo* experiments, we decided against using larger numbers of mice which might have allowed us to detect subtle differences between groups. Adding CTLA4-Ig to cultures had specific effects, targeting CD4^+^ T cell proliferation and production of IL-2 from these cells, and combination of CLTA4-Ig with IC87114 had a major effect on IFN-γ production from CD4^+^ T cells. This was considered promising as IFN-γ production from CD4^+^ T cells that has been shown to be particularly important in the pathology of diabetes in mice as well as men \[[@pone.0146516.ref005], [@pone.0146516.ref030]\]. However, combination treatment with twice daily oral administration of IC87114 and intraperitoneal injections with CTLA4-Ig every other day for 10 days did not lead to protection from diabetes either after transfer of naïve cells or pre-activated Th1 cells. In fact, treatment with IC87114 appeared to attenuate the protective effect afforded by CTLA4-Ig injection, raising the question of whether effects on the regulatory T cell subset may be greater than any effects on anti-inflammatory cytokine production. We found that although IC87114 treatment did not change the percentage of Foxp3^+^ regulatory CD4^+^ T cells present in secondary lymphoid organs, these cells produced less anti-inflammatory IL-10 when activated in the presence of IC87114, and are thus also a feasible targets of IC87114 treatment. Production of anti-inflammatory cytokines such as IL-10 \[[@pone.0146516.ref033]\] and TGF-β is known to suppress development of diabetes in NOD mice \[[@pone.0146516.ref034], [@pone.0146516.ref035]\], and it is not surprising that a reduction in the production of anti-inflammatory cytokines could precipitate disease. Regulatory T cells have been identified as a target of PI3K p110δ inhibitors, and targeting p110δ signalling in these cells increases anti-tumour immune responses by releasing cytotoxic CD8^+^ T cells from Treg control \[[@pone.0146516.ref036], [@pone.0146516.ref037]\] even though lack of p110δ signalling also resulted in decreased CD8^+^ T cell expression of activation marker CD44 as well as granzyme B and perforin. Our findings demonstrate major effects of IC87114 on the production of pro-inflammatory cytokines from diabetogenic T cell clones *in vitro*, but no decrease in their diabetogenicity *in vivo*. In fact, we see that treatment with IC87114 abrogates the protective effects of CTLA4-Ig treatment and causes a quicker progression to diabetes after cell transfer. Our results indicate that IC87114 is not a promising candidate for treatment of type 1 diabetes, even in combination with other drugs such as CTLA4-Ig, but that it leaves the *in vivo* inflammatory response unchanged. Instead, our data adds weight to the proposal that PI3K p110δ inhibition, as a means to reduce the effect of Treg, is a feasible strategy for breaking tumour-specific immune tolerance to achieve improved cancer immunotherapy. Supporting Information {#sec023} ====================== ###### Effects of IC87114 on cytokine production in BDC2.5 CD4^+^ T cells. Cells isolated from the spleens and lymph nodes of BDC2.5 TCR transgenic NOD mice were stimulated with the BDC2.5 mimotope (0.5 μg/mL) with or without increasing concentrations of IC87114 (0.6--10μM) for 48 hours. Cytokines from supernatants were assessed in duplicate using a bead cytokine array, differences between groups were tested using the student t-test. (TIF) ###### Click here for additional data file. ###### Effects of IC87114 on cytokine production in G9C8 CD8^+^ T cells. Cells isolated from the spleens and lymph nodes of G9C8 TCR transgenic NOD mice were stimulated with the insulinB 15--23 peptide (0.5 μg/mL) with or without increasing concentrations of IC87114 (0.6--10μM) for 48 hours. Cytokines from supernatants were assessed in duplicate using a bead cytokine array, differences between groups were tested using the student t-test. (TIF) ###### Click here for additional data file. ###### Effects of IC87114 on the distribution of divisions and activation status. Cells isolated from the spleens and lymph nodes of BDC2.5 TCR transgenic NOD mice were stimulated with the BDC2.5 mimotope (0.5 μg/mL) with or without increasing concentrations of IC87114 (0.6--10μM) for 72 hours, and then stained for CD25 A histogram overlay of representative cultures gated on CD4^+^ cells (A, left) and a graph showing all data (A, right). Cells isolated from the spleens and lymph nodes of G9C8 TCR transgenic NOD mice were stained with CFSE and stimulated with the insulinB 15--23 peptide (0.5 μg/mL) with or without increasing concentrations of IC87114 (0.6--10μM) for 72 hours. A histogram overlay of representative cultures gated on CD8^+^ cells (B, left), and a graph showing all data (B, right). Differences between groups were tested using the student t-test. (TIF) ###### Click here for additional data file. ###### Survival of MHC mis-matched islets in streptozotocin induced diabetic recipients. Wt C57BL/6 mice, CD28 KO, PI3K p110D910A (D910A) and CD28-D910A double deficient mice (DKO) were rendered diabetic through injection of streptozotocin. Diabetic mice received a MHC mis-matched (Cba1-C57BL/6 F1 donor) islet graft under the kidney capsule. Blood glucose was monitored in the recipient mice for up to 215 days. Some DKO mice that remained euglycemic for a long time underwent nephrectomy at the end of the experiment to ascertain that the graft was the cause of the restored euglycemia. The difference in euglycemic survival between wt recipient mice and DKO recipient mice was assessed using the Log Rank survival test, resulting in a p-value of 0.0027 (\*\*). (TIF) ###### Click here for additional data file. ###### Effects of combination of CTLA4-Ig and IC87114 on cytokine production in BDC2.5 CD4^+^ T cells. Cells isolated from the spleens and lymph nodes of BDC2.5 TCR transgenic NOD mice were stimulated with the BDC2.5 mimotope (0.5 μg/mL) in the presence of CTLA4-Ig (100 ng/mL) with or without increasing concentrations of IC87114 (0.6--10μM) for 48 hours. Cytokines from supernatants were assessed in duplicate using a bead cytokine array, differences between groups were tested using the student t-test. (TIF) ###### Click here for additional data file. The authors wish to thank Yvonne Sawyer, Nigel Miller, Lynn Broom and Jonathan Clark for excellent technical assistance, and Nick Holmes for helpful discussion of the data. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: ABB KO MW. Performed the experiments: ABB JE AR MW. Analyzed the data: ABB AR AC MW. Contributed reagents/materials/analysis tools: FSW AC. Wrote the paper: ABB FSW AC KO MW.

1. Introduction {#sec1-sensors-16-00822} =============== The world population is aging rapidly. As time goes on, the proportion of the elderly relative to the total population has increased, and continues to increase, especially in developed countries \[[@B1-sensors-16-00822]\]. Thus, helping seniors live a better life is crucial and has great societal benefits. Although the elders have the option of going to nursing homes or hospice care, most of them would prefer to stay in their own houses where they feel more familiar and comfortable. Limited funding for public healthcare services and the shortage of registered nurses are also driving factors for the adoption of a home-based assisted living paradigm. Therefore, healthy aging at home has become one of the most active research areas \[[@B1-sensors-16-00822]\], especially the problem of abnormal activity detection \[[@B2-sensors-16-00822],[@B3-sensors-16-00822],[@B4-sensors-16-00822],[@B5-sensors-16-00822]\]. The elderly living alone in isolated areas have been in need of emergency attention, and in the worst cases, some were found dead in their homes \[[@B6-sensors-16-00822]\]. Traditionally, abnormal activity detection approaches use cameras to obtain the data of full human body movements \[[@B7-sensors-16-00822]\]. However, there are challenging issues in vision-based methods, such as computational complexity in image processing, data consistency under different illumination conditions, and privacy infringement of the human target \[[@B8-sensors-16-00822]\]. These problems make the practical deployment of vision-based systems difficult. An alternative method is to collect sensing data from wearable motion sensors and detect abnormal activities based on the collected sensing data \[[@B2-sensors-16-00822]\]. Although motion sensors worn on the human body or integrated into human clothing can collect motion data with much less volume of data compared to those from vision-based systems, such wearable devices may make the human subject feel obtrusive. In addition, the elders are prone to forget wearing the devices after they change clothes. In addition, having to recharge the wearable devices regularly, even after deliberate design of power management units, is inconvenient for the user \[[@B2-sensors-16-00822]\]. In order to serve as a reliable and robust abnormal activity detection system for the elderly living alone, the following factors should be considered: robust to the change of environment, especially the light illumination;protective to the residents' privacy;convenient to use, especially for the elderly. Bearing those factors in mind, a Pyroelectric Infrared (PIR) sensing paradigm offers a promising alternative to the optical and wearable counterparts \[[@B3-sensors-16-00822]\]. PIR sensors are non-intrusive sensors and only sensitive to the infrared radiation changes induced by human motion, which makes them robust to interference caused by clustered background and illumination variance. In addition, as PIR sensors are relatively cheap and can be embedded within home environments, such as ceiling-mounted deployment, they are suitable to be used for home-based assisted living. However, there are some challenges facing the PIR based sensing system for abnormal behavior detection. The first one is the design of sensor nodes, which are required to capture the spatio-temporal characteristic of the human motion. Furthermore, as the data generated continuously by ambient PIR sensors, there is an increasing demand to analyze those ever-growing sensing data automatically, with little human intervention. Most importantly, the problem of abnormal activity detection is computationally challenging \[[@B4-sensors-16-00822]\]. Here, we define abnormal activities as events that they have not been expected in advance. Unlike normal activities, the abnormal samples are extremely scarce, or even non-existent. It is impossible to acquire or simulate all kinds of abnormal samples to train the system beforehand. In this paper, we propose a PIR-based sensing system for anomaly detection. We design a PIR sensor node that can capture the spatio-temporal feature of human motion effectively. The key to achieve this target is by leveraging the visibility modulation of each sensor to enhance spatial resolution. We employ the reference structure tomography (RST) paradigm \[[@B9-sensors-16-00822]\] to segment the Field of View (FOV) of each PIR sensor into sampling cells. Thus, different human activities will generate discriminative spatio-temporal signals under the monitor region. Next, we use the Hidden Markov Models (HMMs) \[[@B10-sensors-16-00822]\], one kind of generative models, to profile normal activities. Each training sample is modeled by an HMM, and their dissimilarity is calculated based on the Kullback--Leibler (KL) divergence \[[@B11-sensors-16-00822]\]. A self-tuning spectral clustering algorithm is used to cluster similar training samples, without the need to specify the number of cluster and the distance kernel width manually \[[@B12-sensors-16-00822]\]. Finally, One-Class Support Vector Machines (OSVM) \[[@B13-sensors-16-00822]\] are setup to profile the normal activities, and any unexpected activities will be classified as anomaly. It is worth pointing out that our system is trained in an unsupervised manner, which aims to avoid the laborious and inconsistent manual data labeling process. The rest of the paper is structured as follows: [Section 2](#sec2-sensors-16-00822){ref-type="sec"} introduces the related work. [Section 3](#sec3-sensors-16-00822){ref-type="sec"} provides the design and implement of the PIR sensors. [Section 4](#sec4-sensors-16-00822){ref-type="sec"} offers the overview of our proposed method. [Section 5](#sec5-sensors-16-00822){ref-type="sec"} presents the framework of human activity representation, dissimilarity calculation and clustering. [Section 6](#sec6-sensors-16-00822){ref-type="sec"} depicts the usage of the OSVM algorithm for abnormal activity detection. The experimental results are provided in [Section 7](#sec7-sensors-16-00822){ref-type="sec"}. Conclusions and future work are given in [Section 8](#sec8-sensors-16-00822){ref-type="sec"}. 2. Related Works {#sec2-sensors-16-00822} ================ Traditionally, cameras have been used for human activity classification and abnormal activity detection \[[@B14-sensors-16-00822]\]. The processing of video includes background subtraction, human motion extraction and activity modeling \[[@B15-sensors-16-00822]\]. The video data streams usually contain tens of thousands of pixels in each frame, and their intensity is easily effected by the change of illumination \[[@B5-sensors-16-00822]\]. The excessive computational burden and the feeling of privacy intrusion make it difficult to be employed massively in real home environment. Wearable sensors is another paradigm for anomaly detection. Yin *et al.* \[[@B4-sensors-16-00822]\] propose a two-stage approach for detecting abnormal activities. The first stage is to train an OSVM to model the commonly normal activities, and the second stage is to derive abnormal activity models from the suspicious activities filtered by the first stage. Zhu *et al.* \[[@B2-sensors-16-00822]\] propose using the wearable sensors together with location information provided by the camera to detect abnormal activities. A probabilistic framework is used to model different anomalies, including spatial anomalies, timing anomalies, duration anomalies and sequence anomalies. However, the biggest inconvenience of wearable sensing is that the sensors have to be recharged regularly even after deliberate design of power control units \[[@B2-sensors-16-00822]\]. PIR sensing is a promising choice besides camera-based and wearable-based sensing. In \[[@B16-sensors-16-00822]\], three PIR sensor models which are deployed in a hallway are used to detect the movement of eight human targets, including the two moving directions, three distance intervals and three speed levels. PIR sensor models can also be used to construct wireless sensor networks, which are intended to track and recognize multiple human targets \[[@B17-sensors-16-00822]\]. Using only the binary information obtained by infrared sensors attached to the ceiling of a room, the human positions can be estimated, and even the number of humans in the room changes dynamically \[[@B18-sensors-16-00822]\]. To capture the spatio-temporal feature of human movement, the monitored region is segmented into discrete sampling cells \[[@B19-sensors-16-00822]\]. By leveraging the idea of compressive infrared sampling, the FOV of each PIR sensor is modulated by reference structure \[[@B9-sensors-16-00822]\]. In \[[@B20-sensors-16-00822]\], PIR sensors were used to extract the spatio-temporal feature of the human motion from the infrared radiation domain. Ten aerobic exercises, 360 examples in total, which were performed in front of the PIR sensor node were recorded, and the nearest neighbor classifier was then used to classify different exercises. Furthermore, they presented a PIR-based compressive classification method for recognizing six typical physical activities \[[@B21-sensors-16-00822]\]. Similar to the experimental setup in \[[@B16-sensors-16-00822]\], three sensor models were located on the ceiling, with opposite tripods facing each other. SVM and HMMs were used to evaluate the performance of their system \[[@B21-sensors-16-00822]\]. PIR sensors are also used to detect abnormal activities, especially fall detection. In \[[@B22-sensors-16-00822]\], PIR sensors were deployed in a distributed sensing paradigm, which aimed at capturing the synergistic motion patterns of head, upper-limb and lower-limb. The experiment results of fall detection were encouraging. However, their system was side-view, which means it was easily occluded by other objects, and the falls had to occur perpendicular to the FOV of the PIR sensors. In other words, it was view-dependent. To overcome these limitations, Luo *et al.* \[[@B3-sensors-16-00822]\] proposed using the ceiling-mounted PIR sensor array to implement a fall detection system, *SensFall*. To achieve fall detection, the normal and abnormal training samples had to be collected beforehand. In other words, it was the supervised machine learning paradigm \[[@B23-sensors-16-00822]\]. However, the abnormal detection is clearly a cost sensitive problem \[[@B4-sensors-16-00822]\] because the samples of abnormal behavior were rare, or even non-existent. All types of the anomalies can not be elaborated on in advance. If we can only acquire the sensing data generated from normal activities, how can we train the system for abnormal activity detection automatically? This is the motivation of our study. In this paper, we propose a PIR sensor based sensing paradigm for abnormal activity detection in an unsupervised fashion. To avoid the laborious and inconsistent manual data labeling process, we propose using the self-tuning spectral clustering algorithm to discover the number of normal activities automatically. The KL divergence is employed to measure the similarity between each pair of normal training samples and construct the similarity matrix. HMMs are then utilized to profile each cluster of training samples, and OSVM is trained to detect abnormal activities. The details of our method will be elaborated on in the following sections. 3. Sensing System {#sec3-sensors-16-00822} ================= 3.1. Sensing Model {#sec3dot1-sensors-16-00822} ------------------ In this subsection, we review the design of our sensing model. The task of the sensing model is to capture the discriminative spatio-temporal feature of the human activities. The schematic diagram of our sensing model is shown in [Figure 1](#sensors-16-00822-f001){ref-type="fig"}a. Our model springs from the reference structure tomography (RST), which uses multidimensional modulations to encode the mapping between radiating objects and sensor measurements \[[@B9-sensors-16-00822]\]. The object space refers to the space where humans perform different activities. It is the 3D physical space where human motion will generate varying radiation patterns. The measurement space refers to the space where the PIR sensors are located. Before visibility modulation, the output of all the PIR sensors are the same; they can not be used for activity classification. To capture the spatio-temporal feature of human motion, we segment the object space into discrete cells. The projection of these sampling cells on the ground is shown in [Figure 1](#sensors-16-00822-f001){ref-type="fig"}b. Assume that the object space Ω is divided into *L* discrete non-overlapping sampling cells, denoted as $\Omega_{i}$, then $\Omega = \cup_{i}\Omega_{i},\Omega_{i} \cap \Omega_{j} = \varnothing$, where $1 \leq i,j \leq L$. Assume that there are *M* sensors located in the measurement space. The visibility function $v_{ji}$ is binary valued, depending on whether the sampling cell $\Omega_{i}$ is visible to the *j*th PIR sensor: $$\begin{array}{r} {v_{ji} = \begin{cases} 1 & {{\Omega_{i}~{is}~{visible}~{to}~{the}~j{th}~{PIR}}} \\ 0 & {{otherwise}} \\ \end{cases}} \\ \end{array}$$ The output of the *j*th PIR sensor is given by $$\begin{array}{ccl} {m_{j}{(t)}} & = & {h{(t)}*\sum\limits_{i = 1}^{L}v_{ji}\int_{\Omega_{i}}s{(\mathbf{r},t)}d\mathbf{r}} \\ & = & {\sum\limits_{i = 1}^{L}v_{ji}{\lbrack h{(t)}*\int_{\Omega_{i}}s{(\mathbf{r},t)}d\mathbf{r}\rbrack}} \\ & = & {\sum\limits_{i = 1}^{L}v_{ji}s_{i}{(t)}} \\ \end{array}$$ where "\*" denotes convolution, $h(t)$ is the impulse response of the PIR sensor, and $\Omega_{i} \in R^{3}$ is the *i*th sampling cell. $s(\mathbf{r},t)$ is the thermal density function in the object space, and then $s_{i}{(t)} = h{(t)}*\int_{\Omega_{i}}s{(\mathbf{r},t)}d\mathbf{r}$ is the sensor measurement of sampling cell $\Omega_{i}$. Equation (1) can be equivalently represented in a matrix form as $$\begin{array}{r} {\mathbf{M} = \mathbf{V}\mathbf{S}} \\ \end{array}$$ where $\mathbf{M} = \left\lbrack m_{j}{(t)} \right\rbrack \in \mathbb{R}^{M \times 1}$ is the measurement vector of PIR sensors, $\mathbf{V} = \left\lbrack v_{ji} \right\rbrack \in \mathbb{R}^{M \times L}$ is the measure matrix determined by the visibility modulation scheme, and $\mathbf{S} = \left\lbrack s_{i}{(t)} \right\rbrack \in \mathbb{R}^{L \times 1}$ is the sensor measurement of the sampling cells before visibility modulation. $\mathbf{M}$ can be regarded as a linear measurement of the radiation variation within all cells. The human body can be regarded as an infrared radiative source to the surrounding environment. In comparison with the whole object space, the human body is sparsely distributed. Thus, the change of infrared radiation induced by human motion takes place only in a few sampling cells. This can be regarded as a compressive sensing problem \[[@B24-sensors-16-00822]\], and the activity classification in the object space is projected into an analogous problem in the measurement space. When the signal is sparse or compressible, learning and classification directly in the compressive measurement domain are possible in the compressed sensing framework \[[@B21-sensors-16-00822],[@B25-sensors-16-00822]\]. 3.2. Reference Structure Implementation {#sec3dot2-sensors-16-00822} --------------------------------------- To implement the sensing model described in the previous subsection, which segments the object space into discrete sampling cells, we employ two kinds of masks. These masks play the role of reference structure, which modulates the FOV of PIR sensors. The first type of mask, Type I, is a fan shape, as shown in [Figure 1](#sensors-16-00822-f001){ref-type="fig"}c. After applying such mask, the FOV of the PIR sensor is no longer a full cone, but a partial cone shape, called a fan cone. The second type of mask, Type II, is a ring shape, as shown in [Figure 1](#sensors-16-00822-f001){ref-type="fig"}d. The FOV of the PIR sensor after masked is still a full cone, but its cone angle *β* is less than that of the original cone. These two types of masks provide two degrees of freedom (DOF) spatial partitions. In our system implementation, the performance of our system will improve as the number of PIR sensors increases \[[@B20-sensors-16-00822]\]. Because of the hardware constraint of our sensor node, seven PIR sensors with masks are multiplexing to segment the object space into sampling cells, as shown in [Figure 1](#sensors-16-00822-f001){ref-type="fig"}b. Four PIR sensors are masked by Type I mask, and the remaining three PIR sensors are masked by the Type II mask. In such a configuration, the object space is segmented into 17 sampling cells. Referring to Equation (2), $M = 7,L = 17$, and the measurement matrix *V* is shown in [Figure 2](#sensors-16-00822-f002){ref-type="fig"}. 4. Proposed Algorithm {#sec4-sensors-16-00822} ===================== Based on the implementation of our sensing model, human activity under the object space will generate PIR data streams correspondingly. The measurement of PIR sensors are segmented by the Short-Time Energy method automatically \[[@B3-sensors-16-00822],[@B26-sensors-16-00822]\]. Given a collection of normal samples $\left\{ Y_{1},Y_{2},...,Y_{N} \right\}$, our abnormal activity detection method works in two phases. [Figure 3](#sensors-16-00822-f003){ref-type="fig"} shows a diagrammatic illustration of our method. In the first phase, by applying the self-tuning spectral clustering, the number of activities classes *C* is determined automatically, and the normal traces are grouped accordingly. In the second phase, each class of activity is modeled by an HMM. The equal-length feature vectors are constructed based on the likelihood output of training samples generated by these *C* HMMs. The OSVM is then trained for abnormal activity detection. It shows clearly that the spectral clustering algorithm is the core of our approach. The key components of our approach are explained in detail in the following sections. 5. Spectral Clustering {#sec5-sensors-16-00822} ====================== To profile the normal activities in an unsupervised fashion, we employ the spectral clustering method to cluster similar sequences. However, because the lengths of these sequential data are different and vary greatly in value, it is a challenging issue to model these data for better similarity measures. 5.1. Likelihood Matrix Construction {#sec5dot1-sensors-16-00822} ----------------------------------- Since the training sequences are generated by a hidden mechanism associated with human's underlying activities, it is reasonable to model these sequences using a generative model \[[@B27-sensors-16-00822]\]. In this work, we adopt a set of HMMs to model the training sequences. HMMs are a type of non-deterministic stochastic finite state automata, which are widely employed in signal processing and pattern recognition \[[@B28-sensors-16-00822]\]. The parameters of a continuous HMM with Gaussian mixture emissions can be represented in the following compact form: $$\begin{array}{r} {\lambda = \left\{ \pi,A,\mu,\Sigma \right\}} \\ \end{array}$$ where *π* is the initial state probability distribution, *A* is the state transition probability distribution, *μ* is the mean vector, and Σ is the covariance matrix. The *i*th training sequence $Y_{i}$ can be presented as the output of *M* PIR sensors, $$\begin{array}{r} {Y_{i} = \begin{bmatrix} {m_{1}{(1)}} & {m_{1}{(2)}} & \ldots & {m_{1}{(T_{i})}} \\ \vdots & \vdots & \ddots & \vdots \\ {m_{M}{(1)}} & {m_{M}{(2)}} & \ldots & {m_{M}{(T_{i})}} \\ \end{bmatrix}} \\ \end{array}$$ where $m_{j}{(t)}$ is the output of the *j*th PIR sensor at time *t*, $t = 1...T_{i}$. By using the Baum--Welch algorithm \[[@B10-sensors-16-00822]\], we fit *N* HMMs, one for each individual sequence $Y_{i},1 \leq i \leq N$. To calculate the distance between each pair of these sequences, a probabilistic model-based framework for sequence clustering is proposed in \[[@B29-sensors-16-00822]\]. The likelihood matrix $\mathbf{L} = \left\{ l_{ij} \right\}$, whose $ij$th element is defined as $$\begin{array}{r} {l_{ij} = \log\mspace{720mu} p_{ij} = \frac{1}{{length}(Y_{j})}\log P{(Y_{j};\lambda_{i})},1 \leq i,j \leq N} \\ \end{array}$$ where $Y_{j}$ is the *j*th sequence, $\lambda_{i}$ is the model trained for the *i*th sequence, and $P(Y_{j};\lambda_{i})$ is the likelihood of $Y_{j}$ generated by model $\lambda_{i}$. 5.2. Sequence Distance Measures {#sec5dot2-sensors-16-00822} ------------------------------- After the likelihood matrix $\mathbf{L}$ is constructed, the original variable-length sequence clustering problem is transformed to a typical similarity-based one. The *j*th column of $\mathbf{L}$ represents the likelihood of sequence $Y_{j}$ under each of the trained models. The next step is to define a meaningful distance measure for these sequences. A popular paradigm is to obtain likelihood-based distances between each pair of sequences \[[@B29-sensors-16-00822]\]. Based on this work, several other distance measures have been proposed under a similar philosophy \[[@B27-sensors-16-00822],[@B30-sensors-16-00822],[@B31-sensors-16-00822]\]. However, the main limitation of these methods is that they only consider the distance between two sequences each time, not including the global information of the whole set of data. Hence, we propose using the definition of distance measurement from a probabilistic perspective \[[@B32-sensors-16-00822]\]. We regard the likelihood of each of the sequences under the trained models as samples from the conditional likelihoods of the models given the data, which embeds information from the whole data set \[[@B32-sensors-16-00822]\]. This gives rise to highly structured distance matrices to give a better performance in comparison with aforementioned distance-based methods \[[@B27-sensors-16-00822],[@B29-sensors-16-00822],[@B30-sensors-16-00822],[@B31-sensors-16-00822]\]. According to the definition of likelihood matrix $\mathbf{L}$, the *j*th column of $\mathbf{L}$ can be regarded as the likelihood of the sequence $Y_{j}$ under each of the trained models $\lambda_{i},1 \leq i \leq N$. These *N* models can be regarded as a set of "sampled points" from the model space Λ surrounding the HMMs that actually span the data space. Thus, these *N* trained models become a good discrete approximation $\overline{\Lambda} = \left\{ \lambda_{1},...,\lambda_{N} \right\}$ to the model space of interest. If we normalize the likelihood matrix $\mathbf{L}$, which means each column adds up to one, we get a new matrix $\mathbf{L}_{N}$ whose columns can be regarded as the probability density functions (pdfs) over the approximated model space conditioned on each of the individual sequences: $$\begin{array}{r} {\mathbf{L}_{N} = \left\lbrack f_{\overline{\Lambda}}{(Y_{1})},...,f_{\overline{\Lambda}}{(Y_{N})} \right\rbrack} \\ \end{array}$$ This interpretation leads to the Kullback--Leibler (KL) divergence, which is a natural choice for the measurement of the dissimilarity between two pdfs. The discrete case of the KL divergence formulation is as follows: $$\begin{array}{r} {D_{KL}(f_{P}{||}f_{Q}{) =}\sum\limits_{i}f_{P}{(i)}\log\frac{f_{P}{(i)}}{f_{Q}{(i)}}} \\ \end{array}$$ where $f_{P}$ and $f_{Q}$ are two discrete pdfs. Obviously, the KL divergence is not a proper distance because of its asymmetry; a symmetrized version is used as: $$\begin{array}{r} {D_{KL}^{sym}(f_{P}{||}f_{Q}{) =}\frac{1}{2}\lbrack D_{KL}(f_{P}{||}f_{Q}{) +}D_{KL}(f_{Q}{||}f_{P}{)\rbrack}} \\ \end{array}$$ Thus, the distance between the sequences $Y_{i}$ and $Y_{j}$ can be defined as: $$\begin{array}{r} {d_{ij} = D_{KL}^{sym}(f_{\overline{\Lambda}}{(Y_{i})}{||}f_{\overline{\Lambda}}{(Y_{j})})} \\ \end{array}$$ Distances defined this way are obtained according to the patterns created by each sequence in the probability space spanned by different models, and the distance measured between two sequences $Y_{i}$ and $Y_{j}$ involves information related to the rest of the data sequences. 5.3. Similarity Matrix Construction {#sec5dot3-sensors-16-00822} ----------------------------------- Before applying a spectral clustering algorithm, the distance matrix $\mathbf{D} = \left\{ d_{ij} \right\}$ should be transformed into a similarity matrix $\mathbf{S} = \left\{ s_{ij} \right\}$. A commonly used procedure is to apply a Gaussian kernel, $$\begin{array}{r} {s_{ij} = \begin{cases} {exp( - \frac{d_{ij}^{2}}{2\sigma^{2}})} & {{for}\mspace{720mu} i \neq j} \\ 0 & {{for}\mspace{720mu} i = j} \\ \end{cases}} \\ \end{array}$$ where *σ* is the scaling parameter controlling the kernel width. The value of *σ* is commonly specified manually, or numerous iteration has to be run for a range of *σ* \[[@B33-sensors-16-00822]\]. However, when the input data includes clusters with different local statistics, a single value of *σ* may not work well for all the data. Thus, instead of selecting a single scaling parameter, we propose calculating a local scaling $\sigma_{i}$ for each data point $d_{i}$ \[[@B12-sensors-16-00822]\]. The similarity between $Y_{i}$ to $Y_{j}$ can be revised as $d_{ij}/\sigma_{i}$ while the converse is $d_{ji}/\sigma_{j}$. Therefore, $d_{ij}$ is symmetry, and the Equation (10) can be generalized as: $$\begin{array}{r} {{\hat{s}}_{ij} = \begin{cases} {exp( - \frac{d_{ij}^{2}}{\sigma_{i}\sigma_{j}})} & {{for}\mspace{720mu} i \neq j} \\ 0 & {{for}\mspace{720mu} i = j} \\ \end{cases}} \\ \end{array}$$ where $\sigma_{i} = d{(Y_{i},Y_{K})}$, $Y_{K}$ is the K'th neighbor of $Y_{i}$. The selection of *K* is independent of scale and is a function of the data dimension of the embedding space. Thus, the scaling parameters for each pair of $Y_{i}$ and $Y_{j}$ are not fixed; they are determined automatically according to the local statistics of the neighborhoods. 5.4. Self-Tuning Spectral Clustering {#sec5dot4-sensors-16-00822} ------------------------------------ After the similarity matrix $\hat{\mathbf{S}} = \left\{ {\hat{s}}_{ij} \right\}$ is constructed, we apply spectral clustering methods to partition the training sequences into clusters. For an undirected graph *G* with vertices $v_{i}$ and edges $s_{ij}$, the matrix $\mathbf{S}$ could be considered as an adjacent matrix for *G*, where each element $s_{ij}$ can be viewed as the similarity between the vector $v_{i}$ and $v_{j}$. The target of spectral clustering is to partition the *G* into a distinct sub-graph. It is a tricky problem to specify the number of clusters *C*. One method to discover the number of clusters is to analyze the eigenvalues of the normalized Laplacian matrix $\mathbf{L}_{a}$, which is based on the similarity matrix $\hat{\mathbf{S}}$. The analysis given in \[[@B33-sensors-16-00822]\] shows the number of repeated eigenvalues of magnitude 0 with multiplicity equal to the number of clusters *C*. However, eigenvalues depend on the structure of the individual clusters, and no assumptions can be placed on their values \[[@B12-sensors-16-00822]\]. Once noise is introduced, the eigenvalues deviate from the ideal case, and it is difficult to decide the number of clusters. An alternative approach to discover the number of clusters *C* automatically is to analyze the eigenvectors of Laplacian matrix $\mathbf{L}_{a}$ \[[@B12-sensors-16-00822]\]. Assume the matrix $\mathbf{X} = \left\lbrack x1,...,x_{C} \right\rbrack \in \mathbb{R}^{N \times C}$ is constructed by stacking the largest eigenvectors of $\mathbf{L}_{a}$ in columns. In the ideal case where the data points could be separated distinctly, $\mathbf{X}$ will be strictly block diagonal after sorting the eigenvectors of $\mathbf{L}_{a}$. Nevertheless, in the general case, the $\mathbf{X}$'s off-diagonal blocks are non-zero, and the eigensolver could just pick any other set of the orthogonal vectors; $\mathbf{X}$ could have been replaced by $\hat{\mathbf{X}} = \mathbf{XR}$ for any orthogonal matrix $\mathbf{R} \in \mathbb{R}^{C \times C}$. Now, we have to recover the rotation which best aligns $\mathbf{X}$'s columns with the canonical system with the minimum cost. Let $\mathbf{Z} \in \mathbb{R}^{N \times C}$ be the matrix obtained after rotating the eigenvector matrix $\mathbf{X}$; that is, $\mathbf{Z} = \mathbf{XR}$. We wish to recover the rotation $\mathbf{R}$ for which, in every row in $\mathbf{Z}$, there will be at most one non-zero entry. We thus define the cost function: $$\begin{array}{r} {J = \sum\limits_{i = 1}^{N}\sum\limits_{j = 1}^{C}\frac{Z_{ij}^{2}}{M_{i}^{2}}} \\ \end{array}$$ where $M_{i} = \max_{j}Z_{ij}$. Minimizing this cost function over all possible rotations will provide the best alignment with the canonical coordinate system. The number of clusters, *C*, is taken as the one providing the minimal cost. The spectral clustering algorithm that we apply is similar to the one proposed in \[[@B12-sensors-16-00822]\]. The algorithm works as follows: Define a diagonal degree matrix $\mathbf{D} = \left\{ d_{ij} \right\}$ with $d_{ii} = \sum_{j = 1}^{N}{\hat{s}}_{ij}$, and then construct the normalized Laplacian matrix $\mathbf{L}_{a} = \mathbf{D}^{- 1/2}\hat{\mathbf{S}}\mathbf{D}^{- 1/2}$.Find $C^{\prime}$ principal eigenvectors $x_{1},x_{2},...,x_{C^{\prime}}$ and form the matrix $\mathbf{X} = \left\lbrack x1,...,x_{C}^{\prime} \right\rbrack \in \mathbb{R}^{N \times C^{\prime}}$ by stacking the eigenvectors in columns, where $C^{\prime}$ is the largest possible cluster number.Recover the rotation $\mathbf{R}$ which best aligns $\mathbf{X}$'s columns with the canonical coordinate system using the incremental gradient descent algorithm \[[@B12-sensors-16-00822]\].According to Equation ([12](#FD12-sensors-16-00822){ref-type="disp-formula"}), grade the cost of the alignment for each group number up to $C^{\prime}$. Set the final group number $C_{\text{best}}$ to be the largest group number with minimal alignment cost.Take the alignment result $\mathbf{Z}$ of the $C_{\text{best}}$ eigenvectors, and assign the original point $s_{i}$ to cluster c if and only if $\text{max}_{j}{(Z_{ij}^{2})} = Z_{ic}^{2}$. In our experiments, because the voulunteers will emulate five kinds of activities, $C^{\prime}$ is set to 10, and the self-tuning spectral clustering will determine $C_{\text{best}}$ automatically. 6. One-Class SVM Classifier {#sec6-sensors-16-00822} =========================== 6.1. Feature Extraction {#sec6dot1-sensors-16-00822} ----------------------- After applying the spectral clustering algorithm, we can group the *N* training traces into *C* clusters, which correspond to *C* different types of activities. To train an OSVM, we need to transform the training samples that are of variable lengths into a set of fixed-length feature vectors. Again, we apply HMMs to model these normal activities, one for each cluster. For each learned model with the corresponding parameters ${\hat{\lambda}}_{i}$, $1 \leq i \leq C$, we calculate the log-likelihood of each of the *N* normal traces given the model parameters ${\hat{\lambda}}_{i}$. The log-likelihood value for each pair of trace and HMMs is computed as follows: $$\begin{array}{r} {L{(Y_{i};{\hat{\lambda}}_{j})} = \log P{(Y_{i};{\hat{\lambda}}_{j})},1 \leq i \leq N,1 \leq j \leq C} \\ \end{array}$$ This is calculated by applying the standard forward-backward algorithm \[[@B10-sensors-16-00822]\]. In this way, for each training trace $Y_{i},1 \leq i \leq N$, we can obtain a *C*-dimensional feature vector $\mathbf{x}_{i} = {\langle L{(Y_{i};{\hat{\lambda}}_{1})},...,L{(Y_{i};{\hat{\lambda}}_{C})}\rangle}$. 6.2. One-Class SVM Training {#sec6dot2-sensors-16-00822} --------------------------- After transforming the *N* training traces into a set of feature vector $\mathbf{x}_{1},...,\mathbf{x}_{N}$, we can train the one-class SVM for normal activities. The basic idea is to find a sphere that contains most of the normal data such that the corresponding radius *R* can be minimized: $$\begin{array}{cl} \min\limits_{R,\xi,\mathbf{a}} & { R^{2} + \nu\sum\limits_{i = 1}^{n}\xi_{i}} \\ {s.t.} & {{{||}}\mathbf{x}_{i} - \mathbf{a}{||}^{2} \leq R^{2} + \xi_{i}} \\ & {\xi_{i} \geq 0} \\ \end{array}$$ The slack variables $\xi_{i}$ are introduced to allow some data points to lie outside the sphere, and the parameter $\nu \geq 0$ controls the tradeoff between the volume of the sphere and the number of errors. Using the dual representation of the Lagrangian \[[@B34-sensors-16-00822]\], the objection function is equivalent to $$\begin{aligned} \min\limits_{\alpha} & {\sum\limits_{i,j = 1}^{n}\alpha_{i}\alpha_{j}\left\langle \mathbf{x}_{i} \cdot \mathbf{x}_{j} \right\rangle - \sum\limits_{i = 1}^{n}\alpha_{i}\left\langle \mathbf{x}_{i} \cdot \mathbf{x}_{i} \right\rangle} \\ {s.t.} & { 0 \leq \alpha_{i} \leq \nu,\sum\limits_{i = 1}^{n}\alpha_{i} = 1} \\ \end{aligned}$$ This quadratic programming (QP) problem can be solved using standard optimization techniques \[[@B35-sensors-16-00822]\]. To determine if a testing sample is within the sphere, the distance to the center of the sphere has to be calculated. If the distance is larger than the radius *R*, the testing sample is considered abnormal. Typically, the training samples are not spherically distributed in the input space. Thus, the original data points are first mapped into a feature space so that a better data description can be obtained. Instead of requiring an explicit mapping function from the input space to the feature space, the solution can be obtained by replacing all the inner products $\left\langle \cdot , \cdot , \right\rangle$ in Equation ([15](#FD15-sensors-16-00822){ref-type="disp-formula"}) by a kernel function $k( \cdot , \cdot )$: $$\begin{array}{r} {\min\limits_{\alpha} k{(\mathbf{x}_{i} \cdot \mathbf{x}_{j})} - \sum\limits_{i = 1}^{n}\alpha_{i}k{(\mathbf{x}_{i},\mathbf{x}_{i})}} \\ \end{array}$$ In our context, due to the noisy and nonlinear characteristic of the PIR sensors, the decision boundary of the OSVM is quite complex. Thus, we apply the Gaussian Radial Basis Function (RBF) kernel for the OSVM, which is defined as follows: $$\begin{array}{r} {k{(\mathbf{x}_{\mathbf{i}},\mathbf{x}_{\mathbf{j}})} = \left. exp( - \gamma \middle| \right|\mathbf{x}_{i} - \mathbf{x}_{j}{||}^{2})} \\ \end{array}$$ where $\gamma > 0$ is a scaling factor that controls the width of the kernel function. 7. Experimental Evaluation {#sec7-sensors-16-00822} ========================== In order to evaluate the performance of our proposed method, experiments were carried out on a real data set collected from a wireless sensor network. Our proposed method is referred to as SC+OSVM in the experiments. Two other approaches were also used for comparison. We list these three methods as the following: SC+OSVM---The method proposed in our study, mainly including self-turning spectral clustering and One-Class SVM.SC+iForest---The difference between this method and the SC+OSVM is that we use isolation forest to replace One-Class SVM for abnormal detection. Isolation forest is an alternative algorithm for abnormal detection \[[@B36-sensors-16-00822]\].OneHMM---All the normal training samples are modeled by only one HMM, which corresponds to not applying spectral clustering to the unlabeled samples. A threshold is set to distinguish normal and abnormal activities. 7.1. Experimental Setup {#sec7dot1-sensors-16-00822} ----------------------- The experiments were carried out in a real indoor environment. The monitored region covered by the sensor node was a cone with a 3 m radius. The sensor node is with seven PIR sensors, each of them equipped with fresnel lens arrays and a mask, as shown in [Figure 4](#sensors-16-00822-f004){ref-type="fig"}. The CC2430 module is used for data transmission between the sensor node and the sink based on ZigBee protocol. More details of the sensor node could be found in our previous work \[[@B3-sensors-16-00822]\]. There were eight volunteers that participated in our experiments, including three females and five males. The height of them ranges from 1.64 m to 1.80 m, and the weight of them ranges from 50 kg to 70 kg. Each volunteer emulated five kinds of activities, including falling, sitting down, standing up from a chair, walking and jogging. Every activity was emulated ten times by each volunteer at a self-select speed and strategy, as shown in [Figure 5](#sensors-16-00822-f005){ref-type="fig"}. Totally, we obtained 400 samples, including 80 fall-simulated samples and 320 normal activity samples. In our experiments, fall-simulated samples are regarded as abnormal activities, and other samples are regarded as normal activities. Each sample is segmented automatically by thresholding Short-Time Energy, and all the normal training samples are unlabeled. 7.2. Evaluation Metrics {#sec7dot2-sensors-16-00822} ----------------------- The performance of the abnormal human-activity detection methods can be evaluated in terms of two rates, detection rate and false alarm rate. The detection rate is computed as the ratio of the number of correctly detected abnormal activities to the total number of abnormal activities. The false alarm rate is computed as the ration of the number of normal activities that are incorrectly detected as abnormal activities to the total number of normal activities. Based on the confusion matrix shown in [Table 1](#sensors-16-00822-t001){ref-type="table"}, the two metrics can be defined as follows: $$\begin{aligned} {\text{Detection}\text{Rate}} & {= \frac{TP}{TP + FN}} \\ \end{aligned}$$ $$\begin{aligned} {\text{False}\text{Alarm}\text{Rate}} & {= \frac{FP}{FP + TN}} \\ \end{aligned}$$ The performance of an ideal abnormal human-activity detection algorithm should have a high detection rate and a low false alarm rate. Therefore, we evaluate the performance of the algorithms using an Receiver Operating Characteristic (ROC) curve, which plots the detection rate against the false alarm rate. In addition, we compute the area under the ROC curve (AUC) to compare these algorithms. AUC is a better measurement than accuracy in the evaluation of learning algorithms \[[@B37-sensors-16-00822],[@B38-sensors-16-00822]\], especially in the cost-sensitive problems. A desirable algorithm with a high detection rate and a low false alarm rate should have an AUC value closer to one. 7.3. Experimental Results {#sec7dot3-sensors-16-00822} ------------------------- In our experiments, because the output of the sensor node was a seven-dimensional data stream with continuous values, as shown in [Figure 6](#sensors-16-00822-f006){ref-type="fig"}, we employed HMMs with Gaussian observation density. The number of hidden states and the number of Gaussian models are determined by the log-likelihood of the training samples \[[@B3-sensors-16-00822]\]. Specifically, we use HMMs with two Gaussians and eight states to profile the general human normal activity and each cluster of normal activities. For OSVM, the parameter *ν* was set to 0.01. Experiments were conducted to compare the performance of all three of the algorithms. We randomly selected 240 normal samples for training. The other 80 normal samples and all the 80 abnormal samples were randomly mixed together for testing. [Figure 7](#sensors-16-00822-f007){ref-type="fig"} shows the ROC curve with respect to the detection rate and the false alarm rate. We can see from the figure that SC+OSVM gives the best detection result. This is mainly because the self-turning spectral clustering can estimate the number of different types of activities automatically and then cluster the similar activities accordingly. The discriminative feature vectors are helpful to improve the accuracy of OSVM. We also conducted experiments to investigate the effect of varying the number of training samples on the performance of the three algorithms. In these experiments, we kept the amount of testing data unchanged and reduced the number of training samples. [Figure 8](#sensors-16-00822-f008){ref-type="fig"} and [Figure 9](#sensors-16-00822-f009){ref-type="fig"} show the experimental results using 160 and 80 normal traces for training, respectively. We can see from the figure that, when the number of normal traces for training decreases, the performance of the three algorithms decreases correspondingly. The reason lies in two aspects. First, for HMMs, which are generative models \[[@B3-sensors-16-00822],[@B9-sensors-16-00822]\], sparse training samples affect the accuracy of parameter estimation of the models. Second, for OSVM, when the training samples are sparse, the calculated decision boundary may not exactly capture the characteristics of normal activities. Therefore, the abilities of three algorithms to distinguish normal and abnormal activities degrade. As shown in [Figure 9](#sensors-16-00822-f009){ref-type="fig"}, when there are only 80 normal samples for training, the performance of OneHMM and SC+iForest are comparable to that of SC+OSVM because all of them could not model the normal activities well with less training samples. When the training samples are less than 80, the performance of three algorithms will be worse and unsatisfactory. Again, as shown in [Figure 8](#sensors-16-00822-f008){ref-type="fig"}, when we have 160 normal samples for training, SC+OSVM still performs the best among the three algorithms. To explicitly compare the performance of the three algorithms, we computed the AUC values by calculating the area under the ROC curves depicted in [Figure 7](#sensors-16-00822-f007){ref-type="fig"}, [Figure 8](#sensors-16-00822-f008){ref-type="fig"} and [Figure 9](#sensors-16-00822-f009){ref-type="fig"}. The results are summarized in [Table 2](#sensors-16-00822-t002){ref-type="table"}. We can see from the first column of the table that, when 240 normal samples are used for training, the AUC value for SC+OSVM, SC+iForest and OneHMM are 0.863, 0.375 and 0.354, respectively. SC+OSVM performs better than the other two algorithms. In addition, SC+OSVM is the best among the tree algorithms when we consider 80 and 160 normal samples for training as well. Another observation is that the performance of OneHMM will not be improved as the number of training samples increases. This is because the dissimilarity between different activities will degrade the classification ability of the HMMs; a single HMM to model all normal activities is not discriminative enough. By contrast, after applying the self-turning spectral clustering, each type of normal activities will be profiled by an HMM; it will obviously improve the performance of the OSVM. 8. Conclusions {#sec8-sensors-16-00822} ============== In this paper, we propose a novel approach for detecting a human's abnormal activities. By employing the FOV modulation for PIR sensors, the human activity is encoded into low-dimensional data streams, which can be used to extract the tempo-spatial feature of the human motion. A self-turning spectral clustering algorithm is used to cluster the training samples with modified KL distance. The HMMs are trained to profile each cluster of activity. One-class SVM is setup to classify whether the testing samples are abnormal or not. A major advantage of our approach is that it does not need abnormal samples for training in advance. This is critical in real deployment because it is unrealistic to train the system by providing all kinds of abnormal activities. Another advantage is that our training procedure is an unsupervised learning fashion, which does not need to specify the number of kinds of normal activities. In other words, it is not necessary to label the training samples. This advantage will facilitate the mass deployment in different locations, and it can greatly reduce the human labor spent on training sample preprocessing. We demonstrate the effectiveness of our approach using real data collected from PIR sensors attached to the ceiling of the monitoring region. It shows that, as the number of training samples increases, the performance of our system will improve accordingly. In the future, we wish to continue in the direction of detecting abnormal activities from continuous data streams. We will investigate how to incorporate the location information with the motion information to improve the performance of our system. A robust and reliable abnormal activity detection system is the most important prerequisite of the home-based assisted living paradigm. This work is supported by the National Natural Science Foundation of China (NSFC) under Grant No. 61301294 and 61401174, the Natural Science Foundation of Guangdong under Grant No. 2014A030310462, and the Youth Elite Project of Guangzhou University of Chinese Medicine. Study concept and design: Xiaomu Luo. Acquisition of data: Xiaomu Luo, Qiuju Guan, Tong Liu and Baihua Shen. Analysis and interpretation of data: Xiaomu Luo and Huoyuan Tan. Drafting of the manuscript: Xiaomu Luo. Discussion and reviewing the manuscript: Qiuju Guan and Hankz Hankui Zhuo. All authors read and approved the final manuscript. The authors declare no conflict of interest. {#sensors-16-00822-f001} {#sensors-16-00822-f002} {#sensors-16-00822-f003} {#sensors-16-00822-f004} {#sensors-16-00822-f005} {#sensors-16-00822-f006} {#sensors-16-00822-f007} {#sensors-16-00822-f008} {#sensors-16-00822-f009} sensors-16-00822-t001_Table 1 ###### Confusion matrix. Actual Activity ----------- ---------- --------------------- ------------------------ Predicted Abnormal True Positive (TP) False Postivitive (FP) Label Normal False Negative (FN) True Negative (TN) sensors-16-00822-t002_Table 2 ###### Area under the ROC curve (AUC) values with different algorithms and different numbers of training samples. 240 Samples 160 Samples 80 Samples ------------ ------------- ------------- ------------ SC+OSVM 0.863 0.433 0.710 SC+iForest 0.375 0.404 0.332 OneHMM 0.354 0.379 0.360

1. Introduction {#sec1} =============== Mucormycosis, an uncommon invasive fungal infection, occurs predominantly in debilitated or immunosuppressed hosts. The conditions predisposing to mucormycosis include malignant hematological disease, prolonged and severe neutropenia, poorly controlled diabetes mellitus with or without diabetic ketoacidosis, iron overload, major trauma, prolonged use of corticosteroids, illicit intravenous drug use, neonatal prematurity and malnourishment, and chronic renal insufficiency \[[@B1]\]. However, mucormycosis has been described in previously healthy individuals as well \[[@B2], [@B3]\]. Pulmonary mucormycosis has been reported in renal failure, either as a part of chronic uremia or after transplantation \[[@B2], [@B3]\]. In a literature search spanning 30 years, 13% of the patients with pulmonary mucormycosis had renal disease of which 55% were posttransplant patients \[[@B3]\]. It almost always occurs in patients with an established renal disease. Renal involvement can also occur as a part of a disseminated disease. Mucormycosis has not been reported as the initial presentation of chronic renal failure. The mortality rate is often high, 65% with isolated pulmonary mucormycosis, 96% for those with disseminated disease, and 80% overall. Moreover, mucormycosis is an unusual cause of massive hemoptysis \[[@B4], [@B5]\]. Here we report fatal pulmonary mucormycosis as the initial presentation of chronic renal failure. 2. Case Report {#sec2} ============== A 26-year-old male expatriate was brought to the emergency of Sultan Qaboos University Hospital, Muscat, Oman, in severe respiratory distress. He was having vomiting, shortness of breath, productive cough, and mild fever for almost a week. There was no history of any previous illness. On arrival he was in severe distress with a respiratory rate of 44/minute, heart rate of 103/minute, and blood pressure of 148/97 mmHg. Arterial blood gas analysis while on oxygen showed severe metabolic acidosis {pH---6.9, PCO~2~---12 mmHg, PO~2~---600 mmHg, and HCO~3~---4.4 mmol/L}. Creatinine and urea were very high and the hemoglobin was very low. The basic blood test results were as follows: fasting blood sugar---4.9 mmol/L; creatinine---1327 *μ*mol/L; urea---46.8 mmol/L; bicarbonate---2 mmol/L; sodium---119 mmol/L; potassium---5.8 mmol/L; glomerular filtration rate---4 mL/min/1.73 m^2^; anion gap---22 mmol/L; calcium---2.18 mmol/L; phosphate---3.98 mmol/L; lactate---0.7 mmol/L; hemoglobin---5.4 g/dL; white cell count---52 × 10^9^/L; lactate dehydrogenase---378 U/L; creatine kinase---2548 U/L; INR---1.29; activated partial thromboplastin time---69.2 seconds. Urine dipstick showed the presence of proteins, glucose, and red blood cells. Chest radiograph showed consolidation in the right mid zone and slight blunting of the right costophrenic angle ([Figure 1](#fig1){ref-type="fig"}). The grossly elevated creatinine and urea, low hemoglobin, severe metabolic acidosis, bilateral small kidneys in ultrasound scan of the abdomen, and a high white cell count suggested a primary renal involvement complicated by a pneumonic illness and possibly sepsis. He deteriorated rapidly and was electively intubated and mechanical ventilation was initiated. He received supportive care, fluids, measures to reduce potassium, and broad spectrum antibiotics. Since the acidosis and the renal function did not show any improvement, he was taken up for dialysis later on the same day. Bronchoscopy showed inflamed right bronchi and the subdivisions lined by a thick layer of yellow secretions ([Figure 2](#fig2){ref-type="fig"}). The bronchial washings and brushings showed broad aseptate hyphae with right angled branching consistent with*Mucor* species ([Figure 3](#fig3){ref-type="fig"}). Amphotericin (liposomal amphotericin B, 7 mg/kg/day) was added along with the broad spectrum antibiotics which he was receiving since admission. He continued to have dialysis on a regular schedule. Though there was mild improvement in the clinical and metabolic parameters, he continued to be critically ill. He was extubated on the seventh day of admission and was shifted to the ward once the vitals and the level of consciousness were stable. Two days later during dialysis he suddenly developed hypotension. The patient was conscious and communicating and the blood pressure picked up with inotropes. However, he suddenly developed massive hemoptysis. Though he was reintubated and cardiopulmonary resuscitation was initiated he could not be resuscitated. 3. Discussion {#sec3} ============= Mucormycosis is an invasive fungal infection caused by members of the family Mucoraceae and occurs predominantly in debilitated or immunosuppressed hosts. Mucormycosis is an uncommon disease, even in high-risk patients, and represents 8.3%--13% of all fungal infections encountered in such patients \[[@B1]\]. Six predominant clinical forms of the disease exist, which are, in decreasing frequency, rhinocerebral, pulmonary, disseminated, cutaneous, gastrointestinal, and uncommon rare forms \[[@B1]\]. Mucormycosis has been reported in patients of chronic renal failure on treatment as a complication or a terminal event. However, it is rare in an undiagnosed renal failure. Patients with chronic renal failure on maintenance hemodialysis and those receiving deferoxamine therapy for aluminum toxicity have been reported to be more susceptible to mucormycosis. Renal transplant recipients on conventional immunosuppressive therapy are also more prone to develop mucormycosis with an incidence varying from 0.4 to 2% \[[@B2], [@B6]\]. Mucormycosis usually occurs in the first year after renal transplant \[[@B2]\]. Isolated renal mucormycosis has occurred in intravenous drug users as well as renal transplant recipients in developing countries with warm climates such as India, Egypt, Saudi Arabia, Kuwait, and Singapore \[[@B1]\]. Studies have shown that renal failure portends a poor outcome, and neutropenia in patients with mucormycosis was clearly a predictor of death \[[@B3]\]. Renal involvement has been reported in up to 22% patients with disseminated mucormycosis, but isolated involvement is rare. In a review of 49 patients published in 1971, 10% had uremia. Renal failure is almost universal in patients with bilateral renal involvement \[[@B6]\]. Gupta et al. reported a patient presenting with renal failure and recent GI bleed who had disseminated disease including kidney involvement \[[@B7]\]. In a series of nine cases of fatal disseminated mucormycosis, four patients had chronic renal failure while five had acute renal failure: only two of the latter had proven renal involvement \[[@B8]\]. Primary mucormycosis of the renal allograft is a dreaded disease with a grave prognosis \[[@B9], [@B10]\]. Interestingly, our patient had features of renal failure and mucormycosis on first presentation. An immunodeficient state in renal failure seems to be the major factor responsible for increased vulnerability to invasion by opportunistic infections. Decreased cell-mediated immunity and impaired neutrophil function have been documented in renal failure for a long time \[[@B11]\]. In addition, the accompanying acidosis increases the susceptibility to mucormycosis since the iron required for hyphal growth is released from transferrin as the blood pH drops \[[@B12]\]. Specific host immune defects predispose to different forms of mucormycosis. Patients with diabetic ketoacidosis are prone to develop rhinocerebral form and pulmonary mucormycosis typically affects severely immunocompromised individuals. A hallmark of mucormycosis is extensive angioinvasion with resultant vessel thrombosis and tissue necrosis. Interaction of Mucorales spores with endothelial cells appears to play a critical role in angioinvasion \[[@B13]\]. Sudden, massive hemoptysis is a common fatal complication \[[@B4], [@B5]\]. The most common causes of death are fungal sepsis (42%), respiratory insufficiency (27%), and hemoptysis (13%). Our patient had no apparent previous illness. He thus presented for the first time with features of a pulmonary infection and advanced renal failure. This young patient\'s acute presentation and rapid deterioration are likely due to the pulmonary mucormycosis in the background of chronic renal failure. There was no evidence of renal mucormycosis or disseminated disease from the available evidence. It would be logical to conclude that he had chronic renal failure (undiagnosed/neglected) and the severe fungal infection brought him to the hospital. Tough, sudden, massive hemoptysis is not usual; it is a common cause of mortality in patients with pulmonary mucormycosis. Conflict of Interests ===================== The authors declare that there is no conflict of interests regarding the publication of this paper. {#fig1} {#fig2} {#fig3} [^1]: Academic Editor: Phuong Chi Pham

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Introduction {#s1} ============ The interaction between various myeloid and lymphoid cell populations is crucial to initiate and orchestrate a robust anti-tumor response. By processing tumor associated antigens (TAAs) and migrating to draining lymph nodes (dLN), where T cell priming occurs, dendritic cells (DCs) are considered the most potent professional antigen presenting cells (APCs) to elicit adaptive anti-tumor immunity ([@B1]). In addition to presenting antigens, DCs use soluble molecules such as cytokines and chemokines as well as direct cell-cell contacts to prime and activate TAA-specific T cells. DCs were discovered by Ralph Steinman and Zanvil Cohn in 1973 as an APC population, distinct from macrophages, that initiate adaptive immune responses ([@B2]). As a result of more recent deep-phenotyping, DCs are now recognized to be a heterogenous population comprising several subsets distinguished by their development, phenotypic differences, localization, and functional specialization ([@B2]--[@B6]). This functional specialization of each subset allows DCs to initiate distinct immune responses in different immunological contexts ([@B7]). Here, we review literature supporting the hypothesis that, although one DC subset, conventional DC1(cDC1), has been shown to be crucial for anti-tumor immunity, multiple DC subsets, and interactions with other cells are needed for maximal responses. DC Subsets Are Functionally Specialized --------------------------------------- DCs are broadly classified as classical (or conventional) DCs (cDCs) and plasmacytoid DCs (pDCs), each with specialized functions. cDCs, specialized in antigen presentation to naïve T cells can be further segregated into cDC1s and cDC2s, excelling in MHC class I- and class II-mediated antigen-presentation, respectively ([@B3], [@B6], [@B8]--[@B10]). cDCs are found both as lymphoid and non-lymphoid tissue cells, the latter of which can migrate via the lymph to dLN to present tissue-derived antigens ([@B3], [@B11]). cDC1s, present at lower frequency compared to cDC2s, are identified by the expression of XCR1 ([@B12]), and in humans, also by the expression of CD141 (BDCA3) ([@B5], [@B13], [@B14]). cDC1s possess specialized mechanisms to mediate efficient antigen recognition, antigen transport to appropriate endosomal compartments and subsequent processing for the presentation to CD8 T cells in a process known as cross-presentation ([@B15]--[@B18]). cDC1s can also activate CD4 T cells through MHC class II antigen presentation and can polarize activated CD4 T cells toward a Th1 phenotype through the secretion of IL-12 ([@B19]). cDC2s are specialized in MHC class II-mediated antigen presentation and are the most efficient APCs for activation and expansion of CD4 T cells ([@B5], [@B13], [@B20]). They are the most frequent DC population present in blood, lymphoid organs and tissues and promote a wide range of immune responses including Th1, Th2, and Th17 in specific contexts ([@B13], [@B19], [@B21]--[@B25]). Human cDC2s can be identified by their preferential expression of CD1c (BDCA1) and CD172a (SIRPα) ([@B26]). cDC2s are more heterogenous than cDC1s, and express various receptors that enable them to respond to broad spectrum of microbial products ([@B22], [@B26]--[@B28]). A subset of Notch2-dependent cDC2s specializes in IL-23 production and contributes to innate defense and adaptive immune responses ([@B27], [@B29]). pDCs, distinguished by their ability to produce large amounts of type I IFN upon viral infection ([@B30]--[@B33]) are identified, in humans, by the expression of surface markers CD303 (BDCA-2), CD304 (BDCA-4/Neuropilin) and CD123 ([@B5], [@B13]). They are present mainly in lymphoid organs and can migrate to the LN through blood circulation ([@B5], [@B34]). Mature pDCs can also act as APCs and have distinct regulation of MHC class II surface expression that results in sustained membrane peptide-MHC complex and antigen presentation ([@B30]). A heterogeneity of pDCs is also described in terms of their ability to produce type I IFN and/or antigen presentation ([@B35], [@B36]). Another related but developmentally distinct population, derived from monocytes, termed monocyte DCs (moDC) upregulates certain functional properties of DCs in some contexts and express tumor necrosis factor (TNF)- α and intracellular nitric oxide synthase (iNOS) ([@B37]). More commonly, the term moDCs refers to monocyte isolated from human peripheral blood mononuclear cells (PBMC) that are *in vitro* differentiated in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 into cells sharing several phenotypic and functional features of DCs ([@B26], [@B38], [@B39]). moDCs are the most common *in vitro* model of DCs, yet are quite heterogeneous in both mouse and human, with unclear relationship to *in vivo* cell populations ([@B40]--[@B42]). All DC subsets, including cDCs and pDCs, are found in the tumor microenvironment (TME) ([@B30], [@B43]--[@B47]) and among the cDCs, the cDC2s outnumber cDC1s, with cDC1s being the rarest APCs within the TME ([@B43], [@B48]). The role of pDCs in tumor immunity remains elusive and contradictory. Similarly, the precise role of cDC2s in anti-tumor immunity has been difficult to delineate due to lack of proper genetic tools. On the contrary, mounting evidence suggests cDC1s to be the critical antigen presenting DC subset for the generation of anti-tumor immunity. Here we summarize data supporting the importance of cDC1s in anti-tumor immunity, and then review the recent literature that documents DC crosstalk being necessary for effective immune responses, in other contexts such as anti-viral immune responses, and apply these principles to tumor immunity. cDC1s Are Necessary for Anti-tumor Immunity ------------------------------------------- Since MHC class I molecules are expressed by every cell in the body (not just infected cells and cancer cells), to avoid bystander killing of healthy cells by CTLs, extracellular antigens do not enter the MHC class I-loading machinery ([@B15], [@B18]). Therefore, to generate an immune response, cancer cell antigens need special processing in APCs to be presented to naïve CD8 T cells. Moreover, naïve CD8 T cells primarily circulate through secondary lymphoid organs ([@B15]). Hence, cancer antigens must be brought to secondary lymphoid organs to be presented to naïve CD8 T cells. cDC1s fulfill both functions by patrolling tumor tissues, and by capturing, processing and presenting tumor-antigens on their surface through MHC class I molecules via antigen cross-presentation. cDC1s then migrate to dLN and deliver peptide/MHC class I signal to CD8 T cells which leads to their activation and the initiation of an immune response against tumor cells ([@B15], [@B18]). Although other cell types have been reported to cross-present antigens ([@B11], [@B49]), this specialized function is mostly attributed to the cDC1 subset, owing to their unique adaptations of subcellular molecular machinery and vesicular trafficking ([@B15], [@B18]). Such adaptations include efficient antigen uptake of dying cells, delivery of cell-associated antigen to early endosomes, ([@B15], [@B50]--[@B52]), efficient phagosome-to-cytosol export of an ingested antigen possibly aided by ER-derived translocons and ER-associated degradation (ERAD) components such as Sec61, Derlin, p97 ATPase, Sec22 ([@B15], [@B53]--[@B55]), lower expression of lysosomal proteases ([@B50]) and antagonizing their degradative functions via NOX2-mediated ROS generation ([@B56]--[@B60]). The end result of such lower proteolysis, and therefore, increased antigen retention in cDC1s, is eventually an enhanced ability to carry the antigen all the way from peripheral tissues where the antigen is captured, to the dLN, where priming and activation of CD8 T cells occurs ([@B56]). The importance of cDC1s\' ability to cross-present antigen in its immune functions is recently demonstrated using Wdfy4-deficient mice, which selectively lack cross-presentation ([@B61]). Beyond their role in antigen cross-presentation, cDC1s are the major source of IL-12 production and thus influence anti-tumor immunity by activating NK cells and driving CD4 T cell responses toward Th1 responses ([@B19], [@B62]--[@B64]). The critical role of cDC1s in anti-tumor immunity has been shown using mice deficient in basic leucine zipper transcription factor ATF-like 3 (Batf3), a transcription factor required for cDC1 differentiation ([@B65]). Batf3 knockout mice lack cDC1 cells but not other APCs and display impaired anti-tumor immunity in several models ([@B43], [@B65]--[@B68]). Expansion and activation of cDC1s using fms-related tyrosine kinase 3 ligand (Flt3L) and poly I:C leads to significant enhancement of antitumor responses ([@B45]). Immunotherapies such as PD1/PD-L1 blockade or CD137 agonists are ineffective in Batf3-deficient mice, highlighting the crucial role cDC1s in tumor immunity ([@B68], [@B69]). Furthermore, tumor-resident cDC1s are required for trafficking of adoptively transferred CD8 T cells into tumors through their ability to produce CXCL9 and CXCL10 ([@B67], [@B70]). DC-specific deletion of Sec22b leads not only to impaired cross-presentation of TAAs and reduced anti-tumor immune responses but also abolishes the efficacy of anti-PD1 therapy ([@B53]). In humans, the presence of cDC1s within the TME is associated with better prognosis and response to immunotherapy. Analysis of the cancer genome atlas (TCGA) dataset shows that a higher ratio of a cDC1 gene signature to a signature of all other myeloid cells (including monocyte/macrophage, and not just other DC subsets) is associated with better prognosis across human tumors ([@B44], [@B71]). Abundance of CD8 T cells positively correlates with cDC1 markers in pancreatic tumors ([@B70]). Taken together, these data show that cross-presenting cDC1s are crucial and necessary for the generation of an effective anti-tumor immunity. cDC1 Are Not Sufficient for Maximal Anti-tumor Immunity: Potential Roles of Other DC Subsets -------------------------------------------------------------------------------------------- Tumor immunology is built upon the tenet that cytotoxic CD8 T cells (CTLs) eliminate tumor cells ([@B72]) and the prevailing dogma is that cDC1s are the most potent APCs for the CTL response against tumor. Because of the strong evidence for the importance of cDC1 in tumor immunity, as presented in the previous section, in one scenario it is possible that cDC1s are the sole DC subset sufficient for optimal anti-tumor CTL generation through antigen presentation via MHC class I as well as MHC class II ([Figure 1A](#F1){ref-type="fig"}). A major driver of the current dogma is the studies conducted in mice genetically manipulated to lack cDC1 such as Batf3-deficient or Zbtb46-Cre mice. However, these tools are imperfect. For example, Batf3 is expressed in cDC2 and effector CD4 T cells ([@B65], [@B73]) and Zbtb46 is also expressed in DC2s as well as in endothelial cells ([@B74]--[@B76]), raising the possibility of contributions from additional DC subsets and other cell types. Hence, it is not clear whether the cDC1 subset alone is sufficient to provide the maximal immunity against tumor. Recent evidence in non-tumor settings has demonstrated that cDC1s require significant contributions from other DC subsets and are not sufficient for an optimal CTL response ([@B77]--[@B79]), pointing toward a role for the other cells in shaping a robust and durable anti-tumor immunity. {#F1} Therefore, we describe a second scenario that includes possible roles of other DC subsets for a more robust anti-tumor immunity, directly and indirectly ([Figure 1B](#F1){ref-type="fig"}). This scenario incorporates recent findings of spatiotemporal segregation of cDC1 and cDC2 activation within dLN to activate CD8 and CD4 T cells, respectively, during antiviral response. This robust CTL response requires interactions between multiple DC subsets, including cDCs and pDCs in a two-step priming process ([@B77]--[@B79]). Even though these responses are context dependent and are observed in anti-viral response, the general principals remain the same in anti-tumor immune response. Accordingly, in this scenario, the tumor-derived cDC1 primes CD8 T cells while tumor-derived cDC2 activates CD4 T cells in the first step of the CTL priming process and then in the second step, the activated CD4 T cells licenses a LN-resident cDC1 to relay the help for CTLs. Contributions of activated CD4 T cells to anti-tumor immunity can be more than just providing the help to CTLs, but also include activation of NK cells and macrophages through IFN-γ, modulation of tumor stroma and angiogenesis or direct cytolytic effects ([@B80]--[@B83]). Additionally, during the two-step priming process, pDCs are recruited to cDC1-CD8 T cell priming sites, providing critical licensing signal to cDC1s through type 1 IFN. In this regard, lack of type 1 IFN receptor in cDC1s impairs their ability to reject tumors ([@B84], [@B85]). Furthermore, pDCs are usually weak APCs in the absence of activating signals but direct antigen presentation and T cell stimulation by pDCs has been described ([@B30], [@B86]). In fact, adoptive transfer of tumor-antigen-loaded pDCs induced potent anti-tumor T cell responses in melanoma patients ([@B87]), suggesting the possibility of anti-tumor immunity directly through APC functions by pDCs. In the following sections, we mainly focus on this latter scenario of non-synchronous activation events by cDC1s and cDC2s and the reorganization of pDCs to the sites of CTL priming to describe the crosstalk between DC subsets and propose an integrated model of multi-DC subsets, multi-cell type interactions in achieving full-strength CTL responses in anti-tumor immunity. Crosstalk Between DC Subsets ---------------------------- One of the goals of cancer immunotherapy is to promote tumor-antigen specific T cell responses. The current data supports the notion that cDC1s are well-suited for this purpose and that they are usually necessary for the generation of an anti-tumor response. However, as discussed below, they may not be sufficient for full-strength anti-tumor cytotoxic T cell responses and interactions with other DC subsets contribute to this process. In the following sections, we will review the interactions between each DC subsets separately. ### cDC-pDC Crosstalk cDCs and pDCs are co-localized in many immune contexts, e.g., non-inflamed LNs, skin biopsies from lupus erythematosus patients, thyroid glands from autoimmune thyroiditis patients and spleens of cancer patients ([@B88]--[@B90]). Such close-proximity of pDCs and cDCs suggests possible functional coordination. Indeed, local production of type I IFN by pDCs induces stimulatory molecules on cDCs driving their maturation during an effective immune response ([@B79]). Intravital two-photon microscopic analysis of DC subsets within dLN during vaccinia virus infection showed active, CCR5-mediated recruitment of pDCs to the site of CD8 T cell priming by virus-infected cDC1. The activated CD8 T cells also orchestrate, via XCL1, recruitment of resident, non-infected XCR1^+^cDC1s. pDCs produce type I IFN to induce upregulation of costimulatory molecules including CD40, CD80, and CD86 on non-infected resident-cDC1s ([@B79]), driving their maturation and antigen-presentation functions leading to robust CTL response. pDC help for CTL response, either through type I IFN or other costimulatory molecules such as CD40L has been described in other viral infection models ([@B91]--[@B93]). Depletion of pDCs results in impaired CTL responses in many viral infections, e.g., VSV infection ([@B94]), LCMV infection ([@B95]), and cutaneous herpes simplex virus (HSV) ([@B92]). In the LCMV infection model, pDC-mediated CD4 T cell activation was essential in providing help and generation of anti-viral CTL response ([@B95]). These observations underscore the pivotal role of the crosstalk between DC subsets in maximizing immune response against cell-associated antigens. Similarly, in the context of anti-tumor immune responses, cooperation between pDCs and cDC1s and the resulting synergistic effects dependent on soluble factors such as type I IFN and/or cell-cell contact between the two DC subsets are described ([@B11], [@B47]). The potent anti-tumor T cell responses induced in melanoma patients by adoptive transfer of tumor-antigen-loaded pDCs ([@B87]) could be either a result of direct priming by pDCs or via interactions with other cells, including cDCs. However, tumor infiltrating pDCs exhibit an abnormal or hypofunctional state, most likely due to immuno-suppressive effects of the TME such as TGFβ ([@B96]). The presence of pDCs in tumors is associated with poor prognosis in cancers such as breast and ovarian cancers ([@B97], [@B98]). pDCs are generally thought to contribute to tolerance induction and tumor promotion in this setting, most likely due to Treg induction and expression of immunosuppressive factors such as indoleamine 2,3-dioxygenase (IDO) ([@B98], [@B99]). Thus, the role of pDCs in shaping adaptive tumor immunity remains elusive. It likely depends on their activation status and involves cooperativity with other cells but how pDCs are activated needs further investigation. ### cDC1-cDC2 Crosstalk The two cDC subsets communicate not only through soluble mediators such as IL-12 but also through a third cell viz., activated CD4 T cell. Even though both cDC subsets are adept in priming naïve T cells, cDC2s are more proficient in activating CD4 T cells than CD8 T cells while cDC1s are potent activators of CD8 T cells but present antigen to CD4 T cells less efficiently, both *in vitro* and *in vivo* ([@B8], [@B20], [@B43]). However, recent literature demonstrates that robust and maximal induction of cytotoxic CD8 T cell responses against cell-associated antigens not only requires interactions with cDC1s, but also interactions involving cDC2s ([@B77], [@B100]). Intravital microscopy demonstrated that, in the dLN, the two cDC subsets exhibit differential localization wherein cDC1s are largely segregated to the T cell zone in deep paracortical regions and cDC2s are more peripherally distributed ([@B78], [@B100]--[@B103]) and that CD8 T cells cluster with cDC1s and CD4 T cells cluster with cDC2s during step one of two-step T cell priming event in anti-viral immunity ([@B78], [@B100], [@B104]), suggesting parallel activation of the two T cell subsets by two different cDCs in an asynchronous manner. Such differential localization of the cDC subsets into non-overlapping T cell regions is also reported in the spleen ([@B105]). The peripheral DC subsets also exhibit different kinetics during their migration to dLN ([@B106]), with an implication that cDC2s might access CD4 T cells earlier. The CD4 T is cell activated in the first step of the priming process, then gets recruited to LN-resident, XCR1+ cDC1 during the second step of the priming process and delivers help signals to that cDC1. The receiver-cDC1 then transmits the help signal to CD8 T cell activated in the first step, resulting in a robust expansion of highly effective CTLs. In this regard, it is well-established that, in the absence of CD4 T cell help, CD8 T cell responses are weaker and insufficient to generate long-lasting memory ([@B107]--[@B109]). The CD4 T cell help includes molecules such as CD40L expressed on CD4 T cells, that induces expression of costimulatory molecules including CD70, CD80, CD86, and cytokines such as IL-12, IL-15 by cDC1 ([@B66], [@B110]--[@B112]). The molecular nature of CD4 T cell help in shaping the CTL response is recently reviewed ([@B104]) and will not be discussed here in detail. Signaling though type I IFN is critical for proper functioning of cDC1s ([@B85]) and cDC2s are one of the important sources of this cytokine, as shown by depletion of pDCs using anti-pDCA antibodies in Batf3-deficient mice ([@B84]). cDC1s and cDC2s may also collaborate for optimal Th1 induction. In the context of leishmania infection, targeting antigen to either cDC1 or cDC2 can elicit IFNγ-producing T cells, but interestingly, the cDC2s require IL-12 produced by the cDC1s to induce Th1 responses, whereas the cDC1s induce Th1 responses via CD70, independent of IL-12 ([@B19]). Therefore, each DC subset provides different signals that can contribute to effector T cell responses. Among the activated CD4 T cells, Th1 cells excel in providing the help to cDC1s to prime and expand CTLs through of production large amounts of IFNγ ([@B113]), thus fostering an important crosstalk between the two cDCs. The majority of the experimental data described above originates from studies in anti-viral immunity. However, where and how naïve cancer cell-specific CD4 T cells get activated in a tumor setting is less clear. Lessons learnt on the importance of MHC class II-restricted CD4 T cell responses in autoimmune pathogenesis may shed light on this question in anti-tumor responses as well, since the anti-tumor response is essentially a self-specific response ([@B114]). The highest genetic risk for autoimmunity is conferred by HLA class II genes, with odds ratios \>6, suggesting that CD4 T cell responses are necessary for immunity against self. In the context of autoimmunity, although some priming in the target tissue may occur ([@B115]--[@B117]), most studies suggest that self-specific CD4 T cells are first primed in the dLN, suggesting that a similar phenomenon might be happening in the generation of an anti-tumor immune response. Evidence for the Importance of Tumor-Derived cDC2s and Activation of CD4 T Cells in the Draining Lymph Node ----------------------------------------------------------------------------------------------------------- A large body of literature shows that naïve CD8 T cell activation for the generation of anti-tumor immunity occurs in dLN and is mediated by DCs ([@B118]--[@B121]). Interestingly, requirement of CD4 T cell help for optimal CD8 T cell effector functions in the context of tumor immunity is also well-documented, including the ability of CTLs to infiltrate the tumors ([@B8], [@B119], [@B122]--[@B127]). Non-helped CD8 T cells exhibit dysfunctional state with high expression of exhaustion markers in metastatic lung tumor model ([@B127]). In this regard, it is also well-established that the TME contains both cDC1 and cDC2 subsets ([@B43]--[@B46]). But importantly, both cDC1s and cDC2s scavenge tumor antigens ([@B44]) and migrate to dLN in a CCR7-dependent manner ([@B46]). Under right conditions, cDC2s can induce CD4 T cell activation in response to cell-associated antigen ([@B51]). Consistent with this, tumor-derived and dLN-derived cDC2s stimulate CD4 T cells more efficiently, *ex vivo*, in Lewis lung carcinoma model expressing ova as a model antigen ([@B43]). Furthermore, in this experimental setting, while cDC1 efficiently primed CD8 T cells, cDC2s are the most efficient activators of CD4 T cells. In addition, vaccination with the activated cDC2s reduced tumor growth, similar to that observed with cDC1s ([@B43]). Delivery of tumor antigen to cDC2 using dendritic cell immunoreceptor 2 (Dcir2) leads to significant anti-tumor effects in a mouse melanoma model ([@B128]). In a lung adenocarcinoma mouse model engineered to express MHC class II-restricted cytosolic antigen, activated cDC2 are observed both in the tumor and dLN and antigen-specific naïve CD4 T are activated in the dLN ([@B129]). In breast cancer patients gene signature of cDC2s positively correlates with better survival, similar to that observed with cDC1s, ([@B130]) and MHC class II expression predicts response to anti-PD1/PD-L1 therapy in melanoma patients ([@B131]). Collectively, tumor-derived cDC2s are likely to contribute to CD4 T cell activation in the dLN. Integrated Model of DC Crosstalk in Tumor Draining Lymph Node ------------------------------------------------------------- The spatiotemporal nature of DC crosstalk suggests two distinct DC-mediated events for maximal CD8 T cell responses: one after the initial antigen capture and another after the antigen is transferred to dLN-resident cDC1 cells ([@B8]). This sequential CTL activation is demonstrated by the exclusive clustering of migratory cDC1s with CD8 T cells early on during the initiation of an antiviral immune response. Subsequent clustering of activated CD8 T cells with the LN-resident cDC1s acts as a platform for signal relay from pDCs and activated CD4 T cells ([@B79]). According to this "consecutive interaction" model ([@B79], [@B112]), the generation of maximal CTL response and therapeutic anti-tumor immunity requires a multicellular orchestration of events in the tumor dLN ([Figure 2](#F2){ref-type="fig"}) wherein migratory cDC1s capture the antigen in tumors, migrate to the dLN and form the initial priming site to activate CD8 T cells. The activated CD8 T cells produce CCL3/CCL4 and XCL1 to mediate recruitment of CCR5^+^pDCs and XCR1^+^LN-resident cDC1s, respectively. The migratory cDC1s handoff antigen to resident cDC1s in a yet-to-be-described mechanism ([@B44], [@B106]). In parallel, migratory cDC2s that have captured the antigen also move from the tumor to dLN and activate CD4 T cells. The pDCs induce the maturation of newly recruited, LN-resident cDC1s and the activated CD4 T cells licenses them for superior CTL responses. The overall effect of such orchestration and functional-cooperativity of pathways between different DC subsets is the amplification of CTL responses against a given antigen, without potentially missing out on the critical help necessary for CTLs to function at their peak. In fact, vaccine-mediated induction of such coordinated efforts of multiple DC subsets is known to trigger sustained and potent CTL responses while inhibiting immunosuppressive pathways in preclinical models ([@B132]). *Ex vivo* analyses of individual DC subsets might fail to identify such cellular orchestration to appreciate the relative contribution of each interaction between the different DC subsets in the generation of potent immune response. {#F2} DC Crosstalk in Tumors *in situ* -------------------------------- Accumulating evidence suggest that cross-priming by tumor-resident cDC1 *in situ* is also an important phenomenon in the generation of an anti-tumor immune response. Local T cell priming and activation within tumors were observed in mice that lacked LN, or when T cell recirculation was blocked ([@B133]--[@B135]). Furthermore, intratumoral cDCs are required for the tumor regression achieved with adoptively transferred T cells in an experimental setting where migration of T cells to dLN was prevented ([@B44]). Moreover, tumor-resident cDC1s are the predominant sources of CXCL9 and CXCL10 and mediate recruitment effector T cells into the tumor ([@B67]). Similar to the events described for the dLN in the previous section, activated CD8 T cells could potentially orchestrate events *in situ* in the tumor where LN-like structures known as tertiary lymphoid structures (TLS) are present. A hallmark of TLS is the presence of high endothelial venules (HEVs) and expression of CCL19 and CCL21, the ligands for CCR7 ([@B136], [@B137]). DCs migrate in a CCR7-dependent manner ([@B43], [@B45], [@B46], [@B138], [@B139]). Moreover, well-organized TLSs contain B cell and T cells areas with mature DC subsets including cDCs and pDCs. Such organization makes TLS an ideal place to sustain proximity and the crosstalk between various subsets, and orchestrating local events required for maximal tumor immunity ([@B135], [@B136]). In fact, tumor-associated TLSs are functional structures capable of recruiting antigen-specific T cells and facilitating their activation through interactions with DCs ([@B140]). Interestingly, TLSs have been observed in several human tumors and their presence, particularly the ones containing high amounts of DCs and Th1 cells within the TLS, is associated with better prognosis ([@B137], [@B141], [@B142]) and increased TLS density is associated with strong infiltration of effector and memory CD8 T cells within the tumors ([@B141]), reflecting the importance of crosstalk between DC subsets and, CD4 help in increased CTL trafficking. Lung cancer patients with intratumoral CD8 T cells but no TLS had poor survival, indicating the necessity of their *in situ* education within the TLS for better effector functions ([@B141], [@B143]). In a metastatic lung tumor model, administration of TLR9 activator leads to CD8 T cell infiltration concurrent with TLS formation. The presence of TLS in this model was completely dependent on CD4 help ([@B127]). Taken together, these data suggest that TLSs promote DC crosstalk and anti-tumor immunity. Thus, induction of TLS provides another opportunity to promote communication between DC subsets to augment the magnitude of protective immunity, particularly against neoantigens that arise during the later phases of tumor progression ([@B121]). Moreover, induction of simultaneous trafficking and activation of cDCs and pDCs, using a vaccination strategy that combined DC subset-specific adjuvants (e.g., CpG-ODN and GM-CSF) leads to local accumulation of CD8 T cells and superior anti-tumor responses ([@B132]) suggesting that, even in the absence of TLS, evoking appropriate DC-crosstalk within the tumor tissue has the potential to boost superior CTL responses than targeting a single DC subset. Influence of DC Crosstalk With Other Cells in the TME on Anti-tumor Immunity ---------------------------------------------------------------------------- DCs can also engage with other immune cell types in the TME and lymphoid organs. Such interactions can enhance or dampen DC functions and anti-tumor immunity, depending on the cell types involved. For example, DCs interact with Treg cells, resulting in the suppression of CD8 T cell-mediated anti-tumor immunity ([@B144]). Two-photon laser-scanning microscopy analysis showed that Treg cells engage in prolonged physical interactions with DCs, six times longer than that of DC-CD8 T cell interaction in tumor. This extended physical contact between Treg cells and DCs results in upregulation of the immunosuppressive molecules such as IDO and lower maturation molecules on DC surface ([@B144]). Interactions with other immune cell types such as natural killer (NK) cells with DCs can boost the immune response against tumors. It has long been established that, through the secretion of IL-12, cDC1s can license NK cells to kill tumor cells ([@B145]--[@B147]). However, recent studies have shown that NK cells can also influence DC functions in the context of tumors. In fact, NK cells produce XCL1 to recruit XCR^+^cDC1s to the TME ([@B148]). In addition, NK cells are one source of Flt3L within the tumor and dictate intratumoral accumulation of cDC1 cells by supporting DC survival, proliferation or development ([@B71]). Stimulation of NK cells with DC-derived factors such as IL-12, IL-15/IL-15Rα complex or contact--dependent interactions of OX40-OX40L augment NK cell functions to eliminate tumor cells ([@B149]--[@B151]). TCGA analysis suggests that NK cell/XCL1/cDC1 axis is associated with better survival in many cancer indications ([@B148]). DCs also interact with NKT cells, the unconventional T lymphocytes expressing a semi-invariant T cell receptor (TCR) that recognize glycolipids presented by CD1d. ([@B152]). Although CD1d can be expressed by many hematopoietic cell types, DCs constitutively express CD1d and are the most potent APCs for exogenous glycolipids ([@B153]--[@B155]). The NKT cell ligand α-galactosylceramide (α-GalCer) acts as a potent *in vivo* adjuvant for DCs, resulting in increased expression of MHC class II and other costimulatory molecules ([@B155]). In addition, α-GalCer presented by DCs strongly activates NKT cells through CD40/CD40L interaction to induce IFN-γ production ([@B156]). Administration of α-GalCer was efficacious in preclinical tumor models ([@B157]) but not in patients ([@B158]), most likely due to soluble α-GalCer-induced anergy of NKT cell ([@B159]). Administration of α-GalCer, either soluble or loaded in DCs, is currently being explored to enhance anti-tumor immunity ([@B160]). Endogenous glycolipids are known to activate NKT cells ([@B161]) and CD1d expression is observed on tumor cells ([@B162]). In fact, the level of CD1d expression on tumor cells dictates NKT-mediated cytotoxicity ([@B163]). Tumor-associated macrophages, in most carcinomas, are linked to poor prognosis primarily due to their immunosuppressive phenotype ([@B164], [@B165]). Macrophages produce IL-10 and in turn prevent IL-12 secretion of by DCs, resulting in dampened tumor-specific CD8 T cell activation ([@B166]). Among mononuclear phagocytes, monocyte-derived cells (including macrophages) are found at higher frequencies in tumors compared to DCs, and a higher monocyte-macrophage signature is associated with worse clinical prognosis ([@B130], [@B167]). These cells maintain a phenotype similar to *in vitro* M2 macrophages and contribute to the suppressive tumor microenvironment primarily via expression of anti-inflammatory mediators such as IL-10, TGF-β and IDO. Many of these signals dampen the ability of cDCs to present antigen in an immunogenic manner ([@B164]). However, in other contexts, macrophages can be inflammatory and effective APCs for eliciting T cell responses ([@B168], [@B169]). Thus, with the addition of the right signals, tumor macrophages have the potential to contribute to anti-tumor immunity. Additionally, even though B cells have been described to play varied and often contrasting roles in the contexts of tumor immunity, emerging evidence suggests that B cells may also contribute to tumor immunity, both via antibody-mediated effects and by acting as APCs ([@B170]--[@B172]). Specifically, in terms of the crosstalk, DCs engage with B cells to promote their growth and differentiation, resulting in the production of antibodies. pDCs, through type I IFN production, can increase TLR7 expression and other activation markers on B cells ([@B173]). pDCs are specifically capable of inducing differentiation of activated B cells into Ig-secreting plasma cells through the secretion of type I IFN and IL-6 ([@B174]). Additionally, DCs dramatically enhance the secretion of IgG and IgA through the ligation of CD40 ([@B175]). B cells isolated from TLS-containing lung cancers showed significant antibody response against many TAAs ([@B143], [@B176]). Finally, DC crosstalk with cancer cells has tremendous impact on the immune surveillance of the tumors. Cancer cells express several immunosuppressive factors such as PGE2, β-catenin and cytokines such as IL-10. PGE2 renders cDC1s unresponsive to XCL1 and CCL5 by downregulating *XCR1* and *CCR5* expression ([@B148]). β-catenin expression in cancer cells causes ATF3-mediated suppression of CCL4, the ligand for CCR5, leading to defective recruitment of cDC1 to the TME, and adversely affecting CD8 T cell priming against TAAs ([@B177]). Interestingly, PGE2 also induces the expression of β-catenin not only in tumor cells but also in stromal cells such as cancer associated fibroblasts (CAFs). CAFs respond to tumor-derived TNFα and IL-1β to secrete thymic stromal lymphopoietin (TSLP). TSLP is a strong driver of cDCs to activate Th2 CD4 T cells that are considered pro-tumorigenic ([@B178]). CAFs also produce stromal cell-derived factor 1 (SDF1) which drives cDCs toward tolerogenic DCs secreting IDO in a STAT3-dependent manner and promoting the recruitment and differentiation of Treg cells in tumors ([@B179]). However, co-targeting fibroblasts in combination with DC-based vaccine enhances the anti-tumor immune responses ([@B180]), suggesting that DC/stromal cell interactions can be manipulated to improve immunotherapies. Overall, with the property of bridging the innate and adaptive immune cells, DCs have a pivotal role in orchestrating an anti-tumor immune response by engaging interactions with many cell types within the TME. Potential Therapeutic Applications of Tumor DC-Crosstalk -------------------------------------------------------- The field of cancer immunotherapy, energized by the effect of T cell checkpoint inhibitors (CPI) in some patients, is beginning to focus on ways to treat "cold" tumors that lack T cells which can be activated with an anti-PD1 or other CPI. There is a large unmet medical need to increase the proportion of patients who respond to immunotherapy. Enhancing innate immunity, and DC function in particular, is one way to make tumors "warmer" that has tremendous potential. To date, most cell-based DC cancer therapies have utilized moDCs and have shown limited efficacy ([@B121], [@B181], [@B182]). With our current knowledge of both the importance of cross-presenting cDC1s for tumor immunity and the plasticity of monocyte-derived cells, moDCs are likely not the best cell type to use for inducing optimal clinical outcomes against cancer. Most studies show that moDCs have limited capacity for both cross-presentation and migration to draining LN compared to Batf3-dependent cells ([@B43], [@B183]). In addition, most monocyte-derived cells in the TME are immunosuppressive, and even if *ex vivo* moDCs can be activated to sustain cDC1-like properties, these are not likely maintained in the TME ([@B121], [@B181]). Therefore, moDC-based vaccines may not be the answer, and a new generation of DC-focused cancer immunotherapies are needed. Increasing cDC1 function is one important goal, but as described here, some of this can occur indirectly via the cooperative interactions with other cells. In addition, both cDCs and pDCs have the potential to directly activate T cells that can kill cancer cells if exposed to the right activating signals ([Figure 1B](#F1){ref-type="fig"}). Therefore, targeting maturation signals specifically to just cDC1s may not be the optimal therapy, and delivering signals that can enhance the function of all DC subsets may enhance efficacy and durability. For example, although tumor pDCs often correlate with poor prognosis, they are the most efficient producers of type 1 IFN and have the capacity for sustained MHC class II expression; these functions together may inflame the tumor and elicit strong T cell help that in turn could be sustained by newly matured cDC1s. Therefore, identifying signals that target and activate all DC subsets, and the cells that crosstalk with them will help provide novel insights into the cellular and molecular nature of tumor-specific CTL priming. The goal is to design therapies that build a site of sustained, immunostimulatory tumor-antigen presentation and increase the magnitude of anti-tumor immunity, so we can successfully treat a broader set of patients. Author Contributions {#s2} ==================== RN, SM-M, and KT designed and wrote the manuscript. Conflict of Interest Statement ------------------------------ RN, SM-M, and KT are full-time employees of Amgen Inc. We thank Carlos Briseno, Michael Gonzalez, and Jackson G. Egen for critical reading of the manuscript and helpful discussions. **Funding.** The authors, employees of Amgen Inc., declare that this study received funding from Amgen Inc. in its entirety. Amgen Inc. did not have a role in the study design. [^1]: Edited by: Diana Dudziak, Universitätsklinikum Erlangen, Germany [^2]: Reviewed by: Joke M. M. Den Haan, VU University Medical Center, Netherlands; Roxane Tussiwand, Universität Basel, Switzerland [^3]: This article was submitted to Antigen Presenting Cell Biology, a section of the journal Frontiers in Immunology

Introduction {#Sec1} ============ There are almost 425 million diabetes patients worldwide^[@CR1]^. Diabetic nephropathy (DN) is one of the main complications associated with diabetes. It affects one-third of diabetic patients and is the largest cause of end-stage renal disease (ESRD) in the Western world^[@CR2]^. Blood pressure and sugar control, and the blockade of the Renin-Angiotensin-System (RAS) remain the mainstays of therapy for patients with DN^[@CR3]^. However, the incomplete renoprotective effects that current pharmacological interventions may provide - particularly when started at advanced stages of the disease^[@CR4]^ - create the need for more effective therapeutic approaches. One known risk factor for and significant mechanistic contributor to the development of DN is oxidative stress^[@CR5]^. It derives from an imbalance between increased production of reactive oxygen species (ROS) and insufficient antioxidant capacity of the cell, and is a common feature of all cells that are damaged by diabetes-related hyperglycemia^[@CR6]^. Excessive ROS production is associated with, among other factors, the inactivation of antioxidant enzymes, such as superoxide dismutase 2 (SOD2)^[@CR7]^, and mitochondrial dysfunction. Indeed, mitochondria are the main source of intracellular ROS, and changes in mitochondrial morphology and dynamics under hyperglycemic conditions contribute to increased mitochondrial ROS levels^[@CR8],[@CR9]^. In mitochondria, Sirtuin 3 (SIRT3) is the major NAD^+^-dependent deacetylase^[@CR10]^ that controls a plethora of processes, including antioxidant pathway and energy metabolism^[@CR11]--[@CR14]^. In line with these findings, several studies have highlighted the critical role that SIRT3 has in regulating mitochondrial homeostasis in both healthy and diseased kidneys^[@CR15]^. As for diabetes, it has been shown that, in kidney biopsies from patients with diabetic nephropathy, *Sirt3* mRNA expression is downregulated and treatments that aim to reduce oxidative stress are renoprotective in mice with DN^[@CR16]^. However, whether and to what extent treatments that can directly activate SIRT3 are beneficial in DN remains unknown. Recently, it has been demonstrated that honokiol, a major bioactive compound isolated from magnolia bark, effectively limited podocyte damage and the progression of hypertensive nephropathy by activating SIRT3 signaling in mice that were receiving chronic intraperitoneal injections of angiotensin II^[@CR17]^. Based on the above evidence, we sought here to evaluate whether SIRT3 activation through honokiol can confer protection against renal disease progression in BTBR *ob/ob* mice, which reproduce key features of human DN better than most murine models type 2 DN^[@CR18]--[@CR20]^, starting the treatment at an established phase of the disease to better mimic the condition in humans. Results {#Sec2} ======= SIRT3 is reduced in experimental diabetes, associates with increased ROS levels and is normalized by honokiol {#Sec3} ------------------------------------------------------------------------------------------------------------- To investigate whether SIRT3 plays a role in the pathogenesis of DN, we analyzed the gene expression of *Sirt3* in the kidneys of BTBR WT and diabetic BTBR *ob/ob* mice. RT-PCR analysis showed that renal *Sirt3* mRNA expression was markedly lower in BTBR *ob/ob* mice than that observed in WT mice (*p* \< 0.001, Fig. [1a](#Fig1){ref-type="fig"}). Given that SOD2 is a major target of SIRT3 deacetylase activity, we measured the glomerular expression of acetylated SOD2 at Lysine 68, the specific residue regulated by SIRT3^[@CR21]^. A greater expression of acetylated-SOD2 was detected in the glomeruli of BTBR *ob/ob* mice than in those of WT mice (*p* \< 0.001), indicating lower SIRT3 deacetylase activity in the diabetic mice (Fig. [1b](#Fig1){ref-type="fig"}). If reduced SIRT3 activity is actually responsible for the inactivation of SOD2, we expected these changes to be paralleled by an increase in renal oxidative stress in diabetic mice. Indeed, immunohistochemical analysis showed that nitrotyrosine staining -- a marker of peroxynitrite‐mediated oxidative damage -- was upregulated significantly in the glomerular cells of BTBR *ob/ob* mice compared to those of WT mice (Fig. [1c](#Fig1){ref-type="fig"}).Figure 1Reduction of renal expression and activity of *Sirt3* paralleled with an increase of ROS production in diabetic mice. (**a**) qRT-PCR analysis of *Sirt3* mRNA levels at 14 weeks of age in kidney of BTBR wild-type (WT) mice (n = 9) and BTBR *ob/ob* mice treated with vehicle (n = 6) or honokiol (n = 9) from 8 weeks of age. (**b**) Representative images and quantification of glomerular acetylated SOD2 staining in BTBR WT mice (n = 9) and in BTBR *ob/ob* mice treated with vehicle (n = 6) or honokiol (n = 9) at 14 weeks of age. Insets show acetylated SOD2 in podocytes of WT and diabetic mice (arrowheads). Scale bars: 20 μm. (**c**) Representative images and quantification of glomerular nitrotyrosine staining in BTBR WT mice (n = 9) and in BTBR *ob/ob* mice treated with vehicle (n = 6) or honokiol (n = 9) at 14 weeks of age. Insets show nitrotyrosine staining in podocytes of WT and diabetic mice (arrowheads). Scale bars: 20 μm. (**d,e**) qRT-PCR analysis of *Sirt1* (**d**), and *Sirt6* (**e**) mRNA levels in kidney of BTBR WT mice (n = 9) and BTBR *ob/ob* mice treated with vehicle (n = 6) or honokiol (n = 9) at 14 weeks of age. Data are mean ± SEM and were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test, \**p* \< 0.05, \*\*\**p* \< 0.001 vs BTBR WT mice; ^\#^*p* \< 0.05, ^\#\#\#^*p* \< 0.001 vs BTBR *ob*/*ob* + vehicle. At this point we wondered whether the manipulation of the SIRT3 pathway with honokiol could have a renoprotective effect. Treatment with honokiol restored *Sirt3* expression in BTBR *ob/ob* mice to levels comparable to BTBR WT mice (*p* \< 0.05 versus vehicle, Fig. [1a](#Fig1){ref-type="fig"}). This effect was associated with a significant reduction in SOD2 acetylation in the glomeruli of treated BTBR *ob/ob* mice (*p* \< 0.001 versus vehicle, Fig. [1b](#Fig1){ref-type="fig"}) and in nitrotyrosine expression (*p* \< 0.001 versus vehicle, Fig. [1c](#Fig1){ref-type="fig"}). To rule out the possibility that the protective effect is mediated by SIRT3 and not shared by other sirtuins, we evaluated the expression of *Sirt1* and *Sirt6*, as these are the most involved in renal disorders^[@CR15]^. We found that the renal expression of *Sirt1* was unchanged in diabetic mice (Fig. [1d](#Fig1){ref-type="fig"}) while *Sirt6* was downregulated (Fig. [1e](#Fig1){ref-type="fig"}). Honokiol did not affect either *Sirt1* or *Sirt6* expression (Fig. [1d,e](#Fig1){ref-type="fig"}) suggesting selective activation of SIRT3. Honokiol attenuates albuminuria and ameliorates glomerular injury {#Sec4} ----------------------------------------------------------------- To further examine the renoprotective effect of SIRT3 activation through honokiol treatment, we measured urinary albumin excretion and analyzed the histologic lesions in the kidney of BTBR *ob/ob* mice receiving vehicle or honokiol. We observed that 14-week-old vehicle BTBR *ob/ob* mice exhibited significantly higher levels of albuminuria than BTBR WT mice of the same age (*p* \< 0.001). BTBR *ob/ob* mice treated with honokiol had a significantly lower albuminuria levels (*p* \< 0.001) compared to mice receiving vehicle (Fig. [2a](#Fig2){ref-type="fig"}).Figure 2SIRT3 activation through honokiol treatment limits albuminuria, glomerular structural lesions and inflammation in diabetic mice. (**a**) Urinary albumin excretion at 14 weeks of age in BTBR WT mice (n = 9) and BTBR *ob/ob* mice treated with vehicle (n = 6) or honokiol (n = 9). (**b**) Periodic acid-Schiff-stained sections of representative glomeruli and quantification from BTBR WT mice (n = 9) and from diabetic mice treated with vehicle (n = 6) or honokiol (n = 9) at 14 weeks of age showing glomerular lesions, consisting of mesangial matrix expansion (arrowheads) and mesangiolysis (asterisk). Scale bars: 20 μm. (**c,d**) Representative images and quantification of glomerular CD31 (**c**) and α-SMA expressions (**d**) in BTBR WT mice (n = 9) and in BTBR *ob/ob* mice treated with vehicle (n = 6) or honokiol (n = 9) at 14 weeks of age. Nuclei were stained with DAPI and renal structure with lectin FITC-wheat germ agglutinin (WGA). Scale bars: 20 μm. (**e**) Representative images and quantification of glomerular accumulation of Mac-2-positive monocytes/macrophages in BTBR WT mice (n = 9) and in BTBR *ob/ob* mice treated with vehicle (n = 6) or honokiol (n = 9) at 14 weeks of age. Scale bars: 20 μm. Data are mean ± SEM and were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test, \*\*\**p* \< 0.001 vs BTBR WT mice; ^\#^*p* \< 0.05, ^\#\#\#^*p* \< 0.001 vs BTBR *ob/ob* + vehicle. Diabetic BTBR *ob/ob* mice developed renal lesions consisting of mesangial matrix accumulation accompanied by mesangiolysis, resulting in disrupted sites where capillary loops are anchored into the mesangium, which leads to the dilation of the capillary loop (Fig. [2b](#Fig2){ref-type="fig"}). In association with mesangial matrix accumulation, the glomeruli of BTBR *ob/ob* mice on vehicle exhibited a glomerular capillary rarefaction (*p* \< 0.001 versus WT mice), as revealed by immunofluorescence for CD31, a specific endothelial marker (Fig. [2c](#Fig2){ref-type="fig"}), and an increase in α-smooth muscle actin (α-SMA) expression (*p* \< 0.001 versus WT mice), a marker of mesangial cell activation (Fig. [2d](#Fig2){ref-type="fig"}). Tubular damage in BTBR *ob/ob* mice was mild, as reported previously^[@CR18]--[@CR20]^. Honokiol treatment significantly (*p* \< 0.001) limited mesangial matrix expansion and mesangiolysis (Fig. [2b](#Fig2){ref-type="fig"}). As shown in Fig. [2c](#Fig2){ref-type="fig"}, honokiol limited the decrease in CD31 staining (*p* \< 0.05 versus BTBR *ob/ob* + vehicle), and glomerular α-SMA expression was also significantly (*p* \< 0.001) lower in BTBR *ob/ob* mice after honokiol compared with vehicle (Fig. [2d](#Fig2){ref-type="fig"}). Since recent studies have highlighted the critical role that SIRT3 plays in regulating the inflammatory response^[@CR22]--[@CR24]^, we investigated whether SIRT3-activating treatment with honokiol could reduce inflammation in diabetic mice. BTBR *ob/ob* mice that received vehicle exhibited a significantly greater accumulation of Mac-2-positive monocytes/macrophages at the glomerular level compared with BTBR WT mice (*p* \< 0.001, Fig. [2e](#Fig2){ref-type="fig"}), and honokiol effectively reduced the number of glomerular Mac-2-positive infiltrating cells (*p* \< 0.001 versus vehicle, Fig. [2e](#Fig2){ref-type="fig"}). Honokiol did not affect major systemic alterations that had occurred in diabetic mice, such as increased body weight, hyperglycemia and dyslipidemia (Table [1](#Tab1){ref-type="table"}). On the contrary, kidney weight, which had increased significantly in BTBR *ob/ob* mice receiving vehicle (*p* \< 0.01 versus BTBR WT mice), was significantly lower after honokiol treatment (*p* \< 0.05 versus vehicle) (Table [1](#Tab1){ref-type="table"}).Table 1Systemic and laboratory parameters in BTBR WT mice and BTBR *ob/ob* mice at 14 weeks of age.GroupBody weightBlood glucosePlasma cholesterolPlasma triglyceridesKidney weight(g)(mg/dl)(mg/dl)(mg/dl)(g)BTBR WT (n = 9)37 ± 1131 ± 4133 ± 3117 ± 50.58 ± 0.02BTBR *ob/ob*vehicle (n = 6)49 ± 4\*\*\*569 ± 20\*\*\*155 ± 8\*226 ± 15\*\*\*0.95 ± 0.10\*\*\*honokiol (n = 9)49 ± 1\*\*\*559 ± 15\*\*\*150 ± 4\*211 ± 15\*\*\*0.71 ± 0.05^\#^Data are expressed as mean ± SEM. \**p* \< 0.05, \*\*\**p* \< 0.001 vs BTBR WT mice; ^\#^*p* \< 0.05 vs BTBR *ob/ob* + vehicle. By the end of the study period, the following mortality was recorded for the BTBR *ob*/*ob* mice: six out of twelve mice in the vehicle group died, while only three out of twelve mice in the honokiol group died. All BTBR WT control mice were alive. Honokiol limits podocyte injury in diabetic mice {#Sec5} ------------------------------------------------ To clarify what the cellular basis is for the observed reduction in albuminuria achieved by using honokiol in BTBR *ob/ob* mice, we examined the expression of nephrin - an essential component of the podocyte slit diaphragm that is crucial for maintaining slit pore integrity and renal filtration capacity^[@CR25]^ - and of nestin - a podocyte cytoplasmic protein that is involved in the organization of the cellular cytoskeleton and which plays an important role in maintaining normal podocyte function^[@CR26]^. Immunofluorescence analyses showed intense nephrin and nestin signals with the typical epithelial-like staining pattern in the glomeruli of WT mice, while BTBR *ob/ob* mice receiving vehicle exhibited a consistent reduction and altered expression pattern for both markers (*p* \< 0.001 versus WT mice, Fig. [3a,b](#Fig3){ref-type="fig"}). Honokiol treatment ameliorated the defective nephrin and nestin expression and pattern in diabetic mice (*p* \< 0.001 versus vehicle Fig. [3a,b](#Fig3){ref-type="fig"}).Figure 3Increase of SIRT3 counteracts podocyte damage and loss in BTBR *ob/ob* mice. (**a,b**) Representative images and quantification of glomerular nephrin (**a**) and nestin (**b**) expressions (red) in BTBR WT mice (n = 9) and in BTBR *ob/ob* mice treated with vehicle (n = 6) or honokiol (n = 9) at 14 weeks of age. (**c**) Representative images and morphometric analysis of glomerular volume, WT1-positive podocytes, expressed as number per glomerulus, and podocyte density in BTBR WT mice (n = 9) and in BTBR *ob/ob* mice treated with vehicle (n = 6) and honokiol (n = 9) at 14 weeks of age. Scale bars: 20 μm. Data are mean ± SEM and were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test, \*\*\**p* \< 0.001 vs BTBR WT mice; ^\#^*p* \< 0.05, ^\#\#\#^*p* \< 0.001 vs BTBR *ob/ob* + vehicle. We then evaluated glomerular volume and podocyte number and density. Morphometric analysis of kidney sections revealed that glomerular volume increased in BTBR *ob/ob* mice that received vehicle (*p* \< 0.001 versus WT mice, Fig. [3c](#Fig3){ref-type="fig"}). The number of WT1-positive podocytes per glomerulus was lower than in WT mice (*p* \< 0.001), which translated into a significant reduction in podocyte density (*p* \< 0.001, Fig. [3c](#Fig3){ref-type="fig"}). Honokiol reduced glomerular hypertrophy (*p* \< 0.05 versus vehicle, Fig. [3c](#Fig3){ref-type="fig"}) and limited podocyte depletion (*p* \< 0.05 versus vehicle, Fig. [3c](#Fig3){ref-type="fig"}). Honokiol maintains mitochondrial homeostasis in the glomeruli of BTBR *ob/ob* mice {#Sec6} ---------------------------------------------------------------------------------- Given the role of SIRT3 in regulating mitochondrial structure and function, we investigated whether SIRT3 activation through honokiol translated into improvements in mitochondrial wellness. We therefore analyzed mitochondrial morphology using transmission electron microscopy. Podocytes of BTBR *ob/ob* mice given vehicle exhibited mitochondrial swelling that was associated with disarrangement of cristae, in contrast with the podocytes of WT mice with normal mitochondria morphology (Fig. [4a,b](#Fig4){ref-type="fig"}). Honokiol treatment reversed mitochondria abnormalities induced by diabetes (Fig. [4a,b](#Fig4){ref-type="fig"}).Figure 4Modulation of SIRT3 preserves mitochondria ultrastructure and homeostasis in podocytes. (**a**) Representative electron microscopy images of podocytes from BTBR WT mice and from BTBR *ob/ob* mice treated with vehicle or honokiol at 14 weeks of age. Scale bars: 2 μm. (**b**) Enlarged images show mitochondria structure in podocytes of WT and diabetic mice. Scale bars: 500 nm. (**c**) Representative images and quantification of glomerular PGC-1α expression in BTBR WT mice (n = 9) and in BTBR *ob/ob* mice treated with vehicle (n = 6) and honokiol (n = 9) at 14 weeks of age. Honokiol increases the percentage of PGC-1α-positive nuclei in diabetic mice (arrowheads and insets). Scale bars: 20 μm. Data are mean ± SEM and were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test, \*\*\**p* \< 0.001 vs BTBR WT mice; ^\#\#^*p* \< 0.01 vs BTBR *ob/ob* + vehicle. To further assess the protective role of honokiol treatment, we investigated the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) -- the master regulator of mitochondrial function^[@CR27],[@CR28]^. We found that PGC-1α was significantly lower in the glomerular cells of diabetic mice (*p* \< 0.001), while its expression was restored to control levels through treatment with honokiol (*p* \< 0.01 versus vehicle, Fig. [4c](#Fig4){ref-type="fig"}). Honokiol restores tubular expression of Sirt3 {#Sec7} --------------------------------------------- The modulation of total *Sirt3* mRNA expression and its upregulation by honokiol in diabetic mice prompted us to assess *Sirt3* mRNA localization in the kidney through *in situ* hybridization experiments. We found that *Sirt3* was predominantly expressed in the tubular compartment of WT mice (Fig. [5a](#Fig5){ref-type="fig"}). In BTBR *ob/ob* mice given vehicle, the tubular expression of *Sirt3* was significantly lower compared with WT mice (*p* \< 0.05), but it was restored (*p* \< 0.01 versus vehicle) through honokiol treatment (Fig. [5a,b](#Fig5){ref-type="fig"}). In contrast, a weak *Sirt3* mRNA signal was found in the glomeruli of WT mice, which was not altered in either diabetic mice given vehicle or in those receiving honokiol (data not shown). To test whether increased *Sirt3* mRNA levels, after honokiol treatment, were associated with its increased activity, we analyzed SOD2 acetylation status also in the tubules. Similar to the glomerular compartment, we observed an increased acetylation of SOD2 in the proximal tubules of BTBR *ob/ob* mice given vehicle, which was reduced in mice after honokiol treatment (*p* \< 0.01 *vs* vehicle, Fig. [5c](#Fig5){ref-type="fig"}). To investigate whether the increased expression and activity of *Sirt3* in tubular cells of honokiol-treated diabetic mice can contribute also to increased SIRT3 activity in the glomerular compartment, as it has been established with regard to SIRT1^[@CR29]^, we evaluated the expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for the biosynthesis of nicotinamide adenine dinucleotide (NAD^+^), the essential co-factor of SIRT3 activity. Our results showed that *Nampt* mRNA expression was significantly lower in the kidneys of BTBR *ob/ob* mice given vehicle than in those of WT mice (*p* \< 0.05), and treatment with honokiol restored *Nampt* levels (*p* \< 0.01 versus vehicle) in the diabetic mice (Fig. [5d](#Fig5){ref-type="fig"}).Figure 5Glomerular SIRT3 activation is associated with enhanced tubular *Sirt3* expression. (**a**,**b**) Representative images (**a**) and quantification (**b**) of *in situ* hybridization for *Sirt3* in kidney cortex from BTBR WT mice and BTBR *ob/ob* mice treated with vehicle or honokiol at 14 weeks of age. Scale bars: 20 μm. Insets show the localization of *Sirt3* in tubular cells. Image-based quantitative software analysis was performed to evaluate tubular expression of *Sirt3*. Fiji Image J software (<https://imagej.net/Fiji>) was used for the quantification of the number of *Sirt3* mRNA dots (representing single mRNA molecules). Orbit Image analysis software (<http://orbit.bio>) was used to count the number of cells. *Sirt3* mRNA levels were expressed as average number of dots per cell. (**c**) Quantification of tubular acetylated SOD2 staining in BTBR WT mice (n = 9) and in BTBR *ob/ob* mice treated with vehicle (n = 6) or honokiol (n = 9) at 14 weeks of age. (**d**) qRT-PCR analysis of *Nampt* mRNA levels in kidney of BTBR WT mice (n = 9) and BTBR *ob/ob* mice treated with vehicle (n = 6) or honokiol (n = 9). (**e**) Representative images of NRF2 expression in BTBR WT mice (n = 9) and in BTBR *ob/ob* mice treated with vehicle (n = 6) or honokiol (n = 9) at 14 weeks of age. Scale bars: 50 μm. (**f,g**) qRT-PCR analysis of *Nrf2* (**f**) and *Keap1* (**g**) mRNA levels in kidney of BTBR WT mice (n = 9) and BTBR *ob/ob* mice treated with vehicle (n = 6) or honokiol (n = 9). Data are mean ± SEM and were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test, \**p* \< 0.05, \*\*\**p* \< 0.001 vs BTBR WT mice; ^\#\#^*p* \< 0.01 vs BTBR *ob/ob* ^+^ vehicle. In order to identify the potential mechanism through which honokiol treatment could promote SIRT3 expression and exert anti-oxidant activities, we investigated the expression of nuclear factor erythroid 2-related factor 2 (NRF2), the master regulator of anti-oxidant response^[@CR30]--[@CR32]^. As shown in Fig. [5e](#Fig5){ref-type="fig"}, we found that NRF2 protein expression was reduced in the nucleus and cytoplasm of BTBR *ob/ob* mice given vehicle compared to BTBR WT mice. Treatment with honokiol restored the expression of NRF2 (Fig. [5e](#Fig5){ref-type="fig"}). These data were confirmed by real time RT-PCR experiments that showed a significant reduction of *Nrf2* in BTBR *ob/ob* mice given vehicle compared to BTBR WT mice (Fig. [5f](#Fig5){ref-type="fig"}). Treatment with honokiol normalized *Nrf2* expression (Fig. [5f](#Fig5){ref-type="fig"}). Conversely, the expression of the NRF2 adaptor protein *Keap1* was unchanged in any of the experimental groups (Fig. [5g](#Fig5){ref-type="fig"}), possibly suggesting that KEAP1 is not involved in the maintenance of the homeostatic levels of NRF2. Discussion {#Sec8} ========== In this study, we demonstrated that in experimental diabetes 1) renal SIRT3 expression and activity are reduced and are associated with increased oxidative stress; 2) selective manipulation of the SIRT3 pathway using honokiol protects against albuminuria, glomerular lesions and inflammation; 3) honokiol reduces podocyte damage and loss by preserving mitochondrial structure and function. The first finding of this study is that *Sirt3* mRNA expression was lower in the kidneys of BTBR *ob/ob* mice, which is in line with earlier observations in experimental type 1 diabetes^[@CR33]^ and in kidney biopsies from DN patients^[@CR16]^. The reduction in *Sirt3* expression in BTBR *ob/ob* mice was associated with an impairment in its deacetylase activity towards SOD2 and an increase in ROS levels. Oxidative stress is a well-known driver of diabetic kidney disease^[@CR5]^, and in this regard our study shows that SIRT3-SOD2 axis is a critical modulator of renal oxidative stress in diabetic mice and that this molecular pathway could be targetable through honokiol. Indeed, we observed that honokiol enhanced *Sirt3* mRNA levels in BTBR *ob/ob* mice, and this had a positive impact on SOD2 activity and oxidative stress. Previous studies have elegantly demonstrated that in the heart honokiol can enter mitochondria and physically bind to SIRT3, enhancing its affinity for NAD^+^ with immediate consequences on deacetylase activity and ROS production^[@CR34]^. However, whether honokiol's action is exclusively directed towards the activation of SIRT3 and not shared with other sirtuins has not been addressed in these studies. The finding here that honokiol did not modulate the renal expression of either *Sirt1* or *Sirt6*, which are, along with SIRT3, involved in kidney disease progression^[@CR15]^, clearly demonstrates the beneficial effects of honokiol on the selective restoration of SIRT3 in the kidney. Improvements in albuminuria and glomerular damage after treatment of BTBR *ob/ob* mice with honokiol appear to be relatively consistent. Furthermore, the large reduction in glomerular inflammation we observed in these mice demonstrates that SIRT3 has a potent anti-inflammatory effect on the diabetic kidney that has never been reported before. Glomerular hypertrophy has been identified as a characteristic feature of diabetic glomerular injury in experimental models and in humans. This is accompanied by the inability of podocytes to maintain complete coverage of the glomerular filtration surface area, which results in podocyte hypertrophic stress, leading to podocyte detachment^[@CR35]^. Podocytes are critical players in maintaining the integrity of the glomerular filtration barrier, and podocyte injury and loss have been identified as the initial event in the development of proteinuria and glomerular lesions, ending in glomerulosclerosis. The evidence indicates that the podocyte is the earliest target of diabetic injury, and indeed podocyte number correlates with albuminuria, and glomerular podocyte density appears to be one of the best predictors of albuminuria and disease progression^[@CR36]^. In this study, the anti-albuminuric effect of honokiol was associated with an amelioration of the defective expression of podocyte proteins and a reduction in podocyte loss in BTBR *ob/ob* mice. Increasing evidence indicates that the disruption of mitochondrial bioenergetics in podocytes is crucial for dictating the development and progression of DN^[@CR37]--[@CR39]^, and recent findings have provided novel correlations between podocyte mitochondrial morphology and the progression of DN^[@CR40],[@CR41]^. Here, treatment with honokiol improved podocyte mitochondrial abnormalities in diabetic mice and also restored the expression of PGC-1α, a known modulator of mitochondrial homeostasis, thereby reinforcing the hypothesis that increasing SIRT3 activity is crucial in the preservation of mitochondrial integrity^[@CR42],[@CR43]^. According to a recent study^[@CR44]^, it is conceivable that the ability of honokiol to modulate PGC-1α could be dependent on the activation of the AMPK-CREB pathway by SIRT3, ultimately resulting in PGC-1α stimulation. To further investigate the mechanisms underlying honokiol-induced SIRT3 activation in DN in greater depth, we analyzed the localization of *Sirt3* expression in the kidneys of BTBR *ob/ob* mice. We observed that *Sirt3* levels in tubular cells were lower in diabetic mice and were restored by honokiol treatment, accompanied by a reduction in the acetylation of the SIRT3 target SOD2. The possible mechanism through which honokiol stimulates SIRT3 protein expression relies on NRF2 that we found increased in the kidney of treated diabetic mice. That NRF2 can upregulate SIRT3 expression is supported by the evidence that NRF2 induces SIRT3 expression through the direct binding of NRF2 subunit to the SIRT3 promoter^[@CR45]^. On the other hand, we found that *Sirt3* expression was not modulated by honokiol in the glomeruli of BTBR *ob/ob* mice. These results reminded us of those reported in an interesting study by Hasegawa *et al*.^[@CR29]^, in which the selective upregulation of tubular SIRT1 mediated a retrograde interplay between tubules and podocytes, which resulted in the amelioration of diabetes-associated glomerular disease. In that study, the authors reported that the upregulation of NAMPT in tubular cells, mediated by increased SIRT1 levels, maintained adequate nicotinamide mononucleotide (NMN) concentrations around glomeruli to promote podocyte function, ultimately translating into an anti-albuminuric effect in diabetes. NAMPT is also a critical regulator of SIRT3 activity, as it produces NMN, the intermediate product for NAD^+^ biosynthesis^[@CR46],[@CR47]^. Consistent with the interpretation provided by Hasegawa^[@CR29]^ we suggest that the protective effect of SIRT3 on albuminuria and glomerular changes in BTBR *ob/ob* mice could be due to tubule-glomerulus retrograde interplay. In particular, the upregulation of SIRT3 and NAMPT in tubular cells may provide the required amount of NMN to diabetic podocytes and other glomerular cells, ultimately supplying glomerular NAD^+^ to further increase SIRT3 activity in a virtuous cycle. In conclusion, here we have shown that SIRT3 plays a crucial role in the pathogenesis of DN and that its specific activation through honokiol reduces diabetes-induced oxidative stress and protects podocytes and, generally, the glomerulus from diabetes-induced damage. In the context of the great interest that antioxidant therapies have generated, as promising treatments for DN and other chronic diabetic complications^[@CR48]^, our data provide evidence of a hitherto unknown protective effect of SIRT3 against diabetic glomerular disease. This suggests that the pharmacological activation of SIRT3 activity may be an effective, novel approach for treating DN. Methods {#Sec9} ======= Experimental design {#Sec10} ------------------- Male BTBR (black and tan, brachyuric) Lep^*ob/ob*^ and BTBR wild-type (WT) mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and were kept on specific pathogen-free facility with constant temperature on a 12:12-hour light-dark cycle with free access to standard diet and water. At 8 weeks of age, when they had already developed albuminuria, BTBR *ob/ob* mice were randomly allocated to receive (n = 12 mice/group): vehicle (DMSO in saline solution) or honokiol (10 mg/kg in DMSO; BioVision, Milpitas, CA, USA) by once-daily intraperitoneal injection. The dose of honokiol was chosen according to available data in the literature^[@CR49]^. Treatment lasted until mice were 14 weeks old. BTBR WT mice (n = 9) were followed for the same length of time as controls. Mice were euthanized through CO~2~ inhalation and their kidneys were collected and processed for analysis. Before sacrifice, mice were housed in metabolic cages for 24-hour urine collection for albuminuria assessment. Blood samples were collected for glucose, cholesterol and triglyceride measurements. The experimenters were not blind to the treatment, but they were blind for measurement of experimental outcomes. All animal experiments were conducted in accordance with institutional guidelines in compliance with national (D.L.n.26, March 4, 2014), and international laws and policies (directive 2010/63/EU on the protection of animals used for scientific purposes) and were approved by the Institutional Animal Care and Use Committees of Istituto di Ricerche Farmacologiche Mario Negri IRCCS. Biochemical parameters {#Sec11} ---------------------- Biochemical parameters were assessed as we previously described^[@CR20],[@CR50]^. Blood glucose levels were assessed with a reflectance meter (OneTouch UltraEasy, LifeScan, Milpitas, CA, USA). Plasma cholesterol and triglycerides were measured using the Reflotron test (catalog 10745065202 and catalog 10745049202, Roche Diagnostic Corporation, Indianapolis, IN, USA). Urinary albumin excretion was measured with the ELISA test using the Bethyl test kit (catalog E101, catalog A90-134A and catalog A90-134P, Bethyl Laboratories Inc, Montgomery, TX, USA). Quantitative (q) RT-PCR {#Sec12} ----------------------- Total RNA was isolated from whole kidney tissue as we previously described^[@CR51]^. Briefly, TRIzol Reagent (catalog 15596026, Thermo Fisher Scientific, Waltham, MA, USA) was used according to the manufacturer's instructions. After treatment with DNase (catalog 6101, Promega Madison, WI, USA), cDNA was prepared using the SuperScript VILO cDNA synthesis kit (catalog 11754050, Thermo Fisher Scientific). qRT-PCR analyses were performed on ABI ViiA 7 Real-Time PCR system (Thermo Fisher Scientific). *Sirt3, Sirt1*, *Sirt6* and *Nampt* were assessed using TaqMan Gene Expression Master Mix (catalog 4369016, Thermo Fisher Scientific) and the following TaqMan assays (Thermo Fisher Scientific): Mm01275638_m1, Mm01168521_m1, Mm01149042_m1 and Mm01293560_m1, respectively. Mouse ACTB Endogenous Control (VIC/MGB probe) was used to evaluate the housekeeping gene *β-actin*. Gene expression levels of *Sirt3, Sirt1*, *Sirt6* and *Nampt* were normalized to *β-actin* levels. *Nrf2* and *Keap1* were assessed using SYBR Green PCR Master Mix (catalog 4367659, Thermo Fisher Scientific) and the following primers (300 nM): *Nrf2* forward 5′-CCCAGCAGGACATGGATTTGA-3′ and reverse 5′-CATAGTCCTTCTGTCGCTGACT-3′; *Keap1* forward 5′-ACGTCCTCGGAGGCTATGAT-3′ and reverse 5′- GGGTCACCTCACTCCAGGTA-3′ *β-actin* (forward 5′-CACTGTCGAGTCGCGTCC-3′ and reverse 5′-TCATCCATGGCGAACTGGTG -3′) was used as endogenous control. Relative quantities were calculated by the 2^-ΔΔCt^ method. Data are presented as fold change relative to WT control group. Renal histology {#Sec13} --------------- Kidney samples were fixed in Duboscq-Brazil (catalog P0094, Diapath, Bergamo, Italy), dehydrated, and embedded in paraffin, as we previously described^[@CR50]^. Three-micrometer sections were stained with periodic acid-Schiff (PAS) reagent, and at least 50 glomeruli were examined per animal. The degree of glomerular mesangial matrix expansion was quantified using a score between 0 and 3 (0 = no mesangial matrix expansion; 1 = minimal; 2 = moderate; 3 = diffuse mesangial matrix expansion). The number of glomeruli exhibiting mesangiolysis in an entire kidney section was counted and expressed as a percentage. All biopsies were reviewed by a blinded pathologist. Samples were examined using ApoTome Axio Imager Z2 (Zeiss, Jena, Germany). Immunohistochemistry {#Sec14} -------------------- For immunoperoxidase experiments, as we previously performed^[@CR50]^, formalin-fixed, 3-μm paraffin-embedded kidney sections were incubated with Peroxidazed 1 (catalog PX968H, Biocare Medical, Pacheco, CA, USA) to quench endogenous peroxidase, after antigen retrieval in a decloaking chamber with DIVA, Rodent or Borg decloaker buffer (catalog DV2004MX, catalog RD913M and catalog BD1000MM, Biocare Medical) to increase the reactivity of antibodies to antigens. After blocking for 30 minutes with Rodent Block M (catalog RBM961G, Biocare Medical), sections were incubated with rabbit anti-SOD2/MnSOD (acetyl K68) (catalog ab137037, Abcam, Cambridge, UK, 1:50 and 1:200), rabbit anti-nitrotyrosine (catalog 06-284, Merck Millipore, Burlington, MA, USA, 1:100), rat anti-Mac 2 (clone M3/38, Cedarlane, Burlington, ON, Canada, 1:600), rabbit anti-PGC-1α (catalog ab54481, Abcam, 1:100) and rabbit anti-NRF2 (catalog ab31163, Abcam, 1:100) antibodies, followed by Rat on Mouse HRP-Polymer or Rabbit on Rodent HRP-Polymer (catalog RT517 and catalog RMR622G, Biocare Medical) for 30 minutes at room temperature. Stainings were visualized using diaminobenzidine (catalog BDB2004H, Biocare Medical) substrate solutions. Slides were counterstained with Mayer's hematoxylin (catalog MHS80-2.5 L, Bio Optica, Milan, Italy), mounted with Eukitt mounting medium (catalog 09-00250, Bio Optica) and finally observed using light microscopy (ApoTome, Axio Imager Z2, Zeiss). Negative controls were obtained by omitting the primary antibody on adjacent sections. Glomerular acetylated SOD2 and nitrotyrosine stainings were quantified with a semiquantitative score between 0 and 3 (0: absent glomerular staining, 1: weak staining in a few glomerular cells, 2: moderate glomerular staining, 3: intense glomerular staining). At least 15--20 glomeruli/section for each animal were randomly analyzed. Tubular acetylated SOD2 was quantified with a semiquantitative score between 0 and 3 in proximal tubules (0: absent staining, 1: weak staining, 2: moderate staining, 3: intense staining). At least 10-15 fields/section for each animal were randomly analyzed. Mac-2-positive monocyte/macrophages within glomeruli were counted in a minimum of 50 glomerular cross-sections and expressed as average number of cells per glomerulus. Glomerular PGC-1α was evaluated by expressing the positive-PGC-1α nuclei as a percentage of the total nuclei per tuft (ImageJ software). At least 15--20 glomeruli/section per animal were randomly analyzed. OCT-frozen kidney sections were fixed with cold acetone, blocked in 1% bovine serum albumin (BSA) and then incubated with the following primary antibodies: rat anti-mouse CD31 (catalog 550274, BD Pharmingen, San Jose, CA, 1:100), Cy3-conjugated mouse anti- α-smooth muscle actin (α-SMA) (catalog c6198, clone 1A4, Sigma Aldrich, St. Louis, MO, USA, 1:200), goat anti-nephrin (catalog sc-19000, clone N-20, Santa Cruz Biotechnology Inc, Dallas, TX, USA, 1:100) or rat anti-nestin (catalog ab81462, clone 7A3, Abcam, 1:300) followed by appropriate Cy3-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, Cambridge, UK). For nephrin staining, the antigen retrieval in citrate buffer was performed. Negative controls were obtained by omitting the primary antibody on adjacent sections. Samples were examined using an inverted confocal laser microscope (LSM 510 Meta, Zeiss). Glomerular CD31-positive staining was quantified in 15 glomeruli in each section and expressing the positive glomerular areas as a percentage of the total area (ImageJ software)^[@CR20]^. As we previously analyzed^[@CR20]^, α-SMA, nephrin and nestin stainings were quantified by score from 0 to 3 (α-SMA 0--0.5: absent or weak signal, 1: mild, 2: moderate, 3: intense signal; nephrin, 0--0.5: absent or weak and fragmented signal, 1: linear and thin signal, 2: linear signal, 3: linear and intense signal; nestin, 0--0.5: absent or weak staining in a few podocytes, 1: moderate staining in podocytes with altered distribution, 2: moderate podocyte staining, 3: intense podocyte staining). At least 20 glomeruli/section for each animal were randomly analyzed. Immunohistochemical analysis and scoring assays were done by individuals unaware of sample identity. Glomerular podocyte count {#Sec15} ------------------------- Formalin-fixed, 3-μm paraffin-embedded kidney sections were incubated with Peroxidazed 1, after antigen retrieval in a decloaking chamber with Rodent decloaker buffer. After blocking for 30 minutes with Rodent Block M, sections were incubated with rabbit anti-WT1 (catalog ab89901, Abcam, 1:600) antibody followed by Rabbit on Rodent HRP-Polymer for 30 minutes at RT. Stainings were visualized using diaminobenzidine substrate solutions. Slides were counterstained with Mayer's hematoxylin, mounted with Eukitt mounting medium and finally observed using light microscopy (ApoTome, Zeiss). Negative controls were obtained by omitting the primary antibody on adjacent sections. At least 20 glomeruli/section for each animal were randomly acquired. The estimate of the average number of podocytes per glomerulus and the glomerular volume were determined through morphometric analysis, as previously described^[@CR52]^. Ultrastructural analysis {#Sec16} ------------------------ Mitochondrial morphology was observed by transmission electron microscopy, as we previously performed^[@CR51]^. Fragments of kidney tissue were fixed overnight in 2.5% glutaraldehyde (catalog 340855, Sigma Aldrich, Darmstadt, Germany) in 0.1 M cacodylate buffer (pH 7.4) (catalog 11652, Electron Microscopy Sciences, Hatfield, PA, USA) and washed repeatedly in the same buffer. After postfixation in 1% OsO~4~, specimens were dehydrated through ascending grades of alcohol and embedded in Epon resin. Ultrathin sections were stained with uranyl acetate replacement (catalog 22405, UAR, Electron Microscopy Sciences, Hatfield, PA 19440, USA) and lead citrate (catalog 22410, Electron Microscopy Sciences) and examined using transmission electron microscopy (Fei Morgagni 268D, Philips, Hillsboro, OR, USA). *In situ* hybridization {#Sec17} ----------------------- *Sirt3 in situ* hybridization (ISH) was performed on 3-µm formalin-fixed and paraffin-embedded kidney sections using the RNAscope 2.5 HD Brown Assay kit (catalog 321720, ACD Bio-techne, Minneapolis, MN, USA) according to the manufacturer's instructions (<https://acdbio.com/manual-assays-rnascope>). Briefly, deparaffinized slides were treated with hydrogen peroxide, heat and protease before hybridization with *Sirt3* target probe (40 °C for 2 hours; HybEZ Oven). *Sirt3* target probe (catalog 300031, ACD Bio-techne, Minneapolis) is designed on mouse *Sirt3*, transcript variant 3, mRNA reference sequence (NM_001177804 at the National Center of Biotechnology Information) and detects all the murine *Sirt3* transcript variants. After hybridization, slides were washed and processed for standard signal amplification steps. Chromogenic detection was performed using 3, 3'-diaminobenzidine (DAB) followed by counterstaining with 50% Mayer's hematoxylin (Bio Optica, Milan, Italy). Probes against the housekeeping gene PPIB (peptidylprolyl isomerase B) and the bacterial gene DapB were used as positive and negative controls, respectively. *Sirt3* mRNA molecules were visualized as brown, punctate dots. Ten fields of tubule cells and twenty glomeruli for each animal were randomly acquired using bright-field microscopy (ApoTome, Axio Imager Z2, Zeiss). Image-based quantitative software analysis was performed to evaluate tubular and glomerular expression of *Sirt3*. Fiji Image J software (<https://imagej.net/Fiji>) was used for the quantification of the number of *Sirt3* mRNA dots (representing single mRNA molecules). Orbit Image analysis software (<https://orbit.bio>) was used to count the number of cells. *Sirt3* mRNA levels were expressed as average number of dots per cell. Statistical analysis {#Sec18} -------------------- Results were expressed as mean ± SEM and in bar chart with individual data points. Data analysis was performed using Graph Pad Prism software (Graph Pad, San Diego, CA, USA). Comparisons were made using one-way ANOVA with Tukey's multiple comparisons post hoc test, and the statistical significance was defined as a *p* value of \<0.05. **Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. These authors contributed equally: Monica Locatelli, Carlamaria Zoja, Ariela Benigni and Paola Cassis. The authors thank D. Rottoli for technical assistance with renal histology. They also wish to thank M. Passera for helping to prepare the article and K. Mierke for English language editing. R. Novelli, L. Perico and M. Locatelli are the recipients of a fellowship from Fondazione Aiuti per la Ricerca sulle Malattie Rare (ARMR), Bergamo, Italy. This study was supported by Fondazione Cariplo (grant Giovani Ricercatori 2016 Rif. 2016-0500). M.L. contributed to the research plan, performed the experiments, discussed the data, and contributed to writing the article; Ca.Z. contributed to the research plan, discussed the data, and wrote the article; Cr.Z., Da.C., S.V. and S.B. performed the experiments and analyzed data; R.N. and L.P. analyzed the data and contributed to writing the article; G.R. discussed the data, and critically revised the manuscript; A.B. conceived and designed the study, discussed data and wrote the article; P.C. conceived and designed the study, performed the experiments, discussed the data and wrote the manuscript. All authors approved the final version of the manuscript. A.B. is the guarantor of this work. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. The authors declare no competing interests.

Neurodegenerative diseases, including dementia, primarily affect elderly people, a steadily growing strata of the population.[@B1] In Peru, one in ten people were older than 60 years old in 2015 and it is estimated that this number will increase to 13% of the total population of Peru by 2025.[@B2]^,^[@B3]Amnestic mild cognitive impairment (aMCI) is recognized as a condition that precedes Alzheimer\'s disease (AD), the most common type of dementia. aMCI is characterized by a cognitive alteration so subtle that it does not alter the patient\'s functioning, and can be overlooked in medical care.[@B4]^,^[@B5] AD severely affects the patient\'s cognitive functions and quality of life, and has a high social and economic impact.[@B1]^,^[@B6] It is also estimated that 10% of patients with aMCI will progress to dementia every year.[@B7]^-^[@B9] The prevalence of this condition ranges from 1% (Canada) to 28.3% (New York, USA) in older adults.[@B10] Studies from Latin America (Colombia, Argentina, Mexico) have reported frequencies of 9.1-10.8%.[@B11]^-^[@B13] By contrast, in Peru, the reported crude prevalence of aMCI was 3.1%.[@B14] However, given that the prevalence of dementia in a Peruvian urban population is 6.5%, the prevalence of this prior stage is expected to be higher.[@B15] The heterogeneity of these results in similar studies is probably due to the use of different parameters to define aMCI criteria. For example, these studies used different methods to objectively assess this condition, ranging from the use of different neuropsychological questionnaires to an extensive evaluation by a multidisciplinary team. One of the tools to evaluate aMCI is the Memory Alteration Test (M\@T). This is a brief neuropsychological verbal test that does not require extensive training and can be applied by primary care professionals.[@B16]^,^[@B17] This test has been validated in Peru for adults with a minimum of 6 years of education, and has shown high sensitivity (98.3%) and specificity (97.8%) for the detection of aMCI, surpassing the commonly used Mini-Mental State Examination (MMSE; sensitivity: 83.89%; specificity: 68.89%).[@B18] The availability of sensitive and easy-to-use tests like the M\@T could provide general practitioners with an accessible tool in scenarios where the lack of specialized professionals and resources prevent the treatment of the elderly population at risk.[@B16] Known risk factors for this condition are age, history of stroke, and level of education.[@B19]^,^[@B20] Level of education is highly related to socioeconomic status, which has also shown an association with aMCI.[@B21] Cardiovascular risk factors (CVF), such as arterial hypertension, type 2 diabetes mellitus, obesity, hypercholesterolemia and tobacco smoking, have also shown a positive association.[@B20] Variation in risk factors according to sex has been observed. In women, the risk factors which contributed most were having a lower level of education and not attending meetings of a religious nature. In males, these factors were older age and a history of stroke.[@B11]^,^[@B19]^,^[@B22]^-^[@B24] Lifestyles play an important role in the development of this condition. The protective factors identified were regular physical activity, a healthy diet, higher education and routinely performing mental activities, like reading, resolving puzzles (crossword, Sudoku, etc.) or frequently using the computer.[@B19]^,^[@B20] In our milieu, risk factors for aMCI have not yet been described. Given the growing proportion of population at risk of dementia in Peru, it is important to describe this 'pre-dementia\' stage in an effort to recognize modifiable factors and implement effective intervention in the near future. aMCI early detection and the delay of progression to AD could have a crucial impact on the well-being of patient and their family members, as well as on the Peruvian economy. The aim of this study is to determine the frequency of aMCI in the elderly population from four senior citizen clubs (SCC) in Lima, Peru using the M\@T and to explore factors (age, sex, marital status, years of education, body mass index (BMI), history of hypertension and district of residence) associated with this condition. METHODS ======= Participant selection --------------------- This is a cross-sectional study that included adults older than 60 years old, with complete basic education (6 years of schooling), attending SCC from 4 districts in Lima, Peru (La Molina, Jesus Maria, Carabayllo and Cercado de Lima). The clubs were chosen according to their socioeconomic differences between their respective locations[@B25] and the accessibility they provided. The sample size was 71 subjects per SCC and was calculated based on the approximately 2500 subscribed members in each SCC (1000 in total), for an estimated prevalence of 5%, based on a previous study in Peru that reported 3.1%,[@B11]^,^[@B14] and a confidence interval (CI) of 95% with an estimated loss of 5%. The enrolment period was 1 week per SCC and the sampling was by convenience. People interested in participating were interviewed for more information before participation (See *Ethics* section). Five interviewers (the first three authors and two psychology students) were trained by experts to apply the test with similar consistency and to explain the informed consent form to the participants. Participants that failed to complete all the interview or had a condition limiting test application (overt cognitive impairment, language or hearing disability that impeded the application/completion of the tests, or previously confirmed dementia diagnosis) were not included. Senior Citizen Clubs (SCC) -------------------------- The Peruvian Ministry of Women and Social Development implemented *Centros Integrales del Adulto Mayor* (translated as Senior Citizen Clubs, SCC) all over Peru.[@B26] These centers are dedicated to organizing recreational and health promoting activities for the local elderly citizens. Although the same goal is shared across centers, different activities and resources are used according to the district income. The investment in each SCC activities and resources differ according to socioeconomic status of the district. We selected 4 SCC from Carabayllo, Cercado de Lima, Jesus Maria and La Molina districts (from the lowest to the highest socioeconomic status of the four).[@B2] aMCI criteria ------------- Once enrolled, the subjects had to meet to following criteria (Petersen\'s criteria) in order to establish aMCI status:[@B10] \(i\) Subjective loss of cognitive function: reported as cognitive complaints in the last month (memory loss). This was reported by the subject during the interview.(ii) Preserved functioning. We applied the Pfeffer Functional Activities Questionnaire (PFAQ) to those participants who reported cognitive complaints and had preserved functioning .(iii) Objectively measured cognitive impairment. We applied the M\@T to those participants who reported cognitive complaints and met the previous two criteria.(iv) Non-dementia. Reported by the patient and caregiver and corroborated by the results of the M\@T. Patients with a M\@T score below 28 were excluded from analysis for marked cognitive impairment compatible with dementia and were referred to a neurologist. **Memory Alteration Test (M\@T) -** Appendix 1 shown at: <http://www.demneuropsy.com.br/imageBank/pdf/Artigo-09-(Appendix-01-02).pdf> This test contains 40 questions with a maximum score of 50. It evaluates 5 areas: encoding (5 points), orientation (10 points), semantic (15 points), free recall (10 points), and cued recall (10 points). If the total score is \<28, 28-36 or \>36, the subject is classified as having dementia according to the M\@T, having MCI (aMCI+) or having no amnestic cognitive impairment according to the M\@T (aMCI-), respectively. Patients that were classified as having dementia according to the M\@T were not included in the analysis and were referred to a neurologist for further studies. Interviewers were trained before applying this test.[@B15] **Pfeffer Functional Activity Questionnaire (PFAQ) -** Appendix 2 shown at: <http://www.demneuropsy.com.br/imageBank/pdf/Artigo-09-(Appendix-01-02).pdf> The PFAQ consists of 10 items, each scored from 0 (independent) to 3 (fully dependent). A total score of 9 or more is classified as impairment in functioning. Interviewers were trained before applying the test. Associated factors ------------------ During the interview, sociodemographic data were collected, including age, sex, marital status, years of education, district of the SCC. Any previous history of hypertension was also registered. In order to calculate BMI, we measured height (H, in meters) and weight (W, in kilograms) were measured and the formula W divided by H squared (units: kg/m[@B2]). Statistical analysis -------------------- The data collected was transferred to a database in Microsoft Excel 2013. The sample size was calculated with the open source program *Open-epi.* Descriptive statistics were used to determine frequencies of the condition and characteristics of the population. Means and median with standard deviations and interquartile ranges were reported. Bivariate (Chi-squared and *t-*test) and multivariate analysis (logistic regression) were used to explore associated factors using the statistical software STATA 15. Ethics ------ This study was reviewed and approved by the Institutional Review Board (IRB) of the Universidad Peruana Cayetano Heredia (UPCH), Lima - Peru, in coordination with the municipalities of Jesus Maria, Cercado de Lima, La Molina, and Carabayllo districts in Lima, Peru. All participants gave their written informed consent before participation (See Appendix). This document was also reviewed and approved by the same IRB that approved the protocol. RESULTS ======= Twenty-six of the 378 participants were excluded for scoring below 28 on the M\@T (see aMCI criteria in methods section) or for failing to complete all the interview. The mean age of the remaining 352 participants was 70.91±7.07 years. Three quarters (82.9%) were women. More than half of all participants (51.3%) were married. About one in ten (11.9%, n=42) were older than 80 years. Two thirds (63.1% n=214) had a BMI greater than 25 kg/m[@B2] and 33.2% (n=116) reported a history of arterial hypertension. In the overall group, participants had similar proportions of educational level, although this was slightly higher in the group with 11-15 years of education ([Table 1](#t1){ref-type="table"}). The mean number of years of education was 11.9±3.7 years. Of all participants, 32.1% (n=113) belonged to the SCC La Molina; 24.2% (n=85) Jesus Maria; 23.6% (n=83) Carabayllo and 20.2% (n=71) to the Cercado de Lima. Participants from the SCC Carabayllo had the lowest mean years of education (9.42±3.44 years) while La Molina had the highest (13.08±3.17 years). ###### Sociodemographic characteristics of participants (n=352). Characteristic   Total n (%) --------------------------------------------- ------------ ------------- Age (years)[\*](#TFN1){ref-type="table-fn"}   70.91±7.07 Age group 60-64 69 (19.6) 65-69 98 (27.8) 70-74 77 (21.9) 75-79 65 (18.5) ≥80 42 (11.9) Sex Female 292 (82.9) Male 60 (17.1) Marital status Single 51 (14.5) Married 180 (51.3) Widowed 93 (26.5) Divorced 27 (7.7) Educational level 6-10 91 (25.8) 11-15 152 (43.2) \>16 109 (31.0) History of arterial hypertension   116 (33.2) BMI \< 25 125 (36.9) ≥25 214 (63.1) aMCI Yes 63 (17.9) mean±SD Participants from SCC La Molina and Jesus Maria had more years of education than the individuals from the other two clubs. Also, there were more people older than 80 years of age in the aforementioned SCC. Age, sex, marital status and BMI of participants showed no statistically significant difference between different SCC locations (p\>0.05, see [Table 4](#t4){ref-type="table"}). ###### Baseline sociodemographic characteristics of sample according to presence/absence of aMCI (n=352). Characteristics   aMCI+ (n=63\*) aMCI- (n=289\*) p-value ---------------------------------- ------------- ---------------- ------------------------------------------ ------------------------------------------ Age (years)\*   75.21±7.20 69.99±6.70 \<0.001^[a](#TFN3){ref-type="table-fn"}^ Age group 60-64 2 (2.9) 67 (97.1) \<0.001^[b](#TFN4){ref-type="table-fn"}^ 65-69 14 (14.3) 84 (85.7) 70-74 11 (14.3) 66 (85.7) 75-79 19 (29.2) 46 (70.8) ≥80 16 (38.1) 26 (61.9) Sex Female 52 (82.5) 240 (83.0) 0.923^[c](#TFN5){ref-type="table-fn"}^ Male 11 (17.5) 49 (17.0) Marital status Single 11 (21.6) 40 (78.4) 0.339^[b](#TFN4){ref-type="table-fn"}^ Married 28 (15.6) 152 (84.4) Widowed 21 (22.6) 72 (77.4) Divorced 3 (11.1) 24 (88.9) Years of education   11 (6-11) 13 (11-16) \<0.001^[d](#TFN6){ref-type="table-fn"}^ Educational level 6-10 31 (49.2) 60 (20.8) 11-15 23 (36.5) 129 (44.6) \<0.001^[c](#TFN5){ref-type="table-fn"}^ \>16 9 (14.3) 100 (34.6) History of arterial hypertension Yes 28 (44.4) 88 (30.4) 0.037^[c](#TFN5){ref-type="table-fn"}^ No 35 (55.6) 198 (68.5) Body mass index \<25 34 (53.9) 180 (62.3) 0.187^[c](#TFN5){ref-type="table-fn"}^ ≥25 27 (42.9) 98 (33.9) District of origin Jesús María 14 (16.5) 71 (83.5) 0.002^[c](#TFN5){ref-type="table-fn"}^ La Molina 9 (8.1) 104 (92.0) Carabayllo 21 (25.3) 62 (74.7) Cercado de Lima 19 (26.8) 52 (73.2) Participants with missing values for some characteristics were not included in analysis. t-test; Fisher; Chi-squared; Mann-Whitney tests were used for bivariate analysis because the variable did not have a normal distribution. ###### Factors associated with presence of aMCI, determined by bivariate analysis and multiple regression (n=352). Variables Bivariate analysis   Multiple regression -------------------- -------------------- ------- --------------------- ------- --- ------- ------------ ------- Sex Female Ref.       Ref.       Male 1.03 0.57-1.86 0.923   0.73 0.39-1.34 0.311 Age 60-64 Ref.               65-69 4.93 1.15-21.04 0.031   5.64 1.38-22.96 0.016   70-74 4.93 1.13-21.51 0.034   4.71 1.08-20.56 0.039   75-79 10.08 2.44-41.68 0.001   9.23 2.25-37.91 0.002   ≥80 13.14 3.17-54.4 0.000   15.33 3.79-61.99 0.000 Years of education 6-10 Ref.       Ref.       11-15 0.44 0.27-0.71 0.001   0.56 0.34-0.93 0.024   \>16 0.24 0.12-0.48 0.000   0.32 0.15-0.66 0.002 SCC location Jesús María Ref.       Ref.       La Molina 0.48 0.22-1.07 0.071   0.52 0.25-1.08 0.080   Carabayllo 1.54 0.84-2.82 0.165   1.20 0.65-2.22 0.560   Cercado de Lima 1.62 0.88-3.01 0.122   1.46 0.80-2.67 0.216 \*Adjusted for sex, years of education and SCC location. ###### Characteristics of study participants by SCC location. Characteristics   SCC Jesús María n=85 (24.2%) SCC La Molina n=113 (32.1%) SCC Carabayllo n=83 (23.6%) SCC Cercado de Lima n=71 (20.2) p ------------------------- ----------- ------------------------------ ----------------------------- ----------------------------- --------------------------------- --------- Age (years)\*   71.78 ± 7.30 70.89 ± 7.13 69.98 ± 7.58 70.99 ± 5.99   Age group 60-64 12 (14.1) 20 (17.7) 22 (26.8) 15 (21.1) \<0.000 65-69 26 (30.6) 35 (30.9) 24 (29.3) 13 (18.3) 70-74 20 (23.5) 27 (23.9) 13 (15.9) 17 (23.9) 75-79 13 (15.3) 15 (13.3) 14 (17.1) 23 (32.4) ≥80 14 (16.5) 16 (14.2) 9 (10.9) 3 (4.2) Sex Female 68 (80.0) 98 (86.7) 65 (78.3) 61 (85.9) 0.923 Marital status Single 38 (45.2) 54 (47.8) 58 (69.9) 30 (42.3) 0.341 Married 18 (21.4) 7 (6.2) 12 (14.5) 14 (19.7) Widowed 22 (26.2) 41 (36.3) 11 (13.3) 19 (26.8) Divorced 6 (7.1) 11 (9.7) 2 (2.4) 8 (11.3) Years of education   14 (11-16) 15 (11-16) 9 (6-11) 11 (7-14) Educational level 6-10 9 (10.6) 11 (9.7) 45 (54.2) 26 (36.6) \<0.000 11-15 42 (49.4) 52 (46.0) 30 (36.1) 28 (39.4) \>16 34 (40.0) 50 (44.3) 8 (9.6) 17 (23.9) History of hypertension   35 (41.7) 25 (22.3) 26 (31.7) 30 (42.3) 0.037 Nutritional status \<25 26 (30.9) 46 (45.1) 28 (33.7) 25 (35.7) 0.187 ≥25 58 (69.1) 56 (54.9) 55 (66.3) 45 (64.3) aMCI Yes 14 (16.5) 9 (7.9) 21 (25.3) 19 (26.8) 0.002 We found that 17.9% (n=63) participants had aMCI. The mean age in this group was higher than in the aMCI-group (75.2 vs. 69.9 years old, respectively, p\<0.001). No difference was found in aMCI frequency between males and females. Almost four out of five were women in both aMCI and aMCI- groups while in males the proportion was around 17% in both groups (p=0.923, see [Table 2](#t2){ref-type="table"}). Regarding marital status, 15.6% (n=28) of those with the condition were married, while in the group without cognitive impairment, the percentage of married couples was 84.4% (n=152) of the conglomerate of groups that included single, widowed and divorced subjects (p=0.02, OR 0.55, CI 0.30-0.98). The mean years of education was higher in the aMCI-group (13 vs 11 years, p\<0.001). In the aMCI group, when divided by subgroup, 49.2% (n=31) had 6 to 10 years of education (lowest level of education in our study population) and the proportion of individuals with more than 16 years of education was 14.3% (n=9). In the aMCI-group, 44.6% of participants had 11 to 15 years of education (n=129), closely followed by participants with more than 16 years of education, representing 34.6% (n=100). Differences between these subgroups were statistically significant (p\<0.001). There was no significant difference in the presence of aMCI for the BMI index. In terms of location, the group with the lowest frequency of aMCI was from the district of La Molina with 8.1% (n=9), followed by Jesús María with 16.5% (n=14). The districts with the highest frequency of aMCI were Carabayllo with 25.3% (n=21) and Cercado de Lima with 26.8% (n=19) (p=0.002, see [Table 2](#t2){ref-type="table"}). Being older was associated with the condition. On bivariate analysis, aMCI was 10 times more frequent in participants aged over 75 years (p=0.001, OR 10.08, CI 2.44 - 41.68). Also, having more than 16 years of education was associated with not having the condition (p\<0.001, OR 0.24, CI 0.12-0.18). Belonging to the SCC from La Molina district was associated with a lower frequency of aMCI, but was not statistically significant (p=0.071, OR=0.48, CI=0.22-1.07, see [Table 3](#t3){ref-type="table"}). Multivariate analysis showed similar results (See [Table 3](#t3){ref-type="table"}). All analyses were adjusted for sex, years of education and SCC location. DISCUSSION ========== The frequency of aMCI in this study was 17.9%, similar to the prevalence found in Tremembé, Brazil.[@B27] In other Latin American countries, a prevalence of around 10% was reported.[@B2]^-^[@B4] A multicenter study that included Peru reported a prevalence of 3.1%, low compared to other countries such as the United States, Italy and Spain (15.7%, 18.2%, 18.5%, respectively).[@B5]^-^[@B28] This variability is probably due to the lack of homogeneity of the criteria that defined this entity, the type of sampling and the neuropsychological tests used.[@B9]^-^[@B12]^,^[@B29]^,^[@B30] In addition, the lack of epidemiological studies in our region and the potential underdiagnosis of this entity should be considered. Participants who presented aMCI had an average age greater than those without impairment, which is in agreement with previous literature. This condition was found in 10% of individuals aged 70-79 years and 25% in those aged 80-89 years.[@B5]^,^[@B11]^,^[@B13]^-^[@B15] The subgroup of those over 75 years old had a ten times higher risk of having this condition. Considering that this subgroup is at greater risk of developing AD, this finding highlights the importance of screening for aMCI in older adults, acting on risk factors, delaying both onset and progression of dementia and taking family and social health measures related to dementia care.[@B6]^,^[@B12]^,^[@B16] The other associated factor was educational level. Higher-educated elderly (\>11 years of education) had half the risk of having aMCI compared to those with basic education (6-10 years of education). This finding agrees with several studies, which reported that a higher level of education is a protective factor for cognitive impairment, as well as for the subsequent development of dementia.[@B15]^,^[@B17]^,^[@B31]^-^[@B35] This reinforces the idea that intellectual activity delays the degeneration of cognitive function. There is evidence that a higher cognitive reserve, which refers to differences between individuals that will be more resistant to brain changes, provides more tolerance to neurodegenerative disease. Intellectual and physical activity at all stages of ageing, even in later life, can increase cognitive reserve and may be the reason for the delay in progression of dementia.[@B36] Also, being married was associated with less deterioration, corroborating previous reports. This can be attributed to the fact that married people, when not alone, have greater social activities than those of other marital status and are less predisposed to develop depression. However, factors such as years of marriage and quality of interaction between the couple were not studied. Double the risk in single people has been reported compared to married couples at midlife and a strong sociogenetic role could play a role in the development of AD.[@B37] Further studies are suggested to evaluate their possible role as a protective factor.[@B10]^-^[@B12] Attending the SCC La Molina was also a factor associated with not having the condition, even after adjusting for years of education. This may be due to the previously discussed socioeconomic differences between districts. In addition, this SCC had better infrastructure, human resources that included psychologists, and intellectual activities such as reading clubs, artistic activities, among others, which were also run on a more regular basis. Inequality among districts should be highlighted, this time related to access to public services of higher quality for the elderly, limited by the socioeconomic development of the SCC location. Factors related to the interaction of the elderly for the center such as years of membership and regularity of attendance were not evaluated because of lack of reliable records. One of the strengths of this study is the multicenter approach that included participants from different socioeconomic backgrounds. These centers are potential places for intervention where the public district offices can participate. Another strength of the present study was the use of the M\@T. This instrument is brief and easy to apply, designed as a screening test for use by the professionals of the primary care centers and that has shown high diagnostic performance compared to the MMSE.[@B16] We recognize that secondary causes of aMCI, such as B12 deficiency, hypothyroidism, depression and drug use, were not determined. Also, we relied solely on the M\@T ,which serves more like a tool for initial screening than for establishing a conclusive diagnosis of this condition. It is also important to point out that the majority of participants in this study were female, at a ratio of 4:1 compared to male participants. Several studies show this disproportion, but not to this magnitude, therefore caution should be exercised when attempting to extrapolate the study findings to males. We conclude that the frequency of this entity in the geriatric population of the SCC was 17.9%. Associated factors were age, history of hypertension, and years of education. Risk factors should be identified and screening tests carried out among elderly in primary care to establish an early diagnosis and implement interventions in this predementia stage, thereby slowing or preventing transition to AD. This study was conducted at the Faculty of Medicine, Universidad Peruana Cayetano Heredia, Lima, Peru. **Acknowledgements.** We are grateful for the collaboration and kindness of the personnel from the Municipalities of Carabayllo, Cercado de Lima, Jesus Maria and La Molina districts. [^1]: Disclosure: The authors report no conflicts of interest. [^2]: **Author contributions.** Sofía S. Sánchez Boluarte: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, supervision, validation, visualization, writing-original draft, writing-review & editing. Jesus Abanto, Arantxa Noelia Sanchez Boluarte, Nilton Santos Custodio Capuñay and Frine Samalvides: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, software, supervision, validation, visualization, writing-original draft, writing-review & editing. Alicia Boluarte Carbajal: data curation, formal analysis, funding acquisition, investigation, methodology, resources, software, supervision, writing-review & editing. Danilo Sanchez Coronel: conceptualization, data curation, funding acquisition, methodology, project administration, resources, supervision, validation, visualization.

All relevant data are within the paper and its Supporting Information files. 1. Introduction {#sec001} =============== Bearing capacity of spudcan foundations for offshore mobile jack-up rigs is currently assessed using methods recommended by ISO \[[@pone.0206626.ref001]\]. These include methods recommended by Skempton \[[@pone.0206626.ref002]\], Houlsby & Martin \[[@pone.0206626.ref003]\] and Hossain & Randolph \[[@pone.0206626.ref004], [@pone.0206626.ref005]\]. However, large, rapid post-installation spudcan settlements have occasionally been reported before and after the removal of preloading. In many cases, the cause is spudcan punch-through failure occurred in stiff soil deposit overlying soft soil deposit \[[@pone.0206626.ref006], [@pone.0206626.ref007]\]. Recently, Menzies and Roper \[[@pone.0206626.ref008]\] reported additional spudcan settlements under the maximum preloading at seven locations in the Gulf of Mexico, which however did not appear to be caused by punch-through. They attributed this to an unaccounted-for loading possibly arising from soil "backflow" into the cavity. This postulate is, however, not proven. ISO \[[@pone.0206626.ref001]\] indicates that spudcan settlement may consist of four components, i.e. elastic settlement, consolidation settlement, settlement due to cyclic loading, and settlement due to seabed instability. The elastic settlement is the component which occurs immediately on loading application. This component is often not critical post-installation since it would have taken place upon application of preload. The other three settlement components are still not fully understood. Of these three, seabed instability-induced settlement is likely to be highly dependent upon specific site conditions. This paper examines three components of the spudcan settlement, i.e elastic settlement due to cavity collapse, consolidation settlement and settlement due to cyclic loading, using both centrifuge model tests and finite element analyses. Most importantly, effects of lattice legs on these spudcan settlement components are incorporated. 2. Field data study-settlement due to cavity collapse {#sec002} ===================================================== The effect of cavity collapse on spudcan settlement is first estimated for seven sites reported by Menzies and Roper \[[@pone.0206626.ref008]\]. As shown in [Table 1](#pone.0206626.t001){ref-type="table"}, the reported additional spudcan settlements range from 0.13 to 0.31 times the spudcan diameter D, while maintaining the maximum preload in seven Gulf of Mexico sites. In this study, cavity depths after the completion of spudcan preloading were estimated using Hossain et al.\'s \[[@pone.0206626.ref009]\] method and Li et al.\'s \[[@pone.0206626.ref010]\] method. The former method does not consider the effect of lattice leg whilst the latter does. In Li et al.\'s \[[@pone.0206626.ref010]\] method, the cavity depth within the confines of the lattice leg is dependent on the area and opening ratios of the lattice leg. The area ratio *A*~*a*~ is defined as the ratio of the cross-sectional area of lattice leg to the maximum spudcan footprint, while the opening ratio *e* is defined as the ratio of the area of the openings at the sides of the lattice leg to the total area of the lattice sides. The lattice leg configurations in the seven sites were not specified by Menzies and Roper \[[@pone.0206626.ref008]\]. However, Li et al. \[[@pone.0206626.ref010]\] noted that area and opening ratios typically range from 0·3 to 0·7 and 0·6 to 0·8, respectively, for three- and four-chorded lattice legs. Hence, a lower- and upper-bound area ratio of 0.3 and 0.7 were assumed herein. The opening ratio was assumed to be 0.75 since the range of its variation is much smaller. 10.1371/journal.pone.0206626.t001 ###### Comparison between back-calculated settlements due to full cavity collapse and the measured additional settlements in seven Gulf of Mexico sites. {#pone.0206626.t001g} Site Penetration depth d (m) Spudcan diameter D (m) Soil strength s~u~ (kPa) Predicted cavity depth (m) Back-calculated normalized settlements due to full cavity collapse *w*~*cav*~ / D Measured maximum additional settlements w/D ------ ------------------------- ------------------------ -------------------------- ---------------------------- ----------------------------------------------------------------------------------- --------------------------------------------- ---------- ----- ------ ------------ ------ 1 37.5 13.5 2.4+1.35z^4^ 3.4 1.5 3.9--4.0 1.2--2.8 3.0 0.04 0.06--0.10 0.15 4 14.2 12.0 19.2+1.46z 5.8 2.5 6.6--6.6 2.0--4.7 2.6 0.08 0.10--0.18 0.23 5 17.3 12.0 15.6+1.24z 5.5 2.4 6.6--6.3 1.9--4.4 2.6 0.09 0.10--0.18 0.23 7 22.3 12.0 5.7+1.18z 3.7 1.6 4.3--4.3 1.3--3.0 2.6 0.06 0.08--0.13 0.23 8 23.6 12.0 8.6+1.02z 4.2 1.8 4.8--4.9 1.4--3.4 2.6 0.08 0.09--0.17 0.31 9 8.4 12.0 23.0+1.26z 6.1 2.6 6.9--7.0 2.1--4.9 2.6 0.12 0.13--0.24 0.13 12 10.3 14.6 18.2+2.09z 7.3 3.1 8.3--8.4 2.5--5.8 3.2 0.29 0.31--0.41 0.17 ^1^ Volume-averaged cavity depth H~ave~ is determined assuming a vertical inclined angle of 36^0^ for the cavity slope according to Hossain et al. (2005)'s PIV test results. ^2^ The predicted lower and upper cavity depths are due to different area ratios of lattice leg being assumed, lower and upper area ratios A~a~ of 0.3 and 0.7 are used here, respectively. For both cases, a typical lattice leg opening ratio e of 0.75 is assumed. ^3^ Based on Li et al. (2017(a)), an average cavity outer depth (i.e. the cavity outside of the lattice leg) H~o~ of 0.22D is used for spudcan enclosed with typical lattice leg (A~a~ = 0.6, e = 0.75) to determine the outer cavity volume within the spudcan footprint. Hossain et al. \[[@pone.0206626.ref009]\] proposed a cylindrical cavity shape to be used. However, their centrifuge observations showed that cavities for spudcans without lattices in normally consolidated soil have an inverted shallow conical shape. This was also noted by Li et al. \[[@pone.0206626.ref010]\]. For this reason, a shallow conical cavity is assumed instead to evaluate the cavity volume when Hossain et al.\'s \[[@pone.0206626.ref009]\] method is used. For spudcan with lattices, Li et al. \[[@pone.0206626.ref010]\] noted that, within the confines of the lattice leg, the cavity is almost vertical and co-planar with the sides of the lattice cage. Outside the confines of the lattice legs, the cavity is much shallower and the gradient of the cavity wall is also gentler than that inside the lattice leg. Since the lattice leg is unlikely to have significant effect on the cavity outside it, the shape of the cavity outside the lattice is assumed to be the same as that without the lattice, which is a shallow inverted cone \[[@pone.0206626.ref009], [@pone.0206626.ref010]\]. By assuming that the cavity volume calculated above is completely back filled, the induced additional loads can be used to back-calculate the additional settlement using an appropriate spudcan bearing capacity method. Menzies and Roper \[[@pone.0206626.ref008]\] noted that Hossain & Randolph\'s \[[@pone.0206626.ref004]\] method provides an upper bound of the observed bearing capacity, whereas Skempton\'s \[[@pone.0206626.ref002]\] and Hansen\'s \[[@pone.0206626.ref011]\] methods give good average prediction of the thirteen sites examined by Menzies and Roper \[[@pone.0206626.ref008]\]. If Hossain & Randolph\'s \[[@pone.0206626.ref004]\] bearing capacity factor was 20% reduced, then it gives very similar prediction to Skempton\'s \[[@pone.0206626.ref002]\] and Hansen\'s \[[@pone.0206626.ref011]\] predictions. Following Hossain and Randolph \[[@pone.0206626.ref005]\], the spudcan bearing capacity was estimated using Hossain & Randolph\'s \[[@pone.0206626.ref004]\] bearing capacity factor with a 20% reduction to back-calculate the additional settlement due to cavity collapse. The detailed back-calculating process on spudcan settlement due to full cavity collapse has been described in a flow chart in [Fig 1](#pone.0206626.g001){ref-type="fig"}. {#pone.0206626.g001} As shown in [Table 1](#pone.0206626.t001){ref-type="table"} and [Fig 2](#pone.0206626.g002){ref-type="fig"}, the back-calculated settlements due to backfill of cavity volume are generally smaller than measured settlements except for Sites 9 and 12. For Site 9, the back-calculated settlement using Hossain et al.\'s \[[@pone.0206626.ref009]\] cavity depth lies just below the measured settlement; while that obtained using Li et al.\'s \[[@pone.0206626.ref010]\] lower-bound cavity depth is also in good agreement with the measured data. For Site 12, using Hossain et al.\'s \[[@pone.0206626.ref009]\] and Li et al.\'s \[[@pone.0206626.ref010]\] cavity depth formulae lead to settlement values which significantly exceed the measured data. As [Table 1](#pone.0206626.t001){ref-type="table"} shows, with the exception of Sites 9 and 12, spudcan penetration depths in other five sites are much larger than the stable cavity depths estimated by Li et al. \[[@pone.0206626.ref010]\]. On the other hand, for Sites 9 and 12, spudcan penetration depths are only slightly larger than stable cavity depths. Given the uncertainty in the area and opening ratios and its effect on the stable cavity depth, it is possible that, in these two sites, the stable cavity depth might not have been reached post-installation. For instance, for Site 12, Li et al.\'s \[[@pone.0206626.ref010]\] method estimates a stable cavity depth of about 8.3m, which implies that the spudcan depth required for stable cavity formation must be larger than this depth. In this Site, the spudcan penetration depth is 10.3m, this implies that the distance between the mudline and the top of spudcan is only about 6.1m. Hence, the final stable cavity might have not been formed at this Site at full preload. Using the embedment depth of spudcan top as the cavity depth gives a better agreement with the measured additional settlement in Site 9, which however still exceeds the measured settlement in Site 12. This may suggest that the cavity is not fully backfilled in Site 12. {#pone.0206626.g002} The above discussion would suggest that cavity collapse may be able to account for the measured additional settlements in Sites 9 and 12. On the other hand, cavity collapse is able to account for only part of the measured additional settlements in the other five sites. Compared with the back-calculated settlements using Li et al.\'s \[[@pone.0206626.ref010]\] method, the remaining measured settlements of approximately 0.05D to 0.22D in the five sites are likely to be due to other causes. 3. Consolidation settlement {#sec003} =========================== 3.1 Numerical methodology {#sec004} ------------------------- Coupled consolidation small strain finite element analysis was conducted in ABAQUS/Standard version 6.14 to assess the consolidation settlement of the spudcan in a single clay layer, in which effect of spudcan consolidation load ratio was examined. Spudcan consolidation load ratio is defined as the ratio of the vertical load hold during consolidation over the maximum preload. [Fig 3](#pone.0206626.g003){ref-type="fig"} shows the two dimensional (2D) axisymmetric finite element mesh. To validate against the centrifuge model test \[[@pone.0206626.ref012]\], the soil geometric dimensions (27m in depth and 30m in radial extent) and boundary conditions adopted here were consistent with those used in the test. As shown in [Fig 3](#pone.0206626.g003){ref-type="fig"}, soil displacement normal to the axisymmetric line and the right vertical line was constrained, soil flow in the vertical direction was also constrained to be zero at the soil base. Drained was only allowed at the top of the soil surface. A finer mesh (element length 0.4m) was used one spudcan diameter laterally from spudcan center while a coarse mesh was used far away. This mesh density was found to yield consistent spudcan consolidation settlements. Four-node axisymmetric quadrilateral bilinear displacement and pore pressure elements were used for the soil domain. The soil was modelled using a modified Cam-clay (MCC) material, MCC parameters shown in [Table 2](#pone.0206626.t002){ref-type="table"} are consistent with soil properties used in the centrifuge model test \[[@pone.0206626.ref012]\]. By using Wroth's \[[@pone.0206626.ref013]\] Eq ([1](#pone.0206626.e001){ref-type="disp-formula"}), the simulated soil undrained shear strength *s*~*u*~ = 1.46*z* kPa could be obtained from MCC parameters. The soil was fully saturated with its initial stress being prescribed under a *K*~*0*~ condition, in which *K*~0~ = 1 − sin *φ*\' is an earth pressure coefficient at rest and *φ*\' is a soil critical state friction angle. The initial average void ratio and average density were set to be 1.3 and 1.06, respectively. {#pone.0206626.g003} {#pone.0206626.e001g} ( s u σ v \' ) N C = ( sin φ \' 2 a ) ( a 2 \+ 1 2 ) λ − κ λ Where $a = \frac{3 - \sin\varphi'}{2(3 - 2\mspace{2mu}\sin\varphi')}$, *σ*~*v*~\' is soil effective vertical stress, *λ* and *κ* are the slopes of soil isotropic compression and swelling lines. 10.1371/journal.pone.0206626.t002 ###### Parameters used in the modified Cam-clay model. {#pone.0206626.t002g} -------------------------------------------------------------- ------------ Slope of critical state line *M*~*2*~ 0.9 Slope of isotropic normal compression line *λ* 0.244 Slope of isotropic swelling and re-compression line *κ* 0.0523 Specific volume of soil at critical state (*p*\' = 1*kPa*) Γ 3.221 Effective Poisson's ratio *υ*\' 0.33 Effective unit weight *γ*\'(*kN*/*m*^3^) 6.0 Coefficient of earth pressure at rest *K*~*o*~ 0.6 Coefficient of permeability *k* (m/s) 2.0×10^-8^ Soil critical state friction angle *φ*\' 23^0^ -------------------------------------------------------------- ------------ Noted that the modelled soil strength falls to be the lower bound of the soil strength profiles in the Gulf of Mexico sites \[[@pone.0206626.ref008]\]. Soil bed with a lower soil undrained shear strength is usually correlated with a higher water content \[[@pone.0206626.ref014]\], and hence a higher void ratio. In view of this, spudcan embedded at soil with a lower undrained shear strength is likely to have a larger consolidation settlement. The soil strength profile modelled here would hence provide an upper bound estimation on spudcan consolidation settlements in the Gulf of Mexico sites. To examine spudcan post-installation settlements during jack-up rig operation period, three stages were involved in the numerical simulation, i.e. penetration-unloading-consolidation. The first two stages were usually completed under undrained condition. In view of this, total time for each of the two stages was set to be 1000s, which is relatively short and not likely to induce significant soil consolidation. A total time of 1.6e8s (5 years) was set for the consolidation stage to allow full consolidation. In this numerical simulation, a full-Newton method was used as the convergence criteria. Following Templeton's \[[@pone.0206626.ref015]\] method, the spudcan was firstly wished into a position ([Fig 3](#pone.0206626.g003){ref-type="fig"}, *d* = 13.6 m) at a small distance above the target depth. It was then penetrated over a distance of 1.5 m to achieve plastic penetration and establish the penetration stress field beneath the spudcan. The spudcan was then unloaded to a prescribed consolidation load ratio to commence the consolidation stage. Li *et al*. \[[@pone.0206626.ref012]\] noted that this method gives a good estimation of the measured post-consolidation penetration resistance and consolidation settlement when compared with centrifuge model test data, the latter will be further illustrated in [Fig 4](#pone.0206626.g004){ref-type="fig"}. {#pone.0206626.g004} 3.2 Numerical result analysis {#sec005} ----------------------------- The working load for jack-up rig is typically half of the maximum preload; this corresponds to a consolidation load ratio of 0.5. On the other hand, Menzies and Roper's \[[@pone.0206626.ref008]\] observations were made at a consolidation load ratio of 1.0. To cover this range, three consolidation load ratios 1.0, 0.75 and 0.5 were examined herein. [Fig 4](#pone.0206626.g004){ref-type="fig"} compares the computed consolidation settlements versus normalized consolidation times *T*~*c*~ *= c*~*v*~*t/D*^*2*^ at different consolidation load ratios with other results. *c*~*v*~ is the soil vertical coefficient of consolidation surrounding spudcan consolidation depth, which is about 40m^2^/year. [Fig 4](#pone.0206626.g004){ref-type="fig"} shows that the computed consolidation settlement agrees well with Li et al.\'s \[[@pone.0206626.ref012]\] centrifuge test result at a spudcan consolidation depth of d/D = 1.26 and a consolidation load ratio α of 0.75. Furthermore, the computed consolidation settlements compare well with Wang and Bienen\'s \[[@pone.0206626.ref016]\] large deformation finite element (LDFE) results at consolidation load ratios of 0.75 and 1.0, respectively. This would suggest the feasibility of this numerical method on estimating spudcan consolidation settlement. Wang and Bienen's \[[@pone.0206626.ref016]\] LDFE results also show that spudcan consolidation settlement is also influenced by spudcan embedment depth, which is however much less significant as that of consolidation load ratios. Bienen and Cassidy's \[[@pone.0206626.ref007]\] centrifuge test results also didn't show spudcan embedment depths effect on consolidation settlements. In this respect, spudcan embedment depth effect will not be studied here. [Fig 4](#pone.0206626.g004){ref-type="fig"} illustrates that a larger consolidation load ratio tends to yield a larger consolidation settlement. For each consolidation load ratio, the consolidation settlement increases rapidly at early consolidation stage and finally converges to a constant value. Take spudcan settlement under the consolidation load ratio of 1.0 for instance, about 50% of the consolidation settlement, that is about 0.05D, has taken place by *T*~*c*~ = 0.04 (for *c*~*v*~ = 40m^2^/year and *D* = 12m, the corresponding consolidation time is *t* = 54 days). This consolidation settlement magnitude at *T*~*c*~ = 0.04 is equal to the minimum of the excess settlement (ranging from 0.05D to 0.22D) which could not be accounted for by cavity collapse. For offshore sites with a lower coefficient of consolidation, a much longer consolidation time *t* will be needed to reach this settlement magnitude. In the Gulf of Mexico sites, additional spudcan settlements were however monitored upon the completion of preloading. This, together with that the computed settlement falls to be an upper bound site estimation, consolidation settlement is likely to explain only a small part of the measured additional settlements by Menzies and Roper \[[@pone.0206626.ref008]\]. 4. Settlement due to cyclic rocking {#sec006} =================================== 4.1 Centrifuge model test description {#sec007} ------------------------------------- Yang *et al*. \[[@pone.0206626.ref017]\] and Yang \[[@pone.0206626.ref018]\] reported centrifuge model test data relating to spudcan settlement due to cyclic rocking in normally consolidated (NC) Malaysia kaolin clay ([Table 2](#pone.0206626.t002){ref-type="table"}). Their centrifuge tests were conducted under a model gravity of 100g. The spudcan was penetrated into the consolidated soil bed with a constant velocity of 0.6mm/s to an equivalent prototype soil depth of about 15m (1.25D). Based on Finnie & Randolph's \[[@pone.0206626.ref019]\] dimensionless velocity, this rate of penetration is sufficient to ensure undrained condition during spudcan penetration. The spudcan load was then reduced to 50% of its preload at that depth, to simulate removal of preload. For "no-dissipation" tests, spudcan cyclic rocking commenced immediately after preload removal; this simulates the condition wherein the spudcan is subjected to environmental loads in the offshore field. For "full-dissipation" tests, soil surrounding spudcan footing was allowed to consolidate under the remaining vertical load to allow a full dissipation of excess pore pressure. Two rocking amplitudes were used. The first is a small amplitude rocking with angular amplitude of 0.2°, which represents low-level environmental loading that is unlikely to cause large-scale yielding of the spudcan foundation. The second is a large-amplitude rocking with angular amplitude of 2.3° (spudcan lateral distance around 1m) which represents larger wind and wave loading. [Table 3](#pone.0206626.t003){ref-type="table"} includes a selection of tests reported by Yang et al. \[[@pone.0206626.ref017]\]. As this Table shows, Yang et al.\'s \[[@pone.0206626.ref017]\] lattice legs have opening ratio of 0.75, which is also within the typical range of values of opening ratios used in prototype legs \[[@pone.0206626.ref010]\]. [Fig 5](#pone.0206626.g005){ref-type="fig"} illustrates the typical model lattice leg configuration and its dimensions, which has an opening ratio of *e* = 0.75 and an area ratio of *A*~*a*~ = 0.6. Yang et al. \[[@pone.0206626.ref017]\] also tested model spudcans with fully enclosed sleeves, but these will not be analyzed herein since they do not bear close resemblance to prototype legs. {#pone.0206626.g005} 10.1371/journal.pone.0206626.t003 ###### Centrifuge testing program in normally consolidated Malaysia kaoline clay. {#pone.0206626.t003g} ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Testing No Opening ratio Area ratio Leg shape Dissipation before cyclic rocking Amplitude\ Number of cycles Rocking settlement V~ult~ at 1.3D (MN) M~1~ at 1^st^ cycle (MNm)[^1^](#t001fn001){ref-type="table-fn"} M~1~/DV~ult~ (°) ------------------------------------------- --------------- ------------ ----------- ----------------------------------- ------------ ------------------ -------------------- --------------------- ----------------------------------------------------------------- -------------- SN1 1 0 / No dissipation 0.2 1000 0.030D 29 17.02 0.05 SN2 1 0 / Full dissipation 0.2 1000 0.005D 29 22.91 0.07 RO1 0.75 0.61 Square No dissipation 0.2 1000 0.029D 31 / / RO2 0.75 0.61 Square Full dissipation 0.2 1000 0.002D 31 / / SN3 1 0 / Full dissipation 2.3 70 0.076D 29 45.3 0.13 SN4[^2^](#t003fn002){ref-type="table-fn"} 1 0 / No dissipation 2.3 2 / 29 16.35 0.05 RO3 0.75 0.61 Square Full dissipation 2.3 70 0.077D 31 55 0.15 CO3 0.75 1.0 Circular Full dissipation 2.3 70 0.071D 31 33.5 0.09 CO4 0.75 1.0 Circular No dissipation 2.3 30 0.127D 31 14.8 0.04 ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ^1^The bending moment measured from the straingage installed at the lowest place of the shaft, closing to the spudcan footing, which is roughly assumed to be the moment experienced by the spudcan footing. ^2^Test SN4 was terminated after 2 cycles of loading owing to excessive settlement. Prefixes • SN refers to spudcan model without lattice leg • RO refers to spudcan model with square cross section lattice leg with opening ratio of 0.75 and area ratio of 0.61 • CO refers to spudcan model with circular cross section lattice leg with with opening ratio of 0.75 and area ratio of 0.61. Suffix numerals • 1 refers to "no-dissipation" test with small amplitude rocking • 2 refers to "full-dissipation" test with small amplitude rocking • 3 refers to "full-dissipation" test with large amplitude rocking • 4 refers to "no-dissipation" test with large amplitude rocking. 4.2 Spudcan settlement under small amplitude rocking {#sec008} ---------------------------------------------------- [Fig 6](#pone.0206626.g006){ref-type="fig"} plots normalized spudcan settlements during small amplitude rocking \[[@pone.0206626.ref017]\]. As can be seen, for both spudcans with and without lattice legs, settlements increase gradually with the numbers of cycles and approach stable values after about 1000 cycles. The "no-dissipation" tests show much larger settlements than those of "full-dissipation" tests, and the settlement differences between "no-dissipation" and the corresponding "full-dissipation" tests are about 0.026*D* and 0.025*D* for spudcans with and without lattice legs, respectively. These values are somewhat larger than, but of the same order as the computed consolidation settlement of 0.015*D* at a consolidation load ratio of *α* = 0.5 and a similar consolidation depth of 1.26*D*, [Fig 4](#pone.0206626.g004){ref-type="fig"}. Furthermore, the lattice leg does not significantly influence spudcan settlement under small amplitude rocking. {#pone.0206626.g006} [Fig 7](#pone.0206626.g007){ref-type="fig"} illustrates Yang et al.\'s \[[@pone.0206626.ref017]\] pore pressure dissipation data, which shows a small increase in excess pore pressure over the first \~20cycles of rocking, followed by monotonic decrease. This indicates that while there might have been some initial increase in excess pore pressure due to rocking, the amount is relatively small and pore pressure evolution is dominated by the decrease in excess pore pressure from an initially high value caused by spudcan penetration. This would suggest that much of the measured settlement differences between "no-dissipation" and "full-dissipation" tests can be explained by consolidation during the rocking episode. There might have been a small amount of cyclic-induced settlement, but this is generally small, typically between 0.03D and 0.005D, and are much smaller than the observed spudcan settlement increments reported by Menzies and Roper \[[@pone.0206626.ref008]\]. {#pone.0206626.g007} 4.3 Spudcan settlement under large amplitude rocking {#sec009} ---------------------------------------------------- Spudcan settlement under large amplitude rocking is manifestly different from that under small amplitude rocking. As shown in [Fig 8](#pone.0206626.g008){ref-type="fig"}, spudcan settlement increases rapidly with successive cycles of rocking until the vertical movement reaches the limit of the centrifuge modelling equipment. Settlement had clearly not stabilized at this point. For "full-dissipation" tests, after 70 cycles, additional settlements ranging from 0.071D to 0.077D were recorded for spudcans with and without lattice legs. For "no-dissipation" tests, the additional settlement increases to 0.13D after 30 cycles for spudcan with lattice leg (CO4). In test SN4, settlement exceeds the equipment limit just after 2^nd^ cycles. This magnitude of settlement is much larger than that incurred under 1000 cycles of small amplitude rocking, and can explain the discrepancy range of 0.05D to 0.22D between back-calculated and measured settlements in cases 1, 4, 5, 7 and 8 reported by Menzies and Roper \[[@pone.0206626.ref008]\]. {#pone.0206626.g008} For "full-dissipation" tests, to distinguish the contribution of possible cavity collapse on spudcan settlements, back-calculation of spudcan settlements due to cavity collapse for spudcans with square lattice leg (RO3) and full circular lattice leg (CO3) were carried out. Using Li et al.\'s \[[@pone.0206626.ref010]\] method, averaged cavity depths of 0.57m and 0.91m are obtained for the two cases in NC clays, respectively. Full cavity collapse would lead to additional spudcan settlements of about 0.02D and 0.03D for RO3 and CO3, respectively. Compared to their measured settlements of 0.077D and 0.071D ([Table 3](#pone.0206626.t003){ref-type="table"}), there remains a significant proportion of settlements which cannot be adequately explained by cavity collapse. Furthermore, the durations of the large amplitude tests were much shorter than those of the small amplitude tests, owing to much smaller number of cycles. As [Fig 7](#pone.0206626.g007){ref-type="fig"} show, the amount of pore pressure dissipation which occurs over the first 70 cycles are relatively insignificant and the spudcan foundation is still in a relatively undrained condition. Hence, consolidation is also unlikely to be a cause. Various other factors have been postulated, such as soil degradation effects \[[@pone.0206626.ref001], [@pone.0206626.ref020]\]. The settlement discrepancy between full dissipation test (CO4) and no dissipation test (CO3) is about 0.09D at 30 cycles for spudcan with full circular lattice leg. This discrepancy is much larger than the computed consolidation settlement of 0.015D at a load ratio α of 0.5. Furthermore, undrained condition should still prevail over the first 30 cycles, so much of this settlement discrepancy is likely to be caused by cyclic rocking rather than consolidation. As [Fig 8](#pone.0206626.g008){ref-type="fig"} shows, for full dissipation test, the lattice leg does not significantly influence spudcan settlement. However, comparison of SN4 and CO4 suggests that, for no dissipation test, the presence of the lattice leg appears to reduce spudcan settlement and enhance spudcan stability significantly. In Yang\'s \[[@pone.0206626.ref018]\] spudcan model, a full-bridge strain gauge was installed along the central shaft near the spudcan to measure the bending moment. [Fig 9](#pone.0206626.g009){ref-type="fig"} plots the measured spudcan bending moment under the large amplitude cyclic rocking, together with the moment-vertical load yield envelope from SNAME \[[@pone.0206626.ref021]\], using the maximum preload as the ultimate vertical load *V*~*ult*~. For "no-dissipation" tests, the maximum bending moment is roughly half of the yield moment at the load ratio of 0.5. The spudcan bending moment appears to be slightly reduced by the presence of the full circular lattice leg (CO4), which is consistent with the response of the spudcan settlement as discussed above. Taking into account the increase in vertical loading in the event of cavity collapse leads to only a slight right-shift in the corresponding point. Hence, large settlements observed by Yang et al. \[[@pone.0206626.ref017]\] in "no-dissipation" tests cannot be readily attributed to exceedance of the yield locus. For "full-dissipation" tests, the maximum moment is larger, ranging from just below to about 1.5 times the yield moment. However, since the ultimate vertical load *V*~*ult*~ relates to that before consolidation, the increase in the post-consolidation ultimate vertical load \[[@pone.0206626.ref010], [@pone.0206626.ref022]\] may also lead to an increase in moment capacity and therefore size of the yield locus. {#pone.0206626.g009} 5. Implications of results {#sec010} ========================== Menzies and Roper's \[[@pone.0206626.ref008]\] field observations showed that large additional spudcan settlements can occur at maximum preloads. Part of these additional settlements can be explained in terms of cavity collapse. However, in Sites 1, 4, 5, 7 and 8, full cavity collapse and consolidation settlement appear to be unable to account for the additional settlement. Moreover, there is still an outstanding question on what caused the cavity collapse. The issue of additional settlement was also examined by Yang et al. \[[@pone.0206626.ref017]\] using centrifuge models in which spudcans were subjected to rocking perturbation. Yang et al.\'s \[[@pone.0206626.ref017]\] models were conducted under a load ratio of 0.5 and therefore cannot be compared directly with Menzies and Roper's \[[@pone.0206626.ref008]\] cases. Nonetheless, Yang et al.\'s \[[@pone.0206626.ref017]\] results still contain some significant qualitative indicators. Firstly, small amplitude rocking leads to relatively small settlement which is not only unlikely to explain the large settlement but is also much less than the settlement that would have resulted from cavity collapse. Hence, cavity collapse may not readily occur under small amplitude rocking. Secondly, Yang et al.\'s \[[@pone.0206626.ref017]\] data shows that large-amplitude rocking can lead to large settlement over relatively small number of cycles. Although the measured settlement is still smaller than that observed by Menzies and Roper \[[@pone.0206626.ref008]\], this is due to limitation of the equipment, which was unable to track larger displacements. As [Fig 9](#pone.0206626.g009){ref-type="fig"} shows, in spite of the large settlement, the maximum moment-vertical load combination for the no-dissipation cases lie well within the yield locus. Hence, by all accounts, the model should have been well-within operating envelope. [Fig 9](#pone.0206626.g009){ref-type="fig"} shows that if the additional load from cavity collapse is added, the load point shifts rightwards slightly but still remains well within the yield locus. The above discussion suggests that, even if the load point is within the yield locus, large amplitude cyclic rocking can still lead to large spudcan settlement. One possible reason may be the build-up cyclically generated excess pore pressure surrounding the spudcan footing, leading to a loss of soil effective stress, and thereby soil strength and stiffness. Ho \[[@pone.0206626.ref023]\] reported that cyclic triaxial loading can lead to a significant loss of effective stress if the cyclic stress ratio (q/p\') exceeds a phase transformation stress ratio \[[@pone.0206626.ref024]\], which is typically about 60% of the critical state stress ratio for Malaysia kaolin clay and Singapore upper marine clay. As shown in [Fig 10](#pone.0206626.g010){ref-type="fig"}, the mean effective stress for the Malaysia kaolin clay is approximately 80% lost after 100 cycles loading \[[@pone.0206626.ref023]\]. To identify the stress state of soil surrounding the spudcan footing during cyclic rocking, a small strain finite element simulation of a single cycle of large amplitude rocking on the spudcan was performed. This method has been demonstrated to provide a reasonable estimation on Yang's \[[@pone.0206626.ref018]\] centrifuge model test results regarding the short-term and long-term spudcan fixities \[[@pone.0206626.ref022]\]. {#pone.0206626.g010} [Fig 11](#pone.0206626.g011){ref-type="fig"} presents the three dimensional (3D) finite element mesh, its boundary lengths and boundary conditions coincide with those used in Yang et al.'s \[[@pone.0206626.ref017]\] centrifuge model test. In general, the modelling methodology in 3D is the same as that described in 2D finite element analysis. Four stages were involved in the 3D finite element analysis, i.e. penetration-unloading-consolidation-rocking. To replicate centrifuge model tests by Yang et al. \[[@pone.0206626.ref017]\], the spudcan was initially wished-in-place at a depth of 13.6 m, this was followed by an additional penetration of 1.5m. After that, the spudcan preload at 15.1m was half removed before commencing consolidation. For "full-dissipation" test, a consolidation time of about five years was prescribed; for "no-dissipation" test, rocking was conducted immediately after unloading with no consolidation being allowed. To simulate the large amplitude cyclic rocking, a maximum lateral distance of 1m was applied to the spudcan. [Fig 12](#pone.0206626.g012){ref-type="fig"} shows that the computed stress ratios of soil elements within 0.5D beneath the spudcan footing can reach 94% and 83% of the critical state stress ratio, for "no-dissipation" test and "full-dissipation" test after the first rocking cycle, respectively. Both of these values are sufficient to generate large excess pore pressure with accompanying loss of soil strength and stiffness. Hence, a conservative approach may need to be taken into account for such cyclic loading effects. {#pone.0206626.g011} {#pone.0206626.g012} 6. Conclusions {#sec011} ============== The forgoing discussion explored the possible causes on additional spudcan settlements after the completion of preloading in the Gulf of Mexico sites, the findings show that full cavity collapse could only explain part of the measured additional settlements. Consolidation settlement is likely to account for a small proportion of the spudcan settlement, indeed probably less significant than that caused by cavity collapse. Centrifuge cyclic rocking tests demonstrate that large spudcan settlement can still occur if the spudcan is subjected to large amplitude cyclic loading, even after a 50% removal of the maximum preloading and the yield envelope was not exceeded. This leads to the possibility that stiffness and strength degradation of the soil in the vicinity of the spudcan footing and the leg might have occurred due to cyclic loading. The centrifuge results also show that the lattice leg has a significant effect on reducing the spudcan settlement during cyclic loading, possibly by taking up some of the moment arising from the loading on the jack-up rig \[[@pone.0206626.ref018]\]. In view of these findings, a conservative approach is recommended in instances where large amplitude cyclic rocking, such as that arising from storm loading, is expected shortly after preloading. A~a~ : area ratio, ratio of the cross-sectional area of lattice leg to the maximum spudcan footprint d : spudcan penetration depth, determined from the lowest point of the largest spudcan cross-sectional area to the mudline D : spudcan diameter at the largest cross section e : opening ratio, ratio of the area of the openings at the sides of the lattice leg to the total area of the lattice sides H~max~ : cavity depth determined from the mudline to the bottom of the cavity H~ave~ : averaged cavity depth with a cross sectional area of the maximum spudcan footprint H~i~ : cavity depth inside the lattice leg H~o~ : cavity depth outside the lattice leg K~o~ : coefficient of earth pressure at rest k : coefficient of permeability M~1~ : measured maximum spudcan bending moment at the 1^st^ rocking cycle M~2~ : slope of critical state line p\' : mean effective stress q : deviator stress s~u~ : soil undrained shear strength Tc : normalized consolidation time t : consolidation time V~ult~ : maximum preload V : vertical load hold during consolidation and cyclic rocking V~s~ : spudcan volume w~cav~ : spudcan settlement due to cavity collapse w~con~ : spudcan settlement due to consolidation w~cyclic~ : spudcan settlement due to cyclic loading w : measured additional settlement α : the ratio of the vertical load hold during consolidation over the maximum preload λ : slope of isotropic normal compression line κ : slope of isotropic swelling and recompression line Γ : specific volume of soil at critical state (*p*\' = 1*kPa*) *υ*\' : effective Poisson's ratio *γ*\' : soil effective unit weight *σ*~*v*~\' : soil effective vertical stress *φ*\' : soil critical state friction angle [^1]: **Competing Interests:**The authors have declared that no competing interests exists.

Background {#Sec1} ========== Postoperative pulmonary complications (PPCs) affect morbidity, mortality, length of hospital stay \[[@CR1], [@CR2]\] and are at least as frequent as cardiovascular complications \[[@CR2]\]. Therefore, PPCs are one of the most serious problems during perioperative period \[[@CR2], [@CR3]\]. The incidence of PPCs depends on patients' co-morbidity, surgical procedures and anesthetic factors \[[@CR1], [@CR3]\]. Among these, intraoperative ventilator settings are suggested to be one of the most crucial factors \[[@CR4]\]. To prevent the occurrence of PPCs, intraoperative lung protective ventilation, mainly comprised of low tidal volume (V~T~), slight degree of positive end-expiratory pressure (PEEP), and limited airway pressure, has been reviewed \[[@CR5]--[@CR8]\]. According to several studies in open abdominal surgery, this approach improved not only postoperative respiratory function \[[@CR8]\] but also clinical outcomes \[[@CR5], [@CR7]\]. This lung protective strategy has been steadily filtering into our ventilation strategy as a standard clinical practice. In one-lung ventilation (OLV), it is indicated that high V~T~ and inspiratory airway pressure are risk factors for acute lung injury after thoracic surgery \[[@CR9]--[@CR11]\], while high ventilator support is sometimes needed during OLV to maintain patient's oxygenation and eliminate carbon dioxide. However, the evidence for optimal ventilator settings during OLV remains insufficient. Consequently, there are numerous variations of ventilator settings, including inspired oxygen fraction (F~I~O~2~) as well as V~T~ and PEEP, due to specific pathophysiology and historical background \[[@CR12]--[@CR15]\], especially for the management of oxygen concentrations \[[@CR13]--[@CR16]\]. In this clinical study, we investigated the current practice of intraoperative ventilation during OLV in adult patients undergoing thoracic surgery. Furthermore, we tested whether the intraoperative ventilator settings were associated with the incidence of PPCs after thoracic surgery. Methods {#Sec2} ======= Study design, setting, and participants {#Sec3} --------------------------------------- A two-center prospective observational study was conducted from April 2014 to October 2014 in Japan. Participating hospitals included an academic tertiary care hospital and a community hospital. This study was approved by the institutional ethics review board (IRB) of Okayama University Hospital (No. 1922) and Fukuyama City Hospital (No. 182). The requirement for written informed consent was waived by each IRB. We screened consecutive patients over the age of 20 who were scheduled for a thoracic surgical procedure and required general anesthesia with OLV. We excluded emergency surgery, re-operative surgery, and patients who did not receive OLV. There was no specific protocol for perioperative management at the participating hospitals. Data source and collection {#Sec4} -------------------------- We investigated perioperative information, including preoperative characteristics, details of surgery and anesthesia, and postoperative course. Demographics and clinical data were extracted from electronic medical records. The preoperative data included sex, age, Assess Respiratory Risk in Surgical Patients in Catalonia (ARISCAT) score \[[@CR17]\], preoperative respiratory function, and preoperative percutaneous oxygen saturation (SpO~2~). We collected anesthetic and surgical information, such as surgical procedures, types of general anesthesia, use of epidural anesthesia, and airway management as well as duration of procedure, anesthesia, and OLV. Total blood loss and volume of infusion were also collected. Minimum SpO~2~ throughout the course of anesthesia was recorded. During OLV (0, 30, 60, and 120 min after the start of OLV and at the end of OLV), the following variables were recorded: ventilator mode, F~I~O~2~, V~T~ corrected for predicted body weight (PBW), driving pressure (ΔP) (peak inspiratory pressure minus PEEP on both pressure control and volume control ventilation), and PEEP. These data were collected by attending anesthesiologists. PBW was calculated as follows: for men, 50 + 0.91 (height (cm) - 152.4); and for women, 45.5 + 0.91 (height (cm) - 152.4) \[[@CR18]\]. Quantitative variables and bias {#Sec5} ------------------------------- To avoid surveillance bias, time weighted average (TWA) of ventilation parameters was calculated for the first 2 h of OLV. TWA was determined by summing the mean value between consecutive time points (0, 30, 60, and 120 min after the start of OLV) multiplied by the period of time between consecutive time points and then divided by the total time. We calculated and assessed TWA of F~I~O~2~, V~T~, ΔP, and PEEP during OLV. Outcome measures {#Sec6} ---------------- The primary outcome was the incidence of PPCs occurring within 7 days of thoracotomy. PPCs included pneumonia, pleural effusion, atelectasis, prolonged air leakage, pulmonary embolism and respiratory failure diagnosed according to the definitions (Table [1](#Tab1){ref-type="table"}), which referred to previous studies \[[@CR17], [@CR19], [@CR20]\]. In each center, a predetermined researcher evaluated all patients in accordance with the definitions of PPCs. To investigate the length of hospital stay (LOS) and mortality, patients were followed-up until hospital discharge or death (whichever occurred first).Table 1The definition of PPCsPPCsDefinitionPneumonia \[[@CR19]\]1. Presence of new or progressive infiltrates on chest radiograph\ 2. Fever (\> 38 °C) or leukocyte count (\< 4000, ≥12,000 WBC/mm^3^)\ 3. New or changed sputum, tachypnea, impaired gas exchangePleural effusion \[[@CR17]\]Chest radiograph demonstrating blunting of the costophrenic angle or loss of the sharp silhouette of the hemidiaphragm on the nonoperative sideAtelectasis \[[@CR17]\]Opacities evidenced on chest radiograph with a shift of the mediastinum, hilum, or hemidiaphragm toward the affected areaProlonged air leakage \[[@CR20]\]Air leak requiring insertion of new chest tube or ≥7 days of postoperative chest tube drainagePulmonary embolism \[[@CR20]\]Pulmonary arteriogram or ventilation/perfusion radioisotope scan documenting thrombusRespiratory failure \[[@CR20]\]Postoperative ventilator dependence ≥24 h or Need of reintubation or noninvasive ventilation*PPCs* postoperative pulmonary complications Statistical analysis {#Sec7} -------------------- Variables were assessed for normality. Categorical data were compared using chi-square tests or Fisher exact tests and reported as n (%). Continuous normally distributed variables were compared using Student *t* tests and reported as means (standard deviation), while non-normally distributed data were compared using Wilcoxon rank-sum tests and reported as medians (interquartile range). Univariate analysis was performed to compare perioperative characteristics between patients with and without PPCs. A multivariate logistic regression analysis was performed to estimate the associations between intraoperative ventilator settings and PPCs, adjusting for ARISCAT score and all univariate relevant factors that discriminate between the two groups. To explore subgroup differences in associations between the ventilator settings and PPCs, the same multivariate analyses were performed for subgroups classified according to the ARISCAT score, preoperative SpO~2~ and surgical procedures, respectively. All analyses were performed using JMP version 8.0.2 (SAS Institute, Cary, NC, USA). *P* \< 0.05 was considered statistically significant. This manuscript adheres to the applicable Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines. Results {#Sec8} ======= Participants characteristics {#Sec9} ---------------------------- Overall, 212 cases underwent thoracic surgery with OLV during the study period. Two patients were younger than 20 years old, and 13 cases underwent thoracic surgeries twice during the study period. Thus, 197 patients met the eligibility criteria (Fig. [1](#Fig1){ref-type="fig"}).Fig. 1Study flow diagram. *OLV* one-lung ventilation Baseline characteristics and intraoperative procedures of all patients are noted in Additional file [1](#MOESM1){ref-type="media"}. Most patients (*n* = 190, 96.4%) had an intermediate or high risk of having PPCs according to the ARISCAT score. More than 80% of patients underwent lung resections; however, there was no patient who underwent pneumonectomy. Main results {#Sec10} ------------ Pressure control ventilation (PCV) was utilized in most cases (*n* = 181, 92%). At the start of OLV, median F~I~O~2~ was 1.0 (0.8-1.0). Specifically, an F~I~O~2~ of 1.0 was applied as an initial setting for more than 60% of all patients. In other initial settings, median V~T~ was 6.1 (5.2-7.3) ml/kg, and median ΔP was 16 (14-20) cm H~2~O. PEEP was applied in 171 patients (87%) at a median level of 4 (4-5) cm H~2~O. The distributions of ventilator settings throughout OLV are shown as TWA values in Fig. [2](#Fig2){ref-type="fig"}. Median TWA F~I~O~2~ was 0.8 (0.65-0.94), and 83% of patients received TWA F~I~O~2~ ≥ 0.6. Other median TWA values, such as V~T~, ΔP, and PEEP, were at almost similar levels as the initial settings (V~T~, 6.1 (5.3-7.0) ml/kg; ΔP, 17 (15-20) cm H~2~O; and PEEP, 4 (4-5) cm H~2~O). As a rescue therapy, oxygen therapy to the non-ventilated lung was adopted in only five cases.Fig. 2Distribution of ventilator settings during one-lung ventilation. Each graph represents the distributions of TWA values during one-lung ventilation: (**a**) F~I~O~2~, (**b**) V~T~, (**c**) ΔP, and (**d**) PEEP. *TWA* time weighted average, *F*~*I*~*O*~*2*~ inspiratory oxygen fraction, *V*~*T*~ tidal volume, *ΔP* driving pressure, *PEEP* positive end-expiratory pressure PPCs occurred in 51 of 197 cases (25.9%). Atelectasis developed in 35 patients (17.8%), prolonged air leakage in 10 (5.1%), pneumonia in 3 (1.5%), pleural effusion in 3 (1.5%), and respiratory failure in 2 (1.0%). Two cases with respiratory failure occurred with atelectasis or pleural effusion. None of the patients were diagnosed with pulmonary embolism in this period. Only one patient died during hospital stay, and overall mortality was 0.5%. Baseline characteristics and intraoperative procedures in patients with and without PPCs were shown in Table [2](#Tab2){ref-type="table"}. There were no significant differences in preoperative baseline characteristics, surgical procedures, and intraoperative management regarding anesthesia.Table 2Baseline characteristics and intraoperative information of patients with and without PPCsPatients with PPCs (*N* = 51)Patients without PPCs (*N* = 146)*P* valuePreoperative baseline Age - years67.4 ± 12.963.7 ± 13.20.094 Sex (male) - no. (%)33 (64.7)88 (60.3)0.57 ARISCAT score50 (43-50)50 (27-50)0.44 Preoperative SpO~2~ - %97 (96-98)98 (96.25-99)0.055 %VC - %101 ± 18104 ± 180.31 FEV1.0% - %75 ± 1375 ± 100.72Anesthesia & Operation Lung resection (+) - no. (%)45 (88.2)123 (84.2)0.64 TIVA - no. (%)31 (60.8)78 (53.8)0.39 Epidural anesthesia - no. (%)37 (72.6)109 (74.7)0.77 Oxygen therapy to the non-ventilated lung - no. (%)3 (5.9)2 (1.4)0.08 Duration of anesthesia - min285 (185-362)263 (162-333)0.17 Duration of operation - min205 (118-276)194 (102-258)0.15 Duration of OLV - min173 (96-240)167 (77-224)0.33 Total volume of infusion - ml1660 (1250-2190)1550 (958-2100)0.14 Total blood loss - ml40 (10-100)15 (10-93)0.37 Minimum SpO~2~ - %94 (91-96)95.5 (93-97)0.0053Baseline and procedural characteristics are shown as n (%), means ± standard deviation or medians (interquartile range)*PPCs* postoperative pulmonary complications, *ARISCAT* Assess Respiratory Risk in Surgical Patients in Catalonia, *%VC* % vital capacity, *FEV1.0%* forced expiratory volume in one second %, *TIVA* total intravenous anesthesia, *OLV* one-lung ventilation Among ventilator settings, only TWA F~I~O~2~ in patients with PPCs was significantly higher than that in patients without PPCs (0.85 (0.73-1.0) vs. 0.77 (0.63-0.89); *P* = 0.0032) (Table [3](#Tab3){ref-type="table"}). There was no significant difference in TWA V~T~, TWA ΔP, and TWA PEEP between the two groups. Throughout the anesthesia, minimum SpO~2~ in patients with PPCs was significantly lower than that in patients without PPCs (94 (91-96) % vs. 95.5 (93-97) %; *P* = 0.0053). Finally, the postoperative LOS was longer in patients with PPCs (13 (8-16) days vs. 8 (7-11) days; *P* \< 0.001).Table 3Ventilator setting during OLV of patients with and without PPCsPatients with PPCs (*N* = 51)Patients without PPCs (*N* = 146)*P* valueVentilator setting during OLV Mode (PCV) - no. (%)46 (90.2)135 (92.5)0.62 TWA F~I~O~2~0.85 (0.73-1.0)0.77 (0.63-0.89)0.0032 TWA V~T~ - ml/kg6.2 (5.2-7.4)6.1 (5.4-7.0)0.8495 TWA ΔP - cmH~2~O18 (15-21)16 (15-18)0.0717 TWA PEEP - cmH~2~O4 (4-5)4 (4-5)0.1504Ventilator settings are shown as n (%) or medians (interquartile range)*OLV* one-lung ventilation, *PPCs* postoperative pulmonary complications, *PCV* pressure control ventilation, *TWA* time weighted average, *F*~*I*~*O*~*2*~ inspiratory oxygen fraction, *V*~*T*~ tidal volume, *ΔP* driving pressure, *PEEP* positive end-expiratory pressure In multivariate logistic regression model (Table [4](#Tab4){ref-type="table"}), which was adjusted for ventilator settings (TWA F~I~O~2~, TWA ΔP, and TWA PEEP), ARISCAT score, and minimum SpO~2~, only TWA F~I~O~2~ during OLV was independently associated with the occurrence of PPCs. Odds ratio (OR) per TWA F~I~O~2~ increase of 0.1 was 1.30 (95% confidence interval (CI): 1.04-1.65, *P* = 0.0195). Other variables (TWA ΔP, TWA PEEP, ARISCAT score, and minimum SpO~2~) were not related to the occurrence of PPCs in this model.Table 4Multivariate Analysis of risk factor for PPCsOdds Ratio*P* valueARISCAT score (per 1 point)1.02 (95% CI: 0.99-1.05)0.3038Minimum SpO~2~ (per 1%)0.89 (95% CI: 0.79-1.00)0.0544TWA F~I~O~2~ (per 0.1)1.30 (95% CI: 1.04-1.65)0.0195TWA ΔP (per 1 cmH~2~O)1.03 (95% CI: 0.91-1.16)0.6436TWA PEEP (per 1 cmH~2~O)1.09 (95% CI: 0.86-1.40)0.4994*PPCs* postoperative pulmonary complications, *ARISCAT* Assess Respiratory Risk in Surgical Patients in Catalonia, *CI* confidence interval, *TWA* time weighted average, *F*~*I*~*O*~*2*~ inspiratory oxygen fraction, *ΔP* driving pressure, *PEEP* positive end-expiratory pressure Subgroup analyses {#Sec11} ----------------- There were significant associations between F~I~O~2~ and PPCs in patients with low or intermediate risk of having PPCs according to the ARISCAT score (OR, 1.48; 95% CI, 1.00-2.40; *P* = 0.0496), or undergoing lung resection (OR, 1.31; 95% CI, 1.03-1.70; *P* = 0.0278) (Additional file [2](#MOESM2){ref-type="media"}). Other subgroups including patients with high risk for PPCs and high or low preoperative SpO~2~, also indicated that higher F~I~O~2~ tended to be associated with higher incidence of PPCs. Discussion {#Sec12} ========== Key results {#Sec13} ----------- We conducted a prospective observational study to investigate the current practice of intraoperative ventilation and to evaluate the associations between ventilator settings during OLV and PPCs in patients undergoing thoracic surgery. We found that F~I~O~2~ of ≥0.8, V~T~ of approximately 6 ml/kg, and PEEP of approximately 4 cm H~2~O were common. Patients with PPCs received higher F~I~O~2~ during OLV, while they had lower minimum SpO~2~ than those without PPCs. However, in multivariate logistic regression analysis adjusting for ventilator settings, ARISCAT score, and minimum SpO~2~, only TWA F~I~O~2~ was associated with the occurrence of PPCs, and the adjusted OR per F~I~O~2~ increase of 0.1 was 1.30. Therefore, an increase in oxygen concentration of 10% was associated with approximately 30% increase in the risk of PPCs. Interpretation {#Sec14} -------------- We found that V~T~ was around 6 ml/kg, and PEEP was set around 4 cm H~2~O in most patients. These findings were consistent with recent studies or textbook oriented lung protective strategy \[[@CR15], [@CR21], [@CR22]\]. We also found that high F~I~O~2~ was frequently used during OLV. These findings, however, were inconsistent with recent recommended management \[[@CR22]\]. An F~I~O~2~ of 1.0 was classically a routine component of OLV \[[@CR15], [@CR23]\]. However, the incidence of hypoxemia during OLV has been decreasing \[[@CR15], [@CR22]\], and the harmful effects of high F~I~O~2~, including absorption atelectasis \[[@CR24]--[@CR27]\], production of reactive oxygen species, and increased lung injury \[[@CR28], [@CR29]\], have been reported. Therefore, this classic practice has been questioned and avoidance of excessive F~I~O~2~ has been proposed \[[@CR15]\]. The latest textbook suggests that F~I~O~2~ should be titrated to maintain a stable saturation level above 92-94% during OLV \[[@CR22]\]. However, some reports revealed that relatively high F~I~O~2~ was still applied as a common practice during both two-lung ventilation \[[@CR30], [@CR31]\] and OLV \[[@CR13]--[@CR16]\]. In our survey, intraoperative minimum SpO~2~ was ≥95% in 111 patients (56%), with 83% of them receiving TWA F~I~O~2~ of ≥0.6 (Additional file [3](#MOESM3){ref-type="media"}). These findings indicated that almost half of the patients may have received excessive oxygen regardless of their SpO~2~. There was low compliance with recommended standards to maintain a SpO~2~ above 92-94% during OLV. According to our results, high F~I~O~2~ during OLV was independently associated with the increasing incidence of PPCs, and patients with PPCs had a longer LOS in the hospital. Worse clinical outcomes due to high F~I~O~2~ were previously reported in critically ill adults, including patients with chronic obstructive pulmonary disease, myocardial infarction, cardiac arrest, stroke, and traumatic brain injury \[[@CR32]--[@CR35]\]. Given the above concern, a conservative oxygenation strategy has been shown to be feasible, safe, and effective for mechanically ventilated patients in recent decades \[[@CR36], [@CR37]\]. Notably, conservative oxygen therapy could be associated with decreased evidence of atelectasis as well as earlier weaning from mandatory ventilation in the ICU \[[@CR38]\]. Additionally, a recent randomized control trial of conservative oxygen therapy in ICU showed lower mortality \[[@CR39]\]. Only a few studies investigated the effect of intraoperative F~I~O~2~ on clinical outcomes in thoracic surgery with OLV. Yang et al. reported a lower incidence of postoperative lung dysfunction and satisfactory gas exchange was provided by the lung protective strategy using F~I~O~2~ of 0.5 compared to the conventional strategy using F~I~O~2~ of 1.0 during OLV \[[@CR40]\]. However, F~I~O~2~ was one of components in this lung protective strategy, because V~T~, PEEP, and mode of mechanical ventilation were also different between the groups. Thus, it remains uncertain whether a conservative approach to oxygen therapy during OLV is beneficial or not. To our knowledge, this is the first study to demonstrate an association between high F~I~O~2~ during OLV and the occurrence of PPCs. To confirm and dissect these findings, additional studies should be performed in different settings. Moreover, our findings support the need for randomized control trials to evaluate the safety and feasibility of conservative oxygen therapy during OLV. Limitations {#Sec15} ----------- There were several limitations in this study. First, because this was an observational study, causality was not determined. It should be noted that higher F~I~O~2~ might be confounded by the incidence of hypoxemia, which could cause PPCs. Thus, the role of F~I~O~2~ is difficult to differentiate between "unnecessary use" and "need for higher support." However, after adjusting by ARISCAT score, minimum SpO~2~, ΔP, and PEEP to reduce potential confounding, only higher F~I~O~2~ remained statistically significant as an independent risk factor for PPCs. In subgroup analyses, F~I~O~2~ has been associated with the incidence of PPCs even in patients with comparatively lower risk for PPCs. Additionally, the present study indicated that patients might receive excessive oxygen during OLV. Therefore, we believe that intraoperative F~I~O~2~ could be titrated safely even during OLV. Second, the incidence of PPCs could have heavily depended on our definition. There are various definitions of PPCs. For instance, pneumonia was diagnosed based on radiologic images, symptoms, laboratory findings, or antimicrobial treatment used. The diagnosis of atelectasis was based on images or bronchoscopy. In our study, we used definitions of PPCs from previous studies \[[@CR17], [@CR20]\] and CDC guidelines \[[@CR19]\] as shown in Fig. [1](#Fig1){ref-type="fig"}. As a result, the incidence of PPCs in our study (25.9%) was similar to that of previous works \[[@CR17], [@CR20]\]. Conclusions {#Sec16} =========== In conclusion, liberal oxygen therapy as well as lung protective ventilation comprising low V~T~ and slight PEEP were common for patients undergoing thoracic surgery with OLV in our cohort. Our findings indicated that high F~I~O~2~ during OLV was associated with an increased incidence of PPCs, which is related to prolonged LOS in the hospital. These results suggested that current practices of oxygen therapy during OLV may be suboptimal and warrant further investigation. Additional files ================ {#Sec17} Additional file 1:Baseline characteristics and intraoperative procedures of all patients. (DOCX 21 kb) Additional file 2:Adjusted odds ratio of TWA F~I~O~2~ during OLV for the incidence of PPCs in subgroup analyses. (PPTX 79 kb) Additional file 3:The correlation between TWA F~I~O~2~ and minimum SpO~2~. (PPTX 84 kb) %VC : \% vital capacity ARISCAT : Assess Respiratory Risk in Surgical Patients in Catalonia CI : Confidence interval FEV1.0% : Forced expiratory volume in one second % F~I~O~2~ : Inspired oxygen fraction IRB : Institutional ethics review board LOS : Length of hospital stay OLV : One-lung ventilation OR : Odds ratio PBW : Predicted body weight PCV : Pressure control ventilation PEEP : Positive end-expiratory pressure PPCs : Postoperative pulmonary complications SpO~2~ : Percutaneous oxygen saturation TIVA : Total intravenous anesthesia TWA : Time weighted average V~T~ : Tidal volume ΔP : Driving pressure **Electronic supplementary material** The online version of this article (10.1186/s12871-018-0476-x) contains supplementary material, which is available to authorized users. Not applicable. Funding {#FPar1} ======= None Availability of data and materials {#FPar2} ================================== The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. SO contributed to the study conception and design, data acquisition, statistical analysis and interpretation, and drafting of the manuscript. KS and SS contributed to statistical analysis, interpretation and revised the manuscript. KI contributed to the study design, data acquisition and revised the manuscript. HM contributed to the study conception and design, interpretation and revised the manuscript. All authors read and approved the final version of this manuscript. Authors' information {#FPar3} ==================== All the co-authors approve the publication of this manuscript. This work is to be attributed to the Department of Anesthesiology, Okayama University. Ethics approval and consent to participate {#FPar4} ========================================== This study was approved by the institutional ethics review board of Okayama University Hospital (No. 1922) and Fukuyama City Hospital (No. 182). Due to observational study, the requirement for written informed consent was waived by each IRB. Consent for publication {#FPar5} ======================= Not applicable Competing interests {#FPar6} =================== The authors 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.

Cervical cancer, a common malignant tumor, is an vital cause of morbidity and mortality among women worldwide.^[@bib1]^ Paclitaxel, the targets of which are the microtubules of cancer cells, is one of the most useful anticancer drugs against cervical cancer.^[@bib2]^ Paclitaxel can destroy the dynamic equilibrium of tubulin between soluble dimers and polymerized form to make the microtubule structure stable. In addition, it is a strong inhibitor of chromosomal replication by obstructing tumor cells in the mitotic phases or late G2.^[@bib3]^ However, acquired chemoresistance to paclitaxel obviously limits the successful treatment of cervical cancer. One main explanation on tumor cell resistance to paclitaxel is the overexpression of P-glycoprotein (P-gp, MDR-1), which works as a drug efflux pump. However, clinical utility of P-gp inhibitors are often ineffective or toxic at the doses required to attenuate P-gp function.^[@bib4],\ [@bib5],\ [@bib6]^ Other possible mechanisms of action contain alterations in the drug-binding affinity of the microtubules,^[@bib7]^ changes of tubulin structure and cell cycle deregulation.^[@bib8],\ [@bib9],\ [@bib10],\ [@bib11]^ Thus, paclitaxel-resistant mechanisms are complicated and still not entirely clear until now. As a self-proteolysis procedure in almost all eukaryotic cells, autophagy activated by adverse cellular environment contributes to the breakdown of intracellular components within lysosomes to supply an alternative source of energy and thus sustain cell survival.^[@bib12],\ [@bib13]^ However, it has been shown that cell death could be inhibited by suppressing expression of some vital autophagy-associated genes, underscoring the functional role of autophagy in the cell death.^[@bib14],\ [@bib15]^ Several important autophagy-associated proteins, such as Beclin 1 and PtdIns3K class I, have important roles in the control of both autophagy and apoptosis.^[@bib16],\ [@bib17],\ [@bib18]^ Thus, the function of autophagy has been described as a double-edged sword that can work both as a protector and killer of cells, which depends on the developmental stage of the disease or the surrounding microenvironment.^[@bib19]^ It has been reported that anticancer treatment (such as radiotherapy, chemotherapy and molecular targeted therapy) could induce autophagy in cancer cells. In addition, impaired autophagy could make cancer cells sensitize to these therapies, indicating a hopeful strategy to better the efficacy of cancer treatment.^[@bib11],\ [@bib20],\ [@bib21],\ [@bib22]^ However, few studies on the underlying molecular mechanism of chemoresistance-associated autophagy were carried out. In this study, increased levels of autophagy were observed in paclitaxel-resistant HeLa sublines (HeLa-R cell lines). 3-Methyladenine (3-MA) or ATG7 siRNA, autophagy inhibitors, restored sensitivity of HeLa-R cells to paclitaxel. In addition, a group of metabolic proteins with significant alteration were identified by proteomics, which may suggest the switch of cellular metabolism from tricarboxylic acid cycle to glycolysis. Moreover, inhibition of glycolysis by 2-deoxy-[D]{.smallcaps}-glucose (2-DG) or koningic acid (KA) could inhibit autophagy and enhance sensitivity of HeLa-R cells to paclitaxel. In addition, HIF1-*α* could be activated by glycolysis and HIF1-*α* siRNA could decrease autophagy and resensitize HeLa-R cells to paclitaxel. In conclusion, a possible Warburg effect activated HIF1-*α-*mediated signaling-induced autophagic pathway is proposed, which may provide new insight into paclitaxel chemoresistance. Results ======= Characterization of paclitaxel-resistant HeLa-R cells ----------------------------------------------------- MTT and neutral red uptake assay were performed to confirm resistance to paclitaxel. The inhibitory concentration 50 (IC50) value for HeLa parental cells was as low as 1.87 nM paclitaxel. However, the IC50 value (29.06 nM) of HeLa-R cells for paclitaxel was far higher than in HeLa cells, indicating approximately 16-fold greater resistance. Examples of the concentration-response curves are shown in [Figures 1A and B](#fig1){ref-type="fig"}. Clonogenic assay showed 2 nM paclitaxel obviously inhibited cell proliferation in HeLa parental cells ([Figure 1C](#fig1){ref-type="fig"}), while no statistically significant differences were found in the number of HeLa-R cells after treatment with 2 nM paclitaxel ([Figure 1D](#fig1){ref-type="fig"}). The quantitative analysis of sub-G1 cells by flow cytometry was carried out to estimate the number of apoptotic cells. HeLa parental cells treated with 2 nM paclitaxel for 48 h had an obvious sub-G1 peak compared with the untreated group. However, HeLa-R cells treated with 2 nM paclitaxel did not have a conspicuous sub-G1 peak ([Figure 1E](#fig1){ref-type="fig"}). In order to study the role of P-gp in paclitaxel resistance of HeLa-R cells, the expression of P-gp was examined by western blot. No difference in protein expression level was observed between HeLa and HeLa-R cells (data not shown). Moreover, inhibition of P-gp by verapamil (verap) could not restore HeLa-R cells sensitivity to paclitaxel ([Figure 1F](#fig1){ref-type="fig"}). Thus, in this study P-gp did not have an important role in paclitaxel resistance of HeLa-R cells. Autophagy was activated in HeLa-R cells --------------------------------------- Transmission electron microscopy (TEM) was used to analyze ultrastructure of HeLa and HeLa-R cells. Increased autophagosomes were found in the cytoplasm of HeLa-R cells, while autophagosomes could rarely be observed in HeLa parental cells ([Figure 2a](#fig2){ref-type="fig"}). LC3 immunofluorescence was conducted to assess if recruitment of LC3-II to autophagosomes in HeLa-R cells. Punctate patterns of LC3 immunofluorescence were observed in HeLa-R cells by a fluorescence microscope, whereas it could not be seen in HeLa parental cells ([Figure 2b](#fig2){ref-type="fig"}). LC3-I protein (18 kDa) can be cleaved by autophagic stimuli to LC3-II (16 kDa), which is localized in autophagosome. As shown in [Figure 2c](#fig2){ref-type="fig"}, LC3-II protein was detectable in HeLa-R cells, whereas the expression of LC3-II was undetectable in HeLa parental cells. Furthermore, western blot analysis indicated that P62 (also called SQSTM1, it is degraded when autophagy is induced and may be used as a marker to study autophagic flux) was downregulated, while Beclin 1, Atg7 and Atg12-Atg5 conjugate were upregulated in HeLa-R cells compared with HeLa parental cells ([Figure 2c](#fig2){ref-type="fig"}). These data showed that autophagy was activated in HeLa-R cells. Inhibition of autophagy restored HeLa-R cells sensitivity to paclitaxel ----------------------------------------------------------------------- In order to study whether autophagy results in chemoresistance of HeLa-R cells, we treated HeLa-R cells with 3-MA (a widely used autophagy inhibitor, specific inhibitor of PI3K) to inhibit autophagy, then assessed the cells sensitivity to paclitaxel. Both data from electron microscopy and LC3 immunofluorescence showed that 3-MA (5 mM) effectively inhibited autophagy activation of HeLa-R cells ([Figure 3A](#fig3){ref-type="fig"}). Corresponding to decreased autophagosome, apoptosis was markedly enhanced in 3-MA plus paclitaxel-treated group, compared with treatment with paclitaxel alone (2 nM), 3-MA alone (5 mM) and PBS control ([Figure 3B](#fig3){ref-type="fig"}). Cell viability and number of HeLa-R cells were also significantly reduced in 3-MA plus paclitaxel-treated group compared with treatment with paclitaxel alone (2 nM), 3-MA alone (5 mM) and PBS control ([Figures 3C and D](#fig3){ref-type="fig"}). In order to further confirm inhibition of autophagy could restore HeLa-R cells sensitivity to paclitaxel, we used ATG7 siRNA to inhibit autophagy. ATG7 silencing by siRNA can inhibit the generation of autophagosomes, and thus prevent the induction of autophagy.^[@bib23]^ As shown in [Figures 3E and F](#fig3){ref-type="fig"}, cell viability and number of HeLa-R cells were also significantly reduced in ATG7 siRNA plus paclitaxel-treated group compared with the controls. In addition, parental HeLa cells were tested for response to 3-MA and ATG7 siRNA. As shown in [Figures 3G and H](#fig3){ref-type="fig"}, cell viability and number of HeLa cells were not significantly reduced in 3-MA plus paclitaxel-treated group compared with treatment with paclitaxel alone. Furthermore, ATG7 siRNA could not increase paclitaxel sensitivity in parental HeLa cells ([Figures 3I and J](#fig3){ref-type="fig"}). In conclusion, our data suggested autophagy could have an important role in paclitaxel resistance of Hela-R cells. Differentially expressed proteins between HeLa and HeLa-R cells identified by 2-DE and ESI-Q-TOF MS/MS ------------------------------------------------------------------------------------------------------ In order to study the molecular mechanisms underlying chemoresistance-associated autophagy, two-dimensional gel electrophoresis (2-DE) for HeLa and HeLa-R cells was repeated three times, respectively. The image assessment was conducted by using PDQuest 6.1 software and demonstrated that these 2-DE pictures were reproducible ([Figure 4a](#fig4){ref-type="fig"}). Proteins that were differentially expressed were defined as statistically significant based on two principles: (1) recurrence more than two times in the three repeated tests and (2) protein intensity changes \>2.0-fold (Student\'s *t-*test, *P*\<0.05). Based on these principles above, 42 unique proteins were identified by electrospray ionization quadrupole time-of-flight (ESI-Q-TOF)-MS/MS, as shown in [Table 1](#tbl1){ref-type="table"}. According to their biological functions and subcellular localization, the classification of altered proteins was shown in [Figures 4b and c](#fig4){ref-type="fig"}. Significant alterations were found in a group of metabolic proteins (35% of total 42 proteins). These proteins function in diverse metabolic processes, such as glycolysis, tricarboxylic acid cycle, oxidative phosphorylation and so on. To study whether the proteomics identification of these metabolic proteins corresponded to alteration at the translational level, six proteins (PKM2, LDHA, ALDOC, TPI1, MDH1 and UQCRC1) were chosen for validation by western blot. The expression levels of these proteins agreed with the changes in 2-DE maps ([Figure 4d](#fig4){ref-type="fig"}). The data suggested that there may be the switch of cellular metabolism from tricarboxylic acid cycle to glycolysis in HeLa-R cells (Warburg effect). Inhibition of glycolysis decreased autophagy and resensitized HeLa-R cells to paclitaxel ---------------------------------------------------------------------------------------- To investigate whether glycolysis affected autophagy and facilitated paclitaxel resistance, we treated HeLa-R cells with 2-DG (20 mM), an inhibitor of glycolysis, for 48 h in the presence or absence of paclitaxel. We found 2-DG decreased autophagy ([Figure 5a](#fig5){ref-type="fig"}). Corresponding to decreased autophagosomes, cell viability of HeLa-R cells were reduced in 2-DG plus paclitaxel-treated group compared with treatment with paclitaxel alone (2 nM), 2-DG alone (20 mM) and PBS control ([Figure 5b](#fig5){ref-type="fig"}). In addition, apoptosis was enhanced in 2-DG plus paclitaxel-treated group, when compared with the controls ([Figure 5c](#fig5){ref-type="fig"}). In order to further confirm inhibition of glycolysis could decrease autophagy and restore HeLa-R cells sensitivity to paclitaxel, we used KA (a GAPDH inhibitor) to inhibit glycolysis. As shown in [Figure 5d](#fig5){ref-type="fig"}, KA could decrease autophagy of HeLa-R cells. Furthermore, cell viability of HeLa-R cells was significantly reduced and apoptosis was increased in KA plus paclitaxel-treated group compared with the controls ([Figures 5e and f](#fig5){ref-type="fig"}). In addition, parental HeLa cells were tested for response to inhibitors of glycolysis. As shown in [Figures 5g--j](#fig5){ref-type="fig"}, glycolysis inhibitors (2-DG or KA) could not increase paclitaxel sensitivity in parental HeLa cells. Thus, our data showed inhibition of glycolysis decreased autophagy and resensitized HeLa-R cells to paclitaxel. Glycolysis activated HIF1-*α* and inhibition of HIF1-*α* restored HeLa-R cells sensitivity to paclitaxel -------------------------------------------------------------------------------------------------------- It has been reported that end products of glycolysis could activate HIF1-*α*-inducible gene expression. The 2-DE data above showed that HIF1-*α* ([Figure 4a](#fig4){ref-type="fig"}, [Table 1](#tbl1){ref-type="table"} Spot NO. 4) was upregulated. In order to validate the expression of HIF1-*α* in HeLa-R cells, western blot was conducted. As shown in [Figure 6a](#fig6){ref-type="fig"}, HIF1-*α* was obviously upregulated in HeLa-R cells. Moreover, inhibition of glycolysis by 2-DG could decrease the expression of HIF1-*α* in HeLa-R cells ([Figure 6b](#fig6){ref-type="fig"}). In addition, we used HIF1-*α-*specific siRNA to transfect HeLa-R cells to inhibit the expression of HIF1-*α*, then examined the expression of a critical molecule in autophagy pathway-Beclin 1 by western blot. As a result, Beclin 1 was downregulated significantly after inhibition of HIF1-*α* ([Figure 6c](#fig6){ref-type="fig"}). Moreover, data from electron microscopy and LC3 immunofluorescence also showed similar results ([Figures 6d and e](#fig6){ref-type="fig"}). So HIF1-*α* is involved in the regulation of chemoresistance-associated autophagy in HeLa-R cells. MTT assay revealed that HIF1-*α* siRNA plus paclitaxel-treated group could increase paclitaxel sensitivity compared with the controls ([Figure 6f](#fig6){ref-type="fig"}). Flow cytometry showed the significant increase of apoptotic cells in the HIF1-*α* siRNA plus paclitaxel group compared with the controls ([Figure 6g](#fig6){ref-type="fig"}). These findings showed that glycolysis activated HIF1-*α* and downregulation of HIF1-*α* could resensitize HeLa-R cells to paclitaxel. Increased autophagy and upregulation of glycolytic molecules in clinical tissues from patients who were resistant to paclitaxel chemotherapy -------------------------------------------------------------------------------------------------------------------------------------------- In order to study whether these results above were consistent with clinical samples, fresh tumor tissues were obtain from 6 cervical cancer patients who were resistant to paclitaxel-based chemotherapy and 15 cervical cancer patients who were clinically responsive to paclitaxel-based chemotherapy. This study was approved by the Institutional Ethics Committee of Sichuan University and informed consents were obtained from all patients before analysis. TEM showed autophagy was activated in paclitaxel-resistant tumor tissues when compared with paclitaxel-sensitive tumor tissues (*P*\<0.05) ([Supplementary Figure 1sA](#sup1){ref-type="supplementary-material"}). Real-time quantitative RT-PCR was conducted to examine the expression level of PKM2, LDHA, ALDOC and TPI1. As shown in [Supplementary Figure 1sB](#sup1){ref-type="supplementary-material"}, the mRNA expression level of PKM2, LDHA, ALDOC and TPI1 in paclitaxel-resistant cervical cancer was significantly higher compared with paclitaxel-sensitive tumor tissues (*P*\<0.05). The data were consistent with our results from HeLa-R cell lines. Thus, Warburg effect activated HIF1-*α*-mediated signaling-induced autophagic pathway may have an important role in paclitaxel chemoresistance. Discussion ========== Paclitaxel is an extensively used anticancer drug for the treatment of many types of cancers, including lung cancer, breast cancer, cervical cancer and so on. Paclitaxel resistance may contribute to the consecutive recurrence and metastasis of cancer, finally causing death.^[@bib24],\ [@bib25]^ Although many studies have been conducted about the paclitaxel resistance, the specific mechanisms of action involved are still poorly understood. In this report, paclitaxel-resistant cervical cancer cells (HeLa-R cells) were induced by a stepwise selection of various doses of paclitaxel. Our data showed that autophagy was activated in HeLa-R cells, which was confirmed by formation of autophagosomes, appearance of punctate patterns of LC3 and activation of autophagy genes, including LC3-II, Beclin 1, the Atg12-Atg5 conjugate and Atg7, which are necessary for autophagosome formation. Furthermore, inhibition of autophagy by 3-MA or ATG7 siRNA restored HeLa-R cells sensitivity to paclitaxel. However, 3-MA or ATG7 siRNA could not increase paclitaxel sensitivity in parental HeLa cells. Thus, autophagy, a prosurvival way, could facilitate the development of acquired resistance of paclitaxel. In order to study the underlying mechanism of chemoresistance-associated autophagy, 2-DE combined with ESI-Q-TOF MS/MS identification was utilized to profile differentially expressed proteins between HeLa and HeLa-R cells. In our proteomic study, a series of enzymes involved in energy metabolism were identified with obviously changed expression levels, which may display the switch of cellular metabolism from tricarboxylic acid cycle to glycolysis (known as Warburg effect). Furthermore, our findings showed that inhibition of glycolysis decreased autophagy and resensitized HeLa-R cells to paclitaxel. As it was reported that Warburg effect could promote HIF1-*α* protein stability and activate HIF1-*α*-inducible gene expression,^[@bib26],\ [@bib27]^ it was of particular interest to examine whether expression of HIF1-*α* was altered in HeLa-R cells. We found HIF1-*α* was obviously upregulated in HeLa-R cells. Moreover, inhibition of glycolysis by 2-DG could decrease the expression of HIF1-*α* in HeLa-R cells. HIF1-*α-*specific siRNA could decrease autophagy and resensitize HeLa-R cells to paclitaxel. Finally, increased autophagy and upregulation of glycolytic molecules were found in clinical tissues from patients who were resistant to paclitaxel chemotherapy when compared with paclitaxel-sensitive tumor tissues. The findings need to be further confirmed by more clinical specimens in our next experiments. Taken together, Warburg effect activated HIF1-*α-*mediated signaling-induced autophagic pathway may have an important role in paclitaxel chemoresistance. Tumor cells are different from normal somatic cells in their metabolic mode, with normal somatic cells depending mainly on mitochondrial oxidative phosphorylation, making use of glucose and oxygen to generate energy. In tumor cells, energy production mainly depends on glycolysis even though oxygen is rich.^[@bib28],\ [@bib29],\ [@bib30]^ Although the molecular mechanisms of action underlying the Warburg effect remain obscure, it has been well accepted that increased glycolysis in tumor cells is a very key process to sustain malignant phenotypes.^[@bib31]^ It has been shown that targeting the key enzymes of glycolysis can sensitize cancer cells to chemotherapy.^[@bib24],\ [@bib32],\ [@bib33]^ For example, LDH-A is one of the primary isoforms of LDH, which controls the transition of pyruvate to lactate of glycolysis and has a vital role in sustaining energy metabolism of tumor cell. If its action is repressed, the energy-producing task will be transferred to cellular mitochondria that can contribute to increased oxidative stress and promote apoptosis of mitochondrial pathway. Targeting LDH-A by siRNA or oxamate (a competitive inhibitor of LDH-A) could resensitize chemoresistant cells to paclitaxel.^[@bib24]^ PKM2, a very vital enzyme that can regulate aerobic glycolysis, has an important part in cancer metabolism and proliferation. The combination of shRNA targeting PKM2 and cisplatin or docetaxel suppressed lung carcinoma growth and induced more apoptosis of tumor cells both *in vitro and in vivo*.^[@bib32],\ [@bib33]^ A finding by proteomic analysis in our study is that the key proteins of tricarboxylic acid cycle (such as MDH1 and UQCRC1) were downregulated, while the key proteins of glycolysis (such as PKM2, LDHA, ALDOC and TPI1) were upregulated, which may display the switch of cellular metabolism from tricarboxylic acid cycle to glycolysis. HIF1 is a heterodimer composed of two subunits, HIF1-*α* and HIF1-*β*, both of which are constitutively expressed in mammalian cells.^[@bib34]^ The regulation of the HIF1 complex is mainly dependent on the degradation of the HIF1-*α* subunit. HIF1-*α* activation is highly associated with tumor cell growth, metastasis and poor clinical prognosis.^[@bib35],\ [@bib36]^ It has been reported that end products of glycolytic metabolism can promote HIF1-*α* protein stability and activate HIF1-*α*-inducible gene expression.^[@bib27]^ Moreover, glycolysis can contribute to induction of HIF1-*α* in a hypoxia-independent way by several endocrine agents and environmental toxins.^[@bib37],\ [@bib38]^ Our findings agreed with what was reported before. HIF1-*α* was obviously upregulated in HeLa-R cells and HIF1-*α*-specific siRNA could resensitize HeLa-R cells to paclitaxel. In conclusion, a possible novel Warburg effect activated HIF1-*α*-mediated signaling-induced autophagic pathway is proposed, which may provide new insight into paclitaxel chemoresistance. Materials and Methods ===================== Cell culture and reagents ------------------------- HeLa (from American Type Culture Collection, Manassas, VA, USA) and HeLa-R cells were cultured at 37 °C in DMEM in a humid incubator with 5% CO~2~. Reagents used in this study were: 3-MA (M9281) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine 2000 reagent was purchased from Invitrogen, Karlsruhe, Germany. 2-DG (D8375) was obtained from Sigma, St. Louis, MO, USA. KA (57710-57-3) was obtained from TMS Co. Ltd (Suwon, South Korea). Antibodies used in this study were: P62 antibody (Abcam, Cambridge, UK; ab56416), Beclin 1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-11427), LC3 (Abcam, ab58610), Atg12-Atg5 (Abcam, ab78073), Atg7 (Abcam, ab53255), PKM2 (Abcam, ab58610), LDHA (Abcam, ab47010), ALDOC (Abcam, ab71294), TPI1 (Abcam, ab96696), MDH1 (Abcam, ab76616), UQCRC1 (Abcam, ab96333), HIF-1*α* (Abcam, ab16066), horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Santa Cruz Biotechnology, sc-2004), HRP-conjugated anti-mouse secondary antibody (Santa Cruz Biotechnology, sc-2005). Establishment of HeLa-R ----------------------- HeLa cells were seeded in six-well plates and were allowed to reach about 80% confluency in fresh medium before treating with paclitaxel. The dose of paclitaxel begun with 0.04 nM (about 1/50 IC50, IC50: 1.87 nM in HeLa cells), and it was increased by a dose gradient that was 25--50% of the previous dose. The next dose was given until the cells were stable in proliferation without significant death. Cell viability and proliferation assays --------------------------------------- For the proliferation assay, cells were seeded at 5 × 10^3^ cells per well in 96-well plates. Afterward, medium containing agents were added to each well and cells were further cultured at 37 °C for the indicated times. Cell viability was examined by MTT test and neutral red uptake cytotoxicity assay. The results were from three independent experiments of each group. Cells were trypsinized and replated in 24-well plates at a density of 10 000 cells per well. Cells were incubated for 24 h to allow for attachment, after which a zero time point was determined. Afterward, medium containing agents were added to each well and cells were counted at days 0, 3, 6 and 9 with a Coulter Counter (Coulter Electronics, Inc., Hialeah, FL, USA). All assays were performed at least three times in duplicate. Flow cytometry -------------- Flow cytometric analysis was performed to identify sub-G1 cells/apoptotic cells and to measure the percentage of sub-G1 cells after propidium iodide (PI) staining in hypotonic buffer as described.^[@bib39],\ [@bib40]^ Briefly, cells were suspended in 1 ml hypotonic fluorochrome PI solution (50 *μ*g/ml PI in 0.1% sodium citrate plus 0.1% Triton X-100) containing 0.5 mg/ml RNase A, and the cells were analyzed by the use of a flow cytometer (ESP Elite, Beckman Coulter, Fullerton, CA, USA). Apoptotic cells appeared in the cell cycle distribution as cells with DNA content of less than that of G1 cells. Electron microscopy ------------------- Cells were harvested, pelleted and fixed in paraformaldehyde, 0.1% glutaraldehyde in 0.1 M sodium cacodylate for 2 h, postfixed with 1% OsO~4~ for 1.5 h, washed and finally stained for 1 h in 3% aqueous uranyl acetate. The samples were then rinsed with water again, dehydrated with graded alcohol (50%, 75% and 95--100% alcohol) and embedded in Epon-Araldite resin (Canemco, St. Laurent, QC, Canada; 034). Ultrathin sections were cut on a Reichert Ultramicrotome, counterstained with 0.3% lead citrate and examined on a Philips EM420 transmission electron microscope (Philips, Eindhoven, The Netherlands). Values for the area occupied by autophagic vacuoles and the cytoplasm were obtained with Image Pro Plus version 3 (Media Cybernetics, Silver Spring, MA, USA) and used for the calculation of the cytoplasmic area occupied by the autophagic vacuoles. Immunofluorescence of LC3 ------------------------- Treated cells were washed with PBS and then fixed with paraformaldehyde (4% w/v). After rinsing in PBS, the cells were blocked with 0.1% Triton X-100 containing 1% bovine serum albumin in PBS for 1 h. This was followed by incubation in LC3 antibody (Abcam, ab58610) for 24 h at 4 °C in a humidified chamber. After three washes in PBS, the cells were incubated in secondary antibody conjugated to HRP for 2 h at room temperature. Finally, cells were rinsed in PBS, coverslipped and examined with fluorescence microscopy. siRNA preparation and transfection ---------------------------------- Atg7 and HIF1-*α* knockdowns were accomplished by transfecting cells with siRNA. Atg7 and control siRNA were synthesized by RiboBio (Guangzhou, China). HIF1-*α* and control siRNA were synthesized by Santa Cruz Biotechnology. The siRNA sequences against Atg7 or HIF1-*α* were previously described.^[@bib41],\ [@bib42]^ Cells were transfected with 1 *μ*g siRNA using 2.5 *μ*g Lipofectamine 2000 reagent (Invitrogen). Scrambled siRNA, which consists of a scrambled sequence that will not lead to specific degradation of any known cellular mRNA, was used as negative control. Western blot ------------ Proteins were extracted in RIPA buffer (50 mM Tris-base, 1.0 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF) and quantified by the DC protein assay kit (Bio-Rad, Berkeley, CA, USA). Samples were separated by 12% SDS-PAGE and transferred to PVDF membranes (Amersham Biosciences, Piscataway, NJ, USA). After blocking with 5% non-fat milk in Tris-buffered saline, 0.1% Tween 20 for 1 h, the membranes were incubated overnight at 4 °C with respective primary antibodies. After that, the blots were incubated with secondary antibody conjugated to HRP for 2 h at room temperature. Target proteins were detected by enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Piscataway, NJ, USA). 2-DE and image analysis ----------------------- Cells were lysed in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 100 mM DTT, 0.2% pH 3--10 ampholyte, Bio-Rad) containing protease inhibitor cocktail 8340 (Sigma). Protein samples (2 mg) were applied to IPG strip (17 cm, pH3--10 NL, Bio-Rad) using a passive rehydration method. After 12--16 h of rehydration, the strips were transferred to an IEF Cell (Bio-Rad). IEF was performed as follows: 250 V for 30 min, linear; 1000 V for 1 h, rapid; linear ramping to 10 000 V for 5 h and finally 10 000 V for 4 h. The second dimension was performed using 12% SDS-PAGE at 30 mA constant current per gel after equilibration. The gels were stained using CBB R-250 (Merck, Darmstadt, Germany) and scanned with a Bio-Rad GS-800 scanner. Three independent runs were made to ensure the accuracy of analyses. The maps were analyzed by PDQuest software Version 6.1 (Bio-Rad). The quantity of each spot in a gel was normalized as a percentage of the total quantity of all spots in that gel and evaluated in terms of OD. For statistical analysis, paired *t*-test was performed to compare data from the three repeated experiments. Only spots that showed consistent and significant differences (±over twofold, *P*\<0.05) were selected for analysis with MS. Mass spectrometry analysis -------------------------- Gel bands were cut out and prepared for mass spectrometry analysis with trypsin digestion. Peptides were analyzed using a Q-TOF mass spectrometer (Micromass, Manchester, UK) fitted with an ESI source (Waters, Manchester, UK). Real-time RT-PCR ---------------- mRNA was isolated from frozen tumor tissue using RNeasy total RNA kits (Qiagen, Hilden, Germany). RNA concentration was evaluated by photometric measurement at 260/280 nm. One microgram RNA was used for cDNA synthesis using the QuantiTect Reverse Transcription Kit (Qiagen). PCR reactions were performed by using the Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen) in the 7300 real-time PCR system (Applied Biosystems, Darmstadt, Germany). The paired forward and reverse primers are as follows: PKM2 (forward): 5′-ATTATTTGAGGAACTCCGCCGCCT-3′, and (reverse): 5′-ATTCCGGGTCACAGCAATGATGG-3′ LDHA (forward): 5′-CTCCTGTGCAAAATGGCAAC- 3′, and (reverse): 5′-CCTAGAGCTCACTAGTCACAG-3′ ALDOC (forward) 5′-GCCAAATTGGGGTGGAAAACA-3′, and (reverse) 5′-TTCACACGGTCATCAGCACTG-3′ TPI1 (forward): 5′- CGGGATCCAGCGCCTCGGCTCCAGCG-3′, and (reverse): 5′-GGGGTACCAGTTGTTTGGCATTGATGATGTCCACG-3′. The experiment was repeated three times. Statistical analyses -------------------- Statistical comparisons of mean values were performed using an analysis of variance. Statistical significance was defined as \**P*\<0.05; \*\**P*\<0.01. This work was supported by the grants from National Natural Sciences Foundation of China (81301794), Postdoctoral 52nd Foundation of China (2012M521701), Technology innovation seedling cultivation project of Sichuan province (0030405301429) and National science and technology major projects (2009ZX09503-020). HeLa-R : paclitaxel-resistant HeLa sublines IC50 : inhibitory concentration 50 TEM : transmission electron microscopy 2-DE : two-dimensional gel electrophoresis ESI-Q-TOF : electrospray ionization quadrupole time-of-flight 2-DG : 2-deoxy-[D]{.smallcaps}-glucose KA : koningic acid [Supplementary Information](#sup1){ref-type="supplementary-material"} accompanies this paper on Cell Death and Disease website (http://www.nature.com/cddis) Edited by GM Fimia The authors declare no conflict of interest. Supplementary Material {#sup1} ====================== ###### Click here for additional data file. {#fig1} {#fig2} {#fig3} {#fig4} {#fig5} {#fig6} ###### Identified proteins by MS/MS analysis **Spot no.** **Protein description** **Gene name** **Function** **Accession no.** **Theoretical Mr/pI**[a](#t1-fn1){ref-type="fn"} **Score** **No. of pep**[b](#t1-fn2){ref-type="fn"} **Fold change**[c,](#t1-fn3){ref-type="fn"}[d,](#t1-fn4){ref-type="fn"}[e](#t1-fn5){ref-type="fn"} -------------- ---------------------------------------------------------- --------------- ------------------------------------- ------------------- -------------------------------------------------- ----------- ------------------------------------------- ---------------------------------------------------------------------------------------------------- 1 Peptidyl-prolyl cis-trans isomerase A PPIA Metabolism P62937 17 881/7.82 198 22 ↑2.3±0.5 2 Ubiquitin-conjugating enzyme E2 N UBE2N Proteolysis P61088 17 184/6.13 91 2 ↓3.2±0.7 3 Triosephosphate isomerase TPI1 Metabolism P60174 26 538/6.51 634 17 ↑2.9±0.4 4 Hypoxia-inducible factor 1-alpha HIF1A Metabolism Q16665 92 670/5.17 145 12 ↑NA 5 Nucleoside diphosphate kinase B NME2 Apoptosis regulation P22392 17 298/8.55 45 1 ↓2.0±0.2 6 Peroxiredoxin-5 PRDX5 Apoptosis regulation P30044 17 030/6.73 320 17 ↑NA 7 Low-molecular-weight phosphotyrosine protein phosphatase ACP1 Metabolism P24666 17 911/6.35 117 8 ↑NA 8 Peroxiredoxin-2 PRDX2 Apoptosis regulation P32119 17 660/6.84 84 4 ↑NA 9 Prefoldin subunit 2 PFDN2 Molecular chaperone Q9UHV9 16 695/6.20 161 6 ↓3.7±0.9 10 Programmed cell death protein 5 PDCD5 Apoptosis regulation Q2HJH9 14 094/6.21 123 3 ↓2.9±0.6 11 Cytochrome c oxidase subunit 5A, mitochondrial COX5A Metabolism P20674 16 935/6.30 69 1 ↑2.1±0.4 12 Galectin-7 LGALS7 Cell proliferation/differentiation P47929 15 123/7.03 437 13 ↓2.3±0.5 13 Pyruvate kinase isozymes M1/M2 PKM2 Metabolism P14618 57 805/7.95 213 10 ↑3.3±0.7 14 Proteasome subunit beta type-2 PSMB2 Proteolysis P49721 22 836/6.52 436 36 ↑2.5±0.3 15 Proteasome subunit beta type-4 PSMB4 Proteolysis P28070 25 204/5.47 428 15 ↑4.3±1.4 16 Superoxide dismutase (Cu-Zn) SOD1 Electron transport/redox regulation P00441 15 804/5.70 64 6 ↑2.2±0.8 17 Splicing factor, arginine/serine-rich 7 SFRS7 mRNA splicing Q16629 27 578/11.83 134 5 ↓3.9±1.1 18 3-Hydroxyacyl-CoA dehydrogenase type-2 HSD17B10 Metabolism Q99714 27 134/7.66 298 6 ↓2.0±0.5 19 Superoxide dismutase (Mn) SOD2 Electron transport/redox regulation P04179 24 722/8.35 52 3 ↓2.1±0.3 20 Malate dehydrogenase, cytoplasmic MDH1 Metabolism P40925 36 426/6.89 391 9 ↓2.3±0.6 21 Eukaryotic translation initiation factor 4H EIF4H Transcription regulation Q15056 27 425/6.67 276 8 ↓5.3±1.8 22 28-kDa Heat- and acid-stable phosphoprotein PDAP1 Cell proliferation/differentiation Q13442 20 630/8.84 153 7 ↑2.3±1.1 23 Phosphatidylethanolamine-binding protein PEBP1 Signal transduction P30086 21 057/7.01 106 3 ↓2.0±0.4 24 26S proteasome non-ATPase regulatory subunit 8 PSMD8 Proteolysis P48556 30 005/6.85 121 12 ↑2.4±0.9 25 Myosin regulatory light polypeptide 9 MYL9 Calcium ion binding P24844 19 695/4.80 68 3 ↑2.0±0.2 26 Ig kappa chain C region IGKC Immune response P01834 11 773/5.58 378 11 ↑3.4±0.7 27 Chromobox protein homolog 3 CBX3 Transcription regulation Q13185 20 811/5.23 256 8 ↑NA 28 Heat shock protein beta-1 HSPB1 Molecular chaperone P04792 22 783/5.98 188 15 ↑2.0±0.6 29 Rho GDP-dissociation inhibitor 2 ARHGDIB Signal transduction P52566 22 988/5.1 68 7 ↑2.1±1.3 30 Charged multivesicular body protein 4b CHMP4B Protein binding Q9H444 24 950/4.76 268 9 ↑2.4±0.8 31 Inorganic pyrophosphatase PPA1 Metabolism Q15181 32 660/5.54 106 6 ↑2.3±0.5 32 Heterogeneousnuclear ribonucleoprotein H3 HNRPH3 mRNA splicing P31942 36 960/6.37 134 4 ↓2.7±0.6 33 NADH dehydrogenase flavoprotein 2 NDUFV2 Electron transport/redox regulation P19404 27 392/5.22 241 7 ↓NA 34 LIM and SH3 domain protein 1 LASP1 Zinc ion binding Q14847 30 097/6.61 100 4 ↑2.3±0.5 35 Tumor-suppressor candidate 2 TUSC2 Protein binding O75896 11 942/9.69 36 2 ↓2.0±0.7 36 Alcohol dehydrogenase (NADP) AKR1A1 Metabolism P14550 36 892/6.32 421 29 ↑2.0±0.9 37 Annexin A5 ANXA5 Calcium ion binding P08758 35 937/4.94 217 6 ↑3.6±1.5 38 Cytochrome b-c1 complex subunit 1, mitochondrial UQCRC1 Metabolism P31930 52 646/5.43 423 24 ↓2.9±1.2 39 Creatine kinase M-type CKM Metabolism P06732 43 302/6.77 708 21 ↑5.2±1.4 40 UDP-glucose 4-epimerase GALE Metabolism Q14376 38 656/6.26 152 7 ↓2.1±0.4 41 [L]{.smallcaps}-lactate dehydrogenase A chain LDHA Metabolism P00338 36 689/8.46 467 14 ↑2.9±0.8 42 Fructose-bisphosphate aldolase C ALDOC Metabolism P09972 39 324/6.46 413 5 ↑2.0±0.4 All protein spots identified by ESI-Q-TOF MS/MS as significantly changed in average expression level in Hela-R cells compared with parental Hela cells Theoretical Mr/pI: theoretical molecular weight (kDa) and pI from the ExPASy database No. of pep: number of unique peptides identified by MS/MS sequencing Upward arrows: upregulated Downward arrows: downregulated NA: the spots on one of the paired gels were too weak or nondetectable

1. Introduction {#s0005} =============== A network represents a formal mathematical model in which a complex system can be decomposed into elements (i.e., nodes or vertices) and their interactions (i.e., edges, links, or connections). The comprehensive structural description of the brain as a network of neural elements and their interconnections is known as the connectome [@b0005]. Structural scales in the nervous system range from molecules to the whole brain [@b0010], and the association between these elements in the brain network is generally described by its structural (or functional) connections at four different scales: *macroscale* at the level of gray matter, *mesoscale* at the level of neuronal subgroups, *microscale* at the level of individual neurons, and *nanoscale* at the level of synapses [@b0015]. From a network perspective, brain functions are considered to be highly dependent on the brain's structural network architecture at each scale [@b0020]. Accordingly, several fields in modern network neuroscience utilize their own approach to studying brain connections depending on the acquired level of the dataset (i.e., elements). Recent developments in noninvasive techniques for mapping brain connectivity enable better characterization of the structural and functional properties of a specific neuronal system [@bib691]. The structure of networks has been analyzed within a mathematical framework known as *graph theory* ([Fig. 1](#f0005){ref-type="fig"}). Using graph theory, networks including neural systems such as the brain can be described as a quantitative and comparative model of real-world systems at all scales (i.e., macro, meso, micro, and nano) and modalities (e.g., single-cell recording, neuronal tracing, and neuroimaging) [@b0025]. An early finding from the macroscopic brain network perspective was that the human brain is organized in a highly efficient manner for integrated information transfer [@b0030], known as *small-world topolog*y, as anticipated in several biological, technical, and social networks [@b0035]. Two assumptions are postulated to form a small-world network. First, a subgroup of network elements should form dense, interconnected clusters to confirm local network segregation, as defined by a *clustering coefficient* [@b0035]. A higher clustering coefficient of each network element often leads to network communities or modules ([Fig. 2](#f0010){ref-type="fig"} and [Table 1](#t0005){ref-type="table"}). Second, lengths or distances between any pairs of network elements, often defined by the reciprocal of the connectivity strength, should be shorter for a greater degree of global integration, resulting in a lower *shortest path length* [@b0035]. The small-world topology designates networks in which the clustering coefficient is significantly larger than (and the shortest path length is similar to) those of randomly connected networks, defined as *small-worldness* [@b0040]. However, the existence of small-world topology provides limited information on network architecture and has several pitfalls in terms of its evaluation, utility, and interpretation [@b0025], [@b0045]. As such, more appropriate network measures such as *modularity* have been proposed to characterize local and global network architecture [@b0050]. Network modules (i.e., communities or clusters) are defined by a set of network elements with a number of interconnections within each module and fewer connections among modules [@b0055], [@b0060].Fig. 1The construction of brain networks using neuroimaging and neurophysiological data. For a structural network, the brain is parcellated into a number of segregated regions, often using high-resolution anatomical magnetic resonance imaging (MRI). Subsequently, the fiber tracts are generated using diffusion tensor imaging. Structural connectivity may indicate the existence or weight of a connection between two parcellated regions, resulting in a connectivity matrix. For a functional network, the time-varying brain activity measured with electrophysiological signal measurements or functional MRI can be used to compute functional interdependence leading to functional connectivity. Graph theoretical measures ([Table 1](#t0005){ref-type="table"}) can be computed using the structural and functional connectivity matrix.Fig. 2Basic graph theoretical measures. (A) Networks are described as a set of nodes and edges, in which nodes can be a set of neurons, brain regions, or specific recording sites, and edges can be either structural connections or functional relationships between two nodes. (B) Degree is defined by the number of connections of each node. (C) In a weighted network, the sum of the weights connected to the node represents the strength. (D) The highly clustered node (red) has three neighboring nodes (blue), and they are also connected (orange) to form a cluster. (E) Path length represents the sum of steps (or connection distances) required to travel from one node to another. For example, four steps are required from node *a* (red) to *b* (blue) for the left network, while two steps are required for the right network. (F) Highly clustered nodes are likely to have more mutual connections to form a module. (G) Some nodes in a network may play central roles as hubs for network information transfer. (H) Rich-club nodes represent a set of hub nodes that are also highly connected with each other. (I) Rich-club, feeder, and local connections represent the connections only among rich-club nodes, between rich-club and non-rich-club nodes, and only among non-rich-club nodes, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)Table 1Description of graph theoretical measures.MeasureDescriptionGraphA set of nodes and edges (often called a network).NodeIn a brain network, a node can be a single neuron, set of neurons, brain region, or specific recording site (e.g., electroencephalography electrode position). Each node has a unique topological location within a network.EdgeAn edge, also called a connection or link, is defined by an interrelationship between two nodes. It may be structural or functional. Simply, it could indicate the existence of a connection (i.e., binary, 0 or 1) or strength (i.e., weight; e.g., neural density or functional correspondence). It may have a direction (e.g., positive to the efferent connection and negative to the afferent connection).NeighborhoodConnected nodes to a node by an edge forming a subnetwork.DegreeThe number of edges attached to a node.StrengthSum of the edge weights attached to a node, when all network edges have their own weights (i.e., weighted network).Clustering coefficientThe probability that the neighborhood (i.e., connected) nodes for a node are also connected to each other. The *global* clustering coefficient for a network is computed by averaging clustering coefficients across all nodes in the network.DistanceTopological length, often defined by an inverse of the edge weight (i.e., if the weight = 0, then the distance → ∞).Shortest path lengthDistance between two nodes. It may be the number of steps for a binary network or the sum of connection distances for a weighted network along the shortest path from one node to another.Characteristic path lengthAverage shortest path length across all nodes in the network.Small-worldnessRatio of clustering coefficient and characteristic path length, which are normalized relative to those of the random networks. A small-world network is more clustered with a similar characteristic path length than degree-preserved random networks.EfficiencyAverage of the "inverse" of connection distances from a node to all other nodes refers to *nodal* efficiency. *Global* efficiency represents average of the nodal efficiencies of all nodes. *Local* efficiency is computed at a node and its neighborhood subnetworks. Contrary to the path length, efficiency is less influenced by isolated nodes (i.e., if the path length → ∞, then the efficiency = 0).ModuleA set of highly connected nodes. In general, the number (or strength) of connections within modules is more than that between modules.HubNodes with topologically important roles in a network. A hub can be a node with high degree or strength (often called a core).Rich-clubA set of hub nodes with more connections to each other. A subnetwork with only rich-club nodes should have more connections than a random network with the same degree and edge distributions. A macroscopic structural brain network derived from anatomical magnetic resonance imaging (MRI) has revealed groups of cortical regions that are morphologically connected, subserving distinct brain functions such as language, motor, and visual functions [@b0065]. More detailed analyses using diffusion spectrum imaging and higher resolution of cortical parcellation have identified six structurally distinct modules comprising posterior medial and parietal cerebral cortices, and several distinct temporal and frontal modules [@b0070]. Several findings have suggested the following: (1) brain network modules possess a large number of relatively short connections among adjacent brain regions, and (2) these modules are interconnected via a limited set of network nodes termed *network hubs,* which play central roles in neuronal information flow. Hub regions in brain network modules are defined by network nodes with a larger number of connections and have been identified in the cat, macaque [@b0075], and human brain [@b0070]. Hubs are classified into two categories: *connector hubs* corresponding to the interconnection among network modules and *provincial hubs* within each network module. Damage to connector hub regions (e.g., lesions) causes larger disturbances across widespread brain networks [@b0080], [@b0085]. The concept of the *rich-club* phenomenon within a network arose due to the observation that certain hub nodes of a network are more densely interconnected among themselves than other non-hub nodes [@b0090]. Evidence for the rich-club phenomenon in other biological networks such as the protein interaction network is insufficient, suggesting a higher level of functional specification solely of densely connected network nodes. In this regard, several networks including scientific collaboration networks [@b0090] and power grids [@b0095] exhibit rich-club properties, implying a critical role of rich-club nodes for global communication in the most efficient way. In the human brain network, the existence of rich-club organization has been detected, and certain hub regions are considered rich-club members which form tight subnetworks by themselves [@b0100]. Following the initial discovery of rich-club organization in macroscopic structural networks of the human brain, several studies have demonstrated the characteristics of the rich-club phenomenon in terms of cost-efficiency theory, and brain development and diseases [@b0105], [@b0110], [@b0115], [@b0120], [@b0125]. In the present review, we examine the key contribution of rich-club approaches to understanding its potential roles in brain networks. We first briefly summarize the mathematical background underpinning key concepts of rich-club topology. We then clarify explicit findings in *Caenorhabditis elegans*, the only organism for which the complete neuronal wiring diagram has been mapped. We also assess relevant rich-club data for mammals such as mice, rats, and cats. We then review the background and recent advancements in network studies focusing on the rich-club and structural neuroimaging in human brain development and psychiatric/neurological applications, which constitute one of the most extensively researched areas of the rich-club. We conclude this review by highlighting future prospects in relation to the effectiveness and potential use of this computational and theoretical tool. 2. Construction of neuronal networks {#s0010} ==================================== Serial reconstruction of electron micrographs was initially used to elucidate the complete connectome, particularly for *Caenorhabditis elegans* [@b0130]. This worm has a relatively small number of neurons, which enabled manual reconstruction of its complete neuronal wiring diagram. Nevertheless, this approach suffered from its labor-intensive nature, in particular for neural systems with a larger number of neurons such as the mammalian brain. As a suitable alternative to investigate macroscale neural networks, MRI has been used extensively to define whole brain wiring diagrams *in vivo* for larger neural systems, enabling the comparison of network characteristics across species. Since the introduction of white matter fiber tractography, an MRI-based 3D reconstruction technique that visualizes neural tracts using water diffusivity in the brain predominantly with diffusion tensor imaging (DTI) [@b0135], noninvasive neuroimaging methods such as DTI have been utilized for macroscopic brain network analysis. Other methods for constructing complete whole-brain networks include functional modalities such as functional MRI (fMRI), electroencephalography (EEG), or magnetoencephalography (MEG), by computing the statistical interdependency as a measure of the network edge between spatial locations. Of these, structural neuroimaging modalities such as DTI have overarching benefits from several aspects [@b0140], [@b0145]. First, the DTI technique is specialized for extracting white matter connections and is suitable for macroscale network analysis to investigate larger functional and structural brain subregions (e.g., Brodmann areas). Second, it can reduce computational complexity, which often arises due to the large number of network elements and interactions. For example, the human brain comprises approximately 16 × 10^9^ neurons in the cerebral cortices and 69 × 10^9^ neurons in the cerebellum, with more than 10^12^ neuronal connections [@b0150], [@b0155]. DTI has a 1--2-millimeter resolution, comprising millions of neurons in an acquired voxel, which enables whole-brain network analysis with a reasonable number of network constituents (e.g., 52 distinct Brodmann regions with distinct anatomical parcellations). Third, when combined with functional neuroimaging techniques (e.g., resting and task fMRI), an intuitive approach for examining structural and functional associations is afforded [@b0020], [@b0160]. 3. Detection of the rich-club {#s0015} ============================= Zhou and Mondragon [@b0165] defined rich-club elements as a set of highly interconnected nodes forming a tight subnetwork within a network. The mathematical description of the rich-club[1](#fn1){ref-type="fn"} phenomenon is provided by the rich-club coefficient *ϕ* [@b0090] as follows:$$\phi\left( k \right) = \frac{2E_{> k}}{N_{> k}{(N_{> k} - 1)}}$$where *k* represents the number of connections attached to each network node (the *degree*); *N~\>k~* represents the number of nodes whose degree is larger than a given value *k*; and *E~\>k~* represents the number of connections of a subnetwork comprising *N~\>k~* nodes. It should be noted that the rich-club coefficient *ϕ* is a function of the degree *k,* because it corresponds to the measure of the density of the subnetwork comprising nodes with degree greater than *k*. Practically, the detection procedure involves the following steps: (1) for an *N* × *N* network matrix where *N* is the number of all network nodes, the degree (*k*) is computed at each node; (2) for each *k* (where 1 ≤ *k* \< *N*), all nodes with degrees less than or equal to *k* are removed to construct a new *N~\>k~* × *N~\>k~* subnetwork; (3) the number of existing connections and possible maximum connections of the subnetwork are denoted by *E~\>k~* and calculated by *N*~≥~*~k~*(*N*~≥~*~k~* − 1)/2, respectively; and (4) the rich-club coefficient *ϕ*(*k*) is computed as the ratio of *E~\>k~* and *N*~≥~*~k~*(*N*~≥~*~k~* − 1)/2. Higher degree nodes in a randomly connected network (e.g., the Erdos-Renyi network, a random network with a Poisson degree distribution) tend to have a higher probability of being interconnected to each other by chance [@b0090], [@b0100]. Therefore, to evaluate the statistical significance of *ϕ*(*k*), the coefficient is typically normalized to the rich-club coefficient *ϕ~random~*(*k*) computed from a set of random uncorrelated networks with preserved degree distribution:$$\phi_{\mathit{normalized}}\left( k \right) = \frac{\phi\left( k \right)}{\langle\phi_{\mathit{random}}{(k)}\rangle}$$where $\langle\rangle$ represents the average. If the rich-club coefficient *ϕ*(*k*) of a network is larger than the average rich-club coefficient across randomized networks (i.e., *ϕ*(*k*) \> $\langle\phi_{\mathit{random}}{(k)}\rangle$ or *ϕ~normalized~*(*k*) \> 1), the density of the subnetwork for *k* is considered to be higher than that of its randomized networks, and the network is considered to have a rich-club architecture. For a weighted network, the weighted rich-club coefficient *ϕ^w^* is calculated using the following equation:$$\phi^{w}\left( k \right) = \frac{W_{> k}}{\sum_{l = 1}^{E_{> k}}w_{l}^{\mathit{rank}}}$$where *W~\>k~* represents the sum of weights in the *N~\>k~* × *N~\>k~* subnetwork, $w_{l}^{\mathit{rank}} \geq w_{l + 1}^{\mathit{rank}}$ with 1 ≤ *l* ≤ *E* represents the ranked weights of the links of the network [@b0170], and the coefficient *ϕ^w^* should be normalized over a set of random networks. A unifying framework for the weighted network has been proposed by Alstott and colleagues [@b0175]. In addition to using a network structural attribute (e.g., network degree) to compute rich-club phenomenon, Cinelli [@b0180] recently suggested a generalized rich-club framework using non-structural information (e.g., social or technical attributes related to network nodes). In his work, instead of using only the network degree, any structural measures distinct to degree (e.g., node centrality measures) could also be used to evaluate rich-club ordering. Furthermore, when network nodes have a certain attribute which is not directly derived from the network structure itself (i.e., node metadata such as the wealth of each person in a social network), he suggested two types of network randomization:$$\phi_{\mathit{normalized}}^{\mathit{rewiring}}\left( m \right) = \frac{\phi\left( m \right)}{\langle\phi_{\mathit{random}}^{\mathit{rewiring}}\left( m \right)\rangle}\mspace{6mu}\mspace{6mu}\phi_{\mathit{normalized}}^{\mathit{reshuffling}}\left( m \right) = \frac{\phi\left( m \right)}{\langle\phi_{\mathit{random}}^{\mathit{rereshuffling}}\left( m \right)\rangle}$$where the value *m* corresponds to the value of the node metadata, and $\phi_{\mathit{random}}^{\mathit{rewiring}}$and $\phi_{\mathit{random}}^{\mathit{reshuffling}}$ represent rich-club coefficients with degree-preserving rewiring and metadata reshuffling, respectively. This generalization may be useful for investigating the importance of node metadata in a network and the association between topological and nontopological properties. 4. Rich-club of *Caenorhabditis elegans* {#s0020} ======================================== In 2013, Towlson and colleagues investigated the neural network of the nematode worm *Caenorhabditis elegans*, anatomically defined at a cellular scale with 2287 synaptic connections of 279 neurons [@b0185], [@b0190]. Towlson and colleagues determined 11 neurons as rich-club members, in which eight neurons (AVAR/L, AVBR/L, AVDR/L, and AVER/L) were located in the lateral ganglia of the head and three neurons in the lumbar (PVCR/L) and dorsorectal (DVA) ganglia. The efficiency of the subnetwork with only 11 rich-club neurons (i.e., *network efficiency* [@b0195]) was 0.92, comparable to the efficiency of 268 non-rich neurons (i.e., 0.38). Most of the rich-club neurons (i.e., 10 of 11 rich-club neurons) are command interneurons with functional roles in forward or backward locomotion circuits [@b0185], while DVA is a proprioceptive interneuron which modulates sensorimotor integration during locomotion [@b0200]. Notably, 4% (\~11/297) of these elite neurons were involved in 48% of the total connection distance and 52% of inter-modular connections, suggesting a critical role of rich-club neurons in communication between distant network modules. Towlson and colleagues also reported that rich-club neurons were generated in earlier developmental stages before the main phase of developmental elongation of the body, findings that were replicated by Ma and Mondragon [@b0205]. The importance of the small number of modulatory neurons was revealed in the full hermaphrodite *C. elegans* network with all 302 neurons using the aminergic signaling map [@b0210], in which the monoamine network contains a distinct rich-club comprising dopamine, serotonin, and tyramine-releasing neurons corresponding to sensory and motor activities. These findings were distinct to those of Towlson and colleagues [@b0185] (i.e., interneurons) but further suggested a distinct functional rich-club phenomenon of extrasynaptic networks from synaptic networks in the same nematode worm. More recently, distinctive transcriptional properties of rich-club neurons in *C. elegans* alongside coupled gene expression have been reported, in which rich-club neurons exhibit similar gene expression regulating higher-order behaviors (e.g., locomotion by command interneurons) [@b0215]. Thus, rich-club analyses using neural, chemical, and genetic transcription of this nematode worm may support investigations of higher-order functions and species-conserved mechanisms in the behavioral repertoire of other animals. The rich-club studies on *C. elegans* are valuable as they contribute to deriving fundamental hypotheses on biological network formation. For example, an important aspect of network formation revealed by studies on *C. elegans* is the minimization of network costs (i.e., biological networks are likely to have less connections with shorter paths to minimize the spending of neural resources). However, to reduce costs in a "global" network, revised connection strategies may be more beneficial to facilitate more efficient information propagation. A selected set of network nodes (e.g., rich-club) enables the network to rewire certain connections with increased topological paths and to consequently reduce overall connection costs. Rich-club neurons in *C. elegans* are predominantly command interneurons related to locomotion, suggesting that the existence of rich-club members in a network is crucial to optimize the most important network function (e.g., movement in the case of *C. elegans*). 5. Rich-club of mammals {#s0025} ======================= 5.1. Cat {#s0030} -------- The cerebral cortex of mammals such as cats has a functionally subdivided modular structure (i.e., four main modules of modally-related areas with visual, auditory, somatosensory-motor, and fronto-limbic modules) [@b0220], [@b0225]. Gómez-Gardeñes and colleagues suggested a new module with highly connected but not necessarily module-related areas forming a rich-club connectivity pattern in the cat, in which the rich-club regions of the cat's cerebral cortex (i.e., 53 network nodes + 826 cortico-cortical neural projections) [@b0230] consist of 11 cortical areas (three visual areas: 20a, 7, and anterior ectosylvian sulcus; one auditory area: posterior part of posterior ectosylvian gyrus; two somatosensory-motor areas: medial area 6 and lateral area 5A; and five fronto-limbic areas: agranular and granular insula, posterior cingulate cortex, area 35, and area 36) [@b0235]. Their findings emphasized that these rich-club areas enable a network transition in terms of its dynamics from a simple modular structure to global synchronization, related to higher cognitive tasks in mammals such as planning and integration. The impact of these rich-club regions has been investigated by Lameu and colleagues, who elucidated that a rich-club is highly related to network suppression and global neural synchronization of a network [@b0240]. More detailed analyses of the cat's cerebral network were performed in 2013 by de Reus and van den Heuvel, who defined rich-club members as the top 15 (23%) highest degree nodes with 11 regions based on a previous study [@b0235] and four additional regions (i.e., suprasylvian fringe, dorsolateral division of the prefrontal cortex, anterior part of cingulate cortex, and anterior limbic cortex) [@b0245]. Based on rich-club and non-rich-club nodes, existing connections in the cat network were classified into three categories: (1) rich-club connections only linking rich-club nodes, (2) feeder connections linking rich-club and non-rich-club nodes, and (3) local connections only linking non-rich-club nodes. Importantly, even with the lower connection density of rich-club connections (i.e., 14%, which was comparable to 48% and 38% for feeder and local connections, respectively), approximately 86% of the inter-modular communications were related to rich-club connections, which extended the role of rich-club brain regions to form a larger infrastructure for global and modular communication between different domains in the mammalian brain. The connections of the cat's cerebral network were recently described according to two independent factors: relative cytoarchitectonic differentiation and spatial distance of brain regions, in which a linear combination of these two factors predicted the existence or absence of connections with \>85% accuracy in the cat brain [@b0250]. 5.2. Rat and mouse {#s0035} ------------------ Using an open-access tract-tracing connectivity dataset on the rat [@b0255], van den Heuvel and colleagues demonstrated that the white matter network of rats (with 67 nodes and 1396 connections) contained 14 rich-club members (\~21%) with secondary motor, infralimbic, piriform, dorsal anterior cingulate, prelimbic, medial orbital, posterior agranular insular, temporal association, ectorhinal, perirhinal, lateral entorhinal, medial dorsal entorhinal, lateral amygdalar, and posterior basolateral amygdalar areas [@b0260]. Similar to those in the cat, rich-club connections in the rat constituted 11% of total connections in the network, of which 75% were inter-modular, corresponding to significantly longer distances when compared to feeder and local connections. The association between large-scale network topology and molecular function in the mouse was reported in a transcriptional coupling study [@b0265], in which the highest coupling observed in rich-club connections was driven by genes regulating oxidative synthesis and metabolism of ATP. This study suggested that the connections between brain hub regions are characterized by tightly coupled gene expression related to the regulation of oxidative metabolism. Specifically, approximately 46% of neuronal types comprised the rich-club in the rodent hippocampal neuronal network; indeed, this set had substantially tighter connections, termed *the richest of the rich* club [@b0270]. 5.3. Macaque {#s0040} ------------ The cortical connectivity matrix (binarized with 242 nodes and 4090 connections) defined from 410 neural tracing studies of the macaque revealed rich-club regions in the prefrontal, paracingulate, anterior cingulate, parietal, and temporal cortices [@b0275]. Consistent with those in other species, rich-club regions in the macaque have inter-modular connections rather than isolated subnetworks, and global information flow is mediated by these regions [@b0280]. Moreover, microscale cortical neuronal complexity is associated with macroscale network topology (e.g., degree), particularly in rich-club regions [@b0285], highlighting the importance of neuronal architecture within macroscopic brain networks. Furthermore, structural connectivity is positively associated with resting-state functional connectivity, in which rich-club regions exhibit the greatest functional stability over time depending on their structural topology [@b0290]. More specifically, a small number of neurons in a network seem to be strongly interconnected with oscillatory synchrony to form a rich-club [@b0295]. These neuronal regions may eventually lead to more complex network dynamics in terms of brain function [@b0300], in which connectivity strength could enhance neuronal synchronization, particularly between rich-club regions [@b0305]. 5.4. Rich-club implications in mammals {#s0045} -------------------------------------- While rich-club neurons in *C. elegans* consist primarily of command interneurons, macroscopic rich-club regions are consistently detected in mammals such as the rat, cat, and macaque; i.e., a set of highly connected and central brain regions forming a densely connected rich-club. In mammals, rich-club regions are more highly spatially distributed across the whole brain to facilitate inter-modular communications rather than intra-modular connections. Further, mammalian rich-club regions exhibit greater connection strength among themselves than feeder and local connections ([Fig. 3](#f0015){ref-type="fig"}). While caution should be exercised in cross-species comparisons [@b0310], the considerable overlap in rich-club organization across mammals suggests the existence of "common" biological substrates across mammalian species that may contribute to the integration of neural information resulting in optimal behavioral functioning. Of note, the detailed aspects of rich-club distribution may be related to the characteristics of a species. For example, the rich-club members in the macaque include more prefrontal regions than those in the rat and cat ([Fig. 3](#f0015){ref-type="fig"}), which may be related to the higher-order behavioral functions in this species.Fig. 3Rich-club regions in different species. Brown colors represent the cortical rich-club regions in the rat [@b0260], cat [@b0245], macaque [@b0285], and human [@b0100]. The rat connectivity data were obtained from the open-access BAMS-II database (<https://bams1.org>) [@b0255] and parcellated into 67 regions and 1397 directed axonal projections. The cat cortex was parcellated into 65 regions and 1139 axonal projections as reported by Scannell and colleagues [@b0220], resulting in a binarized connectivity matrix. The macaque axonal projections are based on the open-source CoCoMac database (<http://cocomac.g-node.org/main/index.php>) with 39 × 39 unweighted directed connectivity matrices. The human brain is parcellated using Freesurfer (<http://freesurfer.net>) into 34 (low resolution) and 1170 (high resolution) cortical regions, and the corresponding rich-club regions are depicted in dark and light brown colors. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 6. Rich-club of humans {#s0050} ====================== Rich-club organization in the human brain was first examined using DTI, in which 12 bilateral rich-club regions from 82 cortical and subcortical parcellations were consistently detected in the superior parietal lobule, precuneus, superior frontal cortex, putamen, hippocampus, and thalamus [@b0100], [@b0315]. Consistent with those in the macaque, cat, and rat, all brain regions had at least one link connected to rich-club members, suggesting a key role in global information communication in distant regions via crucial areas in the brain. Simulations of the extent to which the brain network is damaged from an "attack," defined by an arbitrary decrease in connection strength, to a certain set of connections (e.g., (1) "targeted attack" only to rich-club connections, (2) "hub attack" to feeder connections, and (3) "random attack") have revealed that measures of network efficiency are decreased, particularly in rich-club attacks. These findings underscore a potential framework for connectivity disturbances linked to neuropathology. Rich-club regions constitute approximately 69% of all connection paths with 40% of the total communication cost (defined by the number of streamlines based on physical length between two regions), implying the high cost and capacity of these regions for global brain communication [@b0110]. Moreover, structural rich-club regions are strongly associated with resting-state functional networks and regional volume, metabolic energy use, maturation, temporal variability, and structural--functional associations [@b0105], [@b0320], in which the involvement of each functional network of the rich-club varies from a minimum of 3--9% (e.g., extrastriate visual, motor, sensory, and auditory networks) to a maximum of 22% (e.g., default-mode network) [@b0325]. These findings highlight the relevance of centrally collective brain structures in the flexible and reconfigurative nature of brain community organization for integrative cognitive function [@b0325], [@b0330], [@b0335]. Key findings from human rich-club neuroimaging studies are summarized in [Table 2](#t0010){ref-type="table"}.Table 2Summary of outcomes from human rich-club neuroimaging studies.Refs.Sample sizeAge (mean ± SD years)ModalityParcellation: Number of network nodesConnectivityRich-club findingsvan den Heuvel et al. [@b0100]2129.95 ± 8.3DTI82 and 1170 for low and high resolutionDeterministic fiber tractography. SC is binary or weighted (*N~f~*, *N~f~* normalized by regional volumes, and *N~f~* × FA).First RC analysis in the human brain. RC exists in the bilateral superior parietal, precuneus, superior frontal cortex, putamen, hippocampus, and thalamus.Kocher et al. [@b0315]4337.1 ± 11.7DTI82*N~f~* corrected by their length and regional volume.RC nodes are symmetrically distributed across all brain regions. Marked anatomical consistency of RC regions exists.van den Heuvel et al. [@b0110]80Set 1: 28.6 ± 7.9, Set 2: 27.0 ± 6.9DTI1170*N~f~*Network cost (defined as *N~f~* × path length) of RC connections was high (\~40% of the total cost). About 69% of connection paths pass through RC.van den Heuvel et al. [@b0320]7728.2 ± 8.0DTI and rs-fMRIDTI was processed as in [@b0110]. RSNs were extracted with ICA to rs-fMRI.RC nodes are present in all RSNs and coincide in regions with multiple RSNs. Inter-RSN connections are involved in RC.Collin et al. [@b0105]4229.0 ± 8.0DTI, MTI, and rs-fMRI68 cortical regionsSC = FA, MD, TD, and PD. FC = CCRC regions and connections have high levels of volume, white matter organization, metabolic energy usage, long maturational trajectories, more variable regional time-series, and more inter-regional functional couplings.Crossley et al. [@b0325]Meta-analysis (\>1600 studies)rs-fMRI638 brain regionsCoactivation matrixFunctional RC is located in the parietal and prefrontal cortices. It is connected over long distances and coactivated by multiple tasks.Baggio et al. [@b0330]3066.2 ± 10.4DTI68*N~f~*RC connectivity is associated with general cognitive performance.Miŝic, et al. [@b0335]156 subjects in HCPDTI and rs-fMRI114FA and *N~f~*Structural RC connections are more involved in network-level SC--FC associations. RC regions are disproportionately involved in network-wide communication.Ball et al. [@b0120]4627^+1^ (24^+4^--34^+5^) gestational weeksDTI\~500 regions, probabilistic fiber tractography, and binarized SC matrix.1 for connected and 0 for disconnected regionsRC is present by 30-week gestation. Connections between RC and the rest of the brain proliferate until the time of normal birth. RC organization remains intact following premature birth, but both cortical--subcortical connectivity and short-distance corticocortical connections are disrupted.Kim et al. [@b0345]1478.12 ± 1.35DTI90 cortical and subcortical regionsFA of interconnecting fiber tractsConnectivity strength is higher: (1) among RC nodes in children with longer gestation, and (2) in RC nodes compared to the feeder and local connections.Grayson et al. [@b0340]14 adults and 15 childrenAdults: 24--35, children: 7--11DTI and rs-fMRI219 cortical regions*N~f~*Both adults and children have similar structural RC organization, but the adults have greater functional RC organization.Perry et al. [@b0350]11576--94DTI512 cortical and subcortical regions*N~f~* corrected by their lengthRC organization is consistent in both elderly and young adults.Zhao et al. [@b0355]11338.2 ± 21.4, 9--85DTI1024 cortical and subcortical regions*N~f~*Structural RC connectivity has an inverted U-shaped trajectory across the lifespan. RC regions are distributed in the medial frontal, parietal, and occipital cortices in children and young adults, but RC connections in the frontal regions are reduced in older age.Cao et al. [@b0360]12636.8 ± 21.2, 7--85rs-fMRI1024 cortical and subcortical regionsCCFunctional RC connectivity has an inverted U-shaped lifespan trajectory.Zhao et al. [@b0365]7725.0--41.4 weeksDTI58 cortical regions*N~f~* × FAEfficiency of RC networks is increased more rapidly than that of non-RC networks in term-born brain networks.Baker et al. [@b0370]3116.58 ± 0.54DTI80 cortical and subcortical regions*N~f~* and FARC connectivity between subcortical regions decreased over time. Frontal--subcortical and frontal--parietal hub--hub connectivity is increased over time.Kim et al. [@b0375]997.80 ± 1.22DTI90 cortical and subcortical regionsFA of interconnecting fiber tractsRC connectivity has a positive association with children\'s intelligence score. Children\'s ability for visuo-motor spatial reasoning showed significant correlations with RC.van den Heuvel et al. [@b0380]2730--42 gestational weeksDTI and rs-fMRI56 cortical regionsSC = FA, MD, TD, and PD. FC = CCNeonatal RC regions are located in the left superior frontal, left lateral front-orbital, left precentral, left postcentral, left and right superior parietal, left and right cingular, left and right angular, and left and right fusiform areas.Kim et al. [@b0385]496.2--9.4DTI1015 cortical regionsFAPositive associations between prenatal maternal cortisol level and network cost are found for RC connections of girls only at 31 gestational weeks.Scheinost et al. [@b0390]12 preterm and 25 term neonatesPreterm: 27 ± 2.2,Term: 40 ± 1 weeksrs-fMRI95 cortical and subcortical regionsCCBoth preterm and term neonates have RC organization, but preterm neonates have reduced RC connectivity.Fischi-Gomez et al. [@b0395]5126.7--32.5 gestational weeksDTI82*N~f~* corrected by their length and regional volumeNo RC differences between extreme prematurity (EP) and intrauterine growth restriction (IUGR) groups. RC organization after premature birth is maintained even at school age.Karolis et al. [@b0400]51 preterm and 60 controls38--42 gestational weeksDTI82 cortical and subcortical regions*N~f~*Very preterm brains exhibit stronger RC architecture.[^1] 6.1. Application to brain development {#s0055} ------------------------------------- Rich-club organization is established prior to the time of birth [@b0120]. It develops throughout childhood, adolescence, and adulthood [@b0340], [@b0345], [@b0350], [@b0355]; and is sustained throughout the lifespan [@b0360]. Specifically, the connection density between rich-club regions and the rest of the cortex increases during the third trimester [@b0120] in neonates [@b0365], and even in late adolescence [@b0370]. The intelligence quotient of typically developing children exhibits a stronger positive association with rich-club connectivity than with feeder and local connectivity [@b0375]. Although developmental trajectories in the structural and functional rich-clubs are similar in neonates and adults [@b0380], adults exhibit greater functional rich-club organization compared to that in the younger population [@b0340]. The cost of rich-club connections is strongly associated with prenatal maternal cortisol levels, a measure of maternal stress during pregnancy; this effect is unique to women, suggesting sex-specific rich-club contributions during development [@b0385]. The development of term and preterm children using rich-club analyses is an active research area. Preterm neonates were reported to have reduced connectivity of rich-club regions [@b0390], and children with longer gestation exhibit more efficient structural networks with higher rich-club connectivity when compared to children with shorter gestation [@b0345]. In contrast, rich-club characteristics associated with preterm births have been reported to be maintained in school-age children [@b0395] or to be stronger in adults [@b0400]. These findings emphasize that the overall topological characteristics in the brains of children are associated with longer gestation, and shorter gestation does not significantly impact rich-club organization particularly in children ([Table 2](#t0010){ref-type="table"}). In summary, the structural and functional rich-club framework provides a method to map the age-dependent patterns of brain development and to answer several key questions: (1) What are the critical roles of "core" brain regions in brain development? (2) Can improved development of rich-club organization lead to improved brain function? (3) Are peripheral connections merely supplementary to brain functions when compared to rich-club connections? Empirical studies have demonstrated that brain network evolution is particularly centered with rich-club regions in the developing brain. However, critical challenges related to the network development should be addressed in future studies, which include: (1) the precise mapping of developmental stages in both structural and functional rich-clubs, (2) the neural substrates of abnormal rich-club development, and (3) the predominance of rich-club versus non-rich-club regions in network development. 6.2. Clinical findings: Psychiatric disorders {#s0060} --------------------------------------------- Both structural and functional brain networks exhibit rich-club organization in schizophrenia [@b0115], [@b0405], [@b0410], [@b0415], suggesting that the effects of altered brain connectivity are more concentrated in rich-club connections than in feeder or local connections in patients with schizophrenia. The connection density of rich-club regions derived from DTI and white matter tractography is often observed to be reduced in patients with schizophrenia, while rich-club density is intermediate in unaffected siblings relative to that in patients and healthy controls, and is lower in their offspring than in healthy controls [@b0125], [@b0420], [@b0425]. These findings have been replicated in subjects with high clinical risk for psychosis [@b0430]. Furthermore, decreased rich-club connection density is associated with lower levels of global communication capacity (i.e., network efficiency), resulting in a stronger association between structural and resting-state functional connectivity in patients [@b0115]. Importantly, the increased structural--functional association in patients is interpreted as more stringent and less dynamic brain function from the illness that is more directly associated with the underlying structural connectivity. Additionally, higher connectivity strength of rich-club connections is associated with positive changes in general functioning over time in schizophrenia [@b0435], [@b0440]. Rich-club connections among rich-club nodes are lower in major depressive disorder (MDD) and late-life depression patients than in healthy controls, in which higher rich-club connectivity is associated with lower symptom severity score (i.e., Hamilton Depression Rating Scale) [@b0445], [@b0450]. However, the remission of MDD patients is more strongly associated with feeder-local subnetworks than with rich-club connections [@b0455], suggesting that compensatory effects from treatment may be more distinct in non-core brain regions in patients with this psychiatric disorder. Additionally, the development of age-related rich-club organization has been reported in typically developing adolescents but not in patients with autism spectrum disorder (ASD) [@b0460]. Ball and colleagues reported that the rich-club connections in ASD patients exhibited an inverted U-shaped association with age [@b0465], which is similar to that in healthy controls [@b0355]. While the phenotypes of impaired neurodevelopmental disorders such as attention deficit hyperactivity disorder (ADHD) and ASD overlap substantially in terms of clinical comorbidity, ADHD and ASD children exhibit distinct patterns of rich-club and non-rich-club connections [@b0470]. Global network efficiency has been reported to be decreased in bipolar disorder (BD) patients, but no significant differences have been noted in the strength of brain hub connections with rich-club regions [@b0475], [@b0480], [@b0485], suggesting that aberrant network organization may not be specific to the central core system of BD. However, more recent studies have indicated that BD patients possess decreased rich-club and feeder connectivity density [@b0490] and increased rich-club connectivity [@b0495]. 6.3. Clinical findings: Neurological disorders {#s0065} ---------------------------------------------- Rich-club organization is more predominant in patients with Alzheimer's disease (AD), and a recent study suggested that rich-club connectivity (as measured by the fiber density interconnecting two regions) is decreased in the early-onset AD [@b0500]. However, low-degree regions, and not rich-club regions, have been found to be more strongly associated with network disruption in AD patients [@b0505], [@b0510], [@b0515]. This suggests that peripheral connections may be more vulnerable and contribute to cognitive decline in this neurodegenerative disease. Moreover, patients with generalized tonic-clonic seizures have reduced rich-club connectivity, which is associated with longer durations of illness and seizure frequencies [@b0520]. Structural connectivity of rich-club regions is decreased in patients with multiple sclerosis, in whom decreased rich-club connectivity is associated with mobility, hand function, information processing speed [@b0525], and cognitive impairments [@b0530]. In patients with traumatic brain injury (TBI), the strength of local connections is increased, but rich-club connectivity is decreased [@b0535]. These results have been replicated in cognitively impaired and nonimpaired active professional fighters [@b0540]. These findings suggest that peripheral subnetworks may compensate for biologically high-cost rich-club subnetworks after TBI. Finally, decreased structural connectivity has been observed in rich-club regions in patients with cerebral small vessel disease, which was positively associated with psychomotor speed and executive function [@b0545]. However, connectivity disruption in rich-club regions did not have specific effects over time, as observed in a longitudinal study [@b0550]. 6.4. Rich-club implications for brain disorders {#s0070} ----------------------------------------------- Highly interconnected network hub regions often form rich-clubs. A coactivation network *meta*-analysis using functional neuroimaging data revealed that topological characteristics such as network module, small-worldness, and rich-club are often consistent across psychiatric and neurological brain disorders [@b0555]. However, pathological lesions are likely to be found in hub regions rather than peripheral regions, whereby rich-club regions have lesions twice as often as peripheral network nodes. These findings suggest that: (1) brain regions do not function equally in brain network architecture, (2) brain disorders are more strongly associated with damage to central brain regions such as rich-clubs, and (3) the disruption of network rich-clubs may be common across various brain diseases. Of note, the relationship between a specific brain dysfunction and network rich-club regions is dependent on the "location" of lesions. For example, while schizophrenia and AD share hub-specific distributions of lesions, regions more strongly implicated in each disease are located in the frontal and cingulate regions for schizophrenia, and in the medial temporal and parietal regions for AD. 7. Summary and outlook {#s0075} ====================== The functional roles of biological network elements vary according to their anatomical locations. This differentiated functional organization of the central nervous system has often been associated with specific functions of the network system such as sensorimotor function, mental activities, and behaviors [@b0560]. The concept of "functional specialization" is supported by various neurophysiological, anatomical, and noninvasive neuroimaging findings, and has formed a theoretical neural substrate underlying cognition. However, the complex nature of human cognition prompts the following question: How do functionally specialized units communicate with each other optimally? The conceptual framework used by researchers to understand neural systems such as the brain emerged from the idea that individual neural elements are functionally integrated and orchestrate higher-order brain activities such as sensory recognition, emotion, language processing, and social cognition in a coordinated manner [@b0565]. The organization of functionally segregated and anatomically integrated biological systems has been investigated from the perspective of complex network theory [@b0570]. Network science or graph theory has revealed a structural basis for the dynamic functional interactions emerging from a diverse set of neural elements and defined how structural topology gives rise to modular brain function ranging from *C. elegans* to mammals and humans. A key organizational feature is the existence of crucial elements that attribute functional specialization to neural networks, known as *hubs.* Hub elements have been found to produce efficient neural information flow at the expense of neural cost [@b0575]. They have more connections (i.e., higher degree) or higher levels of connectivity, particularly for long-distance connections exhibiting the "rich" aspects of hub elements, forming the "rich-club" [@b0100], [@b0110]. Several important issues arise from the current rich-club perspective and network analysis. It is necessary to develop optimal computational and mathematical frameworks for each data modality in each neuronal system in terms of network construction, statistics, and their interpretations [@b0580]. First, defining network nodes and edges is crucial for modeling neural systems [@b0585] and depends strongly on the research domain (e.g., anatomical and functional networks acquired from neuronal, physiological, and neuroimaging datasets). Regarding macroscale brain networks (i.e., brain regions), parcellation techniques have been used to subdivide the whole brain into anatomically distinct areas resulting in segregated nodes of the brain network. Such parcellations include predefined anatomical segregations (e.g., Brodmann areas, Desikan-Killiany atlas [@b0590], and anatomical automatic labeling map [@b0595]), atlas-independent random cortical segmentation [@b0120], and data-driven clusters, which are often derived from functional correspondence [@b0600], [@b0605], [@b0610], [@b0615] with individualized state-specific parcellations [@b0620], [@b0625] (refer to the study by Arslan and colleagues [@b0630] for a comparison of each parcellation scheme). While the predefined parcellation templates integrate network properties across each individual leading to unbiased group comparisons, it should be noted that the size and distribution of each parcel modulate quantitative topological properties including the rich-club organization of a brain network [@b0635], [@b0640]. The significance of network edges is also considered important. Anatomical neuroimaging techniques represent diverse definitions of structural connectivity between two brain regions (e.g., the number of streamlines generated from fiber tractography, tract-based diffusion characteristics such as fractional anisotropy and mean diffusivity, cortical thickness, and the amount of myelination in white matter) [@b0100], [@b0645], [@b0650], while functional edges are designated by functional similarity such as Pearson correlation coefficient, covariance, coherence, and mutual information measured with fMRI, EEG, and MEG recordings. In addition to the definitions of structural and functional connectivity, functional networks are intrinsically more dynamic than structural networks and act as determinants of brain function and dysfunction that are constrained by brain structure [@b0655]. Furthermore, weighted structural and functional connectivity measures are often noisy due to physiological and methodological limitations. Binarization or thresholding techniques may be applicable to enhance the contrast between relevant and irrelevant connectivity values [@b0660]. However, binarization or thresholding is highly dependent on whether the connectivity is absolute (retaining values over a threshold) or proportional (retaining a fixed percentage of values) and may have a greater impact on certain global network measures particularly derived from functional connectivity [@b0665]. In addition, a general consensus on the definition of network threshold is lacking, and researchers use empirical values to determine thresholds. These binarization or thresholding techniques may diminish or exaggerate connectivity values below or above the threshold, leading to under- or over-estimated network characteristics, respectively. It is challenging to determine whether individual or group-wise variations in rich-club regions are indicative of methodological limitations resulting from the aforementioned computational and technical challenges, or whether they truly reflect additional biological information in terms of inter- and intra-individual variability. For instance, rich-club regions identified in an initial report [@b0100] consist of the superior parietal and frontal cortex, precuneus, putamen, hippocampus, and thalamus. However, more recent studies have identified the insula as another rich-club region [@b0105], [@b0120], [@b0370], [@b0385]. This may arise from the arbitrary threshold for rich-club detection based on statistical rich-club coefficients *ϕ*(*k*) (e.g., top 10% [@b0120], [@b0385], 12% [@b0105], and 18% [@b0370]). Since the number of connections across nodes in the brain network often increases gradually and not distinctively, a more liberal or conservative definition of the rich-club may affect subsequent analyses. Thus, although the rich-club organization exists regardless of definitions of brain parcellation and connectivity, these may affect qualitative characteristics of the rich-club, reflecting distinct aspects of rich-club regions in the brain network. In this regard, a robust and consistent rich-club characterization remains unsolved in brain network science. Rich-club analysis and whole brain anatomical network analyses are often based on neuroimaging techniques such as DTI and fiber tractography. While technological advancements in these techniques are rapidly increasing, the intrinsic nature of neuroimaging remains an open question. First, fiber tractography is a deterministic approach which often provides one-to-one connections from a seed point that may miss crossing, splitting, and/or branching tracts. Although probabilistic algorithms have been applied as an alternative to resolve this issue, other false-positive connections may be detected. This approach may not comprehensively assess rich-club detection and connections because fiber tracts related to the rich-club are relatively insensitive to false-positive and false-negative tracts. However, feeder and local connections are highly dependent on the quality of fiber tractography and may have a larger impact on peripheral associations to rich-club regions. The development of better qualified tractography algorithms is required. Second, similar to conventional graph theoretical analysis, the rich-club organization is relatively dependent on the definition of structural connectivity derived from DTI. For example, the number of streamlined fiber tracts is commonly used for the detection of the rich-club and other network measures. In addition to variations in streamlines related to parameter adjustment during fiber-tracking, investigating the association between the connections with neuronal tracing as true connectivity and the number of streamlines or other variations (e.g., scalar measures along fiber tracts such as fractional anisotropy) is recommended. Third, the structural and functional rich-club organization extracted from neuroimaging is strongly associated with genetic variation [@b0265], [@b0670], structural--functional coupling [@b0115], [@b0420], and metabolism [@b0675]. Therefore, higher cost and central roles of rich-club regions may be established based on the high metabolic energy consumption in these core regions and coupled with certain patterns of gene expression related to metabolism. This may lead to higher levels of functional and structural connections with larger gray matter volume and improved white matter microstructure, thereby establishing a closer relationship between brain structure and function. To establish the consequences of rich-club formation in the brain, various modalities including genetic, metabolic, microbiological measurements, and neuroimaging are required to elucidate the convergent implications of brain rich-club organization. Fourth, graph theoretical brain network analysis, which enables the investigation of rich-club organization, is dependent on the scales-of-interest at multiple levels for a given network (i.e., global, modular, or local network organization). Thus, detection of the rich-club architecture at the level of individual regions is often associated with modular or global brain network properties. In addition to the global communication efficiency derived from rich-club architecture (i.e., role as connector hubs), rich-club nodes often form subnetwork communities or modules with a relatively sparsely connected set of nodes (i.e., peripheral nodes). The implications of each rich-club node (as in psychiatric and neurological disorders) may exert its influence on the participating module within the network rather than on the individual node itself (i.e., role as provincial hubs). Therefore, analyses at the level of intermediate-networks (i.e., modules) and individual rich-club nodes are crucial to extend the utility of these central and rich network units. Of importance, detection of network modules or communities is dependent on the applied optimization algorithm, often resulting in non-unique network subdivisions, which may restrict the unbiased interpretation of consistent modular structures related to specific rich-club nodes. Deriving strategies for optimal modular detection techniques will be crucial to understand the resilience and vulnerability of rich-club nodes at the level of higher-order network structures. Fifth, the rich-club comprises a set of core brain regions, which is relatively invulnerable to external attacks (i.e., brain disease) because the brain has several neural resources (i.e., high cost). This suggests that rich-club abnormalities are more easily detected when the attack exceeds the resistance and tolerance to external insults (i.e., critical point). Therefore, beyond conventional graph theoretical measures, rich-club investigations of the brain network should pay more attention to determining thresholds at which external attacks exceed a critical point (i.e., occurrence of a certain disease). In this review, we have described how rich-club organization has been applied to neural systems to reveal neuroanatomical correlates with brain development and disorders. Network neuroscience investigates brain structure, function, behavior, and cognition. Considering the weaker statistical power of typical clinical studies, systematically collected large-scale datasets from multicenter and multimodal data collection (e.g., Human Connectome Project [@b0680], [@b0685], [@b0690]) will be essential to comprehensively assess the phenomenon of rich-club organization and understand the neural architecture underlying brain development and disorders. Making sense of these brain network datasets presents an exciting challenge of bridging the gap between topological findings related to core brain regions and the biological significance of computational interpretations. Declaration of Competing Interest ================================= The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. This work was supported by the 10.13039/501100002642Korea University Future Research Grant (grant number K2006911 to B.K.M.), the Basic Science Research Program (grant number 2018R1A2B6004084 to B.K.M.), the Convergent Technology R&D Program for Human Augmentation (grant number 2020M3C1B8081319 to B.K.M.), and the Information Technology Research Center (10.13039/501100008122ITRC) support program (grant number 10.13039/501100010418IITP-2020-2016-0-00464 to B.K.M.), funded by the Korean government (MSICT) through the 10.13039/501100003725National Research Foundation of Korea. The authors declare no competing interests. Network rich-club can be computed with BCT (<https://sites.google.com/site/bctnet/>) in MATLAB, braingraph (<https://github.com/cwatson/brainGraph>) in R, or NetworkX (<https://networkx.github.io/documentation/networkx-1.9.1/index.html>) and bctpy (<https://pypi.org/project/bctpy/>) in Python. [^1]: *DTI*, diffusion-tensor imaging; *FA*, fractional anisotropy; *CC*, correlation coefficient; *SC*, structural connectivity; *FC*, functional connectivity; *RC*, rich-club; *N~f~*, the number of fiber tracts between two regions; *rs-fMRI*, resting-state fMRI; *RSN*, resting-state networks; *ICA*; independent-component analysis; *MTI*, magnetic transfer imaging; *HCP*, human connectome project; *MD*, mean diffusivity; *TD*, transverse diffusion; *PD*, parallel diffusion.

INTRODUCTION {#sec1-1} ============ Cerebral hyperperfusion after carotid endarterectomy (CEA) is defined as a major increase in ipsilateral cerebral blood flow (CBF) after surgical repair of carotid stenosis that is well beyond the metabolic demands of the brain tissue.\[[@ref4][@ref25][@ref29][@ref31][@ref33][@ref37]\] However, prompt and precise assessment of cerebral hyperperfusion is often difficult because of limitations in the methodology of CBF measurement during the perioperative period.\[[@ref4][@ref25][@ref29][@ref31][@ref33][@ref37]\] Previous reports have demonstrated cerebral hyperperfusion after CEA with transcranial Doppler, Xe-enhanced computed tomography (CT), or single-photon emission CT (SPECT) using 99mTc-ethylcysteinate dimer (ECD) or N-isopropyl-\[123I\] b-iodoamphetamine (IMP).\[[@ref9][@ref10][@ref13][@ref20][@ref24][@ref25][@ref26][@ref32][@ref37]\] The criteria for cerebral hyperperfusion vary among these studies.\[[@ref25][@ref29][@ref37]\] Diagnosis of cerebral hyperperfusion syndrome (CHS) is based not only on evidence of hyperperfusion on these CBF studies but also on the presence of clinical symptoms such as seizures, deterioration of consciousness (including restlessness), and the development of focal neurological signs such as motor weakness.\[[@ref25][@ref28][@ref29][@ref37]\] Among the various CBF studies performed to date, SPECT has been widely used in Japan to assess the cerebral hemodynamic state and quantify the regional CBF and hemodynamic reserve by measuring the patient\'s cerebrovascular reactivity (CVR) to acetazolamide loading.\[[@ref17][@ref22][@ref28][@ref34]\] Ogasawara *et al*.\[[@ref28]\] reported that IMP-SPECT can be used to preoperatively measure the CVR and identify patients at risk for cerebral hyperperfusion; in addition, post-CEA monitoring of CBF, including immediately after CEA, allows for timely and reliable identification of patients at risk for CHS. However, repeating SPECT studies is practically difficult because administration of the exogenous radioisotope is required. A new magnetic resonance (MR) perfusion imaging technique with arterial spin labeling (ASL) was recently developed to assess the regional CBF without the need for contrast administration.\[[@ref2][@ref5][@ref6][@ref7][@ref8][@ref21][@ref32]\] Because ASL uses magnetically-labelled blood water as an endogenous tracer,\[[@ref30]\] it is a completely noninvasive and repeatable perfusion imaging technique. Previous authors demonstrated a close correlation between ASL and SPECT imaging in terms of assessment of the CBF status.\[[@ref19][@ref27][@ref32][@ref35][@ref36]\] However, ASL has some drawbacks. It has a time delay called the "post-labeling delay" (PLD) between inversion of blood spins passing through the labeling plane in the neck and collection of images in the head after the labelled blood flows into the cerebral tissue.\[[@ref2]\] An ASL sequence with no delay compensation is considerably susceptible to the arrival time of labelled blood in tissue; namely, the arterial transit time (ATT).\[[@ref6][@ref7][@ref8]\] Recent ASL studies have generally used a single PLD that typically ranges from 1.5 to 2.0 s as a trade-off between maintaining adequate diagnostic quality and allowing sufficient delay for visualizing tissue perfusion in clinical MR imaging machines.\[[@ref11][@ref21][@ref39]\] In steno-occlusive cerebrovascular disease, however, the ATT is generally prolonged due to stenosis or occlusion of the major arteries.\[[@ref39]\] Labelled blood that travels via collateral pathways of the circle of Willis or secondary collateral pathways, including those associated with leptomeningeal anastomosis, may also exhibit an increased ATT.\[[@ref11][@ref20][@ref26]\] In this situation, conventional ASL measurements using a single PLD may lead to an underestimation of tissue perfusion.\[[@ref11]\] Conversely, following CEA, improvement of the delayed anterograde flow in the internal carotid artery (ICA) may shorten the ATT, and ASL may lead to an overestimation of the CBF.\[[@ref36]\] In this report, we demonstrate the chronological changes in ASL signals with a single PLD of 1.5 s immediately after CEA, and compare these findings with preoperative clinical conditions and hemodynamic states. A previous study reported signal changes on ASL with a single PLD of 1.5 s at 3 months postoperatively.\[[@ref3]\] The purpose of this study was to determine whether ASL can assess post-CEA hemodynamic changes and identify patients at risk for CHS. PATIENTS AND METHODS {#sec1-2} ==================== Patients {#sec2-1} -------- Thirty-two consecutive patients with ipsilateral ICA stenosis underwent CEA at Kyushu Rosai Hospital from November 2011 to April 2016. The inclusion criteria for CEA used in our institute are carotid stenosis of ≥70% or 50--69% with repeated ischemic cerebrovascular events, activities of daily living corresponding to a modified Rankin Scale (mRS) score of ≤2 in asymptomatic patients and 3 or 4 in symptomatic patients with repeated ischemic cerebrovascular events, small or no infarction on MR imaging, and absence of major occlusive disease (≥70% in diameter) distal to the carotid stenosis. Thirty-one of the 32 patients were men, and 1 was a woman. The mean age of the patients was 73.0 years (range, 46--85 years). We reviewed all medical records. Informed consent was obtained from all the patients. Cerebral blood flow measurements {#sec2-2} -------------------------------- ### Arterial spin labeling measurements {#sec3-1} Routine MR imaging and ASL perfusion imaging were performed using a 3T-MR unit (Signa HDxt 3.0T version 23; GE Healthcare, Milwaukee, WI, USA). ASL was prepared using a three-dimensional (3D) spiral fast-spin echo sequence with background suppression for perfusion imaging covering the entire brain. A pulsed continuous scheme was employed. Other acquisition parameters were as follows: Four arms with 1004 points in each spiral arm, phase encoding in the z direction = 32, section thickness = 4 mm, Time to repeat (TR) = 4728 (AUTO) s, post-label wait = 1.525 s (1.5 s), and number of excitation (NEX) = 3. The acquisition time was 2 min 22 s. All patients underwent preoperative ASL examination with routine MR examination. In all patients, a second ASL examination was performed on postoperative day 1 (POD1); i.e., immediately after extubation of the orotracheal tube. At that time, routine MR imaging was also performed to rule out the appearance of perioperative *de novo* ischemic events. Follow-up ASL was performed when needed. The timing of follow-up ASL varied in this study. Evaluation of decreased or increased ASL signals was based on visual inspection by two experienced radiologists who were blind to the clinical and imaging data. No differences in the radiologists' interpretations were noted on independent assessments. ### Single photon emission computed tomography measurements {#sec3-2} Using SPECT (Headtome SET-031; Shimadzu Co., Kyoto, Japan), CBF was semiquantitatively measured before and 15 min after intravenous injection of 10 mg/kg of acetazolamide (ACZ) on separate days at an interval of 2 to 3 days. Regions of interest were placed automatically in the target area (e.g. in the middle cerebral artery \[MCA\] territory) using commercially available software (E. CAM Signature; Toshiba Medical, Tokyo, Japan/GMS7700R). The CVR to ACZ was calculated as follows: CVR (%) =100 × (CBF~ACZ~ − CBF~REST~)/CBF~REST~, where CBF~REST~ and CBF~ACZ~ represent CBF before and after intravenous injection of ACZ, respectively. A CVR of \<20% was considered to be a reduced CVR according to a previous report.\[[@ref28]\] Twenty-nine patients underwent preoperative SPECT imaging at rest. In 24 patients, CVR was assessed with an ACZ challenge. Of these 24 patients, 21 were assessed with IMP-SPECT and 3 with ECD-SPECT. Electroencephalography {#sec2-3} ---------------------- Twenty-nine of the 32 patients underwent routine EEG recording preoperatively and on POD1. Follow-up EEG was performed in 3 patients who developed CHS. Routine EEG recordings were obtained from an 18-channel digital EEG machine (Neurofax; Nihon-Kohden, Tokyo, Japan) with electrode placement according to the International EEG 10-20 system. The EEG recordings were performed for at least 30 min for each patient at rest. Perioperative management of patients {#sec2-4} ------------------------------------ Twenty-eight patients underwent CEA more than 1 month after the last ischemic event, and 4 patient underwent CEA urgently. All CEA procedures were performed under an operative microscope with the use of internal shunts. After CEA, general anesthesia with propofol was continued under controlled ventilation until the next morning. Blood pressure was maintained at \<130 mm Hg (systolic) and \<90 mm Hg (diastolic) in all patients using intravenous nicardipine. Immediately after extubation, all patients underwent routine MR imaging examination including ASL, as described above. In patients with increased ASL signals, arterial blood pressure was more closely monitored and strict control of blood pressure using intravenous nicardipine and diltiazem and an oral angiotensin II receptor blocker was continued until POD7. Diagnosis of CHS was based on the appearance of clinical symptoms and EEG abnormalities that could explain the clinical findings in addition to evidence of increased ASL signals. In Case 5, postoperative ECD-SPECT was also performed to confirm the cerebral hyperperfusion. Functional outcomes were assessed at discharge using the mRS.\[[@ref38]\] RESULTS {#sec1-3} ======= Relationship between preoperative arterial spin labeling signals and preoperative single photon emission computed tomography {#sec2-5} ---------------------------------------------------------------------------------------------------------------------------- \[[Table 1](#T1){ref-type="table"}\]. Preoperative ASL signals mostly coincided with the CBF map on resting SPECT with visual inspection. However, ASL had a tendency to underestimate the CBF. On preoperative ASL, 19 of 32 patients exhibited decreased ASL signals on the ipsilateral side. On preoperative SPECT of these patients, an ipsilateral decrease in the CBF was noted in 12 patients, no decrease was noted 5, and a bilateral decrease was noted in 1. Nine of the 12 patients with no decreased signal on ASL also exhibited no decreased CBF on SPECT. One other patient showed a decreased CBF of the ipsilateral side on SPECT. In a patient with decreased ASL signals on the contralateral side, no decreased CBF was noted on SPECT. ###### Clinical profile of 32 patients with carotid stenosis who underwent carotid endarterectomy  Arterial spin labeling signals on postoperative day 1 and their chronological course {#sec2-6} ------------------------------------------------------------------------------------ On POD1, 22 (68.8%) of 32 patients exhibited increased ASL signals on both sides (Group A) \[[Figure 1a](#F1){ref-type="fig"}\]. All patients except two (Case 5; detailed clinical course described below and Case 18) did not develop a *de novo* neurological deficit attributed to increased ASL signals on both sides. The increased ASL signals were improved in all 15 patients who underwent re-examination of their CBF by follow-up ASL. The increased ASL signals had disappeared by POD7 in six patients, by POD14 in 5, by postoperative month 2 in 1, by postoperative month 6 in 1, and by postoperative month 14 in 2. {#F1} Four (12.5%) patients demonstrated increased ASL signals only on the operated side (Group B) \[[Figure 1b](#F1){ref-type="fig"}\]. All patients except one (Case 23; detailed clinical course described below) did not develop a *de novo* neurological deficit attributed to the ipsilateral increased ASL signals. Two patients underwent re-examination of their CBF by follow-up ASL; the increased ASL signals had disappeared by POD8 in one patient and by postoperative month 18 in the other. Six (18.8%) patients showed no change in their postoperative ASL signals compared with the preoperative ASL images (Group C) \[[Figure 1c](#F1){ref-type="fig"}\]. None of these patients developed *de novo* neurological deficits. Three patients underwent re-examination of their CBF by follow-up ASL, and the ASL signals exhibited no change in all 3 patients. Relationship between arterial spin labeling signals on postoperative day 1 and pre and postoperative clinical conditions {#sec2-7} ------------------------------------------------------------------------------------------------------------------------ Preoperatively, 13 of 22 patients in Group A were asymptomatic or had a history of transient ischemic attack or transient monocular blindness. Nine patients had experienced small but symptomatic infarctions. The preoperative mRS score was 0 in 13 patients, 1 in 8 patients, 2 in 1 patient. Their mRS scores did not change postoperatively except one (Case 16; improved). In contrast, all patients in Groups B and C had old infarctions. In Group B, the preoperative mRS score was 1 in 2 patients and 3 in 2 patients. In Group C, the preoperative mRS score was 1 in 2 patients, 2 in 2 patients, 3 in 1 patient, and 4 in 1 patient. The mRS scores in Groups B and C did not change postoperatively. Relationship between arterial spin labeling signals on postoperative day 1 and preoperative hemodynamic state {#sec2-8} ------------------------------------------------------------------------------------------------------------- There was no apparent correlation between the ASL signals on POD1 and the preoperative hemodynamic state, as indicated by preoperative ASL, CBF detected by SPECT at rest, and CVR. Among 22 patients in Group A, the preoperative ASL signals exhibited an ipsilateral decrease in 13 patients, no decrease in 8, and a contralateral decrease in 1 with contralateral internal carotid artery occlusion. Preoperative SPECT imaging showed an ipsilateral decrease in the CBF in seven patients and no decrease in twelve. CVR impairment was observed on the ipsilateral side in 7 of 15 patients, bilaterally in 3, and on the contralateral side in 1 with contralateral ICA occlusion. Four patients demonstrated no CVR impairment. In 4 patients in Group B, the preoperative ASL signals exhibited an ipsilateral decrease in 3 patients and no decrease in 1. Preoperative SPECT images showed an ipsilateral decrease in the CBF in 2 patients, a bilateral decrease in one, and no decrease in one. CVR impairment was observed on the ipsilateral side in 3 patients, and 1 patient demonstrated no CVR impairment. In 6 patients in Group C, the preoperative ASL signals exhibited an ipsilateral decrease in 3 patients and no decrease in 3. Preoperative SPECT images showed an ipsilateral decrease in the CBF in 4 patients and no decrease in 2. CVR impairment was observed on the ipsilateral side in 5 of the 6 patients. Conversely, in 15 patients with impaired ipsilateral CVR, increased ASL signals on POD1 were seen on both sides in 7 patients and unilaterally in 3. However, in the remaining 5 patients, increased ASL signals were not seen. In 5 patients without ipsilateral CVR impairment, increased ASL signals on POD1 were seen on both sides in 4 patients and unilaterally in 1. The lack of a correlation between the ASL signals on POD1 and CVR was statistically significant (*P* = 0.2062, Chi-square test using JMP Pro 10.0.2 \[SAS Institute Inc., Cary, NC, USA\]). There was also no apparent correlation between ASL signals on POD1 and patency of the anterior communicating artery-A1 portion of the anterior cerebral artery (ACOM-A1) and the ipsilateral posterior communicating artery (PCOM) on MR angiography. The ACOM-A1 was not patent in only 2 patients in Group A. The ipsilateral PCOM was patent in 17 of 22 patients in Group A, 3 of 4 patients in Group B, and 3 of 6 patients in Group C. Development of cerebral hyperfusion syndrome {#sec2-9} -------------------------------------------- Three out of 32 (9.4%) patients exhibited CHS. These 3 patients were Case 5 and Case 18 in Group A, and Case 23 in Group B. The detailed clinical courses and hemodynamic as well as EEG findings of Case 5 and Case 23 are described below as representative cases because the clinical profiles and EEG findings of Case 18 were quite similar to those of Case 5. Representative cases {#sec2-10} -------------------- ### Case 5 (Group A) {#sec3-3} An 83-year-old man presented with asymptomatic severe stenosis of the right ICA. He had a history of smoking, hypertension, hyperlipidaemia, aortic regurgitation, and chronic heart failure. His neurological examination revealed no remarkable findings. 3D-CTA revealed severe stenosis of the right ICA at the bifurcation of the common carotid artery \[[Figure 2a](#F2){ref-type="fig"}\]. Preoperative ASL showed decreased signals in the right MCA territory \[[Figure 2b](#F2){ref-type="fig"}\]. IMP-SPECT imaging at rest demonstrated mild CBF reduction in the right MCA territory \[[Figure 2c](#F2){ref-type="fig"}\]. CVR impairment in the right anterior cerebral artery and MCA territory was noted with ACZ loading \[[Figure 2d](#F2){ref-type="fig"}\]. ![Case 5 (Group A). (a) Preoperative three-dimensional computed tomographic angiography (3D-CTA) revealed severe stenosis of the right internal carotid artery (ICA) at the bifurcation of the common carotid artery. (b) Preoperative magnetic resonance perfusion image with arterial spin labeling (ASL) showed decreased signals in the right middle cerebral artery (MCA) territory (white dotted arrows). (c) Single-photon emission computed tomography with N-isopropyl-\[123I\] b-iodoamphetamine at rest demonstrated mild reduction of cerebral blood flow in the right MCA territory (white dotted arrows). (d) With acetazolamide loading, impairment of cerebrovascular reserve in the right anterior cerebral artery (ACA) and MCA territories was noted (white dotted arrows). (e) On POD1, diffusion-weighted imaging failed to reveal any *de novo* ischemic lesions. (f) ASL on POD1 clearly showed increased signals in the bilateral ACA and MCA territories, especially on the right side (white arrows). (g) Electroencephalography on POD1 showed slow-wave activities in the bilateral frontal regions (Fp1, Fp2, F3, and F4 of International EEG 10-20 system, black lines) with poorly organized background activities. Asterisks indicate motion artefact due to restless confusion. (h) ASL on POD14 showed disappearance of the increased signals. The preoperative decreased ASL signals in the right MCA territory were also improved. (i) Postoperative 3D-CTA confirmed that the ICA stenosis was improved](SNI-7-1031-g003){#F2} On POD1 after CEA, the patient exhibited mildly restless confusion and a talkative state. Although no *de novo* ischemic events were observed on diffusion-weighted imaging \[[Figure 2e](#F2){ref-type="fig"}\], ASL clearly showed increased ASL signals on both sides, especially the right \[[Figure 2f](#F2){ref-type="fig"}\]. EEG on POD1 showed slow-wave activities in the bilateral frontal regions (Fp1, Fp2, F3, and F4 of International EEG 10-20 system) with poorly organized background activities \[[Figure 2g](#F2){ref-type="fig"}\], indicating diffuse dysfunction, especially concentrated in the bilateral frontal regions. Under a diagnosis of CHS, further intensive control of systolic blood pressure within 100 to 120 mm Hg was performed with intravenous nicardipine, and a free radical scavenger was administered. On POD3, his restlessness was fully resolved. ASL on POD14 showed disappearance of the ASL signal increase \[[Figure 2h](#F2){ref-type="fig"}\]. The preoperatively decreased ASL signals in the right MCA territory were also improved. Furthermore, 3D-CTA confirmed that the ICA stenosis had improved \[[Figure 2i](#F2){ref-type="fig"}\]. EEG on POD14 showed disappearance of slow waves in the bifrontal regions. The patient was discharged without neurologic deficits on POD16. His mRS score was 0 (unchanged from the preoperative state). ### Case 23 (Group B) {#sec3-4} A 46-year-old man presented with transient weakness of his right limbs. He had a history of smoking, hypertension, hyperlipidemia, diabetes mellitus, atrial fibrillation, and chronic heart failure. He had no abnormal neurological findings, although he had an old infarction in the white matter of the left frontal lobe. 3D-CTA revealed severe stenosis of the left ICA \[[Figure 3a](#F3){ref-type="fig"}\]. Preoperative ASL showed decreased signals in the left MCA territory \[[Figure 3b](#F3){ref-type="fig"}\]. A preoperative ECD-SPECT image obtained at rest demonstrated mild reduction of CBF in the left MCA territory \[[Figure 3c](#F3){ref-type="fig"}\]. ACZ challenge depicted CVR impairment in the left MCA territory \[[Figure 3d](#F3){ref-type="fig"}\]. {#F3} On POD1, the patient was found to have motor aphasia. Diffusion-weighted imaging failed to reveal *de novo* ischemic events, although T2 shine-through of the old infarction in the white matter of the left frontal lobe was observed \[[Figure 3e](#F3){ref-type="fig"}\]. ASL demonstrated increased ASL signals in the operated left hemisphere \[[Figure 3f](#F3){ref-type="fig"}\]. A perfusion defect of the old infarction lesion in the white matter of the left frontal lobe became prominent with increased ASL signals in its surroundings. EEG on POD1 showed atypical negative-positive-negative triphasic waves in the left frontotemporal region (Fp1, F3, and F7) on diffuse slow-wave activities \[[Figure 3g](#F3){ref-type="fig"}\]. The triphasic wave in the left frontotemporal region exhibited "sharp-wave and slow-wave" complex morphologies. The patient was diagnosed with nonconvulsive status epilepticus associated with CHS based on our previous reports.\[[@ref8][@ref12][@ref23][@ref24][@ref36][@ref37]\] Fosphenytoin (750 mg) was administered. Intensive systolic blood pressure control at 100--120 mm Hg with intravenous nicardipine and the use of a free radical scavenger relieved the patient\'s symptoms. On POD2, his aphasia had fully resolved. ECD-SPECT on POD2 still demonstrated hyperperfusion in the left MCA territory \[[Figure 3h](#F3){ref-type="fig"}\]. On POD8, the increased ASL signals in the left hemisphere were improved \[[Figure 3i](#F3){ref-type="fig"}\], and EEG showed disappearance of the atypical triphasic waves with physiologically normal background activities without subsequent oral administration of any anticonvulsants. The patient was discharged with no neurologic deficits on POD20. His mRS score was 1 (unchanged from the preoperative state). DISCUSSION {#sec1-4} ========== Preoperative ASL signals with a single PLD of 1.5 s mostly coincided with the CBF map on resting SPECT, supporting the findings of previous reports.\[[@ref27][@ref30][@ref32][@ref35][@ref36]\] However, ASL had a tendency to underestimate the CBF probably because of the prolonged ATT in patients with carotid stenosis.\[[@ref11]\] The incidence of post-CEA hyperperfusion detected with SPECT reportedly ranges from 0.5 to 40.0%.\[[@ref13][@ref16][@ref19][@ref28][@ref29][@ref36][@ref37]\] These contradictory results are probably attributed to different timing of post-CEA CBF measurements and the various quantitative criterion of hyperperfusion.\[[@ref25][@ref37]\] Ogasawara *et al*.\[[@ref28]\] strictly defined post-CEA hyperperfusion as a ≥100% increase in CBF on IMP-SPECT compared with preoperative values and observed ipsilateral hyperperfusion immediately after CEA in 8 (15.6%) out of 51 patients. In the present study, we measured ASL signals immediately after extubation on POD1 because general anesthesia using propofol was continued until the morning of POD1. Twenty-two (68.8%) and 4 (12.5%) of the 32 patients exhibited increased ASL signals bilaterally and on the operated side, respectively. Follow-up ASL showed improvement in these findings. The higher incidence of transiently increased ASL signals in our study is apparently attributed to the shortening of the ATT not only to the increased CBF. Immediately after CEA, improvement in anterograde ICA perfusion on the operated side and no need for slow streaming collaterals resulted in shortening of the ATT not only in the ipsilateral hemisphere but also in the contralateral hemisphere. Previous authors have clearly demonstrated that patients with poor preoperative CVR have a potentially increased risk of post-CEA hyperperfusion.\[[@ref17][@ref28]\] This is because the cerebral vessels are expanded and the cerebral blood volume is elevated in conditions of poor cerebrovascular reactivity, in which the cerebral vessels cannot constrict if the perfusion pressure is normalized by revascularization. Cerebral perfusion will rise as a result.\[[@ref17][@ref28]\] In the present study, the absence of a correlation between preoperative CVR impairment and cerebral hyperperfusion (Groups A and B) may support the idea that ASL with a single PLD does not accurately reflect the CBF. To overcome these shortcomings of ASL with a single PLD and to achieve quantitative measurements of CBF and ATT, a multiple-PLD ASL approach has been used.\[[@ref39]\] Wang *et al*.\[[@ref39]\] presented a multi-PLD ASL protocol using four PLDs of 1.5, 2.0, 2.5, and 3.0 s to achieve simultaneous measurements of CBF and ATT in a total scan time of 11.2 min in patients with moyamoya disease. In addition to the longer scan time, calculation of CBF and ATT requires special software and a workstation; it is not practical for clinical use. Uchihashi *et al*.\[[@ref36]\] demonstrated the usefulness of another ASL quantification approach named quantitative star labeling of arterial region (QUASAR) in the assessment of cerebral hyperperfusion.\[[@ref18][@ref36]\] However, it also requires offline data processing with special software and a workstation. In contrast, the acquisition time of our single-PLD method was as short as 2 min 22 s, and a special calculation was not required. Although visual inspection was subjective, no assessment difference was noted with independent interpretations by two radiologists in the present study. Thus, post-CEA hemodynamic changes, including an increased CBF and shortening of the ATT, could be promptly estimated immediately after routine MR examination. In the present study, 3 patients (Case 5 and Case 18 in Group A, Case 23 in Group B) developed CHS. In Case 5 and 18, on POD1, increased ASL signals were noted on both the sides, and EEG showed slow-wave activities in the bilateral frontal regions with poorly organized background activities, indicating diffuse dysfunction, especially in the bilateral frontal regions. Disinhibition of the bilateral frontal lobes caused by cerebral hyperperfusion explained the patient\'s restlessness. In our previous report,\[[@ref24]\] EEG findings demonstrated that the pathophysiological mechanism of CHS following superficial temporal artery--MCA anastomosis is also cortical dysfunction. In Case 23, atypical triphasic waves were observed in the frontal region of the ipsilateral side. Atypical triphasic waves are now generally accepted as one of the EEG features in patients with nonconvulsive status epilepticus, although there is still argument against true ictal discharges.\[[@ref24]\] A number of reports of patients with CHS have not examined epileptic ictal hyperperfusion\[[@ref12][@ref21]\] despite epilepsy being one of the most common clinical manifestations of CHS.\[[@ref12][@ref23][@ref24]\] As described in our previous reports,\[[@ref12][@ref24]\] ictal hyperperfusion could be included as a pathophysiological condition of CHS. In these three cases, coincidence in the localization of the increased ASL signals and EEG abnormalities was noted. We speculate that the pathophysiological mechanism of CHS is not straightforward and that the marked increase in the CBF on the ipsilateral side was not the sole factor involved in the development of CHS. Increased flow velocity (i.e. shortening of the ATT) could be an additional factor, as measured with transcranial Doppler by previous authors.\[[@ref9]\] Because a limitation of our study is the small number of patients (*n* = 32), further studies in a large cohort should be conducted with a more sophisticated ASL method. We recently developed dual PLD method, instead of single PLD method that we used in this study.\[[@ref1][@ref11]\] To assess the slowly streaming collateral pathway in chronic steno-occlusive disease, we selected another PLD of 2.5 s, in addition to the routinely used PLD of 1.5 s. In our previous reports,\[[@ref1][@ref11]\] we validated the setting of the dual PLDs of 1.5 and 2.5 s by measuring the ATT and identifying the angiographic circulation, including the collateral network, in the target area using digital subtraction angiography. On the contrary, shortening of the ATT is demonstrated immediately after CEA and adequate another PLD setting should be explored. Another promising ASL technique is superselective ASL that enables the measurement of flow territories. This ASL method is able to selectively label various sized vessels from the major arteries down to smaller intracranial vessels supplying brain territories of various sizes.\[[@ref14][@ref15]\] This regional perfusion image can track alterations in perfusion territories upon CEA.\[[@ref15]\] CONCLUSION {#sec1-5} ========== In conclusion, owing to the effect of the ATT, ASL with a single PLD does not accurately reflect the CBF value and cannot identify patients at risk of CHS. However, ASL is completely noninvasive and can be performed as a part of routine MR imaging examination. Furthermore, ASL can promptly evaluate post-CEA hemodynamic changes, including both an increased CBF and shortening of the ATT. Although a more sophisticated ASL method should be explored, ASL with a single PLD may be used as screening test for cerebral hyperperfusion at present. Financial support and sponsorship {#sec2-11} --------------------------------- Nil. Conflicts of interest {#sec2-12} --------------------- There are no conflicts of interest. The authors thank Dr. Hiroyuki Nomiyama and Dr. Seitaro Shin for interpreting the SPECT and ASL images. <http://surgicalneurologyint.com/Signal-changes-on-magnetic-resonance-perfusion-images-with-arterial-spin-labeling-after-carotid-endarterectomy/>

1. Introduction {#sec1-ijms-18-02110} =============== Sea cucumber (*Colochirus robustus*), which belongs to *Echinodermata*, *Holothuroidea*, *Aspidochirotida*, has long been used as a tasty, nutritious food as well as a medicinal remedy in some Asian countries (China, Korea and Japan) because of their various health benefits \[[@B1-ijms-18-02110],[@B2-ijms-18-02110]\]. Sea cucumber has been known to improve wound healing and reduce arthritis pain, thus it is widely used in folk medicine for many centuries in China \[[@B3-ijms-18-02110],[@B4-ijms-18-02110]\]. Previous studies have demonstrated that sea cucumber has several bioactivities, such as anti-microbial, anti-oxidant, anti-cancer, anti-angiogenic, anti-hypertension anti-coagulant, and anti-inflammatory functions \[[@B5-ijms-18-02110],[@B6-ijms-18-02110]\]. These proposed therapeutic properties and potential health benefits of sea cucumbers can be attributed to the presence of bioactive compounds including vitamins, minerals, cerebrocides, peptides, and lectins, as well as some unique molecules such as chondroitin sulfates, polysaccharides, sterols, cerebrosides, and saponins \[[@B7-ijms-18-02110]\]. Thus far, however, little attention has focused on the bioactivities of the constituent compounds in sea cucumber, especially for the peptides. Peptides originated from food proteins can be developed into nutraceuticals which are natural and safe alternatives to synthetic drugs \[[@B8-ijms-18-02110]\]. Peptides, containing 3--20 amino acids in length, form protein primary structure with molecular weight distribution at 100--2000 Da \[[@B9-ijms-18-02110]\]. Multiple biological properties of peptides have been reported, which include anti-microbial and anti-oxidant activities, and as angiotensin-converting enzyme (ACE) inhibitors \[[@B10-ijms-18-02110]\]. Peptides extracted from scorpion venom are effective in recovering immuno-surveillance and intervening immune escape of lung cancer through multi-pathway \[[@B11-ijms-18-02110]\]. Moreover, it has been reported that the peptides from *Pleurotus eryngii mycelium* may be a potential functional food with immunomodulation activity \[[@B12-ijms-18-02110]\]. Recently, Song et al. \[[@B13-ijms-18-02110]\] demonstrated that SCP exerted anti-inflammatory function through inhibiting NF-κB and MAPK activation and inducing HO-1 expression in macrophages. While these results suggest that SCP may modulate innate immune cell functions, it is still elusive as for whether SCP can impact functions of specific immune responses, the more efficient arm of immune system. A host's specific immune responses to pathogens include both cellular and humoral immunity. The humoral immune response is induced by B cells and cell-mediated immune defense by T cells \[[@B14-ijms-18-02110],[@B15-ijms-18-02110]\]. It is generally known that plant lectin Concanavalin A (Con A) or T cell receptor (TCR) antibodies anti-CD3/CD28 stimulate T cell proliferation, whereas bacterial endotoxin lipopolysaccharide (LPS) stimulates B cell proliferation. Although many cell types participate in immunoregulation, Th lymphocytes play a critical role in regulating immune responses. Th cells can be further classified into several subsets, including Th1 and Th2, according to differences in their corresponding cytokine expression profiles \[[@B16-ijms-18-02110]\]. Upon TCR stimulation, the ζ-chain interacts with the Src-family tyrosine kinases Lck and Fyn, becomes phosphorylated on its immunoreceptor tyrosine-based activation motifs (ITAM), and recruits the Syk-family protein tyrosine kinase (PTK) ZAP-70 \[[@B17-ijms-18-02110]\]. The most important member of the CD3 family is CD3ζ, to which ZAP-70 binds. CD3ζ and ZAP-70 can facilitate the lymphocyte to proliferate and secrete cytokines. Given all this, in this study, after we defined the effect of SCP from *Colochirus robustus* on T cell effector functions in the in vivo model, we further investigated effect of SCP on T cell activation with a focus on signaling molecules CD3ζ and ZAP-70 in the in vitro model to help understand the working mechanism of SCP. 2. Results {#sec2-ijms-18-02110} ========== 2.1. Effect of Sea Cucumber Polypeptides (SCP) on the Body Weight {#sec2dot1-ijms-18-02110} ----------------------------------------------------------------- Oral gavage of SCP was well tolerated by mice and no abnormal behavior and side effects were observed throughout the experiment. During the first two weeks of the study, animals in all groups had a slight weight loss of 0.2--0.3 g, probably due to the intragastric excitability ([Figure 1](#ijms-18-02110-f001){ref-type="fig"}). After that, body weight steadily increased throughout the study with a comparable rate across all groups so that no treatment-related difference was observed. 2.2. Molecular Weight Distribution and Amino Acid Composition {#sec2dot2-ijms-18-02110} ------------------------------------------------------------- As shown in [Table 1](#ijms-18-02110-t001){ref-type="table"}, molecular weight distribution of SCP ranged from 100 to 2000 Da (94%) ([Table 1](#ijms-18-02110-t001){ref-type="table"}). Analysis of amino acid composition of SCP indicated that glycine was the most abundant amino acid present in SCP (18.54 g per 100 g protein), followed by glutamic acid, alanine, arginine, and aspartic acid, accounting for 11.23, 9.75, 7.55 and 6.92 g per 100 g protein, respectively ([Table 2](#ijms-18-02110-t002){ref-type="table"}). 2.3. Effect of SCP on Cellular Composition of Spleen {#sec2dot3-ijms-18-02110} ---------------------------------------------------- Compared to the control, mice treated with 75 mg/kg SCP showed a significant increase in percent CD4^+^ and CD8^+^ cells, and mice treated with SCP 50 or 75 mg/kg also showed a higher percent B cells (CD45R^+^) and NK cells (NK 1.1^+^) ([Table 3](#ijms-18-02110-t003){ref-type="table"}). There was no significant difference in percent regulatory T cells (CD4^+^/CD25^+^) between mice treated with SCP and the control. 2.4. Effect of SCP on Lymphocyte Proliferation and Serum Albumin (ALB) Levels {#sec2dot4-ijms-18-02110} ----------------------------------------------------------------------------- Anti-CD3/CD28-induced lymphocyte proliferation was significantly enhanced in mice treated with SCP at all doses (25, 50, 75 mg/kg) with highest increase seen in those receiving 50 mg/kg SCP (*p* \< 0.05) ([Figure 2](#ijms-18-02110-f002){ref-type="fig"}A). A very similar pattern of enhancement related to SCP treatment was also observed in the proliferative response of lymphocytes induced by T cell mitogen Con A (*p* \< 0.05) ([Figure 2](#ijms-18-02110-f002){ref-type="fig"}B). We also found that SCP treatment enhanced B cell proliferation elicited by B cell mitogen LPS in a dose-dependent pattern up to 50 mg/kg, after which the increase leveled off (*p* \< 0.05) ([Figure 2](#ijms-18-02110-f002){ref-type="fig"}C). Serum ALB concentrations were higher in a dose-dependent manner in SCP-treated groups compared to the control group ([Figure 3](#ijms-18-02110-f003){ref-type="fig"}). 2.5. Effect of SCP on Natural Killer (NK) Cell Activity {#sec2dot5-ijms-18-02110} ------------------------------------------------------- The cytotoxic activity of NK cells takes part in tumor cell elimination. The cytotoxic activity of splenocytes against NK cell-sensitive K562 cells was measured using an Accuri C6 flow cytometer. The cytotoxic activity of SCP-treated mice was significantly higher than that of phosphate buffer solution (PBS)-treated mice in a dose-dependent manner (*p* \< 0.05) ([Figure 4](#ijms-18-02110-f004){ref-type="fig"}). 2.6. Effect of SCP on Cytokine Production {#sec2dot6-ijms-18-02110} ----------------------------------------- Overall, SCP-treated mice had higher CD3/CD28-stimulated production of IL-2, IL-4, IL-6 and TNF-α than those in the control group ([Table 4](#ijms-18-02110-t004){ref-type="table"}), while IFN-γ was higher only in high dose of SCP treatment (75 mg/kg BW). In addition, higher cytokine production was found in Con A-stimulated cultures from SCP-treated mice compared to the control mice, and it appeared that the effect of SCP was more pronounced in 50 mg/kg BW than in lower (25 mg/kg BW) or higher (75 mg/kg BW) group ([Table 5](#ijms-18-02110-t005){ref-type="table"}). LPS-stimulated production of IL-1β, IL-6 and TNF-α was higher in SCP groups with similar dose-related pattern as seen in Con A-stimulated cultures ([Table 6](#ijms-18-02110-t006){ref-type="table"}). 2.7. Effect of Hydrolytic Amino Acids (HAA) on Lymphocyte Proliferation {#sec2dot7-ijms-18-02110} ----------------------------------------------------------------------- To verify that SCP-derived HAA (consisted of 16 types of amino acids) contribute to the immuno-enhancing effect of SCP on lymphocyte proliferation, we stimulated splenocytes with anti-CD3/CD28 in the presence of HAA at 0, 0.25, 0.5 and 1 mg/mL. The results showed that HAA significantly enhanced lymphocyte proliferation ([Figure 5](#ijms-18-02110-f005){ref-type="fig"}). 2.8. Effect of HAA on Cytokine Production {#sec2dot8-ijms-18-02110} ----------------------------------------- For the same reason, we also determined effect of in vitro HAA supplementation on cytokine production in comparison with SCP. Similarly, we found that HAA enhanced IL-2 ([Figure 6](#ijms-18-02110-f006){ref-type="fig"}A), IL-10 ([Figure 6](#ijms-18-02110-f006){ref-type="fig"}B) and IFN-γ ([Figure 6](#ijms-18-02110-f006){ref-type="fig"}C) production in splenocytes stimulated with anti-CD3/CD28. 2.9. Effect of HAA on CD3ζ and ζ-Chain-Associated Protein Kinase 70 (ZAP-70) Expressions {#sec2dot9-ijms-18-02110} ---------------------------------------------------------------------------------------- CD3ζ and ZAP-70 expressions in T cells are essential steps and thus are used as relevant indicators for T cell activation. To determine whether HAA-induced enhancement in T cell proliferation and cytokine production are related to early activation events in T cells, we tested expression of CD3ζ ([Figure 7](#ijms-18-02110-f007){ref-type="fig"}A,C) and ZAP-70 ([Figure 7](#ijms-18-02110-f007){ref-type="fig"}B,D) in splenocytes stimulated by anti-CD3/CD28 in the presence of HAA. The results indicated that HAA significantly upregulated CD3ζ and ZAP-70 expression. 3. Discussion {#sec3-ijms-18-02110} ============= Previous studies demonstrated that SCP, a 100--2000 Da biological compound, has a wide spectrum of biological effects, including ACE-inhibitory \[[@B8-ijms-18-02110]\], anti-hypertensive \[[@B18-ijms-18-02110],[@B19-ijms-18-02110]\], and antioxidant activities \[[@B20-ijms-18-02110]\]. While very limited information has suggested that SCP may possess bioactivity in modulating immune function \[[@B21-ijms-18-02110]\], there is a lack of comprehensive verification about this and, in particular, the working mechanism for the proposed immuno-stimulatory properties of SCP has not been well elucidated. In this study, we demonstrated that oral administration of SCP increased serum albumin concentrations, lymphocyte proliferation, NK cell activity, and cytokine production, which may be associated with upregulated signaling of CD3ζ and ZAP-70 as indicated in the in vitro mechanistic experiments. These results suggest that SCP may have a potential of serving as a nutraceutical to improve immune system functions. Lymphocyte proliferation is one of the effective immune responses of T- and B-lymphocytes upon stimulation (such as infection). It has been shown that acidic or neutral peptide fractions stimulated lymphocyte proliferation \[[@B22-ijms-18-02110]\]. In the in vivo study, we robustly stimulated T lymphocyte proliferation with anti-CD3/CD28 ([Figure 2](#ijms-18-02110-f002){ref-type="fig"}A) and Con A ([Figure 2](#ijms-18-02110-f002){ref-type="fig"}B), and B lymphocyte proliferation with LPS ([Figure 2](#ijms-18-02110-f002){ref-type="fig"}C). T-cell-mediated immune response is indispensable for intracellular, in particular the Th cells-derived cytokines that are thought to play a key role in immune function \[[@B23-ijms-18-02110]\]. Th1 type cells are responsible for cell-mediated immune response, while Th2 type cells promote humoral response \[[@B24-ijms-18-02110]\]. The functions of these subsets of Th cells are defined by the cytokines they predominantly produce, for example, IL-2, IFN-γ and TNF-α by Th1 type cells in contrast to IL-4, IL-6 and IL-10 by Th2 type cells \[[@B25-ijms-18-02110]\]. In this study, we found that oral SCP administration increased production of both Th1 and Th2 cytokines. Together, these results suggest that SCP may potentially promote both cellular and humoral immune functions by increasing T cell expansion and secretion of Th1 and Th2 cytokines. Cytotoxic activity of immune cells is import defense against infectious diseases and cancer \[[@B26-ijms-18-02110]\]. NK cells are a group of specialized cytotoxic lymphocyte characterized by their ability to spontaneously kill tumor cells and virus-infected cells \[[@B27-ijms-18-02110],[@B28-ijms-18-02110]\]. This function of NK cells is mediated and regulated by the immunoregulatory cytokines produced by NK cells themselves as well as other cells such as T cells \[[@B29-ijms-18-02110],[@B30-ijms-18-02110]\]. Consistent with the results of by He et al, who reported that sea cucumber oligopeptides improved NK cell activity \[[@B21-ijms-18-02110]\], in the current study, we observed that oral SCP administration significantly increased NK cell activity. Since we also found an increase in the percentage of NK cells in splenocytes used NK activity assay, it is possible that increased NK activity is largely attributed to increased number of NK cells after SCP treatment. The positive effect of SCP on NK cells suggests that SCP may enhance this innate immune response to potentially prevent viral infection and strengthen the surveillance for tumor development. ALB is an abundant multifunctional non-glycosylated, negatively charged plasma protein, and its biological functions include ligand-binding and transporting, antioxidant activity, regulating enzymatic activity, and maintaining colloid osmotic pressure and substance metabolism \[[@B31-ijms-18-02110]\]. Health care practitioners have used the ALB level as an index to evaluate nutrition status, specifically protein nutrition status \[[@B32-ijms-18-02110]\]. In this study, we found that oral SCP administration resulted in an elevation in blood ALB concentrations ([Figure 3](#ijms-18-02110-f003){ref-type="fig"}). Deficiency in dietary protein or amino acids is known to impair immune function and increase the susceptibility to infection in both animals and humans. Amino acids are important energy substrates for immune cells, and they are essential for intact functions of immune cells because of their distinct facilitative characteristic \[[@B33-ijms-18-02110]\]. These amino acids include arginine, leucine, isoleucine, valine, glutamine, lysine, threonine, and tryptophan. Increasing evidence have shown that dietary supplementation of specific amino acids to animals and humans with malnutrition and infectious disease can improve their immune status, thereby reducing morbidity and mortality \[[@B34-ijms-18-02110]\]. Arginine supplementation has been reported to enhance T cell response to mitogens \[[@B35-ijms-18-02110]\]. High levels of glutamine, which can result from damaged tissues, modulate lymphocyte proliferation and production of IL-2, IL-10 and IFN-γ in response to stimuli by polarization of the T helper effector response \[[@B36-ijms-18-02110],[@B37-ijms-18-02110]\]. It has been shown that high doses of arginine increase IL-4, IL-10 and TNF-α secretion of T cells, increased concentrations of lysine and leucine promote IL-10 secretion and proliferative activity of T cells, and threonine enhances TNF-α secretion \[[@B38-ijms-18-02110]\]. SCP contains many important amino acids ([Table 1](#ijms-18-02110-t001){ref-type="table"}), which can be released after SCP is hydrolyzed in a simulated gastrointestinal digestion system in the in vitro study. Using the amino acids (HAA) generated from SCP digestion in the in vitro study, we found that HAA improved the lymphocyte proliferation ([Figure 5](#ijms-18-02110-f005){ref-type="fig"}), and production of IL-2 ([Figure 6](#ijms-18-02110-f006){ref-type="fig"}A), IL-6 ([Figure 6](#ijms-18-02110-f006){ref-type="fig"}B) and IFN-γ ([Figure 6](#ijms-18-02110-f006){ref-type="fig"}C). These results of the in vitro studies further support the results of lymphocyte proliferation ([Figure 2](#ijms-18-02110-f002){ref-type="fig"}) and cytokines production ([Table 4](#ijms-18-02110-t004){ref-type="table"}) in the in vivo study. Thus, we speculate that SCP may enhance the immune function of mice by increased intestinal absorption of the amino acids derived from SCP. At present, the underlying mechanism about immunomodulatory effect of SCP remains unclear. Activation of T lymphocytes is induced by binding of MHC-associated peptides with TCR, transduction of CD3-complex, and expression of CD3ζ and ZAP-70 molecules. The T cell receptor ζ chain (CD3ζ) is the principal signal transduction element of the T cell antigen receptor (TCR) \[[@B39-ijms-18-02110]\]. CD8+ T lymphocytes from chagasic donors display reduced proliferative capacity, which might be associated with CD3ζ down-regulation \[[@B40-ijms-18-02110]\]. ZAP-70 is essential for TCR-mediated activation of mature T cells, and it also plays a critical role in T cell maturation. A recent study has demonstrated that deletion of ZAP-70 affects CD2- and CD3-mediated proliferation as well as cytokines production of TNF-α and IFN-γ in T cells \[[@B41-ijms-18-02110]\]. In this study, our results showed that HAA could enhance CD3ζ ([Figure 7](#ijms-18-02110-f007){ref-type="fig"}A) and ZAP-70 ([Figure 7](#ijms-18-02110-f007){ref-type="fig"}B) expression in vitro. T cell proliferation is known to depend on the presence of amino acids in culture and TCR complex expression \[[@B42-ijms-18-02110]\]. Conversely, amino acids depletion causes diminished T cell proliferation, cytokine production, and CD3ζ chain expression \[[@B43-ijms-18-02110]\]. T cells cultured in the absence of amino acids exhibit a sustained down-regulation of CD3ζ preventing the normal expression of TCR, a decreased proliferation, and a significantly diminished production of IFN-γ, IL-5 and IL-10 \[[@B44-ijms-18-02110]\]. Taken together, our results suggest that SCP-induced immuno-enhancement may be because that SCP is digested in the intestine to release its constituent amino acids, which are absorbed into the body and induce up-regulation of CD3ζ and ZAP-70 leading to enhanced T cell proliferation and cytokine production. 4. Materials and Methods {#sec4-ijms-18-02110} ======================== 4.1. Reagents {#sec4dot1-ijms-18-02110} ------------- The body wall of sea cucumber (Colochirus robustus) was obtained from Ningbo Bofeng Biological Science and Technology Co., Ltd. (Ningbo, China). RPMI-1640 medium and fetal bovine serum (FBS) were from Hyclone (Logan, UT, USA). Con A, trypan blue, Dimethyl sulfoxide (DMSO), and LPS were from Sigma (St. Louis, MO, USA). Cell Counting Kit-8(CCK-8) was from Dojindo (Kumamoto, Kyushu, Japan). K562 cell line (Human chronic myelocytic leukemia) was from Bioscience-iCell (Shanghai, China). 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE), all primary antibodies, and ELISA kits were purchased from eBioscience (SanDiego, CA, USA). 4.2. Preparation of Sea Cucumber Polypeptides (SCP) {#sec4dot2-ijms-18-02110} --------------------------------------------------- SCP was obtained as previously described with some modifications \[[@B13-ijms-18-02110]\]. The fresh body wall of sea cucumber (Colochirus robustus) was rinsed with deionized water. The body wall of sea cucumber was dried and pulverized in order to obtain the powder. The powder was added to PBS and the Flavourzyme of 1% of the body mass of sea cucumber. Then the solution was hydrolysed for 12 h (50 °C, pH 6.8\~7.2). The solution was boiled at 90 °C for 10 min to stop enzyme reaction. Subsequently, the solution was added into a 3-fold volume of ethanol solution for 24 h. The supernatant solution of peptides was obtained by centrifugation at 4500 rpm for 25 min. The peptides solution was purified by G10 gel chromatography. Finally, after freeze-dried, SCP was stored at −20 °C until use. 4.3. Analysis of Amino Acid Composition and Molecular Weight Distribution {#sec4dot3-ijms-18-02110} ------------------------------------------------------------------------- Amino acid composition was measured by an automatic amino acid analyzer following the protocol previously described \[[@B45-ijms-18-02110]\]. The molecular weight distribution of SCP was determined using high performance size exclusion chromatography (HPSEC) as previously described \[[@B46-ijms-18-02110]\]. Briefly, the concentrated SCP dispersion was diluted with 30 volumes (*v*/*v*) of 90% Me2SO, and an aliquot of 50 μL was injected into an HPSEC system with Me2SO as the mobile phase. The raw data were collected using Millennium software and then exported to and processed in MS Excel. 4.4. Animal Treatment {#sec4dot4-ijms-18-02110} --------------------- Six-week-old male C57BL/6 mice weighed 18--22 g were purchased from the Laboratory Animal Centre at the West China Center of Medical Sciences, Sichuan University (Chengdu, China). After 2-week of acclimation, mice were randomly divided into four groups (10/group) to receive daily gavage of PBS (control), SCP in PBS at 0.25, 0.50 or 0.75 g/kg body weight for 8 week. Mice were individually housed in wire-bottomed cages with free access to drinking water and the AIN-93 diet. Environmentally controlled animal rooms provided a constant temperature at 24 °C, relative humidity at 60--70%, and a 12-h-light/-dark cycle (7:00 am/7:00 pm). All procedures of handling the animals were conformity with the National Institutes of Health (NIH) guidelines (Pub. No. 85-23, revised on 1 September 1996) and was approved by Animal Care and Use Committee of the Sichuan Agricultural University. 4.5. Hydrolytic Amino Acids (HAA) Preparation {#sec4dot5-ijms-18-02110} --------------------------------------------- SCP (100 mg) was added to a hydrolysis tube and then sealed with 50 mL of 6 mol/L HCl solutions. After the hydrolysis tube was incubated at 110 °C in an incubator for 24 h, the solution was concentrated by rotary evaporator to remove HCl solutions. The resulting dried free amino acids were collected with constant volume PBS as HAA to be used for the in vitro study. 4.6. Analysis of Splenocyte Phenotype {#sec4dot6-ijms-18-02110} ------------------------------------- After mice were sacrificed by CO2 asphyxiation, spleens were aseptically removed and placed in sterile plates containing RPMI 1640. Single cell suspensions were isolated by gently disrupting spleens, and passed through a 200-mesh stainless steel sieve. After red blood cells were removed using red blood cell lysis buffer (8.29 g/L NH4Cl, 1 g/L KHCO~3~ and 37.2 mg/L Na2EDTA), splenocytes were washed twice and then suspended in 1 mL complete RPMI-1640 medium containing 10% (*v*/*v*) FBS, 100 kU/L penicillin and 100 mg/L streptomycin. Cell viability was assessed by the trypan blue exclusion method. To determine the cellular composition of spleen, 1 × 10^6^ splenocytes were blocked with 0.5 µL Anti-CD16/32 (Fcγblock) (0.5 mg/mL) for 30 min at 4 °C, followed by 3 times of wash with PBS. Splenocytes were then stained in 3 sets of combinations: FITC-conjugated anti-mouse CD3, PE-conjugated anti-mouse CD4 and APC-conjugated anti-mouse CD8 to identify total T cells, CD4+ and CD8+ T cells; APC-conjugated anti-mouse CD45R, FITC-conjugated anti-mouse CD3 and PE-conjugated anti-mouse NK1.1 were used to identify B cells and natural killer cells; FITC-conjugated anti-mouse-CD4 and PE-conjugated anti-mouse-CD25 were used to identify regulatory T cells. Stained cells were analyzed by an Accuri C6 flow cytometer (BD Accuri Cytometers, Ann Arbor, NJ, USA) and acquired data were analyzed using CFlow software (BD Accuri Cytometers, Ann Arbor, NJ, USA). 4.7. Lymphocyte Proliferation Assay {#sec4dot7-ijms-18-02110} ----------------------------------- The lymphocyte proliferation was performed as previously described \[[@B47-ijms-18-02110]\]. Briefly, splenocytes (1 × 10^5^ cells/well) in 96-well flat-bottom plates (Costar^®^ Assay Plate, Corning Incorporated, Corning, New York, NY, USA) were cultured with or without the presence of T cell mitogen Con A at 1.5 mg/L, LPS at 1 mg/L, or plate-coated anti-CD3 (5 mg/L) plus soluble anti-CD28 (1 mg/L) (CD3/CD28). Plates were incubated for 72 h at 37 °C and 5% CO2. During the last 4 h, 10 μL/well of CCK-8 solution was added to plates. The absorbance was measured at 450 nm using a Synergy HT plate reader (BIO-TEK, Winooski, VT, USA). For the in vitro lymphocyte proliferation assay, splenocytes isolated from C57BL/6 mice were incubated with HAA at concentrations of 0, 0.25, 0.5 and 1 mg/mL for 4 h before stimulated by anti-CD3 (5 mg/L)/anti-CD28 (1 mg/L) for 72 h. Cell proliferation was measured as OD 570 nm using a plate reader. 4.8. Serum Albumin (ALB) Concentration Assay {#sec4dot8-ijms-18-02110} -------------------------------------------- At the end of the 8-week oral SCP administration, blood samples were collected into EDTA Eppendorf tubes by retro-orbital venous plexus puncture from mice under anesthesia. Blood samples were centrifuged at 2000 rpm for 5 min at 4 °C and obtained serum samples were stored at −80 °C. ALB concentrations were measured using sandwich ELISA kits following the manufacturer's instructions. The absorbance of the solutions was measured at 450 nm using a plate reader. 4.9. Measurement of Cytokines {#sec4dot9-ijms-18-02110} ----------------------------- Splenocytes (1 × 10^6^ cells/well) in 24 well plates were cultured in the presence of Con A (1.5 mg/L) or anti-CD3 (5 mg/L)/anti-CD28 (1 mg/L) for 72 h at 37 °C in 5% CO2 for cytokines IL-2, IL-4, IL-6, IL-10, IFN-γ and TNF-α production, or in the presence of LPS (1 mg/L) for 72 h for IL-1β, IL-6 and TNF-α. Cell-free supernatants were collected at the end of incubation and stored at −20 °C for later analysis. Cytokine production was measured using sandwich ELISA kits following the manufacturer's instructions. For the in vitro cytokine production assay, splenocytes were incubated with HAA at 0, 0.25, 0.5 or 1 mg/mL for 4 h before being stimulated by CD3 (5 mg/L)/CD28 (1 mg/L) for 48 h for examining IL-2, IL-10 and IFN-γ production. 4.10. Natural Killer (NK) Cell Activity Assay {#sec4dot10-ijms-18-02110} --------------------------------------------- NK cell activity was determined as previously described with some modifications \[[@B14-ijms-18-02110],[@B48-ijms-18-02110]\]. Briefly, K562 cells as target cells were labeled with CFSE (2.5 µM). The splenocytes were used as the effector cells. Splenocytes and K562 cells were mixed at ration 50:1 (effector: target) in 96-well plates. After mixed cells were incubated for 4 h at 37 °C and 5% CO2, 0.25 µL PI solution (1 mg/mL) was added into each well and incubation continued for additional 10 min. CFSE-stained cells and PI-stained cells were determined by an Accuri C6 flow cytometer. NK cells activity was calculated using the following formula: NK cells activity (%) = \[dead K562 Cells (%) − spontaneously dead K562 Cells (%)\] × 100/\[100 − spontaneously dead K562 target cells (%)\]. 4.11. CD3ζ and ZAP-70 Expression {#sec4dot11-ijms-18-02110} -------------------------------- CD3ζ and ZAP-70 expression was measured using a protocol as previously described \[[@B49-ijms-18-02110]\]. In brief, splenocytes (5 × 10^6^ cells/ml) were pre-incubated with RPMI/10% FBS at 4 °C for 10 min and HAA (0, 0.25, 0.5, 1 mg/mL) was added to incubation for 4 h before stimulated by CD3 (5 mg/L)/CD28 (1 mg/L) for 48 h. Cells were stained with PerCP-Cy5.5 conjugated anti-CD3 for 30 min at 4 °C. After washed 3 times, cells were incubated in permeabilization buffer for 10 min. Cells were then re-suspended in PBS, and stained with anti-CD3ζ-FITC or anti-ZAP-70-FITC antibody at 4 °C for 2 h, followed by washing 5 times before analysis. Analysis was performed using CFlow software and expression levels of CD3ζ and ZAP-70 were measured as mean fluorescence intensity (MFI) and percent of positive cells. 4.12. Statistical Analysis {#sec4dot12-ijms-18-02110} -------------------------- All results were expressed as the mean ± standard deviation (S.D.). Statistical analysis was conducted using SPSS software version 23.0 (SPSS Inc., Chicago, IL, USA). One-factor analysis of variance (ANOVA) was used to analyze the data. Values with *p* \< 0.05 were considered statistically significant. 5. Conclusions {#sec5-ijms-18-02110} ============== In summary, in this study, we have shown that oral SCP administration can enhance immune response in mice. This effect of SCP may be associated with increased intake of SCP-derived amino acids which upregulate signaling pathways involving CD3ζ and ZAP-70 activation. These results suggest that SCP has a promising potential as a functional food to improve body's immune function and resistance to infection. Future studies are needed to confirm these findings and, more importantly, to determine its translational value in disease prevention and application in humans. On the other hand, the mechanistic study presented here represents only a preliminary attempt and further expansion to this end is warranted. This work was supported in part by Two Sides Supporting Plan in Sichuan Agriculture University (00770103), Fostering Fund of Academic and Technological Leading Scientist in Sichuan Province (03120235) and Sichuan Education Commission (17ZA0300). Xiaogang Du, Fangliang Lian and Xianyin Zeng conceived and designed the experiments; Xiaogang Du, Fangliang Lian, Yunkun Li, Dong Li, Zhijiang Feng, Yun Li and Qunli Feng performed the experiments; Fangliang Lian, Guixian Bu, Dayong Wu, Fengyan Meng, Xiaohan Cao and Zhiyu Chen analyzed the data; Xiaogang Du and Fangliang Lian wrote the paper; and Xiaogang Du had primary responsibility for final content. All authors read and approved the final manuscript. The authors declare no conflict of interest. {#ijms-18-02110-f001} {#ijms-18-02110-f002} {#ijms-18-02110-f003} {#ijms-18-02110-f004} {#ijms-18-02110-f005} {#ijms-18-02110-f006} {#ijms-18-02110-f007} ijms-18-02110-t001_Table 1 ###### Molecular weight distribution of Sea Cucumber Polypeptides (SCP). Molecular Weight Range \% ------------------------ ------- \<100 1.50 100\~300 28.50 300\~600 30.00 600\~1000 20.00 1000\~2000 15.50 2000\~3000 3.00 \>3000 1.50 ijms-18-02110-t002_Table 2 ###### Amino acid composition of SCP. Amino Acid g/100 g Protein ------------ ----------------- Gly 18.54 Glu 11.23 Ala 9.75 Arg 7.55 Asp 6.92 Pro 5.90 Thr 4.68 Ser 4.50 Leu 4.39 Val 3.83 Lys 3.33 Ile 2.29 Tyr 2.10 Phe 1.80 Met 1.77 His 1.57 ijms-18-02110-t003_Table 3 ###### Effect of oral administration with the SCP on lymphocyte cell phenotype of C57BL/6 mice. Group (mg/kg BW) CD4^+^ T Cells (%) CD8^+^ T Cells (%) B Cells (%) NK (%) Treg (%) ------------------ -------------------- -------------------- ------------------ ----------------- ----------------- 0 29.24 ± 3.32 ^b^ 15.61 ± 2.91 ^b^ 55.12 ± 3.71 ^a^ 2.23 ± 0.46 ^a^ 3.34 ± 0.68 ^a^ 25 28.11 ± 3.12 ^a^ 14.96 ± 2.37 ^a^ 55.52 ± 4.34 ^a^ 2.41 ± 0.62 ^a^ 3.21 ± 0.53 ^a^ 50 29.55 ± 3.73 ^b^ 15.37 ± 2.14 ^ab^ 56.78 ± 5.16 ^b^ 2.96 ± 0.77 ^b^ 3.28 ± 0.76 ^a^ 75 30.47 ± 4.21 ^c^ 16.16 ± 2.74 ^c^ 56.43 ± 4.83 ^b^ 3.37 ± 0.85 ^c^ 3.14 ± 0.52 ^a^ Values are means ± SD, *n* = 10. For each variable, means in a row without a common letter differ by one-factor ANOVA, *p* \< 0.05. NK: natural killer; BW: body weight. ijms-18-02110-t004_Table 4 ###### Effect of oral administration with the SCP on CD3/CD28-induced cytokines production by lymphocyte of C57BL/6 mice. Group (mg/kg BW) IL-2 pg/mL IL-4 pg/mL IL-6 pg/mL IL-10 pg/mL TNFα pg/mL IFNγ pg/mL ------------------ ------------------ ----------------- ---------------- ------------------ ------------------ ------------------ 0 310.4 ± 28.1 ^a^ 62.3 ± 6.1 ^a^ 44.5 ± 3.8 ^a^ 425.3 ± 51.2 ^c^ 215.7 ± 16.5 ^b^ 204.2 ± 17.1 ^c^ 25 412.8 ± 32.7 ^b^ 110.5 ± 8.2 ^b^ 61.5 ± 5.5 ^c^ 386.4 ± 32.5 ^a^ 228.1 ± 18.2 ^c^ 140.6 ± 10.7 ^a^ 50 488.1 ± 25.7 ^c^ 130.1 ± 7.9 ^c^ 65.4 ± 4.7 ^d^ 414.9 ± 49.1 ^b^ 240.1 ± 27.3 ^d^ 157.4 ± 16.3 ^b^ 75 400.2 ± 34.1 ^b^ 100.9 ± 5.6 ^b^ 50.3 ± 4.1 ^b^ 429.5 ± 46.4 ^c^ 203.6 ± 13.2 ^a^ 228.3 ± 25.2 ^d^ Values are means ± SD, *n* = 10. For each variable, means in a row without a common letter differ by one-factor ANOVA, *p* \< 0.05. IFN: Interferon. ijms-18-02110-t005_Table 5 ###### Effect of oral administration with the SCP on Concanavalin A (Con A)-induced cytokines production by lymphocyte of C57BL/6 mice. Group (mg/kg BW) IL-2 pg/mL IL-4 pg/mL IL-6 pg/mL IL-10 pg/mL TNFα pg/mL IFNγ pg/mL ------------------ ------------------ ---------------- ---------------- ------------------ ------------------ ------------------ 0 182.2 ± 25.3 ^b^ 12.1 ± 0.9 ^a^ 26.3 ± 2.1 ^b^ 70.9 ± 5.6 ^a^ 551.5 ± 22.4 ^a^ 58.3 ± 4.3 ^a^ 25 157.6 ± 19.1 ^a^ 16.3 ± 1.8 ^c^ 22.1 ± 1.3 ^a^ 72.4 ± 3.2 ^a^ 575.1 ± 31.6 ^b^ 72.2 ± 5.1 ^b^ 50 207.3 ± 32.4 ^c^ 16.6 ± 1.6 ^c^ 30.2 ± 3.1 ^c^ 125.5 ± 10.8 ^c^ 641.4 ± 56.3 ^d^ 100.2 ± 15.2 ^d^ 75 189.5 ± 23.6 ^b^ 13.3 ± 1.4 ^b^ 31.1 ± 2.5 ^c^ 80.2 ± 7.5 ^b^ 592.3 ± 41.1 ^c^ 84.4 ± 8.6 ^c^ Values are means ± SD, *n* = 10. For each variable, means in a row without a common letter differ by one-factor ANOVA, *p* \< 0.05. ijms-18-02110-t006_Table 6 ###### Effect of oral administration with the SCP on lipopolysaccharide (LPS)-induced cytokines production by lymphocyte of C57BL/6 mice. Group (mg/kg BW) IL-1β pg/mL IL-6 pg/mL TNFα pg/mL ------------------ ------------------ ---------------- ------------------ 0 129.2 ± 10.1 ^a^ 53.2 ± 3.8 ^a^ 140.6 ± 34.3 ^a^ 25 134.5 ± 17.3 ^a^ 61.7 ± 5.8 ^c^ 159.9 ± 51.2 ^c^ 50 157.8 ± 26.2 ^b^ 67.7 ± 8.1 ^d^ 202.3 ± 62.5 ^d^ 75 131.1 ± 12.7 ^a^ 56.2 ± 4.9 ^b^ 149.4 ± 44.3 ^b^ Values are means ± SD, *n* = 10. For each variable, means in a row without a common letter differ by one-factor ANOVA, *p* \< 0.05. [^1]: These authors contributed equally to this work.

Background {#Sec1} ========== Congenital missing teeth, or hypodontia, refer to the lack of development of one or more teeth in the primary dentition, and/or in the permanent dentition \[[@CR1], [@CR2]\]. In the primary dentition, the agenesis has been found to be less frequent being between 0.1 and 2.4% and it is usually followed by permanent tooth missing. The prevalence of agenesis in the permanent dentition, excluding the third molars, ranges between 0.15 and 16.2% \[[@CR3]\] with a higher prevalence in females than males \[[@CR4]--[@CR6]\]. Teeth that erupt in critical terminal areas of the dental lamina (such as the upper lateral incisor, second premolars, third molars) and those located in the embryonic fusion areas are most frequently affected by agenesis, following the so-called end of series \[[@CR7]\]. Two forms of hypodontia have been described: syndromic hypodontia refers to tooth agenesis in subjects who have an underlying recognizable clinical syndrome \[[@CR8]\] while the non-syndromic (or familiar) hypodontia is the most common form and different inheritance modes were found for this condition \[[@CR9], [@CR10]\]. Clinically, hypodontia of the lateral incisors is often observed in association with displacement of maxillary canines (DMC), a condition in which a maxillary canine does not follow its normal eruption path. The prevalence of maxillary canine impaction reaches 1 to 5% \[[@CR11]--[@CR13]\]. Recently, several studies aimed at identifying specific and nonspecific etiological factors or causative genes that can explain the underlying mechanisms involved in the impaction and eruption of maxillary canines \[[@CR14]\]. Two hypotheses related to the etiology of DMC have been proposed. The first one suggests that when the lateral incisor is absent or abnormal, the canine will not find the guidance that would enable it to descend along its normal eruption path \[[@CR15]\]. A 3-dimensional study, conducted by Kim \[[@CR16]\] on 89 CT images of subjects with DMC, indicated that the root and the crown of the maxillary lateral incisor play a key role in the eruption of the maxillary canine. The second hypothesis states that abnormal maxillary canine eruption is genetically determined. Svinhufvud and coworkers in 1988, in a family study of 406 children that received orthodontic treatment, suggest a common genetic origin for DMC and hypodontia \[[@CR17]\]. According to this theory, Peck in 2009 included hypodontia and DMC in the Dental Anomaly Pattern which comprises tooth agenesis, microform teeth, delayed tooth development, palatal displaced maxillary canines, infraocclusion of deciduous molars, and mandibular second premolar distal angulation \[[@CR18]\]. The correlations between PDC and dentoskeletal characteristics in the sagittal plane (molar relationships and sagittal maxillomandibular discrepancy) have been studied in the past \[[@CR19], [@CR20]\]. The literature does not provide information regarding the skeletal relationships in association with PDC but an increased prevalence of III class craniofacial patterns and an occlusal deep bite characteristic has been described in PDC subjects \[[@CR21], [@CR22]\]. However, no consensus has been reached as regards the genetic association of dental anomalies, and the genes responsible for DMC have not yet been reached. For this reasons, the DMC's etiology is still a controversial topic. Pirinen et al. \[[@CR23]\] analyzed the pattern of hypodontia on 106 subjects who had had surgical and orthodontic treatment for DMC. Incisor-premolar hypodontia and peg-shaped incisors were found to be strongly associated with DMC \[[@CR23]\]. Jang et al. \[[@CR24]\], in a radiographic study conducted on 187 patients with DMC, observed a significant association between DMC and maxillary second premolar and lateral incisor agenesis. Considering this background, epidemiological and cross-sectional studies on a large sample might reveal more information about the phenomena. The aims of the present study were to (1) investigate prevalence, characteristics, sex distribution, and significant associations with DMC in a group of growing subjects affected by tooth agenesis; (2) analyze whether the severity (mild, moderate, or severe) of the tooth agenesis has an effect on dental development and presence of DMC; and (3) evaluate whether hypodontia can be a factor involved in maxillary canine impaction when isolated in the maxilla or in the mandible. The null hypotheses for this study were that (1) there is no significant difference about occurrence of canine displacement in subjects with dental agenesis compared with a control group and (2) hypodontia does not have a role in maxillary canine impaction. Methods {#Sec2} ======= An initial sample of digital panoramic radiographs (DPR) of 4706 Caucasian subjects aged between 9 and 12 years, with no genetic syndromes or craniofacial malformations (e.g., cleft lip/palate) and no history of extraction, trauma, or previous orthodontic treatment drawn from the files of the Department of Orthodontics of the University of Rome "Tor Vergata", was examined. All DPRs were collected from January 2006 to July 2015 and presented with a good quality to allow the assessment of crown and root development. The study project was approved by the Ethic Committee at the University of Rome "Tor Vergata", and written consent was obtained from all subjects' parents. From the initial sample of 4706 DPRs, 325 DPRs of subjects (161 females, 164 males) with a mean age of 11.3 ± 1.2 years were selected; the inclusion criteria considered for the study group (SG) were at least one missing tooth (excluding the third molars) and root development of maxillary canines nearly completed (9: root almost completed, 10: root completed) \[[@CR25]\]. The diagnosis of tooth agenesis was based on when no sign of crown calcification and no evidence of loss attributable to orthodontic treatment, caries, periodontal disease, or trauma were found \[[@CR26]\]. The SG was divided into three subgroups according to agenesis severity: mild agenesis with 1--2 absent teeth, moderate agenesis with 3--5 absent teeth, and severe agenesis or oligodontia with 6 or more absent teeth \[[@CR27]\]. The control group (CG) consisted of 325 (1:1 ratio) subjects, selected from the same initial sample of 4706 DPRs. The CG matched the SG as to gender and stage of the dentition. After the selection of both SG and CG, using the method described by Ericson and Kurol \[[@CR28]\] on the panoramic radiograph to assess DMC, the following linear and angular measurements were measured for each subject of the study and control groups:Alpha angle defined as the angle formed by the long axis of the canine and the midline;*d* distance defined as the distance in millimeters from the canine cusp tip to the occlusal plane;Sector: mesiodistal crown position in sector 1--5. As previously described by Naoumova and Kjellberg \[[@CR29]\], DMC was assessed in both groups only when all the conditions for severe DMC were present: an alpha angle ≥ 30°, *d* distance ≥ 15 mm, canine tip cusp in sector 4 or 5. All images were evaluated independently by two different operators (N.V. and G.L.) on a computer monitor in a quiet room with subdued ambient lighting. If there was disagreement between the investigators, consensus was reached after discussion. Statistical analysis {#Sec3} -------------------- The data were analyzed by using SPSS software package (Statistical Package for Social Sciences, version 16.0, SPSS Inc., Chicago, USA). Descriptive statistics were used to describe both sample groups (SG and CG) in terms of age, sex, and prevalence rate of missing teeth. The associations between gender distribution, location in the arch, severity of agenesis, and DMC were analyzed by chi-square test. DPRs in the SG and CG were compared for the prevalence rate of DMC using odds ratio. Stepwise multiple logistic regression using the backwards elimination and the Wald (W) test method was performed to identify the best combination of DMC and dental agenesis. Backward logistic regression is desirable when multiple parameters that are correlated are combined. In backward stepwise logistic regression, all the independent variables are initially entered into the regression equation. The impact of the removal of the single independent variable that reduces the variables by the smallest amount is ascertained. If the decrease is not statistically significant, that variable is permanently eliminated. The process is continued until all remaining variables are significant. This process minimizes the interaction between independent variables and therefore identifies the best combination of variables for predicting the dependent factor or outcome. The level of *P* \< 0.05 was considered statistically significant. Results {#Sec4} ======= The SG aged between 10.7 and 11.8 years has a mean age of 11.3 years and SD of 1.2 years. The sample sizes and gender distribution are presented in Table [1](#Tab1){ref-type="table"}. The male/female ratio of dental agenesis in both groups was about 1:1. In the SG, the largest category was mild agenesis with 284 subjects (87.4%); moderate and severe agenesis were observed in 36 subjects (11.0%) and in 5 subjects (1.5%), respectively. Significant association between the number of missing teeth and prevalence of DMC was not assessed.Table 1Descriptive analysis of the sample*N*°Age (years)SD (years)Mild HModerate HSevere HStudy group (SG) M16411.0± 1.2145202 F16111.6± 1.3139163 TOT32511.3± 1.2284 (87.4%)36 (11.0%)5 (1.5%)Control group (CG) M16411.2± 1.2 F16111.4± 1.3 TOT32511.3± 1.2*M* male, *F* female, *SD* standard deviation, *H* hypodontia The most frequent missing teeth were mandibular second premolars (*n* = 148; 45.5%), maxillary lateral incisors (*n* = 134; 41.2%), and maxillary second premolars (*n* = 60; 18.4%) (Table [3](#Tab3){ref-type="table"}). The overall prevalence of DMC was higher in the SG than in CG, respectively, 13.5% (*n* = 44) and 5.2% (*n* = 17) with an odds ratio value of 1139 and *P* value of 0.0004 (Table [2](#Tab2){ref-type="table"}).Table 2Distribution of displacement of maxillary canine in subjects with tooth agenesis compared with control groupStudy groupControl group*Z* Gauss*P* valueStandard deviationOdds ratioCI 95%DMC13.5%5.2%3.1580.0004^a^0.2871.1391.43--4.15*DMC* displacement of maxillary canines^a^Sig \< 0.05, stepwise multivariate logistic regression Agenesis diagnosed in the two arches registered for 13.8% (*n* = 45). Maxillary hypodontia was observed in 151 subjects and mandibular hypodontia was detected in 129 subjects. The prevalence of DMC in the maxillary hypodontia group (14.8%) was higher than that in the mandibular hypodontia group (9.6%), with a chi-square value of 1.679 and *P* value of 0.192. After the stepwise selection process, during which 11 different models were assessed, only one variable was left in the final model (Mx.I2 agenesis) as shown in Table [3](#Tab3){ref-type="table"}, which lists the regression coefficients, standard error, Wald index, degree of freedom, statistical significance, odds ratio, and confidence interval of variable in the final model. Agenesis of maxillary first premolars (Mx.P1), maxillary second premolars (Mx.P2), maxillary second molars (Mx.M2), mandibular first incisors (Mn.I1), mandibular second incisors (Mn.I2), mandibular first premolars (Mn.P1), mandibular second premolars (Mn.P2), and mandibular second molars (MnM2) was dropped from the model during backward stepwise deletion.Table 3Distribution of missing teeth and statistical association with displacement of maxillary canine*P* (%)*β*SE*W*DF*S*ORCI 95%Variable in the final model Mx.I2 agenesis41.22.1740.30728.89910.000^a^9.2434.070--20.301Variables removed during backward stepwise deletion Mx.P1 agenesis13.8 Mx.P2 agenesis18.4 Mx.M2 agenesis2.4 Mn.I1 agenesis1.5 Mn.I2 agenesis2.4 Mn.P1 agenesis2.4 Mn.P2 agenesis45.5 Mn.M2 agenesis5.2*P* prevalence, *β* regression coefficients, *SE* standard error, *W* Wald index, *DF* degree of freedom, *S* statistical significance, *OR* odds ratio, *CI* confidence interval, *Mx*.*I2* maxillary second incisors, *Mx*.*P1* maxillary first premolars, *Mx*.*P2* maxillary second premolars, *Mx.M2* maxillary second molars, *Mn.I1* mandibular first incisors, *Mn.I2* mandibular second incisors, *Mn.P1* mandibular first premolars, *Mn.P2* mandibular second premolars, *Mn.M2* mandibular second molars^a^Sig \< 0.05, stepwise multivariate logistic regression Analysis showed DMC is significantly associated with the agenesis of maxillary lateral incisors, whereas the mandibular second premolar or other types of agenesis did not show any significant association (Table [3](#Tab3){ref-type="table"}). Discussion {#Sec5} ========== The present retrospective epidemiological study focused on the possible associations between tooth agenesis and DMC in a large sample of Caucasian subjects, when compared with a CG of subjects without agenesis (Additional files [1](#MOESM1){ref-type="media"} and [2](#MOESM2){ref-type="media"}). The chronological age in this type of investigations is a crucial factor as it directly affects the detection of DMC \[[@CR30]\]. Previous studies observed that when the lateral incisor is not yet fully developed, panoramic radiographs more commonly show overlapping of the canine and lateral incisor. Thus, when lateral incisor root development is almost complete and overlapping of canine with lateral incisor is observed, a greater mesial inclination of the canine is present \[[@CR31]\]. Moreover, hypodontia and DMC are often associated with delayed dental development \[[@CR27], [@CR32]\]. For these reasons, in order to minimize false-positive results, it was decided to use root development of maxillary canines nearly completed according to the root development stage method described by Nolla, as inclusion criteria for both SG and CG. To our knowledge, no previous studies analyzed the correlation between tooth agenesis and DMC in a so large group of 325 growing subjects with at least one missing tooth, selected by means of DPR, and compared with a CG derived from the same initial sample. Garib et al. \[[@CR33]\] examined a sample of 126 patients with agenesis to reveal the pattern of associations among DMC and other dental anomalies. Al-Abdallah \[[@CR34]\] used a sample of 106 subjects with maxillary hypodontia and 70 with mandibular hypodontia to evaluate the association between DMC and other dental anomalies in an orthodontic population. The current study found that mild hypodontia was the most common form (87.4%) followed by moderate hypodontia (11.0%), and finally followed by severe hypodontia (1.5%). Mandibular second premolars (45.5%), maxillary lateral incisors (41.2%), and maxillary second premolars (18.4%) resulted as the most frequently missing teeth. These findings are in agreement with a recent systematic review conducted by Khalaf and co-workers \[[@CR4]\] that registered similar prevalence for mild, moderate, and severe hypodontia and a similar distribution for most affected teeth analyzing a total of 93 studies carried out between 1936 and 2012. Several studies reported a prevalence of DMC between 3 and 5%; according to this, the prevalence of DMC in the CG of our study was 5.2%. Moreover, an increasing of DMC in SG (13.5%, *P* value \< 0.05) was observed. A general consensus has been reached in finding that there exists a significant correlation between tooth agenesis and DMC. Sacerdoti and Baccetti \[[@CR12]\] reported an increased prevalence of DMC in a sample of subjects with maxillary lateral incisor agenesis, compared with a control group. Moreover, Camilleri \[[@CR35]\] observed 106 subjects with DMC and stated a strong connection with hypodontia. The most important findings of this study were about the association between tooth agenesis and DMC: it was statistically attributable only at the lack of maxillary lateral incisors. Stepwise multiple logistic regression by the backwards elimination and the Wald test method dropped from the initial model all variables except for agenesis of maxillary lateral incisors whereas the mandibular second premolar or other types of agenesis did not show any significant association. The presence of a substantial relationship between agenesis of maxillary lateral incisors and DMC could be explained by the guidance theory. If the lateral incisor is absent, the canine will not find the guidance that would enable it to descend along its normal eruption path and move down in a more palatal path until it comes close to the periosteum of the medial aspect of the alveolar process \[[@CR36]\]. According to our results, several studies reported that DMC and agenesis of lateral incisors could be a strong predictor of maxillary canine impaction \[[@CR37]--[@CR39]\]. Jang and coworkers \[[@CR24]\] described similar findings of higher correlation with maxillary lateral incisor agenesis and DMC, enrolling 187 cases of DMC and comparing them with a control group. They also report that the correlation between DMC and agenesis of the mandibular second premolar did not reach statistical significance \[[@CR24]\]. In contrast, Peck et al. \[[@CR40]\] found a strong association of DMC with third molar agenesis and second premolar agenesis, whereas upper lateral incisor agenesis was not significantly interrelated. Furthermore, Garib et al. \[[@CR41]\] observed that 21% of the patients with second premolar agenesis had other permanent teeth missing, excluding the third molars, and an increased prevalence of DMC compared with the general population. Discrepancies among studies may be also due to sample size or selection, regional ethnic population, genetic variability, environmental factors, and different diagnostic evaluating methods of DMC. The same-race contemporary population composed both groups of our study allowing a reliable genetic association evaluation. The definition based on alpha angle, *d* distance and displaced sectors \[[@CR28]\], used in our study, is the most widely applied diagnostic criterion for determining canine displacement. Moreover, the large sample of our study was advantageous in assessing a significant difference about a specific trend of a certain type of agenesis. Conclusions {#Sec6} =========== Significant association between the severity of dental agenesis and prevalence of DMC was not assessed. Mild hypodontia was the most common form (87.4%) followed by moderate hypodontia (11.0%), and finally followed by severe hypodontia (1.5%). Mandibular second premolars (45.5%), maxillary lateral incisors (41.2%), and maxillary second premolars (18.4%) resulted as the most frequently missing teeth.Increasing of DMC (respectively, 13.5% in SG and 5.2% in CG, *P* value \< 0.05) was observed in SG group compared with CG.The association between tooth agenesis and DMC was statistically attributable only at the lack of maxillary lateral incisors. Stepwise multiple logistic regressions dropped from the initial model all variables except for agenesis of maxillary lateral incisors whereas the mandibular second premolar or other types of agenesis did not show any significant association.The clinician has to be aware that the absence of maxillary lateral incisors may be considered an early diagnostic marker of the development of DMC. Additional files ================ {#Sec7} Additional file 1:Control Group (subjects without agenesis). (PDF 60 kb) Additional file 2:Study Group (subjects with at least one missing tooth). (PDF 71 kb) CG : Control group DMC : Displacement of maxillary canines DPR : Digital panoramic radiographs Mn.I1 : Mandibular first incisors Mn.I2 : Mandibular second incisors Mn.M2 : Mandibular second molars Mn.P1 : Mandibular first premolars Mn.P2 : Mandibular second premolars Mx.I2 : Maxillary second incisors Mx.M2 : Maxillary second molars Mx.P1 : Maxillary first premolars Mx.P2 : Maxillary second premolars SG : Study group **Electronic supplementary material** The online version of this article (10.1186/s40510-018-0226-0) contains supplementary material, which is available to authorized users. Data used in our research has been provided in Additional files [1](#MOESM1){ref-type="media"} and [2](#MOESM2){ref-type="media"}. GL and NV designed the study; acquired, analyzed, and interpreted the data; and were major contributors in writing the manuscript. RL and PC contributed in drafting the manuscript and interpretation of the data and its critical revision. CC and CD performed the statistical analysis of the data. All authors read and approved the final manuscript. Ethics approval and consent to participate {#FPar1} ========================================== The study project was approved by the Ethic Committee at the University of Tor Vergata (protocol number: 24759). A written consent, signed by the parents of all the subjects involved in this study, was recorded. Consent for publication {#FPar2} ======================= A written consent, signed by the parents of all the subjects involved in this study, was recorded in order to use the anonymous data for scientific publication purpose. Competing interests {#FPar3} =================== The authors declare that they have no competing interests. Publisher's Note {#FPar4} ================ Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

The O-linked β-*N*-acetyl-[d]{.smallcaps}-glucosamine (O-GlcNAc) transferase (OGT) and the hydrolase O-GlcNAcase (OGA) together orchestrate protein O-GlcNAcylation, a dynamic co/posttranslational modification cycling on thousands of nucleocytoplasmic proteins ([@r1], [@r2]). OGT catalyses the covalent attachment of the monosaccharide O-GlcNAc to serine or threonine. This modification has been suggested to affect transcription ([@r3][@r4][@r5]--[@r6]), translation ([@r1]), protein stability ([@r7], [@r8]), and subcellular localization ([@r9], [@r10]). O-GlcNAcylation plays a key role in regulating stress response ([@r11][@r12]--[@r13]), differentiation ([@r14], [@r15]), nutrient sensing ([@r16], [@r17]), and autophagy ([@r18]). Genetic studies have highlighted the importance of OGT and protein O-GlcNAcylation in development. OGT is essential for embryonic stem cell viability ([@r19], [@r20]) and mouse embryonic development ([@r21]). Zebrafish lacking OGT function exhibit shortened body axis and smaller brains ([@r22]). The *Drosophila melanogaster* OGT encoded by the Polycomb group gene *super sex combs* (*sxc*) plays a critical role in establishing *Drosophila* segment identity ([@r23], [@r24]). The O-GlcNAc modification is particularly abundant in the brain ([@r25], [@r26]), where it controls memory formation ([@r27][@r28]--[@r29]), circadian rhythm ([@r30], [@r31]), and appetite ([@r32], [@r33]). Numerous neuron-specific proteins are O-GlcNAcylated, such as the Microtubule-associated proteins tau ([@r34]) and CRMP2 ([@r35]), synaptic vesicle proteins ([@r36]), the transcriptional factor cyclic-AMP response element binding protein (CREB) ([@r28]), and the AMPA receptor GluA2 subunit ([@r27]). Growing evidence suggests that O-GlcNAcylation is essential for normal development and function of the mammalian nervous system ([@r21], [@r33], [@r37][@r38]--[@r39]). Although O-GlcNAcylation has been long implicated in chronic metabolic diseases, such as type II diabetes mellitus, neurodegeneration, and cancer, its role in neurodevelopmental disorders has only recently become apparent. In the past year, a small number of missense mutations within the human OGT gene have been discovered in patients with X-linked intellectual disability (XLID) ([@r40][@r41][@r42]--[@r43]). Intellectual disability (ID) refers to a broad range of developmental disorders characterized by limited intellectual capacity, IQ below 70, and poor adaptive behavior with onset before the age of 18, affecting 1 to 3% of the population worldwide ([@r44]). Genetic factors are the major cause of this developmental condition involving over 700 ID genes ([@r45]). These genes encode for proteins that are required for neuronal development and activity, contributing to neuronal structure and function through several signaling pathways ([@r46]). Mutations in genes located on the X-chromosome account for ∼5 to 10% of all ID causative genes affecting predominantly male individuals ([@r44], [@r47]). To date, XLID-associated mutations in the OGT gene have only been reported for male patients, causing developmental delay and severe cognitive disability. Accompanying clinical phenotypes were dysmorphic features, such as clinodactyly, eye abnormalities, and microcephaly. A common molecular trait of the missense XLID OGT variants is that they appear to retain substantial OGT catalytic activity. This is in agreement with these mutations all being localized to the C-terminal noncatalytic tetratricopeptide repeat (TPR) domain of OGT that is responsible for substrate specificity and scaffolding ([@r40][@r41][@r42]--[@r43]). In cultured cells, global O-GlcNAc levels on proteins were unaltered ([@r41][@r42]--[@r43]), and a decrease in OGA protein expression to compensate for the (presumed) moderate loss of OGT activity was only apparent in patient-derived fibroblast cells ([@r43]). In addition to its glycosyltransferase activity, mammalian OGT is involved in the unusual proteolytic maturation of Host cell factor 1 (HCF1) ([@r48][@r49]--[@r50]), a transcriptional cofactor implicated in cell cycle control ([@r51]), also identified as an ID gene ([@r52][@r53]--[@r54]). HCF1 is O-GlcNAc--modified and subsequently cleaved by OGT using the same active site for both enzymatic activities ([@r49]). Among XLID variant OGT models, a moderate effect on HCF1 processing was only detected for one of the mutations ([@r41], [@r43]). From the currently available OGT XLID mutations, it remains unclear if the patient phenotypes observed are linked to changes in the O-GlcNAc proteome, loss of protein--protein interactions, or misprocessing of HCF1. Here, we characterize an OGT missense mutation in the catalytic domain as observed in female twins with ID and developmental delay. In vitro biochemical analysis of recombinant N567K OGT missense variant protein revealed that this mutation abrogates OGT and HCF1 proteolytic processing activity. Protein crystallography data explain the molecular basis of the observed loss of substrate binding. Introduction of this mutation into *D. melanogaster* by genome editing reveals global effects on the O-GlcNAc proteome. In mouse embryonic stem cells (mESCs) carrying this mutation, loss of OGA protein expression was observed, as well as defective HCF1 processing. The mutation caused defects in neurite outgrowth during neuronal differentiation. Taken together, our data provide evidence that loss of OGT activity could contribute to the XLID phenotype observed in the patients. Results and Discussion {#s1} ====================== Monozygotic Twins with ID and Developmental Delay Possess a De Novo OGT Mutation. {#s2} --------------------------------------------------------------------------------- Twin girls (twin 1 and twin 2) were born at 33-wk gestation by semielective caesarean section for maternal preeclampsia. Conception was unassisted. Twin 1 weighed 1,670 g at birth and required continuous positive airway pressure (CPAP) to aid breathing for 3 wk and supplemental oxygen for 58 d. Twin 2 weighed 2,150 g and needed CPAP for 2 d and supplemental oxygen for 20 d. Both babies were tube-fed initially but bottle feeding was established from 3 to 4 wk of age. The twins showed delay in reaching developmental milestones, especially in areas of speech and language development. By 4 y of age, twin 1 only used 3 to 4 single words; at 10 y she was able to put 2 to 3 words together. Language development of twin 2 was more advanced than her sister; at 4 y she was using short phrases and at 10 y she was speaking in sentences and able to recognize letters of the alphabet. Their movement and physical development were also affected: twin 1 by age 4 y and twin 2 by the age of 2 y were able to walk independently. However, twin 2 had significant gross motor difficulties and clumsiness at the age of 8 y. Both twins were diagnosed with cerebral visual impairment, twin 1 more severe than twin 2. They have attended an additional needs nursery and school. The twins ([Fig. 1 *A* and *B*](#fig01){ref-type="fig"}) appeared to be affected by a similar developmental syndrome, including small nose, high arched palate, and fifth finger clinodactyly. Twin 1 has ataxic gait with knees bent, walking on toes with inverted feet. An MRI brain scan showed prominence of the lateral ventricles and an enlarged cistern magna in twin 1 at age 2 y, suggesting possible hypoplasia of the inferior cerebellar vermis. Although both siblings have kept generally good health, hypotonia was apparent in infancy and they required iron supplementation intermittently. Twin 1 had an adenoidectomy and treatment for gastro-esophageal reflux and constipation. {#fig01} Sanger sequencing was performed as part of the Deciphering Developmental Disorders initiative, which currently lists genomic and clinical data from over 14,000 child patients with severe undiagnosed developmental disorders ([@r55]). This revealed a single pathogenic missense mutation in the OGT gene (X:70779215 T \> A, N567K). The mutation was found absent in control population of 141,456 samples, comprising 125,748 exome sequences and 15,708 whole-genome sequences from unrelated individuals reported in the genome aggregation database, *gnomAD* ([@r56]). The mutation affected both monozygotic twins, whereas both of their parents were noncarriers. A 98:2 skew in X-inactivation was detected in both children using polymorphic markers that are differentially methylated on the active and inactive chromosome ([*SI Appendix*, Fig. S1](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900065116/-/DCSupplemental)). However, being a de novo mutation, which 1 of the 2 chromosomes is predominantly inactivated remains unclear. Taking these data together, we have identified monozygotic twins with ID and developmental delay that possess a de novo OGT mutation. The N567K Mutation Abrogates OGT Activity In Vitro. {#s3} --------------------------------------------------- To understand the potential effects of the OGT N567K (OGT~N567K~) mutation, we next investigated changes in catalytic activity compared with full-length wild-type OGT (OGT~WT~) protein. Steady-state kinetics of OGT glycosyltransferase activity was measured against 2 acceptor peptide substrates, P1 (KKVPVSRA) and P2 (KKVAVSRA), that only varied at the −2 position adjacent to the O-GlcNAcylation site ([@r57]). OGT~N567K~ activity was 12-fold reduced against P1 ([Fig. 2*A*](#fig02){ref-type="fig"} and [Table 1](#t01){ref-type="table"}) and not detectable against P2 ([Fig. 2*A*](#fig02){ref-type="fig"}). Next, OGT activity was measured against an intact protein substrate, TAK-1 binding protein (TAB1), where the reaction was followed with a TAB1 O-GlcNAc site-specific antibody ([@r58]). OGT~N567K~ showed negligible glycosyltransferase activity ([Fig. 2*B*](#fig02){ref-type="fig"}). ![The N567K mutation abrogates OGT activity due to disrupted acceptor binding site. (*A*) Michaelis--Menten kinetics of OGT glycosyltransferase activity against P1 and P2 peptides. (*B*) Immunoblots showing OGT glycosyltransferase activity against TAB1. (*C*) Immunoblots showing OGT glycosyltransferase and proteolytic activities against HCF1-rep1. HCF1-rep1: GST-tagged host cell factor-1 fragment containing the first PRO repeat. W: wild type. E: E1019Q. (*D*) Crystal structures of OGT ternary complexes of OGT~WT~ and OGT~N567K~ active site in complex with UDP-5S-GlcNAc (U5G, turquoise, orange atoms) and acceptor peptide. OGT~WT~ is shown in complex with RB2 (Retinoblastoma-like protein 2 peptide, light gray carbon atoms) \[PDB ID code: 5C1D ([@r57])\]; OGT~N567K~ is shown with P2 peptide (pink carbon atoms) (PDB ID code 6IBO). K567 is shown in red. An unbiased *F*o--*F*c difference map before inclusion of the peptide is shown as a mesh.](pnas.1900065116fig02){#fig02} ###### Michaelis--Menten kinetics of OGT activity against P1 peptide substrate Enzyme *K*~M~ (nM⋅s^−1^) *k*~cat~ (s^−1^) *k*~cat~/*K*~M~ ------------ ------------------- ------------------ ----------------- OGT~WT~ 99 ± 19 119 ± 11 1.2 ± 0.3 OGT~N567K~ 144 ± 63 20 ± 4 0.1 ± 0.2 OGT possesses a second catalytic activity in the form of promoting autocatalytic cleavage and activation of HCF1, which itself is an ID-associated gene ([@r52]). We next investigated the effects of the N567K mutation on OGT-mediated HCF1 processing. OGT proteolytic activity was measured using a GST-fusion construct of a minimal HCF1 fragment (HCF1-rep1) containing one of the PRO repeats that are known to be the targets of OGT-mediated proteolysis ([@r49]). Wild-type OGT not only promotes HCF1 proteolysis but was also able to hyperglycosylate this protein fragment ([Fig. 2*C*](#fig02){ref-type="fig"}). HCF1~E1019Q~, which lacks the key glutamate required for proteolytic processing, was not cleaved by wild-type OGT~.~ Strikingly, even after 8 h of reaction time, OGT~N567K~ was able to neither glycosylate nor cleave HCF1-rep1 ([Fig. 2*C*](#fig02){ref-type="fig"}). Taken together, the data show that the N567K mutation abrogates OGT activity. The N567K Mutation Disrupts the OGT Acceptor Binding Site. {#s4} ---------------------------------------------------------- To understand the molecular basis of the observed loss of OGT catalytic activity, we next initiated structural characterization of OGT~N567K~. Inspection of the wild-type OGT structure reveals that Asn567 maps to a loop in the N-terminal lobe of the catalytic domain that is completely conserved through evolution from *Caenorhabditis elegans* to man ([Fig. 1*C*](#fig01){ref-type="fig"}). The Asn567 side chain borders the −2 subsite as part of the OGT acceptor substrate binding cleft ([Fig. 2*D*](#fig02){ref-type="fig"}). Therefore, we hypothesized that the N567K mutation would affect OGT acceptor substrate binding, without affecting the overall OGT fold. Recombinant OGT~N567K~ was obtained from *Escherichia coli* using construct boundaries previously employed to crystallize OGT~WT~-substrate complexes ([@r57]). OGT~N567K~ was then cocrystallized with a donor analog, UDP-5S-GlcNAc, and 2 OGT acceptor peptide substrates, P1 (KKVPVSRA) and P2 (KKVAVSRA), previously used for activity measurements ([Fig. 2*A*](#fig02){ref-type="fig"}). Only the condition containing P2, which contains an alanine at the −2 subsite, yielded crystals that diffracted to 2.3 Å and allowed structure solution by molecular replacement and refinement ([Table 2](#t02){ref-type="table"}). The OGT~N567K~ structure superposed onto that of OGT~WT~ with an rmsd of 0.4 Å across 675 C~α~ atoms, suggesting the mutation does not induce large conformational changes in the catalytic domain. Initial unbiased difference electron density maps revealed partial density corresponding to the acceptor peptide ([Fig. 2*D*](#fig02){ref-type="fig"}). Surprisingly, this only covered the P2 backbone in the +2 through to −2 subsites, suggesting that the N567K mutation may induce increased flexibility at the N-terminal tail of the acceptor peptide ([Fig. 2*D*](#fig02){ref-type="fig"} and [*SI Appendix*, Fig. S2](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900065116/-/DCSupplemental)). In agreement with this, a comparative B-factor analysis suggested that the acceptor peptide bound to OGT~N567K~ is substantially more flexible than that bound to OGT~WT~ ([*SI Appendix*, Fig. S2](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900065116/-/DCSupplemental)). Close inspection of the OGT~N567K~ acceptor substrate binding cleft revealed that the protrusion caused by the N567K mutation partially occupies the position of the −2 proline frequently observed in OGT acceptor peptides ([@r57], [@r59], [@r60]). In summary, the N567K mutation appears to disrupt the OGT acceptor binding site. ###### X-ray diffraction data processing and refinement statistics of the OGT~N567K~ ternary complex OGT~N567K~ ------------------------- -------------------------- Data collection  Space group *F*222  Cell dimensions   α, β, γ (°) 137.7, 150.6, 200.0   A, B, C (Å) 90.0, 90.0, 90.0  Resolution (Å) 45.31--2.17 (2.17--2.23)  *R*~sym~ or *R*~merge~ 0.07 (0.91)  *I*/σ*I* 11.3 (1.3)  Completeness (%) 100 (99)  Redundancy 5.2 (5.3) Refinement  Resolution (Å) 45.31--2.17 (2.17--2.23)  No. reflections 286,887 (23,302)  *R*~work~/*R*~free~ 0.20/0.25 No. atoms  Protein 5,530  Nucleotide sugar 39  Peptide 35  Water 328 *B*-factors  Protein 53.2  Nucleotide sugar 46.6  Peptide 91.9  Water 51.3 Rmsd  Bond lengths (Å) 0.02  Bond angles (°) 1.8 Numbers in brackets show the highest resolution bin. The *sxc*^*N595K*^ Mutation Leads to a Reduction of Protein O-GlcNAcylation in *D. melanogaster*. {#s5} ------------------------------------------------------------------------------------------------- Lack of functional OGT causes lethality and developmental arrest in most metazoan model organisms, including mice, frogs, zebrafish, and fruit flies ([@r19], [@r22], [@r24], [@r61]). To probe if the N567K mutation directly affects OGT catalytic activity in vivo, we exploited the genetically tractable system *D. melanogaster*, where phenotypes arisen due to null and hypomorphic alleles of *sxc* (the fly *ogt* ortholog) have been described ([@r62], [@r63]). *Drosophila sxc* null mutants (*sxc*^*1*^ and *sxc*^*6*^) die at the pharate adult stage with homeotic transformation defects ([@r62]). A hypomorph allele, *sxc*^*H537A*^, having substantially reduced OGT catalytic activity ([@r64]), shows significantly decreased protein O-GlcNAcylation levels ([@r63]) yet produce fertile offspring. Crucially, the fruit fly provides a platform to dissect the different effects of the OGT N567K mutation. In the fly, feedback between proteome O-GlcNAcylation levels and OGA/OGT protein/mRNA levels has not been observed. Furthermore, although *Drosophila* Hcf1 is heavily O-GlcNAc modified, proteolytic processing is catalyzed by the Taspase I protease instead of OGT ([@r65]). We introduced the OGT N567K equivalent mutation (N595K) into *D. melanogaster* using a CRISPR/Cas9 gene-editing toolkit. Vasa::Cas9 embryos were injected in-house with a mixture of plasmids coding for the guide RNA and repair template DNA. In total, 100 potential candidate flies were screened exploiting restriction fragment-length polymorphism arising from the loss of a restriction digestion site introduced in parallel with the N595K mutation ([*SI Appendix*, Fig. S3](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900065116/-/DCSupplemental)). We recovered 2 independent knockin lines (10.3 and 19.1) carrying the mutation, *sxc*^*N595K*^. Presence of the mutation was confirmed by sequencing the region ∼250 base pairs upstream and downstream from the mutation. Additionally, the lines were further validated by sequencing the full-length *sxc* mRNA. Both lines developed to adulthood without apparent homeotic defects. Transheterozygous *sxc*^*1*^*/sxc*^*H537A*^, *sxc*^1^/*sxc*^*N595K-19.1*^, and *sxc*^1^/*sxc*^*N595K-10.3*^ flies with presumably further reduced OGT activity were viable ([*SI Appendix*, Fig. S4](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900065116/-/DCSupplemental)). To probe the effect of the N595K mutation on OGT enzymatic activity and stability in vivo, we subjected adult fly head and embryo lysates to Western blotting with an O-GlcNAc antibody, RL2 ([Fig. 3*A*](#fig03){ref-type="fig"} and [*SI Appendix*, Fig. S5](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900065116/-/DCSupplemental)). O-GlcNAc levels were reduced to levels comparable to those in the hypomorph *sxc*^*H537A*^ flies ([Fig. 3*A*](#fig03){ref-type="fig"} and [*SI Appendix*, Fig. S5](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900065116/-/DCSupplemental)). OGT protein levels are expressed at wild-type level ([Fig. 3*A*](#fig03){ref-type="fig"} and [*SI Appendix*, Fig. S5](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900065116/-/DCSupplemental)). Taken together, phenotypic and molecular characterization of *sxc*^*N595K*^ *Drosophila* lines revealed that the mutation leads to reduction of protein O-GlcNAcylation. {#fig03} The N567K Mutation Leads to a mESC Neuronal Differentiation Phenotype. {#s6} ---------------------------------------------------------------------- The twins carrying the N567K OGT mutation, and the previously published male patients carrying OGT mutations in the TPR domain, all suffered from neurodevelopmental delay. We next explored the early events of neuronal development by studying effects on pluripotency and differentiation in mESCs, a cellular system amenable to genetic modification. We generated male mESC lines expressing C-terminal triple hemagglutinin (3HA) -tagged version of wild-type OGT (3HA-OGT^WT^) and carrying the N567K mutation (3HA-OGT^N567K^) using 2 rounds of CRISPR/Cas9-mediated gene editing at the endogenous locus ([*SI Appendix*, Figs. S6 and S7](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900065116/-/DCSupplemental)). At each step, at least 2 clones with the correct genotype were isolated and selected for further analysis. Next, we examined whether neuronal differentiation was altered in 3HA-OGT^N567K^ cells. Expression of pluripotency markers Sox2 and Oct4 was unaffected during the first 6 d of differentiation ([Fig. 3 *C* and *D*](#fig03){ref-type="fig"}). Neurite outgrowth was assessed after 8 d. Neurite length was significantly reduced in 3HA-OGT^N567K^ cells compared with 3HA-OGT^WT^ control cells immune-labeled against the neuron-specific β-tubulin III protein ([Fig. 3 *E* and *F*](#fig03){ref-type="fig"}). Thus, the N567K mutation leads to an mESC neuronal differentiation phenotype. The N567K Mutation Leads to Misprocessing of HCF1. {#s7} -------------------------------------------------- We next attempted to uncover the molecular mechanisms underpinning the observed differentiation phenotypes. We tested the levels of protein O-GlcNAcylation using Western blotting ([Fig. 4*A*](#fig04){ref-type="fig"}). Surprisingly, levels of protein O-GlcNAcylation in pluripotent mESCs appeared comparable in 3HA-OGT^N567K^ and 3HA-OGT^WT^ cells. Previous studies have shown that (an as yet to be discovered) feedback mechanism regulates OGA and OGT expression levels in response to protein O-GlcNAcylation levels ([@r42], [@r43], [@r66], [@r67]). Indeed, while OGT levels appeared to be unaltered, OGA levels were abrogated in 3HA-OGT^N567K^ pluripotent mESCs ([Fig. 4 *A* and *B*](#fig04){ref-type="fig"}), presumably to compensate for reduced OGT activity, but appeared normal during differentiation ([Fig. 3 *C* and *D*](#fig03){ref-type="fig"}). {#fig04} Next, we investigated the effects of the N567K mutation on the posttranslational maturation and cellular localization of HCF1 in pluripotent mESCs. Western blot analysis on cytoplasmic and nuclear fractions revealed an elevated ratio of full-length versus total HCF1 in the nuclear fractions of 3HA-OGT^N567K^ compared with 3HA-OGT^WT^ mESCs, while the levels of HCF1 proteolytic products were reduced ([Fig. 4 *C* and *D*](#fig04){ref-type="fig"}). This is in agreement with the impaired OGT-mediated proteolytic cleavage of HCF1 observed in vitro ([Fig. 2*C*](#fig02){ref-type="fig"}). HCF1 contributes to the regulation of transcriptional programs associated with controlling cell cycle and pluripotency ([@r68], [@r69]). Interestingly, the expression of some of the HCF1 interactors are also regulated by HCF1 itself ([@r51], [@r66], [@r69]). As an initial attempt to uncover possible candidate genes affected by HCF1 misprocessing, we investigated the expression levels of known HCF1 partners by qRT-PCR ([Fig. 4*E*](#fig04){ref-type="fig"}). The Ets transcription factor GA-binding protein subunit α (GABPA), a component of the nuclear respiratory factor-2 (NRF2) complex, showed a 2.3-fold increase in mRNA level in 3HA-OGT^N567K^ cells compared with 3HA-OGT^WT^ control samples (*P* = 0.002). GABPA/NRF2 regulates mitochondrial biogenesis and cell cycle progression ([@r70], [@r71]). Taking these data together, the OGT N567K mutation leads to misprocessing of HCF1 in mESCs. Concluding Remarks {#s8} ================== Previous studies have established a link between missense mutations in OGT and XLID in young males. These patients are hemizygous for the mutant OGT allele, with this genetic defect being present in all cells from early development to later life ([@r41][@r42]--[@r43]). However, residing exclusively in the OGT TPR domain, it was not clear whether these mutations led to the observed phenotypes through loss of glycosyltransferase activity, HCF1 misprocessing, or disruption of the OGT interactome ([@r41][@r42]--[@r43]). Here, we have presented female twins with XLID who are heterozygous for an N567K mutation in the OGT catalytic domain. Despite a wild-type *ogt* allele being present in all cells of these patients, its expression is arbitrarily silenced due to X chromosome inactivation at the late blastocyst stage of embryogenesis to compensate for gene dosage ([@r72]). Thus, these females represent an example of mosaic expression of an OGT XLID variant causing developmental abnormalities. Previous examples of female carriers of X-linked genetic disorders showed correlation between the X inactivation pattern and the severity of the condition ([@r73][@r74]--[@r75]). Hence, it is possible that the different etiology of ID in twins is the consequence of distinct spatial patterns and skew of X-inactivation. The N567K variant of OGT retains negligible levels of catalytic activity. Thus, a possible mechanism underpinning the XLID phenotype is reduced protein O-GlcNAcylation on a subset of OGT target proteins at specific time points of development or differentiation. Previous genetic studies in mice have highlighted the vital role of OGT in both neuronal development and adult life ([@r21], [@r33], [@r37][@r38]--[@r39]). OGT is capable of transferring GlcNAc onto over 4,000 different substrate proteins; it possesses an acceptor-peptide specificity with a preference to a degenerated sequon motif ([@r57], [@r76], [@r77]). Our structural analysis has uncovered that the N567K mutation changes the surface of the substrate binding pocket, thereby altering substrate binding ability of the N567K OGT. Previously reported XLID associated mutations lie in the TPR domain of OGT ([@r40][@r41][@r42]--[@r43], [@r78]), so potentially influencing its scaffolding function or reducing O-GlcNAcylation on an as yet unknown, subset of substrates. The N567K mutation may affect the O-GlcNAc proteome globally yet leads to a similar clinical phenotype, suggesting it is the catalytic activity/dosage of OGT that is important. We have identified one of the OGT substrates, HCF1, as a potential candidate that could at least partially explain some of the OGT N567K patient phenotypes. We showed that O-GlcNAcylation and proteolytic activation of HCF1 is significantly decreased, including in mESC nuclei. The HCF1 protein plays an important role in cell growth and cell cycle control through interacting with transcriptional complexes and epigenetic regulators ([@r79][@r80][@r81][@r82]--[@r83]). The HCF1 gene itself has been identified as an intellectual disability gene causing cobalamin type X deficiency, craniofacial abnormalities, and prenatal onset of microcephaly in the most extreme cases ([@r52], [@r53], [@r84]). Less-severe HCF1 mutations have been associated with ID and autism spectrum disorder without cobalamin deficiency ([@r54]). Patients with N567K OGT variant did not suffer from cobalamin deficiency and expressed a much milder form of congenital anomalies than the severe HCF1 group, suggesting that a degree of HCF1 activity is retained. Interestingly, we observed an increase in full-length HCF1 protein level and decreased level of the mature proteolytic fragment in nuclei, showing that HCF1 processing is affected *in cellulo*, albeit with retention of some activity. Surprisingly, the expression of the transcriptional factor GABPA downstream of HCF1 was increased indicating that the N567K mutation in OGT could translate into moderate molecular changes downstream of HCF1. The opposite effect, decreased expression of GABPA, was detected upon overexpression of the HCF1 N-terminal fragment (HCF~N~) ([@r68]). GABPA belongs to the E-26 family of DNA binding factors and regulates expression of several proteins required for mitochondrial DNA transcription and replication ([@r85]). Moreover, GABPA modulates the expression of genes involved in cell cycle control, apoptosis, differentiation, and hormonal regulation. Its importance in early embryonic development was demonstrated in knockout mouse studies ([@r86]). However, there is no established link between GABPA and neurodevelopment. Furthermore, expression of several other transcriptional factors targeted by HCF1---such as *Creb1*, *E2F4*, *Krox-2*, *Sin3a*, *Thap2*, and *Zfp143*---were found unaltered, suggesting that partial abrogation of HCF1 cleavage may affect just a small subset of targets, whose expression is influenced by the altered ratio of proteolytically cleaved and uncleaved HCF1. While OGT is critical for mESC maintenance of pluripotency and differentiation, we have detected no difference at the level of pluripotency markers, Sox2 and Oct4, between 3HA-OGT^WT^ and 3HA-OGT^N567K^ mESCs. The pluripotency factor Sox-2 is O-GlcNAc modified at Ser248 ([@r87]) and this posttranslational modification is indispensable for sustaining pluripotency. However, its O-GlcNAc modification is not required for differentiation, with cells expressing an O-GlcNAc--deficient mutant version of Sox2 exhibited enhanced reprogramming ability ([@r87]). Furthermore, mESCs expressing O-GlcNAc--deficient Sox2 displayed changes in their transcriptional network, specifically increasing the expression of genes vital to maintain pluripotency ([@r87]). It is feasible that a similar mechanism is responsible to compensate for pluripotency defects induced by the N567K OGT mutation. A *Drosophila* model of the N567K OGT mutation showed reduced protein O-GlcNAcylation. However, the abundance of O-GlcNAc--modified proteins was similar in 3HA-OGT^WT^ and 3HA-OGT^N567K^ mESCs. Interestingly, we observed decreased OGA protein levels as a compensatory regulatory mechanism responsible for maintaining protein O-GlcNAc levels in undifferentiated mESCs. Our finding is in line with previous data reported on patient-derived cell lines carrying XLID mutations in the TPR domain of OGT ([@r42], [@r43]). Although there is no evidence for a link between OGA and XLID, given the role of OGA in regulating transcription ([@r88][@r89][@r90]--[@r91]), it cannot be excluded that changes in OGA levels directly contribute to the XLID phenotype. In agreement with previous studies implying a requirement for OGT function in normal neuron differentiation, morphology, and health ([@r27], [@r37], [@r92][@r93]--[@r94]), abnormal OGT activity in 3HA-OGT^N567K^ cells resulted in reduced neurite outgrowth. These data suggest a possible mechanistic link between the mutation and the microcephaly and neurodevelopmental cognitive impairments observed in the patients. In summary, we have shown that impaired catalytic activity of OGT leads to neurodevelopmental defects in humans, through combined downstream effects of impaired OGT activity and reduced HCF1 proteolytic processing. Further studies are required to define the mechanisms downstream of impaired OGT catalytic activity that affect neurodevelopment resulting in intellectual disability. Materials and Methods {#s9} ===================== The study was approved by the University of Dundee Institutional Risk Assessment committee, and 1 of each participants' parents provided written, informed consent. The DDD study has UK Research Ethics Committee (REC) approval (10/H0305/83, granted by the Cambridge South Research Ethic Committee, and GEN/284/12 granted by the Republic of Ireland REC). X-Inactivation Analysis. {#s10} ------------------------ To determine if X chromosome inactivation occurred randomly or nonrandomly, the ratio of methylated and unmethylated maternal and paternal X chromosomes was determined. ([*SI Appendix*, Fig. S1](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900065116/-/DCSupplemental)). Sites are methylated on the inactive X chromosome and unmethylated on the active X chromosome. The method utilizes the androgen receptor sequence that is encoded on the X chromosome (Xq11.2-q12) and contains a highly polymorphic polyglutamine (CAG) trinucleotide repeat in exon 1. There are 20 alleles of androgen receptor, with 11 to 31 repeats reported in the general population and 85 to 90% of patients are heterozygous for this locus, thus suitable for X-inactivation analysis. For experimental procedure, see [*SI Appendix*, *SI Materials and Methods*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900065116/-/DCSupplemental). Structure Solution. {#s11} ------------------- Crystallization of truncated OGT~WT/N567K~ (residues 323--1044) was performed as described previously ([@r57]) (for protein expression, purification, and crystallization, see [*SI Appendix*, *SI Materials and Methods*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900065116/-/DCSupplemental)). The structure was solved by molecular replacement using the structure for OGT~WT~ \[PDB ID code 5C1D ([@r57])\] as the search model. The resulting model was manually refined using Coot ([@r95]) and REFMAC ([@r96]), respectively. The editing and refinement of the model was iterated until it was in complete agreement with the data. Scaling and model building statistics can be seen in [Table 2](#t02){ref-type="table"}. Enzyme Activity Assays. {#s12} ----------------------- Michaelis--Menten kinetics of OGT were measured using a fluorometric assay, as described previously ([@r97]), with the exception of reduced reaction volume of 25 µL and usage of 384-well plate. As acceptor substrate, P1 (KKVPVSRA) and P2 (KKVAVSRA) were used. Additional O-GlcNAcylation assays were performed on TAB1 protein (residues 7--420). Proteolytic assays were performed as described previously ([@r43]), except a noncleavable HCF1-rep1 fragment was used instead of OGA treatment as the negative control. For further details, see [*SI Appendix*, *SI Materials and Methods*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900065116/-/DCSupplemental). mESC Culture. {#s13} ------------- mESC AW2 line was acquired from the MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, University of Edinburgh ([@r98]). mESCs were cultured in an undifferentiated state in 0.1% gelatin (wt/vol)-coated plates in GMEM BHK-21 supplemented with 10% FBS (vol/vol), 0.1 mM MEM nonessential amino acids, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, and 100 units/mL LIF at 37 °C in 5% CO~2~. For differentiation assays, cells were cultured following a previously published protocol ([@r99]). Briefly, cells were cultured in DMEM and F12 media mixed at 1:1 ratio (vol/vol), supplemented with modified N2 (25 mg/mL insulin, 100 μg/mL apo-transferrin, 6 ng/mL progesterone, 16 μg/mL putrescine, 30 nM sodium selenite, and 50 μg/mL BSA fraction) and neurobasal supplement B27. Medium was renewed every 2 d. For generation of CRISPR/Cas9 lines, Immunocytochemistry, Western blotting, and qPCR analyses details are provided in [*SI Appendix*, *SI Materials and Methods*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900065116/-/DCSupplemental). *Drosophila* Husbandry, Microinjection, and Genetics. {#s14} ----------------------------------------------------- Fly stocks were maintained on Nutri-Fly Bloomington Formulation fly food at 25 °C. Vasa::Cas9 (\#51323), *sxc*^*1*^*,bw*^*1*^*,sp*^*1*^*/SM5* (\#3058) and *Df(2R)BSC630/CyO* (\#25705) stocks were obtained from the Bloomington *Drosophila* Stock Centre. The *sxc* hypomorphic allele, *sxc*^*H537A*^, was described in ref. [@r63]. The *sxc*^*N595K*^ *Drosophila* lines were generated by CRISPR/Cas9 mutagenesis, as described in [*SI Appendix*, *SI Materials and Methods*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900065116/-/DCSupplemental). Western Blotting from *Drosophila* Samples. {#s15} ------------------------------------------- Sample preparation of embryos and fly heads and Western blotting were performed as described in [*SI Appendix*, *SI Materials and Methods*](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900065116/-/DCSupplemental). Supplementary Material ====================== This work was funded by a Wellcome Trust Investigator Award (110061) (to D.M.F.v.A.). V.M.P. was supported by the SPRINT/MND-MS Scholarship (University of Edinburgh). The X-ray diffraction experiments were performed on beamline ID23-2 at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. We are grateful to Local Contact at the ESRF for providing assistance for using beamline ID23-2. We acknowledge Dr. Louise A. Young for the aid in the X-inactivation analysis and Dr. Andrew Smith for the AW2 mouse embryonic stem cell line. We acknowledge the Deciphering Developmental Disorders study, presenting independent research commissioned by the Health Innovation Challenge Fund (grant no. HICF-1009-003), a parallel funding partnership between Wellcome and the Department of Health, and the Wellcome Sanger Institute (grant no. WT098051). The research team acknowledges the support of the National Institute for Health Research, through the Comprehensive Clinical Research Network. The authors would also like to thank the Genome Aggregation Database (gnomAD) and the groups that provided exome and genome variant data to this resource. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, [www.pdb.org](http://www.pdb.org) (PDB ID code [6IBO](6IBO)). This article contains supporting information online at [www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900065116/-/DCSupplemental](https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1900065116/-/DCSupplemental). [^1]: Edited by Carolyn R. Bertozzi, Stanford University, Stanford, CA, and approved June 17, 2019 (received for review January 29, 2019) [^2]: Author contributions: V.M.P., V.M., M.G., and D.M.F.v.A. designed research; V.M.P., V.M., and M.G. performed research; A.T.F., P.S.K., V.V., A.C.W., V.S.B., and S.J. contributed new reagents/analytic tools; V.M.P., V.M., M.G., M.P.S., and D.M.F.v.A. analyzed data; and V.M.P., V.M., M.G., and D.M.F.v.A. wrote the paper. [^3]: ^1^V.M.P., V.M., and M.G. contributed equally to this work.

{#sp1 .358}

{#sp1 .146}

In 1909, Moreschi was the first to report that caloric restriction inhibits the development of tumours in mice ([Moreschi, 1909](#bib5){ref-type="other"}). This was subsequently found to be highly reproducible in other rodents, and to involve a variety of tumours ([Weindruch and Walford, 1988](#bib10){ref-type="other"}). Already in the early days in this field of research, scientists recognised that caloric restriction might act via neuro-endocrine mechanisms to inhibit tumour initiation and promotion ([Pomerantz and Mulinos, 1939](#bib7){ref-type="other"}). Since little is known about caloric restriction and cancer risk in humans, we investigated whether exposure to the 1944--1945 Dutch famine in young adulthood influenced urinary sex hormone concentrations in postmenopausal women. Given the lower risk of cancer in rodents fed a calorie-restricted diet, famine might be expected to be associated with lower endogenous sex hormone levels in women, since women with lower levels of such hormones have been found to have a decreased risk for breast cancer ([The Endogenous Hormones and Breast Cancer Collaborative Group, 2002](#bib9){ref-type="other"}; [Onland-Moret *et al*, 2003](#bib6){ref-type="other"}). The famine occurred at the end of World War II in the densely populated western parts of the Netherlands with food supplies deteriorating rapidly from October 1944 onwards: official daily rations dropped from about 1500 kcal in September 1944 to below 700 kcal in January 1945. After 6 months of starvation, the Netherlands were liberated on 5 May 1945, abruptly ending the famine ([Burger *et al*, 1948](#bib2){ref-type="other"}). SUBJECTS AND METHODS ==================== As part of the Diagnostisch Onderzoek Mammacarcinoom (DOM) project, a population-based breast cancer-screening project was conducted between 1974 and 1986 in Utrecht, the Netherlands ([de Waard *et al*, 1984](#bib3){ref-type="other"}), which from 1983 included questions about hunger, cold and weight loss experienced during the 1944--1945 Dutch famine, with answers to these questions as absent, moderate or severe exposure. We combined the answers into a three-level individual famine score as follows: women with severe or absent exposure to at least two aspects of famine (hunger, cold, or weight loss) were classified as severely or, respectively, unexposed to the famine. All the remaining women were classified as having been moderately exposed to the famine ([Elias *et al*, 2002](#bib4){ref-type="other"}). Demographic details and reproductive history were also obtained by questionnaire. Anthropometrical details were collected at recruitment and participants donated a first morning urine sample on the day of their first mammographic examination. These urine samples were stored at −20°C in 250 ml plastic polypropylene jars, without preserving agents, until analysis. A random sample of 424 women with a natural menopause at recruitment and no history of breast cancer was drawn from 27 718 women, born between 1911 and 1945, as a control group for a study addressing the relation of endogenous sex steroids with postmenopausal breast cancer risk ([Onland-Moret *et al*, 2003](#bib6){ref-type="other"}). At the introduction of the famine questionnaire in 1983, 203 women from this random sample still participated in the DOM cohort, of whom 182 resided in the occupied Netherlands during the famine and had sufficient data to compute the famine score. Women using hormone replacement therapy or oral contraceptives at the time of urine sampling were excluded (*n*=19). Urine samples were analysed at the Hormones and Cancer Group, at the International Agency for Research on Cancer in Lyon, France. The hormones oestrone, oestradiol, testosterone and 5*α*-androstane-3*α*,17*β*-diol (3*α*D) were measured by radioimmunoassay after enzymatic hydrolysis, solid-phase extraction and high-performance liquid chromatography purification of the urine samples. The method used in this study and measures of its reproducibility have been described in detail elsewhere ([Rinaldi *et al*, 2003](#bib8){ref-type="other"}). Intra- and interassay coefficients of variation were 8.7 and 17.2% for oestrone, 12.2 and 14.8% for oestradiol, 8.3 and 15.5% for testosterone and 9.0 and 11.4% for 3*α*D. Creatinine was measured in each sample by kinetic Jaffé reaction (Hitachi 717, Roche, Central Laboratory for Biochemistry, Hôpital de l\'Antiquaille, Lyon, France). Hormone concentrations, expressed in ng per mg creatinine to adjust for the differences in urine dilution, were logarithmically transformed to achieve normal distributions. The mean hormone concentrations according to famine exposure categories were then estimated by analysis of covariance, and adjusted for potential confounders (age, body mass index, years since menopause, and cigarette smoking (yes/no) at the time of donation of the urine sample, as well as socio-economic status (high/low) and parity (parous/nulliparous)). Since pregnancy is known to alter -- in some aspects permanently -- the neuro-endocrine milieu ([Bernstein, 2002](#bib1){ref-type="other"}), we tested whether any famine effects depended on parity. We used linear regression to evaluate the linear trends for the effect of famine exposure (quantitatively scored as 1, 2 or 3 with increasing exposure) and to test the interaction terms. Statistical analyses were performed with SPSS 11. All tests of statistical significance were two-sided. Backtransformation of the logarithmic means resulted in geometrical means, which we report together with the corresponding 95% confidence intervals (CIs). RESULTS ======= In total, 29 of the women were severely, 71 moderately and 63 unexposed to the famine. The median age during the famine was 26 years (10--90th percentile: 19--32 years). Overall, we found that famine was modestly associated with oestrone, with lower levels in the unexposed group compared to the moderately and severely exposed (*P*~trend~=0.07). The famine was apparently not related to oestradiol; levels of testosterone and 3*α*D were increased in the famine-exposed groups with highest levels in women with moderate famine exposures ([Table 1](#tbl1){ref-type="table"} Table 1Geometric means^a^ and 95% confidence intervals (CI) of oestrone, oestradiol, testosterone and 5*α*-androstane-3*α*,17*β*-diol levels in urine after correction for creatinine according to the famine score **Famine score**  **Unexposed (*n*=63)Moderately exposed (*n*=71)Severely exposed (*n*=29)**  **Geometric mean95% CIGeometric mean95% CIGeometric mean95% CI** Oestrone1.53(1.32--1.78)1.81(1.57--2.08)1.91(1.54--2.38)*P*~trend~=0.07^b^Oestradiol0.51(0.44--0.59)0.51(0.44--0.58)0.54(0.44--0.67)*P*~trend~=0.68Testosterone2.20(1.81--2.67)2.57(2.14--3.08)2.50(1.88--3.33)*P*~trend~=0.355*α*-androstane-3*α*,17*β*-diol18.20(15.10--21.94)22.69(19.02--27.07)20.26(15.39--26.66)*P*~trend~=0.32[^1][^2]). Parity seemed to modify the associations (interaction tests: oestrone: *P*=0.04; oestradiol: *P*\<0.01; testosterone: *P*=0.09; 3*α*D: *P*=0.70). Famine was not related to any of the hormones in parous women, but showed some evidence of a dose--response relation with oestrone and oestradiol in women who never gave birth ([Table 2](#tbl2){ref-type="table"} Table 2Geometric means^a^ and 95% confidence intervals (CI) of estrone, estradiol, testosterone, and 5*α*-androstane-3*α*, 17*β*-diol levels in urine after correction for creatinine according to the famine score and parity **Famine score**  **UnexposedModerately exposedSeverely exposed**  **Geometric mean95% CIGeometric mean95% CIGeometric mean95% CI** Nulliparous women(*n*=10)(*n*=20)(*n*=6)  Oestrone1.24(0.92--1.67)1.61(1.31--1.99)3.36(2.26--5.01)*P*~trend~=0.001^b^ Oestradiol0.37(0.27--0.51)0.57(0.46--0.72)0.91(0.60--1.39)*P*~trend~=0.001 Testosterone2.04(1.05--3.96)2.51(1.58--4.00)4.19(1.74--10.10)*P*~trend~=0.21 5*α*-androstane-3*α*,17*β*-diol24.98(16.52--37.78)20.88(15.63--27.90)29.80(17.20--51.64)*P*~trend~=0.78Parous women(*n*=53)(*n*=51)(*n*=23)  Oestrone1.60(1.35--1.89)1.87(1.57--2.22)1.69(1.31--2.18)*P*~trend~=0.51 Oestradiol0.54(0.45--0.63)0.49(0.41--0.58)0.48(0.37--0.62)*P*~trend~=0.37 Testosterone2.30(1.89--2.79)2.51(2.05--3.05)2.20(1.64--2.95)*P*~trend~=0.98 5*α*-androstane-3*α*,17*β*-diol17.03(13.85--20.95)23.49(19.01--29.04)18.50(13.52--25.33)*P*~trend~=0.34[^3][^4]). The association with testosterone showed the same tendency, although weaker. Inspection of the data revealed that outliers or between-batch variation did not explain the results. DISCUSSION ========== In conclusion, we found a dose--response increase of postmenopausal urinary oestrone and oestradiol levels with increasing severity of famine exposure in young adulthood among women who had never given birth, but not among parous women. Similar effects were seen for testosterone, whereas famine was not related to 3*α*D. Although a black episode in the history of the Netherlands, the 1944--1945 Dutch famine has enabled us to investigate the long-term effects of a short but severe famine in an otherwise well-nourished human population. We were able to individually classify women in terms of their famine exposure status on the basis of their experiences of weight loss, hunger and cold during the 1944--1945 winter. There was some indirect evidence that the individual famine scores were accurate: the proportion of severely famine-exposed women increased with the degree of residential urbanisation during the famine, reflecting differences in severity of the famine between rural and urban areas ([Burger *et al*, 1948](#bib2){ref-type="other"}). Since famine scores were based on recall, misclassification is a concern. However, we believe that this misclassification is unlikely to be related to the hormone concentrations under investigation, and, if anything, would result in an underestimation of the observed effects. Our findings may be explained by long-lasting effects on the hypothalamo--pituitary--gonadal axis, although adrenal involvement cannot be ruled out. Contrary to our hypothesis, the direction of the effects would imply an increased breast cancer risk in nulliparous, famine-exposed women. We thank Beatrize Vozar and David Achaintre for their assistance in the hormone measurements and Bernard Slotboom and Bep Verkerk for processing and handling of data. The work was supported by the Dutch Cancer Society (grants KWF UU-1999-1935 and KWF UU-2000-2314). [^1]: Expressed in ng per mg creatinine and adjusted for age, body mass index, years since menopause and cigarette smoking at time of donation of urine sample as well as socio-economic status and parity. [^2]: *P* for linear trend. [^3]: Expressed in ng per mg creatinine and adjusted for age, body mass index, years since menopause and cigarette smoking at time of donation of urine sample as well as socio-economic status and parity. [^4]: *P* for linear trend. Tests for linear interaction of famine exposure with parity: *P*=0.04 (oestrone); *P*\<0.01 (oestradiol); *P*=0.09 (testosterone); *P*=0.70 (5*α*-androstane-3*α*,17*β*-diol).

Related literature {#sec1} ================== The title compound was synthesized as an inter­mediate in the search for a new synthetic route to silodosin, an adrenoceptor antagonist, see: Asselin *et al.* (2000[@bb1]); Bremner *et al.* (2000[@bb2]); Elworthy *et al.* (1997[@bb6]); Sorbera *et al.* (2001[@bb10]). For related structures, see: Moreno *et al.* (1998[@bb8]); Wang *et al.*(2007[@bb12]). Experimental {#sec2} ============ {#sec2.1} ### Crystal data {#sec2.1.1} C~13~H~16~ClNO*M* *~r~* = 237.72Triclinic,*a* = 6.9041 (5) Å*b* = 8.4887 (6) Å*c* = 10.6463 (7) Åα = 76.423 (1)°β = 86.955 (1)°γ = 89.969 (1)°*V* = 605.61 (7) Å^3^*Z* = 2Mo *K*α radiationμ = 0.29 mm^−1^*T* = 173 K0.46 × 0.41 × 0.22 mm ### Data collection {#sec2.1.2} Bruker AXS SMART 1000 CCD diffractometerAbsorption correction: multi-scan (*SADABS*; Sheldrick, 2008*a* [@bb13]) *T* ~min~ = 0.877, *T* ~max~ = 0.9384719 measured reflections2343 independent reflections1915 reflections with *I* \> 2σ(*I*)*R* ~int~ = 0.016 ### Refinement {#sec2.1.3} *R*\[*F* ^2^ \> 2σ(*F* ^2^)\] = 0.041*wR*(*F* ^2^) = 0.113*S* = 1.072343 reflections163 parameters5 restraintsH-atom parameters constrainedΔρ~max~ = 0.35 e Å^−3^Δρ~min~ = −0.19 e Å^−3^ {#d5e423} Data collection: *SMART* (Bruker, 2001[@bb3]); cell refinement: *SAINT-Plus* (Bruker, 2003[@bb4]); data reduction: *SAINT-Plus*; program(s) used to solve structure: *SHELXTL* (Sheldrick, 2008*b* [@bb9]); program(s) used to refine structure: *SHELXL97* (Sheldrick, 2008*b* [@bb9]); molecular graphics: *ORTEPIII* (Burnett & Johnson, 1996[@bb5]), *ORTEP-3 for Windows* (Farrugia, 1997[@bb7]) and *PLATON* (Spek, 2009[@bb11]); software used to prepare material for publication: *SHELXTL*. Supplementary Material ====================== Crystal structure: contains datablocks global, I. DOI: [10.1107/S1600536810050476/dn2631sup1.cif](http://dx.doi.org/10.1107/S1600536810050476/dn2631sup1.cif) Structure factors: contains datablocks I. DOI: [10.1107/S1600536810050476/dn2631Isup2.hkl](http://dx.doi.org/10.1107/S1600536810050476/dn2631Isup2.hkl) Additional supplementary materials: [crystallographic information](http://scripts.iucr.org/cgi-bin/sendsupfiles?dn2631&file=dn2631sup0.html&mime=text/html); [3D view](http://scripts.iucr.org/cgi-bin/sendcif?dn2631sup1&Qmime=cif); [checkCIF report](http://scripts.iucr.org/cgi-bin/paper?dn2631&checkcif=yes) Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: [DN2631](http://scripts.iucr.org/cgi-bin/sendsup?dn2631)). We thank Mr Feng Xiaolong for his kind help. Comment ======= In searching for new synthetic route of silodosin, a adrenoceptor antagonist (Sorbera *et al.* 2001; Elworthy *et al.* 1997; Asselin *et al.* 2000; Bremner *et al.* 2000), we synthesized the title compound as racemic intermediate. In the title compound, C~13~H~16~ClNO, the acetylindoline moiety is mainly planar with the largest deviation from the plane being 0.0076 (14)Å at C2. The chloropropane being out of the plane with the C12 atom located 1.0254 (0.0028)Å above the plane (Fig. 1). The chloropropane moiety is statistically disordered over two positions. Indeed, the Cl and CH~3~ located on the stereogenic carbon exchange each other. The geometry within the 1-acetylindoline fragment compares well with related structures as 1-acetylindoline (Moreno *et al.*, 1998) or 1-(trifluoro)acetylindoline (Wang *et al.*, 2007). Non-classical C---H···O hydrogen bonds (Table 1) link the molecules forming layers parallel to the (1 0 0) plane (Figure 2). Experimental {#experimental} ============ 1 g of (*R*/*S*)-1-(1-acetylindolin-5-yl)-2-chloropropan-1-one was dissolved in 50 ml of trifluroacetic acid, and then 1.067 g of triethylsilane was added dropwise within 20 min in -5¯C. The system was stirred overnight in ambient temperature, then extra trifluroacetic acid was distilled out in reduced pressure. To the resulting oil was added 20 ml of water and 5 ml of n-hexane, and stirred for 10 min. The white precipitate was collected through filtration, washed by n-hexane and dried to get 1.24 g of the targeting product. Crystals suitable for X-ray diffraction were obtained by slow evaporation of an ethyl acetate solution. Spectroscopic analysis: ^1^H NMR (CDCl~3~,δ, p.p.m.): 1.519--1.542(d,3*H*), 2.236(s,3*H*), 2.882--3.093(t,2*H*), 3.171--3.227(t,2*H*), 4.037--4.124(t,2*H*), 4.147--4.213(t, 1H), 7.009--7.0977(s,2*H*), 8.111--8.140(d,1*H*). Refinement {#refinement} ========== All H atoms attached to C atoms and N atom were fixed geometrically and treated as riding with C---H = 0.95Å (aromatic), 0.98 Å (methyl), 0.99 Å (methylene) and 0.96Å (methine) with *U*~iso~(H) = 1.2*U*~eq~(Caromatic, Cmethine, Cmethylene) or *U*~iso~(H) = 1.5*U*~eq~(Cmethyl). The Cl and CH3 substituents on the stereogenic carbon are exchanging each other and such disorder induces two configurations. Two sets of positions were defined for the atoms of this group and the site occupation factor of each conformation were refined while restraining their sum to unity and using restraints on C---C and C---Cl distances with the help of SAME and PART instructions within *SHELXL97* (Sheldrick, 2008). In the last stage of refinement, the disordered Cl and C atoms were anisotropically refined but the anistropic thermal parameters of the C atoms were restrained to have similar atomic displacement parameters within a tolerance s.u. of 0.01 Å^2^. Figures ======= {#Fap1} ![Partial packing view of compound ( I ), showing the formation of chains along \[010\] built from hydrogen bonds represented as dashed lines. H atoms not involved in hydrogen bonding have been omitted for clarity. \[Symmetry code: (i) x, y-1, z\]](e-67-00o51-fig2){#Fap2} Crystal data {#tablewrapcrystaldatalong} ============ ----------------------- --------------------------------------- C~13~H~16~ClNO *Z* = 2 *M~r~* = 237.72 *F*(000) = 252 Triclinic, *P*1 *D*~x~ = 1.304 Mg m^−3^ Hall symbol: -P 1 Mo *K*α radiation, λ = 0.71073 Å *a* = 6.9041 (5) Å Cell parameters from 2890 reflections *b* = 8.4887 (6) Å θ = 2.5--27.0° *c* = 10.6463 (7) Å µ = 0.29 mm^−1^ α = 76.423 (1)° *T* = 173 K β = 86.955 (1)° Block, colourless γ = 89.969 (1)° 0.46 × 0.41 × 0.22 mm *V* = 605.61 (7) Å^3^ ----------------------- --------------------------------------- Data collection {#tablewrapdatacollectionlong} =============== --------------------------------------------------------------- -------------------------------------- Bruker AXS SMART 1000 CCD diffractometer 2343 independent reflections Radiation source: fine-focus sealed tube 1915 reflections with *I* \> 2σ(*I*) graphite *R*~int~ = 0.016 ω scans θ~max~ = 26.0°, θ~min~ = 2.0° Absorption correction: multi-scan (*SADABS*; Sheldrick, 2008) *h* = −8→8 *T*~min~ = 0.877, *T*~max~ = 0.938 *k* = −10→10 4719 measured reflections *l* = −13→13 --------------------------------------------------------------- -------------------------------------- Refinement {#tablewraprefinementdatalong} ========== ------------------------------------- ------------------------------------------------------------------------------------------------- Refinement on *F*^2^ Primary atom site location: structure-invariant direct methods Least-squares matrix: full Secondary atom site location: difference Fourier map *R*\[*F*^2^ \> 2σ(*F*^2^)\] = 0.041 Hydrogen site location: inferred from neighbouring sites *wR*(*F*^2^) = 0.113 H-atom parameters constrained *S* = 1.07 *w* = 1/\[σ^2^(*F*~o~^2^) + (0.0478*P*)^2^ + 0.2707*P*\] where *P* = (*F*~o~^2^ + 2*F*~c~^2^)/3 2343 reflections (Δ/σ)~max~ = 0.002 163 parameters Δρ~max~ = 0.35 e Å^−3^ 5 restraints Δρ~min~ = −0.19 e Å^−3^ ------------------------------------- ------------------------------------------------------------------------------------------------- Special details {#specialdetails} =============== ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes. Refinement. Refinement of *F*^2^ against ALL reflections. The weighted *R*-factor *wR* and goodness of fit *S* are based on *F*^2^, conventional *R*-factors *R* are based on *F*, with *F* set to zero for negative *F*^2^. The threshold expression of *F*^2^ \> σ(*F*^2^) is used only for calculating *R*-factors(gt) *etc*. and is not relevant to the choice of reflections for refinement. *R*-factors based on *F*^2^ are statistically about twice as large as those based on *F*, and *R*- factors based on ALL data will be even larger. ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å^2^) {#tablewrapcoords} ================================================================================================== ------ -------------- -------------- -------------- -------------------- ------------ *x* *y* *z* *U*~iso~\*/*U*~eq~ Occ. (\<1) O1 0.2493 (2) 0.69510 (15) 0.57618 (13) 0.0462 (4) N1 0.25327 (19) 0.49053 (16) 0.47345 (13) 0.0306 (3) C1 0.2670 (3) 0.4283 (2) 0.35441 (16) 0.0355 (4) H1A 0.3925 0.4604 0.3057 0.043\* H1B 0.1605 0.4711 0.2977 0.043\* C2 0.2505 (3) 0.2437 (2) 0.40071 (17) 0.0371 (4) H2A 0.1365 0.2019 0.3654 0.045\* H2B 0.3686 0.1912 0.3739 0.045\* C3 0.2275 (2) 0.21325 (19) 0.54586 (16) 0.0293 (4) C4 0.2056 (2) 0.0682 (2) 0.63687 (17) 0.0328 (4) H4 0.2030 −0.0309 0.6101 0.039\* C5 0.1872 (2) 0.0670 (2) 0.76852 (17) 0.0331 (4) C6 0.1927 (2) 0.2141 (2) 0.80423 (16) 0.0340 (4) H6 0.1817 0.2138 0.8936 0.041\* C7 0.2137 (2) 0.3621 (2) 0.71377 (16) 0.0320 (4) H7 0.2165 0.4614 0.7403 0.038\* C8 0.2304 (2) 0.35962 (19) 0.58388 (15) 0.0278 (4) C9 0.2613 (2) 0.6503 (2) 0.47457 (18) 0.0337 (4) C10 0.2848 (3) 0.7693 (2) 0.34518 (19) 0.0404 (4) H10A 0.2923 0.8797 0.3582 0.061\* H10B 0.1734 0.7598 0.2940 0.061\* H10C 0.4042 0.7460 0.2992 0.061\* C11 0.1537 (3) −0.0898 (2) 0.86977 (18) 0.0422 (5) H11A 0.0257 −0.1352 0.8581 0.051\* H11B 0.1485 −0.0653 0.9563 0.051\* C12 0.3060 (3) −0.2180 (2) 0.86693 (18) 0.0400 (4) H12A 0.3065 −0.2493 0.7859 0.048\* Cl1 0.5409 (4) −0.1512 (3) 0.8927 (2) 0.0525 (4) 0.50 C13 0.245 (2) −0.3659 (13) 0.9768 (12) 0.100 (5) 0.50 H13A 0.2548 −0.3380 1.0605 0.150\* 0.50 H13B 0.3311 −0.4566 0.9725 0.150\* 0.50 H13C 0.1111 −0.3970 0.9672 0.150\* 0.50 Cl1B 0.2300 (4) −0.4048 (3) 0.97881 (18) 0.0532 (4) 0.50 C13B 0.4978 (16) −0.1668 (14) 0.9079 (13) 0.102 (5) 0.50 H13D 0.5386 −0.0621 0.8516 0.152\* 0.50 H13E 0.5958 −0.2483 0.9009 0.152\* 0.50 H13F 0.4834 −0.1567 0.9977 0.152\* 0.50 ------ -------------- -------------- -------------- -------------------- ------------ Atomic displacement parameters (Å^2^) {#tablewrapadps} ===================================== ------ ------------- ------------- ------------- ------------- ------------- ------------- *U*^11^ *U*^22^ *U*^33^ *U*^12^ *U*^13^ *U*^23^ O1 0.0608 (9) 0.0299 (7) 0.0505 (8) 0.0026 (6) −0.0098 (6) −0.0135 (6) N1 0.0303 (7) 0.0277 (7) 0.0333 (7) 0.0010 (5) −0.0030 (6) −0.0053 (6) C1 0.0388 (9) 0.0357 (9) 0.0312 (9) 0.0025 (7) −0.0018 (7) −0.0063 (7) C2 0.0447 (10) 0.0343 (9) 0.0334 (9) −0.0024 (7) 0.0009 (7) −0.0106 (7) C3 0.0261 (8) 0.0299 (8) 0.0328 (9) 0.0017 (6) −0.0018 (6) −0.0092 (7) C4 0.0309 (8) 0.0268 (8) 0.0409 (10) 0.0031 (6) −0.0008 (7) −0.0087 (7) C5 0.0260 (8) 0.0341 (9) 0.0357 (9) 0.0046 (7) −0.0005 (7) −0.0016 (7) C6 0.0304 (9) 0.0420 (10) 0.0293 (9) 0.0067 (7) −0.0021 (7) −0.0075 (7) C7 0.0287 (8) 0.0330 (9) 0.0366 (9) 0.0045 (7) −0.0044 (7) −0.0122 (7) C8 0.0218 (7) 0.0279 (8) 0.0335 (9) 0.0024 (6) −0.0033 (6) −0.0063 (6) C9 0.0262 (8) 0.0279 (8) 0.0473 (10) 0.0011 (6) −0.0070 (7) −0.0081 (7) C10 0.0332 (9) 0.0302 (9) 0.0534 (11) 0.0003 (7) −0.0039 (8) −0.0005 (8) C11 0.0401 (10) 0.0401 (10) 0.0408 (10) 0.0038 (8) 0.0034 (8) 0.0006 (8) C12 0.0495 (11) 0.0321 (9) 0.0361 (10) 0.0033 (8) −0.0037 (8) −0.0034 (7) Cl1 0.0513 (8) 0.0619 (10) 0.0492 (7) 0.0144 (6) −0.0190 (6) −0.0190 (7) C13 0.118 (9) 0.030 (6) 0.136 (8) 0.027 (5) 0.001 (5) 0.009 (4) Cl1B 0.0777 (10) 0.0273 (10) 0.0482 (8) 0.0008 (7) −0.0027 (6) 0.0036 (5) C13B 0.081 (7) 0.049 (5) 0.161 (10) 0.028 (4) −0.026 (6) 0.006 (5) ------ ------------- ------------- ------------- ------------- ------------- ------------- Geometric parameters (Å, °) {#tablewrapgeomlong} =========================== -------------------- -------------- ----------------------- -------------- O1---C9 1.227 (2) C7---H7 0.9500 N1---C9 1.360 (2) C9---C10 1.506 (3) N1---C8 1.417 (2) C10---H10A 0.9800 N1---C1 1.482 (2) C10---H10B 0.9800 C1---C2 1.531 (2) C10---H10C 0.9800 C1---H1A 0.9900 C11---C12 1.517 (3) C1---H1B 0.9900 C11---H11A 0.9900 C2---C3 1.505 (2) C11---H11B 0.9900 C2---H2A 0.9900 C12---C13B 1.511 (10) C2---H2B 0.9900 C12---C13 1.543 (10) C3---C4 1.379 (2) C12---Cl1 1.774 (3) C3---C8 1.395 (2) C12---Cl1B 1.804 (3) C4---C5 1.398 (2) C12---H12A 0.9604 C4---H4 0.9500 C13---H13A 0.9800 C5---C6 1.389 (3) C13---H13B 0.9800 C5---C11 1.513 (2) C13---H13C 0.9800 C6---C7 1.395 (2) C13B---H13D 0.9800 C6---H6 0.9500 C13B---H13E 0.9800 C7---C8 1.387 (2) C13B---H13F 0.9800 C9---N1---C8 125.79 (15) H10A---C10---H10B 109.5 C9---N1---C1 124.26 (14) C9---C10---H10C 109.5 C8---N1---C1 109.95 (13) H10A---C10---H10C 109.5 N1---C1---C2 105.54 (13) H10B---C10---H10C 109.5 N1---C1---H1A 110.6 C5---C11---C12 114.95 (15) C2---C1---H1A 110.6 C5---C11---H11A 108.5 N1---C1---H1B 110.6 C12---C11---H11A 108.5 C2---C1---H1B 110.6 C5---C11---H11B 108.5 H1A---C1---H1B 108.8 C12---C11---H11B 108.5 C3---C2---C1 104.39 (13) H11A---C11---H11B 107.5 C3---C2---H2A 110.9 C13B---C12---C11 110.8 (5) C1---C2---H2A 110.9 C13B---C12---C13 103.1 (7) C3---C2---H2B 110.9 C11---C12---C13 106.5 (6) C1---C2---H2B 110.9 C13B---C12---Cl1 7.7 (6) H2A---C2---H2B 108.9 C11---C12---Cl1 112.22 (16) C4---C3---C8 120.53 (15) C13---C12---Cl1 109.3 (6) C4---C3---C2 129.25 (15) C13B---C12---Cl1B 107.8 (5) C8---C3---C2 110.22 (14) C11---C12---Cl1B 109.45 (16) C3---C4---C5 120.04 (15) C13---C12---Cl1B 7.6 (6) C3---C4---H4 120.0 Cl1---C12---Cl1B 113.43 (15) C5---C4---H4 120.0 C13B---C12---H12A 117.2 C6---C5---C4 118.45 (15) C11---C12---H12A 109.8 C6---C5---C11 120.51 (16) C13---C12---H12A 108.8 C4---C5---C11 120.99 (16) Cl1---C12---H12A 110.2 C5---C6---C7 122.45 (16) Cl1B---C12---H12A 101.2 C5---C6---H6 118.8 C12---C13---H13A 109.5 C7---C6---H6 118.8 C12---C13---H13B 109.5 C8---C7---C6 117.80 (15) H13A---C13---H13B 109.5 C8---C7---H7 121.1 C12---C13---H13C 109.5 C6---C7---H7 121.1 H13A---C13---H13C 109.5 C7---C8---C3 120.73 (15) H13B---C13---H13C 109.5 C7---C8---N1 129.36 (15) C12---C13B---H13D 109.5 C3---C8---N1 109.90 (14) C12---C13B---H13E 109.5 O1---C9---N1 121.52 (16) H13D---C13B---H13E 109.5 O1---C9---C10 121.67 (16) C12---C13B---H13F 109.5 N1---C9---C10 116.80 (16) H13D---C13B---H13F 109.5 C9---C10---H10A 109.5 H13E---C13B---H13F 109.5 C9---C10---H10B 109.5 C9---N1---C1---C2 −179.87 (15) C4---C3---C8---N1 −179.69 (14) C8---N1---C1---C2 −0.04 (18) C2---C3---C8---N1 0.25 (18) N1---C1---C2---C3 0.17 (17) C9---N1---C8---C7 −0.8 (3) C1---C2---C3---C4 179.67 (16) C1---N1---C8---C7 179.34 (16) C1---C2---C3---C8 −0.26 (18) C9---N1---C8---C3 179.70 (14) C8---C3---C4---C5 −0.4 (2) C1---N1---C8---C3 −0.13 (18) C2---C3---C4---C5 179.70 (16) C8---N1---C9---O1 0.2 (3) C3---C4---C5---C6 −0.4 (2) C1---N1---C9---O1 −179.96 (16) C3---C4---C5---C11 177.16 (15) C8---N1---C9---C10 −179.55 (14) C4---C5---C6---C7 0.7 (2) C1---N1---C9---C10 0.3 (2) C11---C5---C6---C7 −176.83 (15) C6---C5---C11---C12 −125.81 (18) C5---C6---C7---C8 −0.3 (2) C4---C5---C11---C12 56.7 (2) C6---C7---C8---C3 −0.4 (2) C5---C11---C12---C13B 68.4 (6) C6---C7---C8---N1 −179.86 (15) C5---C11---C12---C13 179.9 (6) C4---C3---C8---C7 0.8 (2) C5---C11---C12---Cl1 60.3 (2) C2---C3---C8---C7 −179.27 (14) C5---C11---C12---Cl1B −172.81 (15) -------------------- -------------- ----------------------- -------------- Hydrogen-bond geometry (Å, °) {#tablewraphbondslong} ============================= -------------------- --------- --------- ----------- --------------- *D*---H···*A* *D*---H H···*A* *D*···*A* *D*---H···*A* C4---H4···O1^i^ 0.95 2.45 3.388 (2) 168\. C12---H12A···O1^i^ 0.96 2.44 3.388 (2) 169\. -------------------- --------- --------- ----------- --------------- Symmetry codes: (i) *x*, *y*−1, *z*. ###### Hydrogen-bond geometry (Å, °) *D*---H⋯*A* *D*---H H⋯*A* *D*⋯*A* *D*---H⋯*A* -------------------- --------- ------- ----------- ------------- C4---H4⋯O1^i^ 0.95 2.45 3.388 (2) 168 C12---H12*A*⋯O1^i^ 0.96 2.44 3.388 (2) 169 Symmetry code: (i) .

The author of this paper recently discussed the findings on cardiovascular safety of the controversial use of chloroquine and hydroxychloroquine for the treatment of COVID-19 reported in observational studies, stressing the need of high quality large randomized controlled trials in order to assess the effectiveness and safety of these drugs and other potential therapies for COVID-19.[@bib0001] One of the commented studies,[@bib0002] which reported a decrease in the in-hospital survival and an increased frequency of de-novo ventricular arrhythmias with the use of chloroquine or hydroxychloroquine, was recently retracted by 3 of the 4 authors, causing controversy in the scientific community and raising serious concerns on the reliability of published papers and the transparency and accountability of researchers particularly in the midst of this global health crisis. The reasons that lead the retraction of the aforementioned study as well as the analysis of other studies with implications for cardiovascular safety that have also been retracted or subjected to an expression of concern, are worthy of consideration. In a recent comment, Mehra et al[@bib0002] stated that after an unsuccessful attempt to conduct an independent peer review of the database on which their findings were based, they can no longer assure the veracity of their conclusions thus, they requested the retraction of their publication. Likewise, a different study conducted by Mehra et al[@bib0003] assessed the relationship of cardiovascular disease and drug therapy with in-hospital mortality among patients with COVID-19. In this study the authors reported no increased risk of in-hospital mortality associated with the use of angiotensin-converting--enzyme inhibitors and angiotensin-receptor blockers. However, in a subsequent letter the authors argued that they were unable to access to the raw data and the database was not available to a third-party auditor validation therefore, the authors asked for retraction of the paper.[@bib0003] At this time, 15 studies about COVID-19 have been retracted, 2 temporarily retracted and 1 subjected to an expression of concern.[@bib0004] The rush for showing results and publishing papers despite its lack of validation, as health professionals and patients desperately seek treatment options, illustrate the obvious need for strengthening the review process of papers for accuracy and reliability before publication and a call to follow the standards of the International Committee of Medical Journal Editors and the Committee on Publication Ethics. Considerations regarding veracity and scientific integrity are of utmost importance. As previously stated by the author of this paper, the current findings on efficacy and safety of the potential therapies for COVID-19 require validation from high-quality large randomized controlled trials.[@bib0001] Funding: None. Conflicts of interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

An optimally efficient anticancer therapy can be achieved by destroying each cancer cell. However, we know that a long-lasting disease-free survival is not easy to achieve, presumably because some tumor (stem) cells escape from therapy and may remain dormant for months, years and sometimes decades. One strategy that might improve therapeutic outcome relies on combination of potent cytotoxic chemotherapeutics and the induction of antitumor immune responses that control residual disease. At the theoretical level, chemotherapy and immunotherapy are difficult to be reconciled because chemotherapy with DNA-damaging agents often induces the massive destruction of immune effectors. Owing to this immunosuppressive side effect, it is intrinsically difficult to elicit immune responses against tumor antigens in patients after several cycles of chemotherapy.^[@bib1]^ Moreover, apoptosis seems to be the principal cell death mechanism induced by chemotherapy, and apoptosis is mostly viewed as a non-immunogenic (or even tolerogenic) cell death modality. Indeed, billions of cells succumb to apoptosis every day in healthy individuals yet do not provoke any signs of autoimmunity.^[@bib2]^ However, a recent series of papers has demonstrated that some chemotherapeutic agents, in particular anthracyclines and oxaliplatin, are unique in their capacity to induce immunogenic cancer cell death in mice. These studies unraveled the molecular mechanisms that distinguish immunogenic from non-immunogenic cell death. The immunogenicity of cell death relies on at least three independent events, namely (i) the early exposure of calreticulin (CRT) on the cell surface of stressed cells (ecto-CRT)^[@bib3]^ and the subsequent (ii) adenosine tri-phosphate (ATP)^[@bib4]^ secretion and (iii) high mobility group box 1 (HMGB1) release^[@bib5]^ by dying tumor cells. Membrane-exposed ecto-CRT favors the engulfment of the apoptotic bodies by dendritic cells (DCs), while HMGB1 and ATP modulate DC-mediated tumor antigen presentation and T-cell polarization.^[@bib4]^ Of note, the vast majority cytotoxic agents fail to induce CRT exposure, while only a few such as anthracyclines and oxaliplatin are able to induce ecto-CRT. Knockdown of CRT, blockade of ecto-CRT or inhibition of the pathway leading to CRT exposure abolishes the immunogenicity of cell death elicited by anthacyclins or oxaliplatin.^[@bib3]^ Normally, CRT is present in the lumen of the endoplasmic reticulum (ER). In response to specific ER stresssors, the eukaryotic translation initiation factor 2*α* (eIF2*α*) becomes phosphorylated, thus enabling the so-called integrated stress response that is linked to a translational arrest.^[@bib6]^ This eIF2*α* phosphorylation is also essential for CRT exposure because cells that lack the eIF2*α* kinase, hyperactivate the eIF2*α* phosphatase or contain a non-phosphorylatable eIF2*α* mutant fail to expose CRT in response to anthracyclines or oxaliplatin.^[@bib7],\ [@bib8]^ The induction of T cells responses against leukemic cells has been shown to ameliorate the therapeutic outcome in patients with acute myeloid leukemia (AML) who were treated with allogenic hematopoietic stem cells transplantation (HSCT).^[@bib9]^ The success of HSCT is linked to a specific immune response, the so-called graft-*versus-*leukemia reaction in which transplanted T cells recognize leukemia-specific antigens as well as minor histocompatibility antigens. It has been suspected, yet remains to be confirmed, that natural autologous immune response may influence the clinical course of AML. Thus, antibodies specific for leukemia-associated antigens (LAAs) have been described, for example, in promyelocytic leukemia.^[@bib10]^ Specific anti-leukemic T lymphocytes have been identified in a few studies involving AML patients.^[@bib11]^ It has been suspected that the presence of these specific autologous T lymphocytes predicts a favorable outcome.^[@bib11]^ Conversely, a high frequency of regulatory T cells (CD4^+^CD25^high^Foxp3^+^ Treg) is associated with a poor outcome of AML therapy.^[@bib12]^ As compared with normal volunteers, AML patients possess an elevated frequency of Treg cells, which have an activated phenotype (with an elevated expression of CTLA-4, GITR, perforin and granzyme B), inhibit conventional T-cell proliferation *ex vivo* via IL-10 and TGF*β*1, and have an elevated capacity to hydrolyze ATP (because of a high expression of the ecto-enzymes CD39 and CD73).^[@bib12]^ Driven by these premises, we examined ecto-CRT expression on malignant blasts from AML patients, before and after chemotherapy with anthracyclines. Importantly, we found that a therapy-independent CRT exposure correlated with the specific immune response of T lymphocytes against autologous blast-derived DC. These results underscore the clinical relevance of CRT exposure in the context of cancer immunology. Results ======= AML blasts can expose CRT (ecto-CRT) at their cell surface regardless of chemotherapy ------------------------------------------------------------------------------------- Anthracyclines are capable of inducing immunogenic cancer cell death in mice.^[@bib2]^ Obeid *et al.*^[@bib3]^ showed that anthracyclines induce the translocation of CRT from its orthotopic localization within the lumen of ER vesicles to the surface of tumor cells (ecto-CRT). Ecto-CRT is essential for the chemotherapy-mediated induction of adaptive immune responses and the rejection of tumors in mice. We therefore examined the membrane translocation of CRT on malignant blasts from patients treated with anthracyclines. The flow cytometry analysis of CRT expression on blast cells at the diagnosis, before any treatment, revealed significant (with a cut-off of 5%) expression of membrane CRT in 10 of the 20 patients ([Figure 1a](#fig1){ref-type="fig"}). The percentage of positive cells was rather variable from one patient to the other, ranging from 5 to 95%. In contrast, no upregulation of ecto-CRT expression was observed on malignant blasts after anthracycline-based chemotherapy ([Figures 1b and c](#fig1){ref-type="fig"}), except for two patients ([Figure 1b](#fig1){ref-type="fig"}). Similar results were found at different time points for several patients (data not shown), suggesting that CRT exposure reflects a stable alteration of blasts. Moreover, different leukemia cell lines (HL60, K562) or primary AML blasts incubated with anthracylines (either doxorubicin or idarubicin) *in vitro* for 1 to 4 h, failed to translocate and/or upregulate CRT to the plasma membrane (data not shown). Peripheral blood mononuclear cell (PBMC) from healthy volunteers (HVs) did not display any significant ecto-CRT expression, meaning that \<5% of normal lymphocytes or monocytes exposed CRT. No clinical or biological characteristic of the AML patients was significantly associated with ecto-CRT expression in terms of age, white blood count or the FAB type ([Table 1](#tbl1){ref-type="table"}). An ELISA assay, which was performed on 16 of the 20 patients\' sera, detected high concentrations of soluble CRT, compared with HVs, for 6 of the AML patients, all with high white blood counts. No significant correlation between the CRT concentration and CRT exposure on the membrane of the blasts could be highlighted (354±370 and 202±221 ng/ml for negative and positive ecto-CRT groups respectively, *P*\>0.05). Moreover, no change of CRT concentration before or after chemotherapy could be demonstrated (275±300 *versus* 180±200 ng/ml respectively, *P*\>0.05). In contrast, we detected a positive correlation between serum CRT levels and white blood cell counts (*r*=0.5699, *P*=0.0169), which may reflect the spontaneous mortality of malignant blasts or, alternatively, a nonspecific release of CRT from such cells. The phosphorylation of eIF2*α* is associated with CRT exposure of on the blasts membrane ---------------------------------------------------------------------------------------- The phosphorylation of eIF2*α* is a crucial event in the molecular pathway that dictates the translocation of ER-resident CRT to the cell surface,^[@bib8]^ we therefore analyzed the phosphorylation state of malignant blast from AML patients that show spontaneous CRT exposure and those that failed to expose CRT even after treatment. Immunodetection of the phosphoneoepitope using a specific antibody revealed that the spontaneous CRT exposure on malignant blasts was associated with a hyperphosphorylation of eIF2*α* ([Figure 1d](#fig1){ref-type="fig"}). CD47 expression on leukemic blasts correlates inversely with ecto-CRT --------------------------------------------------------------------- CD47 is an immunoglobulin-like protein that interacts functionally with integrins and thrombspondin-1 (Gao *et al.*^[@bib13]^) and that participates in functions as diverse as neutrophil migration,^[@bib14]^ axon extension^[@bib15]^ and T-cell costimulation.^[@bib16]^ In addition, CD47 is capable of interacting with its receptor signal-regulatory protein alpha (SIRP*α*)^[@bib17]^ on macrophages to negatively regulate phagocytosis. Blockade of CD47 expression results in phagocytosis of red blood cells,^[@bib18]^ as well as T cells and whole bone marrow cells in a transplant setting.^[@bib19]^ Thus, CD47 functions as a 'don\'t eat me\' signal to ensure that autologous cells are not inappropriately phagocytosed. Intriguingly, two recent studies revealed that CD47 is upregulated on leukemia cells^[@bib20]^ and represent an adverse prognosis factor in AML.^[@bib21]^ Confirming these data, we found that in our cohort that CD47 expression was more abundant on the surface of malignant blasts than on normal monocytes ([Figure 2a](#fig2){ref-type="fig"}). However, by comparing the density of CD47 expression between the two groups of patients that were either ecto-CRT^pos^ or ecto-CRT^neg^, we found that CD47 was weakly expressed among patients expressing ecto-CRT ([Figure 2b](#fig2){ref-type="fig"}). In conclusion, the 'eat me\' signal ecto-CRT correlated inversely with the expression of the 'don\'t eat me\' signal CD47 in AML. Ecto-CRT is associated with an enhanced cellular immune response against tumor antigens --------------------------------------------------------------------------------------- To determine the influence of ecto-CRT on anti-leukemic immune responses, we took advantage of DC derived from malignant blasts ([Figure 3a](#fig3){ref-type="fig"}). Such leukemic DCs (AML-DC) constitutively express tumor-associated antigens (TAAs)/LAAs as, for example, the antigen preferentially expressed in melanoma (PRAME), the Wilms tumor gene 1 or the receptor for hyaluronic acid-mediated motility (CD168), which all can elicit antigen-specific T-cell immune responses.^[@bib22],\ [@bib23],\ [@bib24]^ AML-DC can stimulate autologous T cells for proliferation, interferon-*γ* (IFN*γ*) secretion and cytotoxicity.^[@bib25]^ We comparatively assessed the capacity of autologous T cells to secrete IFN*γ* on exposure to AML-DC in ecto-CRT^pos^ and ecto-CRT^neg^ patients. Purified CD3^+^ T lymphocytes from ecto-CRT^pos^ patients did produce significant levels of IFN*γ* in responses to AML-DC ([Figures 3b and c](#fig3){ref-type="fig"}, middle panel, *P*=0.0078) but failed to do so in response to non-differentiated blasts ([Figures 3b and c](#fig3){ref-type="fig"}, middle panel, *P*\>0.05). This contrasts with the ecto-CRT^neg^ group, in which autologous T cells were unable to upregulate IFN*γ* production in response to AML-DC ([Figures 3b and c](#fig3){ref-type="fig"}, right panel, *P*\>0.05). Most HVs group exhibited a significant auto-reactivity of their T cells against autologous monocyte-derived DCs (mDCs; [Figures 3b and c](#fig3){ref-type="fig"}, left panel, *P*=0.0313). Altogether, these data suggest a significant link between ecto-CRT expression on malignant blasts at diagnosis and the induction of Th1 responses. Impact of ecto-CRT on the clinical evolution of patients -------------------------------------------------------- Considering the small effective and the heterogeneity of our cohort, we could not expect a significant association to one or another evolution parameter. Accordingly, the overall survival of the patients enrolled in this study did not correlate significantly with ecto-CRT levels ([Figure 4a](#fig4){ref-type="fig"}), although there was a trend toward longer relapse-free survival for the ecto-CRT^neg^ group ([Figure 4b](#fig4){ref-type="fig"}). As expected,^[@bib21]^ we found a trend for accelerated relapses in patients with high CD47 expression (mean fluorescence intensity (MFI) ≥100; median relapse-free survival for patient reaching complete remission after induction chemotherapy 11.23 months *versus* unreached for patients with low CD47 expression (MFI\<100)), although our cohort was too small to detect a significant effect of CD47 (*P*=5229). Five patients relapsed in the ecto-CRT^neg^ group (62.5%) *versus* two in the ecto-CRT^pos^ group (28.5%) with a median follow-up of 40 months among patients who had achieved a complete response after induction chemotherapy (*n*=7 for ecto-CRT^pos^ group and *n*=8 for ecto-CRT^neg^ group, [Table 2](#tbl2){ref-type="table"}). This trend in the relapse-free survival for ecto-CRT^pos^ patients prompts us to extend this study to a larger cohort. Discussion ========== This clinical study has explored the putative link between CRT cell surface expression and anthracycline-based chemotherapy in AML patients. Unexpectedly, we failed to produce evidence that anthracyclines would induce CRT on the surface of malignant blasts ([Figure 1](#fig1){ref-type="fig"}). Indeed, in half of the AML cases, leukemic cells spontaneously exposed CRT on their surface, correlating with the constitutive phosphorylation of eIF2*α* ([Figure 1d](#fig1){ref-type="fig"}). This suggests that an ER stress response that culminates in CRT exposure is constitutively activated in a fraction of AML. Half of patients\' tumor cells have activated this crucial mechanism favoring their uptake by antigen-presenting cells (APCs).^[@bib3],\ [@bib4],\ [@bib5]^ This hypothesis is strengthened by the observation that CD47 molecules are less abundant on the surface of ecto-CRT^pos^ than on ecto-CRT^neg^ blasts ([Figure 2](#fig2){ref-type="fig"}). Indeed, the reduced expression of the 'don\'t eat me\' signal (CD47), together with the enhanced expression of the 'eat me\' signal (ecto-CRT), suggests that leukemic cells are efficiently engulfed by APCs that then trigger an immune response against TAAs. Recent reports have highlighted the fact that SIRP*α* could represent a myeloid sensor for the 'self\' molecule CD47.^[@bib26]^ SIRP*α* is an inhibitory immunoreceptor that is selectively expressed by myeloid lineage cells (macrophages, granulocytes and myeloid DC), as well as neuronal cells.^[@bib27],\ [@bib28],\ [@bib29]^ The ligation of SIRP*α* on macrophages (or other phagocytic cells expressing this molecule) by CD47 expressed on non-phagocytic cells generates an SHP-1-dependent inhibitory signal that suppresses phagocytosis.^[@bib30]^ Thus, low expression of CD47 on malignant blasts should be correlated with a better engulfment by SIRP*α*-expressing cells. Moreover, ecto-CRT can favor the uptake of dying cells by DC, leading to efficient T-cell priming *in vivo*.^[@bib3]^ It is still unknown whether the candidate CRT receptors CD91 and scavenger receptor class-A are required for the DC-specific engulfment of CRT-exposing tumor cells.^[@bib31]^ Taken together, these data provide a rational to suggest that in AML, leukemic cells that express ecto-CRT and low levels of CD47 could be engulfed by APCs thus favoring an adaptive immune response against tumor antigens. Interestingly, in the subgroup of patients who failed to express ecto-CRT at the baseline level, anthracyclines were unable to induce ecto-CRT either *in vivo* ([Figure 1d](#fig1){ref-type="fig"}) or *in vitro* (data not shown). This suggests that these tumor cells have a defective CRT exposure pathway ([Figure 1d](#fig1){ref-type="fig"}) that could not be induced and/or restored by induction chemotherapy. In analogy, a previous observation was made on neuroblastoma cells that were also refractory to anthracyclin-induced CRT exposure unless the ER Ca^2+^ channel SERCA was blocked.^[@bib32]^ Although we were unable to identify any effect of anthracyclines on CRT exposure on leukemic cells, we tested the hypothesis that ecto-CRT might be linked to antitumor Th1 immune responses. As shown in [Figure 3](#fig3){ref-type="fig"}, this link exists. Indeed, purified T lymphocytes from the ecto-CRT^pos^ group of patients were capable of secreting IFN*γ* in response to autologous leukemic DCs, while ecto-CRT^neg^ group of patients failed to do so. It is to be noted that lymphocytes were isolated at diagnosis before any treatment, demonstrating that this antitumor immune response occurred 'naturally\' (that is before treatment) in AML. How can the differences between ecto-CRT^pos^ and ecto-CRT^neg^ patients with respect to the antitumor immune response be explained? The 'eat me\' and 'don\'t eat me\' signals expressed on the surface of tumor cells might have a crucial role in determining the nature of APC, thus ultimately influencing the polarization of the immune response. Indeed, it is well accepted that distinct DC subtypes not only control immunity but also regulate the advent and the type of responses against self and non-self.^[@bib33]^ In humans, some DC sub-populations have been proposed to secrete high levels of IL-12, thereby favoring Th1 polarization.^[@bib34]^ Human DC selectively expressing 6-sulfo LacNAc, an O-linked carbohydrate modification of PSGL-1, have recently been identified in the blood and have been nicknamed 'slanDC\' in reference to their specific marker (6-sulfo LacNAc).^[@bib35],\ [@bib36]^ SlanDC account for the majority of DC in human blood (0.6--2% of PBMCs) and have a phenotype (6-sulfo LacNAc+, CD1c-, CD11c+, CD16+, CD142, C5aR+ and CD45RA+) that differs from that of previously described DC sub-populations.^[@bib37]^ SlanDC are the quantitatively most abundant IL-12p70-producing cells among blood leukocytes. These findings emphasize the probable role of slanDC in the induction and perpetuation of inflammation, as this is also suggested by the high frequency of slanDC in diseases characterized by local Th1-dominated inflammatory response, such as psoriasis vulgaris and rheumatoid arthritis. Interestingly, the maturation of and IL-12 production by slanDC is controlled by CD47 expressed on erythrocytes and SIRP*α* expressed on slanDC.^[@bib35]^ Hence, slanDC might represent a good candidate for the APC that engulfs CD47^low^ leukemia cells. Nonetheless, the expression of CRT receptors (such as CD91 and scavenger receptor class-A), on slanDC, remains to be studied. It should be noted that another human DC subtype, DC NK lectin group receptor-1^+^ blood DC antigen-3^+^ DCs has recently been identified.^[@bib38]^ This DC subtype efficiently internalizes antigenic material from necrotic cells, produces high levels of IL-12 and cross-presents exogenous antigens to CD8^+^ T cells, thus representing the human equivalent of murine CD8*α*^+^ DC.^[@bib38]^ However, the relationship between these DC subtypes and their capacity to internalize tumor cells expressing ecto-CRT remains elusive. To conclude, we have identified cell surface exposure of CRT on primary blasts from approximately half of the AML patients. This CRT exposure occurs before chemotherapy and is not further enhanced by anthracycline-based chemotherapy. Conversely, half of the patients\' blasts lacked CRT exposure both before and after anthracyline treatment. We demonstrated for the first time a link between ecto-CRT on the surface of malignant blasts and the secretion of IFN*γ* by autologous T lymphocytes confronted with them. On the basis of these encouraging results, we will launch a large prospective study to determine the prognostic and predictive impact of ecto-CRT expression and that of its surrogate marker, eIF2*α* phosphorylation. Patients and Methods ==================== Patients -------- Twenty patients, who were diagnosed with AML, were consecutively admitted in the Hematology Department of the Institut Gustave Roussy in Villejuif, France, between March 2008 and March 2009. Informed consent was obtained, according to the Declaration of Helsinki, and the study was approved by the local ethics committee (Comité de Protection des Personnes (CPP) Hôpital Bicêtre -- *CALEX* protocol, n° ID RCB 2007-A01074-49, date 29 February 2008). The main clinical and biological characteristics of the patients are summarized in [Table 3](#tbl3){ref-type="table"}. Induction chemotherapy comprised one anthracycline (idarubicine or daunorubicine) associated with cytarabine (except for two patients: one not eligible for an intensive chemotherapy, and one who died in intensive care unit before initiation of the treatment), followed by one to four courses of consolidation. One patient, who was diagnosed with promyelocytic leukemia, also received ATRA. An allograft of hematopoietic stem cells was performed for eligible patients (relapse situation and high risk of relapse, based on cytogenetic and biomolecular results), for whom a (genotypically or phenotypically) compatible donor was found. Blood samples ------------- Peripheral blood samples were obtained before the onset of chemotherapy in all patients. A second sample was drawn 2 to 6 h after the initiation of the intravenous chemotherapy. PBMC were separated on a Ficoll-gradient. Flow cytometry -------------- PBMCs were first fixed briefly in 0.25% paraformaldehyde on ice. Cells were directly labeled with an anti-CD45 monoclonal antibody (coupled to APC, BD Biosciences, Le Pont de Claix, France). CD45 is highly expressed on normal hematopoietic cells, yet poorly expressed on malignant blasts (CD45^dim^). The labeling of the membrane CRT was performed in two-step procedure: (i) incubation with a rabbit anti-CRT antibody (Ozyme, Saint-Quentin-en-Yvelines, France) (or rabbit serum as an isotypic control), then, after a washing step in cold PBS, (ii) labeling with an anti-rabbit immunoglobulin coupled to FITC (BD Biosciences). All these steps were performed on ice. The labeled PBMCs were analyzed on a FACS Calibur cytofluorometer (Becton Dickinson, Le Pont de Claix, France) with a simultaneous discrimination of the live--dead cells using 7AAD (amino-actinomycin D, BD Biosciences). CD47 expression was measured using an anti CD47-phycoerythrine conjugate (BD Biosciences) while gating on CD45^high^ and CD45^dim^ cells in order to discriminate normal from blast cells. FACS data were analyzed with the help of FlowJo software (Tree Star, Inc., Ashland, OR, USA). ELISA assay for the dosage of soluble CRT in sera of patients ------------------------------------------------------------- A sandwich ELISA assay was performed to detect CRT in the sera of patients before and after chemotherapy. Briefly, anti-CRT polyclonal (rabbit anti-CRT, Ozyme) diluted to 1/1000^e^ in PBS (pH=7.2) supplemented with 100 mM bicarbonate, was incubated in 96-well Maxisorp plates at 4 °C overnight. After washing (PBS supplemented with 0.05% Tween), PBS 2% human serum albumin was added for 1 h for saturation. After washing, samples (diluted 1 : 10 in PBS plus 2% albumin) were added to the wells for 2 h at room temperature. After washing, a secondary anti-CRT (mouse anti-CRT, Abcam, Paris, France; dilution 1/5000^e^) was added (1 h at room temperature). A goat anti-mouse antibody coupled to horseradish peroxidase (Jackson Immunoresearch, Marseille, France; dilution 1/5000^e^) was added for 1 h at room temperature and subsequently washed. Revelation was done using TMB substrate (tetramethylbenzidine OptEIA, BD Biosciences). The reaction was stopped with 50 *μ*l of 1 M H~2~SO~4~, and the optical density was measured at 450 nm. Patient samples were analyzed in triplicate and compared with sera from HVs. Leukemic DCs ------------ DCs were derived from PBMC as previously described.^[@bib39]^ Briefly, PBMCs were incubated at 37 °C in a flask at 1.10^6^ cells/cm^2^ in culture medium (AIM-V, Invitrogen, Cergy Pontoise, France); after 2 h, the supernatant was discarded, and the adherent cells (mostly blasts cells) were cultured for 4 days in AIM-V medium containing IL-4 (200 IU/ml, Cellgenix, Clermont L\'Herault, France) and GM-CSF (1000 IU/ml, Cellgenix). Lipopolysaccharide (1 *μ*g/ml, Sigma, Lyon, France) was added on the last day before collection to induce DCs maturation. The cells were collected and their immunophenotype was checked by immunofluorescene and flow cytometry to confirm the presence of DC markers (anti-CD11c PC7, anti-HLA-DR Pacific Blue, anti-CD40 FITC, anti-CD80 PE, all from BD Bioscience). Isolation of T lymphocytes -------------------------- PBMCs from the diagnosis (before treatment) were frozen after collection and isolation by Ficoll density gradient centrifugation. After thawing, cells were labeled with anti-CD3 antibody (coupled to FITC, BD Bioscience), and their viability was assessed using 7AAD. The labeled cells were washed and sorted using a cell sorter cytometer (MoFlow, Beckman, Villepinte, France), while discriminating the cells based on their size, granularity and fluorescence (CD3^+^ and 7AAD^−^) allowing for the isolation of \>95% pure T lymphocytes. *In vitro* stimulation of T lymphocytes --------------------------------------- The leukemic DCs were incubated with autologous T lymphocytes (LT) in 96-wells plates in triplicates, at a LT/DC ratio of 10 to 1. IL-2 (10 IU/ml, Proleukine from Novartis, Rueil-Malmaison, France) and IL-7 (5 ng/ml, R&D system, Minneapolis, MN, USA) were added in the medium. As a control, mDCs from HVs were incubated with autologous T lymphocytes. After 2 days, supernatants were collected and frozen at −80 °C until measurement of IFN*γ* by ELISA. We managed to realize this stimulation protocol for 15 of the 20 patients (*n*=8 for the ecto-CRT-negative group and *n*=7 for the ecto-CRT-positive group of patients); for the five other patients, we were unable to derive DC from blasts or failed to obtain a sufficient number of T lymphocytes from peripheral blood because of a major lymphopenia at diagnosis. The control experiment was performed on five HVs. Statistical analyses were performed using the Wilcoxon test for non-parametric paired values, with the help of the Graphpad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA). Western blot analysis --------------------- Frozen blasts were thawed and washed with PBS before lyses as previously described.^[@bib40]^ The protein content was measured using the Protein Assay Kit (Bio-Rad, Marnes-la-Coquette, France) following the manufacture\'s instruction and 50 *μ*g of protein were separated onto NuPAGE gels (Invitrogen) and transferred to Immobilion-PVDF membranes (Millipore Corp., Billerica, MA, USA). The membranes were incubated for 1 h in TBS-Tween 20 (0.05%) containing 5% BSA. Primary antibodies specific for phosphorylated eIF2*α* (Cell Signaling Technology, Danvers, MA, USA) and eIF2*α* (Cell Signaling Technology) were incubated overnight at 4 °C and revealed with the appropriate horseradish peroxidase-labeled secondary antibodies (SouthernBiotech, Birmingham, AL, USA) by means of chemoluminiscent substrate (Pierce, Rockford, IL, USA). Confocal microscopy ------------------- Primary cells from an AML patient have been washed with PBS before immunostaining using anti-CD45 antibody (FITC coupled from BD Bioscience). The cells have been washed with PBS and subsequently fixed with 0.25% of PFA before staining of cell surface CRT using mouse monoclonal antibody (clone fmc75, ab22683, Abcam) followed by anti-mouse AlexaFluor 568 (Molecular Probes (Cergy Pontoise, France), Invitrogen) in PBS supplemented with 5% BSA. The nuclei were stained with DAPI before image acquisition by means of a TSC-SPE confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany) equipped with a 63X/1.15 objective (Olympus America, Center Valley, PA, USA). Signals from different probes were acquired in sequential scan mode and overlays have been done with Photoshop. We acknowledge support from la Fondation de France contre la Leucémie, Institut National de la Santé et de la Recherche Médicale (INSERM), Institut Gustave Roussy, l\'Institut National du Cancer (INCa), Ligue Nationale contre le Cancer (équipe labellisé by L Zitvogel), Association pour la Recherche sur le Cancer (ARC), Fondation pour la Recherche Médicale en France (FRM). TP is supported by the Swedish Cancer Society, the Children\'s Cancer Society, the Swedish Royal Academy of Sciences and the Åke Wiberg foundation. CRT : calreticulin DC : dendritic cell APC : antigen-presenting cell PBMC : peripheral blood mononuclear cell HMGB1 : high mobility group box 1 ATP : adenosine tri-phosphate SIRP*α* : signal-regulatory protein alpha The authors declare no conflict of interest. {#fig1} {#fig2} {#fig3} {#fig4} ###### Clinical and biological characteristics in CRT^pos^ and CRT^neg^ group of patients   **All** **CRT^pos^ (*n*=10)** **CRT^neg^ (*n*=10)** ***P-*value** -------------------------------------------- --------- ------------------------------ ------------------------------ --------------- Sex (m/f), *n* 9/11 5/5 4/6 \>0.5 Median age at diagnosis, years (range) 62 62 (26--77) 61 (28--82) \>0.5 Median blasts in blood, % (range) 51 51 (3--95) 48.5 (5--85) \>0.5 Median WBC ( × 10^9^/l) 27 35.5 (2.8--215) 22 (5--84) \>0.5           De novo *AML*, n 16 8 7 \>0.5  Of which *n* relapse AML at the inclusion 3 1 2 \>0.5           *Secondary AML*  MPS/MDS, *n* 2 1[a](#t1-fn2){ref-type="fn"} 1[a](#t1-fn2){ref-type="fn"} \>0.5  Therapy related, *n* 3 1[b](#t1-fn3){ref-type="fn"} 2[b](#t1-fn3){ref-type="fn"} \>0.5           *FAB classification*  M0 1 0 1 \>0.5  M1 3 1 2 \>0.5  M2 3 2 1 \>0.5  M3 1 0 1 \>0.5  M4 7 4 3 \>0.5  M5 5 3 2 \>0.5  M6, M7 0 0 0 \>0.5           FLT3 ITD, *n* 4 3 1 \>0.5           *Cytogenetic profile*  Favorable 4 1 3 \>0.5  Intermediate 9 6 3 \>0.5  Unfavorable 5 1 4 \>0.5  unknown 2 2 0 \>0.5 Abbreviations: AML, acute myeloid leukemia; ecto-CRT^neg^, no calreticulin expression; ecto-CRT^pos^, membrane expression of calreticulin; MPS/MDs, myeloproliferative/myelodysplastic syndrome Two patients with chronic myelomonocytic leukemia Previous chemotherapy and radiotherapy for throat and breast cancers ###### Clinical outcome   **All** **CRT (+) (*n*=10)** **CRT (−) (*n*=10)** -------------------------------------------------------------------- ------------ ---------------------- ------------------------------ Intensive chemotherapy, *n* 18 9 9 CR, *n* 15 7 8         *Consolidation in CR 1*  Chemotherapy only, *n* 11 5 6  HSC allograft, *n* 4 2 2 Relapse, *n* 6 2 4         *Death total,* n 8 4 4  Death without CR, *n* 5 3 2  Death after relapse, *n* 2 1 1  Death of other cause, *n* 1 0 1[a](#t2-fn2){ref-type="fn"} Median follow-up, month (range) 13 (1--42) 29 (1--42) 10.3 (2--30) Relapse-free survival in (CR) patients (*n*=15): median, month^\*^   32.5 10.3 Abbreviations: CR, complete remission; HSC, hematopoietic stem cell Death of infectious cause during consolidation course ^\*^Relapse-free survival: survival without relapse for patients who achieved CR (*n*=15) ###### Baseline clinical and biological characteristics of the patients **Variable**   -------------------------------------------- ---------- *Age*  \<50 years 7 (35%)  ≥50 years 13 (65%)  Median (years) 62  Range (years) 26--82     *Sex*  Male 9 (45%)  Female 11 (55%)     *Peripheral-blood white cell count*  \<30.000/mm^3^ 10 (50%)  ≥30.000/mm^3^ 10 (50%)  Median (10^9^ cells/l) 180  Range (10^9^ cells/l) 2.8--215     *Blasts peripheral blood*  Median (%) 51  Range (%) 3--85     *De novo AML,* n 16 (80%)  Of which *n* relapse AML at the inclusion 3 (15%)     *Secondary AML*  MPS/MDS, *n* 2 (10%)  Therapy related, *n* 3 (15%)     *FAB classification*  AML 0 1 (5%)  AML 1 3 (15%)  AML 2 3 (15%)  AML 3 1 (5%)  AML 4 6 (30%)  AML 5 6 (30%)  AML 6 0 (0%)  AML 7 0 (0%)     *Cytogenetic profile*  Favorable 4 (20%)  Intermediate 9 (45%)  Unfavorable 5 (25%)  Missing data 2 (10%)     *Molecular characteristics*  Isolated FLT3-ITD 3 (15%)  FLT3-ITD and NPM^\#^ 1 (5%)  AML1/RUNX1 1 (5%) Abbreviations: AML, acute myeloid leukemia; AML1/RUNX1, runt-related transcription factor 1; FAB, Franco--Americano--British classification; FLT3-ITD, FLT3 internal tandem duplication; MPS/MDs, myeloproliferative/myelodysplastic syndrome; NPM, nuclephosmine; WBC, white blood count ^\#^One patient presented a NPM (nuclephosmine) mutation, but associated with FLT3-ITD

Background {#Sec1} ========== Radioactive fluorodeoxyglucose (2-\[^18^F\]-fluor-2-deoxy-D-glucose, or ^18^F-FDG) is a radiopharmaceutical widely used in PET/CT (positron emission tomography/computed tomography) exams. ^18^F-FDG is captured by glucose transporters, abundant in neoplastic cells due to their high metabolism, but not consumed by cells and therefore remain in the cytoplasm \[[@CR19]\]. Radioactive fluorine (^18^F) is a positron emitter with a half-life of approximately 110 min. The positron travels a short distance in tissues or water before consuming its kinetic energy and combining with an electron. The interaction, named positron annihilation, results in the simultaneous emission of two photons with both having a high specific energy (511 keV) \[[@CR2]\] that increases the risk to occupationally exposed individuals \[[@CR33]\]. High doses of ionizing radiation (IR) can have deleterious consequences in humans, like the development of cardiovascular disease and cataracts \[[@CR6], [@CR28], [@CR41]\], in addition to producing reactive oxygen species and inducing DNA damage \[[@CR27], [@CR34], [@CR45]\]. The genotoxic effects of IR can cause genomic instability and mutations that lead to the development of cancer in exposed individuals \[[@CR27], [@CR37], [@CR43]\]. Professional radiation protection/shielding is an important method of protection against IR exposure. Protective devices are used in hot laboratory areas during the preparation and handling of radiopharmaceuticals. They are also used when injecting patients with radiopharmaceuticals through syringes or vial shields. These shields are typically made of lead, tungsten, or lead-coated steel. Studies have shown the unprotected radiation dose to be 10 to 20 times higher than the exposure under lead shielding \[[@CR1], [@CR26], [@CR30], [@CR46]\]. The choice of leaded or unleaded aprons, apron thickness, and durability according to the manufacturer's warranty are all important factors to consider. The biggest drawback of protective equipment despite the radiological protection is the heavy weight (especially of those containing lead), which can cause back pain, discomfort, and muscle fatigue, and thus reducing ergonomics. The development of new, more ergonomic shields that provide radiological protection similar to lead aprons could contribute to a solution. Polyvinyl chloride (PVC) consists of long carbon chains, where every carbon atom has a chlorine atom attached to it. PVC is one of the most widely used plastic polymers in the world. Its widespread use in industry is due to its low cost and versatility, with applications ranging from thermoplastics and thermosets, to elastomeric shapes \[[@CR40]\]. Considering the importance of constant radiological biomonitoring in individuals occupationally exposed to IR, the cytokinesis-block micronucleus (CBMN) assay in human peripheral blood lymphocytes has become one of the most commonly used tests to measure numerical and structural chromosomal alterations in human cells in vitro and in vivo \[[@CR22], [@CR36]\]. This assay is a reliable test for assessing radiation-induced chromosome damage and is a valuable biomarker in many biomonitoring studies among individuals that are occupationally or environmentally exposed to IR \[[@CR13], [@CR16], [@CR34], [@CR37]\]. Thus, the objective of this study was to evaluate the radiological protection efficacy of a plastic polymer, in the presence or absence of a lead shield, against the toxicogenomic effects of ionizing radiation emitted by the radiopharmaceutical ^18^F-FDG in human lymphocytes, using the CBMN assay. Methods {#Sec2} ======= Measuring instruments and shields {#Sec3} --------------------------------- Geiger-Müller radiation monitors (which determine the number of counts per minute), model MIR-7028 (MRA Electronic Equipment Industry, Brazil), and Inspector Alert model Nuclear Radiation (International Medcom, USA), which are often used to evaluate radiation levels in workplaces \[[@CR7]\], were used. The monitors were calibrated to measure the equivalent dose (μSv/h) and counts per minute (CPM) rates under standard conditions, according to the National Nuclear Energy Commission (CNEN), Brazil. A benchmarking test was performed to verify the compatibility between the dose rate reading and the expected nominal value for the ^137^Cs. Two types of shields were evaluated, the first consisting of a lead shield (1 mm), similar to that found in lead aprons, and the second of a PVC polymer shield (0.5 mm). Both were tested alone or in combination. Shielding test {#Sec4} -------------- The radioactive activity of the radiopharmaceutical ^18^F-FDG (A = 4.90, 2.60, and 0.68 mCi) was measured at distances of 5, 10, 20, and 60 cm under various shield conditions next to a Geiger-Müller detector, as follows: in the absence of shielding, shielding with lead, shielding with polymer, shielding with polymer + lead, or shielding with lead + polymer. Figure [1](#Fig1){ref-type="fig"} represents the lead + polymer shielding scheme. Fig. 1Schematic illustration of the lead + polymer shielding test Chemicals {#Sec5} --------- RPMI 1640 culture medium (R6504) and cytochalasin-B (C6762) were purchased from Sigma (USA). Fetal bovine serum (12657), phytohemagglutinin (10576), and antibiotics (penicillin and streptomycin - 15,140,122) were purchased from Gibco (USA). Giemsa dye was purchased from Merck (Germany). ^18^F-FDG (lot \#FDG001686) was purchased from IBF Brazilian Pharmaceutical Industry (Brazil), which was registered and authorized for distribution of this radiopharmaceutical (CNEN 0029017459/2018). All other reagents used were obtained from laboratories in Brazil. Selection of volunteers {#Sec6} ----------------------- This work was carried out at the Mutagenesis Laboratory (CNEN Registry 15,012) of the Department of Biotechnology, Genetics, and Cell Biology at the State University of Maringá. All experimental procedures were approved by the Committee on Ethics in Human Research. The purpose, scope, benefits, risks, and procedures of the study were explained to each participant and informed consent was obtained from each donor. Three healthy male donors, aged 22, 28, and 30 years old, were voluntarily recruited to observe the spontaneous and induced frequencies of DNA damage (MN -- micronucleus, NPB -- nucleoplasmic bridge, and NBUD -- nuclear bud) and cytokinesis-block proliferation index (CBPI). The subjects had no history of chronic disease, smoking, chemical abuse, or exposure to toxic substances. No radiation exposure or viral infection for 6 months before the study was documented. Peripheral blood samples (maximum of 20 mL for each donor) were collected by venipuncture and placed in heparinized tubes. Two collections were made 30 days apart. Lymphocyte culture and cytokinesis-block micronucleus assay {#Sec7} ----------------------------------------------------------- Leukocytes isolated from whole peripheral blood (500 μL) by simple decantation (3 h) were initially added to RPMI 1640 medium supplemented with fetal bovine serum (10%), phytohemagglutinin (1%), and antibiotics, and then incubated at 37 °C and 5% CO~2~ for 4 h for stabilization. After exposure to ^18^F-FDG, according to the irradiation protocol described in section 2.6, the cultures were returned to the incubator. The CBMN assay was performed as described by Fenech and Morley \[[@CR12]\] and certified by the Organization for Economic Co-operation and Development \[[@CR29]\]. Cytochalasin-B (6 μg/mL) was added to prevent cytoplasm division after 44 h of phytohemagglutinin stimulation, and cells were harvested within 72 h. Lymphocytes were fixed in a methanol-acetic acid solution. The slides were drip mounted, air-dried, and stained with Giemsa (5%) for 5 min. Irradiation of cultures {#Sec8} ----------------------- Following 4 h of stabilization, the lymphocyte cultures were exposed to a glass vial containing 20 mL of 300 mCi ^18^F-FDG, at a distance of 10 cm for 10 min in an acrylic shelf-mounted device. Under these conditions, the dose absorbed by the lymphocytes' culture was mathematically estimated at 0.06 Gy. This device intended to simulate the real-world conditions of occupational exposure (mainly for hands, forearms, and eyes during manipulation, and lower limbs during radiopharmaceutical transport and positioning of the patient for examination) in the radiology laboratory. The cultures were divided into the following groups: Negative control: no exposure to IR;^18^F-FDG unshielded (positive control): direct exposure to IR;^18^F-FDG + lead: the culture was protected with a 1-mm lead shield;^18^F -FDG + polymer: the culture was protected with a 0.5-mm polymer shield;^18^F -FDG + lead + polymer: the culture was simultaneously protected with a 1-mm lead shield at first, and then by 0.5 mm polymer shield (a schematic illustration is provided in Fig. [2](#Fig2){ref-type="fig"}).Fig. 2Schematic illustration of the lead + polymer shielding during exposition to ionizing radiation Analysis criteria {#Sec9} ----------------- Double-blinded microscopic analysis was performed using a 400-magnification light microscope (Leica DM750). All slides were analyzed for the total number of MN, NPB, and NBUD per 1000 binucleated cells, as well as for the total number of micronucleated cells, according to the criteria previously described by Fenech \[[@CR14]\]. The frequency of binucleated cells containing one or more MN was also determined. Only binucleated cells with a well-preserved cytoplasm were scored. The frequencies of mononucleated, binucleated, and polynucleated cells were also counted at 500 cells per individual. The CBPI was calculated on the same slides using the formula: \[M1 + 2 M2 + 3 (M3 + M4)\]/1000, where M1-M4 represented the number of cells with one to four nuclei, respectively, and M3 and M4 were equally considered in their third division cycle \[[@CR16], [@CR23]\]. Statistical analysis {#Sec10} -------------------- Statistical analysis was performed using the GraphPad Prism 5 software. Unpaired t-test was used to compare the number of MN, NPB, and NBUD between the different groups after irradiation. The Kolmogorov-Smirnov test was used to determine the normal distribution of the data. Results and discussion {#Sec11} ====================== As observed in the shielding test (Table [1](#Tab1){ref-type="table"}) of the IR emitted by the ^18^F-FDG, the lead shield was not effective in reducing the number of counts registered by the radiation monitor. There was an increase in counts for all activities at all distances, except for A = 0.68 mCi, which recorded 4000 counts per minute without shielding, and 3800 with the lead shield, at 60 cm. This increase in counts was due to the photoelectric effect, which is predominant for energies less than 600 keV and chemical elements with a high atomic number, such as lead (Z = 82) \[[@CR5], [@CR39]\]. When the IR reaches the lead shield, the transfer of energy from the ionization electron to the material medium produces a proportional secondary ionization that expresses the incident radiation energy at the end of the process. Due to this increase in counts recorded by the radiation monitor behind the shield, it is expected that the individual exposed to the ^18^F-FDG, for example, will not use any type of lead protection equipment when handling radiopharmaceuticals, increasing exposure to IR. The polymer + lead shield fluctuated in the increase and decrease of the counts. The polymer and lead + polymer shields reduced the counts for all the radioactive activities and distances analyzed, highlighting the novelty of this study. Previous experiments showed a similar efficacy of 0.5 mm and 1 mm polymer thickness (data not presented). Table 1Absolute values (counts per minute ✕ 1000) measured in a Geiger-Müller detector for the radiopharmaceutical ^18^F-FDG, with lead and polymer shields^**18**^F-FDGDistanceUnshieldedLeadPolymerPolymer + LeadLead + Polymer**A =4.90mCi***5 cm*satsatsatsatsat*10 cm*satsatsatsatsat*20 cm*225229,9185226,8171,9*60 cm*3034,827,431,824,7**A =2.60mCi***5 cm*satsatsatsatsat*10 cm*satsat266sat252*20 cm*134142106,213496,8*60 cm*26,828,421,22720,8**A =0.68mCi***5 cm*163176130,2172,4121,3*10 cm*8990,473,38665,1*20 cm*343825,33522,6*60 cm*43,83,83,83,5A: radioactive activity of the element. Sat: saturation of the monitor, making measurement impossible These findings support the simulation performed by Fonsêca et al. \[[@CR15]\]. Using the Monte Carlo N-Particle method, they demonstrated that the use of a 0.5-mm thick lead apron increased the dose absorbed by the individual exposed to IR of the ^18^F-FDG by up to 26%, instead of a reduction, depending on the distance between the radiation source and the individual. This increase occurred due to the generation of secondary and scattered particles from the interaction of the incident photons (511 keV) with the lead apron, which would not exist without the apron. Consequently, these new ionizations led to an increased flow of photons and electrons that increased the dose absorbed by the individual. The CBMN assay results (Table [2](#Tab2){ref-type="table"}) indicated a statistically significant increase in the frequency of MN in binucleated cells (*p* \< 0.0001), compared to the negative control, where the lymphocyte cultures were exposed to the ^18^F-FDG radiopharmaceutical without shielding. Shielding significantly reduced the MN frequencies (lead *p* = 0.0001, polymer *p* = 0.0133, and lead + polymer *p* \< 0.0001) compared to the unshielded group. Similarly, for the NPB and NBUD frequencies, a higher induction of DNA damage was observed in cultures exposed to IR (*p* \< 0.0001 and *p* = 0.0026, respectively) compared to the negative control. The polymer shield differed from the negative control (NPB *p* = 0.0171 and NBUD *p* = 0.0442), despite reducing the incidence of damage (NPB *p* = 0.0001 and NBUD *p* = 0.0108). The lead shield (NPB *p* = 0.6877 and NBUD *p* = 0.4033) and the lead + polymer shield (NPB *p* = 0.1773 and NBUD *p* \> 0.9999) significantly reduced IR-induced damage to the level observed in the negative control (Fig. [3](#Fig3){ref-type="fig"}). Table 2Induction of micronuclei by ionizing radiation of ^18^F-FDG in human lymphocytes in vitroShieldingDistribution of BNC according to the number of MNMN/BNC ± SD (%)CMN/BNC ± SD (%)NPB/CBN ± SD (‰)NBUD/CBN ± SD (‰)CBPI ± SD*1234*Negative Control141001.55 ± 0.191.48 ± 0.181.00 ± 0.630.83 ± 0.752.09 ± 0.05^18^F-FDG Unshielded533227.13 ± 0.70^a^5.92 ± 0.68^a^5.83 ± 0.75^a^4.67 ± 1.75^a^1.60 ± 0.06^a18^F-FDG + Lead392114.78 ± 0.33^ab^4.22 ± 0.19^ab^1.17 ± 0.75^b^0.50 ± 0.55^b^1.99 ± 0.03^ab18^F-FDG + Polymer463116.02 ± 0.20^ab^5.10 ± 0.14^ab^2.50 ± 1.05^ab^1.83 ± 0.75^ab^1.88 ± 0.04^ab18^F-FDG + Lead + Polymer381104.30 ± 0.23^ab^3.98 ± 0.23^ab^1.50 ± 0.55^b^0.83 ± 0.75^b^2.06 ± 0.04^b^Number of binucleated cells analyzed for each individual in each repetition = 1000*BNC* binucleated cells, *MN* micronucleus, *CMN* cell with one or more micronuclei, *NPB* nucleoplasmic bridge, *NBUD* nuclear bud, *CBPI* Cytokinesis-Block Proliferation Index^a^ Statistically significant difference from the negative control (*p* \< 0.05)^b^ Statistically significant difference from the unshielded group (*p* \< 0.05)Fig. 3Cell proliferation and DNA-induced damage in human peripheral blood lymphocytes. **a** mononucleated cell. **b** binucleated cell. **c** polynucleated cell. **d** binucleated cell with one micronucleus. **e** binucleated cell with two micronuclei. **f** binucleated cell with nucleoplasmic bridge. **g** binucleated cell with nuclear bud. Magnification: 1000✕ The increase in the frequency of MN, NPB, and NBUD in lymphocytes reflects the chromosomal instability induced by IR. MN can be formed during anaphase when an acentric chromosome, or chromatid fragments from DNA repair failures are not incorporated into the cell's main nucleus. NPB instead originates from dicentric chromosomes, which can occur due to the inadequate repair of breaks in DNA or terminal fusions of telomeres. NBUD represents the process of eliminating amplified DNA, complex DNA repair mechanisms, possible chromosomes of aneuploid cells, and may also result from NPB disruption \[[@CR9]--[@CR11], [@CR16], [@CR22], [@CR36]\]. The direct interaction of IR with DNA induces single and double-strand breaks, changes in nitrogen bases, and DNA cross-linking \[[@CR27], [@CR45]\]. The genotoxic effects of IR may cause genomic instability and mutations that lead to cancer induction in exposed individuals \[[@CR27]\]. Several studies have reported increased levels of chromosomal aberrations in lymphocytes from workers occupationally exposed to IR, compared to those from unexposed workers \[[@CR3], [@CR8], [@CR21], [@CR25], [@CR35], [@CR42], [@CR47]\]. Additionally, some epidemiological studies have revealed that individuals who are occupationally exposed to IR may have an increased risk of developing leukemia and other cancers \[[@CR4], [@CR18], [@CR20], [@CR38], [@CR44]\]. The CBPI analysis (Table [2](#Tab2){ref-type="table"}) is a useful tool to understand the cell cycle kinetics of lymphocyte cultures, especially after exposure to IR \[[@CR32], [@CR36]\]. Evaluation of this parameter in the present study indicated that direct exposure to IR had a cytotoxic effect on lymphocytes, compared to the negative control (*p* \< 0.0001). The polymer (*p* \< 0.0001) and lead (*p* = 0.0031) shields were not effective in protecting lymphocytes from the cytotoxic effects of radiation, compared to the negative control. Only the lead + polymer shield was effective in protecting the cells, since the CBPI of this group did not present a statistically significant difference compared to the negative control (*p* = 0.2903). The results of the CBMN assay are consistent with the CBPI results, suggesting that the lead + polymer shield was equally effective in protecting the DNA and in maintaining the integrity of the lymphocytes exposed to ^18^F-FDG. IR generates an explosion of free radicals capable of inducing DNA damage in exposed cells, because of the ionization of water molecules and the direct ionization of target molecules \[[@CR24], [@CR37]\]. These free radicals are produced in microseconds. However, their effects persist long after their production, owing to the cascade of events triggered at the molecular and cellular level. This ultimately leads to increased oxidative stress, lipid peroxidation, and genome instability \[[@CR17], [@CR37]\]. All these mechanisms can be explained by the cytotoxic and genotoxic effects observed and reaffirms the uniqueness of this study. Considering the analyzed population (*n* = 3 individuals, 6000 cells for CBMN assay, and 3000 cells for CBPI, in each group), it is essential to consider that results with more blood donors could provide even more relevant observations on interindividual and temporal differences. It is also noteworthy that a more significant number of samples would result in a longer exposure time of researchers to ^18^F-FDG, even with the adoption of all security protocols. Moreover, the harmful effect of IR is not limited to lymphocytes. The radiosensitivity of healthy cells, tissues, and organs depend on several factors, including the ability to repair the damage, hypoxia, cell cycle position, and the effective absorbed dose of radiation. Then, further investigations with different cell types must be carried out. Thus, compliant with the basic guidelines of CNEN (NN 3.01 and 3.05) which aim to optimize radiological protection to provide occupationally exposed individuals with better health care, we found that the use of a lead + polymer shield is more effective than lead aprons. The association of lead + polymer reduces the number of counts and mitigates the biological effects of IR, increasing the protection of individuals working in PET/CT procedures with ^18^F-FDG. Similar to the PVC polymer, Prokhorenko et al. \[[@CR31]\] demonstrated that polystyrene composite (19.1%) combined with tungsten (66.9%) and aluminum (14%) was also effective in the production of radioprotective equipment of low weight and high durability, supporting our results. The results from this study show that the addition of a lightweight, low-cost polymer layer to a lead apron is highly recommended and provides enhanced radiological and biological protection to individuals who are occupationally exposed to radiation, particularly during ^18^F-FDG handling. In cases where lead aprons are avoided due to their low ergonomics, we recommended the use of polymer aprons by qualified service personnel at least. Other polymer-based safety equipment may be developed to reduce the biological effects of IR, like glasses and other types of eye protection, and polymer-coated syringe/vial holders and coatings for radiopharmaceuticals. Furthermore, a minimum distance of 25 cm between healthcare professionals and radiopharmaceuticals is recommended during transportation from hot laboratories to patients' rooms. Workers occupationally exposed to ionizing radiation who are better protected will be able to carry out routine activities safely, allowing them to work longer with less radiation exposure, reducing staff turnover costs in addition to minimizing their risk of exposure to the harmful effects of IR. Conclusions {#Sec12} =========== Ionizing radiation is mutagenic and known to induce cell damage, including the formation of micronuclei, nucleoplasmic bridges, and nuclear buds. The results presented in this study suggest that the coating of conventional radiation shielding equipment, like lead aprons, with a polymer layer could increase the radiological protection of occupationally exposed individuals, particularly during ^18^F-FDG PET/CT examinations. The toxicogenomic biomonitoring of workers exposed to ionizing radiation, the application of radiation protection programs, and physical dosimetry procedures are all crucially important in minimizing occupational radiation exposure. Moreover, the development of new protective equipment is key in terms of biotechnology for the protection of individuals and the environment. Better protective equipment will increase the safety of health workers exposed to radiation, and also that of researchers, technicians, and students exposed to radiation in universities, research centers, and industries. Supplementary information ========================= {#Sec13} **Additional file 1.** ^18^F-FDG : Fluorodeoxyglucose BNC : Binucleated cells CBMN : Cytokinesis-Block Micronucleus CBPI : Cytokinesis-Block Proliferation Index CMN : Cell with one or more micronuclei CNEN : National Nuclear Energy Commission, Brazil CPM : Counts per minute IR : Ionizing radiation MN : Micronucleus NBUD : Nuclear bud NPB : Nucleoplasmic bridge PET/CT : Positron Emission Tomography/Computed Tomography PVC : Polyvinyl chloride **Publisher's Note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary information ========================= **Supplementary information** accompanies this paper at 10.1186/s13014-020-01598-0. Authors would like to thank Grupo São Camilo Medicina Diagnóstica, Brazil, for structural support. All authors contributed equally for the development, execution and analysis of this study. All authors read and approved the final manuscript. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Not applicable. All experimental procedures were approved by the Committee on Ethics in Human Research of the State University of Maringá. The purpose, scope, benefits, risks, and procedures of the study were explained to each volunteer and the informed consent for execute were obtained from each blood donor. The informed consent for publication of data were obtained from each blood donor. The authors declare that they have no competing interests.

Introduction {#S1} ============ Brain consists of spatially distributed regions that have their own function, but these regions are functionally connected, that is, they continuously send information to each other. Recent progress in the acquisition and analysis of functional neuroimaging data has made it possible to explore functional connectivity in the human brain. Functional connectivity is defined as a temporal correlation of neuronal activation patterns between anatomically distant brain regions and has been assessed using various non-invasive functional neuroimaging modalities including functional magnetic resonance imaging (fMRI), magnetoencephalography (MEG) and electroencephalography (EEG). In particular, functional connectivity under resting conditions (resting-state functional connectivity, RSFC) has attracted widespread attention in neuroscience ([@B3]; [@B14]). One of the reasons for this might be that a growing body of studies has reported altered levels of functional connectivity in neurological and psychiatric brain disorders, including Alzheimer's disease, depression, dementia and schizophrenia ([@B31]). Most RSFC studies use fMRI because of its high spatial resolution. For example, the first direct evidence for the default mode network (DMN) was demonstrated using resting-state fMRI data based on seed-based correlation analysis ([@B14]). The dorsal attention network (DAN), the resting-state network antagonistically coupled with the DMN, were also identified using resting-state fMRI data with the seed-based approach ([@B10]). There is a more sophisticated approach than the seed-based correlation analysis, the use of spatial independent component analysis (ICA). Spatial ICA is a widely used method for decomposing fMRI data into signal and noise components and was implemented using the Group ICA of fMRI Toolbox (GIFT)^[1](#footnote1){ref-type="fn"}^. Multiple resting-state networks including DMN can be easily identified by applying spatial ICA to resting-state fMRI data ([@B16]), and therefore the use of this approach is increasing. Despite growing use of fMRI in RSFC studies, fMRI has dis-advantages of high cost and low portability. In addition, fMRI may not be safe or appropriate for people (1) with metal or electronic implants in their bodies (such as pacemakers, cochlear implants, metallic tattoos, etc.) because MRI involves exposure to strong magnetic fields and induced electric fields, (2) with claustrophobia because subjects are required to enter narrow scanner tube and (3) who are or may be pregnant because the risk of exposure to magnetic fields for the fetus is still unknown. Functional near-infrared spectroscopy (fNIRS) is a non-invasive optical imaging technique that measures changes in both oxygenated (oxy-) and deoxygenated (deoxy-) hemoglobin (Hb) concentrations based on changes in light absorption at multiple wavelengths, whereas fMRI mainly measures changes in deoxy-Hb concentrations, referred to as the blood-oxygen-level-dependent (BOLD) signals. Because fNIRS is free of above disadvantages in fMRI, it can be used as an alternative human brain mapping technique for situations in which fMRI is contraindicated. Rather than an alternative to fMRI, fNIRS would provide even additional information, because fNIRS creates images of both oxy- and deoxy-Hb simultaneously (as described above, the BOLD signal is mostly sensitive to deoxy-Hb) and has a higher sampling rate than fMRI does (\>10 Hz with fNIRS, whereas ∼0.5 Hz with fMRI). Thanks to these advantages, fNIRS has been used to investigate RSFC. [@B19], one of the earliest resting-state fNIRS studies, demonstrated that, using seed-based correlation analysis, RSFC maps over the sensorimotor and auditory cortexes were consistent with those of previous fMRI findings. Furthermore, [@B6] and [@B24] examined relationship of RSFCs between fNIRS and fMRI by simultaneously recording these signals and demonstrated that fNIRS can be used to collect information regarding RSFC defined in fMRI. As for the approach to estimate RSFC, [@B36] compared a spatial ICA with the conventional seed-based correlation approach with respect to the estimation of RSFC from fNIRS data and demonstrated the superior performance of spatial ICA with higher sensitivity and specificity, especially in the case of higher noise level ([@B36]). Despite the success these resting-state fNIRS studies achieved, current standard fNIRS imaging (i.e., optical topography that is two-dimensional image based on the spatial interpolation method) has several disadvantages. First, fNIRS imaging uses sparse arrangements of source and detector optodes (and therefore measurement channels are also sparsely arranged) and therefore the positions of the measurement channels do not always overlap the real activation foci. Therefore, the spatial resolution of fNIRS imaging is low compared to fMRI. Second, the positions of the measurement channels relative to brain anatomy vary among subjects, and also among runs within the same subjects when the runs were performed on different days, resulting in reduced reliability of comparison among subjects and runs. Third, fNIRS signals are, in most cases, degraded by the hemodynamic changes in the scalp layer. Changes in scalp hemodynamics sometimes exceed those in cortical hemodynamics ([@B30]). In these three problems, the third one was dealt with by several studies that succeeded to reduce scalp artifacts with the use of principal component analysis (PCA) ([@B37]), independent component analysis (ICA) ([@B18]), short-distance channel regression ([@B34]; [@B7]) or combination of PCA and multi-distance probe arrangement ([@B25]). However, there is a way to solve all the three problems at once, a method called diffuse optical tomography (DOT). Diffuse optical tomography is an advanced technique to reconstruct three-dimensional images showing changes in cerebral hemodynamics. The technique follows the strategy to use high-density DOT grids, which bring about overlapping measurements at multiple source-detector separation distances. The use of overlapping measurements improves spatial resolution in the head surface direction. In addition, different measurement distances provide information about different depths. This is because the penetration depth of the light increases with the source-detector separation distance. Depending on the source-detector distance and the subject's scalp/skull thickness, the light may or may not sufficiently penetrate through the superficial layers (scalp) to the deeper layers (brain tissue) ([@B22]). There are two stages to obtain DOT images. The first stage is forward modeling where the measurement process is simulated using a head model and physical laws. The second stage is image reconstruction where the hemodynamic changes inside the head medium are estimated from fNIRS signals by inverting the forward model. The image reconstruction problem is formulated as a linear inverse problem ([@B4]), which is ill-posed and therefore requires *a priori* information to constrain possible solutions. One approach to solve the inverse problem is the regularization. In the DOT algorithm based on the regularization ([@B34]; [@B7]), a DOT image is obtained by minimizing a cost function consisting of the data fitting term and constraint terms representing *a priori* information. Another is the Bayesian approach, which uses a probabilistic model of observations and constraints called the likelihood function and prior distribution, respectively ([@B15]; [@B28]). Although there were a variety of image reconstruction algorithms, no research had proposed a DOT algorithm to accurately reconstruct both the scalp and cortical activities simultaneously. Most studies took a two-process approach: (1) hemodynamic changes in the superficial layers, including scalp and skull, were removed from all measurements and (2) a DOT image reconstruction method was applied to the denoised data in order to estimate only the cortical activity. Most artifact-removal methods used in the first process were based on an assumption that the temporal patters in the hemodynamic changes of the superficial layers are homogeneous over the whole head (e.g., [@B18]) or they are similar to those of short-distance channels ([@B7]). However, the artifact-removal methods based on the above assumption would not work well if the systemic interference occurring in the superficial layers of the human head is inhomogeneous across the surface of the scalp as reported in [@B12], [@B13] or if hemodynamic changes in the superficial layers are highly correlated with those in the cortex (though see [@B17]; [@B11]; for methods to overcome this). To avoid these weak points in the artifact-removal methods based on temporal information, we recently proposed a method that uses the spatial information of the optical paths of all observation channels, which are not affected by temporal inhomogeneity or correlation, in order to remove the scalp hemodynamics ([@B29]). The method is an expanded version of the previously proposed hierarchical Bayesian (HB) DOT algorithm ([@B28]) and is able to reconstruct both the scalp and cortical activities simultaneously. In the first version of our Bayesian DOT algorithm ([@B28]), we introduced sparse regularization to improve the depth accuracy and the spatial resolution and verified its performance with phantom experiments. Then, in the expanded version ([@B29]), we introduced different types of regularization for the cortex and the scalp, sparse and smooth regularization to cortical and scalp's hemodynamic changes, respectively, and validated the proposed method through both two-layer phantom experiments and MRI-based head-model simulations. Furthermore, we have conducted real human experiments with movement tasks and confirmed the performance of the HB DOT on imaging task-related functional responses ([@B33]). The present study therefore aims to investigate whether the HB DOT is used successfully to estimate spontaneous changes in cortical hemodynamics instead of task-related changes. We conducted real human experiments to record resting-state fNIRS signals from bilateral frontal and parietal areas using high-density probe array with multiple-distance channels, which required a major improvement of measurement (i.e., development of a custom-made holder that stably fits to the scalp). Then, we calculated the RSFC from the estimates of cortical hemodynamic changes with HB DOT. We also acquired resting-state fMRI data and used it as a reference in order to validate the performance of our algorithm. In addition, we compared the performance of our method with that of the two-process approach, which is the currently prevailing method for DOT. As for the DOT algorithms used in the two-process approach, we adopted the modified depth-compensation minimum-norm algorithm (abbreviated as MN) and the current standard method developed in Washington University (named in this study as MN-WU) ([@B7]), in line with our previous study on imaging task-related activities ([@B33]). Because there are still few studies on RSFC using DOT ([@B32]; [@B7]), the present study will provide important information to the relevant research areas. Materials and Methods {#S2} ===================== To obtain resting-state brain activity, fMRI and fNIRS data were recorded during resting state on different days. fMRI data was used as a reference. fNIRS signals were passed through DOT analyses to reconstruct three-dimensional images of changes in cerebral hemodynamics. Three different DOT algorithms, HB, MN and MN-WU were compared in terms of resting-state connectivity. Schematic of the processing stream for fNIRS and fMRI data are shown in [Figure 1](#F1){ref-type="fig"}. {#F1} Subjects {#S2.SS1} -------- Twenty healthy male subjects aged between 21 and 38 participated in both fMRI and fNIRS experiments on different days. All of the subjects, except for one subject who is one of the authors, were paid for their participation. None reported history of neurological or psychiatric disorders. All subjects gave written informed consent to participate in the experimental procedures, which were approved by the ATR Review Board Ethics Committee. Tasks {#S2.SS2} ----- In the fMRI experiment, each subject undergone a 10 min resting state condition in which he/she was instructed to stay still, to stay awake, to fixate on the crosshair, and not to think about specific things. In the fNIRS experiment, each subject undergone a two-back working memory task (took about 15 min) and two 10 min resting state conditions similar to that in the fMRI experiment. The resting state conditions were undergone before and after the WM task condition. We did not use the WM data in the present study. MRI and fMRI Data Acquisition {#S2.SS3} ----------------------------- Subjects lay down in an MRI scanner. Structural MR images were acquired for construction of individual head models, and functional images were acquired for evaluation of the reconstructed DOT images. All MRI data were recorded using a 3T MRI scanners, MAGNETOM Trio Tim, MAGNETOM Verio, MAGNETOM Prisma (Siemens Medical Systems, Erlangen, Germany). The acquisition parameters for T1-weighted images were as follows: repetition time (TR) = 2,300 ms, time of echo (TE) = 2.98 ms, flip angle = 9°, slice thickness = 1 mm, field of view (FOV) = 256 mm, imaging matrix = 256 × 256, inversion time (TI) = 900 ms. The acquisition parameters for echo-planar images (EPIs) were as follows: TR = 2,500 ms, TE = 30 ms, flip angle = 80°, slice thickness = 3.2 mm, FOV = 212 mm, imaging matrix = 64 × 64 mm. NIRS Data Acquisition {#S2.SS4} --------------------- Subjects were seated in a comfortable reclining armchair. fNIRS data were acquired using commercial NIRS equipment (SMARTNIRS, Shimadzu Corp., Japan) with probes whose shapes were customized for high-density (HD) measurements. Using a custom-made holder, 32 source and 32 detector probes were placed on the scalp to cover bilateral frontal and parietal areas. We adopted four 4 × 4 square arrays ([Figures 2A,B](#F2){ref-type="fig"}), where the first- and second-shortest distances between source and detector probes were 13 and 29 mm, respectively. We used all of the first- and second-nearest neighbor measurement pairs, which provided 56 'first' and 96 'second' channels, respectively, resulting in a total of 152 measurement channels. {#F2} Just after the fNIRS recording, the surface image of the subject's face, the positions of the three fiducial markers (nasion, left and right preauricular points) and the probe positions were acquired with a hand-held laser scanner and a stylus marker (FastSCAN; Polhemus, United States), for the co-registration of the fNIRS data to the T1-MRI. Three near-infrared beams (wavelength 780, 805, and 830 nm) were irradiated and detection beams sampled at 18.5 Hz were used to calculate Δ\[oxy-Hb\] and Δ\[deoxy-Hb\]. fMRI Data Processing {#S2.SS5} -------------------- fMRI signals were processed using SPM12 (the Wellcome Centre for Human Neuroimaging). The first four volumes were discarded to allow for T1 equilibration. The remaining data were corrected for slice timing and realigned to the mean image of that sequence to compensate for head motion. Next, the structural image was co-registered to the mean functional image and segmented into three tissue classes (gray matter, white matter, and cerebrospinal fluid) in the standard Montreal Neurological Institute (MNI) space. Using associated parameters, the functional images were spatially normalized into the MNI space and resampled in a 2 × 2 × 2 mm grid. Finally, they were spatially smoothed using an isotropic Gaussian kernel of 8 mm full-width at half maximum (FWHM). fNIRS Data Preprocessing {#S2.SS6} ------------------------ Prior to DOT analysis, the following preprocessing was applied to fNIRS signals. 1. Convert voltage data into log-ratios using a base-10 logarithm. 2. Calculate the coefficient of variation (CV, in%) of each channel for each wavelength, where CV = 100 × σ/μ, σ is the signal standard deviation, and μ is the mean signal level. Then, remove the bad channels which had CVs exceeding 15% ([@B21]) at least one of three wavelength or were saturated \[the mean number of rejected channels was 13.0 ± 12.2 (SD)\]. 3. Apply a high-pass filter (Butterworth filter of order 3, cutoff 0.009 Hz) and a low-pass filter (Butterworth filter of order 7, cutoff 0.08 Hz). 4. Remove scalp hemodynamics from the filtered data, only in case of DOT image reconstruction with the MN and MN-WU algorithms. In this process, the global average of all the 'first' channels other than the bad channels is regarded as the scalp dynamics and regressed out. This process is omitted in case of DOT image construction with the HB algorithm. 5. Remove the bad time points when absolute signal amplitudes exceeded more than three standard deviations from the mean at least one of the "first" channels \[the mean number of removed time points was 590 ± 247 (SD) (roughly equal to 32 s)\]. DOT Forward Model Construction {#S2.SS7} ------------------------------ The DOT forward model was constructed in the following way. First, an individual head model was constructed by segmenting their T1 structural image into five tissue layers \[scalp, skull, cerebrospinal fluid (CSF), gray matter, and white matter\], using FreeSurfer software^[2](#footnote2){ref-type="fn"}^. Positions of the fNIRS probes were then co-registered to the head model using an affine transformation. The rotation and translation parameters of the affine transformation were optimized so that a subject's facial surface measured by the laser scanner fitted that extracted from the T1 anatomical image. Next, the photon migration process inside the head was simulated with Monte Carlo simulation software MCX ([@B9]) with 10^9^ photons. We used tissue optical parameters common to all three wavelengths ([Table 1](#T1){ref-type="table"}), as presented in a previous study ([@B8]). Finally, the sensitivity matrix, which relates the absorption changes in the head tissue voxels to the light intensity changes at the source-detector pairs, was computed with Rytov approximation to the MCX results ([@B28]). For computation of the sensitivity matrix, the 1 × 1 × 1 mm voxel space was down-sampled to 4 × 4 × 4 mm voxel space in the reconstructed images. The image reconstruction region included the scalp and cortical voxels inside a 28-mm-deep cuboid, whose surface was a square along the scalp surface. This was obtained by extending the diagonals of the 5 × 5 NIRS probe square by a factor of 1.5. ###### Optical parameters in various head tissue types (common to all three wavelengths of 780, 805, and 830 nm). **Tissue types** **Absorpt. Coeff. μ~a~ (mm^--1^)** **Scattering Coeff. μ~s~ (mm^--1^)** **Anisotropy (g)** **Refract. Index (n)** ------------------ ------------------------------------ -------------------------------------- -------------------- ------------------------ Scalp and skull 0.019 7.8 0.89 1.37 CSF 0.004 0.009 0.89 1.37 Gray matter 0.02 9.0 0.89 1.37 White matter 0.08 40.9 0.84 1.37 DOT Image Reconstruction With the HB Algorithm {#S2.SS8} ---------------------------------------------- DOT image reconstruction with the HB algorithm has two steps. In the first step, DOT image is reconstructed from the preprocessed fNIRS signals, using the modified version of the depth-compensation minimum norm image reconstruction algorithm (MN). In the next step, the DOT image is refined using the iterative algorithm with the MN DOT image obtained in the first step as an initial value and a prior. Note that the preprocessed fNIRS signals used in both steps are not passed through the scalp hemodynamics removal (the 4th process in section "fNIRS data preprocessing"). For the HB DOT image reconstruction in the second step, we used the HB model presented in [@B33]. The hierarchical prior distribution has mean and confidence (or reliability) parameters. The mean parameter $\overline{\lambda_{0⁢\imath}}$ were the mean square values of the solutions obtained from the MN DOT in the first step. The confidence parameter γ~0~ controls the width of the hierarchical prior distribution (the variance of the hierarchical prior distribution is inversely proportional to the confidence parameter); large γ~0~ narrows the hierarchical prior distribution around the mean value $\overline{\lambda_{0⁢\imath}}$, and the estimation depends more critically on the solutions obtained from MN DOT in the first step. The confidence parameter γ~0~ was set to L × 0.1, where L is data length, on the basis of our experience. But, we also tried the following settings; γ~0~ = L × 0.01, L × 0.001, L × 0.0001. For the detail of the HB algorithm, see [@B33]. DOT Image Reconstruction With the MN/MN-WU Algorithms {#S2.SS9} ----------------------------------------------------- The DOT image with the MN algorithm and that with the MN-WU algorithm were also computed for comparison. As mentioned in section "fNIRS data preprocessing," in both MN and MN-WU algorithms, DOT image is reconstructed from the preprocessed fNIRS signals whose scalp hemodynamics were removed by regressing out the averaged 'first' channel data. As for the MN-WU algorithm, the spatially variant parameter was set to β = 0.1 and the regularization parameter α was automatically determined by maximizing the marginal likelihood of each data set (see [@B5]). As for the MN algorithms, the spatially variant parameter was set to β = *mean~νϵI20mm~*(ρ~2~)~*ν*~ where *I*~*20\ mm*~ is a voxel index set whose depth from the scalp is around 20 mm. And the regularization parameter α was automatically determined by maximizing the marginal likelihood using all the measurements included in the I~task~. See the Appendix of [@B33] for the mathematical details. Calculation of Resting-State Connectivity {#S2.SS10} ----------------------------------------- Irrespective of the DOT algorithm, the reconstructed DOT image was passed through a spatial normalization into the standard MNI space and spatial smoothing with a Gaussian kernel of 8 mm full width at half maximum (FWHM), before calculation of resting-state functional connectivity (RSFC). These processes were done with SPM12. The RSFC for both fMRI and DOT was obtained in the following way, using spatially normalized fMRI and DOT images, respectively. First, all cortical voxels were categorized into 278 regions of interest (ROI), based on functional-connectivity-based atlas ([@B26]). Then, voxels with sensitivity values of more than 0.5 for all subjects were regarded as *sensitive* voxels, and ROIs including more than or equal to 10 *sensitive* voxels were regarded as *available* ROIs ([Figure 2C](#F2){ref-type="fig"} for Shen's atlas). Third, for each *available* ROI, timeseries of all *sensitive* voxels within the ROI were averaged. These mean timeseries were assumed to represent temporal activity of the corresponding ROI. Finally, partial correlations between all pairs of *available* ROIs were computed to make a correlation matrix. Partial correlation was used to reduce the influence of extra-neural components such as physiological noise signals due to spontaneous low-frequency oscillations, respiration and cardiac pulsation ([@B23]). Note that all the following analyses were done for *sensitive* voxels and *available* ROIs. The reason why we used Shen's atlas rather than the widely used Brodmann-based automatic anatomic labeling (AAL) atlas is as follows. The AAL atlas uses Brodmann areas which are based on cytoarchitecture. This atlas is not ideal because of its coarse-grained nature (116 regions for the AAL atlas whereas 278 regions for the Shen's atlas in our data) and the risk of including different functional areas within a single region, with the consequence that the resultant mean timeseries may not accurately represent any of the contributing timeseries. Shen's atlas is developed to avoid this pitfall and will provide meaningful nodes ([@B26]), and therefore is suitable for our case. For reference, we will present the corresponding results for the AAL atlas in the [Supplementary Material](#SM1){ref-type="supplementary-material"}. Comparison of Connectivity Matrices Among DOT Algorithms {#S2.SS11} -------------------------------------------------------- In order to establish the superiority of the HB DOT in the estimation of RSFC, we compared correlation matrices among the DOT algorithms (HB, MN, MN-WU) in the following three ways. Because it seems reasonable to use fMRI data as a reference, we first compared *similarity* of RSFCs between fMRI and DOT among three DOT algorithms, where correlation coefficient was used as a similarity measure. The comparison was done in the following way: (1) the lower triangular portion of correlation matrix, *C*~*low*~, was transformed to *z(C~*low*~)* by using Fisher's z-transformation, (2) correlation coefficient of *z(C~*low*~)* between fMRI and DOT, *R*~*fMRI--DOT*~, was calculated, (3) the correlation coefficient *R*~*fMRI--DOT*~ was transformed to *z(R~*fMRI--DOT*~)* by using Fisher's *z*-transformation, (4) differences in mean *z(R~*fMRI--DOT*~)* among three DOT algorithms were tested using one-way analysis of variance (ANOVA) followed by multiple comparisons with the Tukey--Kramer correction. Second, we compared inter-run *reproducibility* of RSFC estimation among three DOT algorithms. The comparison was done in the following way: (1) the lower triangular portion of correlation matrix, *C*~*low*~, was transformed to *z(C~*low*~)* by using Fisher's *z*-transformation, (2) correlation coefficient of *z(C~*low*~)* between run1 and run2, *R~*ses*1--ses2~*, was calculated, (3) the correlation coefficient *R~*ses*1--ses2~* was transformed to *z(R~*ses*1--ses2~)* by using Fisher's *z*-transformation, (4) differences in mean *z(R~*ses*1--ses2~)* among three DOT algorithms were tested using one-way analysis of variance (ANOVA) followed by multiple comparisons. As a complementary metric to Pearson's correlation, intra-class correlation (ICC; [@B20]) was also used for assessment of reproducibility across runs. Both single and average measures, i.e., ICC(C,1) and ICC(C,k), were calculated using MATLAB function *ICC* by Arash Salarian (available at MATLAB Central File Exchange). Third, we performed additional analysis on intra-run test--retest reliability assessment. In this analysis, resting-state fNIRS data recorded in each 10 min run was firstly divided into two segments of equal length \[i.e., first half (FH) and second half (SH)\]. Note that duration of each half is 5 min at most (the mean is about 4 min and 44 s) because bad time points were removed in the fNIRS data preprocessing (see section "fNIRS Data Preprocessing"). As shown in [Figure 3](#F3){ref-type="fig"}, intra-run reproducibility was assessed using two pairs of datasets; run1-FH and run1-SH (intra_run1), as well as run2-FH and run2-SH (intra_run2). In addition, inter-run reproducibility (denoted by half-length inter-run reproducibility to distinguish it from the previous one using full-length data) was assessed with two pairs of datasets; run1-FH and run2-FH (inter_FH), as well as run1-SH and run2-SH (inter_SH). As measures for these reproducibilities, we used Pearson's correlation, ICC(C,1) and ICC(C,k) again. {#F3} We compared intra-run reliability among functional images (i.e., fMRI vs. HB vs. MN vs. MN-WU). More specifically, differences in mean measures for intra-run reliability were tested using one-way ANOVA followed by multiple comparisons. In addition, we compared intra-run reproducibility and half-length inter-run reproducibility. Because resting-state fNIRS was acquired before and after a WM task, this comparison will serve to investigate whether the task affected the resting-state connectivity. Results {#S3} ======= Similarity Between fMRI and DOT {#S3.SS1} ------------------------------- Mean and SD maps for RSFCs of fMRI and DOT with HB, MN and MN-WU algorithms are shown in [Figures 4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}, respectively. The mean map showed a common tendency between fMRI and fNIRS (i.e., HB, MN, and MN-WU) that most pairs between adjacent ROIs had positive correlations, though some of them (e.g., R.BA7.5-R.BA7.6) had negative correlations. Additional findings from the mean map is that some of frontal-parietal pairs (e.g., R.BA7.8-R.BA9.4) and contralateral counterpart pairs (e.g., R.BA9.4-L.BA10.1) had positive correlations in fMRI, whereas such a tendency was weak for the DOT cases. As for the SD maps, variability across subjects was little in fMRI, whereas it was relatively large especially for the pairs between adjacent ROIs in fNIRS regardless of the DOT algorithms. {#F4} {#F5} Similarity of RSFCs between fMRI and DOT for each run and DOT algorithm is summarized in [Table 2](#T2){ref-type="table"}. One-way ANOVA applied to data combined between runs revealed a significant difference among three DOT algorithms for both oxy- and deoxy-Hb \[*F*~(2~,~117)~ = 4.27, *p* = 0.0162 for oxy-Hb; *F*~(2~,~117)~ = 7.69, *p* = 0.0007 for deoxy-Hb\]. The *post hoc* Tukey's HSD test revealed, for both oxy- and deoxy-Hb, that HB had significantly higher correlation values than both MN and MN-WU did (*p* \< 0.05), but the difference of correlation values between MN and MN-WU was not significant. ###### Similarity of correlation matrices between fMRI and DOT. **HB** **MN** **MN-WU** ---------- ------- ------------- ------------- ------------- oxy-Hb run 1 0.34 ± 0.09 0.29 ± 0.10 0.29 ± 0.10 run 2 0.35 ± 0.08 0.30 ± 0.08 0.30 ± 0.09 deoxy-Hb run 1 0.33 ± 0.08 0.25 ± 0.11 0.26 ± 0.12 run 2 0.34 ± 0.08 0.27 ± 0.11 0.25 ± 0.10 Values of correlation coefficients are presented as mean ± standard deviation (SD). Inter-Run Reproducibility, Intra-Run Reproducibility {#S3.SS2} ---------------------------------------------------- Mean and SD maps for RSFCs are compared between runs in [Figures 6](#F6){ref-type="fig"}, [7](#F7){ref-type="fig"}, respectively. Mean ± SD of inter-run reproducibility (i.e., Pearson's correlation and inter-class correlation of correlation matrices between run 1 and 2) is summarized in [Table 3](#T3){ref-type="table"}. {ref-type="fig"}, [5](#F5){ref-type="fig"}.](fnins-14-00032-g006){#F6} {ref-type="fig"}.](fnins-14-00032-g007){#F7} ###### Inter-run reproducibility. **HB** **MN** **MN-WU** ---------- ---------- ------------- ------------- ------------- oxy-Hb r 0.76 ± 0.06 0.61 ± 0.13 0.66 ± 0.12 ICC(C,1) 0.76 ± 0.06 0.61 ± 0.13 0.65 ± 0.12 ICC(C,k) 0.86 ± 0.04 0.75 ± 0.10 0.78 ± 0.09 deoxy-Hb r 0.76 ± 0.07 0.60 ± 0.13 0.63 ± 0.15 ICC(C,1) 0.75 ± 0.07 0.60 ± 0.13 0.62 ± 0.15 ICC(C,k) 0.86 ± 0.04 0.74 ± 0.10 0.76 ± 0.12 Values of Pearson's correlation (r), ICC(C,1) and ICC(C,k) are presented as mean ± standard deviation (SD). As for Pearson's correlation, one-way ANOVA revealed a significant difference among three DOT algorithms for both oxy- and deoxy-Hb \[*F*~(2~,~57)~ = 11.0, *p* = 9.06 × 10^--5^ for oxy-Hb; *F*~(2~,~57)~ = 10.9, *p* = 9.89 × 10^--5^ for deoxy-Hb\]. The *post hoc* Tukey's HSD test revealed, for both oxy- and deoxy-Hb, that HB had significantly higher correlation values than both MN and MN-WU did (*p* \< 0.05), but the difference of correlation values between MN and MN-WU was not significant. As for the ICC metrices, the following results were obtained. First, for ICC(C,1), one-way ANOVA revealed a significant difference among three DOT algorithms for both oxy- and deoxy-Hb \[*F*~(2~,~57)~ = 11.0, *p* = 9.30 × 10^--5^ for oxy-Hb; *F*~(2~,~57)~ = 9.81, *p* = 0.0002 for deoxy-Hb\]. The *post hoc* Tukey's HSD test revealed, for both oxy- and deoxy-Hb, that HB had significantly higher ICC(C,1) values than both MN and MN-WU did (*p* \< 0.05), but the difference of ICC(C,1) values between MN and MN-WU was not significant. Then, for ICC(C,k), one-way ANOVA revealed a significant difference among three DOT algorithms for both oxy- and deoxy-Hb \[*F*~(2~,~57)~ = 10.5, *p* = 0.0001 for oxy-Hb; *F*~(2~,~57)~ = 8.61, *p* = 0.0005 for deoxy-Hb\]. The *post hoc* Tukey's HSD test revealed, for both oxy- and deoxy-Hb, that HB had significantly higher ICC(C,k) values than both MN and MN-WU did (*p* \< 0.05), but the difference of ICC(C,k) values between MN and MN-WU was not significant. Intra-run reproducibility (i.e., similarity of RSFCs between FH and SH) is summarized in [Table 4](#T4){ref-type="table"}. ###### Intra-run reproducibility. **fMRI** **HB** **MN** **MN-WU** ---------- ---------- ------------- ------------- ------------- ------------- oxy-Hb r 0.61 ± 0.08 0.57 ± 0.11 0.38 ± 0.16 0.41 ± 0.13 ICC(C,1) 0.61 ± 0.08 0.56 ± 0.11 0.37 ± 0.15 0.40 ± 0.13 ICC(C,k) 0.75 ± 0.06 0.71 ± 0.09 0.53 ± 0.16 0.56 ± 0.14 deoxy-Hb r 0.61 ± 0.08 0.53 ± 0.10 0.38 ± 0.15 0.38 ± 0.15 ICC(C,1) 0.61 ± 0.08 0.53 ± 0.10 0.37 ± 0.15 0.37 ± 0.15 ICC(C,k) 0.75 ± 0.06 0.68 ± 0.09 0.52 ± 0.16 0.53 ± 0.15 Values of correlation coefficients ( r ), ICC(C,1) and ICC(C,k) are presented as mean ± standard deviation (across subjects for fMRI; across subjects and runs for DOT). One-way ANOVA revealed that Pearson's correlation value is significantly different among functional images \[*F*~(3~,~136)~ = 27.26, *p* = 7.25 × 10^--14^ for oxy-Hb; *F*~(3~,~136)~ = 23.84, *p* = 1.84 × 10^--12^ for deoxy-Hb\]. The *post hoc* Tukey's HSD test revealed, for both oxy- and deoxy-Hb, that HB had significantly higher Pearson's correlation than both MN and MN-WU did (*p* \< 0.05), but the difference between HB and fMRI was not significant. Similarly, according to one-way ANOVA, ICC(C,1) is significantly different among functional images \[*F*~(3~,~136)~ = 27.41, *p* = 6.28 × 10^--14^ for oxy-Hb; *F*~(3~,~136)~ = 24.59, *p* = 8.94 × 10^--13^ for deoxy-Hb\]. According to the Tukey's HSD test, for both oxy- and deoxy-Hb, HB had significantly higher ICC(C,1) than both MN and MN-WU did (*p* \< 0.05), but HB was not significantly different from fMRI. In addition, one-way ANOVA and *post hoc* analysis revealed that ICC(C,k) had similar tendency \[one-way ANOVA, *F*~(3~,~136)~ = 25.00, *p* = 6.04 × 10^--13^ for oxy-Hb; *F*~(3~,~136)~ = 23.15, *p* = 3.61 × 10^--12^ for deoxy-Hb\]. For the HB case, intra-run reproducibility (i.e., similarity of RSFCs between FH and SH) was compared with half-length inter-run reproducibility (i.e., similarity of RSFCs between run 1 and run 2 for the corresponding half) in [Table 5](#T5){ref-type="table"}. Two-sample *t*-test revealed that intra-run reproducibility was not significantly different from half-length inter-run reproducibility for Pearson's correlation (*p* = 0.31 for oxy-Hb; *p* = 0.42 for deoxy-Hb), ICC(C,1) (*p* = 0.36 for oxy-Hb; *p* = 0.47 for deoxy-Hb), and ICC(C,k) (*p* = 0.33 for oxy-Hb; *p* = 0.48 for deoxy-Hb). Similar tendency was observed for both MN and MN-WU cases (detailed data not shown, but *p* \> 0.30 in any case). ###### Comparison of reproducibility between intra-run and half-length inter-run for HB. **Intra-run** **Half-length inter-run** ---------- --------------- --------------------------- ------------- oxy-Hb r 0.57 ± 0.11 0.55 ± 0.12 ICC(C,1) 0.56 ± 0.11 0.54 ± 0.12 ICC(C,k) 0.71 ± 0.09 0.69 ± 0.10 deoxy-Hb r 0.53 ± 0.10 0.51 ± 0.10 ICC(C,1) 0.53 ± 0.10 0.51 ± 0.10 ICC(C,k) 0.68 ± 0.09 0.67 ± 0.09 Values are presented as mean ± standard deviation. Effect of the Confidence Parameter γ~0~ on Connectivity {#S3.SS3} ------------------------------------------------------- As mentioned in the Materials and methods, in the HB algorithm, the confidence parameter, γ~0~, represents the width of the hierarchical prior distribution, controlling how strong the HB method is affected by the prior information (the depth-weighted minimum norm estimation). In this subsection, the effect of the γ~0~ value on the connectivity estimation was examined for only Pearson's correlation. Similarity of RSFCs between fMRI and DOT for each run and γ~0~ value is summarized in [Table 6](#T6){ref-type="table"}. One-way ANOVA applied to data combined between runs revealed no significant difference among five γ~0~ values for both oxy- and deoxy-Hb \[*F*~(4~,~195)~ = 1.26, *p* = 0.287 for oxy-Hb; *F*~(4~,~195)~ = 1.40, *p* = 0.237 for deoxy-Hb\]. ###### Effect of γ~0~ value on similarity of correlation matrices between fMRI and DOT. **L × 10^0^** **L × 10^--1^** **L × 10^--2^** **L × 10^--3^** **L × 10^--4^** ---------- --------------- ----------------- ----------------- ----------------- ----------------- ------------- oxy-Hb run 1 0.34 ± 0.09 0.34 ± 0.09 0.33 ± 0.09 0.32 ± 0.10 0.30 ± 0.11 run 2 0.35 ± 0.09 0.35 ± 0.08 0.35 ± 0.08 0.34 ± 0.09 0.32 ± 0.09 deoxy-Hb run 1 0.33 ± 0.08 0.33 ± 0.08 0.32 ± 0.08 0.31 ± 0.09 0.29 ± 0.10 run 2 0.35 ± 0.08 0.34 ± 0.08 0.34 ± 0.08 0.33 ± 0.08 0.31 ± 0.08 Values of correlation coefficients are presented as mean ± standard deviation (SD). Each column corresponds to γ 0 = L × 10 -- N , where L is the data length (L = 7774--8723, mean 8285). γ 0 = L × 10 --1 is the default setting. Mean ± SD of inter-run reproducibility (i.e., correlation values of correlation matrices between run 1 and 2) for each γ~0~ value is summarized in [Table 7](#T7){ref-type="table"}. One-way ANOVA revealed no significant difference among five γ~0~ values for both oxy- and deoxy-Hb \[*F*~(4~,~95)~ = 0.58, *p* = 0.679 for oxy-Hb; *F*~(4~,~95)~ = 0.49, *p* = 0.744 for deoxy-Hb\]. ###### Effect of γ~0~ value on inter-run reproducibility. **L × 10^0^** **L × 10^--1^** **L × 10^--2^** **L × 10^--3^** **L × 10^--4^** ---------- --------------- ----------------- ----------------- ----------------- ----------------- oxy-Hb 0.76 ± 0.06 0.76 ± 0.06 0.76 ± 0.06 0.75 ± 0.07 0.73 ± 0.09 deoxy-Hb 0.75 ± 0.07 0.76 ± 0.07 0.75 ± 0.08 0.74 ± 0.09 0.72 ± 0.10 Values of correlation coefficients are presented as mean ± standard deviation (SD). Each column corresponds to γ 0 = L × 10 -- N , where L is the data length. γ 0 = L × 10 --1 is the default setting. Discussion {#S4} ========== The aim of this study was to evaluate our proposed HB DOT algorithm in terms of its performance to estimate the resting-state functional connectivity among brain regions, not task-related brain responses. We used fMRI data as a reference. In addition, we compared our method with other DOT algorithms (the MN and MN-WU), which adopt the two-process approach. Similarity (i.e., correlation coefficient) of the RSFCs between fMRI and DOT showed higher for the HB than both the MN and MN-WU, suggesting that DOT with the HB algorithm is more appropriate to a substitute for fMRI than those with the MN and MN-WU in estimating the resting-state functional connectivity as well as the task-related cortical responses ([@B33]). In addition, inter-run reproducibility (i.e., Pearson's correlation and intra-class correlation coefficients of RSFCs between runs) showed higher for the HB than both the MN and MN-WU, suggesting that DOT with the HB algorithm is more reliable. In addition, mean values of both single- and average-measure ICC are far higher than 0.4, a criterion of sufficient reliability ([@B35]), suggesting that DOT with the HB algorithm is highly reliable and comparable to fMRI. These results were true for not only deoxy-Hb but also oxy-Hb, which cannot be measured by fMRI. We conducted additional analyses relating to intra-run reproducibility (i.e., similarity of RSFCs between FH and SH) for fNIRS data. As for the intra-run test-retest reliability, the HB had significantly higher intra-run reproducibility than both the MN and MN-WU for any intra-run reproducibility measure. In particular, both single- and average-measure ICC values for the HB were far higher than 0.4 and comparable to those for the fMRI. Thus, high reliability of DOT image with the HB algorithm was demonstrated in intra-run as well as inter-run analyses. Meanwhile, comparison of reproducibility between intra-run and half-length inter-run showed no significant difference. This result suggests that the WM task, conducted between the two resting-state fNIRS runs, did not affect RSFCs. According to [@B2], the reliability of the resting-state fMRI connectivity estimates was low for the scan length less than 5 min. However, as described in section "Comparison of Connectivity Matrices Among DOT Algorithms," mean scan lengths of both first and second halves of resting-state fNIRS recording were slightly less than 5 min in the present study. Thus, re-examination of results on intra-run analyses is desirable using data with sufficiently long scan lengths. According to the SD maps for the RSFCs (i.e., [Figure 5](#F5){ref-type="fig"}), variability across subjects was little in fMRI, whereas it was large in fNIRS regardless of the DOT algorithms. This difference suggests that the variability of the estimated DOT images across subjects is larger than that of the fMRI images. Such a large variability in DOT images may be due to errors in forward modeling such as probe coregistration error and head model error, variability in measurement condition (e.g., individual difference in a signal-to-noise ratio) and individual difference in optical parameters which cannot be precisely dealt in forward modeling. In addition, oxy-Hb or deoxy-Hb may not solely correspond to BOLD signals. Further work is required to explore the cause of this difference. In the calculation of RSFCs, we used (1) Shen's atlas rather than the widely used AAL atlas and (2) timeseries averaged across all sensitive voxels within each region. This is because we considered that each region of the AAL includes different functional areas because of its coarse-grained nature \[116 regions for a whole brain, but 9 *available* regions in our case ([Supplementary Figure S1](#SM1){ref-type="supplementary-material"})\], whereas that of the Shen's atlas includes a single functional area due to its fine-grained nature \[order of 300 regions in a whole brain, but 19 *available* regions in our case ([Figure 2C](#F2){ref-type="fig"})\] and therefore mean timeseries represent temporal activity of the ROI. In fact, as described in [Supplementary Material](#SM1){ref-type="supplementary-material"} (Section 3 Kendall's *W* analysis) the chi-squared test suggested that time courses of all voxels in each region of the Shen's atlas were concordant, supporting that the Shen's atlas provides functional subunits and therefore mean timeseries represent temporal activity of the ROI. In addition, the Kendall's *W* for the Shen's atlas is significantly larger than that for the AAL atlas, supporting the validity of using the Shen's atlas in the calculation of functional connectivity. These results are true, regardless of the DOT algorithms. Furthermore, the additional analysis with PCA (Section 4 Principal component analysis in [Supplementary Material](#SM1){ref-type="supplementary-material"}) also confirmed the validity of using both voxel-averaged timeseries and Shen's atlas. As described in the previous paragraph, the AAL atlas is not ideal. However, the chi-squared test also failed to reject the null hypothesis that there is no concordance among all voxels in the region of the AAL atlas (*p* \< *0.05* for all *available* ROIs). We therefore calculated the AAL counterpart on both (1) similarity between fMRI and DOT and (2) inter-run reproducibility, for your reference (see [Supplementary Material](#SM1){ref-type="supplementary-material"} for details). The similarity measure revealed no significant difference among three DOT algorithms (HB vs. MN vs. MN-WU), which is inconsistent with the Shen's case. On the contrary, the inter-run reproducibility for the HB tended to be higher than that for the MN and MN-WU, which is consistent with the Shen's case. Thus, the AAL counterpart was not always consistent with the Shen's results. Although we do not have evidence that each region of the AAL atlas includes different functional areas, it is obvious that the Shen's atlas is more suitable. Thus, the results for the Shen's atlas case seem to be more reliable. The confidence parameter γ~0~ in the HB DOT determines the balance between the data and the prior information. As far as we investigated in the present study, γ~0~ did not significantly affected either similarity of RSFCs between fMRI and DOT or inter-run reproducibility of RSFCs. However, both correlation values slightly decreased by setting γ~0~ the lowest value (L × 10^--4^). This may be consistent with the previous finding that a very low γ~0~ results in incorrectly localized sparse DOT images in some subjects in real experimental data which has a low signal-to-noise ratio ([@B33]). At present, we do not have a technique to determine the optimal γ~0~ value. Further work is required to find it. In the present study, we did not adopt the prevailing approach in the resting-state fMRI studies, spatial ICA. One of the reasons for this is that spatial ICA is suited for whole brain analysis whereas fNIRS data in the present study covered only frontal and parietal areas. Applying spatial ICA to whole brain DOT images will be a challenging future work. Conclusion {#S5} ========== The present study showed that our HB algorithm can be used as an alternative to fMRI, in estimating resting-state functional connectivity as well as task-related responses. We also demonstrated its superiority over the current standard DOT algorithms. Although fNIRS data in the present study covered only frontal and parietal areas due to the experimental limitation of the high-density measurement, it is desired to cover wider areas of the cortex (ideally whole brain). Recently, we showed that multi-directional measurement has the ability to accomplish DOT without requiring high-density measurement ([@B27]). It would be interesting to estimate RSFC for more widespread cortical areas using multi-directional DOT in future work. Data Availability Statement {#S6} =========================== The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher. Ethics Statement {#S7} ================ The studies involving human participants were reviewed and approved by the ATR Review Board Ethics Committee. The patients/participants provided their written informed consent to participate in this study. Author Contributions {#S8} ==================== TA, TS, TO, YO, AI, YI, and OY designed the study. TA, TS, TO, YO, and OY collected the data. TA analyzed the data. TA, TS, and OY wrote and reviewed the manuscript. Conflict of Interest {#conf1} ==================== Although the authors AI and YI were employed by the company Shimadzu Corporation, they were not involved in the comparison of several DOT algorithms and therefore the results in the present study cannot be biased. The remaining 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.** This work was funded by the ImPACT Program of the Council for Science, Technology and Innovation (Cabinet Office, Government of Japan). This research was conducted under contract with the National Institute of Information and Communications Technology, entitled "Analysis of multi-modal brain measurement data and development of its application for BMI open innovation (grant \#209)." Part of this work was presented at the fNIRS2018 conference in Tokyo, Japan, during October 2018 ([@B1]). We thank Yoshiko Itakura for her assistance in data acquisition and Kaori Nakamura for her valuable support. <http://trendscenter.org/software/gift/> <https://surfer.nmr.mgh.harvard.edu> Supplementary Material {#S11} ====================== The Supplementary Material for this article can be found online at: <https://www.frontiersin.org/articles/10.3389/fnins.2020.00032/full#supplementary-material> ###### Click here for additional data file. [^1]: Edited by: Yunjie Tong, Purdue University, United States [^2]: Reviewed by: Han Zhang, University of North Carolina at Chapel Hill, United States; Shijie Zhao, Northwestern Polytechnical University, China [^3]: This article was submitted to Brain Imaging Methods, a section of the journal Frontiers in Neuroscience

1. Introduction {#sec1} =============== Potassium (K^+^) is the most abundant intracellular cation in both prokaryotic and eukaryotic cells reaching concentrations of 100 to 1000 mM inside and 0.1 to 10 mM outside the cell \[[@B1]--[@B3]\]. The high intracellular concentrations of the cation are required by cells for diverse cellular processes which include maintenance of turgor pressure, regulation of cytoplasmic pH, osmolarity, transmembrane electrical potential, activation of enzymes, gene expression, and stress responses \[[@B1]--[@B3]\]. In order to maintain these high concentrations, most prokaryotes possess several K^+^ influx and efflux pumps, which have varying affinities for the cation and are operative over a wide range of extracellular K^+^ concentrations \[[@B4], [@B5]\]. In*Mycobacterium tuberculosis (Mtb)*, the causative agent of tuberculosis (TB) disease, only two major K^+^-uptake systems, have been identified, namely, the Trk and Kdp. However, in addition, the genes,*mbtG*,*Rv3200c*, and*Rv3237c*, encode the putative K^+^ channels, L-lysine 6-monooxygenase, a possible transmembrane cation transporter, and a conserved hypothetical protein, respectively \[[@B6]\]. These have been sequenced in the*Mtb* genome, but their roles have not been described \[[@B6]\]. The Trk system is composed of two TrkA proteins, CeoB and CeoC, encoded by two highly homologous operonic genes,*ceoB* and*ceoC* \[[@B6]\]. TrkA is a low-affinity, K^+^-uptake pump, transporting K^+^ at neutral pH under aerobic conditions \[[@B7]\]. In*Escherichia coli*, in which it has been extensively characterized, it is a constitutively expressed, secondary, active K^+^/H^+^ symporter, operating under similar conditions, but ineffective at acidic pH \[[@B8]\]. The second transporter, Kdp, consists of an active KdpFABC element comprising the KdpF, KdpA, KdpB, and KdpC proteins, as well as the two-component response-regulator KdpDE systems comprising the KdpD and KdpE proteins. The two systems are encoded by the*kdpFABC* and*kdpDE* operons, which differ from those of other bacteria, including*E. coli*, being transcribed in opposite directions from each other and separated by an intergenic promoter region of approximately 192 bp \[[@B6]\]. Similar to those of other bacteria, Kdp is an inducible, high-affinity K^+^ transporter functioning as an emergency scavenger when extracellular K^+^ concentrations are low or when the Trk system is either not functioning or absent \[[@B7], [@B9]\]. It has been characterized as being a primary, active ATP-driven K^+^/H^+^ antiporter \[[@B10]\]. During TB infection, bacteria reside intracellularly and extracellularly in macrophages and necrotic granuloma, respectively, where they are exposed to adverse growth conditions including low pH as well as decreased oxygen and nutrient concentrations \[[@B11], [@B12]\]. However, the possible involvement of the mycobacterial K^+^ transporters in sustaining growth and survival in hostile environments has not been defined. In the current study, the roles of the Trk and Kdp systems of*Mtb* during logarithmic growth, as well as the factors that lead to their induction*in vitro*, have been investigated. These include the relative expression of genes encoding the Trk and Kdp systems in the wild-type (WT) and*trk*-gene knockout strain of*Mtb* (expressing the Kdp system only) in relation to rate of growth, uptake of K^+^, extracellular K^+^ concentration, pH level, and membrane potential under aerobic conditions. 2. Materials and Methods {#sec2} ======================== 2.1. Reagents, Primers, and Chemicals {#sec2.1} ------------------------------------- Primer sequences for*trk* and*kdp* target genes, as well as*sigA* used for real-time, reverse-transcriptase polymerase chain reaction (RT-PCR), were designed with Primer 3 Version 0.4.0 \[[@B13]\] and evaluated for dimerization with integrated deoxyribonucleic acid (DNA) technologies (IDT) OligoAnalyzer 3.1 \[[@B14]\]. These primers were synthesized by Inqaba Biotechnology (Pretoria, South Africa). The primer sequences and amplicon sizes of different genes are shown in [Table 1](#tab1){ref-type="table"}. Rubidium-86 chloride (^86^Rb^+^) 37 MBq was purchased from PerkinElmer Radiochemicals, Boston, MA, USA, and used as a surrogate for measurement of K^+^ uptake by*Mtb* strains \[[@B7]\]. Unless otherwise indicated, all other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). 2.2. Strains and Growth Media {#sec2.2} ----------------------------- The bacterial isolates used were WT (H37Rv; ATCC 26518) and the*trk*-gene knockout of*Mtb* \[[@B7]\]. The*trk*-gene knockout strain was constructed from the WT strain using homologous recombination following a two-step strategy \[[@B7]\]. Briefly, the*trk*-encoding genes,*ceoB* and*ceoC*, were sequentially mutated in a p2NIL vector and electroporated into the WT strain, resulting in the formation of the single crossover clone, which subsequently lost the plasmid, forming the double crossover colony, referred to as the*trk*-gene knockout strain. Middlebrook 7H9 broth and 7H10 agar, supplemented with 10% oleic acid, albumin, dextrose, catalase (OADC), and 0.2/0.5% glycerol with/without 0.05% Tween 80, were used as growth media. 2.3. Bacterial Culture Preparation {#sec2.3} ---------------------------------- The bacteria were grown in 7H9 broth and incubated at 37°C under stirring conditions to mid-log phase. Cultures were centrifuged at 3500 ×g at 25°C for 10 min and the pellets resuspended in 7H9 broth to an optical density (OD) of 0.6 at 540 nm (equivalent to 10^7^--10^8^ colony-forming units (cfu)/mL). Approximately 10^5^--10^6^ cfu/mL cells were inoculated into 7H9 broth to early-, mid-, and late-log phases, corresponding to ODs of 0.1--0.3, 0.4--0.6, and 2.0--2.3 at 540 nm, respectively. 2.4. Gene Expression Using RT-PCR {#sec2.4} --------------------------------- ### 2.4.1. RNA Isolation, Purification, and Quantitation {#sec2.4.1} Ribonucleic acid (RNA) was isolated using the Trizol method as described previously \[[@B15], [@B16]\]. Briefly, the bacterial cultures were centrifuged as described above and the cell pellets were resuspended in 1 mL of Trizol (Invitrogen, Carlsbad, CA, USA). The cells were mixed with 0.1 mm zirconia beads (300 *μ*L), lysed for 3 cycles at 45 s each in a Mini-BeadBeater (BioSpec Products, Bartlesville, OK, USA), and centrifuged at 13 000 ×g for 45 s. The supernatants were extracted and mixed with equal volumes of chloroform: isoamyl alcohol (24 : 1 v/v) in a phase-lock gel (Eppendorf, Germany) and centrifuged for 5 min. The recovered supernatants were mixed with isopropanol (600 *μ*L) and incubated overnight at 4°C followed by centrifugation at 13 000 ×g for 20 min at 4°C after which the supernatants were discarded. The RNA pellets were washed once in 70% ethanol (1 mL), air-dried, and treated with DNase I (10 *μ*L RDD buffer, 1 U enzyme; Qiagen, Valencia, CA, USA) for 30 min at 37°C followed by purification using the RNeasy kit (Qiagen) following the manufacturer\'s instructions. The RNA was eluted in 0.1% diethylpyrocarbonate (DEPC) water and its concentration and purity were determined by*A* ~260~/*A* ~280~ ratio reading of \>1.8 using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Its integrity was verified electrophoretically on 2% agarose gel in 1% sodium dodecyl sulfate (SDS). ### 2.4.2. cDNA Synthesis {#sec2.4.2} The complementary DNA (cDNA) was synthesized following a two-step RT-PCR procedure described in the Sigma Enhanced Avian HS RT-PCR kit (Sigma-Aldrich). The annealing mixture for each gene consisting of 100 ng total RNA, 0.5 *μ*M deoxynucleotide triphosphate (dNTP) mixture (dATP, dTTP, dCTP, and dGTP), and 0.5 *μ*M gene-specific reverse primer, made to 10 *μ*L final volume, was incubated at 94°C for 90 s, followed by 65°C for 3 min and 62°C or 57°C for 3 min for the reference or target genes, respectively. The mixture was treated with 0.5 U RNase inhibitor and 0.5 U enhanced avian myeloblastosis virus reverse transcriptase (20 *μ*L) and incubated at 60°C for 30 min and 95°C for 5 min. ### 2.4.3. Real-Time RT-PCR {#sec2.4.3} The RT-PCR reaction was performed using the LightCycler FastStart DNA Master SYBR Green I kit (Roche Molecular Biochemicals, Johannesburg, South Africa) following the manufacturer\'s instructions. A 20 *μ*L mixture consisting of 5 ng cDNA, 0.5 *μ*M of each primer (forward and reverse), 4 mM MgCl~2~, and 1X LC FastStart DNA Master SYBR Green I reaction mixture (FastStart Taq DNA polymerase, reaction buffer, and dNTP mixture, SYBR Green I dye, and MgCI~2~) was added to the LightCycler glass capillary tube and heated at 95°C for 10 min followed by amplification for 40 cycles at 95°C for 10 s, 62°C or 57°C for 30 s, and 72°C for 30 s in a LightCycler 2.0 instrument (Roche Molecular Biochemicals). At the end of the amplification reaction, the melting curves of the PCR products were performed at 65°C for 15 s with single and continuous fluorescence measurement at a heating rate of 0.1°C per second and finally cooled at 40°C to check for the specificity of the products using their melting temperatures (Tm) ([Table 1](#tab1){ref-type="table"}). The primer pairs used for amplicon construction for each gene resulted in a quantification cycle (Cq) \[[@B17]\] value of zero or \>35 for the nontemplate control. ### 2.4.4. Relative Quantification Analysis {#sec2.4.4} The expression levels of the individual genes in the mutant relative to those of the WT strain were determined using the 2^−ΔΔCq^, efficiency-corrected, and the LightCycler methods \[[@B18]--[@B21]\]. The amplification efficiencies were 2 for all the genes using the 2^−ΔΔCq^ method, while those used in efficiency-corrected and LightCycler methods were derived from generated calibration curves for each gene (LightCycler version 4.0). The reference gene was the*sigA* which, based on the Cq values, demonstrated a constant expression throughout the log phase \[[@B22], [@B23]\]. ### 2.4.5. Absolute Quantification Analysis {#sec2.4.5} The calibration curves for each gene, including*sigA*, were generated with genomic standards prepared in 5-fold dilutions containing from 0.0512 to 10^5^  *μ*g/mL DNA per reaction for each gene. 2.5. Bacterial Growth {#sec2.5} --------------------- Approximately 10^5^--10^6^ cfu/mL cells were inoculated into 7H9 broth and incubated at 37°C under stirring conditions for nine days. The cultures were sampled daily, diluted, and plated onto 7H10 agar medium and incubated for three weeks until the colonies appeared. The number of colonies was then counted and compared between the two strains. 2.6. Rubidium Uptake {#sec2.6} -------------------- ^86^Rb^+^ was used as a surrogate tracer for the determination of K^+^ uptake by bacteria. Cells grown in 7H9 broth were harvested, washed, and resuspended in glucose-, K^+^- and Na^+^-free (KONO: 46 mM disodium hydrogen orthophosphate, 23 mM sodium dihydrogen orthophosphate, 0.4 mM magnesium sulphate, and 0.6 mM ferrous sulphate) buffer (pH 7.4). Approximately 10^6^ cfu/mL cells, prewarmed at 37°C, were treated with 1 mCi/L ^86^Rb^+^ in 2 mL KONO buffer containing 22 mM glucose and incubated at 37°C for 90 min. Cells were washed with phosphate-buffered saline (PBS) and lysed with 0.4 mL 5% trichloroacetic acid (TCA). The radioactive content of the lysate was measured using liquid scintillation counter as previously described \[[@B7], [@B24]\]. 2.7. Measurement of pH and Potassium Concentration of the Growth Medium {#sec2.7} ----------------------------------------------------------------------- Bacterial cultures were centrifuged and the supernatants were decontaminated by heating at 95°C for 60 min. The pH of the supernatants was measured directly in the undiluted samples using the CRISON micropH2001 pH meter (Lasec, Johannesburg) and the K^+^ concentrations were measured in the diluted samples by indirect potentiometry utilizing a K^+^ selective electrode in conjunction with a Na^+^ reference electrode (Synchron LX System, Beckman Coulter, Ireland Inc., Gateway, Ireland). In both cases, the bacteria-free 7H9 broth processed as above, as well as unprocessed growth medium, served as control. 2.8. Effects of Alterations in Extracellular pH on Gene Expression, ^86^Rb^+^ Uptake, Membrane Potential, and Growth Rates {#sec2.8} -------------------------------------------------------------------------------------------------------------------------- For these assays, the bacteria, grown in 7H9 broth to mid-log phase (prior to the decrease in extracellular pH and induction of the genes encoding the K^+^ transporters), were washed once and resuspended in Sauton broth, KONO buffer, or indicator-free Hanks balanced salt solution according to the assays being performed, with the pH adjusted to 5.5, 6, 6.5, and 7 with 1 N hydrochloric acid/sodium hydroxide (HCl/NaOH). Similar procedures were followed as described earlier with some minor modifications. Gene expression was performed on RNA isolated from cells grown in pH-adjusted fresh Sauton broth for 24 hours before harvesting. The rate of growth was determined by inoculating the bacteria, resuspended in pH-buffered Sauton broth into fresh culture media and incubated for 14 days under aerobic conditions. The cultures were sampled and plated on Day 14. ^86^Rb^+^ uptake was determined using bacteria suspended in pH-adjusted KONO buffer containing ^86^Rb^+^ and glucose. Membrane potential was determined using bacteria (10^8^ cfu/mL) suspended in pH-adjusted Hanks balanced salt solution (3 mL) in the presence of the membrane potential-sensitive fluorescent dye, dipentyloxacarbocyanine (di-O-C5(3), 400 nM), at 37°C for 5 min. Alterations in fluorescence intensity were monitored using a microplate luminescence spectrophotometer (LS 50: PerkinElmer) with excitation and emission wavelength settings of 460 and 510 nm, respectively. 2.9. Statistical Analysis {#sec2.9} ------------------------- All statistical analyses were carried out with the GraphPad Instat program version 3. The paired Student *t*-test was used to analyze the results for gene expression, while those for ^86^Rb uptake, rates of growth, extracellular pH and K^+^ concentration, and membrane potential were analyzed using an unpaired nonparametric, two-tailed test. The results are expressed as the means ± standard error of mean (SEM) and the significance levels were taken at a *p* \< 0.05. 3. Results {#sec3} ========== 3.1. The Relative Expression of the*trk* and*kdp* Genes {#sec3.1} ------------------------------------------------------- The expression levels of the individual genes in the*trk*-gene knockout relative to those of the WT strain were determined using relative quantification analysis. The Cq values of the*sigA* reference gene were different throughout the log phases, but comparable between the two strains. At the early- and mid-log phases the Cq value for both strains was 22, and as the bacteria progressed into the late-log phase, the values were 18.66 and 18.32 for the WT and*trk*-gene knockout strains, respectively. The efficiencies of calibration curves of the target and*sigA* genes were between 1.8 and 2.3 (SD \> 0.05), with the three methods of analysis generating comparable data. As the efficiency values were close to 2.0, the 2^−ΔΔCq^ method was adopted \[[@B18]--[@B21]\]. The results are shown in [Table 2](#tab2){ref-type="table"}. Mutations of the*ceoB* and*ceoC* genes in the*trk*-gene knockout strain were confirmed by melting curve analysis, resulting in nonspecific fragments. The magnitudes of expression of the*kdp* genes in the WT and*trk*-gene knockout strains throughout the log phase were comparable, with the exceptions of*kdpE* and*kdpF*, the levels of which were moderately increased in the mutant. 3.2. Absolute Expression of*trk* and*kdp* Genes {#sec3.2} ----------------------------------------------- To characterize the expression of individual genes in each strain at the various stages of growth, the absolute expression values of each gene were extrapolated from individual calibration curves. These results are shown in [Table 3](#tab3){ref-type="table"}. The magnitudes of expression of the*sigA* housekeeping gene, during early- and mid-log phases, were comparable between the two strains and were considerably higher than those of the K^+^-uptake encoding genes. The expression levels of the individual K^+^-transporter-encoding genes were also comparable within and between the two strains. The low levels of all the K^+^-uptake encoding genes indicate that these genes are not upregulated during these growth phases and that the observed expression levels probably represent their basal expression. As the growth of the organisms reached the late-log phase, however, the expression levels of all genes, including*sigA*, were elevated, with the highest expression of the*kdp* genes observed in the*trk*-gene knockout strain. The relative fold increase of these K^+^-uptake genes, as ratios of late-log phase in relation to early-log phase for both strains, is shown in [Table 4](#tab4){ref-type="table"}. These fold increases of all the genes indicate that as the bacteria enter into late-log phase, there are environmental factors in the cultures that lead to the upregulation of these genes. However, the fold increase of the*sigA* gene in both strains during late-log phase, in comparison to the K^+^-uptake encoding genes, was very low, indicating that the change in expression of the*sigA* gene in both strains throughout the log phase of growth is minimal. In addition, the induction of the*kdp* genes in the WT strain was low in relation to that of the mutant indicating that the Kdp system is repressed in the presence of the functional Trk system. Gene expression levels calculated as a ratio of the mid- and late-log phases of growth relative to the early-log phase for each gene for each strain are shown in [Table 4](#tab4){ref-type="table"}. Using this format of presentation of data, it is clear that there is a significant upregulation of expression of all the K^+^-related transport genes in the WT strain, especially*ceoB*,*kdpC*, and to a lesser extent*kdpB* and*kdpE*, all of these ratios being much higher than that of the*sigA* gene at the late-log phase. Likewise, the ratios for expression of the*kdp* genes in the mutant strain at the late-log phase were considerably elevated both in relation to*sigA* and the corresponding*kdp* genes in the WT strain, with the*kdpC* and the*kdpB* genes being particularly highly expressed. In addition, the expressions of*kdpA*,*kdpB*, and*kdpC* genes were of a comparable ratio of 1 : 1 : 1 during this growth phase within each strain. 3.3. Rates of Growth {#sec3.3} -------------------- The rates of growth of the two strains were comparable during the early- and mid-log phases, but the*trk*-gene knockout mutant reached the late-log phase earlier than the WT strain. We have previously found that this mutant strain has a faster rate of growth than the WT when using a linear graph presentation \[[@B7]\]. The results are represented as cfu/mL ± SEM and are shown in [Table 5](#tab5){ref-type="table"}. The inoculum sizes at Day 0 were comparable for both strains and were 2 × 10^6^ ± 2 × 10^5^. 3.4. ^86^Rb^+^ Uptake {#sec3.4} --------------------- The efficiencies in K^+^ uptake of the two strains during the mid- and late-log phases were determined by measuring the absolute uptake of ^86^Rb^+^ by the strains during these growth phases. The results are represented as the mean radioactive counts per minute (cpm) values ± SEM and are shown in [Figure 1](#fig1){ref-type="fig"}. During both growth phases, the ^86^Rb^+^-uptake efficiencies of the*trk*-gene knockout strain were higher than those of the WT strain. However, the K^+^-uptake efficiencies of both strains were reduced as the bacteria entered the late-log phase. 3.5. Extracellular pH and Potassium Concentration {#sec3.5} ------------------------------------------------- To characterize the factors that lead to decreased efficiency of uptake of K^+^ and upregulation of the K^+^-uptake genes, presumably as a compensatory mechanism, the possible influences of alterations in extracellular pH and the extracellular K^+^ concentration during the three log phases of growth were evaluated. In the case of both strains, the extracellular pH values of the culture media were comparable to those of bacteria-free media (pH 6.8 ± 0.01) at the early-log stage, increased slightly during mid-log (pH 6.9 ± 0.01), and decreased at the late-log phase (pH 6.5 ± 0.01). With respect to extracellular K^+^ concentrations in the growth media, no significant decreases were detected during the three log phases for both strains, probably reflecting the high K^+^ concentrations of the growth media, as well as possible recycling of K^+^ by the bacteria during growth. The concentrations were 13.3 ± 0.05 mM for the bacteria-free controls, while the paired values for the WT and*trk*-gene knockout strains were 13.4 ± 0.05 and 13.2 ± 0.05, 13.3 ± 0.15 and 13.2 ± 0.15, and 13.5 ± 0.5 and 13.6 ± 0.25 mM during the early-, mid-, and late-log phases, respectively. 3.6. Effect of Manipulation of Extracellular pH on Gene Expression, ^86^Rb^+^ Uptake, Membrane Potential, and Rate of Growth {#sec3.6} ---------------------------------------------------------------------------------------------------------------------------- Because the increase in the magnitude of gene expression of the K^+^ transporters and the decrease in K^+^ uptake ([Section 3.2](#sec3.2){ref-type="sec"}) during late-log phase coincided with a decrease in pH of the growth medium, the possible role played by alterations in extracellular pH on the magnitudes of expression of the K^+^-uptake genes in relation to rate of growth, uptake of ^86^Rb^+^, and membrane potential was investigated in a series of short-duration experiments. For these experiments, based on simultaneous expression of genes from the same operons ([Table 3](#tab3){ref-type="table"}), gene expression was evaluated on one gene of each operon \[[@B7]\] with*sigA* included as a reference gene. All genes, including the*sigA*, were significantly upregulated at pH 5.5 and highest in the*trk*-gene knockout strain. The magnitudes of expression of the*ceoB* and*kdpA* genes were pH-dependent being inversely related to decreasing extracellular pH. Relative to the mutant strain, however, induction of the*kdpA* and*kdpD* genes was less significant in the WT strain ([Figure 2](#fig2){ref-type="fig"}). These results indicate that both K^+^-uptake systems are induced at pH 5.5. However, as the pH increases from 6 to 6.5 the expression of the*trk* genes decreases significantly, while the*kdp* genes remain suppressed in the presence of functional Trk system. In the case of both strains upregulation of gene expression coincided with decreases in the rate of growth, uptake of ^86^Rb^+^, membrane potentials, and extracellular pH which dropped to 5.5 (Figures [3](#fig3){ref-type="fig"}, [4](#fig4){ref-type="fig"}, and [5](#fig5){ref-type="fig"}). Both strains failed to grow at pH 5.5 ([Figure 3](#fig3){ref-type="fig"}). The numbers of bacteria were 5.5 × 10^5^ ± 1.1 × 10^5^ and 4.6 × 10^5^ ± 8 × 10^4^ cfu/mL ± SEM at Day 0 for the WT and*trk-*gene knockout strains, respectively, with corresponding decreases to 2.8 × 10^5^ ± 5.4 × 10^4^ and 1.8 × 10^5^ ± 3.4 × 10^4^ cfu/mL ± SEM on Day 14. As the two K^+^-uptake systems are highly induced at pH 5.5 ([Figure 2](#fig2){ref-type="fig"}), their induction illustrates that these systems are utilized by the bacteria for their survival at this pH. As the extracellular pH increased from 6.0 to 7.0, both strains showed high rates of growth, resulting in at least a 10-fold increase in the number of bacilli in the case of the mutant strain relative to the WT at each pH level. The decrease in K^+^ uptake observed with both strains was pH-dependent being most evident in the case of the*trk*-gene knockout strain ([Figure 4](#fig4){ref-type="fig"}). The membrane potentials of the two strains were comparable at every pH level decreasing in parallel as the pH dropped to 5.5, showing that the membrane potentials are not affected by the presence of Trk system at these pH levels but rather by a change in extracellular pH ([Figure 5](#fig5){ref-type="fig"}). 4. Discussion {#sec4} ============= The results of the current study for the WT strain of*Mtb* demonstrate that the expression levels of the genes encoding the Trk and Kdp K^+^-uptake systems of*Mtb* are extremely low relative to those of the housekeeping gene,*sigA*, during the early- and mid-log phases of bacterial growth. Likewise, in the*trk*-gene knockout strain in which expression of both*ceoB* and*ceoC* was barely detectable, the levels of expression of the*kdp* genes were low and comparable to those of the WT strain. These expression levels suggest that, during these phases of growth in artificial growth medium with a high extracellular K^+^ concentration and neutral pH, utilization of the Trk and Kdp systems is minimal with nonspecific membrane potential-driven uptake of the cation probably predominating, thereby enabling conservation of energy and optimal growth \[[@B25]\]. Alternatively, basal, homeostatic activity of the K^+^ transporters may suffice during the early stages of growth. However, when the bacteria entered the late-log phase of growth striking increases in the levels of expression of both the*trk* and*kdp* genes were noted in the WT strain, which were disproportionately higher than those observed for*sigA*, the respective fold increases being 188, 230, and 77/75 for*ceoB*,*kdpC*, and*kdpB/kdpE*. In the case of the*trk*-gene knockout, a similar pattern in the expression of the*kdp* genes comparable to that of the WT strain was observed, the corresponding fold increases in the*kdpC* and*kdpB* genes being 950 and 539. Presumably the much higher levels of gene expression of the*kdp* genes in the*trk-*gene knockout relative to the WT strain reflect a compensatory strategy to ensure efficient uptake of K^+^ \[[@B7]\]. This pattern in fold increase in*kdp* gene expression has recently been shown for the synthesis of the Kdp subunits of*E. coli* during K^+^-limiting conditions \[[@B26]\]. However, the increased level of expression of the Trk and Kdp systems in*Mtb* during the late-log phase of growth observed in the current study could not be attributed to depletion of extracellular K^+^ since the concentration of the cation in the growth medium was essentially unchanged throughout the log phase of growth. These results are consistent with the existence of additional stressors, such as the decrease in the pH of the growth medium in the late stages of growth that may trigger the upregulation of the K^+^-uptake genes. This apparent relationship was addressed in a separate series of experiments in which the effects of decreasing the extracellular pH of the cell-suspending medium using bacteria harvested at the mid-log phase of growth, prior to the decrease in extracellular pH and upregulation of the K^+^ transport genes, on gene expression, rate of growth, uptake of K^+^, and membrane potential were investigated. In these experiments decreasing the extracellular pH resulted in a concomitant drop in membrane potential, uptake of K^+^, and rate of growth in the setting of upregulation of gene expression, consistent with a mechanistic interrelationship between these events. To summarize, disruption of the membrane potential at low extracellular pH attenuates membrane potential-driven uptake of K^+^, necessitating induction of the genes encoding the active K^+^-uptake systems to enable survival. A similar relationship has also been described in*M. smegmatis* \[[@B27]\]. In addition, other types of microorganism which compensate for decreased K^+^ uptake at low extracellular pH include*Streptomyces lividans*, which possesses a pH-gated K^+^ channel \[[@B28]\], and the Kup transporter of*E. coli*, which functions at low pH \[[@B8]\], as well as the K^+^/H^+^ antiporters operative in most Gram-negative bacteria \[[@B29]\]. In these bacteria deletion of the major K^+^-uptake systems (Trk, Kup, and Kdp) resulted in low membrane potential and poor K^+^ uptake, which occurred concomitantly with failure of maintenance of intracellular pH of 7.4 to 7.8 and attenuation of growth at extracellular acidic pH (pH below 4.5) \[[@B17], [@B27], [@B38], [@B39]\]. At this pH, the growth of*Mtb* is arrested, resulting in a nonreplicating dormant state \[[@B17]\] However, at neutral pH, both cytoplasmic pH and bacterial growth rates are unaffected by the presence of the K^+^-uptake systems, illustrating that transportation of K^+^ is independent of the major K^+^-uptake systems at this pH level \[[@B27], [@B39]\] while at alkaline pH (pH higher than 8), the proton-importing ATP synthase F~1~F~0~ is induced, minimizing the exportation of protons from the cytoplasm \[[@B40]\]. Together with our study, these findings indicate that growth of bacteria at acidic or alkaline pH induces K^+^-uptake genes, which are necessary for the maintenance of internal pH homeostasis and survival of bacteria in these environments \[[@B40], [@B41]\]. During active disease,*Mtb* bacilli are able to survive in acidic environments, found in macrophage vacuoles and necrotic granuloma \[[@B11], [@B12], [@B30]\]. The induction of both the Trk and Kdp systems in acidic environments in the current study indicates that these systems may be required for bacterial adaptation at low pH levels. Following uptake of*Mtb* by macrophages, the phagosome acidifies to pH 6.0, thereafter rising to pH 6.5 within a few hours, enabling the bacteria to survive and multiply \[[@B11], [@B12], [@B31], [@B32]\]. Based on similarities to the TrkA system of*E. coli*, which possesses an NAD^+^-binding motif \[[@B33]\], it is possible, though unproven, that the Trk system of*Mtb*, which has been implicated in intracellular survival \[[@B8], [@B34]\], may function as a K^+^/H^+^ symporter, increasing vacuolar pH. If indeed operative in the intravacuolar milieu, such a mechanism would be expected to interfere with phagosome maturation, thereby promoting intracellular survival. In this growth environment at these pH levels, the Kdp system is likely to be suppressed. However, other factors, such as low K^+^ concentrations, exist in the macrophages that lead to the induction of this K^+^-uptake system \[[@B35]\]. In the granuloma, where the pH ranges between 5.0 and 5.5 \[[@B36], [@B37]\], both K^+^-uptake systems may be required. 5. Conclusion {#sec5} ============= According to the gene expression data, the findings of the current study demonstrate that the Trk and Kdp systems of*Mtb* function at basal levels when conditions, especially high extracellular K^+^ concentration and neutral pH, favour membrane potential-driven uptake of K^+^. However, at low extracellular pH, uptake of K^+^ may become increasingly dependent on induction of both the Trk and Kdp systems as a survival strategy in hostile environments. This study was funded by the South African Medical Research Council and National Research Foundation (Grant no. 87649). Disclosure ========== Part of the work presented herein has been presented at the South African TB Conference, Durban, South Africa, 2014 (<http://www.tbconference.co.za/>). Conflict of Interests ===================== The authors declare that there is no conflict of interests regarding the publication of this paper. {#fig1} {#fig2} {#fig3} {#fig4} {#fig5} ###### Primers for expressed genes in WT and *trk*-gene knockout mutant strains of *Mtb*. Gene name Forward primer (bp) Reverse primer (bp) Fragment length^a^ Tm^b^ ------------ ---------------------------------- ---------------------------------- -------------------- ------- *ceoB* ^c^ CGG CGA CAA CTC CAA CAT (18) CGG CAC ACC GAA GGT TTC (18) 58 86.2 *ceoC* ^c^ CTG CTG GAG TCG ATT CAC CT (20) ACC GCG AAT TCG GTC TTG (20) 107 88.9 *kdpA* GAT TGA ACG GCC TAC TGG TC (20) CTG GAT CTT CTT GCC GAG AT (20) 95 87.5 *kdpB* CTG GGC TGA CGA TCA TCT TT (20) CGT GGT CGG AAT GAG ACA C (19) 122 90.6 *kdpC* TAC CGC AAG GAA AAC AAT CTG (21) GGC ATT GAC CAC CGA TATG (19) 102 90.2 *kdpF* AAC ATC GTC GGG TTG GTG (18) GCG AAT AGG AAC GCC ATT AG (20) 50 83.7 *kdpD* AGT CCA TCG ACC AAC TCA CC (20) CAC CGC TTC CTC CAG GTA T (19) 110 91.4 *kdpE* TGG AAT GGA CGA GTT TCT GG (20) *GAC GGT GAA*TGA ATC GGT TT (20) 103 91.4 *sigA* CAA GGA CGC CGA ACT CAC (18) CTT GCC GAT CTG TTT GAG GT (20) 64 87.4 ^a^Size of the amplicon synthesized by RT-PCR in base pair (bp). ^b^Melting temperature derived from melting curve. ^c^Gene encoding Trk system. ###### Expression of the *trk* and *kdp* genes in the *trk*-gene knockout strain of *Mtb* relative to those of the WT. ---------------------------------------------------------------------------------------------------------------------------- Gene name Early-log ratio\ CI^b^ Mid-log ratio\ CI^b^ Late-log ratio\ CI^b^ (2^−ΔΔCq^)^a^ ± SEM (2^−ΔΔCq^)^a^ ± SEM (2^−ΔΔCq^)^a^ ± SEM ----------- --------------------- ------------- --------------------- -------------- --------------------- ----------------- *ceoB* 0.02 ± 0.01 0.01--0.03 0.016 ± 0.001 0.013--0.02 0.0003 ± 0.001 0.0002--0.00048 *ceoC* 0.01 ± 0.01 0.001--0.01 0.003 ± 0.001 −0.001--0.01 0.002 ± 0.0001 −0.0007--0.01 *kdpA* 1.23 ± 0.11 0.95--1.52 1.03 ± 0.02 0.99--1.07 1.10 ± 0.09 −0.81--1.39 *kdpB* 1.16 ± 0.10 0.9--1.44 1.54 ± 0.16 1.13--1.96 1.30 ± 0.21 0.76--1.85 *kdpC* 1.37 ± 0.14 1.03--1.72 1.00 ± 0.12 0.61--1.23 1.28 ± 0.26 0.62--1.94 *kdpF* 0.82 ± 0.27 0.13--1.51 2.15 ± 0.63 0.52--3.77 1.13 ± 0.18 0.66--1.59 *kdpD* 0.76 ± 0.12 0.48--1.05 0.65 ± 0.13 0.32--0.98 1.39 ± 0.48 0.15--2.63 *kdpE* 1.29 ± 0.19 0.82--1.77 1.84 ± 0.46 0.65--3.02 1.63 ± 0.26 0.96--2.3 ---------------------------------------------------------------------------------------------------------------------------- ^a^Ratio of quantification using delta delta Cq method \[[@B13], [@B14]\]. ^b^Confidence interval. ###### Absolute expression of the *trk* and *kdp* genes in WT and *trk*-gene knockout strains of *Mtb*. Gene name H37Rv (*μ*g/mL) *trk*-gene knockout (*μ*g/mL) ----------- ----------------- ------------------------------- ---------------- -------------- -------------- ----------------- *ceoB* 16.3 ± 6.6 14.7 ± 1.1 3071.0 ± 602.1 0.43 ± 0.1 0.49 ± 0.1 2.34 ± 0.7 *ceoC* 25.7 ± 6.5 25.8 ± 1.3 653.8 ± 68.6 0.13 ± 0.10 0.01 ± 0.01 2.11 ± 0.7 *kdpA* 22.5 ± 8.7 16.4 ± 3.2 618.8 ± 115.7 22.5 ± 5.2 14.7 ± 4.3 3410.6 ± 1192.0 *kdpB* 6.7 ± 3.0 8.2 ± 1.1 519.9 ± 84.4 6.5 ± 2.6 9.2 ± 1.0 3452.3 ± 1266.4 *kdpC* 3.4 ± 1.2 5.4 ± 0.5 787.3 ± 103.3 3.5 ± 0.9 3.3 ± 0.8 3332.2 ± 1109.1 *kdpF* 28.0 ± 9.1 19.6 ± 8.6 1523.8 ± 180.2 11.3 ± 0.5 15.1 ± 1.2 2640.8 ± 648.9 *kdpD* 38.4 ± 5.8 40.2 ± 3.0 1539.5 ± 193.0 34.6 ± 3.0 33.6 ± 5.2 4672.9 ± 1452.0 *kdpE* 9.5 ± 1.7 9.1 ± 2.0 716.8 ± 100.7 10.6 ± 0.4 9.3 ± 1.6 2776.6 ± 805.3 *sigA* 254.5 ± 45.3 208.0 ± 8.1 1088.6 ± 68.2 249.5 ± 36.0 201.6 ± 10.6 2545.8 ± 728.2 ###### Ratio of gene expression of both strains of *Mtb* during mid- and late-log phases in relation to the corresponding early-log phase. Gene name H37Rv *trk*-gene knockout ----------- ------- --------------------- ------- ------- *ceoB* 0.9 188.8 NA^a^ NA^a^ *ceoC* 1.0 25.4 NA^a^ NA^a^ *kdpA* 0.7 27.5 0.7 151.4 *kdpB* 1.2 77.7 1.4 534.4 *kdpC* 1.6 235.0 0.9 952.1 *kdpF* 0.7 54.5 1.3 233.7 *kdpD* 1.1 40.1 1.0 135.3 *kdpE* 1.0 75.7 0.9 262.4 *sigA* 0.8 4.3 0.8 10.2 ^a^Not available since the expression levels in the mutant for these two genes were not detectable. ###### Rate of growth of both strains of *Mtb*. Growth phase ODs Number of colonies cfu/mL ± SEM Days achieved -------------- ---------- --------------------------------------------------------- --------------- Early-log 0.1--0.3 6.0 × 10^6^ ± 6.2 × 10^5^--9.5 × 10^7^ ± 2.1 × 10^7^ 1--3 Mid-log 0.4--0.6 6.8 × 10^8^ ± 3.6 × 10^7^--7.0 × 10^10^ ± 1.7 × 10^9^ 4-5 Late-log 2--2.3 5.0 × 10^11^ ± 7.0 × 10^9^--2.0 × 10^14^ ± 8.0 × 10^12^ 8-9 [^1]: Academic Editor: Bo Zuo

INTRODUCTION ============ Cellular adaptation to a shortage of oxygen is mainly governed by transcriptional regulation. Hypoxia-inducible factors (HIFs) are key players in the hypoxic cell and orchestrate the expression of hundreds of downstream target genes, adapting the cellular metabolism to a low oxygen environment ([@gkr978-B1]). Heterodimeric HIFs consist of a tightly O~2~-regulated α-subunit (HIF-1α, HIF-2α or HIF-3α) and a constitutively expressed β-subunit (HIF-1β). At oxic conditions, HIF α-subunits are continuously marked for proteasomal degradation through hydroxylation of two key prolyl-residues by prolyl-4-hydroxylase domain (PHD) oxygen sensor proteins ([@gkr978-B2]). PHD hydroxylation activity fades as a direct function of oxygen, thus reciprocally controlling the nuclear accumulation of HIFαs. Stabilized HIF-complexes bind to a *cis*-acting HIF binding site (HBS) conformed by a highly conserved core 5′-RCGTG-3′ motif present in all direct target genes ([@gkr978-B1],[@gkr978-B3],[@gkr978-B4]). In analogy to destabilizing proline hydroxylation, the transcriptional activity of HIF is tuned by factor inhibiting HIF (FIH), which hydroxylates a distinct asparagine residue within the HIF-α carboxy terminus and consequently hinders its association with the transcriptional 300-kilodalton coactivator protein (p300) and cAMP response element-binding protein (CREB) binding protein (CBP) ([@gkr978-B5; @gkr978-B6; @gkr978-B7]). Among the three characterized HIF prolyl-4-hydroxylases, PHD2 is widely accepted as the most crucial isoform controlling basal activity of the HIF pathway in oxic cells ([@gkr978-B8]). Underlining its dominant role, disruption of the *Egln1* locus, encoding mouse PHD2, results in prenatal lethality, while PHD1 and PHD3 knock out mice are born normally ([@gkr978-B9]). Broad-spectrum conditional deletions of all three PHDs in mice revealed a global hyperproliferative vascular phenotype uniquely when targeting PHD2, demonstrating an absolute requirement for PHD2, which is not confined to embryonic development ([@gkr978-B10]). Accordingly, PHD2 abundance is considered as a critical factor in tumor angiogenesis, although divergent roles of stromal and tumor cell-derived PHD2 have been discussed ([@gkr978-B11; @gkr978-B12; @gkr978-B13]). As PHD2 protein is strikingly stable and the *de novo* translated enzyme outlasts a period of transient hypoxia by more than 48 h, transcriptional regulation of the *PHD2* locus must be considered as the main process defining cellular levels of PHD2 ([@gkr978-B14],[@gkr978-B15]). Expression of PHD2 itself is delicately influenced by HIF transcriptional activity, forming a negative feedback loop which facilitates dynamic oxygen sensing ([@gkr978-B16; @gkr978-B17; @gkr978-B18]). To identify upstream regulatory pathways affecting *PHD2* gene expression in an unbiased system, we developed a screening approach that allows the identification of transcriptional interactions with DNA-bound HIF complexes and HIF-independent promoter regulation at the same time. The herein described synthetic transactivation screening led to the identification of several members of the E-twenty six (ETS) and FBJ murine osteosarcoma viral oncogene homolog (FOS) families of transcription factors as novel activators of the human *PHD2* promoter (P2P). Among those, ETS translocation variant 4 (ETV4; also known as E1A enhancer binding protein, E1AF, or polyoma-enhancing activator 3, PEA3), was found to be a potent coactivator of HIF-1-dependent transcription. MATERIALS AND METHODS ===================== Cell culture ------------ Human HeLa cervix carcinoma and U2OS osteosarcoma cells were grown in Dulbecco\'s modified Eagle\'s medium (DMEM, Sigma). Human PC3 prostate cancer cells were cultured in Roswell Park Memorial Institute medium (RPMI-1640, Sigma). Media were supplemented with 10% fetal calf serum (FCS) and antibiotics (penicillin 50 IU/ml and streptomycin 100 µg/ml; Gibco-BRL). Hypoxic cell culture was carried out at 0.2% O~2~ (if not indicated differently) using a gas-controlled InvivO~2~ 400 workstation (Ruskinn Technologies). Transfections were performed using polyethyleneimine (PEI; Polysciences) as described earlier ([@gkr978-B17]). P2P constructs -------------- P2P constructs containing the wild-type and mutant HBS in the pGL3basic luciferase vector (Promega) were generated in earlier work ([@gkr978-B16]). Serial 5′-truncations of P2P and a start codon fusion to the luciferase open reading frame (ORF) were used for both promoter versions using standard cloning techniques. Within the scope of the screening approach, the *firefly* reporter gene of pGL-P2P(−607/+3) variants was replaced with the *renilla* luciferase ORF cloned into NcoI and XbaI sites. Transfection and synthetic transactivation screening ---------------------------------------------------- Reverse transfection ([@gkr978-B19]) and screening were carried out using an arrayed expression library containing 704 transcriptionally relevant human full-length cDNAs from the Origene collection (FTCW 19603, GFC-transfection array in a 96-well format). An annotated list of all genes covered by this array is provided online by the manufacturer. Dried DNA (100 ng) of a distinct expression construct spotted per well was reconstituted at room temperature with 20 µl of serum-free medium containing a mixture of pGL-P2P(−607/+3) HBSwt *firefly* and pGL-P2P(−607/+3) HBSmut *renilla* reporter plasmids (100 ng DNA/each). Subsequently, 20 µl of diluted TransIT-LT1 transfection reagent (3:1, µg DNA/ µl TransIT-LT1; Mirus Bio LLC) was added and complex formation was allowed for 30 min at room temperature before 60 µl of a cell suspension containing 1 × 10^4^ U2OS cells in DMEM supplemented with 10% FCS were plated in each well. Plates were incubated at 20% O~2~ for 24 h before being subjected to the screening conditions of 0.2% O~2~ for an additional 24 h. Cultures were lysed in 20 µl of passive lysis buffer (Promega) and luminescence was immediately analyzed with a microplate luminometer (Berthold) using a standard dual luciferase reporter assay system (Promega). Luciferase activities were normalized to the median calculated individually for each plate and luminescence source and expressed as induction factors (IF) according to the equation , with *N*~i~ indicating individual luciferase activity value of each well of plate *i* and median~i~ indicating median of luciferase activities of all 96-wells on plate *i*. To compare the distribution of replicate assays a standard *z*-score evaluation was performed following the equation , with , plate mean of respective luminescence values and *σ*~i~, standard deviation (SD) of plate mean of luminescence values. Reporter gene assays and mammalian two-hybrid analyses ------------------------------------------------------ Construction of pGLTfHRE wt and pGLTfHRE mut reporter plasmids carrying a hypoxia-responsive enhancer element derived from the human *Transferrin* gene was described previously ([@gkr978-B20]). Transfections for standard reporter gene experiments were carried out on 100 mm culture plates essentially as described earlier ([@gkr978-B17]). In brief, U2OS cells were cotransfected with 3 µg reporter plasmid or a mix of 1.5 µg reporter and 1.5 µg expression plasmids, respectively. Transfection efficiency was controlled by cotransfection of 20 ng pRL*SV40 renilla* luciferase reporter vector (Promega). RNA-interference (RNAi) mediated knock down of HIF-1α, FIH-1 or p300 was achieved by transiently transfecting once (HIF-1α, p300) or twice (FIH-1) U2OS cells with 100 nM stealth RNAi duplexes (HIF-1α, 5′-caggacaguacaggaugcuugccaa-3′; FIH-1, 5′-gaaacauugagaagaugcuuggaga-3′; p300, 5′-ggauucgucugugauggcuguuuaa-3′; control, 5′-gcuccggagaacuaccagaguauua-3′; sense strands) using Lipofectamine 2000 according to the manufacturer\'s instructions (Invitrogen). Combined knock down and reporter gene analyses were performed by sequentially transfecting cells with stealth RNAi duplexes 24 h before subjecting them to polyethyleneimine (PEI)-mediated DNA transfection. Mammalian two-hybrid analyses were performed using the mammalian Matchmaker system (Clontech) as described previously ([@gkr978-B14]). Expression vectors of HIF-1α NAD and carboxy-terminal activation domain (CAD) fused to Gal4 DBD ([@gkr978-B21]) were a kind gift of Dr Sang (Drexel University, Philadelphia, PA, USA). U2OS cells were transiently cotransfected with 1.5 µg of Gal4 DBD and 1.5 μg of VP16 AD fusion protein vectors together with 500 ng of *firefly* luciferase reporter vector pGRE5xE1b and 20 ng of pRL-SV40. Total transfected DNA amounts were equalized in each experiment using the corresponding empty vector. Luciferase reporter gene activities were determined using the dual-luciferase reporter assay system (Promega). Fluorescence resonance energy transfer -------------------------------------- The full-length ORF of human ETV4 was cloned into pENTR4 and subsequently recombined with pECFP-C1-DEST ([@gkr978-B15]) to obtain the expression vector for a cyan fluorescent ETV4 fusion protein. U2OS cells were transiently transfected with the pECFP-ETV4 and pEYFP-HIF1 or pEYFP-HIF2 plasmids as recently described ([@gkr978-B22]). Fluorescence resonance energy transfer (FRET) was monitored under normoxic or hypoxic conditions (1% O~2~ for 4 h) 24--48 h post-transfection. Protein extraction and immunoblot analysis ------------------------------------------ Cells were washed twice and scraped into ice-cold phosphate-buffered saline. Soluble cellular protein was extracted with a high salt extraction buffer containing 0.4 M NaCl, 0.1% Nonidet P-40, 10 mM Tris--HCl (pH 8.0), 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride and 1× protease inhibitor cocktail (Sigma). Protein concentrations were determined by the Bradford method and 50--80 µg of cellular protein were subjected to immunoblot analysis using the following antibodies: mouse monoclonal antibody (mAb), anti-human HIF-1α (clone 54/HIF-1α; BD Transduction Laboratories), mAb anti-ETV4 \[PEA3 ([@gkr978-B16]); Santa Cruz Biotechnology\], rabbit anti-ETV4 (sdix20580002; Novus Biologicals), rabbit anti-human PHD2 (NB100-137; Novus Biologicals), mAb anti-FIH-1 (NBP1-30333; Novus Biologicals), mAb anti-p300 (554215; BD Pharmingen) and mAb anti-β-actin (clone AC-74; Sigma). Primary antibodies were detected with respective polyclonal anti-mouse or anti-rabbit sera conjugated to horseradish peroxidase (Pierce). Chemiluminescence signals were developed using Supersignal West Dura substrate (Pierce) and images were acquired with a digital light imaging system (LAS 4000; Fuji). mRNA quantification ------------------- Complementary DNA was generated by reverse transcription (RT) of 1--5 µg of total RNA using AffinityScript reverse transcriptase (Agilent). Transcript levels were determined by real-time quantitative (q) polymerase chain reaction (PCR) using a SybrGreen qPCR reagent kit (Sigma) in combination with the MX3000P light cycler (Agilent). All RT-qPCR data are presented as ratios relative to ribosomal protein L28 mRNA values. Primer sets for human PHD2, CA9 and L28 have been described earlier ([@gkr978-B17],[@gkr978-B23]). TMA analysis ------------ Clinicopathological characterization and immunohistochemical analyses of selected components of the HIF-pathway using a tissue microarray (TMA) consisting of 282 invasive breast cancer cases diagnosed at the Institute of Surgical Pathology (University Hospital, Zürich, Switzerland) have been described recently ([@gkr978-B13]). Sections of the same TMA were stained with a rabbit polyclonal anti-ETV4 antibody (HPA005768, Sigma) in a 1:100 dilution using an automated immunohistochemistry platform (Ventana BenchMark, Roche). An immunoreactive score for ETV4 staining was calculated by multiplication of staining intensity (graded between 0 and 3) and the percentage of positive cells (graded between 0 and 4 with 0, nil; 1, \<10%; 2, 10--50%; 3, 51--80%; 4, \>80%) as quantified by a senior pathologist (G.K.). Nonparametric correlations between ETV4 expression and HIF-1/2α or HIF target genes were analyzed by calculating Spearman\'s rank correlation coefficient using predictive analysis software (IBM SPSS Statistics 18). Short hairpin RNA constructs and lentiviral infections ------------------------------------------------------ Expression vectors encoding short hairpin RNA (shRNA) sequences targeting human ETV4 and a noncoding control driven by the U6 promoter in a pLKO.1-puro plasmid were purchased from Sigma. Viral particles were produced in HEK293T cells using the ViraPower lentiviral expression system according to the manufacturer\'s instructions (Invitrogen). Infected PC3 cells were cultured in RPMI-1640 supplemented with 0.5 μg/ml puromycin. Chromatin immunoprecipitation ----------------------------- Chromatin immunoprecipitation (ChIP) assays from parental PC3 cells exposed to 20% or 0.2% O~2~ for 4 and 24 h were performed essentially as described previously ([@gkr978-B24]). The following antibodies were used for immunoprecipitation: rabbit anti-HIF-1α (ab2185; Abcam) and rabbit anti-ETV4 (sdix20580002; Novus Biologicals). Rabbit serum (011-000-001; Jackson ImmunoResearch) served as unspecific control. Enrichment of P2P chromatin was determined by PCR using the following oligonucleotides: *PHD2* forward 5′-gtatgccctgcgctcctc-3′, reverse 5′-gctgagagaatagggcctgtg-3′. Gene array analysis ------------------- Total RNA was extracted from pools of shRNA expressing PC3 clones with RNeasy (Qiagen). RNA integrity was evaluated using the Agilent 2100 Bioanalyzer. Genome-wide RNA levels were quantified using the human gene expression ShurePrint GE3 (8 × 60 K) microarray according to the manufacturer\'s instructions (Agilent). All data were deposited in NCBI\'s Gene Expression Omnibus (GEO) and are publicly accessible through GEO accession number GSE32385 (<http://www.ncbi.nlm.nih.gov/geo/>). Statistical analysis -------------------- If not otherwise indicated, results are presented as mean values ± standard error of the mean of at least three independent experiments. Column statistics applying paired Student\'s *t*-tests were calculated using GraphPad Prism version 4.0 (GraphPad Software). RESULTS ======= A single HBS is sufficient for hypoxic induction of the human P2P but dispensable for basal promoter activity ------------------------------------------------------------------------------------------------------------- Previously, we have reported on a functional, single HIF-binding site located in the 5′-regulatory region of the human *PHD2* gene ([@gkr978-B16]). Using a series of 5′-truncated luciferase reporter genes (schematically depicted in [Figure 1](#gkr978-F1){ref-type="fig"}A), the minimal hypoxia-responsive region of the P2P was further mapped to an element spanning nucleotides −424 to +3 relative to the translational start site. While pGL-P2P(−424/+3) still revealed high hypoxic inducibility when transiently transfected into U2OS or HeLa cells, normoxic promoter activity of this region was largely lost when compared with the longer promoter variants, regardless of whether the HBS was wild-type or mutant ([Figure 1](#gkr978-F1){ref-type="fig"}B). Elongation of the promoter by at least 183 nts \[construct pGL-P2P(−607/+3)\] showed robust normoxic activity in both cell lines ([Figure 1](#gkr978-F1){ref-type="fig"}B). As basal induction of HBS-lacking constructs was observed between nucleotides −424 and −607 without any further effects upstream, the existence of transcriptionally active elements required for oxic expression of the *PHD2* gene in this particular region is suggested ([Figure 1](#gkr978-F1){ref-type="fig"}B). Of note, the fold of hypoxic activation of pGL-P2P(−607/+3) resembled the oxygen-dependent IF of endogenous PHD2 mRNA and protein in both cell lines ([Figure 1](#gkr978-F1){ref-type="fig"}C). Figure 1.Identification of the minimal human P2P. (**A**) Schematic representation of P2P 5′-truncations and their cloning strategy as used in this study. The translational start site is designated '+1'. (**B**) Regulatory DNA regions of the human *PHD2* gene were cloned into luciferase reporter vectors that were transiently transfected into human U2OS osteosarcoma cells. One day after transfection, cells were incubated for 24 h at 20 or 0.2% O~2~. Hypoxic IF (mean values ± SD) of relative luciferase activities were calculated from three independent experiments performed in triplicates. Mutation of a single HBS (black rectangles in A) completely abrogated hypoxic inducibility of all constructs. (**C**) HeLa and U2OS cells were incubated at 20 or 0.2% O~2~ for 4--24 h and protein levels of HIF-1α, PHD2 and β-actin were analyzed by immunoblotting. Total RNA was isolated from cultures treated as in (B) and mRNA levels of PHD2 and CA9 were determined by RT-qPCR. Transcript levels of CA9 served as positive control to confirm continuous hypoxic responses. Gene expression levels were expressed in relation to ribosomal L28 mRNA (rel. levels) calculated from three independent experiments (±SD). A differential screening approach to identify site-specific transcription factor interplay ------------------------------------------------------------------------------------------ Multiplexed transfection of *firefly* and *renilla* reporter genes controlled by either wild-type or HBS-mutant P2Ps, respectively, allows to classify any reporter-modulating event as HIF-dependent or self-sufficient. Moreover, the dual reporter system provides a read-out that permits to screen under HIF-stabilizing conditions (e.g. hypoxia) while simultaneously assessing the noninduced background levels (simulated 'normoxia') in the very same cells, thereby reducing intra-assay variabilities and screening complexity. Nucleotides −607 to +3 of the P2P containing either the wild-type or mutant HBS were used to drive transcription of *firefly* or *renilla* luciferase cDNAs, respectively. The two luciferase reporter genes showed identical hypoxic responses in the respective reporter vectors ([Figure 2](#gkr978-F2){ref-type="fig"}A). As a proof of principle, this system was tested by coexpressing *mIpas*, encoding an inhibitory isoform of HIF-α ([@gkr978-B25]), with the two reporter plasmids as described above. Indeed, a marked downregulation of hypoxic *firefly* versus *renilla* luciferase activity was observed when compared with cotransfection with the empty vector or an unrelated transcription factor (mHes-1). As expected ([@gkr978-B17]), forced expression of PHD2 or PHD3 strongly attenuated the hypoxic activation of the reporter system, confirming that also post-translational mechanisms impairing the activation state of the HIF-pathway can be assessed by this method ([Figure 2](#gkr978-F2){ref-type="fig"}B). Figure 2.Synthetic transactivation screening allows to discriminate HBS-dependent and independent promoter activation. (**A**) P2P(−607/+3) driven *firefly* (blue) and *renilla* (red) luciferase reporter genes transfected into U2OS cells show similar hypoxic activation (0.2% O~2~ for 24 h, black bars) when compared with normoxic (20% O~2~, open bars) cultures. HBS mutation completely abrogated hypoxic induction of both constructs. (**B**) Coexpression of transcription factors (mIpas, mHes-1) or PHD enzymes with P2P(−607/+3)wt HBS driven *firefly* and P2P(−607/+3)mut HBS-driven *renilla* luciferase reporters, respectively. Dual luciferase activities were determined after 24 h of hypoxic exposure (0.2% O~2~) and expressed as relative luciferase activity normalized to controls receiving the empty expression vector. (**C**) Schematic overview of the P2P-luciferase constructs used for synthetic transactivation screening. Expected readouts for HBS-dependent (*firefly* reporter activation only, blue circle) and independent reporter gene activation (simultaneous activation of *firefly* and *renilla* reporter genes indicated by blue and red circles, respectively) are illustrated by a cross table. (**D**) Workflow of synthetic transactivation screening analyzing a commercial transcription factor expression library. (**E**) *Z*-scores of two independent screening experiments were plotted for both reporters. Thresholds for *z* \> 2 are indicated by dotted lines. (**F**) Paired values for individual coexpressed cDNAs for *firefly* (blue) and *renilla* (red) read-outs are depicted. A fold of induction \>2 was considered as lead for a re-screening. Positions of HIF-1α and seven new transcription factors positively re-evaluated at a secondary screening level are indicated. Data are given as the mean of two independent screening experiments. For calculations and full gene names refer to the text. Members of the FOS and ETS transcription factor families transactivate the human P2P ------------------------------------------------------------------------------------ An array of 704 cDNA expression vectors representing all commonly known transcription factor families was screened using the HBS-specific synthetic transactivation readout. Possible HIF coregulators alter *firefly* but not *renilla* luciferase expression, whereas HIF-independent factors interfere with both reporters (schematically depicted in [Figure 2](#gkr978-F2){ref-type="fig"}C). A work flow of the transfection and screening procedure is given in [Figure 2](#gkr978-F2){ref-type="fig"}D. Two independent screens were performed and reproducibility was visualized by plotting *z*-scores ([Figure 2](#gkr978-F2){ref-type="fig"}E). Solid correlations were observed for the vast majority of the coexpressed transcription factors, confirming that no significant deviation between the two experiments occurred. Reporter gene inductions by each coexpressed transcription factor were calculated as multiples of the respective 96-well plate median individually for *firefly* and *renilla* activities, averaged over the two experiments, and ranked according to the IF of the HBS-mutant *renilla* reporter ([Figure 2](#gkr978-F2){ref-type="fig"}F). The cut-off was defined as an increase in luciferase activity by a factor of at least 2, tolerating a deviation of 0.05. Factors that showed a reproducible increase in either *firefly* luminescence ([Supplementary Table S1A](http://nar.oxfordjournals.org/cgi/content/full/gkr978/DC1)) or *firefly* and *renilla* luminescence together ([Supplementary Table S1B](http://nar.oxfordjournals.org/cgi/content/full/gkr978/DC1)) were considered as leads. Underlining the validity of this approach, the only known transcriptional activator of PHD2 expression, namely HIF-1α, was among the cDNAs identified ([Figure 2](#gkr978-F2){ref-type="fig"}E and F). For reevaluation, the cDNAs of 43 leads were retrieved and partially sequenced to verify their identities. Seven of these cDNAs, namely ETS-variant 4 (ETV4), Spi-C transcription factor (SPIC), ETS homologous factor (EHF), the proto-oncogenes JUN and FOSB, v-crk sarcoma virus CT10 oncogene homolog (CRK) and Jumonji domain-containing protein 2A resulted in a reproducible HBS-dependent regulation of the P2P. Most of these factors can be attributed to two major groups: the ETS (ETV4, EHF and SPIC) and the FOS (JUN and FOSB) families. ETV4 activates *PHD2* and *transferrin* promoters synergistically with HIF-1 ---------------------------------------------------------------------------- Expression vectors of the seven newly identified factors were cotransfected together with pGL-P2P(−607/+3) HBS wild-type or mutant reporter genes, cloned into identical backbones to exclude false positive effects, which may have resulted from the two different luciferase cDNAs in the original screen. In the presence of a functional HBS, coexpression of ETV4 resulted in a striking super-induction of the P2P under hypoxic conditions only ([Figure 3](#gkr978-F3){ref-type="fig"}A, left panel), which was fully lost with the mutant construct ([Figure 3](#gkr978-F3){ref-type="fig"}A, right panel). As ETV4 was the strongest hit identified, subsequent work focused on the role of this transcription factor as a putative transactivator of PHD2. Of note, the P2P region used in this screen lacks a consensus 5′-^A^/~C~GGAAGT-3′ ETV4 binding site ([@gkr978-B26]). Thus, direct binding of ETV4 to P2P appears unlikely, although not fully excluded regarding the small residual stimulation of constitutive P2P activity following HBS mutation. However, the complete lack of hypoxic P2P stimulation by ETV4 suggests that HIF-1 might actually recruit ETV4 to enhance hypoxic induction. Accordingly, knock down of HIF-1α by RNA interference in U2OS cells abrogated hypoxia and ETV4-mediated induction of the wild-type P2P ([Figure 3](#gkr978-F3){ref-type="fig"}B, right panel), while an unrelated control siRNA had no effect ([Figure 3](#gkr978-F3){ref-type="fig"}B, left panel). A heterologous reporter construct driven by the minimal *SV*40 promoter in conjunction with either a wild-type ([Figure 3](#gkr978-F3){ref-type="fig"}C, left panel) or mutant ([Figure 3](#gkr978-F3){ref-type="fig"}C, right panel) hypoxia response element derived from the human *Transferrin* gene ([@gkr978-B20]) recapitulated the strong hypoxic superinduction by exogenous ETV4, proposing a more general model of synergistic interaction between ETV4 and HIF-1. In line with our screening data, overexpressed ETV4 significantly upregulated endogenous PHD2 mRNA and protein levels in hypoxic U2OS cells, while PHD1 mRNA levels remained unaffected ([Figure 3](#gkr978-F3){ref-type="fig"}D and E). Figure 3.Hypoxic transactivation of the P2P by ETV4 requires HIF-1α activity. (**A**) Standard dual luciferase reporter gene assays of seven reevaluated hits from the transcription factor overexpression array. Wild-type (left panel) or HBS mutant (right panel) P2P regions controlling *firefly* luciferase reporter plasmids were cotransfected into U2OS cells together with expression constructs of the aforementioned factors. Transfection of an empty expression vector (empty) served as negative control and differences in transfection efficiency were controlled by cotransfecting *SV40* promoter driven *renilla* luciferase. Cells were cultured at 20% or 0.2% oxygen for 24 h before dual luciferase activities were determined. (**B**) Transient RNAi mediated knock down of HIF-1α fully abrogated hypoxic activation of the P2P by ETV4. U2OS cells were transiently transfected with siRNA oligonucleotides targeting HIF-1α (siHIF1α, right panel) or a control sequence having no human target (siControl, left panel). Reporter gene experiments using the P2P reporter construct with only wild-type HBS were performed as described in (A). The inset shows an immunoblot confirming the robust knock down of HIF-1α in U2OS cells. (**C**) ETV4 and HIF-1 synergism in hypoxic gene activation is not restricted to the P2P. A heterologous hypoxia responsive reporter gene containing two functional HBS from the human *Transferrin* hypoxia response element (pGL-TfHRE wt) was tested in luciferase reporter assays as described in (A). Mutation of both HBS (pGL-TfHRE mut) caused an abrogation of the signal as seen in (A). (**D** and **E**) Forced expression of ETV4 in U2OS cells upregulates endogenous PHD2 protein and transcript levels. (**D**) Whole cell lysates were prepared from cells exposed for 16 h to 20 or 0.2% oxygen and analyzed for HIF-1α, ETV4, PHD2 and β-actin levels by immunoblotting. (**E**) Total RNA was extracted of similarly treated cells and mRNA levels of PHD1, PHD2 and L28 were quantified by RT-qPCR. Data are shown in relation to ribosomal L28 mRNA (rel. levels) calculated from three independent experiments (\*\**P* \< 0.01, paired Student\'s *t*-test). ETV4 interaction with the carboxy-terminal transactivation domain of HIF-1α depends on corecruitment of p300 ------------------------------------------------------------------------------------------------------------ Intrigued by the HIF-dependent ETV4 effects, we aimed for the characterization of a putative physical interaction between ETV4 and HIF-1α. Using a mammalian two-hybrid system, expression plasmids encoding for ETV4 fused to the activation domain (AD) of viral protein 16 (VP16-ETV4) were cotransfected with HIF-1α oxygen regulatory domains ([@gkr978-B21],[@gkr978-B27]) fused to a Gal4-DNA binding domain (DBD; [Figure 4](#gkr978-F4){ref-type="fig"}A). Due to its intrinsic transactivation activity, constructs containing the CAD of HIF-1α \[amino acids 775-826 ([@gkr978-B27])\] were sufficient to activate the Gal4-responsive promoter ([Figure 4](#gkr978-F4){ref-type="fig"}B). Coexpression of ETV4 strikingly superinduced GH1α740-826 and GH1α786-826, particularly under hypoxic conditions, suggesting that HIF-1α CAD and ETV4 cooperate to transactivate target genes ([Figure 4](#gkr978-F4){ref-type="fig"}B). ETV4 effects on the aminoterminal activation domain ([@gkr978-B27]) (NAD; HIF-1α amino acids 549-582) were negligible. Both, HIF-1α and ETV4 have been demonstrated to interact with the ubiquitous transcriptional coactivators p300/CBP ([@gkr978-B28],[@gkr978-B29]). To address the question whether the two factors directly interact or whether a ternary complex between HIF-1, p300/CBP and ETV4 is formed (schematically depicted in [Figure 4](#gkr978-F4){ref-type="fig"}C), binding of HIF-1α CAD to p300 was disrupted by forced overexpression of CBP/p300-interacting transactivator 2 (CITED2), known to negatively regulate HIF function ([@gkr978-B30]). Structural analyses revealed that CITED2 and HIF-1α share an overlapping binding interface in the p300 cysteine--histidine-rich 1 (CH1) domain and competition assays showed a 33-fold higher affinity of CITED2 for binding to p300 CH1 than a corresponding HIF-1α CAD peptide, indicating that CITED2 is a dominant inhibitor of HIF-1α:p300/CBP complex formation ([@gkr978-B31]). HIF-1α CAD:ETV4 interplay was totally abrogated by CITED2 in mammalian two-hybrid experiments ([Figure 4](#gkr978-F4){ref-type="fig"}D), underscoring the assumption that ETV4 coactivation of HIF-1 requires functional interaction of the latter with p300/CBP. Figure 4.Transcriptional cooperation between ETV4 and HIF-1 is disrupted by CITED2. (**A**) Schematic representation of HIF-1α and ETV4 domain structure and fusion constructs used in mammalian two-hybrid assays. PAS, PER-ARNT-SIM; bHLH, basic helix--loop--helix domain; ODD, oxygen-dependent degradation domain; NRR, negative regulatory region; NAD and CAD, amino-carboxy-terminal activation domain and CADs, respectively. A GAL4-DBD was fused to regions encompassing the HIF-1α NAD and CAD. Full-length ETV4 bearing two activation domains (AD, acidic domain; Ct, carboxy-terminal tail) flanking a central ETS domain was fused to a VP16 activation domain (VP16-AD). Numbers indicate the amino acids present in the respective constructs. (**B**) U2OS cells were cotransfected with a Gal4-responsive reporter plasmid and Gal4-HIF-1α (GH1α) constructs alone or in combination with VP16-ETV4. The GH1α fusion constructs are specified by the aminoterminal starting amino acid of the truncated HIF-1α regions (530, 740 and 786, respectively). Following transfection, cells were evenly split and incubated at 20 or 0.2% O~2~ before luciferase activities were determined 24 h later. Noninteracting Gal4 DBD-p53 and VP16-AD-CP1 served as negative control (neg. ctrl.), while the interactions between Gal4 DBD-PHD2 and VP16-AD-HIF-2α(ODD) or VP16-AD-FKBP38 were used as positive controls (pos. ctrl. 1 and pos. ctrl. 2, respectively). (**C**) Scheme of the potential interactions between HIF-1, p300/CBP and ETV4 as assessed by mammalian two-hybrid assays. Both CITED2 and FIH can block the interaction between HIF-1α and p300/CBP. (**D**) Cotransfection of the indicated amounts of a CITED2 expression construct together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). (**E**) Cotransfection of siRNA directed against p300 together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). The p300 knock down efficiency of different siP300 oligonucleotides was analyzed by immunoblotting (upper panel) and siP300\#1 was chosen for further experiments. (**F**) Cotransfection of siRNA directed against FIH together with the mammalian two-hybrid expression vectors followed by hypoxic exposure and luciferase activity determination as described for (B). The efficiency of the siFIH mediated FIH knock down was confirmed by immunoblotting as shown in the inset. (**G**) ChIP of normoxic or hypoxic PC3 cells using antibodies directed against HIF-1α or ETV4, or control serum. The amount of coprecipitated chromatin derived from the human P2P region (encoded by *EGLN1*) containing the HBS was determined by PCR followed by agarose gel electrophoresis. While transient knock down of p300 slightly reduced the intrinsic transactivation activity of GH1α786-826 in mammalian two-hybrid experiments, robust superinduction of this construct occurred when VP16-ETV4 was cotransfected, irrespective of the presence of p300 ([Figure 4](#gkr978-F4){ref-type="fig"}E). Thus, we assume that both p300 and CBP can function as bridging molecules for HIF-1α CAD:ETV4 interplay. Underlining the essential requirement of p300/CBP for HIF-1α:ETV4 interaction, normoxic transactivation activity of cotransfected GH1α786-826 and VP16-ETV4 was similar to hypoxic activation levels in U2OS cells transiently depleted of FIH ([Figure 4](#gkr978-F4){ref-type="fig"}F). FIH has been described previously as oxygen-dependent negative regulator of HIF-α:p300/CBP interaction ([@gkr978-B7]). ChIP experiments using either anti-HIF-1α or anti-ETV4 antibodies revealed oxygen-dependent enrichment of the HRE-containing *PHD2* (*EGLN1*) promoter region in both precipitations ([Figure 4](#gkr978-F4){ref-type="fig"}G), providing further evidence for corecruitment of the two transcription factors to the endogenous *PHD2* locus. Both HIF-α isoforms are capable of forming a complex with ETV4 -------------------------------------------------------------- Fluorescence resonance energy transfer (FRET) analyses of coexpressed ETV4 and HIF-1α marked with cyan or yellow fluorescent protein tags (CFP and YFP, respectively) resulted in a robust energy transfer between both factors. Similar FRET efficiencies were observed when YFP-labeled HIF-2α was used together with CFP-ETV4 ([Figure 5](#gkr978-F5){ref-type="fig"}A and B). The intracellular distance of the two nuclear proteins was calculated to be 5.6--5.7 nm and did not differ in oxygenated or hypoxic cells, which might be explained by saturation of the HIF-α degradation pathways by exogenous overexpression of the transcription factors. Notably, efficient energy transfer between HIF-1α and p300 at ambient oxygen tensions has been reported previously ([@gkr978-B32]). Figure 5.Both HIF-1α and HIF-2α colocalize with ETV4 to the nucleus within molecular proximity. U2OS cells were transiently transfected with the indicated CFP or YFP plasmids, and FRET analysis was performed at 20% O~2~ or 1% O~2~ 24--48 h post-transfection. (**A**) Microscopic images showing the subcellular localization of the exogenous proteins. Fluorescence intensity of FRET signals is visualized by false colors on a color bar from low (blue) to high (white) intensity. (**B**) FRET efficiencies for CFP-ETV4 and YFP-HIF-1α (upper panel) or YFP-HIF-2α (lower panel) fusion protein pairs were calculated from 20 to 40 randomly selected cells which displayed various fluorescent acceptor/donor ratios. Scatter plots were fit to a single-site binding model. FRET efficiencies are given as the percentage of transferred energy relative to the energy absorbed by the donor. A broad role for ETV4 in hypoxic gene expression ------------------------------------------------ Recent work reported high ETV4 expression levels in the human prostate cancer cell line PC3 that lacks the constitutive photomorphogenic protein COP1 acting as E3 ubiquitin ligase for a variety of ETS proteins ([@gkr978-B33],[@gkr978-B34]). As endogenous ETV4 expression levels in U2OS cells were close to the detection limit, the PC3 cell model was chosen to study the involvement of ETV4 in the hypoxic response by applying a genome-wide expression array screening. PC3 cells lentivirally infected with shRNA expression constructs targeting ETV4 (shETV4) revealed a robust knock down of mRNA and protein levels, while a nontarget control shRNA (shNTC) did not affect ETV4 expression ([Figure 6](#gkr978-F6){ref-type="fig"}A and B). ETV4 depleted PC3 cells showed robustly reduced mRNA levels of the known ETV4 target gene cyclooxygenase-2 (*COX2*) ([@gkr978-B29],[@gkr978-B35]), confirming loss of ETV4 function in these cells ([Figure 6](#gkr978-F6){ref-type="fig"}B). Total RNA was isolated from PC3 shETV4 and shNTC control cells exposed to 20 or 0.2% oxygen for 24 h and samples from three independent experiments were labelled for microarray analysis. When compared with normoxic control cells, 977 mRNAs and large intergenic noncoding RNAs (lincRNAs) were more than twofold downregulated (*P* \< 0.05) in normoxic PC3 cells lacking ETV4 ([Figure 6](#gkr978-F6){ref-type="fig"}C; green). Hypoxia alone upregulated 608 mRNAs/lincRNAs by more than twofold (*P* \< 0.05; [Figure 6](#gkr978-F6){ref-type="fig"}C; red). Interestingly, 450 mRNAs/lincRNAs showed a more than twofold reduction (*P* \< 0.05) of the hypoxic expression levels in cells lacking ETV4 when compared with hypoxic control cells ([Figure 6](#gkr978-F6){ref-type="fig"}C; blue), out of which a group of 47 mRNAs/lincRNAs was found to be simultaneously hypoxia-inducible. Individual expression levels of these 47 transcripts centered on the mean of the three normoxic control samples (PC3 shNTC) are depicted in a heatmap in [Figure 6](#gkr978-F6){ref-type="fig"}D*.* Array data were validated by RT-qPCR of four randomly chosen transcripts ([Figure 6](#gkr978-F6){ref-type="fig"}E). Figure 6.Genome-wide microarray expression analysis reveals a broad role for ETV4 in HIF mediated hypoxic gene regulation. (**A**) Efficient knock down of ETV4 in human PC3 prostate cancer cells. PC3 cells were stably transduced with lentiviral shRNA expression vectors encoding either a nontarget control (shNTC) or shETV4. Following 24 h of exposure to 20% O~2~ or 0.2% O~2~, ETV4, HIF-1α, PHD2 and β-actin protein levels were analyzed by immunoblotting. (**B**) Total RNA was isolated from cultures treated as in (A) and mRNA levels of ETV4 and its target gene COX2 were determined by RT-qPCR. Gene expression levels were expressed in relation to ribosomal L28 mRNA (rel. levels) calculated from three independent experiments. (**C**) Venn diagram showing the number of transcripts regulated by either an at least twofold induction by hypoxia alone (red), an at least twofold reduction in normoxic cells by the knock down of ETV4 (green), or an at least twofold reduction in hypoxic cells by the knock down of ETV4 (blue), respectively. (**D**) Heatmap of the individual expression levels of the 47 transcripts that required ETV4 for efficient hypoxic induction. (**E**) Expression levels of four randomly chosen transcripts shown in (D) were confirmed by RT-qPCR as described for (B). HIF target genes divide into two groups, either ETV4 coactivated or independent ------------------------------------------------------------------------------- Because hypoxically upregulated gene sets are highly variable between different cellular models, we next focused on a predefined gene set of 61 hypoxia-inducible transcripts. This gene set has previously been reported based on established HIF target genes ([@gkr978-B36]). In line with this publication, the majority of these genes was found to be hypoxically upregulated in PC3 shNTC control cells (green dots in [Figure 7](#gkr978-F7){ref-type="fig"}A, left panel). Comparing hypoxic expression levels of the same genes in PC3 shETV4 knock down with shNTC control cells, the group of established HIF target genes roughly clustered into two halves, representing transcripts which either remained unaffected or which did not respond to hypoxia anymore in the absence of ETV4 ([Figure 7](#gkr978-F7){ref-type="fig"}A, right panel). Interestingly, following ranking of the HIF target genes according to their requirement for ETV4, the HIF-dependent PHD3 oxygen sensor (encoded by *EGLN3*) showed the highest ETV4 sensitivity for hypoxic induction in PC3 cells ([Figure 7](#gkr978-F7){ref-type="fig"}B and C), demonstrating that ETV4 plays a major role in the feedback control of mammalian oxygen sensing. However, a number of established HIF-responsive genes was only slightly affected (e.g. PAI1, encoded by *SERPINE1*) or completely resistant (e.g. GLUT1, encoded by *SLC2A1*) to ETV4 depletion (see [Figure 7](#gkr978-F7){ref-type="fig"}B and C), suggesting that the HIF pathway can be divided into two branches according to the requirement for ETV4. Figure 7.Role of ETV4 in the regulation of established HIF target genes *in vitro* and *in vivo*. (**A**) Dot plots showing the correlation between transcripts in normoxic versus hypoxic control cells (left panel) or in hypoxic control versus hypoxic ETV4 knock down cells (right panel) as derived from the gene array data (grey dots). Red dots refer to internal controls and the blue dot shows ETV4 which is downregulated in shETV4 cells. Green dots indicate the positions of a predefined set of 61 well-established HIF target genes. (**B**) Heat map of the 61 HIF target genes ranked by the magnitude of ETV4 requirement for hypoxic induction according to differences in hypoxic expression levels with Δhyp = log~2~(shNTC_hypoxia) − log~2~(shETV4_hypoxia) and mean hypoxic expression levels centered to the mean of normoxic control cells. (**C**) Exemplary mRNA levels of HIF target genes which either require ETV4 for efficient hypoxic induction (PHD3 and CA9) or which remain unaffected by the ETV4 knock down (GLUT1 and PAI1). mRNA was quantified as described for [Figure 6](#gkr978-F6){ref-type="fig"}B. (**D** and **E**) Correlation between ETV4 and established markers for tissue hypoxia in human breast cancer. (**D**) Independent specimens (spec.) of immunohistochemical evaluation of ETV4 expression in primary mammary carcinoma with high (upper panel) or low (lower panel) ETV4 expression levels. (**E**) Rank-order correlations (Spearman\'s rho) for ETV4 and PHD2 as well as known markers reflecting tissue hypoxia (HIF-1α, HIF-2α, PAI1, GLUT1 and CA9) are summarized in a cross table. The number of cases where both of the correlated markers could be assessed is displayed in parentheses. Asterisks indicate statistical significance with \**P* \< 0.05 and \*\**P* \< 0.01. ETV4 expression levels correlate with HIF-α accumulation in human breast cancer ------------------------------------------------------------------------------- Elevated ETV4 transcript levels have been reported in a variety of human neoplastic diseases including breast cancer ([@gkr978-B37]). Moreover, the onset of spontaneous mammary tumor development has been shown to be profoundly delayed in MMTV-*neu* transgenic mice which express a dominant negative variant of the mouse homologue of ETV4, suggesting that ETV4 may possess tumor promoting effects ([@gkr978-B38]). We recently characterized protein levels of a variety of hypoxic marker genes, including HIF-1α, in tumor samples of 282 patients diagnosed with primary breast carcinoma ([@gkr978-B13],[@gkr978-B24]). When the same specimens were immunostained for ETV4 protein levels, a strong and highly significant correlation between ETV4 and PHD2 was observed in samples where both factors were assessed (*P* \< 0.01, Spearman\'s rho, *N* = 243). Consistent with our model of synthetic action of ETV4 and HIF-α, a solid association was also observed between these transcription factors and three well described target genes (PAI1; GLUT1; CA9) of the HIF pathway. Correlation coefficients between ETV4, PHD2 and HIF-1α and HIF-2α were considerably higher than those observed among both HIF-αs and the three markers for tissue hypoxia ([Figure 7](#gkr978-F7){ref-type="fig"}D and E), demonstrating a putative role for ETV4 in the regulation of PHD2 expression also *in vivo*. DISCUSSION ========== Unique responsiveness to altered oxygen environments and broad conservation of all components of the PHD/HIF oxygen sensing pathway in multicellular life clearly indicate the central role of HIFs as literally hypoxia-inducible transcription factors ([@gkr978-B39]). However, eukaryotic gene expression is a multistep process requiring the complex transcription machinery to interact with promoter DNA and initiate transcription ([@gkr978-B40]). Not surprisingly, numerous studies have identified other nuclear regulators that contribute to the full spectrum of transcriptional changes in response to hypoxia. Yet, general patterns of direct interplay among HIFs and other transcriptional regulators are largely unknown and interactions were often found to rely on specific cell models ([@gkr978-B41]). Here, we report on a novel screening approach that in combination with overexpression of arrayed transcription factors aimed for the systematic analysis of HBS-specific transcription factor interplay. The core P2P was used as a paradigm for HIF-dependent gene regulation, as it embeds a single HBS conferring hypoxic activation and because the endogenous locus is ubiquitously expressed. We identified various members of the activating protein-1 (AP-1) family as novel activators of the *PHD2* gene, although JUN/FOS has been found to enhance hypoxic gene expression previously ([@gkr978-B41]). However, to the best of our knowledge, our study is the first to link ETV4 with HIF-dependent transcription. As shown by the use of different reporter genes, ETV4 function as facilitator of HIF-1 transactivation activity is not restricted to the P2P. While nuclear distances between ETV4 and HIF-1/2α, as calculated by FRET experiments, support a close interaction, our data favor a model where p300/CBP serves as essential bridging molecule between the two factors. This conclusion is based on the following features of the interaction between ETV4 and HIF-1α: (i) oxygen sensitivity in the absence of the oxygen-dependent degradation domain; (ii) mapping to the C-terminal activation domain; (iii) competition by CITED2; and (iv) requirement of FIH for oxygen sensitivity. Such a ternary complex is still in line with the FRET data, as previous findings suggest binding of HIF-1α to both CH1 and CH3 domains of p300 ([@gkr978-B42]). ETV4 is known to interact with p300 at its CH3 domain and, thus, might well get into close or even physical contact to HIF-1α ([@gkr978-B43]). Interestingly, out of 371 known interactors of human p300/CBP ([@gkr978-B44]), 101 (27.2%) were present in the synthetic transactivation screen, but only 10 (9.9%) of them met the criteria to be considered as reproducible activators of the P2P. Apparently, there is no simple redundancy among the p300/CBP interactors to serve as transcriptional coactivators, and the target gene context is thought to play an important role in p300/CBP complex formation ([@gkr978-B44]). The latter is of particular importance, as it provides some reliability regarding the specificity of our screening approach, underlining its general applicability. Based on literature searches, ETV4 and HIF pathways share several common target genes, including matrix metalloproteases (MMP) 1, MMP-3, MMP-7, MMP-9, iNOS and COX-2 ([@gkr978-B45; @gkr978-B46; @gkr978-B47; @gkr978-B48]). Hence, one might speculate that the two factors directly cooperate at regulatory elements of these genes, jointly boosting invasive properties of malignant cells. Notably, ETV4 and its close relatives ETV1 and ETV5 are frequently overexpressed in prostate cancer due to gene fusion with androgen-responsive gene loci ([@gkr978-B49]). Similarly, loss of the tumor suppressor PTEN causes high normoxic expression levels of HIF-1α, a key feature of invasive prostate cancers ([@gkr978-B50]). Recent studies using animal models propose a mutational sequence, where early loss of PTEN and overactivation of ETS target genes collectively promote prostatic cancer progression ([@gkr978-B34],[@gkr978-B51]). Thus, it will be highly interesting to explore a putative synthetic HIF-1/ETV4 role in these pathologies. Due to its high endogenous expression levels in the PC3 prostate cancer cell line, gene array analyses in ETV4 wild-type and knock down PC3 cells were undertaken to explore the general role of ETV4 in hypoxic gene regulation. Remarkably, 47 of 608 hypoxically induced transcripts depend on ETV4 for efficient upregulation. Further analysis concentrating on a set of 61 established HIF target genes revealed 14 genes whose hypoxic induction was at least twofold higher in the presence of ETV4 than in its absence. For example, hypoxic induction of carbonic anhydrase 9 (*CA9*) was strongly dependent on the presence of ETV4, as it was largely absent in ETV4 knock down cells. This finding further explains the unusually strong hypoxic inducibility of *CA9* which has previously been attributed to the cooperation between HIF and ATF-4 ([@gkr978-B52]), another transcription factor that we found to be involved in oxygen signaling ([@gkr978-B53]). Somewhat unexpected, PHD2 did not fulfil, at least in PC3 cells, our stringency criteria for ETV4-dependent hypoxically induced genes. Individual inspection revealed a 2.7-fold hypoxic induction that was only reduced by 16% in the absence of ETV4. Low hypoxic inducibility in this cell type was also seen on the protein level (see also [Figure 6](#gkr978-F6){ref-type="fig"}A) and might explain the rather weak response of this gene to ETV4 depletion, despite the fact that forced expression of ETV4 strongly induced the P2P in the U2OS cell model. However, out of 61 established HIF target genes, PHD3 was most sensitive to ETV4 depletion, suggesting that in PC3 cells PHD3 rather than PHD2 might represent the primary oxygen sensor targeted by ETV4. *In vivo*, ETV4 has been implicated in kidney branching morphogenesis, differentiation of spinal motor neurons and mammary gland development ([@gkr978-B54; @gkr978-B55; @gkr978-B56; @gkr978-B57]). Importantly, ETV1 (ER81), ETV4 (PEA3/E1AF) and ETV5 (ERM) are highly similar and constitute the PEA3 subfamily among the ETS-domain family of transcription factors ([@gkr978-B58]). ETV4 and ETV5 are functionally highly redundant and a double knock out was required to reveal the role of ETV4 in kidney development ([@gkr978-B54],[@gkr978-B55]). Thus, we tested the ability of these additional subfamily members to induce the P2P. While ETV1 did not have any effect, ETV4 and ETV5 super-induced the hypoxic PHD2 and transferrin promoters in a HBS-dependent manner, suggesting functional redundancy of these two subfamily members in hypoxic gene regulation ([Supplementary Figure S1](http://nar.oxfordjournals.org/cgi/content/full/gkr978/DC1)). As the DNA-binding/ETS domain is highly conserved between all three PEA3 subfamily members, we further conclude that the interaction with HIFα takes place outside of the ETS domain of ETV4. Because these developmental processes often occur in tissues with low oxygenation, our data point to a role of tissue hypoxia in physiological ETV4/ETV5 function. Similarly, high expression levels of ETV4 have been linked to metastasis or bad prognosis in a variety of human cancers ([@gkr978-B59]). These clinical features are well-known for hypoxic tumors expressing high levels of HIF-1 ([@gkr978-B60]). Supporting our screening results, we found a strikingly good correlation between ETV4 and PHD2 protein levels in breast cancer tissues, in line with a potentially relevant function of ETV4 in hypoxic tissues *in vivo*. In summary, synthetic transactivation screening as exemplarily demonstrated for HIF-dependent gene expression proved to be a powerful tool to unravel novel interactions among common signaling pathways. The general setup of this method may be easily adapted to study other transcriptional pathways. A multiplexed single-well readout system predestines this approach for extensive screening projects, including small molecule library analyses and genome-wide gene silencing approaches, where inter-well variances are technically difficult to control. ACCESSION NUMBER ================ NCBI\'s Gene Expression Omnibus (GEO) accession number GSE32385. SUPPLEMENTARY DATA ================== [Supplementary Data](http://nar.oxfordjournals.org/cgi/content/full/gkr978/DC1) are available at NAR Online: Supplementary Table 1, Supplementary Figure 1. FUNDING ======= Deutsche Forschungsgemeinschaft Grants FA225/22 (to U.B.-P. and J.F.) and GRK1431/2 (to J.F.); Hartmann Müller-Stiftung (to D.P.S.); Forschungskredit of the University of Zürich (to D.P.S.); Swiss National Science Foundation Grant 31003A_129962/1 (to R.H.W. and D.P.S.). Funding for open access charge: Regular budget of the Wenger group at the Institute of Physiology of the University of Zurich. *Conflict of interest statement*. None declared. Supplementary Material ====================== ###### Supplementary Data We thank N. Sang, L. Poellinger and J.-L. Baert for providing expression plasmids; S. Behnke and M. Storz for assistance with TMA analyses; S. Hafen-Wirth for the labeling of the microarray samples and P. Spielmann for general technical assistance.

Introduction ============ There is a widespread consensus that nearly all women feel the societal pressure of having an ideal female body ([@B73]; [@B71]). This pressure often translates into body image dissatisfaction, which has been labeled as normative in heterosexual women ([@B62]). Despite the flourishing body of research, which has examined sexual orientation-based differences in women's body image perceptions and attitudes, it remains unclear whether lesbian women experience body dissatisfaction similar to their heterosexual female counterparts. Research carried out to date points in two directions. Some studies have found that lesbian and heterosexual women share the same body issues when it comes to level of dissatisfaction ([@B67]; [@B3]; [@B41]; [@B74]); concerns over weight and appearance ([@B27]; [@B74]); and their internalization of thin and beauty ideals ([@B41]). Given that all women in Western cultures find themselves immersed in heteronormative societies, which follow specific beauty and appearance "norms," these findings might indicate that lesbian women are exposed to the same body dissatisfaction risks as heterosexual women ([@B20]). Other studies, meanwhile, have found that lesbian women are less influenced by oppressive beauty standards because they experience less body dissatisfaction ([@B65]; [@B13]; [@B59]; [@B45]; [@B1]); hold a broader ideal of body shape ([@B13]; [@B1]; [@B51]); are less concerned about weight and the drive for thinness ([@B59]; [@B45]); and possess more flexible beauty standards ([@B65]; [@B56]; [@B31]) compared to heterosexual women. In the latter case, these findings may indicate that lesbian women move within a subculture, which is likely to shield them from mainstream heteronormative beauty standards ([@B10]). In the classic meta-analysis conducted by [@B55], significant body dissatisfaction differences were observed between lesbian and heterosexual women: lesbian women were slightly more satisfied with their bodies than heterosexual women; however, the effect size was so small that it did little to clear up the issue. The contradictory results regarding the differences between heterosexual and lesbian women in their body experiences may stem from the fact that the lesbian community is considered a homogeneous group. In a recent study, [@B30] suggested that lesbian gender expression within the lesbian culture may account for their body dissatisfaction differences. They found that lesbians with more stereotypically feminine traits were most at risk of body dissatisfaction, whereas lesbians with more stereotypically masculine traits, that is, "butch" women, were less likely to be affected. These findings coincide with previous literature demonstrating that greater masculinity is associated with less body dissatisfaction ([@B39]; [@B37]; [@B6]), and stronger identification with femininity is related to higher body dissatisfaction ([@B57]; [@B44]). Feminine gender expression could imply a greater risk of body dissatisfaction and drive for thinness ([@B53]), whereas a "butch" gender expression may protect women from sexual objectification, although some research suggests that this "butch-identity" has more to do with "authenticity" and less to do with aesthetic desires ([@B48]). Furthermore, objectification theory has been used to explain differences between heterosexual and lesbian women in their body image concerns. Specifically, sexual objectification, which sees women more likely to be treated as sexual objects, can make them more vulnerable to developing eating disorder symptomatology. This sexual objectification is related to the internalization of the thin-ideal standard of beauty, and predisposes women to closely monitor their body image and become more body dissatisfied ([@B23]). Objectification theory has received robust support, primarily in relation to heterosexual women ([@B54]; [@B2]); however, mixed results have been found for lesbian women. [@B43] observed how higher self-surveillance levels were predictive of increased body shame, which, in turn, predicted eating disorder symptomatology in lesbian women and indicated a direct link between levels of body surveillance and negative eating attitudes. [@B26] presented an independent replication of Kozee and Tylka's modeled direct path between surveillance and negative eating attitudes in lesbian women, yet they also found that body self-surveillance and feeling shame about one's appearance led to increased negative eating attitudes and depression in this group. These findings support the notion that models developed for heterosexual women may not fully apply to lesbian women. In fact, [@B21] found that lesbian women share similar experiences of sexual objectification to heterosexual women, but report less body surveillance. The authors pointed to a facet of lesbian identity as something that may mitigate the effects of being objectified by others, and that the sexualized evaluation of one's body by another woman, compared to the male gaze, could be experienced as quite different by lesbian women. Although there is limited research on sexual minority women, the findings posit that sexual objectification experienced by these minorities may differ from that of heterosexual women. With regard to bisexuality, we know even less about bisexual women's body experiences. Research into the influence of sexual orientation on female body image has either excluded bisexual women or grouped lesbian and bisexual women together for comparison with heterosexual women. However, because they attract both sexes, bisexual women occupy a unique social position in terms of how they move in heterosexual and LGBT cultural environments, thus deserving of individualized attention. There is increasing awareness of the distress that bisexual women may experience compared to their exclusively gay or heterosexual-identified peers. Bisexual women are interested in attracting men and may feel the pressure to fit the beauty standards defined by the male gaze. However, they are also interested in women, which can align them with lesbian women, both groups separating themselves from the dominant heterosexist culture. As a result of this dichotomy, bisexual women's body image experiences seem to be unique. [@B12] found that bisexual women experience a tension between their resistance to adopt sexist ideals, characteristic of feminist women, and the assumption of the thin ideal of feminine beauty. Previous studies have reported that bisexual women worry about their body image and conform to the feminine ideal (e.g., thinness) more so in relationships with men, and may enjoy greater freedom in defining their own standards of attractiveness in same-sex relationships ([@B34]). Research also suggests that bisexual women may have more body image issues than lesbian and heterosexual women ([@B5]; [@B59]); are more prone to developing eating disorders compared to lesbian women ([@B42]); and exhibit higher rates of unhealthy weight-control behaviors than heterosexual women ([@B59]). Furthermore, research into body image and objectification theory involving sexual minority women has primarily focused on lesbian women, excluding their bisexual counterparts. To our knowledge, only one research study has analyzed the sexual objectification experiences of bisexual women. [@B8] examined objectification theory in bisexual women and the roles of two minority stressors, namely antibisexual discrimination and internalized biphobia. The results revealed that antibisexual discrimination and internalized biphobia yielded links with the internalization of sociocultural standards of attractiveness, body surveillance, and body shame, confirming that bisexual women's dichotomous experiences of body image are unique and unusual. Body image is closely related to sexual satisfaction. Feelings of body concern are shown to be associated with a negative impact on females' daily sex lives ([@B72]; [@B11]; [@B60]). Research into sexual satisfaction has mostly centered on white, married, and heterosexual people; again, Lesbian, Gay, Bisexual and Transsexual groups (LGBT) have been underrepresented in these studies. Among those who identify themselves as heterosexual, earlier research confirms that body dissatisfaction is associated with increased sex-related distress and anxiety ([@B4]); feeling less comfortable exposing their bodies during sex ([@B11]); and feeling less sexual desire and engaging in less frequent sexual activity ([@B40]). As for the LGBT community, little research exists, yet findings point in the same direction. [@B64] found that sexual anxiety, relationship commitment, body image, and pride in identity significantly contribute to predicting sexual satisfaction in LGBT groups. [@B58] compared body dissatisfaction and comfort with one's body during sexual activity among the aforementioned groups. They found that men who identify themselves as heterosexual report more positive evaluations of their appearance; less preoccupation with their weight; more positive effects of their body image on their quality of life and the quality of their sex life; and greater willingness to reveal aspects of their body to their partner during sexual activity, compared to those men who identify themselves as gay, heterosexual women, and lesbian women. These findings support the argument that gay men are at greater risk of experiencing body dissatisfaction than heterosexual men, but turn up little evidence of lesbian women's body experiences compared to heterosexual women. Measures of body satisfaction in lesbian and heterosexual women did not differ significantly, with similarities observed in mean *self-rated attractiveness* and *comfort in a swimsuit*, although there was a small trend for more heterosexual women than lesbian women to report hiding at least one aspect of their bodies during sex (52% vs. 44%), especially their stomach. Regarding bisexuality, a more positive body image was found to be associated with higher levels of sexual activity and sexual satisfaction in bisexual women ([@B75]). Women's body experiences and the consequences they have on their sexual satisfaction give us a good opportunity to analyze the impact of body image concerns on female quality of life. Findings from previous literature suggest that the more dissatisfied a woman is with her own body, the more likely she is to report a dissatisfying sex life. However, the scale of this effect may be influenced by sexual orientation. It is during sexual activity when the body is completely exposed to the "others gaze." As such, making comparisons about how body image concerns impact on sexual satisfaction depends on whether the other gaze comes from a woman or a man. The outcomes may yield knowledge about the pressures to fit the beauty standards defined by the male gaze and about the internalization of heterosexist values among heterosexual, lesbian, and bisexual women. Past literature studying the role of body image on sexual satisfaction has focused primarily on heterosexual samples, and the few research studies published on sexual minority women have either compared lesbian and heterosexual women---excluding bisexual women---or have solely focused on bisexual women. To our knowledge, no studies to date have analyzed this topic by simultaneously comparing heterosexual, lesbian, and bisexual women. Thus, our study aimed to quantitatively examine the degree of body image and sexual dissatisfaction experienced by heterosexual, lesbian, and bisexual women to determine (1) whether body dissatisfaction is a reliable predictor of sexual satisfaction; and (2) whether differences by sexual orientation exist. In line with earlier research, we hypothesized that bisexual women would report less dissatisfaction with their bodies compared to their heterosexual and lesbian counterparts, and lesbian women would be slightly more satisfied with their bodies than heterosexual women. In previous studies, lesbian, bisexual, and heterosexual women reported more similarities than differences in their sex life ([@B52]; [@B29]). Therefore, we did not expect to find differences in sexual satisfaction and frequency of sexual activity by sexual orientation. Finally, objectification theory has been widely confirmed in all women, including sexual minority groups, although lesbian and bisexual women have yielded mixed and more complex results. Therefore, we hypothesized that women interested in attracting men (heterosexual and bisexual) would feel more pressure to fit the beauty standards defined by the male gaze, and that their body concerns would have the most impact on their sexual satisfaction, compared to women removed from the male gaze in their sexual relations (lesbian women). Materials and Methods {#s1} ===================== Participants ------------ Three hundred and fifty-four (354) women were initially recruited into the study. However, the final sample was reduced to 333 participants after removing 21 women (13 were underage and eight left some responses blank). *A priori* analyses of power were run to select a sufficient number of participants according to our chosen statistical test. Based on the results for detecting a small-to-medium effect size (0.18), with a statistical power of 0.8, a minimum of 303 participants would be required. The women were asked to complete a survey prompted by online advertisements on different websites. Postings were added to general community forums and websites of interest to lesbian and bisexual women in Spain. Participation was voluntary and completely anonymous; no compensation was offered to survey respondents. A total of 176 women identified themselves as heterosexual; 79 as bisexual; and 78 as lesbian. Ages ranged from 18 to 62 years, with a mean age of 25.43 (*SD* = 7.21). The participants' educational level ranged from "no high-school qualifications" to "higher education" (university-level studies). Measures -------- ### Demographic Variables Participants reported age, educational level, height (in centimeters), weight (in kilograms), relationship status, sexual orientation, and frequency of sexual activity via checkboxes. Relationship status responses were elicited through the item "Currently..." with three options to choose from: "I am in a stable relationship"; "I am casually dating"; and "I am not dating anyone." Respondents were asked about their sexual orientation in the following way: "I consider myself..." and the options: "Heterosexual"; "Homosexual"; "Bisexual." Taking into account the past 6 months, frequency of sexual activity was elicited via the statement: "I have sex\..." and four possible responses: "Daily"; "A few times a week"; "A few times a month; and "I haven't had sex." Body Mass Index (BMI) was calculated as a continuous variable using the standard formula of kilograms over height squared. The Body Shape Questionnaire (BSQ; [@B14]) was used to assess body dissatisfaction. This 34-item self-report instrument measures the degree of concern with body image, especially feelings of fatness (e.g., "Have you thought that your thighs, hips or bottom are too large for the rest of you?"). Respondents rate each item on a scale of 1--6 (1 = *Never*, 6 = *Always*). The total score ranges from 34 to 204, and scores above 105 are indicative of mild to severe body dissatisfaction. The BSQ has been validated with Spanish-speaking populations ([@B61]). This questionnaire shows a high consistency for measuring body dissatisfaction ([@B70]). In the present study, Cronbach's alpha was 0.97. The Index of Sexual Satisfaction (ISS; [@B33]) was used to assess sexual dissatisfaction. This 25-item questionnaire measures current partners' sexual dissatisfaction with their sexual interactions (e.g., "I feel that my sex life is boring"). Respondents rate each item on a scale of 1--5 (1 = *Never*, 5 = *Always*). Some items are worded positively and coded inversely. All 25 items are summed after the positively worded items have been reverse scored and then subtracting 25 from this sum, giving the total score. The scores range from 0 to 100, and those equal to or greater than 30 indicate sexual dissatisfaction. The ISS has been adapted into Spanish ([@B15]) and validated with Spanish-speaking populations, showing a high consistency for measuring sexual dissatisfaction ([@B36]). In the present study, Cronbach's alpha was 0.90. Procedure --------- We used the same procedure as described in [@B30]. Participants were recruited through postings placed on several Facebook pages. The ads featured a brief explanation of the study and a link to the online survey. These ads were shown to Facebook users who indicated they were over 18, lived in Spain and have Spanish nationality, and identified themselves as female. Postings appeared on pages of general interest and pages of specific lesbian interest (e.g., LGTBI associations or private groups in Facebook) Some participants were also recruited by asking them to directly forward the research announcement. Hence, a snowballing sampling technique was employed, whereby links were posted on social media networking sites or emailed to contacts, and respondents were asked to share them with their networks. We used the survey platform Eval&Go. Our study was carried out in accordance with the recommendations outlined in the APA Ethical Guidelines for Human Research and adhering to the principles of Spain's Organic Law 15/1999 for the Protection of Personal Data ([@B47]). Before starting the survey, participants were warned that it contained information of a sexual nature. They were also informed that participation was voluntary and anonymous, and a guarantee was given that the data would only be used for research purposes. Participants consented to completing the survey and confirmed that they understood the purpose of the study. The survey comprised 69 items corresponding to the chosen questionnaires. Participants were of adult age (\>18 years); they received the necessary information to help them decide whether to participate or not; and there were no potential risks associated with the research, therefore the approval of the ethics committee was not applicable according to the institutional ethical committee and national guidelines. All participants signed informed consent in accordance with the Declaration of Helsinki. Data Analyses ------------- A contingency table and a chi-squared test were used to analyze the correlation between frequency of sexual activity, relationship status, and sexual orientation. One-way ANOVAs were performed to determine differences in body dissatisfaction, sexual dissatisfaction, and BMI among heterosexual, bisexual, and lesbian women. In order to examine how the variables interrelate and whether they relate differently depending on sexual orientation, we tested for bivariate correlations between them for each sexual orientation group using Pearson's correlation coefficient for the BSQ score and BMI, and Spearman's rho for frequency of sexual activity because of its ordinal nature. Furthermore, we ran a Fisher's r-to-z transformation to compare correlation indices for each correlation pair (heterosexual vs. lesbian; heterosexual vs. bisexual; lesbian vs. bisexual). Finally, simultaneous multiple regression analyses were performed for each sexual orientation to confirm the predictive value of all independent variables regarding sexual dissatisfaction. This regression method is recommended when no specific predictions are made about the weight of each variable, as in this case. However, there is interest in determining the degree of influence of several variables and the relative influence of each variable under study ([@B38], p.76). The analyses were carried out using statistical package IBM SPSS version 24.0. We used the G^∗^Power software ([@B22]) to calculate statistical power. Results ======= Regarding frequency of sexual activity, the contingency table (see [Table 1](#T1){ref-type="table"}) reveals similar percentages of sexual activity in women by sexual orientation. The chi-squared analyses confirmed no significant correlations between variables\[χ^2^~(6,333)~ = 6.19, *p* = 0.40\]. ###### Frequency of sexual activity by sexual orientation. Never (*n* = 23) Few times/month (*n* = 94) Few times/week (*n* = 188) Daily (*n* = 28) -------------- ------------------ ---------------------------- ---------------------------- ------------------ Heterosexual 5.1% 29.0% 57.4% 8.5% Homosexual 5.1% 26.6% 58.2% 10.1% Bisexual 12.8% 28.2% 52.6% 6.4% In terms of relationship status, 75.4% of women reported having a stable partner; 13.8% said they were casually dating; and 10.8% were not dating anyone. Again, no significant correlation between sexual orientation and relationship status was found \[χ^2^~(4,333)~ = 8.02, *p* = 0.91\]. Given that the primary aim was to determine whether body dissatisfaction might predict sexual dissatisfaction, women who reported not having had sex in the last 6 months were excluded from all remaining analyses. Therefore, the following analyses were run on 310 women. The one-way ANOVA results showed no significant differences among heterosexual, bisexual, and lesbian women in terms of body dissatisfaction (BSQ), sexual dissatisfaction (ISS), and BMI (see [Table 2](#T2){ref-type="table"}). Age^[1](#fn01){ref-type="fn"}^ was also analyzed to check for possible between-group differences, yet no significant differences were found. ###### Descriptive statistics of BSQ, ISS, BMI scores and age, by sexual orientation. *M* *SD* Min Max *F* *p* -------- -------------- ------- ------- ------- -------- ------ ------ BSQ^1^ Heterosexual 88.87 36.49 36.00 182.00 Lesbian 88.76 41.04 34.00 183.00 0.52 0.56 Bisexual 88.38 39.08 34.00 204.00 ISS^2^ Heterosexual 18.96 11.64 2.00 53.00 Lesbian 18.56 11.83 0.00 58.00 0.62 0.94 Bisexual 18.44 11.55 0.00 57.00 BMI^3^ Heterosexual 24.40 5.15 16.00 42.54 Lesbian 25.78 6.51 16.90 51.60 1.80 0.17 Bisexual 24.40 4.91 16.96 38.86 AGE Heterosexual 24.91 0.57 18.00 57.00 Lesbian 26.47 0.74 18.00 57.00 1.26 0.28 Bisexual 25.72 0.93 18.00 62.00 1 Body Shape Questionnaire. 2 Index of Sexual Satisfaction. 3 Body Mass Index. The correlation coefficients of the associations between variables which were later entered into the regression analysis for each group were calculated (See [Table 3](#T3){ref-type="table"}). ###### Correlations between BSQ and BMI scores (Pearson) and frequency of sexual activity (Spearman), with sexual dissatisfaction (ISS) by sexual orientation. Heterosexual (*n* = 167) Lesbian (*n* = 75) Bisexual (*n* = 68) ------------------------------ -------------------------- -------------------- --------------------- BSQ 0.35^∗∗a^ 0.17 0.12^a^ BMI 0.25^∗∗a^ 0.20^b^ −0.15^a,b^ Frequency of sexual activity −0.42^∗∗^ −0.33^∗∗^ −0.27^∗^ ∗ p \< 0.05; ∗∗ p \< 0.01. a,b Matched superscript across rows are those between which are significant differences according to Fisher's r-to-z transformation. Numbers without superscript point to an absence of significant differences with the rest. The results revealed that heterosexual women were the only group in which sexual dissatisfaction was significantly correlated with BSQ scores, BMI, and frequency of sexual activity, indicating that increased body dissatisfaction, high BMI, and low frequency of sexual activity are associated with less sexual satisfaction. In contrast, in bisexual and lesbian women, only frequency of sexual activity was significantly correlated with sexual dissatisfaction, demonstrating how less sexual activity on its own is associated with less sexual satisfaction. Fisher's r-to-z transformation showed significant differences in the BSQ--sexual dissatisfaction correlation between heterosexual and bisexual women (*z* = 1.67, *p* \< 0.05), yielding a stronger correlation in heterosexual women as well as in the BMI--sexual dissatisfaction correlation between heterosexual and bisexual women (*z* = 2.77, *p* \< 0.005) and between lesbian and bisexual women (*z* = 1.89, *p* \< 0.05), stronger in the first instance. Simultaneous multiple regression analyses were conducted to predict sexual dissatisfaction by taking into account body dissatisfaction, BMI and frequency of sexual activity as predictors. Given the ordinal nature of frequency, three dummy variables were created: daily, weekly, and monthly. Different result patterns emerged for each sexual orientation. For heterosexual women, a model explaining 27.0% of the variance was obtained, *F*(4,164) = 16.18, *p* \< 0.001. Body dissatisfaction and monthly sexual activity were significant in terms of predicting sexual dissatisfaction, with the most relevant predictor being monthly sexual activity. In lesbian women, a significant model accounting for 20.2% of the variance was obtained, *F*(4,73) = 5.63, *p* = 0.001; however, in this case, only monthly sexual activity significantly predicted sexual dissatisfaction. The analysis of bisexual women generated a model explaining 13.4% of the variance, *F*(4,67) = 3.60, *p* = 0.011. For bisexual women, body dissatisfaction, BMI, and monthly sexual activity were the variables that significantly predicted sexual dissatisfaction. See [Table 4](#T4){ref-type="table"} for the parameter estimates in the analyses. ###### Parameter estimates in the multiple regression analyses for heterosexual, lesbian, and bisexual women. B S.E. β *t* Sig. C.I. 95% -------------------------- ------------------- ------- ------ ------- ------- ------ ---------- ------- Heterosexual (*n* = 167) Constant 2.70 3.83 0.71 0.48 −4.86 10.28 BSQ 0.08 0.03 0.24 3.14 0.00 0.03 0.13 BMI 0.28 0.17 0.12 1.60 0.11 −0.07 0.62 Monthly frequency 9.50 1.72 0.38 5.51 0.00 6.09 12.90 Daily frequency −3.00 2.87 −0.07 −1.05 0.30 −8.68 2.67 Lesbian (*n* = 75) Constant 5.05 5.24 0.96 0.40 −5.406 15.51 BSQ 0.04 0.03 0.15 1.27 0.21 −0.024 0.10 BMI 0.28 0.21 0.15 1.31 0.20 −0.147 0.71 Monthly frequency 10.69 2.83 0.41 3.78 0.00 5.044 16.33 Daily frequency −4.25 4.24 −0.11 −1.00 0.32 −12.721 4.21 Bisexual (*n* = 68) Constant 24.30 6.74 3.60 0.00 10.82 37.77 BSQ 0.09 0.04 0.31 2.41 0.02 0.02 0.17 BMI −0.68 0.30 −0.29 −2.27 0.03 −1.28 −0.08 Monthly frequency 8.70 2.90 0.36 3.00 0.00 2.91 14.50 Daily frequency 1.91 5.12 0.04 0.37 0.71 −8.32 12.13 Criterion variable: Sexual dissatisfaction (Index of Sexual Satisfaction). Weekly frequency was excluded from all regression analyses. BSQ: Body Shape Questionnaire; BMI: Body Mass Index. Discussion ========== To our knowledge, the present study is the first to quantitatively examine differences in body image and sexual dissatisfaction by sexual orientation, and to determine whether body concerns might predict sexual satisfaction in heterosexual, lesbian, and bisexual women. Findings revealed no differences in levels of body dissatisfaction among lesbian, bisexual, and heterosexual women in line with our predictions. However, based on the premise that sexual orientation may be a protective factor against the negative influence of male gaze-based beauty standards, the hypothesis that heterosexual and bisexual women's body concerns would have the greatest impact on their sexual satisfaction, compared to lesbian women, was confirmed. As expected, no differences in sexual dissatisfaction and frequency of sexual activity by sexual orientation were found. A quantitative examination of the body dissatisfaction differences revealed no sexual orientation-based significant differences. Contrary to our expectations, overall levels of body dissatisfaction were similar in all three groups of women This result coincides with those studies in which no overall differences in level of satisfaction between lesbian and heterosexual women were observed ([@B67]; [@B3]; [@B27]; [@B55]; [@B25]; [@B58]; [@B41]; [@B74]; [@B35]; [@B69]), yet it also contradicts research suggesting that bisexual women may experience more body image issues than their lesbian and heterosexual peers ([@B42]; [@B5]; [@B59]). The absence of significant differences between lesbian, bisexual, and heterosexual women in body dissatisfaction levels could indicate that the pressure to fit beauty standards is widespread across all women, regardless of their sexual orientation ([@B73]). Nearly all women are considered successful in various life domains based on their appearance and thinness ideals ([@B28]; [@B71]). Body evaluation is central to sexual objectification and a woman's appearance is constantly being scrutinized by men and other women. This, therefore, may explain the normative levels of body dissatisfaction reported by the female population, including sexual minority women. The media promotes a thin and curvaceous ideal female body shape, and bisexual and lesbian women experience similar mainstream pressures to conform to this thin ideal; therefore, the similarities regarding the overall level of body dissatisfaction among heterosexual, lesbian, and bisexual women reported in the present study are justified ([@B66]). Our study also confirms that the internalization of heterosexist values defined by the male gaze may prove relevant to identifying the scale of pressure experienced by women of different sexual orientations. After examining the links between body concerns and sex dissatisfaction, we found that both constructs were significant and differently correlated across all groups of women. Correlation analyses revealed that less frequent sexual activity is associated with less sexual satisfaction in all women, while higher levels of body dissatisfaction and BMI and less frequent sexual activity are associated with less sexual satisfaction in heterosexual women only. Furthermore, the multiple regression analyses showed that body dissatisfaction predicts sexual dissatisfaction in heterosexual and bisexual women, but not in lesbian women. In heterosexual women, the regression model was the most explanatory (accounting for almost 30% of the variance of sexual dissatisfaction); frequency of sexual activity and body dissatisfaction were significant, but not BMI. In bisexual women, the regression model explained the least variance compared to the other two groups, but all independent variables predicted sexual dissatisfaction, whereas in lesbian women, only frequency of sexual activity predicted sexual dissatisfaction. Starting from the premise of objectification theory, we hypothesized that the sexual satisfaction of women interested in attracting men (heterosexual and bisexual women) would be more affected by their body concerns compared to those removed from the male gaze in their sexual relations (lesbian women). These findings, taken together, indirectly support this premise. In general terms, they coincide with the study published by [@B58], in which lesbian and heterosexual women did not differ significantly in body dissatisfaction, although there was a small trend for heterosexual women to hide some parts of their body during sex. It could be argued that women sensitive to the male gaze may have strongly internalized heterosexist values about beauty standards and, as a consequence, their body concerns may have more strongly affected their sex lives. Therefore, sexual orientation may be a protective factor for women who are not likely to be judged by the man-defined "attractiveness" standard of beauty. Somehow lesbian women still seem to retain this kind of protective factor with regard to their body concerns, at least when it comes to their sexual activity. Appearance is important in sexual relationships for both women and men ([@B11]). Exclusive lesbians do not engage in sexual relations with men, and this holds partially true for bisexual women. Because lesbians seem to be freer from the male gaze, it could be argued that societal pressure to be physically attractive is less salient for lesbian women, as [@B10] pointed out. Some authors confirm that men value physical attractiveness more than women do. In a large-scale survey, heterosexual and gay men considered appearance and face attractiveness quite important, whereas women ranked personality traits as more relevant than good looks ([@B49]). The women in [@B50] study believe that men have a strong preference for attractiveness and thinness, and expect them to meet these standards. [@B46] found that men, in particular, prefer an attractive partner regardless of their sexual orientation. Interestingly, no differences between lesbian and heterosexual women were observed in levels of body dissatisfaction or internalization of beauty standards; however, a lesbian's preference for attractiveness and thinness in a partner was not influenced by their body shape or weight dissatisfaction. The authors concluded that lesbian women who felt body dissatisfaction were the least affected; they did not expect their respective partners to be excessively critical about their appearance as a male partner would be toward heterosexual women. [@B66] also found that lesbian women were not necessarily protected from mainstream body ideals and appearance norms, despite of their perceptions about being more accepting of larger body types and alternative styles by their partners. In line with these results, our study findings suggest that lesbian women are not shielded from experiencing body dissatisfaction, but they are in terms of how body concerns affect other life domains, such as sexual experiences. Neither heterosexual nor bisexual women enjoy this kind of protection. Our results indicate increased distress among bisexual women compared to exclusively gay or heterosexual women, as the literature shows ([@B42]; [@B5]; [@B59]). Bisexual women represent the only group in which the three main independent variables (body dissatisfaction, BMI, and frequency of sexual activity) predict sexual dissatisfaction. Therefore, it could be argued that sexual satisfaction in bisexual women may be more negatively affected by their body concerns. However, an unexpected result emerged for BMI: higher levels of body dissatisfaction and low sexual activity frequency predict sexual satisfaction, whereas a lower BMI predicts increased sexual dissatisfaction. Data about the role of BMI in the body dissatisfaction-- sexual orientation relationship are equivocal. Some studies have found that BMI is more strongly associated with body dissatisfaction in heterosexual women than in lesbian or bisexual women (e.g., [@B63]), whereas others have not drawn this conclusion (e.g., [@B58]). Nevertheless, [@B16], in a study focused on bisexual population, found that BMI and self-esteem were predictors of body dissatisfaction in bisexual women versus lesbian and heterosexual women. These could mean that bisexual women could be more vulnerable to the influence of BMI in body dissatisfaction, which could affect to sexual dissatisfaction. On the other hand, although the direction of the association 'the higher the BMI, the more body dissatisfaction' is unequivocal in the literature, some studies have found non-linear relationship between BMI and psychopathology. For instance, the results of the study by [@B17] give evidence for a significant U-shaped trend in the association between BMI and depression. Same results were found regarding association between BMI and anxiety ([@B18]). In a similar way, some studies have reported inverse or inverted J-shaped relationship between BMI and completed suicide, such that suicide risk is highest among individuals with BMI \< 20 kg/m^2^ (and, ≥35 kg/m^2^, in those that report a J-shaped relationship), with lowest risk in the overweight and moderate obesity range ([@B19]; [@B24]; [@B9]). Thus, and being cautious because we have not assessed psychopathology, maybe a non-linear relationship which the regression model does not reflect, could explain these results. It is clear that more research is necessary to clarify the role that BMI plays in bisexual women's body dissatisfaction. In line with [@B68], we found no BMI differences among the three groups of women under study, unlike some studies that have observed a higher BMI in lesbian women than in heterosexual women ([@B7]; [@B32]; [@B41]). However, if heterosexual, bisexual, and lesbian women experience similar mainstream pressures to conform to the thin and curvaceous body ideal, then we would expect to observe similarities in their BMI. Special attention would also need to be given to determining whether BMIs tend to equal out among sexual minorities in future studies. Certain limitations have been identified in our study. We had to shorten the survey in order to increase participation; consequently, no additional measures beyond body and sexual dissatisfaction were included. Eating or sexual disorders were not taken into account, nor were measures of relationship satisfaction, which have been found to mediate the link between body satisfaction and sexual satisfaction in lesbian, bisexual, and heterosexual women ([@B51]). Furthermore, considering additional protective and risk factors regarding body dissatisfaction---including, for example, feminine and masculine gender expressions and the internalization of beauty and thinness ideals---would be welcome in futures studies. Because participants were recruited over the Internet, the sample was not fully representative. The socio-economic status of participants was not assessed in the present study: given that the survey was online, participants were women that have access to technology and time to complete the survey, which may compromise the representativeness of the present sample. A certain recruitment bias could be that non-Internet users were not included in the sample. Another bias relates to the sites where the survey was posted: by resorting to specific lesbian-interest web pages, some women from the lesbian community may have been overrepresented, especially those holding a more assertive profile regarding their sexual orientation. Finally, other minorities such as transgender or non-binary persons, were not included in the present study. Determining whether the conclusions we have drawn are applicable to the aforementioned groups could be a valuable next step in future research endeavors. Our study also has several strengths. It represents one of few studies examining differences in body dissatisfaction experience by sexual orientation, encompassing sexual minorities such as lesbians and bisexuals. The differences observed between bisexual and lesbian women indicate that all orientations should be considered, and confirms the importance of including bisexuality in future studies. The absence of differences among lesbian, bisexual, and heterosexual women in the quantitative levels of body dissatisfaction in this and previous studies could be indicative of how factors traditionally seen to protect lesbian women are compromised. Lesbian women are less likely to be judged by male-defined standards of beauty; however, the slight distinction between lesbian and straight female looks could cause traditional beauty standards to become widespread in the lesbian community. A trend toward the homogenization of the beauty canon could trigger an increase in body dissatisfaction levels in women, regardless of their sexual orientation. This study highlights the need to regulate the abuse of the dominant aesthetic canon that harms women of all sexual orientations and identities. To conclude, our study aimed to quantitatively examine the degree of body image and sexual dissatisfaction experienced by heterosexual, lesbian and bisexual women, to determine whether body dissatisfaction can predict sexual dissatisfaction. No sexual orientation-based differences were found for body dissatisfaction, frequency of sexual activity, relationship status, or sexual dissatisfaction. However, body dissatisfaction did exert a lesser influence on sexual dissatisfaction in lesbian women compared to their heterosexual and bisexual counterparts. The fact that lesbian women are uninterested themselves in the male gaze could be interpreted as a protective factor which shields lesbian women from the consequences of feeling concerned about their appearance. The traditional beauty standard may become widespread among all women, confirming the need to regulate the abuse of the dominant aesthetic canon. Ethics Statement ================ This study was carried out in accordance with the recommendations of the Ethical Commitment of the University of Jaén with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the University of Jaén. Author Contributions ==================== SM-D, TR, and PE designed the study. SM-D and TR collected the data. SM-D and PE conducted the analyses in close consultation with TR. SM-D, TR, and PE wrote the first draft of the manuscript. All authors contributed to and have approved the final 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. Given that age was not a primary outcome, it was not included in the regression analyses. [^1]: Edited by: Andrea Sabrina Hartmann, Osnabrück University, Germany [^2]: Reviewed by: Erik M. Benau, Columbia University, United States; Ingela Lundin Kvalem, University of Oslo, Norway [^3]: This article was submitted to Eating Behavior, a section of the journal Frontiers in Psychology

Background {#Sec1} ========== Pulmonary Tuberculosis (PTB) is a serious public health problem facing mineworkers, not only in Zambia but in most developing countries. The tuberculosis (TB) incidence in miners in Southern Africa is reportedly the highest among any working population \[[@CR1]\]. Silica is a major component of sand and rocks and workers at risk of exposure, apart from the mineral ore miners, are tunnel drillers and those involved in quarry work, foundries, and excavation \[[@CR2]\]. Mining activities expose workers to silica dust, which has contributed substantially to the TB epidemics in southern Africa and other low income regions of the world \[[@CR3]\]. About 1.7 million workers in the United States are potentially exposed to silica dust \[[@CR4]\]. Several studies among South Africa goldminers, particularly in studies of ex-miners, have found high prevalences of PTB, ranging from 23.9 to 35 % \[[@CR5]--[@CR8]\] and increased mortality rate from TB among white South Africa gold miners followed up for 20 years compared with the general white population \[[@CR9]\]. Some researchers have noted increased TB prevalence with duration of exposure. Increased duration and levels of exposure have been associated with increased risk among goldminers \[[@CR10]\], and crystalline silica exposed industrial workers in USA \[[@CR11]\]. This risk may persist after exposure ceases \[[@CR12]\]. Despite lower prevalence among South African coal miners (5.2 %), increased risks for TB have also been described \[[@CR13]\]. There is limited reporting in the literature of PTB among copper miners. One study described a high prevalence of silicosis (35 %) in former Brazilian copper miners with 4 to 40 years of exposure, and with 11 % of the miners having TB, emphysema and COPD \[[@CR14]\]. This study was aimed at determining the dust-related dose response risk for PTB among mineworkers exposed to silica dust in the copper mines in Zambia. Methods {#Sec2} ======= Study design and setting {#Sec3} ------------------------ This cross-sectional study was conducted for in-service miners examined between 2005 and 2010 at the Occupational Health and Safety Institute (OHSI), a statutory agency in Kitwe, Zambia where miners report for annual mandatory medical examinations. Any person aged between 18 and 59 years, working or seeking to work in the mine, is subjected to an annual mandatory medical examination. Prospective miners are brought to the institution by companies intending to employ them, while others present themselves for initial medical examinations. Each miner, on first presentation to the Institute (initial medical examinations), is assigned a unique Institute number which becomes his/her personal number for as long as he/she remains working in the mining industry. The medical examinations consist of medical and occupational history, clinical examinations, laboratory investigations and chest radiographs on all prospective miners. Sputum for acid fast bacilli (AFB) and erythrocyte sedimentation rate (ESR) are done on miners with abnormal chest radiographs (CXRs). Additional investigations such as spirometry and audiometry are done depending on the findings from the initial assessment. Prospective miners with a history of TB or radiological scarring suggestive of past TB are not permitted to work underground and are not employed in the mines. At each mandatory annual medical assessment, a 12-month fitness-to-work certificate is issued, obliging workers to return the following year, if continued employment is required. For miners certified with pneumoconiosis or work-related cardiorespiratory TB, additional information such as date of disease certification, age at certification and duration of employment before certification, is entered into the Institute medical files. A miner who has been certified as having PTB is relocated to non-dusty areas of the mine or discharged. A miner found to have PTB can only be certified if he has worked in the scheduled area for more than 12 completed months. PTB, which has been diagnosed in a miner with a work history of less than 12 months in an area with a risk of exposure to silica dust is considered non work-related and is not certified. All work-related diagnosed PTB is certified for compensation purposes. All the information on miners is entered and stored safely onto the OHSI medical files. The study involved the review of miners' medical records maintained at the OHSI. The medical records contained information on health status of the miner and CXR report read from posteroanterior (PA) view of the standard-size chest radiographs. These were read by the medical panel at the time the miner was being screened. The process of screening and examinations at the Institute involves medical and occupational history taking, and clinical examination of miners done by individual medical officers. All the information gathered by the medical officers is forwarded to the medical panel which sits on daily basis. The medical panel is mandated to read all the CXRs with laboratory findings and award medical certificates. The medical panel consists of the screening medical doctors together with medical doctors with B-reader training and one an occupational medicine specialist with substantial experience in reading CXRs, including distinguishing between radiological active and old PTB. Non-TB lesions on CXRs were distinguished from TB by the medical panel after reviewing both the medical and occupational history, clinical findings and radiological findings such as cavitations, military mottling, hilar adenopathy and the presence of opacities in the upper and middle lobes which are more suggestive of TB. The final decision of the medical panel was made by consensus. For the purposes of this study, the outcome used was certified TB, which was defined as sputum smear positive or CXR for active TB, meeting the criteria for work-relatedness as described above. The CXR findings which were reported by the medical panel at the time of examination and was recorded in the file was used in this research. CXRs were not subjected to re-reading. All non-TB abnormalities were aggregated during coding, making it impossible to retrieve data on silicosis specifically. Population and sample selection {#Sec4} ------------------------------- Included in the study, were seven major copper mines with underground operations. The newly opened mines and open pit mines were excluded from the study. The participating mines where chosen because they had a long history of underground mining, a large number of employees (more than a 1000 miners) and no history of mine closures. Three mines met the inclusion criteria and accepted invitation to participate. All in-service miners examined at OHSI between 1 January 2005 and 31 December 2010 from the participating mines and who had worked for 12 completed months or more, were eligible for selection as study subjects (*N* = 5840). Exclusion of ex-miners was necessary as the exposure data for periods of their past employment (1960's and 1970's) was absent. The unique Institute identification numbers was used as sampling frame. The study sample was selected using a 1 in 16 systematic random sampling method, to provide a total sample of 360, which was estimated as being appropriate to determine whether an association between exposure and PTB truly existed among the sample. The medical files were used as the sampling frame and captured through the Institute numbers. Exposure assessment {#Sec5} ------------------- The exposure data from the Mine Safety Department (MSD), the government agency responsible for the monitoring of the mining environment was obtained for the mines under study. The data (*n* = 16678) was in the form of dust counts from various mine worksites such as loading bay and crushers and processes like lashing and drilling, using the Rand Konimeter for 'snap' sampling. It was taken by occupational hygienists and qualified technicians from the ventilation departments of respective mines. Random checks and sampling of dust were carried out by the MSD to monitor compliance and accuracy of the results they received from the mines. The reports contained information on mine name, date of collection, major worksites (surface or underground), sections and processes. These legally mandatory reports are presented on quarterly basis to the MSD. Legislation demands that all work sites are periodically sampled at least every 90 days. Dust reports from 1990 to 2010 were available for the research. The silica dust levels in the work sites and processes were converted to equivalent gravimetric silica estimates by applying the conversion factor of 0.09 mg/m^3^ to convert from million particles per cubic foot (mppcf) to mg/m^3^ (i.e 1ppcc = 0.003 mg/m3) suggested by previous researchers \[[@CR15], [@CR16]\]. These levels were then allocated to each selected miner based on the job title and the area the miner had been operating from within a particular mine in a specified time, using the occupational history from the medical records. The dust levels were allocated for each of these strata, and summed across the entire lifetime of employment in the industry to obtain cumulative dust exposures (CDE), according to the formula:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ CD{E}_i={\displaystyle \sum_{jk}{M}_{jk}{Y}_{ijk}} $$\end{document}$$ where *M*~*jk*~ is the mean dust level for work title, *j* in work area, k and *Y*~*ijk*~ is the time in years spent by subject *i* in work title, *j* and area *k*. No participant was employed in the period prior to 1990. Statistical analysis {#Sec6} -------------------- The data for in-service miners collected were coded and captured in Epidata version 3.1 (EpiData Association, Odense, Denmark). Statistical analysis was conducted using Stata IC version 13.1 software for Windows (StataCorp LP, College Station, TX). Means and standard deviations (SD) were compared for numerical variables using student's *t*-test. Mann-Whitney *U* test was used to compare medians for data not normally distributed. Pearson's chi-square test was used to test for association with PTB for categorical variables. Analysis of variance (ANOVA) test was used to compare means across the three mines. Cumulative respirable silica dust exposure (CDE) was calculated by the product of annual mean concentration of respirable silica dust of the section worked and years worked in that section, summed for all sections, over the lifetime employment in the copper mining industry. It was calculated from the start of mining to the date of certification of PTB, or end of the study period (2010) for non-PTB cases. For analysis, CDE was divided into tertiles: low (cumulative respirable silica dust below 1.0 mg.yrs/m^3^), medium (cumulative respirable silica dust from 1.01 to 1.82 mg.yrs/m^3^) and high (cumulative respirable silica dust more than 1.82 mg.yrs/m^3^). Exposure categories were also used to analyse trends of disease prevalence with increasing cumulative respirable silica dust by comparing the prevalence of certified active TB in the medium and high cumulative respirable silica dust category with low cumulative respirable silica dust category as a reference category. Multivariate logistic regression was used to determine the association between certified active TB, and cumulative dust exposure categories, adjusted for age at certification, gender and smoking status. In a separate model, CDE was also run as a continuous variable to determine the association with certified active TB, adjusting for age at certification, gender and smoking status. Miners with unknown smoking status (*n* = 11) were not included in the model. Odds ratios with 95 % confidence intervals were calculated and presented. All analyses were conducted with a significance level of 5 %. Results {#Sec7} ======= Of the 360 files of miners in the original random sample selected from the mines, 3 (0.8 %) samples comprised of females. Because of the few number of females, they were excluded from analysis. There were no significant differences in the demographics across the mines. Altogether, 51 (14.3 %) of the miners were smokers at the time of employment. There was no data on smoking for 11 (3.1 %) (Table [1](#Tab1){ref-type="table"}). Of the 34 miners diagnosed with PTB, 16 (47.1 %) and 18 (52.9 %) were sputum positive and negative on direct microscopy for AFB respectively.Table 1Demographic factors and clinical findings of underground copper miners from selected mines in ZambiaCharacteristicsMine 1 (*n* = 137)Mine 2 (*n* = 99)Mine 3 (*n* = 121)Age at certification^a^, years, (mean (SD)) (Range)30.8 (5.9) (19.6--49.1)31.9 (6.9) (21.2--54.2)31.3 (6.0) (19--52.2)Sex, n, (%) Male137 (100)99 (100)121 (100)Smoking, n, (%) ^b^ Non-smokers121 (88.3)76 (76.8)98 (81) Smokers14 (10.2)17 (17.2)20 (16.5) Unknown2 (1.5)6 (6.1)3 (2.5)Chest radiographs, n, (%) Normal117 (85.4)82 (82.8)98 (81) Abnormal (PTB) ^c^12 (8.8)7 (7.1)14 (11.6) Abnormal (Non-PTB) ^d^8 (5.8)10 (10.1)9 (7.4)AFB Positive n, (%)4 (33.3)5 (62.5)7 (50)ESR mm/h, mean, (SD) (Range)76.9 (31.0) (35--127)73.3 (25.2) (30--108)85.2 (22.1) (54--133)*ESR* Erythrocyte sedimentation rate (positive if \>20 mm/h)*AFB* Acid-fast bacillus^a^Age at certification -- mean age of the miners at the time of certification compared across the Mines^b^Smoking status --current smokers at employment^c^Abnormal radiographs (PTB) = Chest radiographs suggestive of PTB^d^Abnormal radiographs (Non PTB) = Chest radiographs suggestive of chest pathology other than PTB There were no statistical significant differences across the mines with respect to clinical characteristics (Table [1](#Tab1){ref-type="table"}), duration of service or silica dust exposure (Table [2](#Tab2){ref-type="table"}). The duration of exposure was short, with the mean years of exposure of 5.0 (Mine 2) and 5.2 (Mines 1 and 3) (Table [2](#Tab2){ref-type="table"}). Median exposure levels for both overall mine and for specific job descriptions, exceeded the newly revised Occupational Safety and Health Administration Permissible exposure level (OSHA-PEL) of 0.05 mg/m^3^ (Table [2](#Tab2){ref-type="table"}). In addition, more than two-thirds of all sampling points (*n* = 16678) were also above this standard. With the exception of the high levels recording for crushing and blasting tasks at Mine 1, the median exposure levels for other job descriptions were in a narrow range from 0.2 to 0.4 mg/m^3^.Table 2Exposure characteristics of miners (*n* = 357)CharacteristicsMine 1 (*n* = 137)Mine 2 (*n* = 99)Mine 3 (*n* = 121)Length of service, years, Mean (SD) (Range)5.2 (2.7) (1.0--13.1)5.0 (2.5) (1.1--11.4)5.2 (2.3) (1.2--11.0)Overall respirable silica dust mg/m^3^, Median (Range)0.4 (0.0--1.0)0.2 (0.1--1.3)0.2 (0.1--0.5)Silica dust in sections mg/m^3^, Median (Range) Crusher Attend0.6 (0.1--1.0)0.3 (0.1--0.9)0.3 (0.1--0.5) Blasting Tech/Foreman0.5 (0.2--0.9)0.4 (0.1--1.1)0.3 (0.1--0.4) Lasher0.4 (0.0--1.0)0.2 (0.1--1.0)0.2 (0.1--0.4) Driller0.4 (0.2--0.9)0.2 (0.1--0.9)0.2 (0.1--0.5) Trammer0.4 (0.2--0.8)0.3 (0.1--1.3)0.2 (0.1--0.4) W/shop foreman/Attend0.4 (0.1--0.7)0.2 (0.1--0.6)0.2 (0.1--0.4) Others-workman0.4 (0.1--0.9)0.2 (0.1--1.0)0.2 (0.1--0.4)Cumulative respirable silica dust mg.y/m^3^, Mean (SD) (Range)1.3 (0.7) (0.3--3.2)1.1 (0.5) (0.3--2.2)2.3 (1.0) (0.7--4.7)*SD* Standard deviation, *tech* Technician, *Attend* attendant, *W/shop* workshop Medium and high CDE categories, age at certification, smoking status, and length of service were significantly associated with PTB (Table [3](#Tab3){ref-type="table"}). There was a statistically significant increasing trend (p~trend~ \< 0.01) of disease prevalence with increasing categories of dust exposure (low CDE = 2.5 % (3); medium CDE = 8.7 % (10) and high CDE = 17.1 % (21)).Table 3PTB status across the variables of interestCharacteristicsPTB cases\ (*n* = 34)No PTB\ (*n* = 323)Age at certification, years, Mean, (SD) ^a\*^34 (6.0)31.0 (6.2)Smoking status^b^ Non-smokers, n, (%)22 (64.7)273 (84.5) Current smokers, n, (%)^\*\*^12 (35.3)39 (12.1)Length of service, Mean, (SD) ^a\*^6.1 (2.0)5.1 (2.5)Sex Male, n, (%)34 (9.5)323 (90.5)Cumulative respirable silica Dust Exposure, Mean, (SD) ^a\*\*^2.2 (1.0)1.5 (0.9)Cumulative respirable silica dust categories^b^ Low, n, (%)3 (8.8)116 (35.9) Medium, n, (%)^\*^10 (29.4)105 (32.5) High, n, (%)^\*\*^21 (61.8)102 (31.6)\**p*-value \< 0.05; \*\* *p*-value \< 0.01^a^t-test^b^Chi-square test The high CDE category, compared with the lowest category, was statistically significantly associated with PTB (OR = 6.4 (95 % CI 1.8--23.0)), after adjusting for age, gender and smoking status. Medium CDE showed a non-statistically significant elevated risk but lower than that of the highest category, suggesting a dose-related gradient. Current smoking, as compared to a current non-smoker also presented as a statistically increased risk for the development of PTB (Table [4](#Tab4){ref-type="table"}).Table 4Multivariate logistic regression analyses of independent factors as predictors of PTBVariablesOR95 % CIAge at certification1.11.0--1.1Current smoker\*4.31.9--9.9Cumulative respirable silica dust exposure Low1 Medium2.80.7--10.6 High\*\*6.41.8--23.0Miners with unknown smoking status not includedCurrent smokers compared with current non-smokers*CI* confidence interval, *OR* odds ratio*p* \< 0.01\*, *p* \< 0.05\*\* A model which included cumulative respirable silica dust exposure as a continuous variable was also run, adjusting for age, sex and smoking. A definite dose response was seen: for each unit increase in silica levels for each year of exposure, there was almost a twofold increase in PTB risk (OR: 1.9; 95 % CI: 1.3--2.8). Discussion {#Sec8} ========== In this record review of 360 Zambian underground copper miners, certified active TB was associated with increasing cumulative silica exposure, despite the relatively short duration of exposures on these selected mines. To the best of our knowledge, this is the first such description among copper miners. Our study established a prevalence of certified active TB in underground miners in Zambia of 9.5 %. The higher prevalences of TB found in the gold mines than in the present study in copper mines could probably be due to the differences in the definition of TB used across the studies, and in the varying silica concentration between the gold and copper mines. In the Zambian copper mines, the silica content in the atmospheric dust was reported to be in the range of 19 to 43 % \[[@CR17]\] with respirable silica dust levels established in this study of 0.1--1.3 mg/m^3^, but in the gold mines, the reported routine respirable dust measurements are in the range of 0.02--4.29 mg/m^3^ with mean silica fraction of 16 % \[[@CR18]\]. Other researchers have reported silica concentrations in the gold mines as high as 54 % after acid washing and incineration \[[@CR19]\]. The reported dust levels in the coal mines in South Africa are in the range of 0.9--2 mg/m^3^ with silica content reportedly to be below the regulatory action level of 5 % \[[@CR20]\], with the prevalence of TB on these mines ranging from 3.6 to 5.4 % \[[@CR13]\]. Dust concentrations across studies may vary depending on the source, treatment and methods of analysis and these differences must be cautiously interpreted. It is likely that the differences found in our study could also be related to the age of the miners (mean age = 31.3 years). Additionally, the duration of exposure could provide another explanation. In the current study, the miners had a short mean working history of about 5 years and a relatively younger age group and could explain why there was no association between age at certification and PTB after adjusting for smoking, gender and cumulative respirable silica dust categories, although by bivariate analysis, association was significant. Silica exposure has been well documented to be associated with PTB \[[@CR5], [@CR8], [@CR9], [@CR11], [@CR12], [@CR21], [@CR22]\]. While these findings have been shown in a variety of mineral settings, such as gold \[[@CR5]--[@CR8], [@CR10], [@CR12]\], coal \[[@CR13]\], foundry workers \[[@CR23]\] and quartz stone crushers \[[@CR24]\], this has not been shown previously within copper mines, where levels of silica are not typically as high as in other settings. The risk of PTB in the copper mines is not well established as compared with the gold mines. Our data demonstrated increased risk for certified active TB with increased exposure to silica dust. From the respirable silica dust established by this study, the estimated median quartz levels in the dust on the mines under study ranged from as low as 0.01 mg/m^3^ for the mine with the lowest silica content of 19 % to as high as 0.6 mg/m^3^ for the mine with the highest silica content of 43 %. More than two- thirds of the dust data points were above the now revised OSHA-PEL of 0.05 mg/m^3^. Before the OSHA-PEL was revised downwards to 0.05 mg/m^3^, mines were using 0.1 mg/m^3^ as Occupational Exposure Limit (OEL). Miners were at risk of developing silicosis when exposed to levels at or below 0.1 mg/m^3^ \[[@CR5], [@CR18], [@CR25], [@CR26]\]. Thus, it is unlikely that this level is protective against PTB -- as is indicated by our data. In addition to respirable silica exposure, we found a strong association between smoking and certified active TB. This supports population based studies where smoking has been shown to increase the risk of PTB \[[@CR27]--[@CR32]\]. The risk estimates in these studies range from 1.61 to 4.5, similar to those found in our study. The comparison with non-mining study populations must be viewed with caution, given the differences in case definitions. The strengths of our study were the ability to recreate exposure profiles from a sample of miners from time of employment until certification of disease. This detailed occupational history and exposure data was used to estimate cumulative exposures for each miner for all the years worked as opposed to current exposures and length of service being used as a proxy to exposure to estimate cumulative exposures. This minimized exposure misclassification. The exposure data were collected by qualified technicians and hygienists. In addition, the clinical evaluation conducted by a group of trained and experienced physicians at OHSI provided us with access to quality data. The other merit of this study is that it is the first study to look at the relationship between PTB and exposure to silica dust among underground mineworkers in copper mines in Zambia or elsewhere, as well as the dose response impact of dust exposure on frequency of pulmonary TB. The study limitations included lack of data on other risk factors for PTB, such as previous TB, TB contacts, socioeconomic factors, Human Immunodeficiency Virus (HIV) status and past smoking status which could have influenced the relationship between PTB and exposure. Absence of cultures for TB is another study limitation with a potential of TB misclassification and an effect on the accuracy of active TB prevalence. Past history of TB is likely to have been minimal, as this would have been detected on the initial medical assessment, which includes a worker interview, clinical assessment and chest radiograph, and those with such history or radiological scarring suggestive of past TB are not permitted to work underground or in scheduled areas and were not part of this study. Other factors, such as past smoking status, TB contact, overcrowding and HIV status is unlikely to have had a differential distribution across the exposure categories, and thus only minimally likely to have influenced the estimates of risk. The estimated prevalence of HIV in Zambia among the 15--49 years age group is 12.5 % \[[@CR33]\]. Furthermore, the prevalence of TB in 2012 in Zambia was 388 per 100,000 population \[[@CR34]\], which is lower than that established by this study (9.5 %). Lack of information on the use of personal protective equipment (e.g masks) is another limitation factor but is likely to also have been non-differentially distributed across the sample. The lack of information on silicosis made it impossible to determine the relationship between TB, respirable silica and silicosis in this sample. The other study limitation is the exposure data. Konimeter measurements which are based on optical counting of the number of particles in a portion of sample are prone to analytical variability arising from collection and analytical methods. It is least reliable for high dust count as opposed to low dust count \[[@CR35]\] and could have affected this study. Conclusion {#Sec9} ========== The findings of this study add to the existing evidence that occupational exposure to silica dust poses a high risk of TB in underground miners, even in conditions of short duration of dust exposure. This study underlines the need to maintain the dust levels below international standards to lower the prevalence of TB further through dust control. AFB : Acid fast bacilli CDE : Cumulative silica dust exposure COPD : Chronic obstructive pulmonary disease CXR : Chest x-ray or radiograph ESR : Erythrocyte sedimentation rate HIV : Human Immunodeficiency Virus MSD : Mine safety department OHSI : Occupational Health and Safety Institute PA : Posteroanterior PTB : Pulmonary tuberculosis TB : Tuberculosis Gratitude to the members of staff at the Discipline of Occupational and Environment health, University of KwaZulu-Natal. Appreciation to Dr. William Sitembo, Dr. Connard Mwansa and members of staff at OHSI, and MSD in Kitwe. Funding {#FPar1} ======= Research reported in this publication was supported by the Fogarty International Centre of the U. S. National Institutes of Health under award number 2D43TW000812. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Authors' contributions {#FPar2} ====================== KN conceived and implemented the study design. Collected and analysed the data. Wrote the manuscript drafts. RN helped conceive and implement the study design. Contributed to statistical analyses and edited the final manuscript. Both authors read and approved the final manuscript. Competing interests {#FPar3} =================== The authors declare that they have no competing interests. Consent for publication {#FPar4} ======================= Consent for publication in this study was not applicable. Ethics approval and consent to participate {#FPar5} ========================================== The study was approved by the Biomedical Research Ethics Committee (BREC) of the University of KwaZulu-Natal, South Africa (Reference number BE081/13) and Ethics Review Committee of the Tropical Diseases Research Centre (TDRC-ERC), Zambia (Reference number TRC/ERC/04/10/2013). Consent to participate was not applicable in this study.

Steve Lerner has written a compelling treatise on why the dominant environmental protection paradigm should be overhauled to emphasize prevention, precaution, and equal protection. *Sacrifice Zones* is a significant complement to three decades of environmental justice research; it provides irrefutable empirical evidence that not all American communities are created equal. Over the course of 2 years, the author traveled to 12 communities from New York to Alaska to collect stories from residents who live in communities that are on the front line and in the middle of toxic "sacrifice zones"---some of the most polluted and poisoned places in America. Sacrifice zones are often "fenceline communities" of low-income and people of color, or "hot spots" of chemical pollution where residents live immediately adjacent to heavily polluted industries or military bases. Quite often, this pattern of unequal protection constitutes environmental racism---a pattern first challenged in the courts in a 1979 lawsuit, *Bean v. Southwestern Waste Management*, for which I served as an expert witness. *Bean* was the first lawsuit to use civil rights law to challenge environmental racism. *Bean* also demonstrated that environmental justice was not just a "poverty thing"; this was later confirmed by dozens of empirical studies. A 2008 study (Downey L, Hawkins B, "Race, Income, and Environmental Inequality in the United States," Sociol Perspec 51(4):759--781) found that African Americans experience such a high pollution burden that African American households with incomes between \$50,000 and \$60,000 live in neighborhoods that are, on average, more polluted than the average white neighborhood of households with incomes \< \$10,000. Lerner assesses the diverse strategies used by affected communities to win legal settlements, educate and mobilize their residents, block permits and expansion of polluting facilities, force cleanup of contamination, extract concessions and pollution reduction from fenceline industrial plants, and target enforcement by government. *Sacrifice Zones* graphically describes what life is like for people of color and poor people who live on the "wrong side of the tracks" and in "throw-away communities" whose residents receive unequal protection, if any protection at all; such communities contain locally unwanted land uses, or LULUs, and industries deposit pollutants just outside the factory gates. Even after more than three decades of environmental justice mobilization and activism, Lerner had hundred of communities from which to choose for his study; there is no shortage of poisoned communities. Lerner not only captures the hard, cold, sometimes depressing statistics, but he gives us a close-up and personal account of what it is like to live, work, and sometimes die in these communities. It is not surprising that half of the cases in *Sacrifice Zones* were from the South (all of which I have visited to work with local residents) and conforms to a well-established pattern I documented in *Dumping in Dixie* (Westview Press, 1990). This disturbing pattern was recently documented in the 2007 *Toxic Wastes and Race at Twenty* study (Bullard et al., United Church of Christ), which found that people of color make up the majority (56%) of those living in neighborhoods within 2 miles of the nation's commercial hazardous waste facilities, and nearly double the percentage in areas beyond 2 miles (30%). People of color make up 69% in neighborhoods with clustered facilities. Siting disparities were discovered in 9 of 10 U.S. EPA regions and 40 of 44 states (90%) with hazardous waste. The findings in Lerner's case studies mirror those uncovered in the national *Toxic Wastes and Race at Twenty* report. One of the most important indicators of an individual's health is one's ZIP code. *Sacrifice Zones* joins hundreds of environmental justice books that provide compelling evidence for government to take immediate action rather than waiting and watching while mostly African Americans, Hispanics, Native Americans and Alaska Natives, and working-class whites needlessly suffer. Lerner's case studies illustrate that African Americans and other people of color at various income levels also experience higher levels of pollution when compared with their white counterparts. Grassroots community leaders, most of whom are led by women of color and retired persons, are taking up the leadership mantle of environmental justice and health equity. Leaders in sacrifice zones have used the environmental justice movement to leverage the power imbalance between polluting industries and fenceline residents---understanding that the struggle is not a sprint but is more akin to a marathon. The case studies detail how race and class affect health outcomes. Lerner asks, why do grandmothers have to perform the work government regulators should be doing? Finally, Lerner offers the reader an inside view of environmental justice activism---which emerged from protests in Warren County, North Carolina, in the 1980s---that fuses environmental protection, economic and social justice, public health, and civil rights and human rights. Without a doubt, these and other grassroots struggles have transformed the environmental justice movement in the United States and the global movement for environmental and economic justice and human rights. {#f1-ehp-119-a266a}

With advances in cancer treatments, the numbers of cancer survivors increased quickly.^[@bib1]^ However, a medical survey of 1807 cancer survivors followed for 7 years showed that 33% died of heart diseases.^[@bib2]^ Despite doxorubicin (DOX) effectiveness against cancer, dose-dependent cardiotoxicity restricts its long-term application in chemotherapy, as it diminishes the quality of life of cancer patients.^[@bib3],\ [@bib4]^ For example, one case report described DOX-related congestive heart failure in a patient who had received a 400 mg/m^2^ cumulative dose of DOX.^[@bib5]^ The onset of cardiac complications can occur during treatment with DOX or up to 10 years after cessation of DOX therapy.^[@bib6]^ In fact, cardiovascular-related disease derived from the adverse effects of cancer treatments has become the leading noncancer-related cause of morbidity and mortality in long-term cancer survivors.^[@bib6]^ Despite great achievements gained over the past several decades, the precise mechanisms implicated in DOX-induced cardiomyopathy remain unclear. Oxidative stress, inflammation, and apoptosis have been proposed as the mechanisms of the DOX-induced cardiotoxicity, which at least could result in cardiac remodeling and dysfunction.^[@bib7]^ To date, no targeted strategies are available for preventing DOX-induced cardiotoxicity. Some chemicals and functional factors have been evaluated for their ability to moderate DOX-induced cardiotoxicity, but little success was reported.^[@bib8],\ [@bib9],\ [@bib10]^ Currently, dexrazoxane is the only agent approved by the United States Food and Drug Administration and the European Medicines Agency for the prevention of long-term cardiotoxicity caused by DOX. Nevertheless, dexrazoxane may interfere with the anticancer activity of DOX and lead to a higher latent risk for acute myeloid leukemia in pediatric patients.^[@bib11]^ Thus, a more specific drug or strategy needs to be developed to protect against DOX cardiotoxicity. Fibroblast growth factor 21 (FGF21), as an effective metabolic factor on glucose and lipid metabolism, was predominantly found in adipose and liver tissue. However, based on our and other's studies, FGF21 is expressed in other tissues such as the myocardium.^[@bib12],\ [@bib13],\ [@bib14]^ Numerous studies have demonstrated that FGF21 displays anti-inflammatory and anti-oxidative stress activities,^[@bib15],\ [@bib16]^ and thus has a critical role in protecting against tissue injury from acute toxicity.^[@bib17],\ [@bib18]^ In addition, the anti-apoptotic activity of FGF21 in islet *β*-cells and endothelial cells also was reported.^[@bib19],\ [@bib20]^ More recently, studies demonstrated FGF21-mediated protection against cardiac ischemia and reperfusion injury^[@bib21]^ and isoprenaline-induced myocardial hypertrophy.^[@bib22]^ Still, the exact effect of FGF21 and the detailed underlying mechanism on the cardiac system remain largely unclear. Our previous research demonstrated that FGF21 interacts with its receptors to stimulate sirtuin 1 (SIRT1)-dependent autophagy, which prevents diabetic cardiomyopathy.^[@bib13],\ [@bib14]^ Nevertheless, no study was yet explored the effect of FGF21 on DOX-induced cardiac injury. In the present study, we first examined the protective ability of FGF21 against DOX-induced cardiotoxicity. Furthermore, the mechanisms of FGF21's cardioprotective activity were analyzed using cardiomyoblasts (H9c2 cells), adult mouse cardiomyocytes, and a wild-type mouse model (129S1/SvImJ). The results of these experiments demonstrated that the cytotoxicity of DOX to H9c2 cells, adult mouse cardiomyocytes, and the heart of 129S1/SvImJ mice can be attenuated by FGF21. Further mechanistic studies showed that FGF21 obviously prevented the DOX-induced cardiotoxicity via the suppression of oxidative stress, inflammation, and apoptosis through activating the SIRT1/liver kinase B1 (LKB1)/AMP-activated protein kinase (AMPK) signaling pathway both *in vitro* and *in vivo*. Our findings indicate that FGF21 could be considered as a therapeutic target for the clinical treatment and prevention for DOX-induced cardiac injury. Results ======= FGF21 prevented DOX-induced cardiac remodeling and dysfunction -------------------------------------------------------------- After administration of FGF21 to the DOX (5 mg/ml)- or PBS-treated 129S1/SvImJ mice, cardiac fibrosis was preliminary analyzed with Sirius red staining. Compared with the PBS control treatment, DOX treatment clearly caused collagen accumulation (red staining) in the cardiac slides ([Figure 1a](#fig1){ref-type="fig"}), indicating the induction of cardiac fibrosis. Pre-treatment with FGF21 significantly reduced the degree of collagen deposition induced by DOX. This result was continuously confirmed by quantitative real-time PCR (qRT-PCR) for c*ollagen I* mRNA expression ([Figure 1b](#fig1){ref-type="fig"}) and found that DOX-induced *collagen I* increase was obviously blocked by pre-administration of FGF21. Furthermore, DOX-induced cardiac fibrosis was verified by increased expression of pro-fibrotic mediators, connective tissue growth factor (CTGF) and transforming growth factor *β* (TGF-*β*) ([Figures 1c and d](#fig1){ref-type="fig"}), and these elevations were almost completely prevented by FGF21 pre-treatment. Next, we performed a cardiac functional analysis by echocardiography in the indicated treatment groups. DOX treatment significantly increased the left ventricular (LV) internal systolic diameter, the LV internal diastolic diameter, the LV end diastolic volume, and the LV end systolic volume, but decreased the ejection fraction (EF) and fractional shortening (FS; [Table 1](#tbl1){ref-type="table"}). Overall, pretreatment with FGF21 considerably recovered the alteration induced by DOX ([Table 1](#tbl1){ref-type="table"}). Inflammation, oxidative stress, and apoptosis are reported to participate in DOX-induced cardiotoxicity;^[@bib23]^ therefore, we subsequently investigated the protective activity of FGF21 against DOX-induced inflammation, oxidative stress, and apoptosis in the H9c2 cell line and adult mouse cardiomyocytes. FGF21 attenuated the upregulation of inflammatory cytokines and NF-*κ*B p65 activation in DOX-treated H9c2 cells and adult cardiomyocytes ----------------------------------------------------------------------------------------------------------------------------------------- As shown in [Figures 2a--c](#fig2){ref-type="fig"} and [Supplementary Figure 2A](#sup1){ref-type="supplementary-material"}, DOX significantly increased the mRNA levels of tumor necrosis factor*-α* (*TNF-α*) and interleukin-6 (*IL-6*), but not *IL-1β* compared with levels in the control, which were remarkably inhibited by pretreatment with FGF21. NF-*κ*B p65 is regarded as a major controller of the transcription and expression of several inflammatory cytokines (such as TNF-*α* and IL-6),^[@bib24]^ and IKK/I*κ*B*α* mediates the activation and nuclear translocation of NF-*κ*B p65. Therefore, we detected the effect of FGF21 on phosphorylated IKK (p-IKK) and I*κ*B*α* protein (p-I*κ*B*α*) expression by western blotting ([Figures 2d and e](#fig2){ref-type="fig"}). Interestingly, DOX treatment significantly increased both p-IKK and p-I*κ*B*α* expression, and these alterations were obviously attenuated by pretreatment with FGF21. Continuously, the sub-cellular distribution of NF-*κ*B p65 was analyzed by western blotting to observe whether the alteration in IKK and I*κ*B*α* affects NF-*κ*B p65 nuclear translocation. Clearly, DOX significantly increased the nuclear translocation of NF-*κ*B p65 from the cytosol and this effect was abolished by pre-treatment with FGF21 ([Figure 2f](#fig2){ref-type="fig"} and [Supplementary Figure 2B](#sup1){ref-type="supplementary-material"}). FGF21 efficiently prevented the DOX-induced generation of ROS and oxidative stress ---------------------------------------------------------------------------------- To determine whether FGF21 can prevent the DOX-induced generation of reactive oxygen species (ROS), we measured the ROS levels in different groups. In a dihydrogen ethidium (DHE) staining study ([Figure 3a](#fig3){ref-type="fig"}), FGF21 significantly prevented the DOX-induced ROS formation in H9c2 cells. In addition, levels of the oxidative stress marker proteins 3-nitrotyrosine (3-NT) and 4-hydroxy-2-nonenal (4-HNE) ([Figures 3c and d](#fig3){ref-type="fig"}, and [Supplementary Figures 3A and B](#sup1){ref-type="supplementary-material"}), and malondialdehyde ([Figure 3b](#fig3){ref-type="fig"} and [Supplementary Figure 3C](#sup1){ref-type="supplementary-material"}) were significantly increased in the DOX group, and these increases were obviously reduced by FGF21 in the FGF21/DOX groups in both H9c2 and adult cardiomyocytes. Nuclear transcription factor erythroid 2-related factor 2 (Nrf2), as an antioxidant sensor, can translocate from the cytoplasm to the nucleus, to interact with the antioxidant defense system and mediate the transcription of target genes. According to western blot analysis ([Figure 3e](#fig3){ref-type="fig"} and [Supplementary Figure 3D](#sup1){ref-type="supplementary-material"}), nuclear Nrf2 accumulation and activity via quantification of its downstream gene such as NADPH quinone oxidoreductase-1 (NQO-1), catalase (CAT), and heme oxygenase 1 (HO-1) ([Figure 3f](#fig3){ref-type="fig"} and [Supplementary Figure 3E](#sup1){ref-type="supplementary-material"}) in the DOX group was significantly less than that in the controls, and this effect also was prevented by pre-treatment with FGF21 in both H9c2 cells and adult cardiomyocytes. Taken together, our results indicate that FGF21 attenuated DOX-induced oxidative stress, likely through restoration of a Nrf2-regulated antioxidant capacity. FGF21 attenuated DOX-induced cardiac apoptosis through the mitochondrial cell death pathway ------------------------------------------------------------------------------------------- The anti-apoptotic activity of FGF21 was further examined in the DOX-induced cardiac injury model. Consistent with a previous study,^[@bib7]^ DOX notably induced apoptosis in H9c2 cells and adult cardiomyocytes as detected by TUNEL staining, DNA fragmentation, and caspase-3 and (ADP-ribose) polymerase (PARP) cleavage ([Figures 4a--c](#fig4){ref-type="fig"} and [Supplementary Figures 4A and B](#sup1){ref-type="supplementary-material"}) compared with those levels in control cells. Noticeably, FGF21 completely attenuated these pro-apoptotic activities of DOX. Accumulating evidence indicates that mitochondrial death pathways have a key role in multiple programmed cell death processes. FGF21 was reported to be involved in mitochondrial cell death pathways in diabetes-induced testicular cell death.^[@bib25]^ Here we analyzed the mechanism of the anti-apoptotic activity of FGF21 in DOX-treated H9c2 cells and adult cardiomyocytes. We did not observe any significant differences in the levels of cleaved caspase-8 among the groups treated with DOX ([Figure 4d](#fig4){ref-type="fig"}). Therefore, we focused on the mitochondrial cell death pathways in our subsequent research. Western blot analysis revealed a significant increase in the Bax/Bcl-2 ratio ([Figure 4e](#fig4){ref-type="fig"} and [Supplementary Figure 4C](#sup1){ref-type="supplementary-material"}), an index of the mitochondrial cell death pathway,^[@bib26]^ in the DOX group, and this change was significantly normalized by FGF21 in the FGF21/DOX group. FGF21 prohibited interaction of SIRT1 with LKB1 in DOX-treated H9c2 cells and adult cardiomyocytes -------------------------------------------------------------------------------------------------- Our previous research demonstrated that FGF21 interacts with its receptors to prevent diabetic cardiomyopathy.^[@bib14]^ Thus, the mRNA expression of both FGFR1c and *β*-Klotho receptors, which are reported as two major receptors for FGF21,^[@bib27]^ as well as FGFR2, FGFR3, and FGFR4 were examined by qRT-PCR in DOX-treated H9c2 cells and adult cardiomyocytes. The results demonstrated that only FGFR1 and *β*-Klotho receptors ([Figures 5a and b](#fig5){ref-type="fig"}, and [Supplementary Figure 5A](#sup1){ref-type="supplementary-material"}), but not the other receptors, were significantly upregulated in the FGF21 and FGF21/DOX groups, and slightly decreased (but without statistical significance) in the DOX group. The phosphorylation of the signaling molecules downstream of FGF receptors were analyzed as well following FGF21 stimulation, which demonstrated that FGF21 treatment could significantly trigger the FRS2 and ERK1/2 activation in the FGF21 and FGF21/DOX groups ([Figure 5c](#fig5){ref-type="fig"} and [Supplementary Figure 5B](#sup1){ref-type="supplementary-material"}). To investigate whether FGF21 upregulates deacetylase activity in the DOX-induced cardiac injury, we detected expression of the major nuclear histone deacetylases (HDACs), including SIRT1, SIRT6, and HDAC1. The results showed that FGF21 significantly upregulated either SIRT1 or SIRT6 activities in the FGF21 group. Conversely, DOX obviously decreased the expression of SIRT1 and SIRT6 compared with levels in the control group and these effects could be abolished by FGF21 in the FGF21/DOX group. In addition, HDAC1 expression did not significantly change among all groups ([Figure 5d](#fig5){ref-type="fig"} and [Supplementary Figure 5C](#sup1){ref-type="supplementary-material"}). In a signaling transduction investigation, we found that FGF21 treatment significantly increased phosphorylation of AMPK in DOX-treated H9c2 cells and adult cardiomyocytes ([Figure 5e](#fig5){ref-type="fig"} and [Supplementary Figure 5D](#sup1){ref-type="supplementary-material"}). LKB1 has been verified to be the major upstream kinase of AMPK in a majority of tissues.^[@bib28]^ The deacetylation status of LKB1 directly influences its intracellular localization and AMPK-regulating activity.^[@bib29]^ To determine whether FGF21 mediates SIRT1/SIRT6-regulated LKB1 deacetylation, the acetylation level of LKB1 was analyzed by immunoprecipitation (IP)/western blotting. The results shown in [Figure 5f](#fig5){ref-type="fig"} and [Supplementary Figure 5E](#sup1){ref-type="supplementary-material"} indicate that FGF21 significantly decreased DOX-induced LKB1 acetylation and subsequently increased AMPK activation in the FGF21/DOX group. To explore which HDAC(s) interacts with LKB1, the nuclear extracts of the indicated H9c2 cells and adult cardiomyocytes were subjected to IP assays with SIRT1 or SIRT6 antibody as probes in LKB1-precipitated samples. Clearly, the IP tests showed that DOX prohibited the interaction of SIRT1 with LKB1 (left panel of [Figure 5g](#fig5){ref-type="fig"} and [Supplementary Figure 5F](#sup1){ref-type="supplementary-material"}), and FGF21 could notably restore this interaction of SIRT1 and LKB1 in the FGF21/DOX group. In contrast, no SIRT6-LKB1 interaction was detected among all groups (right panel of [Figure 5g](#fig5){ref-type="fig"}). Anti-inflammatory, anti-oxidative, and anti-apoptotic activities of FGF21 in DOX-stimulated 129S1/SvImJ mice cardiac samples *in vivo* -------------------------------------------------------------------------------------------------------------------------------------- To confirm the above findings *in vivo* in 129S1/SvImJ mice, as shown in the left panel of [Figure 6a](#fig6){ref-type="fig"}, we found that administration of FGF21 significantly attenuated the upregulation of *TNF-α* and *IL-6* mRNA expression induced by DOX in mice by qRT-PCR. Western blot analysis revealed that the DOX-upregulated nuclear NF-*κ*B p65 protein was obviously reduced by FGF21 (right panels of [Figure 6a](#fig6){ref-type="fig"}) as well. These results verified the anti-cardiac inflammatory activity of FGF21 *in vivo*. Continuously, the anti-oxidative stress and anti-apoptotic activities of FGF21 were detected *in vivo* using DOX-treated mice heart samples. In DHE and TUNEL staining analysis, treatment with FGF21 resulted in clear reductions in numbers of DHE-stained ([Figure 6b](#fig6){ref-type="fig"}) and TUNEL-positive ([Figure 6c](#fig6){ref-type="fig"}) cells induced by DOX. In addition, the DOX-upregulated cleaved caspase-3 ([Figure 6d](#fig6){ref-type="fig"}) was returned to normal levels after FGF21 administration, according to western blot analyses. The signaling pathway for the ability of FGF21 to protect against DOX-induced cardiac toxicity was examined *in vivo* as well. Consistent with our previous results *in vitro*, the expression of SIRT1 and the acetylated LKB1 and phosphorylated AMPK were almost normalized by FGF21 in DOX-treated cardiac samples ([Figures 6e and f](#fig6){ref-type="fig"}). SIRT1 gene knockdown restricted the anti-inflammatory, anti-oxidative, and anti-apoptotic activities of FGF21 in DOX-stimulated H9c2 cells and adult cardiomyocytes ------------------------------------------------------------------------------------------------------------------------------------------------------------------- To verify the critical role of SIRT1 in the deacetylation of LKB1 by FGF21, SIRT1 RNAi was employed and efficiently knocked down SIRT1 expression in both H9c2 cells and adult cardiomyocytes ([Figure 7a](#fig7){ref-type="fig"} and [Supplementary Figure 6A](#sup1){ref-type="supplementary-material"}). Interestingly, SIRT1 knockdown by RNAi almost completely recovered the level of acetylated LKB1 ([Figure 7b](#fig7){ref-type="fig"} and [Supplementary Figure 6B](#sup1){ref-type="supplementary-material"}) and subsequently prohibited the AMPK activity induced by FGF21 ([Figure 7a](#fig7){ref-type="fig"} and [Supplementary Figure 6A](#sup1){ref-type="supplementary-material"}), through which the critical role of SIRT1 in FGF21-induced activation of LKB1 and AMPK in DOX-treated H9c2 cells and adult cardiomyocytes was emphasized. Based on this finding, the anti-inflammatory, anti-oxidative, and anti-apoptotic activities of FGF21 continued to be investigated in the SIRT1 gene knockdown H9c2 cells and adult cardiomyocytes treated with DOX. As expected, knocking down of SIRT1 by RNAi restricted the anti-inflammatory, anti-oxidative, and anti-apoptotic activities of FGF21. Compared with the siRNA control data, the capacity of FGF21 to attenuate DOX-induced inflammatory marker protein (NF-*κ*B p65, in [Figure 7c](#fig7){ref-type="fig"} and [Supplementary Figure 6C](#sup1){ref-type="supplementary-material"}), apoptotic representative protein (Caspase 3, in [Figure 7c](#fig7){ref-type="fig"} and [Supplementary Figure 6C](#sup1){ref-type="supplementary-material"}), and ROS ([Figure 7d](#fig7){ref-type="fig"}) was significantly restricted after transfection of H9c2 cells and adult cardiomyocytes with SIRT1-specific siRNA. Discussion ========== This study for the first time revealed that FGF21 can improve cardiac dysfunction and pathological changes induced by DOX. The major finding from both *in vitro* and *in vivo* experiments was that FGF21 exerts anti-inflammatory, anti-oxidative stress, and anti-apoptotic effects to attenuate the cardiac injury induced by DOX via activation of the SIRT1/LKB1/AMPK pathway. The pathogenesis of DOX-induced cardiotoxicity involves complex processes, of which the underlying mechanisms are involved in the activation of various downstream pro-inflammatory, pro-oxidative, and pro-apoptotic processes.^[@bib30]^ The increasing numbers of basic and clinical studies suggest that pro-inflammatory factors, including TNF-*α* and IL-6, participate in the pathogenesis of DOX-induced heart dysfunction.^[@bib31]^ In response to the extracellular stimulation, IKK is converted from a neutral form to the active form and subsequently phosphorylates I*κ*B*α* to result in its degradation. Upon I*κ*B*α* degradation, NF-*κ*B is then released from association with the I*κ*B*α* to translocate into the nucleus where it triggers inflammatory cytokine synthesis.^[@bib32]^ In our DOX-induced cardiac injury model, we preliminary found that DOX clearly elevated *TNF-α* and *IL-6* expression along with phosphorylation of both IKK and I*κ*B*α*. Pre-treatment with FGF21 not only obviously attenuated the DOX-induced upregulation of *TNF-α* and *IL-6* levels, but also decreased the levels of phosphorylated IKK and I*κ*B*α*. FGF21, in addition, significantly blocked the DOX-induced nuclear translocation of NF-*κ*B p65 ([Figures 2](#fig2){ref-type="fig"} and [6](#fig6){ref-type="fig"}, and [Supplementary Figure 2](#sup1){ref-type="supplementary-material"}). Taken together, these results indicate that FGF21 plays an anti-inflammatory role in the DOX-induced cardiotoxicity, and the mechanism may involve activation of IKK by FGF21 to preserve the I*κ*B*α*/NF-*κ*B p65 association. The association of I*κ*B*α*/NF-*κ*B p65 then limits NF-*κ*B p65 translocation into nuclei, thereby inhibiting transcription of inflammatory factors (such as *TNF-α* and *IL-6*). Hearts of cancer patients receiving DOX therapy are highly sensitive to DOX-induced oxidative stress. The heart expresses low levels of antioxidant enzymes, rendering it particularly vulnerable to free radical damage and DOX cardiotoxicity.^[@bib33],\ [@bib34]^ In our model, multiple lines of evidence showed that DOX induced ROS generation and oxidative stress in cardiac tissue, and FGF21 exhibited the capacity to alleviate the degree of oxidative stress induced by DOX. It has been suggested that the occurrence of oxidative injury reflects the generation of ROS, which exceeds the capacity of antioxidant defense systems.^[@bib35]^ Then the expression of antioxidant proteins Nrf2, NQO-1, HO-1, and CAT was found to be decreased in the DOX group, and these reductions were all significantly attenuated by FGF21. These data suggest that FGF21 possesses the free radical scavenging and antioxidant capacity via activation of Nrf2 ([Figures 3](#fig3){ref-type="fig"} and [6](#fig6){ref-type="fig"}, and [Supplementary Figure 3](#sup1){ref-type="supplementary-material"}). The increase in myocardial cell apoptosis is another pathogenic mechanism in the DOX-induced cardiotoxicity.^[@bib36]^ Some researchers believed that cardiomyocyte apoptosis could be the leading cause of cardiac dysfunction in DOX-induced cardiomyopathy.^[@bib37],\ [@bib38]^ To estimate the DOX-induced apoptosis, we conducted a series of assays including TUNEL staining, DNA fragmentation detection, and examination of the Bax/Bcl2 ratio in the DOX group. The results indicated that DOX did induce mitochondrial cell death, whereas FGF21 treatment could abolish these alterations ([Figures 4](#fig4){ref-type="fig"} and [6](#fig6){ref-type="fig"}, and [Supplementary Figure 4](#sup1){ref-type="supplementary-material"}). One of our previous studies showed that FGF21 prevents lipid- or diabetes-induced cardiac apoptosis by activating the AMPK pathway^[@bib13]^ and another study demonstrated that activating the LKB1/AMPK pathway prevents the development of cardiomyopathy in type 2 diabetic mice by improving lipid metabolism.^[@bib39]^ These studies suggest that the LKB1/AMPK axis is an attractive therapeutic target, since it participates in multiple biological processes during cellular growth.^[@bib40]^ Recent studies suggest that the LKB1/AMPK axis is a highly sensitive target of DOX-induced damage in the heart.^[@bib41],\ [@bib42]^ Notably, 2 *μ*M DOX (the peak plasma concentration dose of DOX) decreased LKB1/AMPK protein expression.^[@bib43]^ The detailed mechanisms of LKB1/AMPK inhibition are still unclear; however, the mechanism may involve alteration of acetylation activity. Reportedly, LKB1/AMPK signaling can be controlled through acetylation and de-acetylation by various acetyltransferases and HDACs, respectively.^[@bib44]^ Among the several HDACs, SIRT1 is reported to have a critical role in controlling the cardiac LKB1/AMPK pathway. A study with *in vitro* and *in vivo* models has demonstrated the crucial role of SIRT1 for the control of inflammation and apoptosis through LKB1/AMPK-dependent pathways.^[@bib45]^ In cultured 293 T cells, regulation of the LKB1/AMPK pathway by SIRT1 was also reported.^[@bib29]^ In the present study, we found that FGF21 promotes interaction of LKB1 with SIRT1, then diminishes lysine acetylation of LKB1 and concurrently increases its activity, and subsequently activates AMPK to prevent the DOX-induced inflammation, oxidative stress, and apoptosis in cardiac cells ([Figures 5](#fig5){ref-type="fig"} and [6](#fig6){ref-type="fig"}, and [Supplementary Figure 5](#sup1){ref-type="supplementary-material"}). It is noteworthy that SIRT1 seems to have a critical role in this pathway. To verify this hypothesis, we employed an SIRT1 RNAi to specifically block this pathway to see whether the FGF21 still prevented DOX-induced cardiac cellular inflammation, oxidative stress, and apoptosis. Undoubtedly, the SIRT1 RNAi not only prohibited the FGF21-induced LKB1 and AMPK activity, but also abolished the anti-inflammatory, anti-oxidative stress, and anti-apoptotic activities of FGF21 ([Figure 7](#fig7){ref-type="fig"} and [Supplementary Figure 6](#sup1){ref-type="supplementary-material"}). These results verified our supposition that SIRT1 is a critical factor in FGF21-induced cardiac protection. In conclusion, this study demonstrated that FGF21 can protect against DOX-induced cardiac toxicity. The molecular mechanism responsible for FGF21's cardioprotective activity may involve activation of the SIRT1/LKB1/AMPK signaling pathway, which alleviates DOX-induced inflammation, oxidative stress, and apoptosis to improve cardiac dysfunction. This finding may provide an effective way to protect against or reduce the cardiac adverse effects of anthracycline during anti-cancer therapy. In addition, it delivers theoretic evidence to support the development of some more efficient and safer FGF21-like medicines such as LY2405319.^[@bib46]^ Materials and methods ===================== Cell culture, animal care, and experimental design -------------------------------------------------- The rat cardiomyoblast line H9c2 was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). For the *in vitro* study design, the H9c2 cells were pretreated with FGF21 at 50 ng/ml for 2 h and then incubated with DOX at 5 μg/ml for 22 h. All the experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of the Jilin University, which was in accordance with Guide for the Care and Use of Laboratory Animals, Eighth Edition (Library of Congress Control Number: 2010940400, revised 2011). Eight-week-old male 129S1/SvImJ mice were divided into four groups (Control, FGF21-, DOX-, and FGF21/DOX-treated mice) with 11 mice in each group. In the FGF21/DOX-group, the mice were given an intraperitoneal injection of 100 *μ*g/kg body weight FGF21 5 days/week for 5 weeks. This regimen included 1 week of FGF21 pretreatment before DOX exposure. One week later, the mice were intraperitoneally injected with DOX (5 mg/kg) once a week for the remaining 4 weeks. PBS injection served as a control treatment, and injection of FGF21 or DOX alone was applied in the FGF21 and DOX groups, respectively. Primary cardiomyocyte isolation and treatment --------------------------------------------- Adult mouse cardiomyocytes were isolated and cultured as our previous description.^[@bib13]^ The adult cardiomyocytes were pretreated with FGF21 at 50 ng/ml for 2 h and then incubated with DOX at 5 *μ*g/ml for 22 h. A preliminary study was performed to optimize the transfection efficiency with SIRT1 siRNA in adult mouse cardiomyocytes ([Supplementary Figure 1](#sup1){ref-type="supplementary-material"}). Sirius red staining ------------------- Collagen accumulation was analyzed with Sirius red staining. The sections were incubated with 0.1% Sirius-red F3BA and 0.25% Fast Green FCF and observed under a Nikon microscope. A computer-assisted image-analysis system was applied to evaluate the sections stained for Sirius red as described before.^[@bib14]^ Semi-quantitative analysis was obtained by computerization of the percentage of the positive staining from six samples in each group with two sections for each sample and five images for each section. Echocardiography ---------------- To assess cardiac function, transthoracic echocardiography was performed using a Philips 7500 with a 15-MHz transducer (Sonos 7500, Amsterdam, The Netherlands) as described before.^[@bib47]^ Transthoracic echocardiography at the parasternal long-axis and short-axis views were performed and recorded. Two-dimensional and M-mode echo were employed to detect the wall motion, the chamber dimensions, and the cardiac function. LV dimensions and wall thicknesses were estimated with parasternal short axis M-mode images. Simultaneously, EF, FS, LV mass, and LV end diastolic volume were calculated by Philips7500 software. The final data represented averaged values of 10 cardiac cycles. Quantitative real-time PCR -------------------------- The mRNA levels of *TNF-α*, *IL-6*, *IL-1β*, *collagen-1*, *FGFR1, β-Klotho*, *FGFR2*, *FGFR3*, and *FGFR4* were quantified by qRT-PCR as described previously.^[@bib47],\ [@bib48]^ The primers of *TNF-α* (Mm00443285_m1), *IL-6* (Mm00446190_m1), *IL-1β* (Mm00434228_m1), *collagen-1* (Mm01302043), *FGFR1* (Mm00438930_m1), *β-Klotho* (Mm00473122_m1), *FGFR2* (Mm01269930_m1), *FGFR3* (Mm00433294_m1), *FGFR4* (Mm01341852_m1), and *β-actin* (Mm00607939) were from Applied Biosystems (Carlsbad, CA, USA). The expression levels of the target genes were normalized to that of the housekeeping gene *β*-actin. Western blotting ---------------- To analyze the protein expression, western blot analysis was performed as previously described.^[@bib47],\ [@bib49]^ The primary antibodies to Nrf2, HDAC1, SIRT1, SIRT6, and AMPK were purchased from Abcam (Cambridge, MA, USA). Anti-LKB1 was obtained from Sigma-Aldrich (Sigma, MO, USA), anti-HO-1, anti-NQO-1, anti-CAT, anti-IKK, anti-I*κ*B*α*, anti-*β*-Klotho, anti-CTGF, and anti-TGF-*β* were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p65, anti-caspase-8, anti-caspase-3, anti-PARP, anti-Bax, anti-Bcl-2, anti-FGFR1, and anti-acetylated-lysine were purchased from Cell Signaling (Danvers, MA, USA). Anti-3-NT was from Millipore (Billerica, CA, USA), and anti-4-HNE was from Alpha Diagnostic International (San Antonio, TX, USA). After three washes with Tris-buffered saline (pH 7.2) containing 0.05% Tween 20, the membrane was reacted with appropriate secondary antibodies for 1 h at room temperature. Finally, the probed proteins were stained with enhanced chemiluminescence reagent and visualized using the BIO-RAD ChemiDoc Touch Imaging System (BIO-RAD, Hercules, CA, USA). Preparation of nuclear extract proteins --------------------------------------- Nuclear protein was extracted according to the manufacturer's instructions (Thermo Fisher Scientific, Frederick, MD, USA). The heart tissue, H9c2 cells, or adult cardiomyocytes after different treatments were harvested, centrifuged, and incubated in ice-cold buffer for 10 min. The supernatant (cytoplasmic extract) was immediately transferred to a clean pre-chilled tube and stored at −80 °C. The insoluble (pellet) fraction was resuspended and vortexed for 15 s every 10 min for a total 40 min, and after centrifugation, the nuclear protein was obtained and stored at −80 °C. For western blot analysis, histone H3, and GAPDH were used as internal controls for nuclear and cytoplasmic extracts, respectively. DHE staining ------------ The DHE staining method was applied to measure the generation of ROS. The pre-treated H9c2 cells (1 × 10^6^ cells/well) or heart tissue sections (5 *μ*m thick) were incubated with 5 *μ*M/ml DHE dye (Invitrogen, Grand Island, NY, USA) in Hank's balanced salt solution buffer for 30 min at 37 °C without light. Oxidative stress was examined and captured by immunofluorescence microscopy. The DHE fluorescence intensity (red staining) was quantified using Image Pro Plus software (Media Cybernetics Inc., Bethesda, MD, USA). TUNEL staining -------------- Five-micrometer-thick tissue sections were used for TUNEL staining with ApopTag Peroxidase *In Situ* Apoptosis Detection Kit (Chemicon, Temecula, CA, USA).^[@bib13]^ Briefly, the slides were deparaffinized, rehydrated, and treated with proteinase K (20 mg/ml) for 15 min at room temperature. The slide was preliminarily incubated with a TUNEL reaction mixture containing terminal deoxynucleotidyl transferase and digoxigenin-11-dUTP at room temperature for 2 h. Then hematoxylin was used for counterstaining. The H9c2 cells (1 × 10^6^ cells/well) were seeded on six-well chamber slides. After different treatments, the slides were detected with the *In Situ* Cell Death Detection Kit, TMR red (Roche, Mannheim, Germany). Hoechst 33342 was used for nuclear counterstaining. TUNEL-positive cells were imaged under a fluorescence microscope (Nikon, Tokyo, Japan), and the cell death detection ELISA kit (Roche) was used to measure histone-bound DNA fragments as described before according to the manufacturer's instructions.^[@bib13]^ IP assay -------- The IP assays were performed as described previously.^[@bib39],\ [@bib49]^ Briefly, H9c2 cells, adult cardiomyocytes, or heart tissue were lysed in IP buffer (25 mM Tris, pH 7.6; 150 mM NaCl; 1 mM EDTA; 1% NP-40; protease and phosphatase inhibitors). Then 300 *μ*g of the lysate was immunoprecipitated overnight at 4 °C with the monoclonal antibodies LKB1 (Sigma-Aldrich) and protein G-agarose (Pierce Biotechnology Ltd., Rockford, IL, USA). The immunocomplexes were probed by western blotting with monoclonal anti-LKB1 (Sigma-Aldrich), SIRT1 (Abcam), SIRT6 (Abcam), or acetyl-lysine antibody (Cell Signaling). Statistical analysis -------------------- Data are presented as means±S.D. Two-way analysis of variance for comparisons was performed for the different groups, followed by *post-hoc* pairwise repetitive comparisons with Tukey's test, using Origin 9.0 Lab data analysis and graphing software (OriginLab Co., Northampton, MA, USA). *P*\<0.05 was considered statistically significant. The work was supported by a grant from the National Natural Science Foundation of China (number 81600631 to JG). **Author contributions** SW set up the animal model and performed the research. YW, ZZ, and QL contributed to the data collection and analysis. JG participated in the project design as well as manuscript draft preparation and revision. All authors read and approved the final manuscript. [Supplementary Information](#sup1){ref-type="supplementary-material"} accompanies this paper on Cell Death and Disease website (http://www.nature.com/cddis) **Publisher's Note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Edited by H-U Simon The authors declare no conflict of interest. Supplementary Material {#sup1} ====================== ###### Click here for additional data file. {#fig1} {#fig2} {#fig3} {#fig4} ###### FGF21 prohibited the interaction of SIRT1 with LKB1 in DOX-treated H9c2 cells. The mRNA levels of FGFR1, *β*-Klotho, FGFR2, FGFR3, and FGFR4 (**a**) were detected by qRT-PCR. The protein expression levels of FGFR1, *β*-Klotho (**b**), SIRT1, SIRT6, HDAC1 (**d**), and AMPK (**e**) were analyzed by western blotting. The phosphorylated (P-) and total (T-) protein of FRS2 and ERK1/2 were examined by western blotting (**c**). The LKB1 proteins were immunoprecipitated with anti-LKB1 antibody in the H9c2 cells of the different groups and then probed with acetylated-lysine (Acetyl) antibodies (**f**). The LKB1 proteins were immunoprecipitated with anti-LKB1 antibody in the H9c2 cells of the different groups and then probed with SIRT1 (left panel) and SIRT6 (right panel) antibodies (**g**). Data are presented as means±S.D. of three separate experiments. \**P*\<0.05 *versus* Ctrl group; ^\#^*P*\<0.05 *versus* DOX group   {#fig6} {#fig7} ###### Protective effect of FGF21 against DOX-induced cardiac dysfunction   ***Ctrl*** ***FGF21*** ***DOX*** ***FGF21/DOX*** ---------- ------------ ------------- -------------- ----------------- IVS;d 0.62±0.03 0.61±0.06 0.61±0.02 0.62±0.05 LVID;d 3.72±0.06 3.75±0.08 3.87±0.05\* 3.77±0.07^†^ LVPW;d 0.85±0.08 0.84±0.08 0.82±0.05 0.86±0.03 IVS;s 1.03±0.07 1.07±0.08 0.99±0.08 1.03±0.02 LVID;s 2.01±0.12 2.03±0.11 2.51±0.12\* 2.12±0.10^†^ LVPW;s 1.28±0.10 1.32±0.09 1.28±0.08 1.31±0.11 LV Vol;d 57.90±0.98 58.87 ±1.15 62.32±1.17\* 58.69±1.41^†^ LV Vol;s 13.11±1.10 13.51±0.85 21.39±1.14\* 14.79±1.92^†^ %EF 77.35±2.76 77.05±2.36 65.67±2.43\* 74.70±3.87^†^ \% FS 45.96±1.58 45.89±2.23 35.14±2.14\* 43.76±2.74^†^ LV Mass 88.10±2.52 90.25±2.37 95.01±2.66 91.41±2.96 Abbreviations: EF, ejection fraction; FS, fractional shortening; IVS, interventricular septum; LV mass, left ventricular mass; LVID;d, left ventricular internal diastolic diameter; LVID;s, left ventricular internal systolic diameter; LVPW, left ventricular posterior wall; LV vol;d, left ventricular end diastolic volume; LV vol;s, left ventricular end systolic volume Data are presented as means±S.D. (*n*=11). \**P*\<0.05 *versus* Ctrl group; ^†^*P*\<0.05 *versus* DOX group

1.. Introduction {#S1} ================ Fetal heart rate (FHR) monitoring is an important method in the field of obstetrics. It can help clinicians to obtain timely information on the physiological status of the fetus during pregnancy and childbirth \[[@ref001]\]. Changes in the physiological state of the fetus can often be reflected in the changes of the FHR. The abnormal FHR may mean fetal distress or other problems, and urgent measures are needed for pregnant women. Therefore, FHR monitoring technology has a great application value in clinical practice. There are many ways to monitor the FHR. The methods used early on are fetal heart sound method \[[@ref002]\] and magnetocardiography \[[@ref003]\], but because of their own limitations, these methods have rarely been used in clinical practice. In recent years, photoelectric method has also appeared to detect the FHR \[[@ref004], [@ref005]\], but the technology has not yet matured. At present, the main method of clinical FHR monitoring is the ultrasound Doppler method. This method has a good monitoring effect on the FHR changes, but it has the following three limitations: 1. This method requires a strict position of the ultrasonic probe, so it often requires professional to operate it. 2. Pregnant women need to remain inactive during the monitoring process, so this method is not suitable for long-term monitoring \[[@ref006]\]. 3. At the same time, one study has shown that the long-term use of ultrasound testing will affect the growth and development of the fetus \[[@ref007]\]. The FHR can also be calculated from the fetal electrocardiogram (FECG). The FHR calculated by the FECG and the FHR obtained by the ultrasound Doppler method are highly correlated even in the first stage of labor \[[@ref008]\]. The acquisition method of the FECG is divided into invasive and non-invasive. The invasive method may cause infection to the fetus or the pregnant woman, so it is rarely used in the clinic. The non-invasive method places a set of ECG electrodes on the abdomen of the pregnant woman to collect abdominal ECG (AECG). The FECG can be extracted from the AECG according to the specific FECG signal separation algorithm. In comparison to the ultrasonic Doppler method, the non-invasive method has the following advantages: 1. The ECG electrodes require less accuracy of placement than the ultrasonic probe, so the method can also be performed without professional guidance and pregnant women can do it themselves. 2. This method is less affected by the posture of the pregnant woman than with the ultrasound Doppler method, so it can be used for long-term FHR monitoring. 3. The detection effect of this method is significantly better than the ultrasound Doppler method, especially for obese pregnant women or pregnant women who are in an active state \[[@ref009]\]. 4. One study has shown that the method can detect higher baseline variability in early pregnancy compared to traditional FHR monitoring, so it is possible to perform FHR detection earlier on \[[@ref010]\]. 2.. System structure {#S2} ==================== 2.1. Overall system structure {#S2.SS1} ----------------------------- The FHR calculation system consists of two parts, as shown in Fig. [1](#thc-28-thc209019-g001){ref-type="fig"}: the fetal ECG recorder (MF-HOLTER), which collects the AECG through 4 electrodes, and the fetal ECG monitoring software (FECG-MS), which displays waveform and analysis data. Figure 1.The overall structure of the system. Left: MF-HOLTER. Right: FECG-MS. 2.2. Hardware: MF-HOLTER {#S2.SS2} ------------------------ This study designed the MF-HOLTER with four leads that can continuously acquire the AECG for a long time. It can synchronously collect four channels of the AECG, package data according to the certain data format, and transmit data to the FECG-MS through Bluetooth Low Energy transmission. The amplitude of the MECG signal in the clinic is about 100 $\sim$ 150 $\mu$V, and the amplitude of the FECG signal can only be up to 60 $\mu$V \[[@ref001]\]. With a sampling rate of 250 sps and a 24-bit resolution, the MF-HOLTER can recognize signals with a minimum amplitude of about 0.02 $\mu$V, thus meeting the requirements of clinical signal acquisition. The flowchart of the MF-HOLTER is shown in Fig. [2](#thc-28-thc209019-g002){ref-type="fig"}. Figure 2.The flowchart of the MF-HOLTER. 2.3. Software: FECG-MS {#S2.SS3} ---------------------- The FECG-MS is responsible for controlling the work of the MF-HOLTER, receiving the AECG data collected in real time, and packaging the AECG data according to the specific format to the FECG separation algorithm for separation. The R-R interval values in each separation are calculated based on the returned fetal QRS (FQRS) position, and the FHR is further calculated. The collected AECG and the separated FECG are drawn to the screen, the AECG and the FECG and the positions of the FQRS can be saved to the local disk in a CSV file format. At the same time, the FECG-MS provides the function of assessing the accuracy of FHR calculation. The flowchart of the FECG-MS is shown in Fig. [3](#thc-28-thc209019-g003){ref-type="fig"}. Figure 3.The flowchart of the FECG-MS. 3.. Method for calculating the FHR {#S3} ================================== According to the position of the FQRS returned from the FECG separation algorithm, the R-R interval values can be calculated, and the FHR can be further calculated. 3.1. The FECG separation algorithm {#S3.SS1} ---------------------------------- This study is based on the existing FECG separation algorithm in the authors' laboratory. The input of the FECG separation algorithm is a 4-channel AECG signal that has 4S duration data, and the output is the FECG signal and the position of the FQRS. The basic idea of the algorithm is as follows: 1. The AECG is pre-processed to remove the maternal muscle noise, the baseline drift, the power frequency interference, the electrode noise and so on, leaving only the mixed ECG signal containing the MECG and the FECG. 2. The template signal of the MECG is constructed by identifying the maternal QRS (MQRS). 3. The template signal of the MECG is subtracted from the mixed ECG signal to get the pure FECG, as shown in Fig. [4](#thc-28-thc209019-g004){ref-type="fig"}. 4. The position of the FQRS is identified. Figure 4.The complete FECG separated (120 bpm). However, there are some shortcomings in the current FECG separation algorithm: 1. When the amplitude of the FECG is very low and submerged by noise, the FQRS will not be recognized, so the FQRS will be lost in this position, as shown in Fig. [5](#thc-28-thc209019-g005){ref-type="fig"}. 2. When the MQRS and the FQRS in the AECG are aligned, the elimination of the MECG is not complete, and the MQRS will remain in the FECG, thus the MQRS will be recognized as the FQRS in advance, while the real FQRS will not be recognized, as shown in Fig. [6](#thc-28-thc209019-g006){ref-type="fig"}. Figure 5.The separated FECG with FQRS lost (120 bpm). Figure 6.The separated FECG where the MQRS is misjudged as FQRS (120 bpm). 3.2. The FQRS position correction algorithm {#S3.SS2} ------------------------------------------- In order to improve the deviation of the FQRS position recognition in the FECG separation algorithm, the FQRS position correction algorithm is proposed in this study. The specific steps of the algorithm are as follows: 1. The R-R interval values in the current separation are calculated according to the position of the FQRS returned by the FECG separation algorithm. 2. According to the number of occurrences of each value, the value with the most occurrences is regarded as the reference of R-R interval values in the current separation, which is recorded as RRIR, but the RRIR obtained at this time is not necessarily accurate and needs to be improved. 3. Intra-group correction is conducted. 3.1. Find out whether there is a value of half of the RRIR in those R-R interval values obtained from the current separation, and record it as 1/2\*RRIR. If there is one, it means that there are too many FQRS waves lost in the current separation and the RRIR is a pseudo-reference value. At this time, the RRIR is reassigned with the 1/2\*RRIR as the final R-R interval reference value. 3.2. Determine whether there is a value of RRIR$+$$\Delta$t in those R-R interval values ($\Delta$t is an offset greater than 0). If it exists, the RRIR$+$$\Delta$t is reassigned to the RRIR as the final reference for the R-R interval values to correct the deviation caused by identifying the MQRS as the FQRS in advance. 4. If the intra-group correction can not be successfully carried out, that is to say there is no the 1/2\*RRIR or the RRIR$+$$\Delta$t in those R-R interval values, then out-of-group correction is needed. The latest five RRIR values before the current separation are recorded, and the value with the most occurrences is selected as the R-R interval reference value for the out-of-group correction, which is recorded as RRIR-outside. The RRIR-outside is used to reassign the RRIR as the final R-R interval reference value. At the same time, the RRIR in the current separation is also recorded and if the same RRIR value appears three times in a row, it is considered to be a correct value, then the out-of-group correction will not be carried out. The step diagram of the algorithm is shown in Fig. [7](#thc-28-thc209019-g007){ref-type="fig"}. Figure 7.The step diagram. 4.. Data test {#S4} ============= In this study, two different data sets were used to assess the performance of the proposed system. One was the signal provided by the FLUKE's PS320 FETAL SIMULATOR, which is recorded as DB1. The other was the AECG signals of pregnant women collected at the Peking Union Medical College Hospital, which is recorded as DB2. 4.1. The data from the FLUKE's PS320 FETAL SIMULATOR {#S4.SS1} ---------------------------------------------------- The PS320 FETAL SIMULATOR is a FECG simulator, which can provide pure FECG signal, pure MECG signal, and mixed signal of the FECG and the MECG. Some specific parameters are shown in Table [1](#T1){ref-type="table"}. Table 1The parameters of FLUKE's PS320 FETAL SIMULATORECG typeOptional amplitude ($\mu$V)Optional heart rate (bpm)MECG500, 1000, 200060, 80, 100, 120, 140, 160FECG50, 100, 200, 500, 1000, 200030, 60, 90, 120, 150, 180, 210, 240 Figure 8.The three AECG signals. From top to bottom are case 1, case 2 and case 3, respectively. One problem that must be highlighted here is that when the FECG and the MECG are mixed together, the amplitude of the FECG will be reduced to half the set value, which is real and has been officially certified by FLUKE. Specific data testing methods are as follows: 1. The following four sets of data were collected in this study: 120 bpm, 150 bpm, 180 bpm, 120/150/180 evenly mixed. 2. The same points of the four sets of data are as follows: the data acquisition time for each data set is 15 minutes, the maternal heart rate is set to 80 bpm, the MECG amplitude is set to 1000 $\mu$V, and the FECG amplitude is also set to 1000 $\mu$V. The difference is that the first three sets of data select a single FHR value, and the fourth set of data mixes 120 bpm, 150 bpm, and 180 bpm evenly, and the FHR value is switched every one minute, and the specific performance is 120, 150, 180, 150,120 such a cyclical data form. 3. To evaluate the accuracy of the FHR calculation for each group of collected data, a specific evaluation method has been implemented in the FECG-MS. The accuracy of calculation is further obtained by recording the number of the total FHR values calculated and the number of the wrong FHR values obtained in a period of time. 4.2. The data from Peking Union Medical College Hospital {#S4.SS2} -------------------------------------------------------- The DB2 is the AECG signals of pregnant women collected at the Peking Union Medical College Hospital. In this study, three cases of data were selected, and the data of one minute was intercepted respectively to evaluate the performance of the FHR calculation system. Three AECG signals are shown in Fig. [8](#thc-28-thc209019-g008){ref-type="fig"}. 5.. Results {#S5} =========== 5.1. Performance based on DB1 {#S5.SS1} ----------------------------- In order to eliminate the influence caused by uncertain factors such as the external environment, this study randomly conducted two data collections and evaluations of the accuracy of the FHR calculation, then took the average as the final accuracy of each data set. The accuracy of the FHR calculation for each data set is shown in Table [2](#T2){ref-type="table"}. Table 2The accuracy of the FHR calculationFHR value (bpm)Duration (min)Results12015Total: 3616   Wrong: 8     Accuracy: 99.78%15015Total: 3616   Wrong: 40   Accuracy: 98.89%18015Total: 3616   Wrong: 40   Accuracy: 98.89%120/150/18015Total: 3616   Wrong: 72   Accuracy: 98.01% Table 3The average FHR values from the proposed system and Monica AN24DataCase 1Case 2Case 3The proposed system144.13133.06127.53Monica AN24144.18132.95128.27Difference0.050.110.74 The results show that the FHR calculating system can obtain more accurate FHR values for processing the AECG provided by FLUKE's PS320 FETAL SIMULATOR, and still has a higher calculating accuracy when dealing with the variable FHR values. 5.2. Performance based on DB2 {#S5.SS2} ----------------------------- Three cases of data were processed using the FHR calculation system proposed in this study, and the average FHR within one minute duration was calculated. At the same time, this study used Monica AN24 to process the same data set. Monica AN24 is a maternal-fetal holter monitor based on FECG technology developed by the University of Nottingham, UK, which is recognized in clinical applications \[[@ref009]\]. The average FHR values are as shown in Table [3](#T3){ref-type="table"}. The unit is bpm (beat per minute). From Table [3](#T3){ref-type="table"}, we can see that the average FHR values obtained by both the FHR calculation system proposed in this study and Monica AN24 are very close, and the error is less than 1 bpm. 6.. Conclusion and discussion {#S6} ============================= The results show that the FHR calculation system proposed in this study has achieved good performance when processing the simulator signals and clinical data with high signal-to-noise ratio. There are many new ways to continue to explore FECG monitoring. Zhang et al. used the single-channel combined with the adaptive noise canceller to achieve a good result in processing the data of the public database \[[@ref011]\]. Marchon et al. studied the distribution of ECG electrodes \[[@ref012]\]. Li et al. used convolutional neural networks to automatically classify the FHR \[[@ref013]\]. Future works will deal with more clinical data to enhance clinical applicability. At the same time, more features of the FECG in the time domain and frequency domain will be explored to improve the accuracy of the FQRS recognition. This work was supported by the National Key R&D Program of China (no. 2019YFC0119700), the Bill and Melinda Gates Foundation (no. OPP1148910), the Beijing Natural Science Foundation (no. 7172015), and Intelligent Physiological Measurement and Clinical Translation, Beijing International Base for Scientific and Technological Cooperation. Conflict of interest {#sec0100} ==================== None to report. [^1]: Co-first authors.

1. Introduction {#sec1} =============== *Overview*. Ovarian cancer (OC) is a common condition in women scenario and it represents the principal cause of death from gynaecologic cancer in United States \[[@B1]\]. It is estimated that 21.290 cases will have been diagnosed in 2015 and that 14.180 women will have died due to this malignancy. About 90% of OC are epithelial carcinomas and 70% of those have a serous histology \[[@B2]\]. Death rate from OC declined from 1970s to 1990s but it has since then remained stable. In light of these discouraging data, the development of novel therapies for OC has become a priority. Recent better molecular characterization and immune system identification are the starting point of future research in immunotherapy. Undoubtedly, the next decade will see immunotherapy coming to the clinic use alongside standard regimes and it is possible that it could replace cytotoxic chemotherapy in combination strategies. Therefore, in addition to possessing expertise with immunotherapy, oncologists will be expected to conduct trials of novel agents in combination with standard treatment. Thus, it is paramount to focus the attention on maximising the knowledge of the more important component of the immune system. Loss in this challenge could run the risk that oncologists will take a passive role in the development of new strategies. This paper reviews the rationale for immunotherapy and the main approaches under investigation in OC, with a special focus on the role of checkpoint inhibitors. We will briefly describe the human immune system in an attempt to provide a means of understanding how it relates specifically to the clinical practice. 2. Immune System and Cancer Disease {#sec2} =================================== 2.1. The Father of Immunotherapy {#sec2.1} -------------------------------- The role of immunotherapy in cancer treatment has been identified decades ago, because of beneficial effect of severe induced infection on tumour regression. At that time, Coley showed that inoculation with streptococcal organisms resulted in the shrinkage of inoperable bone and soft-tissue sarcomas \[[@B3]\]. However, severe criticisms due to the inconsistency of the method and results emerged in the scientific community, across the years. One explanation was that other physicians, who tested his treatment, did not report the same excellent effect. These results, as well as the concurrent development of radiotherapy and chemotherapy, determined immunotherapy to slowly disappear from treatment cancer scenario \[[@B4]\]. Since Coley\'s death, immunology has represented an active research field and, nowadays, immunotherapy is considered again a valid treatment option in different types of cancer \[[@B5]\]. 2.2. Basic Knowledge of Human Immune System {#sec2.2} ------------------------------------------- The human immune system can be divided in two components: the innate and the adaptive immunity. The innate immune system consists of natural killer (NK) cells, dendritic cells, and macrophages and neutrophils, whereas B cells and T cells, including cytotoxic (CD8+ T or CTL) cells, helper (CD4+ T) cells, and NK T cells, are specific of the adaptive immunity \[[@B6]\]. The innate immunity provides a first line response against pathogens in a nonspecific manner; it has no immunologic memory and it is not able to recognize antigen. Thus, in terms of tumor immunology, its contribution is marginal and limited to secreted cytokines that recruit immune cells. On the other hand, the adaptive immune system plays a central role in the antitumor immune response, due to its capability in processing "nonself" cells. T cell activation requires at least two conditions. Firstly, the presentation of an antigen to a T cell receptor (TCR) by a major histocompatibility complex (MHC) molecule on antigen presenting cells (APCs). Secondly, the interaction of the CD28 receptor on T cells to B7 costimulatory molecules (B7-1 and B7-2) on APCs \[[@B7]\]. Actually, the immune system often recognized the tumor cells as "self," because they are basically expression of patient\'s own cell types. The distinction between "self" and "nonself" is provided by cancer-specific antigens that are expressed by tumor cells. Tumor antigens are traditionally divided in two classes: tumor specific antigens (TSAs) and tumor associated antigens (TAAs). TSAs are exclusively expressed by tumor cells and thus they are easily recognized as "nonself" by the immune system. TSAs represent an ideal target for anticancer immunotherapy. On the contrary, TAAs are normally found on nonmalignant cells. For instance, oncospermatogonal antigens are expressed by tumor cells as well as normal spermatocytes; carcinoembryonic antigen (CEA) is expressed on fetal tissues and in several cancer types. Therefore TAAs are less expected to activate an effective and efficient immune system response \[[@B8]\]. 2.3. Immunoediting Process: The 3Es of the Immune System {#sec2.3} -------------------------------------------------------- Theoretically, the immune system recognizes nascent transformed cells, in order to prevent progression to clinical tumor. If intrinsic tumor suppressor mechanisms failed, the cancer immunoediting is engaged. It is an extrinsic tumor suppressor mechanism that consists of 3 sequential phases: elimination, equilibrium, and escape \[[@B9]\]. The elimination phase was previously known as cancer immunosurveillance. In this phase, transformed cells are recognized and eradicated by the innate and adaptive immune system. CD8+ T cells, CD4+ T cells, natural killer (NK) cells, and NK T cells secrete interferon- (INF-) *γ* to inhibit tumor cell proliferation and angiogenesis, whereas macrophages and dendritic cells are processed to phagocytise and remove tumor cells killed \[[@B10]\]. The cells that are not eliminated in this phase may then enter the equilibrium phase, in which their development is prevented by adaptive immunologic mechanism. CD8+ T cells and dendritic cells secrete INF-*γ* and interleukin- (IL-) 12, respectively, and preserve tumor cells in a steady state. This is a functional state in which latent tumor cells are specifically controlled by the adaptive immunity. This dynamic balance can persist for long period, sometimes exceeding 20 years \[[@B9]\]. In response to immune system, tumor cells can change their characteristics in immune resistant cells and therefore escape from immune system suppression. In this final phase, tumor cells emerge and become clinically apparent, because they are no longer blocked by immunity. The generation of immune resistant tumor cells can occur in several ways: through loss of tumor antigens expression; through downregulation of MHC; through the overactivation of the prooncogenic transcription factor STAT3; through the overexpression of antiapoptotic effector BCL-2; through the expression of inhibitory cell surface molecules, such as programmed cell death 1 ligand 1 (PD-L1), cytotoxic T-lymphocyte associated protein-4 (CTLA-4), and Fas ligand (FasL), which directly kill cytotoxic CD8+ T cells. Otherwise tumor cell escape can be a consequence of an immunosuppressive state established in the tumor microenvironment. This condition may result from the secretion of immunosuppressive cytokines, like IL-4, IL-1*β*, vascular endothelial growth factor (VEGF), and prostaglandin-E2 (PGE2), which recruit regulatory cells. Particularly, the secretion of IL-4 recruits macrophages that inhibit CD4+ T cells, by expressing transforming growth factor-*β* (TGF-*β*), IL-10, and platelet-derived growth factor (PDGF), whereas the secretion of IL-1*β*, VEGF, and PGE2 determines the accumulation of myeloid-derived suppressor cells that blocks T cell function \[[@B11]\]. 2.4. The Rationale {#sec2.4} ------------------ Over the last decade immunotherapy has become a mainstay in anticancer therapy. The aim is to eradicate tumor cells stimulating the normal human immune system. We need to integrate the potential understanding of the immunoediting process from the 3Es and the tumor characteristics to conduct the optimal treatment. It is difficult to define a clear role of immunotherapy; nonetheless it is reasonable to hypothesise that any immune molecule capable of activating this process might have a useful role in eradication of nascent tumor cells. At this time it is paramount that oncologists are familiar with the immunoediting process so that they can have a role in the rational development of innovative clinical trials. Immunotherapy has the potential to guide the future direction of cancer treatment. The stabilization of equilibrium state, as well as the inhibition of tumor escape mechanisms, should be clinical endpoints. 3. Cancer Immunotherapy: The Role of Immune Checkpoint {#sec3} ====================================================== 3.1. Current Immunotherapy Options {#sec3.1} ---------------------------------- Current immunotherapies for cancer treatment include therapeutic vaccines, cytokines, immune modulators, immune checkpoint inhibitors, and adoptive T cell transfer \[[@B12]\]. Therapeutic vaccines are designed to treat established cancers and may be used in the induction of the tumor-directed immune response of the patients through the introduction of tumor antigens. The other approaches such as immune checkpoint inhibitors and adoptive T cell transfer are designed to augment anticancer immunity against cancer \[[@B13]\]. 3.2. Focus on Immune Checkpoint {#sec3.2} ------------------------------- Nowadays, one of the most promising strategies seems to be the takeover of immune cell-intrinsic checkpoints that are induced on T cells activation. The blockade of one of these checkpoints, such as CTLA-4 \[[@B14]\] or the programmed death 1 (PD-1) receptor, has recently been found to be active to achieve an immune-modulation approach in the treatment of solid tumors \[[@B15], [@B16]\]. The immune checkpoint blockade targeted agents might represent breakthrough drugs in the treatment of solid tumors and have generated greater expectations in the field of cancer immunotherapy, even in OC \[[@B17], [@B18]\]. 3.3. Mechanisms of Action of Immunomodulators {#sec3.3} --------------------------------------------- T cells activity is regulated by a great number of different molecules, as well as immune-modulatory signals, both costimulatory and coinhibitory \[[@B19]\]. To avoid inappropriate T cell activation, resulting in autoimmunity, negative regulators of T cell immunity, including CTLA-4 and PD-1, are needed. Preclinical models on the blockade of these coinhibitory molecules showed an antitumor immune response \[[@B17]\]. In fact both CTLA-4 and PD-1 are key immune checkpoint proteins and represent a further promising immunotherapeutic target. CTLA-4 is a member of the CD28:B7 immunoglobulin superfamily, typically low-expressed on the surface of naive effector T cells and regulatory T cells (Tregs) \[[@B20]\]. When naive T cells are stimulated through the TCR, CTLA-4 is upregulated and competes with CD28 for B7 and, finally, determines the suppression of T cell activity \[[@B21]\]. It was found that the antitumor effect of CTLA-4 blockade might be obtained also by depletion of Treg \[[@B22]\], as revealed in a model of mouse melanoma, in which both the augmentation of T effector cell function and inhibition of Treg activity through the blockade of CTLA-4 manage to obtain a strong antitumor response. PD-1 is expressed on chronically stimulated T cells, as well as Tregs, activated B cells, and NK cells \[[@B23]\]. Differently from CTLA-4, which regulates T-lymphocytes at the level of initial activation, PD-1 regulates immunity at multiple phases of the immune response, including its effect on effector T-lymphocyte activity in the peripheral tissues. Experimental models showed that PD-1 deficient mice present enhanced immunity with phenotypes characterized by autoimmune cardiomyopathy and a lupus-like syndrome \[[@B24], [@B25]\]. The activity of PD-1 is related to its interaction with its ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC) \[[@B26]\]. Both ligands, especially PD-1, are expressed on many hematologic and nonhematologic human tumors \[[@B27]\]. Generally, in human cancer, when PD-1 binds with cells bearing one of its ligands, T cell activity is attenuated (phenomena known as peripheral tolerance), which prevents these T cells from rejecting the tumor at the tissue level, and tumors can thereby employ the PD-1 inhibitory pathway to silence the immune system \[[@B28]\]. 4. Clinical Trials with Immune Checkpoint Blockade Targeted Agents in OC {#sec4} ======================================================================== Based upon the findings of preclinical studies, suggesting the involvement of these molecules in immune control, various agents blocking CTLA-4, PD-1, or PD-L1 or other immune molecules are currently investigated in ovarian cancer (OC) treatment. Details are shown in [Table 1](#tab1){ref-type="table"}. 4.1. Anticytotoxic T-Lymphocyte Antigens {#sec4.1} ---------------------------------------- The CTLA-4 is currently being investigated as a single or combinatorial therapy in clinical trials involving several cancer types. Ipilimumab and tremelimumab are fully human IgG1 or IgG2 antibodies, respectively, that antagonize the CTLA-4 immune checkpoint. The majority of clinical data derived from studies in patients with melanoma. In these studies CTLA-4 blockade has yielded objective responses to such an extent that ipilimumab was FDA approved to treat metastatic or unresectable melanoma in 2011 \[[@B29], [@B30]\]. It represented the first standard-of-care immune checkpoint inhibitor. Experience in OC is actually based on small population studies but results seem to be interesting. Hodi et al. firstly showed \[[@B31], [@B32]\] antitumor effects in patients with stage IV OC patients. Initially \[[@B31]\], they reported that a single infusion of ipilimumab (3 mg/kg) in two-stage IV OC patients previously vaccinated with granulocyte-macrophage colony-stimulating factor modified irradiated autologous tumor cells (GVAX), was well tolerated, and triggered a decrease or stabilization of CA-125 levels of several months\' duration. In order to clarify the toxicity and antitumor efficacy, they treated additional 9-stage IV OC subjects by using the same antibody dose and schedule (with the exception of one patient) \[[@B32]\]. In one patient, an objective radiographic response was noted and multiple infusions of anti-CTLA-4 antibody every 3 to 5 months have maintained disease control over 4 years; furthermore, 3 out of 9 patients had stable disease of 6 (ongoing at the moment of paper\'s publication), 4, and 2 months\' duration, as measured by CA-125 levels and radiographic criteria, in the absence of serious toxicities. Few patients showed manageable inflammatory toxicities. Tumor regression correlated with the CD8+/Treg ratio, suggesting that other forms of therapy that target Treg depletion might have a synergistic effect when combined with the tumor vaccine and CTLA-4 antibody molecules. These findings prompted a phase II clinical trial to evaluate ipilimumab as monotherapy in platinum-resistant OC patients ([NCT01611558](http://clinicaltrials.gov/ct2/show/NCT01611558)) \[[@B33]\]. Ipilimumab can cause significant immune-related adverse events (AEs), and the more common observed side effects include diarrhea, colitis, and dermatitis. Less common severe immune-related adverse events include hypophysitis, thyroiditis, and hepatitis. Tremelimumab (previously known as ticilimumab) is a fully human IgG2 monoclonal antibody to CTLA-4. In contrast to ipilimumab, a large phase III trial \[[@B34]\] in melanoma did not demonstrate improved PFS or OS compared with cytotoxic chemotherapy although durable responses were observed in some patients. Much speculation have been done about the potential reasons for this clinical result, because both phase III clinical trials \[[@B14], [@B29]\] testing ipilimumab succeeded in showing improved OS. It has been proposed that human IgG1 (the ipilimumab subclass) binds with a higher affinity to Fc*γ*Rs than human IgG2 (the subclass of tremelimumab) does \[[@B35]\], therefore suggesting that tremelimumab might determine a CTLA-4 antibody mediated Treg-cell depletion to a lesser extent \[[@B36], [@B37]\]. The combination of tremelimumab and a PD-1 inhibitor (see below) is currently ongoing, in a phase I study including ovarian and cervical cancer patients \[[@B38]\]. 4.2. PD-1 and PD-L1 Targeting Agents {#sec4.2} ------------------------------------ The therapeutic benefit obtained with CTLA-4 inhibition led to the effort in identifying other potential immune checkpoint inhibitors that should have been more specific and equally efficacious and have less immune toxicity. PD-1 and PD-L1 inhibitors were identified as potentially accomplishing those requirements. Differently from CTLA-4, which regulates T-lymphocytes at the level of initial activation, PD-1 regulates immunity at multiple steps, including exerting its effect on effector T-lymphocyte activity in the peripheral tissues. Several monoclonal antibodies have been developed that block the PD-1 system, either by interactions with the PD-1 receptor or with its specific ligands. Nivolumab (also known as BMS-936558 or MDX1106) is a fully human IgG4 monoclonal antibody that targets PD-1. A phase I/II clinical trial \[[@B39]\] tested the safety and efficacy of nivolumab at doses of 0.1 to 10.0 mg/kg of body weight intravenously every 2 weeks for up to 12 cycles until disease progression or a complete response occurred. Patients with advanced melanoma, non-small-cell lung cancer, prostate cancer, renal cancer, and colorectal cancer were enrolled. Among the 296 patients, those with metastatic melanoma achieved the higher rates of objective responses (27.6%), with a median OS of 16.8 months; conversely responses were not observed in colon and prostate cancer patients. Responses were seen in both PD-L1 positive and negative patients, even if with lower extent. Common treatment-related adverse events included fatigue, diarrhea, pruritus, rash, nausea, and decreased appetite. Grade 3 or 4 treatment-related adverse events were seen in 14% of patients. Treatment-related serious adverse events were noted in 11% of patients and included pneumonitis (3%, and grade 3 or 4 in 1%), colitis, hepatitis, thyroiditis, and hypophysitis. Recently, at the 2014 ASCO meeting \[[@B40]\], the first clinical trial of nivolumab treatment against platinum-resistant OC has been presented. A total of 18 evaluable patients were treated with nivolumab: 10 patients were administered 1 mg/kg and 8 patients were administered 3 mg/kg, each every 2 weeks for 1 year. Starting at week 8, patients were assessed every 8 weeks and patients with disease progression were taken off study. Median treatment duration was 14 weeks. There were two serious treatment-related AEs: one patient in the 1 mg/kg group experienced grade 3 fever, disorientation, and gait disorder and one patient in the 3 mg/kg category experienced grade 3 fever and deep-vein thrombosis. Other grade 3/4 treatment-related AEs included hypothyroidism (two patients, both in the 1 mg/kg group); heart arrhythmia (one patient, in the 3 mg/kg group); and lymphocytopenia (one patient, in the 1 mg/kg group). Interestingly, the overall objective response rate was 17%. The 3 mg/kg dose may be more favourable (25%) than 1 mg/kg (10%). Two patients in the 3 mg/kg group experienced complete response (CR; response rate 25%). Among those receiving 1 mg/kg nivolumab, one experienced a partial response (10% response rate) and two patients experienced stable disease (SD). Further researches are investigating biomarkers predicting response. A further molecule investigated in OC is pembrolizumab (MK-3475, formerly known as lambrolizumab), a humanized IgG4 monoclonal antibody against PD-1. It was found to be active in treating both melanoma and NSCLC \[[@B41]--[@B43]\], similar to nivolumab. Actually, no randomized trial has compared the two agents, which are surely different in binding affinities, nivolumab being a fully human IgG4, and pembrolizumab is humanized. Currently, phase I trials are ongoing with both molecules including OC patients. Recently, an interim analysis with pembrolizumab showed preliminary signal for clinical efficacy in recurrent OC \[[@B44]\]. In addition to antibodies targeting PD-1, several different anti-PD1-L1 monoclonal antibodies, such as BMS-936559, MPDL3280A, MEDI4736, and MSB0010718C, have been developed which might enhance immune function. It was found that the ligand/receptor interaction inhibits the T-lymphocyte response by inhibiting the kinases involved in T-lymphocyte activation via phosphatase activity and other signaling pathways \[[@B45]\]. BMS-936559 is a high-affinity, fully human IgG4 monoclonal antibody that binds PD-L1 and that blocks PD-L1 from binding its two known receptors PD-1 and CD8. It was safe in a phase I trial that included 17 OC patients \[[@B15]\] in escalating doses of 0.3--10 mg/kg iv every 14 days in 6-week cycles for up to 16 cycles or until the patient had a complete response or confirmed disease progression and observed durable tumor regression and prolonged stabilization of disease. Common side effects included fatigue, infusion reactions, diarrhea, arthralgia, pruritis, rash, nausea, and headache. In the trial, only OC patients at the 10 mg/kg dose achieved objective responses: 1 (6%) with a partial response and 3 (18%) with stable disease lasting more than 24 weeks. MSB0010718C is a fully human IgG1 monoclonal antibody targeting PD-L1. Unlike other PD-L1 targeting agents, it is a native Fc receptor, allowing for antibody dependent cell mediated cytotoxicity. In a phase I trial \[[@B46]\] 27 patients with refractory malignancies were treated with MSB0010718C at 1, 3, 10, and 20 mg/kg twice weekly. Eleven patients in the study had received prior treatment with an immunotherapy. At the 3 and 10 mg/kg doses, the drug was found to inhibit 93.8% and 93.2% of the PD-L1 receptor on peripheral leukocytes. Additionally, a linear PK profile was found, with a maximum concentration of the drug achieved at 1.5--2 hours following infusion. At the 20 mg/kg dose, a dose-limiting immune-related adverse event was noted. In this trial also OC patients were included and, interestingly, a larger subsequent meta-analysis of the company developing the drug (Merck) \[[@B47]\], including 23 patients\' OC cohort, showed 48% of patients reaching stable disease and 17% getting a partial response within 30 weeks of treatment start, though 13 had been taken off the drug. Noteworthily, the responses came despite 77% of patients having already failed at least three lines of therapy. More recently \[[@B48]\], efficacy data from the 23 patients followed up for more than 2 months (range 2--8 months) were presented. Four patients (17.4%) achieved an unconfirmed partial response, 11 (47.8%) patients had stable disease, and 2 patients had \>30% tumor shrinkage after progression. Median PFS was 11.9 weeks and the PFS rate at 24 weeks was 33.3%. Toxicity was manageable and only 2 patients (8.7%) experienced grade ≥ 3 drug-related AEs. The most commonly reported AEs were fatigue, nausea, and diarrhea. A larger, phase II clinical trial enrolling 590 patients is ongoing. Other molecules, such as MPDL3280A and MEDI4736, are currently investigated in phase I trials including OC patients. 4.3. Concluding Remarks {#sec4.3} ----------------------- OC is defined as an immunogenic tumor that exhibits a spontaneous antitumor immune response \[[@B49]\]. Tumor tissue can be considered a Darwinian microenvironment that selects the better strategy to elude the immune system. Immune checkpoint pathways are modulated by ligand/receptor interactions. Expression of specific ligands, such as PD-L1 and CTLA-4, in the stroma or in the tumor cells associated, is paramount to improve growth and resistance to immune attack. It depends on both tumor type and histology, and therefore it also represents the major limitation of immunotherapy. Maybe the identification and characterization of similar patients population, as well as tumor histology, could provide data to facilitate the development of novel treatment strategies. Immune check point inhibitors may have a synergic mechanism in multimodality treatment and thus a positive effect on overall survival, with a tolerable toxicity profile. Further randomized trials are paramount to prospectively clarify this hypothesis. 5. Conclusions {#sec5} ============== In the last years, immunotherapy has achieved an important role in the fight against cancer and also, in OC immunological phenomena, has been demonstrated to play a central role. Novel and promising agents have been developed. Immune checkpoint inhibitors have shown clinical activity in several cancers, especially melanoma, and they represent a major step forward in the fight against cancer. These novel therapies will likely play a role also in OC given the potential for rapid, durable responses and their favourable toxicity profiles. Their function in the treatment of patients with OC remains to be defined but initial results seem to be promising. Next challenges should be the clinical development of combinatorial approaches and further defining patients who benefit from immune checkpoint monotherapy and patients who require potentially more active albeit more toxic combination regimens. Finally, the definition of potential biomarkers that can determine which immune checkpoint pathway or pathways dominate in a particular tumor will be crucial to guide the choice of inhibitor. The possibility of using immunotherapy in OC is still restricted to clinical trials but it is reasonable to expect that over the next years important advances in OC immunotherapy will be made, running further phase II and III trials development. Conflict of Interests ===================== The authors declare that there is no conflict of interests regarding the publication of this paper. Authors\' Contribution ====================== Francesca De Felice and Claudia Marchetti are equal contributors. ###### Active trials of checkpoint inhibitor in ovarian cancer. ----------------------------------------------------------------------------------------------------------------------------------------- Drug Antibody type^*∗*^ Notable side effects Study (phase) ------------------------ -------------------- --------------------------------------------------------------------------- --------------- Anti-CTLA-4 antibodies        Ipilimumab IgG1 Diarrhea, colitis, fatigue,\ I; II transaminitis, hypophysitis.  Tremelimumab IgG2 Diarrhea, fatigue, nausea,\ I vomiting, anorexia, rash Anti-PD-1 antibodies        Nivolumab IgG4 Pneumonitis, lymphopenia, fatigue,\ I; II diarrhea, hepatitis, renal insufficiency  Pembrolizumab IgG4-kappa Pneumonitis, fatigue, thyroid problems I Anti-PD-L1 antibodies        BMS-936559 IgG4 Fatigue, hyperglycemia, infusion reaction,\ I; II endocrinopathies, adrenal insufficiency, myasthenia gravis  MEDI4736 IgG1-kappa Diarrhea, fatigue, rash, vomiting I  MPDL33280A IgG4 Hyperglycemia, hypophysitis, pericardial effusion, fatigue I  MSB0010718C IgG1 Laboratory abnormalities, creatine kinase increase, myositis, myocarditis I; II ----------------------------------------------------------------------------------------------------------------------------------------- ^*∗*^All fully human, except pembrolizumab which is a humanized IgG4-kappa. Updated on February 28, 2015. [^1]: Academic Editor: David Kaplan

{#sp1 .641} {#sp2 .642}

In April 2014, the 3rd International Conference ≪Genetics of aging and longevity≫ took place in Sochi, Russia. We are pleased to present a collection of articles based on or related to the topics of the Conference and published in Frontiers in Genetics of Aging in 2015. They reflect understanding complex interactions between genes and genetic pathways underlying aging that is of utmost significance for the future life of humanity. Accordingly, genetic basis of longevity remained the most important topic of the Conference. The role of genes involved in energy supply and mitochondrial integrity and function deserved a close attention in two reviews published in this collection ([Morrow and Tanguay](http://dx.doi.org/10.3389/fgene.2015.00103); [Rogers and Rogina](http://dx.doi.org/10.3389/fgene.2015.00204)). The importance of redox homeostasis in aging was considered in the paper by [Klichko et al.](http://dx.doi.org/10.3389/fgene.2015.00083) Undoubtedly, energy metabolism governed by various genes and environmental factors, such as calorie intake, represents one of the key players in longevity control. [Klichko et al.](http://dx.doi.org/10.3389/fgene.2015.00083) also demonstrated that temporal regulation of redox status is related to the aging-associated loss of a proper circadian modulation with age. Having joined an interesting extramural discussion, [Koliada et al.](http://dx.doi.org/10.3389/fgene.2015.00082) reviewed the current views on the telomere length as a marker and/or a cause of aging and came to the conclusion that telomere may not serve as a "clock" counting cell divisions, but that telomere shortening, rather, reflects the history of exposure to oxidative stress in cell lineages. Thereto, another article described age-associated alterations in the chromosome morphology in a particular type of cells and indicated molecular mechanisms underlying these changes ([Lebedeva et al.](http://dx.doi.org/10.3389/fgene.2015.00274)). Aging is defined as a gradual loss of physiological functions accompanied by decreasing fertility and increasing risk of mortality. An important and challenging question for the world-wide scientific community is whether there are means allowing to combat aging and to prolong health span and life span. Therefore, it is not surprising that anti-aging interventions attracted much attention at the Conference and, therefore, a number of articles in this collection were concentrated on possible pharmacological agents aimed to slow down aging ([Carretero et al.](http://dx.doi.org/10.3389/fgene.2015.00077); [Johnson et al.](http://dx.doi.org/10.3389/fgene.2015.00247); [Semenkov et al.](http://dx.doi.org/10.3389/fgene.2015.00245); [Sycheva](http://dx.doi.org/10.3389/fgene.2015.00184)). Of note, effects of treatments on age-related traits, such as weight, as well as on some pathological states, rather than on aging and longevity were often considered ([Johnson et al.](http://dx.doi.org/10.3389/fgene.2015.00247); [Semenkov et al.](http://dx.doi.org/10.3389/fgene.2015.00245); [Sycheva](http://dx.doi.org/10.3389/fgene.2015.00184)). In two research papers ([Johnson et al.](http://dx.doi.org/10.3389/fgene.2015.00247); [Semenkov et al.](http://dx.doi.org/10.3389/fgene.2015.00245)), anti-aging interventions were to apply chemical (rapamycin) and physical (temperature, UV light) agents targeted at TOR signaling and ROS production; the role of vitamins in reducing cellular damage was regarded by [Sycheva](http://dx.doi.org/10.3389/fgene.2015.00184); five main pharmacological classes of compounds known to extend lifespan were considered in the review written by [Carretero et al.](http://dx.doi.org/10.3389/fgene.2015.00077)). Arguments supporting the idea that the nervous system is an important target for anti-aging prophylaxis were also discussed by [Omelyanchuk et al.](http://dx.doi.org/10.3389/fgene.2015.00089) The wide spectrum of agents and targets considered in a restricted number of articles once again sends us a trivial, albeit important message: modulation and fine tuning of multiple molecular mechanisms are needed to ensure longevity. Likewise, analysis of genome-wide screens for polymorphisms significantly associated with human longevity ([Yashin et al.](http://dx.doi.org/10.3389/fgene.2015.00122)) brought authors to the conclusion that genes involved in the life span control affect various molecular functions but, eventually, these functions converge in the development of aging and key age-related pathologies. In the context of the search for means that are able to prolong the life span, [Baranova and Willett](http://dx.doi.org/10.3389/fgene.2015.00129) suggested to pay a close attention to "the world of metabolites," arguing that metabolome might be the most effective target for anti-aging interventions. Overall, articles from this collection reflect a wide range of current aging research and highlight its prospects and future directions. We hope that they will attract the attention of a broad scientific readership. Author contributions {#s1} ==================== All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication. 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. [^1]: Edited and reviewed by: Blanka Rogina, University of Connecticut Health Center, USA [^2]: This article was submitted to Genetics of Aging, a section of the journal Frontiers in Genetics [^3]: †These authors have contributed equally to this work.

1. Introduction {#sec1-molecules-23-00256} =============== β-Elemene, a natural sesquiterpene and its isomers were isolated for the first time in 1994 from dry curcuma rhizome, and until now rhizome of curcuma (species of *C. Wenyujin* Y. H. Chen & C. Ling, *C. phaeocaulis* Valeton, and *C. kwangsiensis* S. G. Lee & C. F. Liang, Zingiberaceae) is the only plant source that has been used in elemene processing industry \[[@B1-molecules-23-00256]\]. The described content of β-elemene in extract of rhizome curcuma processed during industrial supercritical CO~2~ extraction is up to 27.83%; however, there are several plant species more abundant in β-elemene or its isomers in their essential oils (*Nigella damascena* L., *Magnolia figo* (Lour.) DC, *Alisma plantago-aquatica subsp. orientale* (Sam.) Sam. and *Solidago decurrens* Lour). Among them, *N. damascena* essential oil is the most promising source in which β-elemene can reach up to 73% \[[@B1-molecules-23-00256]\]. Techniques previously used for β-elemene isolation include classical column chromatography: silica gel column chromatography \[[@B2-molecules-23-00256]\], silver ion coordination chromatography \[[@B3-molecules-23-00256],[@B4-molecules-23-00256],[@B5-molecules-23-00256]\], or HP20 macroporous resin chromatography \[[@B5-molecules-23-00256]\] with further purification by means of preparative high performance liquid chromatography \[[@B5-molecules-23-00256]\]. Also, preparative gas chromatography with dimethylpolysiloxane or enantioselective cyclodextrin columns were applied \[[@B6-molecules-23-00256]\]. Modern techniques based on the principle of liquid-liquid extraction (in which no losses of sample occurs), like high-performance centrifugal partition chromatography (HPCPC) \[[@B7-molecules-23-00256]\] or high-speed counter-current chromatography (HSCCC) \[[@B8-molecules-23-00256]\], were used to obtain β-elemene from plant material/essential oil as well. In our study the other modern technique based on the principle of liquid-liquid extraction, high performance counter-current chromatography (HPCCC), was applied, because it enables the achievement of a higher *g* level, and thus higher efficiency, compared to high-speed counter-current chromatography. Furthermore, the rapid purification can be obtained due to providing good retention of a high amount of the stationary phase and possibility to use higher flow rate of the mobile phase \[[@B9-molecules-23-00256],[@B10-molecules-23-00256]\]. To our knowledge, this technique was not previously used for β-elemene isolation \[[@B10-molecules-23-00256],[@B11-molecules-23-00256]\]; however, there are few papers describing successful purification of terpenoids from essential oils with application of HPCCC \[[@B12-molecules-23-00256],[@B13-molecules-23-00256],[@B14-molecules-23-00256]\]. There is little information regarding antimicrobial action of β-elemene. What is more, the increasing incidence of multi drug resistance of microorganisms to different antibiotics and chemotherapeutics is a strong reason for searching for new active agents \[[@B15-molecules-23-00256]\]. Sesquiterpenes isolated from essential oils are among compounds with promising antimicrobial activity \[[@B16-molecules-23-00256],[@B17-molecules-23-00256]\]. Also, β-elemene is natural sesquiterpene; therefore, it is worth to check the antimicrobial activity of this compound against different pathogens including mycobacteria. Hence, in this work, we aimed to elaborate the effective protocol for preparative isolation of β-elemene from a new plant source---*N. damascena* essential oil---using HPCCC technique, and to check its antimicrobial action and compare it with activity of crude essential oil against panel of Gram-positive, Gram-negative bacteria, fungi, and mycobacterial strains. 2. Results {#sec2-molecules-23-00256} ========== 2.1. Chemical Composition of N. damascena Essential Oil {#sec2dot1-molecules-23-00256} ------------------------------------------------------- GC-MS analysis was used to confirm a proper selection of source material for β-elemene isolation. *N. damascena* essential oil (EO) obtained via hydrodistillation constituted 0.436% of dry plant material. 29 compounds representing over 99% of the total EO were identified ([Table 1](#molecules-23-00256-t001){ref-type="table"}), with β-elemene being the main ingredient and accounting for 47.37% in the EO ([Figure 1](#molecules-23-00256-f001){ref-type="fig"}). The other compounds present in the amount above 10% and listed according to the elution time were β-selinene, *α*-selinene, and damascenine. Based on these data, we subjected *N. damascena* EO to HPCCC separation as optimal source for β-elemene isolation. 2.2. Isolation of β-Elemene {#sec2dot2-molecules-23-00256} --------------------------- The selection of solvent systems used for determination of K*~β~*~-elemene~ was based on the previous work of Dang et al., who separated β-elemene along with five other volatiles from *C. wenyujin* essential oil \[[@B7-molecules-23-00256]\]. The K*~β~*~-elemene~ values obtained in our study ([Table 2](#molecules-23-00256-t002){ref-type="table"}) differed from this reported previously (systems 1 and 2); what is more, heptane/acetonitrile/ethyl acetate in the ratio 2:1:1 became a single phase above 23 °C. The additional variants of mixture of petroleum ether/acetonitrile/acetone (systems 3 and 4) gave satisfactory K*~β~*~-elemene~ values; however, the problem with system stability over 23 °C appeared again in case of system 3. The final separation was carried out in a mixture of petroleum ether/acetonitrile/acetone in the ratio 2:1.5:0.5, with a good retention of stationary phase (78%). β-elemene was eluted between 55--70 min ([Supplementary Materials: Figure S1](#app1-molecules-23-00256){ref-type="app"}), and its purity in the fractions ranged from 87 to 96%. The optimization of the sample mass revealed that the best purity (96%) and yield (80%) was obtained when 100 mg of essential oil was used ([Table 3](#molecules-23-00256-t003){ref-type="table"}). Compounds other than β-elemene and damascenine (eluted in first ten minutes of the run) were retained in the column as having higher K values (data not shown). 2.3. Identification of Isolated β-Elemene {#sec2dot3-molecules-23-00256} ----------------------------------------- Spectroscopic data: MS (EI, 70 eV) *m/z* (%): 81 (100); 93 (95); 107 (70); 67 (60); 121 (45); 147 (45); 56 (35); 121 (35); ^1^H NMR (300 MHz, CDCl~3~), δ: 5.86--5.80 (1H, dd, *J* = 11.0, 17.4), 4.93--4.89 (2H, m), 4.83--4.82 (1H, t, *J* = 1.6), 4.73--4.71 (2H, m), 4.60 (1H, bs), 2.04--2.00 (1H, m), 1.96--1.92 (1H, m), 1.75 (3H, s), 1.72 (3H, s), 1.62--1.43 (6H, m), 1.01 (3H, s). ^13^C NMR (9300 MHz, CDCl~3~), δ: 150.4, 150.3, 147.7, 112.1, 109.8, 108.2, 52.7, 45.7, 39.9, 39.8, 32.9, 26.8, 24.8, 21.1, 16.6 Chromatographic profiles ([Supplementary Materials: Figure S2A,B](#app1-molecules-23-00256){ref-type="app"}) and spectroscopic data ([Supplementary Materials: Figure S2C; Figures S3 and S4](#app1-molecules-23-00256){ref-type="app"}) matched the molecular structure of β-elemene, which was also in accordance with previous reports \[[@B5-molecules-23-00256]\]. 2.4. Antimicrobial Testing {#sec2dot4-molecules-23-00256} -------------------------- The antimicrobial activity of isolated β-elemene, as well as *N. damascena* essential oil, was checked against standard panel of microorganisms including Gram-positive, Gram-negative bacteria, fungi, and mycobacterial strains. The MIC reading was normalized against controls with 1% DMSO (with bacteria/yeasts) and blanks (without bacteria/yeasts), and compared with the positive control drugs. Normalization means that antibiotics with MIC values known for the tested set of strains were used. The obtained control values were within the reference ranges, which proves that the MIC test was performed correctly. The control with DMSO solvent confirmed that there is no influence of the solvent itself on the growth of tested organisms; hence, the obtained results are not false positive. Our findings regarding the use of DMSO solvent are supported by earlier study performed by O'Neill et al., who studied the minimal inhibitory effect of this solvent against *M. tuberculosis* in microplate resazurin assay \[[@B18-molecules-23-00256]\] and by Wadhwani et al. who evaluated the influence of different solvents, including DMSO, on a growth of several bacteria species \[[@B19-molecules-23-00256]\]. Both research teams found the 2% DMSO concentration is maximal acceptable with regard to the compromise between solubility of tested compounds and providing sufficient bacteria growth. In our study, 1% DMSO was used, because the solubility of studied samples was not problematic in this concentration. No activity of β-elemene, as well as *N. damascena* essential oil, was observed against *Staphylococcus aureus*, *Staphylococcus epidermidis*, *Escherichia coli*, *Pseudomonas aeruginosa*, *Candida albicans*, *Candida parapsilosis,* and *Mycobacterium smegmatis* (MIC \> 1000 μg/mL); however, the medium activity was noticed for both samples against *M. tuberculosis* (MIC value of 128 μg/mL and 256 μg/mL for β-elemene and EO, respectively). The obtained results clearly indicate that *N. damascena* EO and β-elemene itself exert moderate action against *M. tuberculosis* H37Ra strain ([Table 4](#molecules-23-00256-t004){ref-type="table"}). 3. Discussion {#sec3-molecules-23-00256} ============= 3.1. N. damascena Essential Oil Is a New, Rich Source for β-Elemene Isolation {#sec3dot1-molecules-23-00256} ----------------------------------------------------------------------------- EO used in this study was obtained from seeds of *N. damascena* cultivated in Poland, and it contained 47% of β-elemene. Higher content of β-elemene, reaching up to 73%, was reported previously by D'Antuono et al. \[[@B20-molecules-23-00256]\] and by Moretti et al. \[[@B21-molecules-23-00256]\]. However, results similar to ours were obtained by Fico et al. \[[@B22-molecules-23-00256]\] and by Wajs et al. \[[@B23-molecules-23-00256]\], who also found β-elemene content in the essential oil obtained from *N. damascena* seeds equal to 50% and 59%, respectively. The chemical composition of essential oils and plant extracts is influenced by different factors, among which climate, growing conditions, or harvest time are the most studied \[[@B24-molecules-23-00256]\]. The essential oil used in our study was hydrodistilled form seeds obtained from market in Poland. Wajs et al. also studied polish seeds purchased from the market \[[@B23-molecules-23-00256]\]. However, Fico, who found similar content of β-elemene (49%), studied seeds from plants grown in Italy. Interestingly, they concluded that seeds from wild plants are less abundant in β-elemene compared to commercial seeds (38% vs. 49%, respectively) \[[@B22-molecules-23-00256]\]. This finding is in agreement with higher content of compound of interest found in seeds harvested from plants grown from commercial seeds \[[@B20-molecules-23-00256]\]; hence, not only the plant origin, but what is more more important, the plant cultivation, influence the content of β-elemene in essential oil. According to Wang et al. \[[@B1-molecules-23-00256]\], *N. damascena* essential oil is the richest alternative to *Rhizoma curcuma* source of β-elemene. This source can be used in elemene processing industry delivering β-elemene for antineoplastic medicine. Therefore, we aimed to elaborate the protocol for β-elemene isolation from mentioned essential oil applying HPCCC technique. Application of high performance counter-current chromatography enabled us to obtain β-elemene of high purity and recovery in 70 min. The previously reported isolation of β-elemene is mostly based on silica gel column chromatography \[[@B2-molecules-23-00256]\] or silver ion coordination chromatography \[[@B3-molecules-23-00256],[@B4-molecules-23-00256],[@B5-molecules-23-00256]\]; however, the latter one is more effective due to the formation of coordination complex between silver ion and the double bond of β-elemene \[[@B3-molecules-23-00256]\]. The formation of coordination complex is, therefore, selective, and enabled the removal of all compounds without double bonds. Wei et al. \[[@B5-molecules-23-00256]\] obtained β-elemene with 56.1% purity using AgNO~3~ silica gel column chromatography; however, they also show that HP20 macroporous resin chromatography gives higher purity of target compound (82.9%). To obtain β-elemene with purity similar to obtained in our study (96%), the additional step of preparative HPLC was required after HP20 column chromatography \[[@B5-molecules-23-00256]\]. There were also a few attempts to isolate β-elemene applying counter-current chromatography. High-performance centrifugal partition chromatography (HPCPC) resulted in elution of β-elemene after 400 min \[[@B7-molecules-23-00256]\], whereas our protocol enabled the elution of target compound in 70 min. Also, silver ion coordination high-speed counter-current chromatography resulted in higher elution time of β-elemene (over 80 min) \[[@B8-molecules-23-00256]\] compared to our method. The differences in the elution time come from different separation conditions between these techniques (HPCCC vs. HPCPC or HSCCC; e.g., rotation speed, column volume, solvent system flow rate); however, they also come from the different essential oils used. In both previously applied counter-current chromatographic techniques, *C. Wenyuj in* essential oil was used for β-elemene isolation. Because *C. wenyujin* essential oil contains a lot of constituents, Lu et al. \[[@B8-molecules-23-00256]\] introduced a silica gel column chromatography step before HSCCC separation. This enabled the removal of impurities and increased β-elemene content in the purified fraction \[[@B8-molecules-23-00256]\]. The two steps separation resulted in around 63% recovery \[[@B8-molecules-23-00256]\]. The isolation protocol described in our study is more effective, since no purification step is required to obtain β-elemene with purity up to 96% and 80% yield. Although *C. wenyujin* essential oil served as material for separation of curdione, curcumol, germacrone, curzerene, 1,8-cineole, and β-elemene \[[@B7-molecules-23-00256],[@B25-molecules-23-00256]\], it is not the most abundant source of β-elemene. In our study, we used *N. damascena* essential oil as more cost-effective source of target compound. The chosen essential oil contains less constituents (29) than *C. wenyujin* essential oil (72) \[[@B26-molecules-23-00256]\], and the content of β-elemene in the first essential oil is at least three times higher, which makes the isolation more effective. The source of β-elemene for industry processing is an extract of rhizome curcuma obtained by means of supercritical CO~2~ extraction \[[@B1-molecules-23-00256]\]; however, it contains smaller amount of compound of interest (up to 27.83%) than source used in our study, and supercritical CO~2~ extraction technique itself is much more expensive and far less effective in β-elemene isolation than simple hydrodistillation, which was proved by Sajfrtova et al. \[[@B27-molecules-23-00256]\]. What is more, the β-elemene injection applied clinically as a national second-class anticancer drug in China contains β-elemene accompanied by its structural isomers, γ- and δ-elemene, and small amount of other terpenoids, among them β-caryophyllene as a main impurity. β-, γ-, and δ-Elemene account for 85% in elemene injection \[[@B28-molecules-23-00256]\]. In our study, we obtained β-elemene of better purity, up to 96%, depending on the amount of sample loaded into column, and we proved the usefulness of *N. damascena* essential oil as a source for β-elemene isolation. To our knowledge, this is the first application of HPCCC technique for β-elemene isolation from the plant material and the first one from *N. damascena* essential oil. 3.2. β-Elemene and N. damascena Essential Oil Exert Action against M. tuberculosis H37Ra {#sec3dot2-molecules-23-00256} ---------------------------------------------------------------------------------------- The literature data show the activity of many essential oils containing β-elemene (including EO from *C. wenyujin*) against different pathogenic bacteria \[[@B29-molecules-23-00256],[@B30-molecules-23-00256],[@B31-molecules-23-00256]\]; however, the activity of β-elemene itself was studied only by Zhu et al. \[[@B31-molecules-23-00256]\] and our team. In the previous report, β-elemene was active against Gram-positive bacteria *Propionibacterium acnes*, and *Staphylococcus aureus* (strain different than in our study), as well as against fungus *Malassezia furfur* \[[@B31-molecules-23-00256]\]. The results of this study show no activity of β-elemene against tested bacteria with exception of *Mycobacterium tuberculosis* H37Ra. What is more, only our previous work \[[@B32-molecules-23-00256]\], together with this study, describe the influence of β-elemene on *Mycobacterium tuberculosis* and *Mycobacterium smegmatis*. The investigated earlier, isolated strain of *M. tuberculosis* was more sensitive to β-elemene action than that used in the current study H37Ra reference strain; however, the activity of this compound against *M. tuberculosis* is still noticeable, making β-elemene worthy f further testing. Investigated in this study, *N. damascena* EO showed similar activity to β-elemene activity against *M. tuberculosis* and no activity against other microorganisms. The other studies investigating extracts from *N. damascena* seeds and from callus cultures also showed no activity against Gram-positive and Gram-negative bacterial strains and yeast \[[@B33-molecules-23-00256],[@B34-molecules-23-00256]\]. However, Fico et al. \[[@B35-molecules-23-00256]\] described that EO of *N. damascena* inhibited the growth of several bacteria strains in an agar disc diffusion assay. The differences in the observed activity may be explained by different methodology used to evaluate examined samples. The performed study showed that isolated β-elemene is twofold more potent against *M. tuberculosis* than whole *N. damascena* essential oil, in which β-elemene constitutes almost 50%. It may suggest that the main ingredient is responsible for the activity of EO. Our findings are in agreement with those published previously by Zhu et al., 2013, who also observed that β-elemene is twofold more active against *Propionibacterium acnes* and *Staphylococcus aureus* than *Curcuma wenyujin* EO \[[@B31-molecules-23-00256]\]. This confirms that studied compound is the main one responsible for EO activity. However, the literature data also presents opposite results, suggesting the synergistic action of essential oils constituents, which is expressed in lower MIC values obtained for whole essential oil compared to its main ingredients \[[@B36-molecules-23-00256],[@B37-molecules-23-00256]\]. 4. Materials and Methods {#sec4-molecules-23-00256} ======================== 4.1. Plant Material {#sec4dot1-molecules-23-00256} ------------------- Commercially available seeds of *Nigella damascena* L. (100 g) obtained from Vilmorin Garden (Komorniki, Poland) were subjected to 3 h hydrodistillation in Deryng apparatus to obtain essential oil \[[@B38-molecules-23-00256]\]. 4.2. GC-MS Analysis {#sec4dot2-molecules-23-00256} ------------------- GC--MS was performed with a Shimadzu GC-2010 Plus coupled to a Shimadzu QP2010 Ultra mass spectrometer (Japan). A fused-silica capillary column ZB-5 MS (30 m, 0.25 mm i.d.) with a film thickness of 0.25 mm (Phenomenex, Torrance, CA, USA) was used. GC-MS conditions applied for analysis of the essential oil and obtained fractions followed Sieniawska et al. \[[@B12-molecules-23-00256]\]. The retention indices were determined in relation to a homologous series of *n*-alkanes (C~8~--C~24~) under the same operating conditions. Constituents of studied essential oil were identified by comparison of their mass spectra and retention indices with computer-supported spectral libraries (Mass Finder 2.1 and NIST database) and with authentic standard of β-elemene. 4.3. Isolation and Identification of β-Elemene {#sec4dot3-molecules-23-00256} ---------------------------------------------- Isolation was carried out with Spectrum High-performance counter-current chromatographic (HPCCC) equipment purchased from Dynamic Extraction Co., Ltd. (Slough, Berkshire, UK). The two-phase solvent system suitable for further separation was selected according to the partition coefficient (K) of the target compound ([Table 1](#molecules-23-00256-t001){ref-type="table"}). Procedure of two-phase solvent system and sample solution preparation was described in detail previously \[[@B12-molecules-23-00256]\]. The separation was performed in the solvent system composed of petroleum ether, acetonitrile, and acetone in a ratio of 2:1.5:0.5 (*v/v*), in the reversed phase mode at 26 °C. The rotation of bobbins applied was 1600 rpm, while the flow rate was 1 and 6 mL/min in the analytical and preparative conditions, respectively. The optimized analytical conditions were scaled up for preparative separation (from 22 mL coil to 137 mL, respectively). The fractions monitored at 210 nm were collected per minute and analyzed by GC-MS. The mass of sample (100, 165, and 200 mg of essential oil) was optimized to obtain the best preparative separation. Chromatographic (GC profiles) and spectroscopic (mass and NMR spectra) data were used to confirm identity and purity of isolated compound. Bruker Ultrashield 300 spectrometer (Bruker Corporation, Billerica, MA, USA) was used to perform NMR analysis. 4.4. Antimicrobial Assay {#sec4dot4-molecules-23-00256} ------------------------ ### 4.4.1. Tested Organism and Storing Conditions {#sec4dot4dot1-molecules-23-00256} The following reference bacterial and fungal strains were used in the studies: *Staphylococcus aureus* ATCC 25923, *Staphylococcus epidermidis* ATCC 12228, *Escherichia coli* ATCC 25922, *Pseudomonas aeruginosa* ATCC 27853, *Mycobacterium smegmatis* ATCC 607, *Mycobacterium smegmatis* ATCC 19420, *Mycobacterium tuberculosis* H37Ra ATCC 25177, *Candida albicans* ATCC 10231, and *Candida parapsilosis* ATCC 22019. All microbial strains stocks except mycobacteria were stored in −70 °C in Viabank vials (Medical Wire & Equipment Co. Ltd., Corsham, UK). Mycobacteria were stored in −70 °C in Middlebrook 7H9 broth (Difco Laboratories Inc., Detroit, MI, USA) with 10% albumin dextrose complex (ADC; Becton, Dickinson and Company, Franklin Lakes, NJ, USA) supplemented with 15% glycerol. All microbiological procedures were made according to Clinical and Laboratory Standards Institute \[[@B39-molecules-23-00256],[@B40-molecules-23-00256]\]. ### 4.4.2. Inoculum Preparation {#sec4dot4dot2-molecules-23-00256} ***Mycobacterium tuberculosis*** *M. tuberculosis* H37Ra was grown on Löwenstein-Jensen slopes (BioMaxima, Lublin, Poland) for up to two weeks. Bacterial mass was transferred to 5 mL of the fresh Middlebrook 7H9 broth supplemented with 10% ADC and vortex with 1 mm glass beads for 3 min. After 30 min of incubation (lager clumps sedimentation) in room temperature, upper 2 mL was transferred to a sterile tube and left for next 15 min. One milliliter of supernatant was placed in a sterile tube and was adjusted to 0.5 McFarland standard with ADC-supplemented Middlebrook 7H9 broth. The density of bacterial suspension used for the plate inoculation was 1 × 10^6^ CFU/mL. ***Mycobacterium smegmatis*** The inocula of *M.* *smegmatis* were prepared in the same way as *M.* *tuberculosis*. Instead of the Middlebrook 7H9 with ADS, Mueller-Hinton Broth (MHB) (BioMaxima, Lublin, Poland) was used. **Other bacterial strains** The inocula of all other bacterial strains used in this study were prepared with the direct colony suspension method in MHB. Colonies from 24 h Mueller-Hinton Agar (MHA)(BioMaxima, Lublin, Poland) plate were scraped, suspended in MHB, and adjusted to the turbidity of 0.5 McFarland standard. The density of bacterial suspension used for the microtiter plate inoculation was 1 × 10^6^ CFU/mL. **Yeasts** Colonies of approx. 1 mm in diameter were picked from 24 h Sabouraud dextrose agar plate (BioMaxima, Lublin, Poland), suspended in 5 mL of sterile 0.85% saline, and vortexed for 15 s. The cell density was adjusted to 0.5 McFarland standard with sufficient volume of sterile saline. Resulted suspension was diluted with RPMI 1640 broth medium to obtain twofold test inoculum 5 × 10^3^ CFU/mL used to inoculate microtiter plate. ### 4.4.3. Dilutions of Tested Substances {#sec4dot4dot3-molecules-23-00256} *N. damascena* essential oil and β-elemen were tested in the concentration range 1000--32 µg/mL. Serial twofold dilutions were prepared in dimethyl sulphoxide (DMSO; Sigma-Aldrich, St. Louis, , MO, USA) and diluted with medium suitable for the given organism. The final DMSO concentration in the well did not exceed 1% (*v/v*) and had no influence on growth of the tested strains. ### 4.4.4. References Antimicrobials {#sec4dot4dot4-molecules-23-00256} Ciprofloxacin, gentamicin, cefoxitin, amphotericin B, and ethambutol (Sigma-Aldrich, St. Louis, MO, USA) were used as reference standards. Stock solutions were prepared according to the manufacturer's instructions. Final twofold dilutions from 16 to 0.004 μg/mL were prepared in broth suitable for given microbial. ### 4.4.5. Plates Preparation {#sec4dot4dot5-molecules-23-00256} The round bottom microwell plates were prepared as follows: in each well, 50 μL of inoculum and 50 μL of tested substances were combined. The final density of inoculum in each well was approx. 5 × 10^5^ CFU/mL for all bacterial strains and approx. 2.5 × 10^3^ CFU/mL for yeast strains. The sterility, growth, and 1% DMSO controls were carried out for each tested strain. The plates were closed with sealing foil to prevent liquid evaporation and incubated at 35 °C except *M. smegmatis* incubated at 29 °C. ### 4.4.6. Determination of Minimal Inhibitory Concentration (MIC) {#sec4dot4dot6-molecules-23-00256} **The resazurin microtiter assay for *Mycobacterium tuberculosis*** After 8 days of incubation, 10 µL of resazurin (alamarBlue; Invitrogen, Carlsbad, CA, USA) solution was added to each well, incubated 48 h at 35 °C, and assessed for color development. A change from blue to pink indicates reduction of resazurin to resorfurin and proofs bacterial viability. The MIC was defined as the lowest drug concentration that prevented this color change \[[@B41-molecules-23-00256]\]. The assay was performed in triplicate. **The MIC for other microbials** The MIC was read as follows: *E. coli*, *P. aeruginosa*, *S. aureus*, and *S. epidermidis* after 18 h of incubation; yeasts after 48 h of incubation and *M. smegmatis* after 72 h of incubation. The MIC was defined as the lowest concentration of antimicrobial agent that completely inhibits growth of the organism in the microtitration plate's wells detected by the unaided eye. The assay was performed in triplicate. 5. Conclusions {#sec5-molecules-23-00256} ============== In this work, *N. damascena* essential oil was shown to be a new, abundant source of β-elemene. The high purity β-elemene was successfully isolated from this EO for the first time. High-performance counter-current chromatography was proved to be an efficient technique for this sesquiterpene separation, while antimicrobial testing revealed moderate activity of β-elemene against *Mycobacterium tuberculosis* H37Ra. This work was financially supported by grant No. 2013/11/D/NZ7/01613 from the Polish National Science Centre as well as Ministry of Science and Higher Education in Poland within the DS2 project of the Medical University of Lublin. This work was developed using the equipment purchased within the agreement No. POPW.01.03.00-06-010/09-00 Operational Program Development of Eastern Poland 2007--2013, Priority Axis I, Modern Economy, Operations 1.3. Innovations Promotion. **Sample Availability:** Samples of *Nigella damascena* seeds are available from the authors. Supplementary materials are available online. Figure S1: HPCCC chromatogram of N. damascena essential oil, Figure S2 A: GC chromatogram of N. damascena essential oil, B: GC chromatogram of β-elemene standard, C: GC chromatogram of isolated β-elemene, D: MS spectrum of β-elemene; Figure S3: ^1^H NMR spectrum of β-elemene; Figure S4: ^13^C NMR spectrum of β-elemene. ###### Click here for additional data file. E.S., R.S. and K.S-W. conceived and designed the experiments; E.S., M.S.-O., J.G., and R.S. performed the experiments; E.S. and K.S-W. analyzed the data; E.S. wrote the paper, G.G. and K.S.-W. consulted the work. The authors declare no conflict of interest. {#molecules-23-00256-f001} molecules-23-00256-t001_Table 1 ###### The chemical composition of *N. damascena* L. essential oil. No Compound tr RI Area (%) -------- -------------------------------------------------- -------- ------ ---------- **1** 2,4-Dimethyl-heptane 4.057 850 0.16 **2** Hexanoic acid, izopropyl ester 9.838 1020 0.13 **3** 2-Methoxyl-6-antranilate 13.270 1227 0.10 **4** Sativen 15.500 1352 0.07 **5** Longifolene 15.910 1376 0.09 **6** Iso-β-elemene 16.120 1383 0.13 **7** β-Bourbonene 16.187 1388 2.38 **8** β-Elemene 16.270 1398 47.37 **9** Isocaryophyllene 16.570 1415 0.08 **10** 7-Isoprenyl-1-methyl-4-methylenedecahydroazulene 16.707 1428 0.91 **11** β-Caryophyllene 16.803 1430 0.06 **12** Selina 4,11-diene 17.640 1482 2.68 **13** *γ*-Gurjunene 17.703 1486 0.69 **14** Valencene 17.787 1488 0.69 **15** β-Selinene 17.940 1495 10.10 **16** *α*-Selinene 18.043 1506 13.52 **17** *α*-Bulnesne 18.143 1514 0.43 **18** 8-Isopropenyl-1,5 dimethyl 1,5-cyclodecadiene 18.223 1519 1.18 **19** *δ*-Cadinene 18.320 1524 0.16 **20** 7-Epi-*α*-selinene 18.403 1532 3.26 **21** Metyl 2-amino-3-methoxyl benzoate 18.780 1553 0.06 **22** Elemol 18.807 1557 0.93 **23** Damascenine 19.073 1575 11.97 **24** Elema-1,3-dien-6-α-ol 19.837 1630 0.24 **25** Selina-6-en-4-ol 20.523 1673 0.67 **26** Longifolenaldehyde 20.670 1680 0.07 **27** Oleylalkohol, metyl ether 22.300 1802 0.22 **28** Unknown 22.353 1807 1.07 **29** 1-Octadecanol, metyl ether 22.623 1828 0.32 Total 99.2 tr---retention time, RI---measured retention indices. molecules-23-00256-t002_Table 2 ###### The partition coefficients values for β-elemene in different solvent systems. No Solvent Systems (*v/v*) K ------- -------------------------------------------------- ------ **1** Heptane/Acetonitrile/Ethyl acetate (2:1:1) \* 1.54 **2** Petroleum ether/Acetonitrile/Acetone (7:6:1) 4.46 **3** Petroleum ether/Acetonitrile/Acetone (2:1:1) \* 1.74 **4** Petroleum ether/Acetonitrile/Acetone (2:1.5:0.5) 2.58 K---partition coefficient; \* the solvent system becomes a single phase above 23°C. molecules-23-00256-t003_Table 3 ###### The influence of sample mass on the purity and yield of β-elemene. Mass of Sample Fraction (min) Purity (%) Yield (mg) ---------------- ---------------- ------------ ------------ 200 mg 49--56 87 90 57--61 92 22 165 mg 50--58 86 78 59--64 92 25 100 mg 55--62 89 47 63--68 96 22 69--70 92 11 molecules-23-00256-t004_Table 4 ###### The antimicrobial activity of tested samples. The activity was expressed as minimal inhibitory concentration (μg/mL). CIP GEN FOX ETB AMP ND E ----------------------------------------------- ------- ------- ----- ----- ------ -------- -------- *Staphylococcus aureus* ATCC 25923 0.25 0.25 \- \- \- \>1000 \>1000 *Staphylococcus epidermidis* ATCC 12228 0.06 0.125 \- \- \- \>1000 \>1000 *Escherichia coli* ATCC 25922 0.008 0.5 \- \- \- \>1000 \>1000 *Pseudomonas aeruginosa* ATCC 27853 0.25 2 \- \- \- \>1000 \>1000 *Candida albicans* ATCC 10231 \- \- \- \- 0.03 \>1000 \- *Candida parapsilosis* ATCC 22019 \- \- \- \- 0.25 \>1000 \- *Mycobacterium smegmatis* ATCC 607 0.25 \- 16 \- \- \>1000 \>1000 *Mycobacterium smegmatis* ATCC 19420 0.25 \- 16 \- \- \>1000 \>1000 *Mycobacterium tuberculosis* H37Ra ATCC 25177 0.125 \- \- 2 \- 256 128 CIP---Ciprofloxacin; GEN---Gentamicin; FOX---Cefoxitin; AMP---AmphotericinB; ETB---Ethambutol; ND---*Nigella damascena* EO; E---β-elemene. Drugs were used as positive controls; 1% DMSO was used as a negative control.

Erratum {#Sec1} ======= After the publication of this article \[[@CR1]\] it has come to our attention that the ethical approval receipt number of this study was not included in the article. As such, please note that this study \[[@CR1]\] was approved by the Ethics Committee of Osaka City University (receipt no. 27--8-1). The online version of the original article can be found under doi:10.1186/s12199-017-0658-z

Introduction {#sec1} ============ About 10% of men and 18% of women over 60 y of age have osteoarthritis (OA) ([@bib24]). It is painful for the patient, limits mobility and reduces quality of life. As there is no regenerative therapy for OA, the last treatment option is usually a total joint replacement. Research on development of disease-modifying OA drugs has been active in recent decades ([@bib18]). Currently available drug therapies were developed under the limitation of poor localization, that is, systemic delivery or injections into the joint. However, OA lesions in articular cartilage (AC) can be very localized. Therefore, local administration of the drug could potentially enhance the therapeutic effect within the degenerated tissue while avoiding adverse effects elsewhere in the body ([@bib23]). For instance, although transforming growth factor TGF-β1 speeds up AC matrix regeneration by stimulating chondrocytes, it simultaneously inflames the synovial capsule and promotes osteophyte formation ([@bib21]). Non-destructive localized delivery of drugs into AC could, therefore, lead to new treatment strategies for OA therapy. Small OA drug molecules such as glucosamine (∼179 Da) and diacerein (∼368 Da) can move relatively freely in and out of AC, resulting in short residence times. On the other hand, penetration of large molecules (≥45 kDa) into AC is limited ([@bib11]). A method to force either large molecules (*e.g.*, growth factors) or drug carriers containing small drug molecules into AC could contribute to localization and prolonged drug residence. This would potentially confine drug exposure and permit more personalized treatment than afforded by current drug techniques of delivery into AC. Clinical techniques to deliver drugs locally into AC are still to be developed, but a few studies have already attempted to force agents into AC by means of an electric field ([@bib3]), kilohertz ultrasound ([@bib14]) and acoustic shock waves ([@bib15]). High-intensity ultrasound (HIU) can transport material through such phenomena as acoustic radiation force, acoustic streaming and ultrasound--bubble interactions ([@bib1; @bib7]). All these effects can potentially be harnessed to move agents locally inside porous materials such as AC. It is known that ultrasound can deliver agents into skin ([@bib19]) and release encapsulated agents in tumor tissue ([@bib10]). We have previously reported that low-frequency (20 kHz, 78 W/cm^2^) ultrasound can deliver soot particles (in the nanometer to micrometer range), models for drug carriers, locally into AC ([@bib14]). However, we observed microcratering of the AC surface possibly caused by inertial cavitation ([@bib6]). The risk of encountering this adverse phenomenon can be avoided by increasing the sonication frequency into the megahertz range. Therefore, the aim of this study was to illustrate that intense megahertz ultrasound enhances the penetration of a kDa-sized agent into AC without damaging the tissue. Methods {#sec2} ======= Samples {#sec2.1} ------- Bovine joints were obtained from a local meat refinery (Lihakonttori Oy, Helsinki, Finland). Cylindrical osteochondral plugs (samples 1--5, n = 5, diameter = 28 mm) were prepared from normal-looking femoral condyles, one sample per animal ([Fig. 1](#fig1){ref-type="fig"}). For damage assessment tests, we prepared paired osteochondral strips (samples 6--9\[a, b\], n = 8, width = 7 mm) from four joints with visually normal femoral condyles. Ultrasound experiments and damage assessment {#sec2.2} -------------------------------------------- A custom-made 1.138-MHz focused ultrasound system ([Fig. 2](#fig2){ref-type="fig"}) (peak positive and peak negative pressure, along the acoustic axes, ±3 mm from the sample surface = 2.70 ± 0.06 and 1.18 ± 0.01 MPa, respectively; beam width = 5.7 mm at −6 dB of maximum peak pressure; *I*~sptp~ = 488 W/cm^2^, *I*~sppa~ = 102 W/cm^2^, *I*~spta~ = 5 W/cm^2^; pulse repetition frequency = 285 Hz; cycles per pulse = 200; duty cycle = 5.0%; mechanical index \[MI\] = 1.1; immersion fluid/subchondral bone temperature \<33°C) was used to deliver a drug surrogate (X-ray contrast agent: 1% w/v phosphotungstic acid \[PTA\], a negatively charged molecule, molecular weight = 2.88 kDa) into samples 1--5 as follows: First, the focus of the ultrasound beam was positioned at the AC surface; second, the sample was sonicated for 2.5 h. The contrast agent was dissolved in 70% ethanol for sample 1 because PTA in ethanol is used to label collagen in embryos ([@bib12]). We chose phosphate-buffered saline (PBS) as base medium for samples 2--5 to provide a more physiologic-like environment. For damage assessment (histopathologic assessment), one sample (a) each from sample pairs 6--9 was sonicated in PBS containing no PTA using the protocol described earlier ([Fig. 1](#fig1){ref-type="fig"}). The second sample (b) from sample pairs 6--9 was immersed, but not exposed to ultrasound and acted as a paired control. Samples were then subjected to histologic staining (Masson\'s trichrome and Safranin-O) to evaluate collagen and proteoglycan distributions, respectively. To evaluate potential ultrasound-induced damage to the AC, the excisions were optically imaged at 10× using a light microscope (Aristoplan, Ernst Leitz Wetzlar, Wetzlar, Germany) and camera (MicroPublisher 5.0 RTV, Qimaging, Surrey, BC, Canada), and at 1× using a microscope slide scanner (Pathscan Enabler IV, Meyer Instruments, Houston, TX, USA). Ultrasound beam characteristics (peak positive/negative pressure amplitude, MI, beam width and intensity) were determined using a calibrated needle hydrophone (element diameter = 0.2 mm, tip diameter = 0.67 mm, Model HPM02/1, Precision Acoustics, Dorchester, UK). Intensity values (SPTP, SPPA, SPTA) were determined in ion-exchanged water at a distance from the transducer that corresponded to the position of the AC surface. Detection of delivery {#sec2.3} --------------------- Samples 1--5 were imaged with X-ray microtomography (XMT) to determine PTA distribution within the tissue (Nanotom 180 NF, Phoenix X-ray Systems/GE, Fairfield, CT, USA; settings for sample 1: 75 kV, 190 μA, 1,080 projections, voxel side length = 38.0 μm; settings for samples 2--5: 75--80 kV, 75--110 μA, 1,200 projections, voxel side length = 16.3 to 16.7 μm). To avoid sample drying during XMT, the samples were kept in sealed containers containing cotton balls moistened with the base solution (70% ethanol for sample 1 or PBS for samples 2--5). The PTA distributions in AC were determined on the basis of X-ray attenuation in the XMT images. Raw data from samples presented in [@bib15] (samples 1 and 2) were reanalyzed and pooled into this study. The penetration depth of PTA and thickness of AC were determined from reconstructed XMT image stacks ([Fig. 3](#fig3){ref-type="fig"}). AC without PTA (*i.e*., volume 1) and AC with PTA (*i.e.*, volume 2) were segmented from the XMT image stack based on image contrast (Avizo Fire, Versions 7.1.1--8.0.1, FEI Visualization Sciences Group, Burlington, MA, USA). The local thickness of each volume was calculated with the LocalThickness plugin ([@bib8]) to the ImageJ software (Version 1.4.7, NIH, Bethesda, MD, USA), and projected onto a plane approximately tangential to the AC surface with the VGStudioMAX software (Version 1.3, Volume Graphics, Heidelberg, Germany). We named this the spatial maximum penetration map (SMPM). Within the SMPM of samples 2--5, the delivery depth in the treatment area was defined as the mean ± standard deviation (SD) of the pixel values that were within 1-mm radius from the sample center. The delivery depth into adjacent tissue (control) was defined as the mean ± SD of pixel values that were inside a band 9.5--10.5 mm from the sample center. In addition, the global maximum of delivery depth within the SMPM was determined. Results {#sec3} ======= After sonication, we observed increased X-ray attenuation (*i.e.*, high PTA concentration) inside a circular penetration shape at the location of the ultrasound beam center in all samples ([Fig. 4](#fig4){ref-type="fig"}). For sample 1 (70% ethanol as base medium), the maximum delivery depth was 488 ± 15 μm (mean ± SD), with no delivery in adjacent tissue. This maximum delivery depth corresponded to 39% of the AC thickness and was achieved at an average penetration speed of 195 μm/h. The delivery depths for samples 2--5 (immersed in PTA + PBS solution while sonicated) are summarized in [Table 1](#tbl1){ref-type="table"} and [Figures 4 and 5](#fig4 fig5){ref-type="fig"}. For these samples, the average penetration depth of PTA at the treatment area was 2.6 times the penetration depth in the control tissue ([Fig. 5](#fig5){ref-type="fig"}). For samples with strong PTA penetration at the sample center, that is, samples 2, 3 and 5, the maximum penetration depths were 775, 883 and 754 μm, respectively ([Fig. 4](#fig4){ref-type="fig"}), corresponding to a relative penetration of 53 ± 4% and an average penetration speed of 322 μm/h. Sample 4 appeared to exhibit strong passive PTA diffusion outside the ultrasound beam; nevertheless, a circular PTA penetration pattern is seen at the location of the ultrasound beam, that is, sample center ([Fig. 4](#fig4){ref-type="fig"}f). Subchondral bone temperature remained below 33°C (n = 3). Histologic evaluation of ultrasound-treated AC samples (6a--9a) and control samples (6b--9b) revealed no difference between the groups with respect to superficial tissue fibrillation or collagen (*blue contrast* in Masson\'s trichrome-stained sections) or proteoglycan (*red contrast* in Safranin-O-stained sections) distribution ([Fig. 6](#fig6){ref-type="fig"}). Discussion {#sec4} ========== The results suggest that intense megahertz ultrasound delivered the drug surrogate into AC. Because the applied ultrasound energy (1 MHz, 2.7/1.2 MPa peak positive/negative pressure amplitude at focus, *I*~sptp~ = 488 W/cm^2^, *I*~sppa~ = 102 W/cm^2^, *I*~spta~ = 5 W/cm^2^) attenuates in soft tissue ([@bib9; @bib16]) and body fluids ([@bib13]), it may induce acoustic radiation forces in solids or at solid--fluid interfaces ([@bib22]) and fluid streaming ([@bib17]), potentially contributing to mass transfer into and inside articular cartilage. The radiation pressure generated in water at the location of AC surface is on average ∼680 Pa during one ultrasound burst. The resulting radiation force and PRF correspond to tapping the tissue 2.6 million times during the treatment. Specifically, stable cavitation (threshold for unstable inertial cavitation = 3.9 MPa peak negative pressure at 1 MHz \[[@bib2]\]) is one possible explanation for the delivery; that is, ultrasound may excite micro- or nanobubbles to generate a streaming fluid around the bubbles ([@bib1]). Such streaming could enhance the diffusion within the ultrasound beam. As we detected a temperature rise in the subchondral bone from room temperature to 33°C, it is possible that the temperature increase in AC enhances diffusion of delivered agents into and inside AC ([@bib5]). The PTA delivery depth was relatively similar in all samples (excluding sample 4) and limited to a relatively confined round region ([Fig. 4](#fig4){ref-type="fig"}f). The difference in delivery can be due to biological variation, that is, permeability, which can vary with the biochemical composition of AC and from one joint to another. Permeability affects the speed of passive diffusion. The base solution was 70% ethanol for sample 1 and PBS for samples 2--5, which may impair the comparability of delivery in sample 1 to delivery in samples 2--5. PTA is a negatively charged molecule that is attracted by collagen. Therefore, we expected to see PTA penetration into the control tissue. Despite this, enhanced penetration at the sample center was evident compared with adjacent tissue. Qualitatively, no histologic difference was observed in AC surface structure or in the content of collagen or proteoglycan between sonicated and non-sonicated samples ([Fig. 6](#fig6){ref-type="fig"}). Because of the low MI (1.1), we presumed that the exposure should be tolerable, as the MI does not exceed the U.S. Food and Drug Administration-defined maximum of 1.9 for clinical ultrasound imaging ([@bib6]). Previously we used 20-kHz HIU ([@bib14]) to deliver particles into osteochondral tissue; however, this induced superficial microcratering in the AC, because the pressure threshold for inertial cavitation is much lower at 20 kHz (*e.g.*, 68-kPa peak negative pressure \[PNP\] at 23 kHz \[[@bib4]\]) than at 1.138 MHz (3.9-MPa PNP at 1 MHz \[[@bib2]\]). Absence of evident damage to the AC, therefore, suggests that megahertz ultrasound may be a preferable approach for drug delivery compared with kilohertz frequency ultrasound. In humans, delivering PTA halfway through the cartilage thickness is potentially important, because early OA typically manifests as histopathologic changes (superficial fibrillation and clefts, loss in proteoglycans) in the superficial AC ([@bib20]). The average delivery speed of the agent in this study, ∼5 μm/min, suggests that agent delivery to the very superficial articular cartilage can be achieved with the proposed technique within a clinically acceptable time frame (on average an ∼50 μm penetration depth in 10 min for a molecule or drug carrier equivalent to PTA). With respect to delivery of agent into deeper tissue, the delivery speed is currently too low for clinical relevance. In addition, bovine cartilage properties *in vitro* may differ significantly from human cartilage properties *in vivo*. Although the control tissue (sample edge) is less than one-third of the −6 dB beam width of the ultrasound, control tissue may have been subjected to ultrasound exposure and acoustic streaming affecting the control. Despite these limitations, the results are encouraging, with successful delivery without apparent damage. Further research is needed to identify the mechanisms of delivery, to enhance delivery speed, to confirm cell viability and to test the biological response of the proposed approach using OA drugs. The proposed experimental setup was designed to study the capability of megahertz ultrasound to transport agents into articular cartilage. The technical realization of the potential clinical application could, however, differ significantly from that used in this study. Delivery with megahertz ultrasound could be applied not only extracorporeally, but also intra-articularly. An intra-articular approach would allow local administration of the drug or drug carrier enhanced by ultrasonic delivery. Examples of intra-articular approaches could be a catheter-based high-intensity focused transducer or a fiber optic photo-acoustic ultrasound system, which because of their small size could fit into the joint cavity. Conclusions {#sec5} =========== We have illustrated the ability of intense megahertz ultrasound to transport agents into articular cartilage without causing microscopically discernible histologic damage. Ultrasound, therefore, has the potential to deliver agents such as drugs and drug carriers into articular cartilage in a localized manner. At best, this approach could permit new treatment strategies for OA drug therapy and a paradigm shift in drug development of locally administered agents. We thank the Academy of Finland (Projects 253579, 268378 and 273571) and the European Research Council under the European Union\'s Seventh Framework Programme (FP/2007-2013, ERC Grant Agreement 336267) for financial support. We are grateful to Lihakonttori Oy (Helsinki, Finland) for providing bovine knees. We warmly thank Dr. S. Adam Hacking, Ph.D. and Prof. Kenneth P. H. Pritzker, M.D., FRCPC for their constructive criticism. We are grateful to Mr. Alexander Meaney for assistance with the XMT imaging of sample 1. We thank Mr. Eetu Lampsijärvi, Mr. Jari Rinta-aho and Mr. Nestori Westerlund for assistance with experiments and Mr. Christoffer Fridlund for assistance in sample preparation. We are grateful to Ms. Ida Holopainen and Mr. Anton Nolvi, B.Sc. for their help in technical drawing. {ref-type="fig"}). For damage assessment, osteochondral tissue strips (width = 7 mm) were prepared from femoral condyle (b), one sample pair per joint. One sample from each joint was sonicated with megahertz ultrasound while immersed in PBS (samples 6a--9a) and the adjacent samples (samples 6b--9b) not sonicated acted as controls. Damage assessment samples were subjected to histology that is, Masson or Safranin-O staining and light microscopy.](gr1){#fig1} ![Experimental setup. The sample was immersed in a contrast agent (phosphotungstic acid \[PTA\]), while sonicated with megahertz ultrasound. A thin nitrile-containing membrane prevented PTA penetration into the tissue from the side. PBS = phosphate-buffered saline.](gr2){#fig2} {#fig3} ![Delivery outcome. Surrogate (phosphotungstic acid \[PTA\]) delivery was observed in X-ray microtomography (XMT) images of samples 1--3 and 5 (a--e, g) at the sample center (location of maximum delivery depth indicated with *black arrow*) compared with adjacent tissue (*white arrow*) after the sample was sonicated with intense megahertz ultrasound in surrogate immersion. In sample 4 (f), diffusion (potentially passive) is strong outside the ultrasound beam (*purple arrow*), but deeper delivery is seen at the sample center (*blue arrow*) compared with adjacent tissue (*green arrow*). The result suggests that ultrasound can deliver agents into articular cartilage and that the delivery is confined to the location of sonication.](gr4){#fig4} {ref-type="fig"}). The samples were immersed in phosphotungstic acid (PTA) + phosphate-buffered saline (PBS) solution, while sonicated with intense megahertz ultrasound.](gr5){#fig5} {#fig6} ###### Penetration depth of PTA at the treatment location, the location of the global maximum and the control location for all samples immersed in PBS + PTA solution[∗](#tbl1fnlowast){ref-type="table-fn"} Sample Treatment penetration depth (μm) Control penetration depth (μm) Penetration depth difference: Treatment − Control (μm) Maximum penetration depth (μm) -------- ---------------------------------- -------------------------------- -------------------------------------------------------- -------------------------------------------------- 2 742 ± 4 0 ± 0 742 775 3 869 ± 11 197 ± 211 672 883 4 654 ± 8 653 ± 65 1 734[†](#tbl1fndagger){ref-type="table-fn"} (686) 5 684 ± 18 264 ± 186 420 754 PBS = phosphate-buffered saline; PTA = phosphotungstic acid. The treatment location values were calculated as the mean ± standard deviation of values within 1-mm radius from sample center. The control values were calculated as the mean ± standard deviation within a 1-mm band at a radial distance of 10 mm (from 9.5 to 10.5 mm) from the sample center. The numbers of pixels used to calculate delivery depth statistics were 11,213--11,873 and 183,662--202,668 for treatment and control, respectively. Maximum located outside the sample center. The maximum value at sample center is provided in parentheses.

1. Introduction {#sec1-dentistry-06-00068} =============== Homelessness is a multi-dimensional experience characterised not merely by the lack of a roof over one's head but also by physiological and emotional deprivation \[[@B1-dentistry-06-00068]\]. Being homeless can be "impoverishing and isolating" and is often associated with physical and mental ill-health \[[@B2-dentistry-06-00068]\]. Indeed, as Scottish Government statistics show, 42% of those who were assessed as homeless during 2015--2016 were found to have one or more additional support needs, including mental ill-health, drug or alcohol dependency as well as medical condition(s) \[[@B3-dentistry-06-00068]\]. This suggests that this group of people had experienced multiple exclusion homelessness, which can be defined as the experience of homelessness in addition to one or more of the following: institutional care (e.g., prison, hospital or being a looked after child); street activities such as begging; or substance misuse \[[@B4-dentistry-06-00068]\]. In addition to the physical health of people experiencing homelessness, there is evidence that this population have poor oral health. Previous studies have found that homeless populations have a high prevalence of bleeding gums, calculus and periodontal inflammation \[[@B5-dentistry-06-00068],[@B6-dentistry-06-00068],[@B7-dentistry-06-00068]\]. Research has also found that people experiencing homelessness often have a poor record of dental attendance and unmet treatment needs \[[@B6-dentistry-06-00068]\]. In addition, this population has been found to suffer from dental anxiety and poor oral health related quality of life \[[@B5-dentistry-06-00068]\]. People experiencing homelessness face many health inequalities---for example, they have a higher risk of death than the general population, as well as higher rates of suicide and depression \[[@B8-dentistry-06-00068]\]. Poor oral health could also be seen as a contributing factor to the health inequalities faced by the homeless population: the Groundswell Healthy Mouths report found that "participants were regularly facing issues with their oral health that were making it difficult for them to live fulfilled lives" \[[@B9-dentistry-06-00068]\] (p. 2). In addition, those that had oral health problems reported being "handicapped" with 21% of participants reporting they felt "completely unable to function" because of their oral health, compared to 1% of the general population \[[@B9-dentistry-06-00068]\] (p. 2). Many reported turning to alcohol and drugs to help them cope with their dental pain, which, in turn, exacerbates other health needs \[[@B8-dentistry-06-00068]\]. The Scottish Government recognised the health needs of the Scottish homeless population in 2005, with the Health and Homelessness Standards, designed to improve the health of people experiencing homelessness and to tackle health inequalities \[[@B10-dentistry-06-00068]\]. This was followed in the same year by the Dental Action Plan, which recognised people experiencing homelessness as a priority group that required tailored oral healthcare \[[@B11-dentistry-06-00068]\]. Both documents called for the National Health Service (NHS) to take steps to address the general and oral health of people experiencing homelessness in Scotland. In Scotland, the NHS is comprised of 14 Boards, each linked to a geographical area of the country, and provides primary and secondary healthcare to the whole population, regardless of their ability to pay, and is free at the point of delivery \[[@B12-dentistry-06-00068]\]. This need for tailored health messages was strengthened by evidence from a systematic review and meta-analysis that concluded that messages that were tailored to the health needs of patients were effective in promoting behaviour change \[[@B13-dentistry-06-00068]\]. In 2012, the Scottish Government renewed its commitment to the oral health of people experiencing homelessness in the Priority Groups Strategy, which emphasised the need for accessible dental services and preventive oral health programmes \[[@B14-dentistry-06-00068]\]. In response to the Scottish Government's policies, an oral health intervention, called Smile4life, was developed to address the oral health needs of the homeless population in Scotland. It was developed from the evidence-base gleaned from a national survey of 853 participants that was conducted during 2008--2009. This sample population was shown to have had poorer oral and psychosocial health when compared to the general population of Scotland \[[@B15-dentistry-06-00068]\]. Qualitative interviews also took place with 34 people experiencing homelessness, highlighting that maintaining good oral health was not always practically possible when homeless. Furthermore, oral health was often not a priority, as evidenced by poor dental attendance \[[@B15-dentistry-06-00068]\]. Together, the quantitative and qualitative findings from the Smile4life report led to the development of the Smile4life intervention and accompanying Guide for Trainers \[[@B16-dentistry-06-00068]\], a resource intended to be used by National Health Service (NHS) and Third Sector staff to assist in the training of their staff to deliver evidence-based oral health messages to their service users. For the purposes of this research, the Third Sector refers to voluntary or charity organisations or community groups working with homeless service users, providing support and/or accommodation. The Guide for Trainers provided oral health practitioners and Third Sector staff with: an overview of homelessness and oral health, including barriers and enablers to oral health care; information about oral health (e.g., specific problems, access to care, and preventive care advice); information about the common risk factor approach to oral health; and information about the Smile4life intervention itself, including guidelines for specific roles that practitioners could adopt, motivational interviewing techniques, how to deliver tailored oral health advice, and how to support behaviour change. The Guide for Trainers, and the Smile4life intervention itself, was launched in 2012 \[[@B16-dentistry-06-00068]\]. A theory-based process evaluation of the implementation of the Smile4life intervention began in 2013. Regular telephone interviews took place with NHS staff responsible for the implementation. The evaluation found that it took NHS Boards an average of 16 months to implement the Smile4life intervention following the launch of the Guide for Trainers training package. The results of this evaluation highlighted various barriers to successful implementation, particularly a lack of resources (staffing, time constraints), and poor engagement between the NHS and the Third Sector \[[@B17-dentistry-06-00068]\]. Factors that facilitated implementation included motivated practitioners and a willingness to engage with other organisations \[[@B17-dentistry-06-00068]\]. While this process evaluation illustrated factors that act as barriers and/or facilitators to successful implementation, much remained unknown about how and why NHS and Third Sector organisations implement Smile4life. Therefore, this study aimed to answer the question: how is Smile4life being translated from national guidance into practice? To begin to answer this question, it was crucial to ascertain how the Smile4life intervention was currently being delivered within NHS Boards. In order to do so, it was necessary to observe the behaviours associated with the delivery of Smile4life. 2. Materials and Methods {#sec2-dentistry-06-00068} ======================== 2.1. Sample and Recruitment {#sec2dot1-dentistry-06-00068} --------------------------- Sampling was purposive, with all three NHS Boards being selected because they had taken part in the earlier previous evaluation of Smile4life and had demonstrated contrasting levels of experience and a variety of approaches in delivering oral/dental health services to people affected by homelessness, allowing for a theoretical sampling \[[@B17-dentistry-06-00068],[@B18-dentistry-06-00068],[@B19-dentistry-06-00068]\]. Furthermore, the NHS Boards also varied in the number of people assessed as being homeless, with Board 3 having 7685, Board 2 having 6056 and Board 1 having 2379 in the most recent statistics from the Scottish Government \[[@B20-dentistry-06-00068]\]. The recruited individuals were all NHS employees who worked directly with service users, offered training to Third Sector staff, and delivered oral health education. Their job titles varied depending on the NHS Board of employment and included oral health educators and dental health support workers. Oral health educators promote and raise awareness of oral health issues amongst the homeless population and any Third Sector staff that work in the homelessness sector. They deliver oral health advice, provide support and maintain links with homeless organisations. The role of a dental health support worker is community-based, supporting homeless individuals directly by providing oral health advice, signposting to relevant services, making dental appointments and accompanying patients to the dentist, if required. For the purposes of this article, these participants are referred to as "oral health practitioners". 2.2. Ethical Considerations {#sec2dot2-dentistry-06-00068} --------------------------- Ethical approval was applied for and granted by the University Research Ethics Council at the University of Dundee (UREC 15098). Consent forms had to be read and signed before any observation could take place and all data were anonymised. 2.3. Data Collection {#sec2dot3-dentistry-06-00068} -------------------- Participant observation was chosen as the data collection method for this research, in order to reveal the existing relationships between the oral health practitioners and Third Sector practitioners and the homeless service users they interact with, as well as variations in the working practices of the oral health practitioners. Participant observation would allow the researcher to observe first hand these interactions and variations as they happened, rather than relying on second-hand accounts from the oral health practitioners themselves about their working practices. Furthermore, using participant observation for this research was in line with Taylor-Powell and Steele's guidelines on when participant observation is appropriate, e.g., "when you are trying to understand ongoing behaviour, process, unfolding situation or event" or "when written or other data collection procedures seem inappropriate" \[[@B21-dentistry-06-00068]\] (p. 1). In addition, participant observation is recognised as being a "valuable approach for community health research", providing an opportunity for researchers to become more involved in the community of the person(s) being observed, whereby "the informants are more likely to disclose their real beliefs and perspectives" \[[@B22-dentistry-06-00068]\] (p. 4). Detailed field notes were taken to record what was heard and what was seen. The field notes recorded the date, time and location, as well as any other relevant contextual information, and were written in a narrative style. Notes were made of everything that seemed relevant as Taylor-Powell and Steele noted "in some situations, observing what does not happen may be as important as observing what happens" \[[@B21-dentistry-06-00068]\] (p. 3). Each additional observation session was then informed by the ones that preceded it, allowing for theoretical sampling. As Jorgensen stated, "it is important at the outset of inquiry to remain open to the unexpected" \[[@B23-dentistry-06-00068]\] (p. 82). Therefore, the observation was based on a combination of structured and unstructured formats---there were pre-identified items that should be looked for, as well as space to report anything relevant but unexpected \[[@B21-dentistry-06-00068]\]. This allowed an insight into the existing relationship between the oral health practitioners, Third Sector practitioners and homeless service users, and reveal variations in the working practices of the oral health practitioners. It also revealed whether practitioners were following the national guidance in Smile4life, concerning oral health and homelessness. 2.4. Data Analysis {#sec2dot4-dentistry-06-00068} ------------------ Content analysis was chosen as the method by which to analyse the data collected from the observation sessions. This is a form of data analysis that is understood to be a "systematic and objective means of describing and quantifying phenomena" \[[@B24-dentistry-06-00068]\] (p. 108). It "involves establishing categories, (and) systematic linkages between them" \[[@B25-dentistry-06-00068]\] (p. 467). Before any data were collected, the researcher reflected on the aim of the observation sessions---to explore how Smile4life was being delivered within the NHS Boards---and had identified several key topics that would be specifically looked for. The researcher also considered the recommendations made by Mays and Pope that observers should aim "to record exactly what happened, including his or her own feelings and responses to the situations witnessed" \[[@B18-dentistry-06-00068]\] (p. 184). The key issues the researcher aimed to observe primarily concerned dialogues about oral health, and the role and skills of the practitioner. The researcher also noted the physical context, any general observations that did not fit into any of these questions, and then her own reflections of the session that had been observed. These questions helped to select specific areas to examine and analyse, as part of the preparation stage of content analysis, e.g., how practitioners engaged with service users and Third Sector staff, and how they have chosen to implement Smile4life. The data were then open coded---reading through all the field notes and identifying recurrent categories, based on existing knowledge and reflecting on the research question. Memos, written in the margins, were used to note emerging ideas and reflections concerning the data \[[@B26-dentistry-06-00068]\]. The process was then repeated, to test and refine and revise categories, with similar sub-categories being grouped together where appropriate \[[@B18-dentistry-06-00068],[@B27-dentistry-06-00068]\]. 3. Results {#sec3-dentistry-06-00068} ========== Data were collected over a ten-month period, beginning in November 2015, and ending in August 2016. Three observation sessions were conducted in Board 1, two in Board 3 and four in Board 2. In Boards 2 and 3, these were whole day sessions, where the researcher accompanied the oral health practitioners to a series of different locations as they went about a normal Smile4life working day. In Board 1, this approach was not possible, so sessions lasted approximately 1.5--2 h---the time that a mobile dental unit spent at a location treating patients. Data were collected at the three locations over a series of sessions until saturation had been reached, i.e., when the researcher had witnessed the full range of services offered by the oral health practitioner being observed. More information about each location visited is provided in [Table 1](#dentistry-06-00068-t001){ref-type="table"}. More details about the practitioners are included in [Table 2](#dentistry-06-00068-t002){ref-type="table"}. From the initial coding, one overarching theme emerged: a working alliance between the oral health practitioner and (i) the Third Sector staff, and (ii) the service users. Evidence of this alliance is presented below, followed by an exploration of the barriers and enablers to a positive working alliance. The concept of a working alliance originated in psychoanalysis, where it is understood to be part of a therapeutic relationship between a health professional and patient. More generally, it is the relationship between a person who wants to make a change, and another person who can help them to make that change \[[@B28-dentistry-06-00068]\]. Bordin stated that this working relationship was "key to the change process" \[[@B28-dentistry-06-00068]\] (p. 252), and compared the relationship to that of a parent and child or teacher and pupil. Bordin explained that the working alliance is comprised of three factors: "an agreement on goals, an assignment of task or series of tasks, and the development of bonds" \[[@B28-dentistry-06-00068]\] (p. 253). In relation to Smile4life, these three factors could be interpreted as: improving the oral health of homeless service users; promoting Smile4life and encouraging service users to attend oral health sessions; and a strong relationship between the oral health practitioner and the Third Sector staff. It became clear that, with regard to the delivery of Smile4life, the working alliance went beyond the traditional dyadic relationship of a health professional and a patient---it also involved a third element: the Third Sector staff. Triadic relationships first came to prominence in the work of the sociologist Georg Simmel. While Simmel had written about this in 1908, Hill and McGrath \[[@B29-dentistry-06-00068]\] argued that it did not gain wider attention until 1950 when his work was definitively translated by Wolf. Simmel stated that when three elements are present "each one operates as an intermediary between the other two" \[[@B30-dentistry-06-00068]\] (p. 135). The third person can have three potential roles: (1) a mediator who "deprives conflicting claims of their affective qualities because it neutrally formulates and presents these claims to the two parties involved"; (2) a non-partisan: one party who facilitates the "concord of two colliding parties" or an arbiter who "balances... contradictory claims against one another"; or (3) a "tertius gaudens", a person that can benefit from the conflict of the other two parties within a triad \[[@B30-dentistry-06-00068]\] (pp. 146--154). Simmel also highlighted that, in some situations, a third person joining an existing dyad could be seen as an intruder \[[@B30-dentistry-06-00068]\]. Indeed, when two parties are present, there can be no majority but, when a third party joins, the group dynamic can shift to two against one, "revealing emergent power relations" \[[@B29-dentistry-06-00068]\] (p. 53). Within the Smile4life triadic working alliance, there are three principle relationships: (1) the oral health practitioner and the service user; (2) the oral health practitioner and the Third Sector member of staff; and (3) the Third Sector member of staff and the service user. These are discussed in turn below, and are illustrated in [Figure 1](#dentistry-06-00068-f001){ref-type="fig"}. 3.1. Alliance 1: Oral Health Practitioner and Service User {#sec3dot1-dentistry-06-00068} ---------------------------------------------------------- For the oral health practitioners that were observed, interacting directly with homeless service users was their main role with regards to delivering the Smile4life intervention (as opposed to training Third Sector staff). Therefore, this alliance needed to be strong, in order for the oral health practitioner to engage with the service users and address their oral health needs, and for the service user to be interested and receptive to the oral health information being discussed. However, this alliance was affected by numerous barriers, which are discussed below. ### 3.1.1. Barriers {#sec3dot1dot1-dentistry-06-00068} In many of the locations where the oral health practitioners were observed, it was often the oral health practitioner that had the initiative to approach the service user about their oral health, and this was often opportunistic, with the oral health practitioners seizing upon any opportunity to engage with service users. > "Practitioner 4 would go up to tables with men having breakfast and say who she was and why she was there, but just to make them aware---it was up to the individuals to approach her if they wanted to" > > (Observation 8) However, as this excerpt from the field notes highlights, the oral health practitioners are dependent on the service users taking an interest in what they have to say. Often, this is not the case---there was occasionally disinterest or hostility from the service users, who were usually not interested in receiving oral health advice. In Boards 2 and 3, many of the interactions with service users are opportunistic, with the oral health practitioner approaching the service user to talk about oral health, rather than the other way around. In one instance, a service user at a drop-in did not want the toothbrush pack from the practitioner because the toothpaste contained fluoride which he considered to be a "neurotoxin". The practitioner tactfully explained the benefits of fluoride, but did not stress the point because the service user was getting angry and argumentative with her. An additional barrier that prevented the formation of strong working alliance between the oral health practitioners and the service users was the space the oral health practitioner was given by the service in which to deliver Smile4life. As [Table 1](#dentistry-06-00068-t001){ref-type="table"} shows, in many locations, the oral health practitioner was told to set up in a large communal area, often a canteen---a space in which there is the potential to reach many service users. However, in seven of the services visited, the oral health practitioner was put in a space that meant the service users must approach them, i.e., a medical room, or a meeting room away from the communal spaces. This blocked engagement attempts of any kind, by preventing the oral health practitioner from approaching the service users directly. Furthermore, the rooms the oral health practitioners were put in were predominantly chosen by the staff, and were not always appropriate for the delivery of Smile4life. > *"We were put in the medical room along the corridor from the office, but there was no opportunity for Practitioner 4 to approach any of the residents. We only saw service users if they specifically wanted to talk about their oral health or if they had walked past the room and wanted to see who we were."* > > (Observation 9) In the case of Board 1, the space that the oral health practitioners were in was also not ideal, but this was not due to the staff within the Third Sector services they were attending. All data collection took place within the Mobile Dental Unit, which was a small confined space. Outside of the dental surgery section of the Unit there was a waiting area with enough space for approximately two people---any more than two and it began to feel crowded. This meant that it was not the most appropriate place for impromptu oral health advice or longer discussions about oral health with service users. The observations at the Mobile Dental Unit (MDU) highlighted an additional barrier to the working alliance between the oral health practitioners and the service users---that there are risks involved in delivering Smile4life and that the service users can be unpredictable and occasionally violent. On a number of occasions, disruptive service users were observed barging into the Unit, demanding to be seen by a dentist. In these instances, the oral health practitioner was the one who acted as a gatekeeper, preventing the service users from accessing the dental surgery section of the Unit. > "When I was there today two service users opened the MDU door from the outside and barged in" > > (Observation 3) > "When I arrived there were two women outside the front door (of the MDU) shouting." > > (Observation 4) Perceptions of there being risks associated with Smile4life delivery were strengthened by the repeated use of safety measures, such as alarms or radios, in the services visited while observing the oral health practitioners. In most instances, these devices were given to the oral health practitioner by the Third Sector staff before they proceeded to interact with service users---the researcher never observed an emergency alarm or radio being used. Indeed, it became apparent that the oral health practitioners did not feel they were at risk, despite Practitioner 1 having a good reason to be concerned about her safety. She told the researcher that on a previous visit to a drop-in she had been bitten on the hand by a service user, while she had been discussing oral health with him. This event was then reported to Datix, the NHS's incident reporting tool. However, when she saw that he was present at the session we were attending, she did not avoid him and spoke with him again. At the same session, Practitioner 1 had warned the researcher beforehand to be careful about her handbag, because some of the service users had been known to steal. ### 3.1.2. Enablers {#sec3dot1dot2-dentistry-06-00068} It became clear that there were two key factors in overcoming these barriers and enabling a successful working alliance to be formed between the oral health practitioners and the service users: the skills and attitudes of the oral health practitioners and the use of incentives. With regard to the oral health practitioners themselves, the researcher observed that they needed to be confident, and in some respects, fearless. Indeed, the field notes frequently reflect this: > *"Working in the mobile dental unit would not be for everyone---you need to be confident and thick-skinned"* > > (Observation 3) > *"Practitioner 1 is confident and appears quite fearless, putting up with language/behaviour that would not be tolerated in a normal clinic."* > > (Observation 6) > *"I get the impression that Practitioner 4 and the other practitioners I have observed... do not see the risk involved in their job, or they just see it as part of the job... it is also possible that after a while doing this kind of work they stop seeing it as risky"* > > (Observation 9) It became apparent that working on Smile4life was not something that would suit everyone---the oral health practitioners themselves said as much. > *"The dental team spoke about how they felt that working in the MDU would not suit everyone"* > > (Observation 3) > *"I believe that not everyone is suited to doing Practitioner 1's job---personality aside, you need to be fast-thinking, tough-skinned, a bit fearless, and approachable."* > > (Observation 6) In addition to confidence, the oral health practitioners also had to be flexible in their interactions with service users, in particular, tolerating disruptive behaviour, for a working alliance to be sustained. In Board 1, the oral health practitioner, and the dentist and dental nurse who worked in the Mobile Dental Unit, described some service users as being disruptive, trying to flirt with them and one man who had taken off his t-shirt to show them his tattoos. Other service users swore or used offensive language. The oral health practitioners admitted that they tolerated this kind of behaviour from the patients they saw in the MDU, but would not normally do so, with patients they saw in their usual clinics. In the case where the patient was flirting and removing his clothes, they did not seem fazed by this. It became apparent over the course of the observation sessions that the success of engagement attempts was closely associated with the individual oral health practitioner, and their personality, experience, communication skills and how they perceived their job role and responsibilities. From this, it appears that, for the oral health practitioner to be successful in implementing Smile4life, they needed to be flexible, not just in their working hours (e.g., working in the evenings) but also in tolerating disruptive behaviours from patients, while remaining non-judgmental and not taking undue risks. They must tailor their approach to the needs of the individual service user. > *"Practitioner 1 is very experienced and upfront with all service users---not visibly fazed by service users' admissions or behaviours"* > > (Observation 1) > *"The MDU dentist admitted that she is aware that she acts differently with patients in the MDU than she would with regular patients---she is not as formal, more likely to speak to them in the same way they speak to her... she is quite matter-of-fact"* > > (Observation 3) Furthermore, the researcher observed that the oral health practitioners have to be very sensitive and empathetic, to strengthen the working alliance---often service users will share information about their lives, and their past experiences, and practitioners must listen and respond appropriately. The oral health practitioners at the MDU were observed speaking affectionately about long-term patients, and remembering everyone's names. Others were observed tailoring their advice to the needs of the service users, and letting service users tell their stories about their lives, occasionally attempting to bring the conversation back to oral health. In some instances, the oral health practitioner would share their own life experiences if it was related to the topics that were being discussed: > *"When a service user said he found it difficult to stop smoking, Practitioner 1 admitted that she was an ex-smoker, and explained that she still feels tempted"* > > (Observation 1) The oral health practitioner in Board 3 demonstrated on numerous occasions that she was willing to go the extra mile for the service users she sees. These extra tasks that she does are not because she has been asked to by her managers, but because she cares about the service users, and wants to offer them the best service she can. > *"To me it seems that Practitioner 4 goes above and beyond for the service users she sees. She will offer to phone and make appointments, sends them reminders the day before (even on her day off) and she will even take them to an appointment. It's clear that she cares if they attend or not---she mentioned that she has asked some practices to waive fines, and clearly advocates for the service users when necessary."* > > (Observation 8) Incentives were also used, to encourage service users to engage with the oral health practitioners, aiding in the development and maintenance of the working alliance. These were predominantly toothbrush and toothpaste packs, but in some NHS Boards could also be free samples of a wider range of toothpastes (e.g., Oral-B or Corsodyl), denture-care packs, or toothbrush cases. Indeed, service users were always eager to get the free samples of the branded toothpastes, compared to the more basic NHS-provided packs. These incentives often acted as an icebreaker, particularly in locations where the oral health practitioner had to approach service users to see if they were interested in discussing oral health, rather than interested service users approaching them. In some instances, when a wide range of resources were available, they were also an opportunity for the oral health practitioners to find out more about the service users oral health---they could ask questions under the pretence of ensuring they gave them the most appropriate product. For example: > *"Practitioner 1 would take the time to find free samples that would be specific to the service user, e.g., Corsodyl toothpaste for people with bleeding gums, or denture care items"* > > (Observation 1) 3.2. Alliance 2: Oral Health Practitioners and Third Sector Staff {#sec3dot2-dentistry-06-00068} ----------------------------------------------------------------- While considering Alliance 1, it became apparent that Smile4life delivery may be reduced if there is no alliance between the oral health practitioners and the Third Sector staff. The cooperation of the Third Sector staff is crucial as they allow the oral health practitioner access to their service and their service users. For example, they can choose to promote Smile4life and visits from the oral health practitioner or do nothing to encourage service users to be interested in their oral health. In the observations, the Third Sector staff often acted as a barrier to alliances forming between them and the oral health practitioners, as well as between the oral health practitioners and the service users. Alternatively, in one NHS Board, the oral health practitioners themselves were a barrier to a working alliance with local Third Sector staff. These barriers are discussed in more depth below. ### 3.2.1. Barriers {#sec3dot2dot1-dentistry-06-00068} In Board 1, there was no observed working alliance between the oral health practitioners, service users or Third Sector staff. Unlike the more pro-active oral health practitioners observed in the other Boards, in Board 1, the oral health practitioners did not approach service users---they offered a mobile dental unit and if any service user was interested in getting treatment they had to approach the unit. > *"Practitioner 3 did not really interact with service users beyond telling them if they can be seen by the dentist, or making general conversation. She would encourage people to wait in the drop-in rather than in the MDU before their treatment"* > > (Observation 3) > *"The oral health team do not seem bothered to recruit any patients, even if that means sitting waiting with nothing to do---the feeling seems to be that if a patient wants to be seen then they will come to the MDU."* > > (Observation 4) In the other two NHS Boards, in most instances, the oral health practitioner was reliant on the Third Sector staff at the organisations to promote and advertise the oral health visits, e.g., by putting up posters, flyers, or announcing the visit over the overhead speaker. However, this was often not the case: > *"In all three establishments today, staff did not seem well prepared for Practitioner 2's visit---her poster was only displayed in one of them, and they had not spoken to their service users about her visit...In one place, the room we were offered was in the staff area, so there would never be any passing service users"* > > (Observation 2) This excerpt from the field notes highlights the extent to which the oral health practitioner is at the mercy of the Third Sector staff---during that observation, the oral health practitioner visited three services and only spoke to one service user. At the first service, the oral health practitioner had been advised to attend at a time when there were no service users awake; at the second, the oral health practitioner was given a room to use which was a meeting room in the staff area. There was also no attempt by Third Sector staff at any of the three locations to let their service users know that Practitioner 2 was available to talk to. This suggests that, if the oral health practitioner does not know the staff that well, they are not motivated to prioritise her requests or to put up her posters. Similarly, if they do not understand the importance of oral health then they would be unlikely to encourage their service users to care about their oral health. ### 3.2.2. Enablers {#sec3dot2dot2-dentistry-06-00068} Despite the barriers discussed above, any potential alliance between the oral health practitioner and the Third Sector staff was dependent on both parties engaging with each other. While the previous example demonstrated that, when the Third Sector staff do not help the oral health practitioners, Smile4life cannot be delivered and it is the responsibility of the oral health practitioner to attempt to establish a relationship in the first instance. As with forming an alliance with service users, the oral health practitioners were themselves an enabler to forming relationships with the Third Sector staff. > *"it is clear that Practitioner 1 works hard at maintaining strong relationships with staff at these locations... she makes a point of visiting every 6 weeks and reminds them the day before that she will be visiting."* > > (Observation 1) > *"Practitioner 1 clearly has a good relationship with the staff... she told me that 'keeping the staff sweet' is a major part of her role and really helps with building rapport"* > > (Observation 7) As with service users, incentives also aided in the formation of an alliance with the Third Sector staff---Practitioner 1 said as much when she told the researcher that she needed to "keep the staff sweet" so would purposively keep two packs of PolyGrip aside for one member of staff in particular (Observation 7). 3.3. Alliance 3: Third Sector Staff and Service Users {#sec3dot3-dentistry-06-00068} ----------------------------------------------------- The third alliance that exists between the three key parties involved in Smile4life is the one between the Third Sector staff and the service users. However, interactions between the Third Sector staff and the service users were not observed during the present study as the oral health practitioners were the focus of the participant observation---this, therefore, represents a limitation with regard to dental care policy. The need remains to conduct additional studies to confirm this process to provide dental care for people experiencing homelessness. 4. Discussion {#sec4-dentistry-06-00068} ============= The findings from the observation sessions suggest that key factors in the delivery of Smile4life are the working alliances among the oral health practitioners, the Third Sector staff and the service users. Within this triad, there are three key alliances: (1) oral health practitioners and service users; (2) oral health practitioners and Third Sector staff; and (3) Third Sector staff and service users. When there are strong working alliances, the Third Sector staff can promote and signpost to the oral health practitioner, who in turn can engage directly with service users about their oral health. With regard to Simmel's work concerning coalitions within a triad, in an ideal Smile4life scenario, the Third Sector staff would act as a "non-partisan", facilitating a connection between the oral health practitioner and the service users \[[@B30-dentistry-06-00068]\]. Indeed, this was the case in the more successful interactions that were observed, However, it is apparent from the observation data that this was not always the case: while not necessarily partisan, it would appear that some Third Sector staff were indifferent about oral health. In these instances, it is possible that the Third Sector staff interpreted the oral health practitioners as intruders, disturbing their existing dyadic relationship with their service users, or potentially excluding them from the triad. Due to this possible interpretation, in many cases, it is the Third Sector staff that hold the power within the triad---they can control access to the service and the service users, essentially acting as gatekeepers to protect the service users from what they may perceive as a threat, i.e., the "tertius gaudens" scenario as described by Simmel \[[@B30-dentistry-06-00068]\]. However, in instances where the Third Sector staff are helpful---and perhaps the communication between parties is more effective---there can still be the issue of disinterested or disruptive service users. In these cases, the power distribution shifts and it is the service users that hold the power---it is up to them if they are receptive to Smile4life, or whether they will be rude and disinterested in what the oral health practitioner has to say. For example, in instances such as those observed in Boards 1 and 2, the service users demonstrated their power by being disruptive or argumentative with the oral health practitioners, e.g., arguing about fluoride. Interestingly, in both scenarios, the oral health practitioners are powerless. Indeed, in many respects, they *are* intruders, or outsiders, attempting to infiltrate the service where the Third Sector staff and the service users are based. In this respect, when the oral health practitioner enters, they allow a majority to form, i.e., two against one \[[@B29-dentistry-06-00068]\]. Caplow examined the power dynamics and coalitions present in three-person groups and considered there to be eight types of coalition, dependent on the power held by each of the three parties, e.g., A = B = C, where all parties are equal; or A \< B, B = C, where B and C have equal power, which is greater than that of A---this could be said to be the case when oral health practitioners are seen as intruding upon the existing alliance of the Third Sector staff and the service users \[[@B31-dentistry-06-00068]\]. However, due to the scope of the research and the focus on the oral health practitioners, it was not possible to observe Alliance 3, and therefore not possible to fully explore the different power dynamics present in the three-party group. This is a limitation of this research, and a potential topic for future studies in this area. Observing the relationship between the Third Sector staff and the service users would reveal if oral health is considered a priority for these individuals, particularly in the absence of the oral health practitioners. It would also aid in a deeper understanding of how the triadic working alliance operates, and if the entrance of the oral health practitioners is truly seen as an intrusion, forcing the three-person group into "a pair and an other", with the oral health practitioner being the "other" \[[@B32-dentistry-06-00068]\] (p. 351) \[[@B30-dentistry-06-00068]\]. There are also limitations associated with participant observation as a data collection tool. While it allows the researcher to gain first-hand experience of the topic being studied, it is not repeatable, and is dependent on the researcher's interpretations of what is being observed \[[@B33-dentistry-06-00068]\]. It is also time-consuming, requiring the researcher to spend long periods with the participants being observed \[[@B22-dentistry-06-00068]\]. However, despite these limitations, it was an appropriate tool for this study, as it allowed the authors to immerse themselves in the working lives of the oral health practitioners, which would not necessarily have been possible with other data collection methods, e.g., interviews. While it was time-consuming, this provided the "time to develop an intuitive feel for the particular system studied" \[[@B33-dentistry-06-00068]\] (p. 37). While the observations concerned the delivery of the Smile4life intervention and the relationships between oral health practitioners, homeless service users and Third Sector staff, it is possible that the results could be generalisable to any situation where oral health is being delivered to an excluded or vulnerable population, such as adults with learning disabilities. Many of the barriers faced by the oral health practitioners could be considered organisational barriers, which are not specific to the homelessness context (e.g., uncooperative Third Sector staff and lack of access to service users/patients). Similarly, the factors identified as enablers to the alliances discussed above (e.g., the skills and attitude of the oral health practitioners) would be beneficial to any oral health practitioner attempting to forge a good working relationship with any patients or organisations, not just within homelessness. With regard to recommendations for the continued delivery of the Smile4life intervention, based on the observations, it would appear that there needs to be a strengthened relationship---or alliance---between the oral health practitioners and the Third Sector staff. Specifically, there should be more awareness raising about the benefits of Smile4life and what the oral health practitioners are attempting to do when they visit a Third Sector organisation. This would potentially overcome the issue of the location or physical space the oral health practitioner is given to deliver Smile4life. Additional buy-in from the Third Sector could also facilitate improved access to the service users, if the Third Sector staff see the importance of oral health and encourage their service users to see the oral health practitioner. However, it must be acknowledged that some barriers experienced by the oral health practitioners are organisational issues, such as staffing within the Third Sector organisation, and as such are not straightforward to overcome. In addition, it would be necessary to investigate the barriers and enablers in more detail, to establish what the oral health practitioners themselves think of their experiences and their role in delivering Smile4life, before any changes are recommended regarding the delivery of Smile4life. 5. Conclusions {#sec5-dentistry-06-00068} ============== The observation sessions have demonstrated how Smile4life is implemented in three different NHS Boards across Scotland, and highlighted the variations in practitioners' approaches to their Smile4life-related work. Furthermore, they revealed the three key working alliances that exist among the oral health practitioners, the Third Sector staff and the service users. By referring to theories of triadic coalitions, it was possible to infer the types of relationships that exist within the triad, and the power dynamics that exist within these relationships. To successfully deliver Smile4life to service users, all parties in the triad must work together, and each of the three key alliances must be strong. There can be no Alliance 1 if Alliances 2 and 3 do not already exist. In addition, there were many factors that influenced these alliances, and these acted as barriers and enablers to strong and beneficial relationships. We are grateful to the NHS practitioners for taking part in this research. This work forms part of L.B.'s doctoral research and is taken from her thesis. Conceptualisation, L.B.; Formal analysis, L.B.; Investigation, L.B.; Methodology, L.B.; Supervision, I.A., G.H., A.R. and R.F.; Visualisation, L.B.; Writing---original draft, L.B.; and Writing---review and editing, L.B., I.A., G.H., A.R. and R.F. The Smile4life programme was funded by the Scottish Government and National Health Service Boards (grant number: 121.80.4497). The authors declare no conflict of interest. {#dentistry-06-00068-f001} dentistry-06-00068-t001_Table 1 ###### Details of observation sessions. ----------------------------------------------------------------------------------------------------------------------------------------------------------- Observation Location Services Visited Setting --------------- ---------- ---------------------------------------------------------------------------------------------- --------------------------------- Observation 1 Board 2 3 supported accommodation establishments (1 h each) Shared living room\ Meeting room Observation 2 Board 2 An accommodation for young men (1 h)\ Kitchen\ A long-term accommodation for families and individuals (1 h)\ Meeting room\ A short-term accommodation for young people (1 h) Shared living room Observation 3 Board 1 Mobile dental unit at a drop-in service (2 h) Mobile dental unit waiting area Observation 4 Board 1 Mobile dental unit at a harm reduction service for young people (1 h 30 min) Mobile dental unit waiting area Observation 5 Board 1 Mobile dental unit at a harm reduction service for young people (1 h 30 min) Mobile dental unit waiting area Observation 6 Board 2 Drop-in service providing hot meals (3 h) Canteen Observation 7 Board 2 A space for families and friend of prisoners to wait before entering the prison (4 h 30 min) Visitor centre Observation 8 Board 3 An emergency accommodation for women (1 h)\ Canteen\ A support and drop-in service (1 h 45 min)\ Reception area An emergency accommodation for men (2 h) Observation 9 Board 3 A homeless assessment centre and short-term accommodation (2 h)\ Medical room\ Supported long-term accommodation for women (1 h) Service user's flat ----------------------------------------------------------------------------------------------------------------------------------------------------------- dentistry-06-00068-t002_Table 2 ###### Details of participating oral health practitioners. Practitioner Number Board Job Title Gender --------------------- --------- ------------------------------ -------- Practitioner 3 Board 1 Dental Health Support Worker Female Practitioner 1 Board 2 Oral Health Educator Female Practitioner 2 Board 2 Oral Health Educator Female Practitioner 4 Board 3 Dental Health Support Worker Female

Introduction {#Sec1} ============ Health care workers (HCWs) have a crucial role to play in responses to chemical, biological, radiological, nuclear and explosive (CBRNE) events . These workers often take on serious risks when they do so. The Black Death (bubonic plague) swept through Medieval Europe on various occassions, leading to debate over whether physicians and other public officials had a duty to remain with the sick (Luther [@CR16]). During the 1918 influenza pandemic, many nurses and others who volunteered to care for the infected themselves contracted the deadly influenza (Godderis and Rossiter [@CR12]). After the 1995 Aum Shinrokyo Sarin attacks, 10% of hospital HCWs involved in treating exposed patients ended up needing to be treated themselves because patients' clothes exposed them to Sarin (Rebera and Rafalowski [@CR24]). In the 2002--2003 SARS outbreak, approximately 30% of all cases occurred in HCWs; in Toronto, about half of the cases were in HCWs, three of whom died (Malm et al. [@CR17]). Towards the end of the Ebola virus disease outbreak in West Africa, 898 cases of Ebola infection had occurred in HCWs, of whom 518 died (Statista [@CR28]). The lethality of the virus is well-known, but the HCWs were disproportionately impacted. Liberia was worst hit, where 8.07% of its health workforce died from the virus, compared to 0.11% of its general population (Evans et al. [@CR9]). Not only did caring for patients put HCWs at risk of physical disease and death, but some who survived faced stigmatisation and discrimination afterwards (OHCHR [@CR22]). Yet possibly the most egregious example of the risks HCWs find themselves exposed to has been in the ongoing war in Syria. In spite of the traditional and internationally protected status of health care facilities and workers during conflict and war, they have become common targets of violence (ICRC [@CR35]). In Syria alone, between November 2015 and December 2016, over 400 violent acts against health care facilities have been documented (Elamein et al. [@CR8]). These resulted in 677 injuries and 261 deaths, with one quarter of the victims being HCWs. As a result, health care services are being divided between locations and staff numbers on site are kept to a minimum to reduce the impact of individual attacks. HCWs who respond to CBRNE events typically put themselves in harm's way to an increasing degree compared to responders to other disasters that do not involve infectious agents or contaminants. Disaster responders typically choose to enter disaster zones, and usually have specialised training to prepare for such events. This chapter will not focus on such responders, but on HCWs who find themselves in the midst of a CBRNE event. They may find themselves grappling with whether or not they have an ethical obligation to continue to provide health care to those injured in the event. The ethical question is whether HCWs have a duty to care for any and all patients in the event of a CBRNE crisis. In other words, are HCWs obliged to accept the risks that accompany caring for patients after a CBRNE event? In recent years, 'the issue of duty to care has emerged as a matter of paramount concern among health care professionals, hospital administrators, public policy makers, and bioethicists' (Ruderman et al. [@CR25], 2). The Current Situation {#Sec2} ===================== Before proceeding to examine the ethical issues involved, this discussion must be prefaced by the finding that many HCWs accept the risks of their work and diligently care for their patients in spite of the dangers involved. The heroism apparent during the Ebola outbreak led *Time* to declare these HCWs the 2014 Person of the Year (Von Drehle and Baker [@CR31]). A report into the SARS outbreak in Toronto found that, 'Workers generally showed heroism and altruism in the face of danger during the SARS outbreak' (University of Toronto [@CR29], 10). However, this was not the case for all. The Toronto report continued that 'some balked at caring for people infected with SARS, and a few were dismissed for failing to report for duty. Post-SARS, many health care workers raised concerns about the level of protection to themselves and their families. Some even left the profession' (University of Toronto [@CR29], 10). CBRNE crises lead to ethical challenges that involve individual and societal decisions. On an individual level, workers have to balance their ethical duties towards their patients and the public with those towards themselves and their own health, and their duties towards their families and other dependants. The societal level is seen most clearly by the devastation brought by Ebola onto the healthcare workforce in the affected countries. These systems started with woefully inadequate healthcare workforces, and the deaths of so many HCWs from Ebola have left the systems even more debilitated. Although Ebola was directly devastating in many ways, an additional 25,000 deaths may occur each year because of the deaths of so many HCWs in these countries (Evans et al. [@CR9]). A healthcare system, and society more broadly, must somehow balance its ethical duty to care for those presently sick while knowing that this can increase the risks to the long-term health of the people in their societies who will require care in the future. With the constant risk of an incurable pathological agent appearing, the growing fear of an imminent pandemic, and the threat of terrorists obtaining nuclear materials, the importance of reflecting on the duty to care and other ethical challenges associated with CBRNE has been noted (and was one of the impetuses for the two projects than contributed to this book). Ethical decision-making skills are needed to address such issues. Yet in spite of the acknowledged importance of such topics, the Toronto SARS report found that 'There is currently a vacuum in this field' (University of Toronto [@CR29], 10). In the US, for example, the 2009 H1N1 flu pandemic led to several calls to develop pandemic readiness plans. Such plans identify the duty to care as one of several ethical principles (Koenig et al. [@CR14]). Some US states have pandemic plans, but few address the duty to care explicitly. Louisiana, the state most directly hit by Hurricane Katrina, is one that does, defining the duty to care as 'the obligation of health care professionals to care for patients at all times' (Louisiana Department of Health & Hospitals [@CR15], 10). It notes that during pandemics HCWs will have to balance their duty to individual patients with that to all patients. Yet no discussion is provided about how HCWs should balance this duty with their duties to themselves and their families. At the same time, other developments have occurred within healthcare that challenge the traditional view of this issue. Workers' rights and the importance of creating safe workplaces is increasingly recognised, appropriately so. Yet these developments could raise questions about policies that put HCWs at increased risk of harm. As noted above, the duty to care usually refers to individual patients. During a crisis, like a CBRNE event, a balance may be needed between the duty to care for individuals and for society as a whole. In additional, the duty not to harm is a strong ethical principle. HCWs may see their duty not to harm patients as obliging them to avoid contagious or contaminated patients because of the risk that they could pick up the agent and pass it on to others unknowingly. While the duty to care is a long-standing ethical principle in healthcare, it raises many ethical challenges that require further careful reflection and analysis. Healthcare Readiness for CBRNE {#Sec3} ============================== Some discussion has occurred around the readiness of current healthcare systems for emergency responses to CBRNE. The World Health Organization (WHO) Regional Office for Europe published a checklist to assist hospital administrators and emergency managers prepare for disasters. The guidance states that 'Effective human resource management is essential to ensure adequate staff capacity and the continuity of operations during any incident that increases the demand for human resources' (WHO Regional Office for Europe [@CR33], 17). However, the document does not discuss the ethical issues faced by HCWs, nor does it provide guidance on ethical decision-making . Instead, the focus is on policy and management preparation. A systematic review examined qualitative research into nurses' preparations for ethical issues in public health emergencies and disasters. The authors identified 'a failure to directly address the issue of ethical considerations in planning, preparedness, and response to public health emergencies and disasters by nurses' (Johnstone and Turale [@CR13], 73). The American Nurses Association ([@CR3]) acknowledged the conflict between nurses' duty to care for patients and their own right to self-preservation, and the challenge of finding the right balance. The American College of Chest Physicians (CHEST) has a prominent role in preparing US doctors who will be at the front line of any respiratory pandemic. They issued a Consensus Statement on the ethical issues of caring for patients during pandemics and disasters. This Statement does not address the duty to care (Biddison et al. [@CR5]). With little explicit emphasis on the duty to care in crisis situations, HCWs may not be aware of their obligations or have reflected on how they might respond during a CBRNE crisis. Some surveys have asked HCWs if they would report for work in the event of different types of crises. In a survey in the US, roughly half of the HCWs who responded stated they likely would not report to work during an influenza pandemic (Balicer et al. [@CR4]). Another survey of Emergency Department workers in Chicago asked whether people would work additional hours to help victims of different types of CBRNE events. Among these HCWs, 98% said they would accept extra work after an airplane crash, 85% after a radioactive bomb, and 54% after the release of a biological agent (Masterson et al. [@CR19]). These types of studies have weaknesses because they ask people's opinions about hypothetical situations when they are not in a disaster. Just as questionnaires about past events suffer from recall bias, questionnaires about the future can also be biased. Studies of actual behaviour over a number of decades have found that the vast majority of those in emergency roles fulfilled their duty when called (Scanlon [@CR26]). However, the on-going nature of a pandemic and the lethality of agents like Ebola, sarin and radioactivity, may lead some to question whether their duty to care applies to those patients. Changes to the nature of healthcare are also important to consider here. In some countries, healthcare is increasing viewed and practiced according to a business model. Arguments are made to view healthcare organisations as businesses, patients as consumers, and HCWs as employees contracted to deliver a service. As such approaches come to infuse the ethos of healthcare, they may inadvertently impact responses to CBRNE crises. Sheri Fink's investigation into Hurricane Katrina included an exploration of some of the corporate decision-making that interferred with patient care and rescue (Fink [@CR10]). This sort of approach can lead to a focus on contractual obligations towards workers and patients, which may not address obligations to society as a whole. As will be discussed more below, a contractual approach has particular limitations when altruism and sacrifice are required, which is usually the situation when the duty to care is invoked. The Traditional View {#Sec4} ==================== Granted the limitations of the surveys cited above, the possibility that a significant proportion of the healthcare workforce might not show up in the aftermath of a CBRNE crisis is of concern. This conflicts with what can be called the traditional view of the duty to care in emergencies. Daniel Defoe captured this view dramatically in his fictionalised account of the Great Plague of London which may have claimed up to 20% of the population in 1665. The book examines many of the same ethical issues that challenged the responders to Ebola virus disease."So the Plague defied all Medicine; the very Physicians were seized with it, ... This was the Case of several Physicians, even some of them the most eminent; ... it rather is to their Praise, that they ventured their Lives so far as even to lose them in the Service of Mankind; They endeavoured to do good, and to save the Lives of others (Defoe [@CR7], 35--6)."This traditional view is that doctors and nurses will lay down their lives for their patients and the good of society. This ethic has been lived out in HCWs and disaster responders throughout history. Those who stayed to take care of Ebola patients, or came to offer what they could, acted upon this view (Von Drehle [@CR31]). In battle-torn Syria, the White Helmets formed a volunteer group to provide aid and rescue to those injured by chemical and explosive devices. Their motto is 'Whoever saves one life, saves all of humanity' (Malsin [@CR18], 23). Among responders like these, the duty to care focuses on putting the needs of others above ones own needs. The belief is that those who have specialised training, or more opportunities than others, thereby have a responsibility to help those in need. Martin Luther gave similar advice to those facing the Black Death: 'paid public servants such as city physicians, city clerks and constables, or whatever their titles, should not flee unless they furnish capable substitutes who are acceptable to their employer' (Luther [@CR16], 477). Changing Professional Ethics {#Sec5} ============================ This view was explicitly called for in earlier professional ethics codes. The 1847 *Code of Medial Ethics* of the American Medical Association stated that physicians have a 'duty to face the danger, and to continue their labours for the alleviation of the suffering, even at the jeopardy of their own lives' (American Medical Association [@CR2], 27). Something very similar was included in the 1922 Code of Ethics of the Canadian Medical Association (Ruderman et al. [@CR25]). But things have changed. The 1949 International Code of Medical Ethics of the World Medical Association stated that, 'A doctor must give emergency care as a humanitarian duty unless he is assured that others are willing and able to give such care' (World Medical Association [@CR34]). By the 1970s such strong statements about the duty to care in the face of infectious risks had disappeared from most codes (Ruderman et al. [@CR25]). The traditional obligation that a doctor should accept the risks of practicing medicine has faded. Different reasons have been given for this, including the general belief that infectious diseases had been overpowered, as least in those countries with access to effective antibiotics (Ruderman et al. [@CR25]). The move from healthcare as a calling to one where healthcare is a rewarding and finantially comfortable career has been implicated in this change, as well as the business model for healthcare organisations. The arrival in the 1980s of HIV and AIDS, especially at first when little was known about its transmission and less about its treatment, led to further weakening of the meaning of the duty to care. SARS, H1N1 and Ebola raised further questions about what the duty to care implies, both ethically and practically. In response, nurses in Liberia went on strike during the Ebola outbreak because of low wages and the lack of personal protective equipment (PPE) that guards against infectious diseases like Ebola (Agence France-Presse [@CR1]). Likewise, nurses in the US protested, and some went on strike, to express concern over the lack of preparation and protective equipment for Ebola in their hospitals (Skinner and Johnson [@CR27]). In the 1800s, a doctor knew that caring for some patients entailed serious risks. In some parts of the world, that never changed. Many HCWs have died, and continue to die, in the service of their patients. As one doctor noted during Ebola, many HCWs 'found themselves "fighting a forest fire with spray bottles." They did not give up' (Von Drehle [@CR31]). The prospect of a pandemic has brought things full circle and requires much more extensive discussions about the ethical requirements entailed in the duty to care. Ethical Justification {#Sec6} ===================== The arguments brought up to ethically justify the duty to care can be grouped into five approaches, each with its counter-arguments. They will be summarised here, but are discussed in more detail with an extensive bibliography by Malm et al. ([@CR17]). The first is that HCWs consent to take on certain risks as they go through their professional training. Risks are part of all forms of practice, and can arise physically, such as from needle-sticks, using certain equipment, lifting patients, etc., and also emotionally, such as from watching patients suffer and even die. Training should make people aware of these risks and therefore HCWs consent to accept these risks as part of professional practice. This is especially the case when someone works in infectious diseases, but applies to all HCWs. The criticism of this argument is that SARS, H1N1 and Ebola have changed healthcare practice. Risks from serious infectious diseases did not exist for many HCWs trained in earlier years, and hence people have not in any realistic way consented to take on some of the risks that exist today. The second approach uses an analogy to argue that HCW give implied consent to certain risks. If someone becomes a soldier, she cannot legitimately object to the risks of combat. If someone becomes a firefighter, he cannot object to getting close to fires. As part of the decision to pursue particular careers, certain risks must be accepted even if this is done implicitly or changes over time. The criticism of this argument is that even if accepted in principle, it does not necessarily entail that all HCWs have a duty to care for all patients, particularly those who put HCWs at higher risk. For example, someone with training in infectious diseases may have a duty to care for infected patients, but this does not imply that a psychiatric nurse has the same duty to those patients. Thus, the duty to care should be limited to those who have relevant training and experience in the conditions that ail the patient. The third argument is that someone with specialised knowledge, training and skills has an additional responsibility to use that knowledge to help those in need of that expertise. By their own training, HCWs are better able to care for such patients in ways that reduce their own risks. They also have easier access to the necessary protective equipment. However, objections to this approach point out that HCWs differ widely in their training and skills. If this implies that HCWs have different duties to care, this will lead to a confusing range of obligations. The fourth approach uses that of reciprocity, or a social contract view. Many HCWs receive assistance from society in various ways (through scholarships or research funds) and often receive privileges and status from their careers, not to mention good salaries. As a result of such contributions from society, HCWs should reciprocate by taking care of patients who are in need. Some object that HCWs receive widely varying contributions from society and also vary widely in the privileges they receive. Some claim that the respect or status once given HCWs no longer exists, and in some places HCWs do not receive a commensurate salary. The fifth approach is that many HCWs take professional oaths or accept ethics codes and these often commit them to a duty to care. However, these declarations tend to be very general and also change with time. People entering a professional may view oaths as symbolic rather than a serious commitment to specific ethical values. Further, a review of 61 professional codes found that 85% either did not mention the duty to care or offered no clear guidance on its implementation (Upshur et al. [@CR30]). One further approach is regularly mentioned by those who volunteer to serve: the importance of serving one's community. The White Helmets help their fellow Syrians because, according to their director, 'At the end of the day, this is my country' (Malsin [@CR18], 26). The Ebola fighters regularly mentioned that they wanted to help their neighbours and their communities (Von Drehle [@CR31]). This aspect gets at the importance of the internal ethical motivations of HCWs, and in particular the ethical virtues that will be discussed below. Healthcare ethics in Western contexts has become focused on individuals and their rights. Public health crises and CBRNE events remind us of the need to consider relationships and communities within ethics. At the same time, such approaches do not provide clear mechanisms for balancing the duties HCWs have to the variety of people they are in relationships with: family, neighbours, colleagues, patients. Guidance is needed on how to balance such conflicting obligations to various parties. WHO Guidance {#Sec7} ============ The World Health Organization (WHO) developed general guidance on the ethical issues in public health responses to pandemic influenza (WHO [@CR32]). The document devoted one section to the duty to care, and provided some guidelines. This acknowledged that there is a need for open discussions and agreement between HCWs, their professional organisations, and the public on the duty to care. What was stated after SARS remains the case today: 'the time to address the ethical duty to provide care is at hand -- before the arrival of the next public health emergency' (Ruderman et al. [@CR25], 6). The guidance notes that the duty to care can be addressed in terms of moral, professional, contractual or legal obligations. A strong case is made for approaching this as a moral obligation. At the same time, WHO acknowledges that 'the duty to work notwithstanding risks to one's own health is not unlimited' (WHO [@CR32], 14). Rather than taking a rigid legalistic approach, the guidance offers some flexibility. Specific policies about the duty to care should be developed within jurisdictions through dialogue and consultation with all stakeholders. These should take account of differences in expertise and skills possessed by various HCWs, although as needs develop, people may be asked to work beyond their usual responsibilities. The risks that HCWs are asked to accept should reasonably be expected to make a difference in the pandemic. This highlights the importance of policies being based on the best available evidence. Reasonable accomodations should also be made for those whose own health changes their risk, such as those who may be immunocompromised or pregnant. A mechanism should be available by which risks are distributed among individuals and groups in an equitable, fair and transparent way. Open consultation and dialogue help to show whether policies are viewed as fair and just. As HCWs accept additional risks, employers and governments have reciprocal obligations to reduce risks to HCWs. These include providing training for pandemics, implementing appropriate preventive measures, and having available the necessary equipment to respond adequately, such as personal protective equipment (PPE). When people become ill or injured through their work, treatment should be provided, as well as access to psychosocial support. To uphold the duty to not harm, employers and governments should educate HCWs on their ethical obligation to reduce the spread of infection if they become ill. These recommendations are applicable for HCWs responding to many CBRNE events. However, they were published in 2007 and their uptake has been relatively slow. For example, a CBRNE readiness survey was sent to all hospitals in Belgium and the results published in 2014. Seventy two percent of the hospitals responded, with 11% stating they had decontamination facilities close to the emergency department entrance, and 6% reported having appropriate PPE available for those doing triage and decontamination. At the same time, almost three-quarters of the facilities expressed the belief that they were ready for CBRNE events. The researchers concluded that 'There are serious gaps in hospital preparedness for CBRN incidents in Belgium' (Mortelmans et al. [@CR20], 300). Much further work is needed to engage with HCWs on the duty to treat and the practical obligations this entails. Policies should be developed based on discussions between all parties involved, including the public. At the same time, evidence needs to be collected to support related policy and practice. As changes are made, and as CBRNE events occur, data should be collected on the effectiveness of various approaches, policies and recommendations, including those related to ethical issues. This area thus overlaps with chapters in this book on research ethics so that research conducted to generate evidence is carried out in ethically appropriate ways. Ethical Virtues and the Duty to Care {#Sec8} ==================================== While codes and regulations have their place, they have limitations. In his study of trust in the helping professions, Edmund Pellegrino finds that contractual agreements tend to lead to 'ethical minimalism' ([@CR23], 79). 'The professional's necessity to efface self-interest will be blunted since legalistic and contractual relationships call upon the participants to protect their own interest, not that of the other party---except to the extent the contract requires. The impetus to do the "extra" that requires some compromise of self-interest is blunted if not destroyed entirely' (ibid.). In CBRNE crises, going the extra mile is exactly what needs to be encouraged, and this requires a return to traditional professional values and what are called ethical virtues: the personal character traits that lead people to strive for ethical ideals. When the duty to care is seen as based on ethical virtues, as opposed to on legal or contractual obligations, HCWs will be guided by their personal integrity and consciences on how to balance this duty with their other duties. Concerns have been raised that this may lead to many HCWs failing to report for work during CBRNE events. A small amount of research has been conducted on the factors that motivate people to work during CBRNE events. In one study, the leading motivational factor was a sense of duty to their profession, but other factors included concern for their family's health, personal safety and child care (Masterson et al. [@CR19]). These are very personal factors, rather than ones based on professional and legal obligations. When interviewed for *Time's* 2014 Person of the Year award, Ebola virus disease responders did not refer to regulatory or professional codes. Instead, they were motivated by personal factors. 'Ask what drove them and some talk about God; some about country; some about the instinct to run into the fire, not away.' Some were inspired by those who had come from far away. Some survived Ebola and said 'It looked like God gave me a second chance to help others' (Gibbs [@CR11]). Ways of communicating these personal ethical virtues need to be developed and included in the training of HCWs, especially those more likely to be involved in CBRNE events. The traditional way to encourage virtuous development has been through narratives (O'Mathúna [@CR21]). Stories about courage, heroism and fear in the face of life's dangers have been used throughout human history to encourage reflection on the sorts of character traits that are necessary to deal with life's risks. Such was the purpose of the ancient folk tales and more recent moral fables. The real-life stories of responders such as those recounted in the Ebola issue of *Time* can be used to foster the development of virtues like courage and volunteerism in the face of CBRNE risks (Von Drehle [@CR31]). Leadership and mentoring are other important factors in this area. Dr. Jerry Brown was the Medical Director & General Surgeon of ELWA Hospital in Monrovia, Liberia. As he helped set up an early Ebola treatment unit (ETU) he realised that his leadership and example were going to speak more loudly to the other HCWs than any policy, code or contract. 'He was now forced to ... suit up in Tyvek and go to work in the ETU. Every willing hand was needed, and the fearful staff must see that the boss had enough courage to do as much as he asked of them' (Von Drehle [@CR31]). Leadership by example is more compatible with virtue ethics than leadership by decree or contract. Those leading facilities and developing policies may at times need to take on the same risks as other front-line workers. Dr. Carlo Urbani accepted this as part of his role within the WHO and as a result became exposed to SARS and died from the infection (Ruderman et al. [@CR25]). His leadership and example can motivate others to have the moral courage to serve in the face of CBRNE risks. Conclusion {#Sec9} ========== Stories set in dramatic circumstances sometimes receive much publicity. In this way, the movie *Megan Leavy* tells the story of a US Marine and her dog Rex whose job was to search for explosives to protect their colleagues (Cowperthwaite [@CR6]). In spite of the dangers to themselves, she and other CBRNE responders enact the virtues needed to fulfil the duty to care in crisis situations. The *Time* stories thus reveal modern-day heroes who chose to act on their duty to care as a result of their virtues and in spite of their fears. It is just as important to praise those who in less dramatic ways act on the basis of their moral courage and exemplify the ethical traits needed to promote sacrificial behaviour on behalf of others and society. These are ordinary people who do extraordinary things based on virtues like courage, compassion and commitment to the good of others. As those values and virtues are promoted, the duty to care will once more be seen as part of what it means to be a virtuous healthcare worker.

1. Introduction =============== Hunting is relatively wide spread among many wild primates. Predation is mainly specific to arthropods and small vertebrates \[[@B1-animals-02-00363],[@B2-animals-02-00363]\]. Predatory behavior in wild chimpanzees has been well documented over the last 30 years \[[@B3-animals-02-00363],[@B4-animals-02-00363],[@B5-animals-02-00363],[@B6-animals-02-00363],[@B7-animals-02-00363]\]. Studies have shown that it is a systematic behavior in different populations of chimpanzees \[[@B8-animals-02-00363]\], which may occur either individually or cooperatively. Cooperative hunting has social implications since it helps to maintain and reinforce social relationships among the individuals of a group. The most commonly hunted prey is red colobus (*Procolobus*sp.) at all long-term study sites: Gombe \[[@B9-animals-02-00363]\], Mahale \[[@B10-animals-02-00363]\], Ngogo \[[@B11-animals-02-00363]\], and Taï \[[@B12-animals-02-00363]\], among others. However, hunting episodes of small prey are also well documented \[[@B13-animals-02-00363]\]. Moreover, other primate species can be hunted by chimpanzees: *Galago senegalensis* \[[@B14-animals-02-00363]\], *Papio cynocephalus* \[[@B15-animals-02-00363]\], or *Erythrocebus patas* \[[@B16-animals-02-00363]\], among others. Furthermore, some authors have observed chimpanzees using tools in attempts to extract, capture and kill prey \[[@B17-animals-02-00363],[@B18-animals-02-00363]\]. Predatory behavior in captive primates include reports of opportunistic individual predation of vertebrates by several species of the genus *Galago*, *Saguinus*, *Saimiri*, *Cebus*, *Lagothrix*, *Macaca*, *Mandrillus* and *Cercopithecus* \[[@B19-animals-02-00363]\]. This behavior has also been documented in other captive primates \[[@B20-animals-02-00363]\]. But surprisingly, evidence of predation is rarely documented in captive chimpanzees. The first evidence of this type of behavior came from recent reports \[[@B21-animals-02-00363]\]. These episodes were performed by isolated individuals, never those in groups and mainly focused on birds and small mammals. Recently, Ross and Lonsdorf \[[@B22-animals-02-00363]\] have evaluated the prevalence and nature of interactions between zoo-housed great apes and various North-American wildlife species. However, this study was carried out using a questionnaire of 14 items, but not fully describing the real behavioral observations of the great apes during the interactions toward those species. The lack of records of these kinds of episodes in captivity may be due to the fact that groups are rarely housed in large naturalistic enclosures, but instead they are located close to urban centers and the presence of native wildlife that may enter the enclosures is limited. We documented five episodes of well-recorded predatory behavior carried out by a group of chimpanzees originally coming from strongly humanized contexts and currently housed in a naturalistic environment. We consider that this report is important because the only other similar report described in the scientific literature was published by Videan and colleagues \[[@B21-animals-02-00363]\] at the Primate Foundation of Arizona, and it only referred to individual performances. At the Fundació Mona (FM), we recorded individual and social performances during hunting and in most cases they were associated with fast-moving animals. The question then remains whether non-experienced chimpanzees will exhibit predatory behavior similar to that of wild or other captive chimpanzees. 2. Material and Methods ======================= 2.1. Study Subjects and Facilities ---------------------------------- Since 2000, the Fundació Mona (Girona, northeast Spain; 41°54\'N, 2°49\'E) has devoted itself to the rescue, recovery, rehabilitation, resocialisation and sheltering of primates that have been exploited or mistreated. The FM attempts to provide these primates with better living conditions through naturalistic environments and stable social groups in order to help the animals develop behavioral patterns typical of their species, promoting their well-being and welfare. During the daytime the chimpanzees live in a naturalistic, main outdoor enclosure comprised of a total of 5,640 m^2^. The enclosure is divided into two areas: one is 2,420 m^2^ and the other is 3,220 m^2^, with a total division perimeter of 191 meters. The enclosure is surrounded by a steel fence and a 12 V electrified fence. Inside the enclosure are wooden platforms, towers and structures for climbing, resting and socializing. Two artificial water fountains provide drinking water *ad libitum*. The ground substrate is natural and characterized by a majority of Mediterranean and shoreline herbaceous vegetation. The vegetation is subject to seasonal changes and to the alteration of its physical environment by the chimpanzees. The following plant species are present: *Verbena officinalis*, *Conyza*sp., *Plantago lanceolada*, *Anthemis*sp., *Rumex obtusifolious*, *Portulaca oleracea*, *Verbascum blattaria, Taraxacum officinale* and *Polycarpon tetraphylum*, among others. The primates also have access to artificial termite mounds which are used for environmental enrichment. Occasionally, some native animals rest, feed or take up residence in or near the outdoor enclosure providing the chimpanzees an opportunity to hunt them. The most common species coming into contact with the naturalistic enclosure are: (1) Birds: House sparrow (*Passer domesticus*), Nightingale (*Luscinia megarhynchos*), Grey Heron (*Ardea cinerea*), European robin (*Erithacus rubecula*), Yellow-legged Gull (*Larus michahellis*), Black Kite (*Milvus migrans*), Hoopoe (*Upupa epops*), Common Blackbird (*Turdus merula*), European magpie (*Pica pica*), and Common Kestrel (*Falco tinnunculus*); (2) Mammals: European hedgehog (*Erinaceus europaeus*), European rabbit (*Oryctolagus cuniculus*), wood mouse (*Apodemus sylvaticus*), Brown rat (*Rattus norvegicus*), and European Mole (*Talpa europaea*); and (3) Amphibians and reptiles: grass snake (*Natrix natrix*), ladder snake (*Rhinechis scalaris*), Mediterranean tree frog (*Hyla meridionalis*), Perez's frog (*Rana perezi*), Common toad (*Bufo bufo*), and the Iberian wall lizard (*Podarcis hispanica*); among others. At the beginning of this study in January 2008, the sample group consisted of thirteen individuals, separated into two different groups: group A (Male group) and group B (Family group). The group of chimpanzees is formed of nine males and four females, but only 10 of them took part in the activities described here. They range from 4 to 51 years old. Between 2008 and 2011 some animals changed groups and in 2010 one of the subjects (Romie from the Family group) passed away due to natural causes. A new individual (Africa) was also introduced to group B in 2010. [Table 1](#animals-02-00363-t001){ref-type="table"} shows the additional traits of these individuals: sex, age, place of birth and origin. The chimpanzees are fed juices, fresh fruit, fresh vegetables, boiled rice, nuts and other seeds four times per day and water is always available. During a routine day, animals forage and are fed food which is hidden or scattered throughout the naturalistic enclosure. We conducted this research in accordance with all national and institutional guidelines for the care and management of primates established by FM. animals-02-00363-t001_Table 1 ###### Additional traits of the Fundació Mona (FM) chimpanzees: sex, age, place of birth and origin. Year arrived at Mona Foundation Name Sex Birth date Birth place Background Group Participation in hunting --------------------------------- --------- -------- ------------ ------------- ------------------ ------- -------------------------- 2009 África Female 2000 Wild Pet B YES 2002 Bongo Male 2000 Captivity Circus B/A YES 2001 Charly Male 1989 Captivity Circus, TV A YES 2003 Juanito Male 2003 Captivity Pet B YES 2001 Marco Male 1984 Captivity Circus, TV A YES 2004 Nico Male 2001 Captivity Pet B YES 2001 Romie Female 1979 Wild Breeding, Circus B YES 2004 Sara Female 1998 Captivity Pet, TV B YES 2005 Tico Male 1985 Captivity Pet, Zoo B NO 2001 Toni Male 1983 Wild TV, Zoo A NO 2003 Toto Male 1956 Wild Pet, Zoo B YES 2006 Víctor Male 1982 Wild Pet, Zoo B NO 2002 Waty Female 1996 Captivity Circus B YES 2.2. Behavioral Observations ---------------------------- Five cases of prey capture were observed between January 2007 and September 2011. The episodes of predation were observed during our ongoing Hand Laterality Research Project, which began in 2002 \[[@B23-animals-02-00363],[@B24-animals-02-00363],[@B25-animals-02-00363]\] and our on-going Rehabilitation and Resocialisation Project which began in 2006 \[[@B26-animals-02-00363],[@B27-animals-02-00363]\]. All five predation episodes presented here were recorded while the researcher was present. This factor does not interfere with the results since all the chimpanzees are acclimated to the presence of the FM researchers. Three of the episodes were video and photographically recorded (episodes ID H1, H3 and H4). Two of the episodes were only photographically recorded (episodes ID H2 and H5). Data were recorded on an *ab libitum* basis during the predation events. Species of the animal prey, date, and time were recorded. Animals involved in the predation were also described. Regarding the H1 episode (social hunting), independent observers describe and interpret this observation the same way the original observer (MV) did. This event was videotaped, so it would be possible to test observer reliability by having three naïve observers (unaware of the original description and interpretation). 3. Results ========== Between 2007 and 2011, five occurrences of predatory behavior by the FM chimpanzees were directly recorded ([Table 2](#animals-02-00363-t002){ref-type="table"}). Each of the predatory events happened during the daily routine activities of the group and all of the cases below are described in chronological order. In the first episode, the entire group was involved in the predation (social hunting), while in the other episodes the predation incident was performed individually. Although some other predation incidents occurred during this five-year period, we only registered those in which one of the researchers was present from the beginning to the end of the episode (observed kills). During the study period we discovered some post-kill evidence (n = 10 species) but we discarded it because we did not have specific information about how it was produced. The post-kill evidence refers mainly to the following species: *Passer domesticus*, *Apodemus sylvaticus*, *Natrix natrix*, *Hyla meridionalis*, *Rana perezi*, and *Bufo bufo*([Table 3](#animals-02-00363-t003){ref-type="table"}). animals-02-00363-t002_Table 2 ###### Prey captured by FM chimpanzees between 2007 and 2011. ID Date Species Social/Individual N° individuals involved Predatory strategy Prey consumption System record Chimpanzee group ---- ------------ ------------------------- ------------------- ------------------------- -------------------- ------------------ ----------------- ------------------ H1 2007-01-19 *Oryctolagus cuniculus* Social 4 Coordinated Try Video B H2 2008-05-24 *Turdus merula* Individual 1 Individual Yes Photo B H3 2008-08-02 *Larus michaellis* Social 3 Individual Try Video and photo A H4 2010-10-29 *Oryctolagus cuniculus* Individual 2 Individual Yes Video and photo A H5 2011-09-17 *Oryctolagus cuniculus* Individual 1 Individual Try Photo B animals-02-00363-t003_Table 3 ###### Native wildlife at FM with killing and post-killing evidence. Species Common name Evidence of hunting Chimpanzee group ------------------------- ------------------------- --------------------- ------------------ *Passer domesticus* House sparrow Post-kill A/B *Luscinia megarhynchos* Nightingale No *Ardea cinerea* Grey Heron No *Erithacus rubecula* European robin No *Larus michahellis* Yellow-legged Gull Kill A *Milvus migrans* Black kite No *Upupa epops* Hoopoe No *Turdus merula* Common Blackbird Kill B *Pica pica* European magpie No *Falco tinnunculus* Common Kestrel No *Erinaceus europaeus* European hedgehog Post-kill A/B *Oryctolagus cuniculus* European rabbit Kill A/B *Apodemus sylvaticus* Wood mouse Post-kill A/B *Rattus norvegicus* Brown rat No *Talpa europaea* European mole Post-kill A/B *Natrix natrix* Grass snake Post-kill A *Rhinechis scalaris* Ladder snake Post-kill A *Hyla meridionalis* Mediterranean tree frog Post-kill A/B *Rana perezi* Pere\'s frog Post-kill A/B *Bufo bufo* Common toad Post-kill A/B *Podarcis hispanica* Iberian wall lizard Post-kill A/B 3.1. Case 1 (H1)---19 January 2007. Social Predation of a Rabbit (Oryctolagus cuniculus) ---------------------------------------------------------------------------------------- This episode was recorded on 19 January 2007. It started at 17:10. Four chimpanzees took an active part in the predation: two juvenile females, one young male and one juvenile male ([Figure 1](#animals-02-00363-f001){ref-type="fig"}). A rabbit (*Oryctolagus cuniculus*) came into the enclosure by chance. It was finally captured by the four chimpanzees after two attempts in which the hunters took on different roles in the action. The first attempt was made by three chimpanzees (one juvenile male and two juvenile females). The male (Bongo) pursued the prey while the females (Sara and Waty) positioned themselves for an ambush to block the rabbit's escape route and to capture the animal ([Figure 1](#animals-02-00363-f001){ref-type="fig"}). The second attempt was performed by all four chimpanzees (one young male, one juvenile male and two juvenile females). One of them (Juanito) performed the role of pursuer while the others (Waty, Sara and Bongo) set up an ambush in a semicircular position, blocking and capturing the prey. Once the hunting episode was finished, the other three chimpanzees joined the group: one adult female (Romie), one adult male (Toto) and one young male (Nico). All the individuals gathered around the rabbit, which was taken by its hind legs by the oldest chimpanzee (Toto) and beaten once against the ground with one fatal blow. Immediately, a sequence of exploration and brutal play behaviors started among the five juvenile and young individuals ([Figure 2](#animals-02-00363-f002){ref-type="fig"}). The prey was not consumed, although some individuals tried to bite it. {#animals-02-00363-f001} {#animals-02-00363-f002} 3.2. Case 2 (H2)---24 May 2008. Individual Predation of a Common Blackbird (Turdus merula) ------------------------------------------------------------------------------------------ This episode was recorded on 24 May 2008 between 15:46 and 15:59. Only one chimpanzee (Nico, male adolescent) took part in the episode. No other group individual was present during the predation episode and the prey manipulation. The capture of the prey was done opportunistically while the prey was on the ground. The prey was a young individual from the *Turdus merula* species. Nico hunted the prey with his hands and killed it with a blow. For five minutes Nico manipulated the prey in a playful manner. Later, using his mouth, he tore open the prey's abdomen and ingested part of the guts, ignoring the wings, head and lower extremities ([Figure 3](#animals-02-00363-f003){ref-type="fig"}). After the episode the prey was left in the same place. {#animals-02-00363-f003} 3.3. Case 3 (H3)---2 August 2008. Individual Predation of a Yellow-Legged Gull (Larus michahellis) -------------------------------------------------------------------------------------------------- The episode was recorded on 2 August 2008 between 17:22 and 17:38. Three individuals were involved in the hunting episode. An adult *Larus michahellis* specimen remained in the artificial pool of the enclosure. Charly (Group A, adult male) captured the prey with his hands. We neither recorded the killing strategy nor who was the actual killer. Immediately afterwards Marco (*Alpha*male) took hold of the already dead prey and carried it to one of the enclosure towers accompanied by Bongo (adolescent male) and by Charly ([Figure 4](#animals-02-00363-f004){ref-type="fig"}(a)). Charly left the tower after one minute. Bongo and Marco remained together until the end of the episode. From 17:25 to 17:27, Marco examined the prey. During this time, Bongo simply observed without taking part. At 17:28, Bongo struck the head of the prey with his hands. At 17:29 Marco began ripping off one of the lower limbs of the prey ([Figure 4](#animals-02-00363-f004){ref-type="fig"}(b)). At 17:32 he bit the limb of the prey to pull it apart ([Figure 4](#animals-02-00363-f004){ref-type="fig"}(c)). Marco ended up abandoning the prey in the tower structure. {#animals-02-00363-f004} 3.4. Case 4 (H4)---29 October 2010. Individual Predation of a Rabbit (Oryctolagus cuniculus) -------------------------------------------------------------------------------------------- The episode was recorded on 29 October 2010 between 14:45 and 14:55. Marco (Group A, adult male) was involved in the episode but we neither detected the predation strategy, the exact moment of predation, nor exactly how he hunted. We photographically recorded the episode from 14:49 ([Figure 5](#animals-02-00363-f005){ref-type="fig"}(a)). At that moment, the prey already appeared ripped apart with parts of its body missing, presumably ingested ([Figure 5](#animals-02-00363-f005){ref-type="fig"}(b)). At 14:55, the prey was abandoned on the enclosure substrate. {#animals-02-00363-f005} 3.5. Case 5 (H5)---17 September 2011. Individual Predation of a Rabbit (Oryctolagus cuniculus) ---------------------------------------------------------------------------------------------- An episode was registered on 17 September 2011 between 12:20 and 12:30. Juanito (Group B, male adolescent) was the subject most implicated in the episode but we neither detected his predation strategy, the exact moment of predation, nor exactly how he hunted. Two other individuals (Nico, male adolescent) and (Africa, female adolescent) played secondary roles in the episode. At 12:22, we started to photographically record the hunting episode. At 12:23, the prey was placed on the ground and Juanito took hold of a wooden stick to use it as a spear and began to repeatedly stab the rabbit with it ([Figure 6](#animals-02-00363-f006){ref-type="fig"}). At 12:24 Nico rejoined Juanito. At that moment, Juanito approached the perimeter of the enclosure and threw the rabbit against the electrified fence. At 12:26 he returned transporting the prey in his mouth ([Figure 7](#animals-02-00363-f007){ref-type="fig"}(a)). The abdomen of the rabbit was totally open with the intestines missing. One of the lower limbs was missing its muscular mass (possibly due to ingestion) and it was possible to see exposed bone. At 12:30 Juanito returned holding the prey and brought it up to his mouth, tasting it but without totally ingesting it ([Figure 7](#animals-02-00363-f007){ref-type="fig"}(b)). Finally, the prey was discarded on the ground. {#animals-02-00363-f006} {#animals-02-00363-f007} 4. Discussion ============= Predation on fast-moving animals (*i.e.*, rabbits) or large birds (*i.e.*, seagulls) has not been documented in captive chimpanzees despite several decades of observation of these animals in captive and naturalistic settings. The only report about hunting behavior in captive chimpanzees was published by Videan and colleagues several years ago \[[@B21-animals-02-00363]\]. Even rehabilitated animals and non-experienced subjects, when given the opportunity will hunt and occasionally partially consume prey items. Although wild chimpanzees rarely capture an animal without eating it, in both the wild and in captivity, some individuals (such as at FM) hunt but do not eat prey \[[@B28-animals-02-00363]\]. Once they have killed, some wild chimpanzees prefer to play with rather than consume their prey \[[@B29-animals-02-00363]\] as we reported in all five episodes at Mona. Wild bonobos (*Pan paniscus*) also showed similar exploration and grooming following their capture of infant monkeys which were handled and manipulated like dolls \[[@B30-animals-02-00363]\]. At FM the prey was not eaten entirely but partially consumed \[[@B14-animals-02-00363]\]. According to the roles, juveniles and adolescent individuals showed more interest in the prey, although some adult subjects participated in the hunting events as well. In wild settings the active role of males during the "hunting but not eating" episodes was not found \[[@B28-animals-02-00363]\]. In the case of bird predation we found some similarities between FM and wild chimpanzees. In Bossou \[[@B28-animals-02-00363]\] and in Mahale \[[@B31-animals-02-00363]\] the catching of birds is mainly solitary and opportunistic, not requiring social hunting, and the performance and strategy tend to be repeated in subsequent occasions. From our point of view, the present findings shed light on the behavioral recovery and diversity of rehabilitated chimpanzees and on the ability of these animals to develop species-typical behaviors in these types of naturalistic enclosures, although unusual in that they did not eat much of the meat. Furthermore, we hypothesize that predation could be a form of boredom alleviation despite the extensive methods of environmental enrichment engaged by Fundació Mona \[[@B27-animals-02-00363],[@B32-animals-02-00363],[@B33-animals-02-00363],[@B34-animals-02-00363],[@B35-animals-02-00363]\]. In fact, although with a decreasing trend, inactivity is the most frequent behavior in the study sample \[[@B36-animals-02-00363]\]. It is worth emphasizing that the H1 episode is the only social (likely cooperative) hunting event documented in a naturalistic environment to date. The FM chimpanzees mainly come from isolated environments, born in captivity or captive since youth. Therefore, either by mutual learning during their time spent at the FM or by innate nature, hunting seems to be both common and frequent in naturalised environments. Another plausible interpretation can be found for the social hunting episode. Captive chimpanzees can intercept moving targets (*i.e.*, catch them by moving to a spot where their target will predictably move). So, the behavior described here as cooperative "ambushing" might instead be individual efforts to catch the rabbit independently by interception. Furthermore, two other questions emerge from the evidence described here. Firstly, concerning the roles established by the chimpanzees in social hunting episode (H1), some individuals pursued the prey while others set up ambushes to block and capture it. These roles directly indicate spontaneous social organization for some tasks. However, hunting in FM chimpanzees appears mostly solitary and associated with males and adolescent subjects. Secondly, in the wild predatory behavior usually consists of both hunting and consuming. However, the animals involved in episodes H2, H3, H4 and H5 were partially eaten, but the rabbit from the social episode (H1) was not eaten at all. Therefore, it seems that hunting and consuming may occur and function separately. Also, it is worth noting that in H1, the chimpanzee that took charge and beat the prey before any of the other individuals was the oldest individual (Toto) who did not take part in the hunting episode. Further research will study whether the other chimpanzees implicitly accepted his higher authority in terms of his age. If so, and similar to the wild, predation in captive chimpanzees may be related to the maintenance and demonstration of social relations. This study also has some important implications for the surveillance of outdoor enclosures (and other types of facilities) and the potential for disease or introduction of parasitic infection by captured prey such as: *Cheyletiellosis dermatitis*\[[@B37-animals-02-00363]\], *Fancisella tularensis*\[[@B38-animals-02-00363],[@B39-animals-02-00363]\], *Salmonella*sp. \[[@B40-animals-02-00363],[@B41-animals-02-00363]\] or *Entamoeba histolytica* \[[@B42-animals-02-00363]\], among others. 5. Conclusions ============== We emphasize the following conclusions: (1) predatory events are an important element of chimpanzee's natural behavioral repertoire and should be considered as part of the design of enriched environments; (2) co-operative hunting roles for large prey are an emergent feature of chimpanzee social structures even in small groups; (3) predation may serve important social functions or alleviate boredom; and (4) females are active participants in hunting episodes, challenging some of the hunter/gatherer hypotheses regarding primate and hominin evolution. Finally, we want to underscore the importance of naturalistic outdoor enclosures to enhance species-typical behavioral patterns and welfare \[[@B43-animals-02-00363],[@B44-animals-02-00363]\]. This evidence highlights the significance of comparing the behavior of wild and semi-captive chimpanzees, particularly where hunting is concerned. We consider that further research and monitoring is needed to systematically assess the strategy, consumption and performance of predatory behavior in captive rehabilitated chimpanzees. This study has been supported by both the Spanish Ministerio de Ciencia e Innovación (MICIIN-HAR2009-07223/HIST) and the Universitat Rovira i Virgili (URV2009AIRE-05). David Riba's research is funded by Fundación Atapuerca. Our thanks to Charles Duke and Carl Abrahams for English revision of this article and to the whole FM keeper team. We are also grateful to Richard Wrangham for his helpful comments and suggestions. The authors declare no conflict of interest.

Introduction {#s1} ============ The emergence of molecular therapies for inherited retinal degenerations has highlighted the need for imaging modalities that can sensitively identify structural disease progression. One such technique is short-wavelength fundus autofluorescence (AF) using confocal scanning laser ophthalmoscopy (cSLO), a noninvasive method that evaluates retinal disease. Increased AF is believed to indicate regions of elevated fluorophores, primarily lipofuscin, a product of photoreceptor outer-segment disc shedding and phagocytosis,^[@i2164-2591-5-6-10-b01],[@i2164-2591-5-6-10-b02]^ while decreased AF may signify obstruction of the autofluorescent signal or atrophy of the underlying retinal pigment epithelium (RPE) and/or photoreceptors.^[@i2164-2591-5-6-10-b03][@i2164-2591-5-6-10-b04]--[@i2164-2591-5-6-10-b05]^ In retinitis pigmentosa (RP), AF has been recognized to identify a parafoveal ring of increased AF in some patients^[@i2164-2591-5-6-10-b06]^ and varying degrees of patchy hypofluorescence within the periphery. Classically, a region of preserved normal central AF is surrounded by an annulus of increased AF, which delineates disruption of the photoreceptor inner segment and outer-segment (IS/OS) junction on spectral-domain optical coherence tomography (SDOCT).^[@i2164-2591-5-6-10-b07],[@i2164-2591-5-6-10-b08]^ Gradual constriction of the hyperfluorescent annulus occurs as the disease progresses, while in the peripheral retina, hypofluorescent lesions of varying size have been shown to act as a surrogate for duration of disease.^[@i2164-2591-5-6-10-b09]^ In view of these findings, areas of both hyperfluorescence and hypofluorescence can be combined and analyzed as a useful disease biomarker. Studies exploring the implications of increased AF in retinal dystrophies have demonstrated that hyperfluorescent demarcation arcs correlate with functional parameters. In patients with electrophysiologically confirmed RP, pattern electroretinography (PERG) P50 amplitude is strongly associated with the radius of the hyperfluorescent annulus among patients with RP and normal visual acuity.^[@i2164-2591-5-6-10-b10]^ Psychophysical testing has shown a strong association between both automatic and kinetic perimetry with the parafoveal ring radius, although this was only pertinent in those without any atrophic disease within the vascular arcades, suggesting that AF evaluation may be more useful in early disease.^[@i2164-2591-5-6-10-b09],[@i2164-2591-5-6-10-b11]^ Similar results have been found with microperimetry.^[@i2164-2591-5-6-10-b12]^ Such correlation suggests that AF may provide a useful additional end point in illustrating disease progression. Most published work regarding quantitative AF utilizes luminance data, but special adaptations and processing are required. Delori et al.^[@i2164-2591-5-6-10-b13]^ described a powerful technique where highly repeatable measures of AF can be achieved through installation of an internal fluorescent reference to obviate concerns over cSLO gain sensitivity and laser variation. Alternatively, area measurements are utilized, such as in Best vitelliform macular dystrophy or Stargardt disease; however, these are not appropriate for RP as this may omit part of the disease process.^[@i2164-2591-5-6-10-b14],[@i2164-2591-5-6-10-b15]^ It is not always possible to capture images in a routine clinical setting in a way that is compatible with the quantitative analysis techniques reported. Moreover, the internal fluorescent reference is not commercially available. We included AF analysis in the observational arm of a clinical trial assessing the impact of transcorneal electrical stimulation in participants with RP. As part of that study, we were able to rigorously assess AF changes in the control eyes of participants at regular time points. This provided insight into the baseline characteristics of AF in RP and most importantly on the reproducibility of serial AF analysis in individual patients over a period of time. Here, we report a novel method for serially evaluating AF in patients with RP, which utilizes luminance values in order to interpret distance data and is therefore less influenced by raw values, which might vary from session to session. This method does not require the installation of additional equipment to the cSLO. Such a method could inform patient prognosis through revealing the rate of AF loss as well as provide a useful structural trial end point in subsequent studies. Methods {#s2} ======= Participants with confirmed RP were recruited into an open-label observational trial assessing a novel therapy for RP (NCT01847365) at the Oxford University Eye Hospital and Moorfields Eye Hospital. Participants underwent clinical assessment and AF imaging at the baseline visit and each subsequent 3 months for a total of 1 year. Autofluorescence images of the control eye of a subset of five participants were analyzed to inform the development of this methodology. The study was approved by the National Health Service (NHS) Research Ethics Committee and received institutional approval from the University of Oxford Clinical Trial Research and Governance Unit (12/SW/0293). The study protocol was carried out in accordance with the Declaration of Helsinki (2008) and good clinical practice. All clinical trial participants provided informed consent prior to enrolment. Pupil mydriasis was achieved through administration of 2.5% phenylephrine and 1% tropicamide. Autofluorescence images with a 55-degree field were acquired using a confocal scanning laser ophthalmoscope (Heidelberg Spectralis; Heidelberg-Engineering, Heidelberg, Germany) with an excitation filter of 488 nm in automatic real-time mode, using a minimum average of 25 scans. A standard corneal keratometry (K) measurement of 7.70 was used for each patient. Images were exported as TIFF files in an anonymized format. Geographic Information Systems (GIS) Extract Transform Load software (FME, Safe Software Inc., Surrey, BC, Canada) was then used to evaluate the AF images to allow automated analysis to create a number of outputs. The images were first georeferenced ("georeferencing" is the GIS term for aligning images consistently with a known base) by ensuring the fovea on each AF image was given coordinates of 0,0. The image was then scaled to place the center of the optic cup at a constant x coordinate of 200 units (equivalent to 300-μm distance). Following this, the image was then rotated around the central coordinates (0,0) to give the optic cup center marking a y coordinate of 0, thus giving the optic cup final coordinates of 200,0 consistently in all images. Although it is appreciated that the fovea is inferior to the optic disc, this distance can vary with refractive status, and the direct linear distance at the 200,0 position provides consistent disc--fovea alignment for all images. As the rotation involves interpolation, the red and yellow marks were removed from the images prior to this to stop data contamination. Following the rotation, all image pixels were converted into a square polygon, termed a cell, which contained the hyperfluorescence value for the pixel from which it was derived ([Supplementary Fig. 1](#i2164-2591-5-6-10-s01){ref-type="supplementary-material"}). Four standardized bisecting lines were created, one each for vertical and horizontal and the two diagonals, to create an eight-point star, with the center of the point being 0,0, where it intersected the fovea ([Supplementary Fig. 1B](#i2164-2591-5-6-10-s01){ref-type="supplementary-material"}). The lines were of a defined length and were used for each image to ensure consistency of the area being analyzed across patients and over time. Using a spatial filter, each bisecting line was spatially compared to the cells, and any cells that intersected it (overlapped, touched, contained, crossed, or within) were kept in the sequence they appeared from start to end along the bisecting line. The maximum hyperfluorescence value (\_max) across all cells from each image was then extracted and used as the reference. In order to improve the consistency of analysis of the ring, a series of image refinements were applied to each image. First, a visualization output was created that demonstrated the extent of the hyperfluorescent ring. The cells were then colored depending on whether they were within a varying percentage of the \_max value (set to green) or not (set to red), that is, within that percentage of the maximal luminance value in the image. The green cells were also buffered to make them larger and more obvious before all the cells were output on top of the georeferenced image. The percentage was adjusted between 60% of the maximum luminance to 90% of the maximal luminance in 5% increments, and all input images were processed for each percentage. The resulting images were examined by clinicians to decide which percentage best captured the extent of the hyperfluorescent ring across the cohort, the final decision being 70%, as it equated to a threshold where six of eight meridians were captured in all participant images ([Fig. 1](#i2164-2591-5-6-10-f01){ref-type="fig"}). The pattern of change over time for each percentile is shown in [Figure 2](#i2164-2591-5-6-10-f02){ref-type="fig"}. {#i2164-2591-5-6-10-f01} {#i2164-2591-5-6-10-f02} With the 70% chosen as the cutoff percentage, the FME workspace was expanded. Another output was created that started by removing all cells that had a hyperfluorescence value of less than 70% of the \_max value in order to leave only those points representing the hyperfluorescent ring. Of the cells that passed this test, the distance was calculated in micrometers from the cell to the fovea. This was achieved by standardizing the distance between the optic nerve and fovea to a distance of 300 μm. As the fovea splits each bisecting line in half following the georeferencing process described above, this resulted in distances from eight transects to the middle, each separated by 45 degrees and centered on the fovea. Along each half line, the nearest and farthest cell distances were extracted and the midpoint between them calculated by dividing the difference between them by two. This number represents the distance from the fovea to the hypothetical midpoint of the hyperfluorescent ring at the fovea. By summing together the two midpoint distances for each half of their respective line, a single value was determined that represented the diameter of the midsection of the hyperfluorescent ring on that bisecting line. Finally, the georeferenced image was taken and clipped to a circle, centered on the fovea that was slightly wider than the optic cup ([Supplementary Fig. 2](#i2164-2591-5-6-10-s01){ref-type="supplementary-material"}). This was to ensure the same area of the image and identical features were being analyzed across the cohort regardless of the centration of the original image as shown in [Supplementary Fig. 2](#i2164-2591-5-6-10-s01){ref-type="supplementary-material"}. The luminance value of each pixel was measured, and the percentage of pixels with a value within the lowest 10% was returned. This percentage, highlighted in green, was taken to indicate the degree of atrophy and the optic disc. The optic disc was assumed not to change significantly over this time period, so any change detected in serial analysis can be attributed mainly to progression of atrophy. These processes were conducted on a series of images from six eyes with three different sensitivity measures for each eye. This was conducted to assess the robustness of the measurements in images of varying quality to reflect a routine clinical scenario. Repeated measures ANOVA was used to investigate statistical significance. Results {#s3} ======= The AF images of the control eye for three male and two female participants, aged 18 to 56, with confirmed rod--cone dystrophy over a 12-month period were included ([Table](#i2164-2591-5-6-10-t01){ref-type="table"}). Participant A, B, and C had well-defined parafoveal annuli of increased AF and varying degrees of decreased patchy AF in the periphery. Participant D had advanced rod--cone dystrophy with significant speckled foveal hyperfluorescence. Participant E had a diffuse region of increased AF parafoveally with scalloped peripheral areas of decreased AF ([Fig. 3](#i2164-2591-5-6-10-f03){ref-type="fig"}). ###### Demographics and Mutational Analysis Results of the Five Participants  {#i2164-2591-5-6-10-f03} Sensitivity was set automatically in order to optimize the image captured. To assess the extent to which the sensitivity value impacts the measurements described from AF images, a subset of participants underwent serial testing with three different sensitivity values: 80% to 89%, termed dim; 90% to 99%, termed mid; and 100% to 107%, termed bright. The results are shown in [Supplementary Table 1](#i2164-2591-5-6-10-s01){ref-type="supplementary-material"}. Repeated measures ANOVA showed a significant difference between AF images captured at dim versus mid conditions (*P* = 0.03), while those taken at mid sensitivity were not significantly different from bright images (*P* = 0.32). Similar results were found when highlighting areas below the 10th percentile of brightness---dim versus mid sensitivity neared statistical significance (*P* = 0.09), while those between mid and bright conditions did not (*P* = 0.21). Autofluorescence images over a 1-year period underwent serial analysis at baseline, 6 months, and 12 months for change in AF. [Figure 2A](#i2164-2591-5-6-10-f02){ref-type="fig"}--[E](#i2164-2591-5-6-10-f02){ref-type="fig"}, displays serial analysis at each of the cutoff points. The 70% cutoff displays the most stable results in all the participants and displays small error bars when the results are combined from the participants ([Fig. 2F](#i2164-2591-5-6-10-f02){ref-type="fig"}). Standard deviation of mean radius was 486 μm at 70% versus the highest error at 65% (536 μm). Average constriction of the ring in each meridian at the 70% cutoff was then calculated to provide a mean radial constriction measurement. There was a general constriction in the size of the ring of increased AF over the 1-year duration. Mean change in annulus radius for participant A was −232.7 μm; participant B, −95 μm; participant C, +8.0 μm; participant D, +163 μm; and participant E, −55.4 μm. Collated values for each time point are shown in [Figure 4](#i2164-2591-5-6-10-f04){ref-type="fig"} with no statistically significant difference between time points (*P* = 0.93). {#i2164-2591-5-6-10-f04} Changes in regions of AF below the 10th percentile were evaluated in the same five participants and shown in [Figure 5](#i2164-2591-5-6-10-f05){ref-type="fig"}. Area of the image below the 10th percentile increased in five participants, A, B, C, D, and E, by 0.23%, 3.05%, 1.82%, 1.56%, and 17.7% respectively. {#i2164-2591-5-6-10-f05} Discussion {#s4} ========== With potential treatments such as gene therapy, now entering clinical trials for inherited retinal dystrophies, there has been an impetus to develop robust methods of analyzing disease progression. Autofluorescence may provide additional insight into the structural changes occurring following an intervention and act as an additional clinical trial end point. Our proposed technique uses a pixel-by-pixel percentile grading system to identify desired regions above or below a defined threshold. Georeferencing of the image and establishment of the fovea as the central coordinate in a standard Cartesian plane allows comparison within the same patient between different images while obviating major concerns with variability in image magnification. In our study, the 70th percentile (i.e., all points within 70% luminance of the brightest point in the image) was chosen as the threshold that best highlighted the hyperfluorescent annulus when considering the presence of false positives and false negatives. Despite tailoring the choice of percentile in this study, there were unavoidable points of artefactual fluorescence, and conversely, regions of the AF ring could be missed as the level of AF varies along the circumference. However, the percentile can be adjusted between subjects to optimize capture of the AF ring provided that the same level is used within images of the same individual. Note that as the percentile threshold was increased, the annulus radius decreased. We hypothesize that this may reflect the centripetal progression of the hyperfluorescent annulus where the region of greatest hyperfluorescence lies along the inner aspect. We have also shown the measurement method is robust to changes in the sensitivity setting when capturing images for all values ≥90. This can be used to guide future photography. This methodology leads to reproducible measures between visits, allowing for quantification of disease change. It has been shown that AF can vary among healthy individuals depending on race, sex, and age.^[@i2164-2591-5-6-10-b16]^ This method relies on percentile grading rather than absolute luminance values, mitigating the impact of the general background AF of the subject. Our study benefited from a number of consistencies---device sensitivity did not vary significantly, and images were acquired by the same two experienced operators throughout the study. Subjects had good fixation and were familiar with the device. Exclusion criteria were also such that participants had no other significant retinal pathology besides RP and no substantial media opacity. Undoubtedly, the presence of cataract or corneal pathology would impair the quality of AF while epiretinal membrane and cystoid macular edema might confound pixel capture. Mapping of the AF ring was achieved through four major meridians of equal length traversing the fovea and at 45 degrees to each other, forming a regular octagram star polygon. Thus, the predominant direction of disease progression could be mapped such that superior ring constriction may alert one to the deterioration of the inferior visual field. In our cohort, participant A had greatest reduction in annulus radius in the temporal region, whereas the superonasal aspect was greatest in participant B. There are a plethora of options around meridian position and number: Cases of sectoral RP could be described with further capture lines in the appropriate sector. Similarly, the method could be extrapolated to form longer meridians in wide-field imaging. In theory, the progression of mid-peripheral granular hypofluorescence could also be measured. The potential of this technique can expand beyond rod--cone dystrophies. Holz et al.^[@i2164-2591-5-6-10-b17],[@i2164-2591-5-6-10-b18]^ have shown that the boundaries of geographic atrophy secondary to age-related macular degeneration, which demonstrate increased AF, are more likely to progress. Setting an appropriate percentile threshold could quantify the advancement of the lesion. Our study assessed multiple AF imaging timepoints in detail over a 12-month period in the control arm of an RP interventional trial. Twelve months is a relatively short period in the progression of RP, thus allowing us to explore the repeatability of the method. This was shown effectively over each 6-month period. Although our primary objective was to show reproducibility of the methodology, our study mirrored previous reports in demonstrating that serial AF analysis in RP identifies a progressive non--statistically significant constriction of the hyperfluorescent parafoveal ring toward the end of the observation period.^[@i2164-2591-5-6-10-b19][@i2164-2591-5-6-10-b20]--[@i2164-2591-5-6-10-b21]^ Despite the short duration of our study, the mean annular radius constricted in three subjects. Change in the diameter of the hyperfluorescent ring has important implications as numerous groups have reported on the functional correlation of its constriction. Structurally, the peak AF within the ring is known to correspond to fragmentation of the IS/OS region on SDOCT and demarcates central preservation of this junction and loss peripherally.^[@i2164-2591-5-6-10-b07],[@i2164-2591-5-6-10-b08]^ Correlates with the PERG, multifocal electroretinography, and psychophysical testing are also well described.^[@i2164-2591-5-6-10-b09],[@i2164-2591-5-6-10-b10],[@i2164-2591-5-6-10-b12]^ Autofluorescence cannot replace these other measures but provides an additional objective adjunct in assessing disease progression and potential response to therapeutic intervention. Retinal pigment epithelium atrophy, as illustrated by regions of decreased AF, was quantified by setting a percentile grading to 10% of the image\'s overall fluorescence. Again, this number can be adjusted as required. For instance, a lower threshold could be set to isolate the central homogenous region of decreased AF from the peripheral granular decreased AF that accompanies certain Stargardt disease phenotypes. Our patients all demonstrated reproducible degrees of atrophy throughout the 12-month period, with marked progression in participant E between 0 and 6 months. There are a number of limitations with our methodology. There could theoretically be substantial bias if the detector gain were altered, although the impact would be greater on absolute luminance values than a percentile grading system. Mapping of the AF ring is facilitated by a clearly delineated change in AF, which is not present in all cases of RP. Furthermore, as the ring approaches the fovea in more advanced disease, it becomes less defined, which could complicate its capture. Our study describes a straightforward method for serially analyzing AF images in subjects with RP by utilizing a percentile grading system. It can be applied retrospectively to AF images in a clinical setting and adjusted accordingly for quantifying the hyperfluorescent annulus and peripheral hypofluorescence characteristic of many patients with RP. The technique is most informative in subjects with earlier disease as demarcation arcs retain greater definition. Further work will elucidate how quantifiable progression of AF patterns correlates with or predicts subsequent functional deterioration. Moreover, there is potential for application of this technique in other retinal dystrophies and other imaging modalities. Supplementary Material ====================== ###### Click here for additional data file. With thanks to Jon Brett, medical photographer, for providing the additional serial image sets for further analysis. Supported by the Oxford Biomedical Research Centre based at Oxford University Hospitals NHS Trust, the University of Oxford, and Okuvision, with research infrastructure support from the National Institute for Health Research (NIHR) as a Clinical Research Network Portfolio-adopted study. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR, or the Department of Health. The sponsor and funding organization had no role in the design or conduct of this research. Disclosure: **J.K. Jolly**, R; **S.K. Wagner**, None; **J. Moules**, S; **F. Gekeler**, Okuvision GmbH (P); **A.R. Webster**, None; **S.M. Downes**, None; **R.E. MacLaren**, None

TO THE EDITOR ============= Ikeda T *et al* (2019)[@r1] recently published an article about EBV-positive mucocutaneous ulcers (EBVMCU) in the *Journal of Clinical and Experimental Hematopathology*. The authors explained many clinical and pathological aspects of this disease which pathologists must be attentive to during their diagnostic routine. At the end of the article,[@r1] the authors pointed out that clinicians and pathologists need to be able to distinguish EBVMCU from lymphomas, especially classic Hodgkin lymphoma, because of the Reed-Sternberg cells with positivity for CD30. Although malignancies are important, we must not forget benign lymphoproliferations that may be a differential diagnosis for EBVMCU. The oral cavity is a good example to discuss benign and malignant differentials for EBVMCU. To that end, we wrote this letter and used two real examples of our diagnostic routine. The diagnosis of ulcerative lesions affecting the oral mucosa is usually difficult because different processes may share a similar clinical appearance, such as infectious disorders, autoimmune diseases and tumors, especially oral squamous cell carcinoma.[@r2] In this context, a biopsy may be necessary to rule out a neoplasm. Microscopically, the occasional observation of large atypical lymphoid cells could represent a lymphoproliferative disorder. Although rare, accounting for 5% of all head and neck malignancies,[@r3] oral lymphomas should be included in the differential diagnosis of ulcerative lesions of the oral cavity. The images shown in [Figure 1 (A-F)](#f1){ref-type="fig"} are from a 50-year-old woman with a gingival ulcer on the edge of an implanted tooth. The patient's medical history was remarkable, as she had systemic lupus erythematosus. There was no lymphadenomegaly and PET-CT was negative at the onset of the ulcer. A biopsy was performed and showed a granulation tissue under the ulcerated mucosa associated with an inflammatory infiltrate consisting of small lymphocytes, eosinophils and plasma cells. A dense and diffuse lymphoid proliferation with atypical large cells often with Hodgkin/Reed-Sternberg (HRS) cell-like morphology was also observed below the epithelium. These cells were strongly positive for CD30, LMP-1 and CD20 and negative for CD15. The ulcer regressed spontaneously in three months. On the basis of the history, clinical examination, immunohistochemistry with LMP-1 positivity and good outcome, a final diagnosis of EBV-positive mucocutaneos ulcer (EBVMCU) was made. {#f1} [Figure 1 (G-L)](#f1){ref-type="fig"} represents ulcers on the tongue sides of a 30-year-old man, with periods of improvement and worsening, and a history of traumatic tongue bite. Histologically, there was a polymorphous inflammatory infiltrate with a considerable number of eosinophils, with few lymphocytes and neutrophils, in addition to a population of atypical large mononuclear cells with large nucleoli extending to the deeper muscle fibers and nerves. Immunohistochemistry showed a T cell population greater than the B cell population, and CD3+ cells were also CD4+. Occasional large CD30+ cells were also observed. These cells were LMP-1 and EBER-ISH negative. The lesion improved after treatment with dental guard to avoid persistent mucosal trauma. The diagnosis was an eosinophilic ulcer of the oral mucosa (EUOM). EBVMCU is recognized as a clinicopathological entity in the last WHO classification of hematological malignancies,[@r1]^,^[@r4] with Hodgkin-like features and a self-limited indolent course, generally responding well to conservative management, and associated with iatrogenic immunosuppression or age-related immunosenescence.[@r1]^,^[@r4] EUOM is a rare self-limited oral condition, best regarded as a reactive pattern of unclear etiology, although a local traumatic event has been often incriminated.[@r2] Both entities manifest themselves as an oral ulcer and the observation of large CD30+ cells could simulate an oral lymphoma, such as CD30+ primary cutaneous lymphoproliferative disorders,[@r2] and Hodgkin lymphoma[@r5] ([Table 1](#t1){ref-type="table"}). As there are similar histologic and clinical features, EUOM and EBVMCU have to be possible diagnosis in this context, and the combination of clinical, morphologic and immunophenotypic parameters is imperative to reach the correct diagnosis, thus avoiding aggressive and iatrogenic treatments. ###### Differential diagnosis of CD30+ atypical cells in oral mucosa ------------------------------------- -------------------------------------------------------------- CD30+ lymphoproliferative disorders Oral involvement of anaplastic large T-cell lymphoma (ALCL)\ Oral involvement of lymphomatoid papulosis (LyP)\ Classical Hodgkin lymphoma Reactive inflammatoy disorders Eosinophilic ulcer of the oral mucosa (EUOM)\ EBV-positive mucocutaneos ulcer (EBVMCU) ------------------------------------- -------------------------------------------------------------- When comparing the two cases, we observe that the morphological aspects are very similar to each other, and the differential diagnosis of malignancy is quite important. Immunohistochemistry is a useful tool, but its interpretation needs to be combined with a careful clinical history. Increasingly, pathologists and clinicians need to act together for a more accurate diagnosis of patients. In the case of oral lesions, dentists are important allies as well. With these cases, we show that, in addition to malignancies, EBVMCU also has a differential with benign lesions, for example, non-infectious conditions as we discussed in the second case. Thus, we believe that our letter adds information to the article of Ikeda *et al* (2019),[@r1] improving clinical and pathological reasoning when faced with these challenging diseases. **CONFLICT OF INTEREST:** No funding obtained, No conflict of interest to disclose.

1. Introduction {#sec1} =============== There were over 1.34 million cerebrovascular patients in 2008 reported by the Japanese Ministry of Health, Labour, and Welfare \[[@B1]\]. Constipation is one of the complications seen in poststroke patients. Stratified by stroke severity on the National Institutes of Health Stroke Scale, the reported incidence of constipation in poststroke patients is 38.9% to 88.2% \[[@B2]\]. Functional constipation is thought to originate from decreased gastrointestinal motility as well as from decreased autonomic nervous system efficiency, impaired physical activity, abdominal muscle weakness secondary to hemiplegia, and diet \[[@B3]\]. Conventional therapy to control constipation involves the use of laxatives or stimulant purgatives, and these drugs are often used in the long term in chronic constipation patients \[[@B4]\]. However, patients can develop a tolerance to laxatives or stimulant purgatives, and paralytic ileus occasionally occurs in the clinical setting, even with conventional therapy \[[@B5]\]. DKT has historically been used to treat gastrointestinal dysfunction with abdominal coldness and pain in many East Asian countries, including Japan and China \[[@B6]\]. Recently, it has also been used to prevent ileus after gastrointestinal surgery and to treat irritable bowel syndrome \[[@B7]\]. Horiuchi et al. reported that DKT significantly improved abdominal bloating and pain and reduced intestinal gas volume in patients with intractable functional constipation \[[@B8]\]. Physiological reactions to the administration of DKT have been reported as promoting gastrointestinal motility \[[@B9]--[@B13]\] and increasing intestinal blood perfusion \[[@B14]--[@B19]\]. DKT\'s effectiveness in treating defecation disorders in patients with cerebrovascular disease is commonly observed in the clinical setting. Potential mechanisms underlying the physiological responses to DKT have been investigated in animal models and include elevated levels of plasma vasoactive intestinal polypeptide \[[@B14], [@B17], [@B20]\], substance P \[[@B14], [@B17], [@B21], [@B22]\], motilin \[[@B23]--[@B25]\], and acetylcholine \[[@B10], [@B11], [@B13], [@B26]--[@B28]\], which promote gastrointestinal motility, as well as calcitonin gene-related peptide (CGRP) \[[@B14], [@B15], [@B17], [@B21]\] and adrenomedullin \[[@B15], [@B16], [@B29], [@B30]\], which increase intestinal blood flow. Poststroke patients are at risk for arteriosclerosis and often experience abdominal pain accompanied by a cold sensation in the abdomen associated with low blood perfusion in the mesenteric arteries. DKT has been used to treat defecation disorders with abdominal coldness and pain caused by decreased intestinal motility and blood flow. We previously reported that administration of DKT increased blood flow in the superior mesenteric artery and promoted intestinal peristalsis in healthy subjects \[[@B18], [@B19]\]. Sato et al. reported that DKT significantly increased plasma CGRP levels in healthy subjects \[[@B21]\]. Therefore, plasma CGRP may be a useful biomarker to evaluate the effects of DKT on intestinal blood flow. This study aimed to investigate the efficacy of DKT in treating functional constipation in poststroke patients. In addition, this study investigated the impact of DKT therapy on CGRP concentration. 2. Methods {#sec2} ========== 2.1. Subject Eligibility Criteria {#sec2.1} --------------------------------- Eligible patients were aged 20 to 99 years of both genders, had been diagnosed with functional constipation according to the Rome III criteria \[[@B31]\], and remained stable over a 6-month period from the onset of cerebral hemorrhage, cerebral infarction, and subarachnoid hemorrhage. Patients received nutrition orally or through a nasogastric or gastrostomy tube. Patients with concurrent diabetes were required to have an HbA1c (NGSP) less than 9%. 2.2. Subject Exclusion Criteria {#sec2.2} ------------------------------- Patients meeting or diagnosed with any of the following criteria were excluded: risk of intestinal adhesion following abdominal surgery, inflammatory bowel disease, or malignant gastrointestinal disease; hypoxic encephalopathy or myelopathy; history of interstitial pneumonia; liver and/or kidney dysfunction; cancer; and neurodegenerative disease, such as Parkinson\'s disease or spinocerebellar degeneration. However, patients who underwent laparoscopic cholecystectomy or underwent percutaneous endoscopic gastrostomy were not excluded because the invasiveness of the operation was minimal. 2.3. Patient Recruitment {#sec2.3} ------------------------ From September 2012 to December 2013, eligible subjects were recruited from 6 hospitals: National Yonezawa Hospital, Ishinomaki Rehabilitation Hospital, National Hachinohe Hospital, Hikarigaoka Spellman Hospital, Miyagi Rifu Ekisaikai Hospital, and Wakuya Medical and Welfare Center. 2.4. Logistics {#sec2.4} -------------- Subjects were randomly assigned to the DKT group or the control group. The study protocol was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Boards of Tohoku University Hospital and the 6 collaborating hospitals. Written informed consent was obtained from all patients or their families. 2.5. Trial Methods {#sec2.5} ------------------ The study protocol included an intention to treat analysis. The control group underwent conventional therapy for constipation, such as laxative administration, enemas, and disimpaction. In addition to conventional therapy, the DKT group continuously received 5.0 g of Daikenchuto extract granules (TJ-100, Tsumura & Co., Tokyo, Japan) 3 times a day before meals for 4 weeks. Each clinical parameter was measured before and after the 4-week trial. Fifteen grams of TJ-100 (DKT) extract granules contains a dried herbal extract mixture in the following proportions: Ginseng radix (Araliaceae,*Panax ginseng* C.A. Meyer, Radix) (3.0 g), processed ginger root (Zingiberaceae,*Zingiber officinale* Roscoe, rhizoma) (5.0 g),*Zanthoxylum* fruit (Rutaceae,*Zanthoxylum piperitum* De Candolle, pericarpium) (2.0 g), and saccharum granorum (the candy produced from maltose) (10.0 g). This formulation is registered in the Japanese Pharmacopoeia Sixteenth Edition \[[@B32]\]. The production and supply processes for TJ-100 comply with good manufacturing practice standards for Kampo products and have been approved by the Japanese Ministry of Health, Labour, and Welfare. 2.6. Evaluation of Clinical Symptoms {#sec2.6} ------------------------------------ ### 2.6.1. Activities of Daily Living {#sec2.6.1} The Barthel Index was recorded for each patient at study enrollment to assess activities of daily living \[[@B33]\]. ### 2.6.2. Clinical Constipation Scores {#sec2.6.2} Clinical scores for constipation were recorded before and after the 4-week trial period using the constipation scoring system (CSS, see the appendix) \[[@B34]\]. Questionnaires concerning constipation were administered to patients; however, if the patients could not completely answer the question, their families or nurses evaluated the questions depending on the objective findings (i.e., painful evacuation effort or abdominal pain before defecation was evaluated by family members or nurses using the patients\' facial expressions; feeling of incomplete evacuation was evaluated with abdominal fullness after defecation). Because it was difficult to evaluate Q5 ("Time: minutes in lavatory per attempt") in the CSS for bedridden subjects using diapers, we removed Q5 from the statistical analysis. Evaluations before and after the administration of DKT were performed by the same family member or nurse with blinding of DKT administration. ### 2.6.3. Plain Abdominal Radiography {#sec2.6.3} Plain abdominal radiographs of fasting patients in a supine position were obtained before and after the trial period. The gas volume score (GVS) was calculated by Koide\'s method \[[@B35]\] using ImageJ \[[@B36]\] ([Figure 1](#fig1){ref-type="fig"}). ### 2.6.4. Blood Sampling {#sec2.6.4} General blood counts and biochemistry tests were performed in fasting patients before and after the trial period to assess potential adverse effects. Blood sample portions were stored in EDTA-2Na tubes. Samples were centrifuged (3000 rev/10 min), and 0.5 mL of plasma was collected and stored at −20°C. The concentration of plasma CGRP was quantified using the Human CGRP Elisa Kit (MyBioSource, Inc., San Diego, USA) tested by SRL, Inc., Tokyo, Japan. ### 2.6.5. Statistical Analysis {#sec2.6.5} Statistical analysis was performed using SPSS software (ver. 16, SPSS Japan Inc., Tokyo, Japan). Baseline comparisons of group differences were conducted using the independent samples*t*-test for continuous variables and the chi-square test for categorical variables. Measurement of the mean and standard deviation (SD) was performed at baseline and at the endpoint for all parameters. Comparisons between the DKT and control groups were performed by two-way analysis of variance (ANOVA). Changes within groups before and after the trial period were compared using the paired*t*-test when the intergroup difference was significant. Correlation between age and the CSS points was analyzed by coefficient of product-moment correlation (Pearson correlation coefficient).*P* values \<0.05 were considered significant. 3. Results {#sec3} ========== From September 2012 to December 2013, 34 subjects (17 men and 17 women; mean age: 78.1 ± 11.6 years) at 6 hospitals participated in the study. Patients were randomly assigned to 2 groups (control group or DKT group). The demographic characteristics, CSS, and GVS of each group at baseline are shown in [Table 1](#tab1){ref-type="table"}. There was no significant difference between groups in characteristics, the way of nutrition intake, CSS, or GVS at baseline. 3.1. Changes in Clinical Constipation Scores {#sec3.1} -------------------------------------------- All 34 subjects completed the CCS questionnaire before and after the observation period, and results are summarized in [Table 2](#tab2){ref-type="table"}. There was no significant correlation between age and the CSS points on the baseline (*n* = 34) (*r* = 0.12, *P* = 0.49). Significant differences in the CSS scores were observed between the 2 groups (two-way ANOVA, *P* \< 0.01). In the DKT group, the CSS scores significantly improved from 8.0 ± 3.1 to 6.0 ± 3.1 points (paired*t*-test, *P* \< 0.01). There was no significant correlation between age and the changes of the CSS scores for subjects in DKT group (*n* = 17) (*r* = −0.16, *P* = 0.53). The control group did not show any significant improvement ([Table 2](#tab2){ref-type="table"}). CSS subcategory findings are summarized for both groups in [Table 3](#tab3){ref-type="table"}. Among the CSS subcategories, there were significant differences between the DKT and control groups using two-way ANOVA for the following questions: Q1 (frequency of bowel movements; *P* \< 0.01), Q3 (feeling of incomplete evacuation; *P* = 0.03), and Q6 (need for drugs/enema/disimpaction; *P* = 0.02). In the DKT group, the constipation scores significantly decreased over the trial period for Q1 (*P* \< 0.01), Q3 (*P* = 0.049), and Q6 (*P* = 0.03). The control group, however, did not show any significant changes ([Table 3](#tab3){ref-type="table"}). Overall, the average change of 1 point in the score for Q1 means an improvement in defecation frequency from "once per week" to "2 times per week" or "less than once per week" to "once per week" in the clinical setting. The average change of 0.4 points in the scores for Q3 and Q6 means that digital assistance or enemas were no longer necessary for approximately 30% of the patients in the DKT group. 3.2. Changes in Gas Volume Score {#sec3.2} -------------------------------- [Figure 2](#fig2){ref-type="fig"} summarizes changes in the GVS before and after the observation period for both groups. There was a significant difference between the 2 groups (two-way ANOVA; *P* = 0.03), and the intragroup comparison revealed a significant decrease in the DKT group from 16.3 ± 6.7% to 9.9 ± 6.0% (*P* \< 0.01) while the control group did not show any significant changes (*P* = 0.61). Representative abdominal radiographs of a patient before and after DKT administration show reduced intestinal gas volume (Figures [3(a)](#fig3){ref-type="fig"} and [3(b)](#fig3){ref-type="fig"}). In this case, DKT administration reduced the GVS from 26.0% to 12.3%. 3.3. Changes in Plasma Calcitonin Gene-Related Peptide Concentrations {#sec3.3} --------------------------------------------------------------------- In the DKT group, the initial and final CGRP concentrations were 409 ± 482 pg/mL and 452 ± 574 pg/mL, respectively. In the control group, the initial and final values were 270 ± 172 pg/mL and 251 ± 118 pg/mL, respectively. There was no significant difference between the 2 groups in plasma CGRP (two-way ANOVA; *P* = 0.08). 3.4. Adverse Effects {#sec3.4} -------------------- Notable adverse effects, such as itching, gastrointestinal symptoms, other subjective symptoms, and abnormalities in blood counts and blood biochemistry, were not observed during and after DKT administration. 4. Discussion {#sec4} ============= This study shows that DKT in addition to conventional therapy for functional constipation significantly improved the CSS scores and significantly reduced the GVS in poststroke patients. The incidence of adverse effects associated with DKT extract, such as gastrointestinal discomfort and liver dysfunction, has been reported as 1.9% in prior studies \[[@B37]\], but no adverse effects were observed during the 4-week treatment period in the present study. Functional constipation has a complex pathophysiology, and intestinal function is controlled by the autonomic nervous system; consequently, therapeutic protocols are limited in poststroke patients \[[@B38], [@B39]\]. Several clinical studies of DKT therapy for constipation have been reported, but almost all of these were limited to healthy subjects or were case series. The present study was a prospective randomized controlled trial for functional constipation in patients with stroke-related morbidity and therefore could show stronger evidence than previous reports of the clinical effects of DKT. In a prior clinical study, it was reported that DKT extract improved colorectal function in patients diagnosed with Parkinson\'s disease \[[@B40]\]. Another study reported that administration of DKT to patients with chronic intractable constipation improved abdominal bloating and pain symptoms \[[@B8]\]. The present study similarly found improvement in clinical constipation scores and GVS. Numerous studies have investigated the active ingredients and mechanisms underlying the improved intestinal motility. Intestinal contraction may be induced by DKT through the cholinergic nervous system via serotonin receptors \[[@B13], [@B27], [@B28]\], motilin activity \[[@B23], [@B24]\], and the transient receptor potential vanilloid type 1 channel \[[@B11], [@B41]\]. Satoh et al. reported that*Zanthoxylum* fruit and maltose, ingredients in DKT, improved delayed propulsion in the small intestine.*Zanthoxylum* fruit also improved delayed propulsion in the distal colon. Endogenous cholecystokinin secretion resulting from maltose administration may play a role in the effect of DKT \[[@B42]\]. These reports describe the possible mechanisms through which DKT promotes intestinal movement and explain some aspects of the improvement in the CSS scores and the reduction of GVS noted in our study. Some studies reported that DKT extract increased CGRP in healthy subjects \[[@B21], [@B25]\]. In another report, DKT did not change CGRP levels in patients with constipation secondary to palliative morphine therapy for cancer \[[@B24]\]. In the present study, changes in CGRP did not reach statistical significance. Several mechanisms may explain this lack of change in CGRP levels in the DKT group. Plasma CGRP is notably unstable \[[@B43]\]. An elevation following DKT administration may have been obscured by factors such as testing procedures, individual differences, daily fluctuations, and day-to-day variations. Furthermore, although some studies confirmed elevated CGRP immediately after DKT administration \[[@B21], [@B25]\], the CGRP level may be too unstable to be used as a target factor for evaluating the effects of DKT. DKT is thought to affect the promotion of intestinal motility and intestinal blood flow. Increase in intestinal blood flow is believed to be mediated through adrenomedullin and CGRP or through the transient receptor potential ankyrin 1 channel \[[@B16], [@B29], [@B30]\]. The mechanisms promoting intestinal motility and blood flow have complex interactions, which may be altered further by disease pathology, environment, and individual differences. The present results of improved constipation following DKT administration are overall consistent with the findings of prior studies, despite the lack of significant change in CGRP levels. 4.1. Limitations {#sec4.1} ---------------- The small sample size is the first limitation of the present study. The CGRP level tended to differ between the groups (ANOVA, *P* = 0.08); a larger sample size could determine the significance of this difference. In addition, participants were limited to hospitalized patients; therefore, patients who were hemiplegic, yet stable enough to receive outpatient care, were not included. As a result, the population was skewed toward patients with low activities of daily living. Third, there are no objective parameters for abdominal coldness at present. Ultrasound assessment of blood flow in the superior mesenteric artery was nearly impossible in poststroke patients with constipation owing to the presence of intestinal gas. Finally, the placebo effect of oral administration cannot be overlooked. A randomized double-blind comparative study using a placebo would be ideal and would eliminate the placebo effect. DKT includes 4 crude herbs and has a sweet and hot flavor. It will be difficult to produce a placebo without bioactivity that has a smell and flavor similar to DKT. Accordingly, the present study did not use a placebo control but rather compared the effects of DKT administration plus conventional treatment to conventional treatment alone. 5. Conclusions {#sec5} ============== Administration of DKT extract in conjunction with conventional therapy to treat functional constipation in poststroke patients improved clinical constipation scores and reduced intestinal gas volume. Results of this study show that DKT is effective for defecation control in poststroke patients. The present study was conducted using a Grant-in-Aid for Scientific Research (Academic Research Grant, no. 23590867). The authors sincerely appreciate the individuals who participated in the trial throughout the entire study, as well as their families. They are grateful to the staff at National Yonezawa Hospital, Ishinomaki Rehabilitation Hospital, Miyagi Rifu Ekisaikai Hospital, Hikarigaoka Spellman Hospital, National Hachinohe Hospital, and Wakuya Medical and Welfare Center for their help with data collection. Constipation Scoring System (CSS) \[[@B34]\] {#secA} ============================================ Minimum score, 0; Maximum score, 30, the numbering starting from zero represents the scores. (1) Frequency of bowel movements (0) 1-2 times per 1-2 days (1) 2 times per week (2) Once per week (3) Less than once per week (4) Less than once per month (2) Difficulty: painful evacuation effort (3) Completeness: feeling incomplete evacuation (4) Pain: abdominal pain (0) Never (1) Rarely (2) Sometimes (3) Usually (4) Always (5) Time: minutes in lavatory per attempt (0) Less than 5 (1) 5−10 (2) 10−20 (3) 20−30 (4) More than 30 (6) Assistance: type of assistance (0) Without assistance (1) Stimulative laxatives (2) Digital assistance or enema (7) Failure: unsuccessful attempts for evacuation per 24 hours (0) Never (1) 1--3 (2) 3--6 (3) 6--9 (4) More than 9 (8) History: duration of constipation (yr) (0) 0 (1) 1--5 (2) 5--10 (3) 10--20 (4) More than 20 Conflict of Interests ===================== All authors declare no personal competing financial or nonfinancial interests in this study; however, Tohoku University Graduate School of Medicine received a grant from Tsumura Co. Ltd., the manufacturer of TJ-100. Authors\' Contribution ====================== Takehiro Numata took part in planning the study, performed the data analysis, and wrote the paper. Shin Takayama and Koh Iwasaki were the original proposers of the study and were involved in developing the protocol and paper preparation. Muneshige Tobita, Shuichi Ishida, Dai Katayose, Mitsutoshi Shinkawa, Takashi Oikawa, and Takanori Aonuma took part in recruiting subjects and laboratory management in their hospitals. Soichiro Kaneko, Junichi Tanaka, and Seiki Kanemura helped to plan the study and provided advice related to writing the paper. Tadashi Ishii and Nobuo Yaegashi were responsible for the study design and execution and assisted in writing the paper. All authors read and approved the final paper. {#fig1} {#fig2} {#fig3} ###### Baseline population demographics of DKT and control groups.   Group *P*\* ------------------------------- -------------- ------------- ------ *N* 17 17   Sex     0.73  Female 9 8    Male 8 9   Age (y) 77.5 ± 11.9 78.7 ± 12.1 0.78 Height (cm) 156.3 ± 12.1 154.1 ± 9.3 0.56 Body weight (kg) 48.4 ± 10.2 48.3 ± 9.4 0.99 Diagnoses, *N*     0.31  Brain infarction 10 14    Cerebral hemorrhage 4 2    Subarachnoid hemorrhage 3 1   Illness duration (y) 7.8 ± 6.1 4.8 ± 4.2 0.15 Barthel Index 2.1 ± 3.1 1.2 ± 2.8 0.39 The way of nutritional intake     0.14  Orally 5 1    Through nasogastric tube 2 5    Through gastrostomy tube 10 11   CSS total^b^ (points) 8.0 ± 3.1 8.1 ± 3.7 0.96 CGRP (pg/mL) 408 ± 482 262 ± 170 0.25 GVS (%) 16.3 ± 6.7 14.4 ± 7.8 0.44 ^a^DKT, Daikenchuto; CSS, constipation scoring system; CGRP, calcitonin gene-related peptide; GVS, gas volume score. ^b^CSS total: not including point of Q5. \*Significance designated at *P* \< 0.05. ###### Clinical constipation scores in both groups at baseline and endpoint.   DKT^a^ group (*N* = 17) Intragroup difference Control group (*N* = 17) Intragroup difference Intergroup difference ----------------------- ------------------------- ----------------------- -------------------------- ----------------------- ----------------------- ------ -------- CSS total^c^ (points) 8.0 ± 3.1 6.0 ± 3.1 \<0.01 8.1 ± 3.7 8.2 ± 3.7 0.33 \<0.01 ^a^DKT, Daikenchuto; CSS, constipation scoring system. ^b^Endpoint: after the 4-week trial period. ^c^CSS total: not including point of Q5. \*Significance designated at *P* \< 0.05. ###### Constipation scoring system (CSS) subcategory scores in both groups at baseline and endpoint.   DKT^a^ group (*N* = 17) Intragroup difference Control group (*N* = 17) Intragroup difference Intergroup difference ------------- ------------------------- ----------------------- -------------------------- ----------------------- ----------------------- ------ -------- Q1 (points) 2.2 ± 1.5 1.2 ± 1.4 \<0.01 2.1 ± 1.4 2.1 ± 1.5 0.33 \<0.01 Q2 (points) 0.5 ± 0.9 0.3 ± 0.7 --- 0.6 ± 0.9 0.6 ± 0.9 --- 0.07 Q3 (points) 1.2 ± 1.2 0.8 ± 1.0 0.049 1.5 ± 1.3 1.6 ± 1.4 0.33 0.03 Q4 (points) 0.4 ± 0.8 0.4 ± 0.7 --- 0.7 ± 0.9 0.7 ± 0.9 --- 0.33 Q5 (points) --- --- --- --- --- --- --- Q6 (points) 1.8 ± 0.5 1.4 ± 0.8 0.03 1.7 ± 0.7 1.7 ± 0.7 1.00 0.02 Q7 (points) 0.1 ± 0.2 0.1 ± 0.2 --- 0.1 ± 0.2 0.1 ± 0.2 --- 1.00 Q8 (points) 1.9 ± 1.1 1.9 ± 1.1 --- 1.5 ± 0.9 1.5 ± 0.9 --- 1.00 Intragroup difference was calculated using the paired *t*-test only when the intergroup difference was significant. ^a^DKT, Daikenchuto. ^b^Endpoint: after the 4-week trial period. \*Significance designated at *P* \< 0.05. [^1]: Academic Editor: Toku Takahashi

Editorial Note on the Review Process ==================================== [F1000 Faculty Reviews](http://f1000research.com/browse/f1000-faculty-reviews) are commissioned from members of the prestigious [F1000 Faculty](http://f1000.com/prime/thefaculty) and are edited as a service to readers. In order to make these reviews as comprehensive and accessible as possible, the referees provide input before publication and only the final, revised version is published. The referees who approved the final version are listed with their names and affiliations but without their reports on earlier versions (any comments will already have been addressed in the published version). The referees who approved this article are: 1. Shoichiro Ono, Department of Pathology and Department of Cell Biology, Emory University, Atlanta, GA, USA [^2] 2. Thomas Pollard, Department of Cell Biology, Yale University, New Haven, Connecticut, USA [^3] 3. George von Dassow, Oregon Institute of Marine Biology, University of Oregon, Charleston, Oregon, USA [^4] Activation of RhoA by the RhoGEF ECT-2 directs furrow formation =============================================================== Shortly after mitotic chromosomes separate, the small GTPase, RhoA, accumulates in its active form at the equatorial plasma membrane of animal cells ^[@ref-1]--\ [@ref-4]^. This pool of RhoA-GTP is necessary ^[@ref-5]--\ [@ref-7]^ and sufficient ^[@ref-8]^ for the assembly of an actomyosin-driven furrow that partitions a cell into two daughter cells. As cytokinesis occurs at a specific site at a precise time, RhoA activation is necessarily highly regulated. In metazoa, RhoA activation during cytokinesis is mediated by a Rho guanine nucleotide exchange factor (RhoGEF), ECT-2, which plays a well-conserved role in activating RhoA during cytokinesis ^[@ref-9]--\ [@ref-15]^. However, ECT-2 itself is highly regulated; its activity is suppressed by autoinhibition and phosphorylation, and it is activated by protein-protein interactions and dephosphorylation. Here, we discuss recent progress in defining the role of an atypical Rho GTPase-activating protein (GAP), CYK-4, protein in RhoA activation during cytokinesis with a focus on the early *Caenorhabditis elegans* embryo. CYK-4 protein: a brief introduction =================================== CYK-4, so named in *C. elegans*, comprises an N-terminal coiled coil, a central membrane-binding C1 domain, and a C-terminal RhoGAP domain ( [Figure 1A](#f1){ref-type="fig"}). It is known as MgcRacGAP in mammalian cells and as RacGAP50C and *tumbleweed* in *Drosophila.* CYK-4 exists as a heterotetramer with ZEN-4 (MKLP1 in mammals and *pavarotti* in *Drosophila*) ^[@ref-16]^. This multimeric complex, dubbed centralspindlin, accumulates and drives bundling of antiparallel microtubules in the anaphase spindle midzone ^[@ref-17]--\ [@ref-22]^. It can also be detected in small quantities at the equatorial plasma membrane in anaphase ^[@ref-21],\ [@ref-23]--\ [@ref-25]^, where it is well situated to act as a RhoA activator. {#f1} Prior to anaphase, the activity of the cytokinetic RhoGEF ECT-2 is suppressed by autoinhibition ^[@ref-26]^ and Cdk1 phosphorylation ^[@ref-27]^. Curiously, the RhoGAP protein CYK-4, which ordinarily would be predicted to turn a GTPase "off", interacts with ECT-2 via their respective N-termini in a phospho-regulated manner ^[@ref-12]^. This interaction occurs in a wide variety of contexts, assays, and organisms and recruits the GEF to the midzone ^[@ref-12],\ [@ref-14],\ [@ref-28]--\ [@ref-31]^. Centralspindlin contributes to localization or activation of ECT-2 (or both) at the plasma membrane, where this RhoGEF primarily acts ^[@ref-27]^. Barring the noteworthy *C. elegans* embryo (discussed in detail below), depleting CYK-4 in animal cells blocks the formation of a cleavage furrow, suggesting that it positively regulates RhoA activity ^[@ref-2],\ [@ref-11],\ [@ref-12],\ [@ref-14],\ [@ref-28],\ [@ref-32]^. Does the CYK-4 GAP domain contribute positively or negatively to RhoA regulation? While the case of CYK-4 is pertinent to cytokinesis and GTPase regulation in diverse systems, the function of this protein has been most extensively analyzed in one-cell *C. elegans* embryos. Two pathways for RhoA activation in *C. elegans* ================================================ The one-cell *C. elegans* embryo, despite being a powerful system for studying the cell biology and genetics of cytokinesis, has a complication not observed in other systems. RhoA activation during cytokinesis in the early *C. elegans* embryo, unlike cultured human cells and *Drosophila* cells, depends on the combined action of centralspindlin and a second pathway. This second pathway involves a distinct activator of ECT-2 which has a distinct means for spatial regulation of its activity. While this can be confounding for *cyk-4* analyses, it is not insurmountable. This second pathway can be isolated from the centralspindlin-directed pathway by attenuation of spindle pulling forces ^[@ref-11]^, severing the central spindle with a laser ^[@ref-33]^, and altering spindle position ^[@ref-34]^. Most recently, a specific molecular component of this second pathway, NOP-1, was identified ^[@ref-2]^. Because of their overlapping functions, in order to delineate the role of CYK-4 in cleavage furrow formation, the alternate furrow induction pathway must be eliminated. The simplest means to do so is by inactivating NOP-1. *nop-1* is a non-essential, poorly conserved, nematode-specific gene. Nevertheless, it promotes RhoA activation during polarization of the *C. elegans* zygote ^[@ref-2],\ [@ref-35]^. NOP-1--deficient embryos complete cytokinesis with wild-type kinetics, but embryos defective in both NOP-1 and CYK-4---by RNA interference (RNAi) or any of the point mutants discussed below---do not initiate cleavage furrows ^[@ref-2],\ [@ref-36]^. Imaging with a RhoA biosensor confirms that CYK-4 and NOP-1 coordinately promote RhoA activation upstream of ECT-2 during cytokinesis and that CYK-4 plays the more prominent role ^[@ref-2]^. The NOP-1 pathway appears capable of directing global RhoA activation in the embryo. However, astral microtubules limit the sites of active RhoA accumulation to the anterior of the zygote during polarization and to broad regions at the equator and the most anterior part of the zygote during anaphase. The NOP-1 and centralspindlin pathways can be readily spatially separated by manipulating the spindle to position it at the posterior of the embryo ^[@ref-2],\ [@ref-34]^. At anaphase, an embryo, so modified, produces an anterior furrow that is NOP-1--dependent and a posterior furrow that is centralspindlin-dependent. Although the biochemistry of NOP-1 activation of ECT-2/RhoA is unknown, NOP-1--deficient *C. elegans* embryos resemble other eukaryotic cells in that cleavage furrow formation, and ingression are completely dependent upon CYK-4, thus providing a straightforward way to analyze the function of the latter. Genetic dissection of the CYK-4 GAP domain in *C. elegans* embryos ================================================================== CYK-4 GAP domain mutants ------------------------ While there is compelling evidence that CYK-4 plays a role in RhoA activation, the role of its GAP domain remains contentious. The isolated GAP domain of CYK-4 promotes GTP hydrolysis *in vitro* by Rho family members but has much stronger activity toward Rho GTPases Rac and Cdc42 when compared with RhoA ^[@ref-22],\ [@ref-37],\ [@ref-38]^. Importantly, however, Rac1 and Cdc42 are dispensable for cytokinesis in a wide variety of contexts ^[@ref-36],\ [@ref-39]--\ [@ref-41]^. Thus, do these *in vitro* assays properly reflect the function of full-length CYK-4 during cytokinesis *in vivo*? Does it function as a conventional GAP that inactivates Rac or RhoA or both, or does it participate, unexpectedly, in activating RhoA via ECT-2, or a combination thereof? CYK-4 was first implicated in cytokinesis on the basis of a temperature-sensitive lethal mutation--- *cyk-4(t1689ts)*---in *C. elegans* encoding an S15L substitution ( [Figure 1A](#f1){ref-type="fig"}) in the N-terminus of CYK-4 that disrupts its interaction with ZEN-4 ^[@ref-16],\ [@ref-42]^. Subsequently, Canman and colleagues isolated two additional mutations--- *cyk-4(or749ts)*, yielding CYK-4 ^E448K^, and *cyk-4(or570ts)*, yielding CYK-4 ^T546I^---and both substitutions map to the GAP domain of CYK-4 ^[@ref-43]^. However, these mutations are not in the active site, and the biochemical nature of these alleles has not been characterized. Therefore, it is risky to assume that these mutants are solely defective in CYK-4 GAP activity. Indeed, CYK-4 ^E448K^ exhibits a defect in membrane localization in the gonad ^[@ref-36]^. However, because the mutants assemble normal central spindles, they cannot be entirely unfolded at the restrictive temperature. More recently, a mutant targeting the conserved catalytic arginine in the GAP domain was generated (R459A), as was a variant where two surface residues were mutated (K495E and R499E) to block CYK-4 interaction with Rho family GTPases ^[@ref-36]^. None of the *cyk-4* mutations mentioned here abolishes furrow formation in otherwise wild-type embryos; furrows ingress partially and regress ( [Figure 1](#f1){ref-type="fig"}). The debate regarding CYK-4 GAP domain function was initiated by the discovery that depleting the GTPase Rac1 (CED-10 in *C. elegans*) in CYK-4 ^E448K^ embryos frequently results in complete ingression of the furrow ^[@ref-43]^. This was interpreted to mean that the primary function of the CYK-4 GAP domain during wild-type cytokinesis is to inactivate Rac1/CED-10 ^[@ref-43],\ [@ref-44]^. The experimental result ( [Figure 1B](#f1){ref-type="fig"}) is uncontested and has been confirmed by using either RNAi ^[@ref-36],\ [@ref-43]^ or loss-of-function alleles ^[@ref-44],\ [@ref-45]^ to inactivate Rac1/CED-10. However, there is significant disagreement regarding interpretation of the results and the underlying mechanism of CYK-4 GAP function. Redundant furrow induction pathways obscure CYK-4 function ---------------------------------------------------------- The presence of a parallel pathway for furrow induction contributes to the difficulty in interpreting results concerning the role of the CYK-4 GAP domain. If a given GTPase is the sole, relevant target of the CYK-4 GAP domain during cytokinesis, then its loss should reverse the defects of CYK-4 GAP mutants in a variety of genetic contexts. However, inactivation of Rac1/CED-10 rescues furrow ingression in GAP domain mutants (E448K, R459A, and T546I) ^[@ref-36],\ [@ref-43]--\ [@ref-45]^ only in the presence of NOP-1 ^[@ref-36]^. In NOP-1 mutant embryos, which are cytokinesis proficient, mutations in the GAP domain of CYK-4 block furrow formation altogether and the activity of Rac1/CED- 10 has no bearing on this behavior ( [Table 1](#T1){ref-type="table"}) ^[@ref-36]^. If mutations in the GAP domain of CYK-4 indeed cause Rac1/CED 10 hyperactivation, then inactivation of Rac1/CED-10 should correct this defect regardless of the presence or absence of NOP-1. ###### Summary of available data for RhoA and Rac1 effector accumulation and furrowing phenotypes during cytokinesis in two key *Caenorhabditis elegans cyk-4* GTPase-activating protein mutants. ------------------------------------------------------------------------------------------------------------------------------------ CYK-4\ RhoA\ Rac1 effector\ Furrow\ Furrow\ Furrow\ Furrow\ variant effector\ hyperaccumulation\ ingression ingression in\ ingression in\ ingression in\ accumulation\ at furrow the absence\ the absence\ the absence of\ at furrow of Rac1 of NOP-1 NOP-1 and Rac1 ----------- ------------------------------- -------------------- --------------- ---------------- ---------------- ----------------- Wild-type [+++](#fn2){ref-type="other"} Absent Normal,\ Normal,\ Normal,\ Normal,\ complete complete complete complete E448K [\*](#fn1){ref-type="other"} Not determined Slow, partial Slow,\ Absent Absent complete R459A [+](#fn3){ref-type="other"} Not determined Slow, partial Slow,\ Absent Absent complete ------------------------------------------------------------------------------------------------------------------------------------ \*Conflicting data; see the "cyk-4 GAP mutants have multiple defects" section. +++ Wild-type accumulation of myosin II, RhoA biosensor. \+ Reduced accumulation of myosin II, RhoA biosensor. In addition to NOP-1 inactivation, there are other ways in which the centralspindlin-dependent cytokinetic furrow in *C. elegans* embryos can be isolated from the alternative pathway (see "CYK-4 protein: a brief introduction"). In each case, the CYK-4 ^E448K^ mutation prevents centralspindlin-dependent furrow formation, and, again, inactivation of Rac1/CED-10 does not restore furrowing ^[@ref-45]^. Stated another way, loss of Rac1/CED-10 reverses the furrowing defect in CYK-4 GAP mutants only when the centralspindlin-independent pathway for furrow formation is active (that is, when an alternative pool of locally activated RhoA is available). Strikingly, in their recent publication, Canman and colleagues did not use any of these approaches to test whether Rac1/CED-10 is a direct target of CYK-4 GAP activity ^[@ref-44]^. *cyk-4* GAP mutants have multiple defects ----------------------------------------- To analyze CYK-4 GAP domain function using *cyk-4* mutants in a meaningful way, all aspects of a mutant phenotype must be considered. In addition to being incomplete, furrows in embryos with *cyk-4* GAP domain mutations have slower ingression kinetics and weakly accumulate active RhoA and its effectors. Although Rac1/CED-10 depletion in *cyk-4* GAP mutant embryos permits the completion of furrow ingression, it is unable to restore the other two phenotypes ^[@ref-44],\ [@ref-45]^. If Rac1/CED-10 was the primary relevant target of CYK-4, embryos deficient in both Rac1/CED-10 and CYK-4 GAP activity would be predicted to resemble Rac1/CED-10--deficient embryos (which closely resemble wild-type embryos). Canman and colleagues recently suggested that mutations in the GAP domain of CYK-4 do not impair myosin accumulation ( [Table 1](#T1){ref-type="table"}, asterisk) ^[@ref-44]^, despite Canman and colleagues' earlier work demonstrating a defect in myosin at the furrow tip in *cyk-4* mutant embryos (see Figure S3 in [@ref-43]). A variety of mutations in the CYK-4 GAP domain impair the accumulation of myosin, actin, anillin, and a RhoA biosensor ( [Table 1](#T1){ref-type="table"}, asterisk) ^[@ref-2],\ [@ref-36],\ [@ref-45]^. Again, these defects are not corrected by depleting Rac1. This is particularly dramatic in NOP-1--deficient embryos where myosin and RhoA activation during cytokinesis are entirely dependent upon centralspindlin ^[@ref-2],\ [@ref-36]^. These results cannot be explained if the CYK-4 GAP domain acts on Rac1/CED-10. Rac1/CED-10 activates the Arp-2/3 complex ^[@ref-46],\ [@ref-47]^, a nucleator of branched actin filaments ^[@ref-48]^. If CYK-4 GAP mutations prevent Rac1 inactivation, hyperaccumulation of Rac1/CED-10 effectors or an increase in branched actin on the membrane would be expected in these mutants. But, in fact, *cyk-4* mutations result in a reduction in actin accumulation ^[@ref-45]^. Critically, there is no evidence that mutations in the GAP domain of CYK-4 cause ectopic Rac1/CED-10 activation in the *C. elegans* embryo ( [Table 1](#T1){ref-type="table"} and [Figure 2A](#f2){ref-type="fig"}). Collectively, the available data do not support models in which Rac1/CED-10 is the primary relevant target of CYK-4 GAP activity during cytokinetic furrowing. ![Proposed models for function of the CYK-4 GTPase-activating protein (GAP) domain in *Caenorhabditis elegans* with predicted phenotypes.\ At the top, a diagram depicting Rac1 and RhoA activities in a dividing wild-type or *nop-1* mutant embryo. ( **A**) Genetic pathway and schematic model for cytokinetic furrow formation in *C. elegans* where CYK-4 solely functions to inactivate Rac1, as described by [@ref-44]. In this model, ECT-2 is not subject to activation. However, as discussed in the text, available experimental evidence does not support this model. ( **B**) Genetic pathway and schematic model for cytokinetic furrow formation in *C. elegans* where CYK-4 and NOP-1 function in parallel upstream of ECT-2 in RhoA activation. The diagrams at the bottom of ( **A**) and ( **B**) reflect scenarios where NOP-1 is absent.](f1000research-6-13052-g0001){#f2} An indirect role for Rac1/CED-10 in furrow formation ---------------------------------------------------- Why does loss of Rac1/CED-10 allow CYK-4 GAP mutants to complete furrow ingression? Inactivation of Rac1/CED-10 partially rescues the cytokinesis defect resulting from selected mutations in CYK-4 and only in the presence of a redundant furrow induction pathway. The extremely specific situations in which restoration to full ingression is observed strongly support that this behavior is mediated by bypass suppression. That is, instead of correcting the primary defect resulting from the mutation in CYK-4, the absence of Rac1/CED-10 attenuates the requirement for RhoA activation in furrow ingression. Depletion of ARX-2, a subunit of the ARP-2/3 complex, has a similar effect ^[@ref-43],\ [@ref-45]^. Inactivation of Rac1/CED-10 or ARX-2 also partially remedies the cytokinesis defects caused by weak mutations in ECT-2 ^[@ref-45]^. While RhoA activates formins to generate linear actin filaments at the furrow, the anterior cortex of the embryo is enriched in ARP-2/3 nucleated branched actin ^[@ref-49]--\ [@ref-52]^. Depleting ARX-2 or Rac1/CED-10 induces cortical instability ^[@ref-45],\ [@ref-53]^, which may allow the weak, NOP-1--dependent furrow to ingress further because the cortex is now more pliant. Alternatively, or in addition, depolymerization of this pool of branched F-actin may allow increased actin polymerization in the furrow because of a reduction in competition for actin monomer ^[@ref-54]^. In either case, the suppression is due to enhancement of the centralspindlin-independent pathway. Canman and colleagues have suggested that if Rac1/CED-10 depletion generally promotes ring constriction, then Rac1/CED-10 depletion should also be able to rescue weak contractile rings resulting from temperature-sensitive mutations of the formin CYK-1 and non-muscle myosin II (NMY-2) at intermediate temperatures ^[@ref-44]^, neither of which is observed. However, as both furrowing pathways are active in this experiment and both strictly require formin and myosin for furrow formation, it is not entirely surprising that Rac1/CED-10 depletion does not compensate when major structural components of the contractile ring are crippled. Direct visualization of the sites of accumulation of branched actin in *cyk-4* mutant embryos and assays for the effects of Rac1/CED-10 and the Arp2/3 complex on cortical stability during cytokinesis will help better understand these results. An alternative model for CYK-4 GAP domain function -------------------------------------------------- The data discussed above point to a compelling, albeit counterintuitive, role for CYK-4 in the activation of RhoA during cytokinesis ( [Figure 2B](#f2){ref-type="fig"}). As stated previously, in many model systems, the CYK-4 N-terminus directly interacts with the RhoGEF ECT-2 and is responsible for the relief of autoinhibition and can contribute to the recruitment of the latter to the division plane. Where analyzed, depletion of CYK-4 results in weak or no furrows. This is also the case in *C. elegans* CYK-4 GAP mutants ( [Figure 1](#f1){ref-type="fig"}). Importantly, as stated in the " *cyk-4* GAP mutants have multiple defects" section above, these CYK-4 mutations result in reduced accumulation of RhoA effectors at the furrow. Disabling parallel furrow induction pathways (see "CYK-4 protein: a brief introduction") in CYK-4 GAP mutants abolishes the residual accumulation of active RhoA and its targets. As the above results suggest, if CYK-4 GAP mutants are truly defective in activating RhoA, then experimentally increasing active RhoA levels should restore full RhoA function and cytokinetic furrowing. Importantly, this should occur even in the absence of a redundant furrowing pathway. This prediction was tested in two ways. As mentioned previously, CYK-4 GAP mutants do not furrow in the absence of NOP-1. Remarkably, depletion of RGA-3/4, a conventional RhoGAP that switches off RhoA ^[@ref-55],\ [@ref-56]^, results in wild-type cytokinetic furrowing in CYK-4 GAP mutants in the presence and absence of NOP-1 ^[@ref-36]^. Increasing RhoA activity using an activated allele of the RhoGEF ECT-2 has the same striking effect ^[@ref-36]^. These results together support the hypothesis that CYK-4 GAP domain participates in RhoA activation. The results described herein are compiled in poster format viewable on figshare ( [10.6084/m9.figshare.5325619](http://dx.doi.org/10.6084/m9.figshare.5325619)). In addition to interacting via their N-termini, an association between the CYK-4 GAP and the ECT-2 GEF domain has also been reported ^[@ref-36]^. The function of this interaction remains to be elucidated. The GAP domain may facilitate relief of ECT-2 autoinhibition and participate in a ternary complex with RhoA to fully activate the GTPase. While such a model may explain results from *C. elegans* embryos, CYK-4 function could be highly context-dependent and may vary with diverse cellular parameters. Context dependence: other experimental systems ============================================== Thus far, we have discussed results concerning the GAP domain of CYK-4 in *C. elegans* embryos, the system that has been studied in the most detail. The conservation of the GAP domain suggests that GAP activity is important for the organism; however, this conservation does not imply that the activity is critical in all cell types. Indeed, while the GAP activity of CYK-4 is essential for cytokinesis in early blastomeres, it is dispensable later in embryogenesis, except in the germline ^[@ref-36]^. The mechanisms that are required for RhoA activation employed by a given cell likely depend on features such as cell-cell or cell-substrate adhesion, cell size, spindle size, spindle--to--plasma membrane distance, and relative abundance of proteins. There are a number of other examples where the genetic requirements for cytokinesis depend on the cellular or tissue context ^[@ref-57],\ [@ref-58]^. Experimentally, using optogenetics to control RhoA activity, cleavage furrow ingression can be dramatically slowed by polar or global activation of RhoA ^[@ref-8]^. The requirement for the GAP domain of CYK-4 and its orthologs has been studied in a number of other contexts. While CYK-4 protein is required in all cells examined to date, the requirement for the GAP domain and its catalytic activity varies. In chicken B cells, the CYK-4 GAP domain, but not the catalytic arginine, is required for cytokinesis ^[@ref-59]^, and mutations in the GAP domain strongly impair furrow formation in *Drosophila* ectoderm ^[@ref-60]^. These results are largely consistent with a role for the GAP domain in promoting RhoA activation. The role of CYK-4 GAP activity has been investigated in some detail in HeLa cells. In these cells, catalytically inactive CYK-4 displays a range of phenotypes, including many cells with a late cytokinesis defect in which cells ingress with apparently normal kinetics but fail to undergo abscission ^[@ref-38]^. Inactivation of CYK-4 GAP activity results in a dramatic delocalization of the RhoA effector anillin. In addition, there is increased cell spreading and adhesion to the substrate, both in the midzone where CYK-4 concentrates and in the cell periphery. Cytokinesis failure in these cells could be suppressed by depletion of Rac1 or selected effectors. These results are consistent with a model in which CYK-4 targets Rac1 in this context, although indirect effects involving GTPase crosstalk are possible ^[@ref-61]^. In *Xenopus* embryos, CYK-4 is also essential for RhoA activation. A useful feature of this system is that cytokinetic RhoA activation can be readily measured with a biosensor that reveals a distinct "Rho zone" during cytokinesis. When the GAP domain is deleted, RhoA activation occurs, but the Rho zone appears to move as a wave in the plane of the membrane. Conversely, inactivation of GAP activity results in a broadening of the Rho zone during cytokinesis ^[@ref-62]^. Thus, unlike the case in *C. elegans* embryos, activation of RhoA in *Xenopus* does not strictly depend on the GAP activity or its RhoA-binding activity. The expansion seen in the catalytically inactive mutant in *Xenopus* is consistent with a conventional view of GAP function in which CYK-4 acts to inactivate RhoA, pointing to a requirement for flux in maintaining the size of the Rho zone. If that was the sole role of the GAP domain, the GAP domain deletion mutant would be expected to behave similarly. Rather, the mobility of the Rho zone in the deletion mutant indicates that GAP domain plays a role in anchoring the Rho zone. Evidence from *C. elegans* indicates that the RhoA GAP domain associates with active RhoA ^[@ref-36]^. These two findings in *Xenopus* also fit a model in which binding of CYK-4 to active RhoA promotes positive feedback through ECT-2 recruitment. Mutation of the active site of CYK-4 GAP could induce expansion of the Rho zone by enhancing positive feedback, as the GAP-deficient mutant binds more strongly to active RhoA than wild-type CYK-4. The GAP domain deletion mutant obviously lacks this functionality which could allow for the observed movement of the Rho zone. Summary ======= The results described here reveal that cytokinesis in metazoa is driven by a contractile ring that assembles in response to local RhoA activation at the equatorial cortex during anaphase. This is one of the near-universal features of the process; other aspects vary in different cell types and organisms. The extent of RhoA activation required to divide a cell will depend considerably on its context. Division of a large embryonic blastomere is unlikely to be identical to that of a small somatic cell of the same animal. Similarly, we have described that changes to the properties of the overall cell cortex of the large blastomere can alter the requirements for cytokinesis. Nevertheless, the proteins that mediate RhoA activation are well conserved among metazoa. Many regulatory features are conserved, but the extent to which they are required is more variable. There is ample evidence that caution must be used when extrapolating from one system to another. Here, we have focused on CYK-4 during cytokinesis in the early *C. elegans* embryo. In this system, the available evidence reveals that the GAP activity of CYK-4 promotes RhoA activation. The underlying mechanism will be fascinating to dissect. [^1]: **Competing interests:**The authors declare that they have no competing interests. [^2]: No competing interests were disclosed. [^3]: No competing interests were disclosed. [^4]: No competing interests were disclosed.

Introduction {#Sec1} ============ The paraventricular thalamic nucleus (PVT) is a part of epithalamus having characteristic neural connections. The PVT receives input from serotonergic neurons, CRH (corticotoropin-releasing hormone) neurons in the hypothalamus, and suprachiasmatic nucleus. On the other hand, it sends output to the medial prefrontal cortex, nucleus accumbens, amygdala, and insular^[@CR1]^. Thus, the PVT is connected with many of brain regions implicated in emotion regulation. Recent studies show various emotion-related roles of the PVT such as fear conditioning^[@CR2]^, opiate withdrawal^[@CR3]^, saliency^[@CR4]^, and wakefulness^[@CR5]^. We recently reported that neuron-specific transgenic mice with mutant polymerase γ (*Polg*), a causative gene for mitochondrial diseases, showed recurrent spontaneous depression-like episodes and accumulation of deleted mitochondrial DNA (mtDNA) in the PVT^[@CR6]^, suggesting the possible role of the PVT in mood regulation^[@CR7]^. However, the role of the PVT in mood regulation has not been extensively studied yet. Though the output from the PVT to the nucleus accumbens^[@CR3]^ and amygdala^[@CR2]^ has been extensively studied, the role of the efferent projections from the PVT to the medial prefrontal cortex, which plays a central role in depression-like behavior, has not been well studied yet. In this study, we first investigated the neurophysiological effect of PVT manipulation on mPFC neurons by using genetic and pharmacogenetic approaches. We generated mice infected with adeno-associated virus (AAV) expressing the designer receptors exclusively activated by designer drugs (DREADDs) hM3Dq and Cre recombinase to induce tetanus toxin (TeTX), which cleaved the Vamp2 protein required for synaptic transmission, and then obtained electrophysiological recordings. Thereafter, we evaluated the effects of acute or long-term hM3Dq- or hM4Di-, a Gi-coupled inhibitory DREADD, and TeTX-PVT manipulation on the behavior in the long-term measurements of spontaneous wheel-running activity, forced swimming test (FST) and tail suspension test (TST). Materials and Methods {#Sec2} ===================== Animals {#Sec3} ------- All animal care and experimental procedures were in accordance with the guidelines for proper conduct of animal experiments published by the Science Council of Japan, and all the experiments were approved by RIKEN Wako Animal Experiment Committee and RIKEN Genetic Recombinant Experiment Safety Committee. For presynaptic inhibition in specific neurons by TeTX light chain, *CaMKIIα*-promoter-loxP-STOP-loxP-tTA (Tg2) and *TetO*-TeTX (Tg3) transgenic mice, which were kindly provided by Dr. S. Tonegawa (Massachusetts Institute of Technology, Boston, MA, USA), generated under a C57BL/6 genetic background were used^[@CR8]^. Heterozygous Tg2 and Tg3 were crossed to obtain the double-Tg mutant mice (Tg2/+; Tg3/+) which express TeTX depending on Cre recombinase expression. Other progenies (single mutant of Tg2/+ or Tg3/+, or wild-type+/+) were used as control mice. Behavioral data of the mice were excluded when they were injured or died due to an accident. Chemicals {#Sec4} --------- In the experiments for DREADD, clozapine N-oxide (CNO) (3 µg/g body weight, Enzo Life Sciences, Farmingdale, NY, USA) was administered by intraperitoneal injection at least one hour before behavioral experiments and mouse brain fixation. For long-term modulation of neuronal activity by DREADD, slow-releasing pellets of CNO for 90 days (Innovative Research of America, Sarasota, FL, USA) were implanted into the back subcutaneous tissue of the interscapular region under isoflurane anesthesia. Chow containing doxycycline (10 mg/kg, Oriental Yeast Co., Ltd., Tokyo, Japan) was fed to repress the expression of TeTX for at least two weeks. Virus and its injection into the PVT {#Sec5} ------------------------------------ AAV2-hSyn-HA-hM3D(Gq)-IRES-mCitrine, AAV2-hSyn-HA-hM4D(Gi)-IRES-mCitrine, and AAV8-hSyn-DIO-HA-hM3Dq(Gq)-IRES-mCitrine were purchased from UNC Vector Core (University of North Carolina at Chapel Hill, Chapel Hill, NC, USA). A bicistronic expression vector for Cre recombinase and EGFP (AAV2-Cre-IRES-EGFP) was produced as previously described^[@CR6],[@CR9]^. In brief, a PCR fragment containing Cre-IRES-EGFP was subcloned under the pCMV-β globin intron driver of AAV MCS vector to obtain pAAV-Cre-IRES-EGFP. The AAV2-CMV-hrGFP vector was purchased (Agilent, La Jolla, CA, USA). AAV particles were produced by using HEK293 cells. Before the surgery, mice were anesthetized with isoflurane and fixed in a stereotaxic frame. For the electrophysiological experiments, 1.0 µL of virus mixture containing AAV2-Cre-IRES-EGFP and AAV8-hSyn-DIO-HA-hM3Dq(Gq)-IRES-mCitrine (1:4) was injected into the PVT (AP --1.7 mm, ML 0.0 mm, and DV 3.2 mm from the bregma) by using a pump (UMP3; World precision instruments, Sarasota, FL, USA) with a 10-µL Hamilton syringe attached to a 33-gauge needle at the speed of 1 μl/20 min as described in previous paper^[@CR6]^. For the behavioral tests in the TeTX mice, 1.0 µL of AAV2-Cre-IRES-EGFP was injected in the PVT. Male heterozygous double-Tg mice (28--30 weeks old) Tg2/+;Tg3/+ \[N = 8\] or their other male littermate controls (N = 5:single mutant of Tg2/+ \[N = 2\], Tg3/+ \[N = 1\] and wild-type, +/+ \[N = 2\]) were used. After wheel-running measurements, the FST and TST were performed at 54--66 week of age, and the mice were sacrificed and the injection sites of AAV were verified by immunohistochemistry at the age of 65--70 weeks. For the behavioral tests in DREADD experiments, male C57BL/6 J mice were purchased from CLEA Japan Ltd. (Tokyo, Japan) at 8 weeks of age and used for the experiments. AAV2-hSyn-HA-hM3D(Gq)-IRES-mCitrine (N = 11), AAV2-hSyn-HA-hM4D(Gi)-IRES-mCitrine (N = 14), and AAV2-hSyn-EGFP (N = 12) were injected at 9--11 weeks of age, as described above. From 26 weeks of age, behavioral tests including the FST and TST were performed. At 48 weeks of age, the mice were sacrificed and the injection sites were verified by immunohistochemistry. Electrophysiology {#Sec6} ----------------- Detailed surgical procedures for chronic recording are described in a previous paper^[@CR10]^. In brief, a 32-channel silicon probe (A4x2-tet-5mm-500-400-121, NeuroNexus, Ann Arbor, MI, USA) was attached to a micromanipulator to adjust the desired depth position. Three mice were implanted with a silicon probe whose shanks were aligned along the rostrocaudal axis and a 10 degree angle from vertical axis in the mPFC (AP = 1.30 mm, ML = 0.75 mm, DV = 2.0 mm from the bregma) for data acquisition. Ground and reference wires were connected to screws implanted in the bone above cerebellum. During the recording sessions, animals were connected to a multiple channel amplifier (KJE1001, Amplipex Ltd, Hungary) via a headstage amplifier fixed to the micromanipulator to obtain neurophysiological signals at 20 kHz sampling rate and were allowed to freely move in an acrylic box (30 cm width × 30 cm height × 30 cm depth) covered with aluminum foil and white paper for animal bedding. Spike sorting was performed semi-automatically with KlustaKwik2^[@CR11]^, followed by manual adjustment by using Klusters v2.0.0^[@CR12]^. The clusters that included obvious noise were removed and those that were stable throughout the experiment were used for analysis. For electrophysiological experiments, AAV mixture (described above) was injected to the PVT of Tg2/+; Tg3/+ male mice as described above. Initially, the mice were fed with the chow containing doxycycline. In a single session, at 30 mins after the recording start, CNO or vehicle (phosphate buffered saline, PBS) was intraperitoneally injected into the mice, and the recording was conducted for 120 mins. After that, doxycycline-free chow was given to the mice for 2 weeks, and the same experiments were performed. Immunohistochemistry {#Sec7} -------------------- Mice were deeply anesthetized by intraperitoneal injection of 2.5% avertin (Tribromoethanol) at a dosage rate of 0.2 mL/10 g body weight and were fixed by transcardial perfusion with saline (0.9%), followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB, pH 7.4) and post-fixation with the same fixative overnight. The fixed brains were immersed in 30% sucrose in 0.1 M PB until sinking and embedded in OCT compound (Sakura Finetek Japan Co., Ltd, Tokyo, Japan). Cryosections of 14-µm thickness were prepared on a cryostat and attached to microscope slides. As primary antibodies, rabbit polyclonal anti-c-fos antibody (1:5000, PC38, Calbiochem, San Diego, CA, USA), rabbit polyclonal anti-pCREB antibody (1:500, \#9198, Cell Signaling Technology, Danvers, MA, USA), and chicken polyclonal anti-GFP antibody (1:500, \#GFP-1020, Aves labs, Tigard, OR, USA) were used. For pCREB staining, signals were enhanced by using TSA biotin system (PerkinElmer Japan Co., Ltd., Kanagawa, Japan). The localization of the antigen was visualized by using donkey anti-rabbit IgG Dylight 594, donkey anti-chicken IgY Dylight 488, donkey anti-rabbit IgG biotin, and streptavidine Dylight 594 as secondary antibodies. All secondary antibodies were purchased from Jackson ImmunoResearch (West Grove, PA, USA). The images were captured by confocal microscopy (FV1000; Olympus, Tokyo, Japan). Behavioral analysis {#Sec8} ------------------- ### Forced swimming test {#Sec9} Behavioral analyses were performed as described previously^[@CR6]^. In brief, the mouse was placed in a transparent, acrylic cylinder (11 cm diameter × 21.5 cm height) filled with tap water (22.5 °C ± 0.5 °C) with the depth of 11.5 cm. The cylinder was placed in an opaque box (33 cm × 22 cm × 48 cm), and the behavior of the mouse was recorded by a CCD video camera. In the experiments for TeTX mice, we tried to examine the effect of TeTX-on to TeTX-off switching, and the same animals were subjected to the FST again after doxycycline-chow feeding for more than for 2 weeks. However, it was difficult to compare the difference because of the shortened immobility time due to learned helplessness. In this paper, we describe approximately the initial 6 min in the first session. In the DREADD experiments, a single swimming session for 6 min was performed. ### Tail suspension test {#Sec10} For the TST, the mouse was suspended with its tail attached to a small aluminum plate by adhesive medical tape, and the plate was hooked to an attachment located inside an opaque box (48 × 22 × 33 cm). The behavior of the mouse was recorded by a CCD video camera for 6 min. Long-term analysis of wheel running {#Sec11} ----------------------------------- Wheel-running activity of the female mice was recorded and analyzed as described previously^[@CR6]^. Each mouse was individually housed in a cage equipped with a running wheel (5 cm wide and 14 cm in diameter) and an automatic counter for wheel rotation (O'hara & Co., Ltd., Tokyo, Japan). We prepared three groups of mice, AAV-hM3Dq (N = 12), AAV-hM4Di (N = 12) and AAV-GFP control (N = 6). Among these, 11 mice (N = 3, N = 4, and N = 4, respectively) did not show typical patterns of wheel running. Some did not learn wheel running, and others showed an abnormal pattern due to equipment issues, resulting in a constant light (LL) condition. These mice were excluded from the behavioral analysis. Because the number of the mice with AAV-GFP was too small to apply statistical analyses, the data of this group were shown only as a reference. At 124 days after the initiation of the wheel running, a pellet containing CNO was implanted. The depression-like episodes were defined by the relative strength index (RSI) as reported previously. Since the test period for the effect of DREADD in this study was shorter (3 months) than our previous study (\> = 6 months), we used relaxed criteria for the episodes (minimum 7 days with RSI \< 50, instead of 9 days in the original criteria, and at least one day with RSI \<30, instead of RSI \<25 in the original criteria). When the episode duration overlapped with the day of the start of CNO, episodes with low RSI (\<30) seen before the initiation of CNO treatment were included into the period before CNO, and those with the low RSI that appeared only after the initiation of CNO were included into the period after CNO. Even when the original criteria for the definition of the episode were used, the results were basically similar. Data analysis and statistics {#Sec12} ---------------------------- Statistical analysis was performed using Microsoft Excel (Microsoft Japan, Tokyo, Japan), Prism 4 (GraphPad Software, Inc., La Jolla, CA, USA) and an online website (<http://vassarstats.net/>). In the experiments for the electrophysiology, firing patterns of well-isolated units were analyzed using a custom script for Matlab with signal processing and statistic toolboxes (Mathworks, Natick, MA)^[@CR13]^ and Python 3 included in the Anaconda distribution downloaded from <https://anaconda.org/>. To examine the changes in firing rate (FR) associated with hM3Dq and/or TeTX, the obtained FR from each unit was evaluated as log2 ratio of each quarter to the first quarter in the sessions. Based on the patterns of normalized FR changes, the single units were divided into several clusters by using the "TimeSeriesKmeans" method with the Euclidean metric to calculate distance in the tslearn package in Python 3. The optimal number of clusters was decided by using the "elbow method," in which distortion was calculated as summation of total distances to the nearest cluster centers. We used eight cluster divisions for further analysis (Supplementary Fig. [1C,D](#MOESM1){ref-type="media"}). The significance of FR change was determined if the slopes of the linear regression of the normalized FR in the clusters were not significantly equal to zero (*p \< *0.05) and their absolute values were more than 1.0/120 mins in the sessions. Fisher's exact test was used for analysis of the proportion of the single units. The difference in the average FR at baseline (30 mins before the administration of PBS or CNO) was tested by Student's t-test. The difference in the proportion of inter-spike intervals (ISIs) was tested by the Kolmogrov-Smirnov (KS) test. When equal variance could not be assumed by Levene's test, Student t-test without assumption of equal variances was used. In the behavioral analysis, the immobility time and wheel-running activity were processed by the TimeFZ2 software attached to the equipment (O'hara & Co., Ltd.). In the immunohistochemical analysis, the number of c-fos- or pCREB-positive cells was counted by using ImageJ included in the Fiji distribution downloaded from <https://fiji.sc/>. One-way ANOVA with post-hoc Tukey's test was used for analysis of the number of c-fos- or pCREB-positive cells and immobility time in DREADD experiments. The one-tailed Student's t-test was used for the comparison of control and TeTX mice. The Kruskal-Wallis test was used for the comparison of the values obtained before and after CNO pellet implantation in wheel-running experiments. Statistical analysis was performed as described in Supplementary Methods. All data used for statistical analysis is available upon request. Results {#Sec13} ======= Firing patterns of mPFC neurons after activity modulation of PVT neurons {#Sec14} ------------------------------------------------------------------------ We generated the TeTX transgenic mice that were infected by AAV-Cre and AAV-DIO-hM3Dq and investigated the effect of manipulation of PVT neurons on neuronal activities in the mPFC. These mice enable us to bi-directionally modulate PVT activity by CNO administration for acute activation and by withdrawing doxycycline for chronic inhibition of their synaptic transmission. By using these mice, we conducted extracellular single-unit recordings in the mPFC to examine the effect of acute and chronic activity modulation of PVT neurons by hM3Dq and CNO administration under the TeTX-off or TeTX-on conditions (Fig. [1A,B](#Fig1){ref-type="fig"}). In the well-isolated units, there were two typical types of spike forms demarcated by a border in the trough-to-peak and spike width features indicating the rapid-firing putative interneurons (0.386 ± 0.026 ms \[mean ± standard error of mean (SEM)\]: trough-to-peak, 0.279 ± 0.014 ms: spike width) and regular firing putative pyramidal neurons (0.712 ± 0.012 ms: trough-to-peak, 0.437 ± 0.005 ms: spike width Fig. [1C(a,b)](#Fig1){ref-type="fig"})^[@CR14]^. To evaluate the effect of chronic presynaptic inhibition of PVT neurons, we compared the activity of mPFC neurons during baseline, the first 30 mins in each session, and found that the number of well-isolated units was decreased in the TeTX-on condition, the proportion of interneurons in the units, however, was significantly higher (Fig. [1C(c)](#Fig1){ref-type="fig"}). The average FR of interneurons during baseline was significantly reduced in the TeTX-on conditions, whereas that of pyramidal neurons did not change (Fig. [1D](#Fig1){ref-type="fig"}). In addition to the reduction of FR, the proportion of inter-spike intervals (ISIs) of interneurons in the TeTX-on conditions was biased toward being shorter than that in the TeTX-off conditions, irrespective of PBS or CNO administration, indicating intermittent firing patterns (Fig. [1E](#Fig1){ref-type="fig"}, Supplementary Fig. [1A](#MOESM1){ref-type="media"}). These ISIs in the TeTX-on conditions had a tendency to shorten as the sessions progressed (Supplementary Fig. [1B](#MOESM1){ref-type="media"}). Next, to evaluate the effect of acute activation of hM3Dq-expressing PVT neurons by CNO administration, we observed time course of FR of pyramidal neurons and interneurons (Fig. [1F](#Fig1){ref-type="fig"}). Based on their firing patterns, units were divided into eight clusters and the units in the clusters that showed significant trend were defined as the cell groups showing increasing or decreasing FR (Supplementary Fig. [1C,D](#MOESM1){ref-type="media"}). The activities of mPFC pyramidal neurons in the TeTX-off conditions were altered by CNO administration whereas that in interneurons in the TeTX-off conditions and in both cells in the TeTX-on conditions were not, suggesting that neuronal activity changes in the mPFC by PVT activation had been mediated by synaptic transmission from PVT neurons to mPFC neurons. These experiments indicated that synaptic transmission of PVT neurons affected the activity of mPFC pyramidal neurons and chronic presynaptic inhibition in PVT neurons altered the proportion of excitatory and inhibitory neurons in the mPFC and induced intermittent firing patterns with decreased FR in mPFC interneurons.Figure 1Effect of manipulation of PVT neurons by hM3Dq and TeTX on neuronal activity in the mPFC. (**A**) AAV constructs and genetic background of TeTX mice. After excision of floxed STOP by Cre recombinase, TeTX expression was induced by tTA that was inhibited by doxycycline. TeTX was expressed in doxycycline-free conditions. AAV-hM3Dq with double-floxed inverse open reading frame (DIO) expresses hM3Dq when co-injected with AAV-Cre. (**B**) Schematic drawing of electrophysiological experiments. AAVs were injected into the PVT and electrodes were implanted into the mPFC. Each recording session consisted of first 30 min of baseline and the subsequent 90 min of the test period for acute evaluation of activation of PVT neurons. Recordings were conducted under TeTX-off and TeTX-on conditions to evaluate chronic effect of presynaptic inhibition of PVT neurons. (**C**) (a) Classification of isolated units based on spike waveforms. (b) The two clusters demarcated by a border in the trough-to-peak vs spike width plot indicated rapid-firing putative interneurons (red) and regular-firing pyramidal neurons (black) in the TeTX-off conditions (left) and the TeTX-on conditions (right). (c) The cumulative bar graph indicates the proportion of pyramidal neurons and interneurons in isolated units. Error bars indicate standard error of mean (SEM). \* indicates p \< 0.05 by two-tailed Student's t-test. (**D**) Average FR of pyramidal neurons (left) and interneurons (right) during baseline. \*\*indicates *p* \< 0.001 and NS indicates *p* \> 0.05 by two-tailed Student's t-test. Error bars indicate SEM. (**E**) Probability distributions of inter-spike intervals (ISIs) of pyramidal neurons (upper) and interneurons (bottom). The probabilities in each 1 ms-bin (main graph) and the cumulative probabilities (inset) in PBS (cyan) or CNO (orange) administration in the TeTX-off conditions or PBS (green) or CNO (red) administration in the TeTX-on conditions are shown. Error bars indicate SEM. \* indicates *p* \< 0.05 by 2 sample Kolmogorov-Smirnov test. (**F**) Line plots indicate the time course of normalized firing rate (FR) of pyramidal neurons (upper) and interneurons (bottom) in the indicated experimental conditions. Pie charts indicate the proportion of cells classified into clusters showing stable FR (grey), decreasing FR (blue), and increasing FR (red). Colors in line plots also indicate the cells in each cluster. \*indicates *p* \< 0.05 by two-tailed Fisher's exact test. Details of clustering of all units are shown Supplementary Fig. [1](#MOESM1){ref-type="media"}. Long-term effect of continuous manipulation of PVT neurons by DREADD {#Sec15} -------------------------------------------------------------------- Next, we examined the effect of long-term modulation of the activity of PVT neurons by DREADD on long-term wheel-running activity measurements in this study. For this analysis, we generated female mice infected with AAV-hM3Dq, AAV-hM4Di or AAV-GFP and subcutaneously implanted drug-releasing pellets containing CNO in the middle of wheel-running measurements. The first 4 months of measurements were defined as baseline and the following 3 months after the implantation of CNO pellets were evaluated as a test period for the modulation of neuronal activity. The frequency of the hypoactivity episodes in the mice with AAV-hM3Dq after the CNO implantation was significantly greater than that in all mice before CNO implantation,whereas that was not significantly increased in the group of AAV-GFP and AAV-hM4Di (Fig. [2A,B](#Fig2){ref-type="fig"}). After wheel-running measurements, we conducted immunohistochemical staining for GFP and pCREB and found a significant increase in pCREB-positive cells in the PVT region in AAV-hM3Dq mice (Fig. [2C,D](#Fig2){ref-type="fig"} and Supplementary Fig. 2). These results suggest that AAV-hM3Dq mice implanted with CNO-releasing pellets showed sustained activity of PVT neurons and increased hypoactivity episodes. Unexpectedly, however, AAV-hM4Di mice also showed increased pCREB-positive cells in the PVT (Fig. [2C,D](#Fig2){ref-type="fig"}). There might be uncertain feedback mechanisms caused by chronic hM4Di activation.Figure 2Effect of long-term manipulation of PVT neurons by DREADD on long-term wheel-running activity. (**A**) Wheel-running activity of the mice with AAV-GFP, AAV-hM3Dq, or AAV-hM4Di. Horizontal axis indicates the date from the implantation of CNO pellet (Day 0). Vertical axis shows the movement of the running wheel per day. The thick lines under the record of wheel running indicate the hypo-activity episodes. (**B**) The frequency of episodes per month before (4 months) and after (3 months) the CNO pellet implantation. \*indicates *p* \< 0.05 by post-hoc Dunn's Multiple Comparison test following Kruskal-Wallis test. (**C**) images indicate anti-GFP (green) and anti-pCREB (red) immunostaining and nuclear staining with DAPI (blue) in the mice with AAV-GFP, AAV-hM3Dq, or AAV-hM4Di. Scale bar indicates 100 µm. (**D**) Number of pCREB-positive cells in the three groups, AAV-GFP, AAV-hM3Dq, or AAV-hM4Di. The AAV-GFP group included two mice housed under the LL condition, because they showed similar pCREB staining with mice under the LD condition. Error bars indicate standard error of mean. \* and \*\*indicate *p* \< 0.05 and *p* \< 0.01, respectively. Effect of chronic presynaptic inhibition in PVT neurons by TeTX on FST and TST {#Sec16} ------------------------------------------------------------------------------ We further examined the effect of chronic presynaptic inhibition in PVT neurons by TeTX on the FST and TST (Fig. [3A](#Fig3){ref-type="fig"}). The immobility time of the TeTX-expressing mice was significantly shorter than that of control mice in the FST (Fig. [3B](#Fig3){ref-type="fig"}). There was no detectable change in the TST (Fig. [3C](#Fig3){ref-type="fig"}). On the other hand, acute modulation of PVT neurons by DREADD did not affect FST and TST (Supplementary Fig. 3).Figure 3Effect of presynaptic inhibition in PVT neurons by TeTX on the FST and TST. (**A**) AAV construct and genetic background of the TeTX mice. (**B**,**C**) Bar plots showing the ratio of immobility time in total time (6 mins) in the FST (**B**) and TST (**C**). Error bars indicate SEM. \* indicates *p* \< 0.05 by one-tailed Student's t-test. Discussion {#Sec17} ========== In rodents, several regions of the medial prefrontal cortex (mPFC) are distinguished by their distinct cytoarchitectures such as the anterior cingulate, prelimbic, or infralimbic cortex. Among them, PVT neurons are reported to project to the layers 1 and 5 of the prelimbic and infralimbic cortices in rats, and we also confirmed that the same regions were innervated by the PVT in mice (Supplementary Fig. 4). However, functional significance of the projection from the PVT to the mPFC is not understood yet. In this study, we found that the modulation of the activity of PVT neurons affected firing patterns in mPFC neurons (Fig. [1](#Fig1){ref-type="fig"}). Chronic presynaptic inhibition in PVT neurons increased the proportions of the interneurons that showed intermittent firing patterns with reduced FR in isolated units (Fig. [1C--E](#Fig1){ref-type="fig"} and Supplementary Fig. [1A,B](#MOESM1){ref-type="media"}). In addition, activation of PVT neurons by hM3Dq expression and CNO administration affected the FR of pyramidal neurons in the mPFC, whereas the effect was nullified in the TeTX-on conditions (Fig. [1F](#Fig1){ref-type="fig"}). Although recent reports indicated that CNO or its metabolite clozapine could affect neuronal activities and animal behaviors^[@CR15],[@CR16]^, the observations in the experiments in this study did not correspond with those findings, because this effect was not observed under the TeTX-on conditions, which would mean that alterations of FR in the mPFC depended on synaptic transmission of PVT neurons (Fig. [1F](#Fig1){ref-type="fig"}). In behavioral analysis, we found that long-term activation of PVT neurons by hM3Dq increased the frequency of depression-like episodes (Fig. [2](#Fig2){ref-type="fig"}) and chronic presynaptic inhibition in PVT neurons shortened the immobility time in the FST (Fig. [3](#Fig3){ref-type="fig"}). Our electrophysiological experiments in the mPFC in the mice in which PVT neurons were manipulated by hM3Dq and TeTX revealed that the acute activation of PVT neurons caused FR change (Fig. [1F](#Fig1){ref-type="fig"}). On the other hand, chronic presynaptic inhibition in PVT neurons altered the population of active neurons that was relatively interneuron dominant compared with normal (TeTX-off) conditions and induced intermittent firing patterns (Fig. [1E](#Fig1){ref-type="fig"} and Supplementary Fig. [1A,B](#MOESM1){ref-type="media"}), even though the features obtained from the experiments under normal conditions correspond largely to previous reports described in this region^[@CR10],[@CR14],[@CR17]^. Recently, another group reported that acute inhibition of the mediodorsal thalamus (MD), a region adjacent to the PVT, mostly affected interneurons in the mPFC and selective activation of parvalbumin interneurons in the mPFC by hM3Dq ameliorated not only excitatory and inhibitory imbalance in the mPFC but also cognitive deficits caused by inhibition of MD neurons^[@CR18]^. The MD as well as the PVT mainly consist of glutamatergic neurons, a part of them harboring projections to similar areas in the mPFC^[@CR19],[@CR20]^, and both may have similar functions to regulate the excitatory/inhibitory (E/I) balance in the mPFC, even though they seem to subserve distinct roles associated with "flexibility" or "cognition memory" in the MD, but with "affective function" or "stress response" in the PVT probably due to the different upstream in the neural circuits^[@CR20]^. Meanwhile, it cannot be completely excluded that viral vector affected not only PVT but also the adjacent MD which projects to the mPFC as well. Intermittent firing patterns with the reduction of FR in interneurons in the mPFC were also previously observed in the rats in which the substantia nigra compacta (SNc) was lesioned by 6-hydroxydopamine and that was mediated by serotonin signaling^[@CR21]^. Therefore, the characteristic firing patterns caused by chronic presynaptic inhibition of PVT neurons may be the consequence of involvement of dopamine and serotonin signaling. While the PVT receives dopamine fibers from the hypothalamus (ventrorostral A10, A11, A13 and A15 cell groups) and periaqueductal gray (dorsocaudal A10), the PVT does not receive fibers from the SNc (A9) or ventral tegmental area (VTA, A10) directory. In spite of different locus, the impairments in the PVT induce intermittent firing in the mPFC as well as that in SNc/VTA may be due to direct or indirect connection to 5-HT~3~-expressing interneurons in the mPFC. We unexpectedly found that long-term treatment of CNO in AAV-hM4Di mice increased pCREB-positive cells in PVT neurons (Fig. [2C,D](#Fig2){ref-type="fig"}), whereas acute treatment with CNO showed the tendency to decrease c-fos positive cells in the PVT (Supplementary Fig. [3A,B](#MOESM1){ref-type="media"}). The effect of hM4Di has been shown to be mediated by G-protein coupled inward rectifier K^+^ channels (GIRKs)^[@CR22]^. A previous report showed that thyrotropin-releasing hormone (TRH) enhances excitability in PVT neurons through inactivation of GIRKs by decreasing their conductance^[@CR23]^. Hence, chronic activation of hM4Di in PVT neurons may paradoxically activate PVT neurons in the presence of TRH. Both TeTX and hM4Di are generally used to induce loss-of-function in specific neurons; each of them, however, shows technical limitations depending on the mechanism and in the case of PVT silencing, TeTX is considered to be more beneficial. In this study, we used hSyn promoter for expression of DREADDs and CaMKIIα promoter for expression of tetanus toxin, and thus there is a possibility that different neurons were manipulated in these experiments. Indeed, a recent study showed promoter-specific effects of viral-mediated gene transfer (hSyn promoter vs Camk2a promoter) on synaptic transmission and plasticity in hippocampus. However, the PVT in rodents consists of mainly glutamatergic neurons, and has no GABAergic neurons. The data of Allen Brain Atlas (<https://portal.brain-map.org/>) also shows that the PVT consists of Vglut2 positive cells with no detectable Gad1 expressing cells. Therefore, we can assume that the neurons labelled by CaMKIIα promoter and hSyn promoter do not largely differ. Indeed, our previous study, expression of tetanus toxin in PVT neurons under CaMKIIα promoter caused reduction of Vamp2 in the similar regions to the current experiment; the prelimbic and infralimbic cortices^[@CR6]^. However, we cannot completely rule out that different neurons were manipulated in these experiments. Although several reports have suggested the association between the PVT and in human patients with major depressive disorder or bipolar disorder^[@CR7]^, the roles of the PVT related to manifestation or amelioration of the symptoms remain largely unknown. Many types of stress cause induction of c-fos in the PVT region^[@CR24]^. Induction of c-fos by stress is reversed by antidepressants^[@CR25]^, and reduction of c-fos induction in the PVT region by antidepressants is correlated with the behavioral effect of an antidepressant on the FST^[@CR26]^. Although antidepressants consistently upregulate c-fos in the PVT^[@CR27]^, this is an acute effect and thus may not be related to antidepressive effect. In contrast to the present finding that long-term activation of PVT neurons by hM3Dq caused hypoactivity episode, we previously reported that chronic presynaptic inhibition in PVT neurons by TeTX increased the frequency of such episodes in long-term wheel-running activity^[@CR6]^. There might be the mechanism influencing PVT targets by chronic presynaptic inhibition of PVT neurons in non-cell autonomous manner. Unless innervations of PVT neurons are completely biased to inhibitory neurons or excitatory neurons, the following both scenarios could be induced by inhibition of PVT neurons: activation of excitatory neurons via suppression of inhibitory neurons and suppression of activation of excitatory neurons. Based on our results, chronic presynaptic inhibition of PVT neurons induced dominance of inhibitory neurons in the mPFC. The fact, however, does not necessarily mean that long-term activation of PVT neurons results in opposite reaction. It is also possible that alterations of synaptic plasticity like long-term potentiation or depression in mPFC neurons are involved in regulation of this circuit. Though we did not address the effects of neural transmission from PVT neurons on the activities of mPFC neurons during the mice showing depression like behavior, the fact that PVT neurons can affect the E/I balance of mPFC neurons suggest a possibility that PVT-mPFC pathway might play a role in this behavior. The results of FST and TST were not consistent between chronic inhibition by TeTX and acute inhibition by hM4Di. Because these tests were performed at 26--66 weeks old, the age might not be optimum for these analyses. Whereas chronic inhibition of PVT neurons by TeTX reduced immobility time in FST, it did not in TST. Although both tasks are used to evaluate the effect of anti-depressants in rodents, these two test have differences in many aspects such as time course and drug sensitivity and may have different neurobiological basis which may be associated with heterogeneity of psychiatric symptoms. Thus, it would be rather interesting that only FST was affected by PVT inhibition. In this study, we found that long-term activation of the PVT increased the frequency of depression-like hypoactivity episodes in mice. This finding suggests that proper synaptic transmission of the PVT to the related targets has important roles in depression. Further detailed studies to manipulate more specific neural circuits related to the PVT would contribute to understanding the basis of mood disorders. Supplementary information ========================= {#Sec18} Supplementary Information **Publisher's note** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary information ========================= is available for this paper at 10.1038/s41598-019-52984-y. The work was supported by JSPS KAKENHI Grant Number 18H05435 and 17H01573 to T.K. and the Advanced Genome Research and Bioinformatics Study to Facilitate Medical Innovation (GRIFIN) from the Japan Agency for Medical Research and Development (AMED) (19km0405208h0004) to T.K. T.M.K. and T.K. conceived the study and analyzed the data. T.M.K. and N.F.-T. performed experiments. H.M. and K.O. produced adeno-associated virus vectors. S.F. supervised the electrophysiological experiments and data analysis. T.M.K. and T.K. wrote the paper. The authors declare no competing interests.

Specifications tableSubjectRenewable Energy, Sustainability and EnvironmentSpecific subject areaLignocellulosic Biomass, Biofuels, BioproductsType of dataTable, Image, GraphHow data were acquiredThe data were acquired based on the analytical instrument which included scanning electron microscope (SEM), Fourier Transform Infrared Spectroscopy (FTIR), Electron Dispersive X-ray (EDX), X-ray Diffraction (XRD), High Performance Liquid Chromatography (HPLC) and UV--vis Spectrophotometry.Data formatRaw and analysed.Parameters for data collectionCharacterisations of fresh empty fruit bunch (EFB), degraded EFB (DEFB) and treated DEFB after alkaline ethylene glycol pretreatment and dataset for analysis methods used for the acid hydrolysate obtained from the two-staged acid hydrolysis.Description of data collectionThe characterisations of fresh EFB, DEFB and treated DEFB were conducted by using SEM, EDX, FTIR and XRD. 3,5-Dinitrosaclicylic acid (DNS) and Folin Ciocalteus reagent tests were measured by UV--Vis spectrophotometry. Concentrations of furfural and hydroxymethylfurfural (HMF) were quantified by HPLC.Data source locationInstitution: Lee Kong Chian Faculty of Engineering and Science, Universiti Tunku Abdul Rahman (UTAR)\ City/Town/Region: 43,000 Kajang, Selangor\ Country: MalaysiaData accessibilityThe data were available in this article.Related research articleChin, D.W.K., Lim, S., Pang, Y.L., Lim, C.H., Lee, K.M., 2019. Two-staged acid hydrolysis on ethylene glycol pretreated degraded oil palm empty fruit bunch for sugar based substrate recovery. Bioresour. Technol. 292, 121,967. <https://doi.org/10.1016/j.biortech.2019.121967>. [@bib0001] Value of the Data {#sec0001a} ================= •The presented dataset are useful for researchers to study on different pretreatment methods for biofuel and biochemical production since they are still very rarely accessible in the literature.•The various characterisations tests carried out in this study can serve as an important reference for other researchers intending to perform similar analysis.•The characterisation results after alkaline ethylene glycol pretreatment can be used as a comparison with other pretreatment methods to develop an effective pretreatment for other types of lignocellulosic biomass.•The dataset of the analysis methods for the acid hydrolysate can be used as reference for developments of advanced analysis methods to further improve their accuracy and consistency.•The data in this article provide the transformation of DEFB after subjected to the alkaline ethylene glycol pretreatment and two-staged acid hydrolysis which are essential for researchers and scientific community in this field. 1. Data description {#sec0001} =================== Fresh empty fruit bunch (EFB) is a lignocellulosic biomass waste which is produced during palm oil extraction process. Fresh EFB is highly susceptible to microbial attack when it is exposed to the open environment. The growth of the microorganisms will degrade its physical and chemical properties and affect its reutilisation. However, the degraded empty fruit bunch (DEFB) remains to be a potential source for biofuels and biochemical productions through alkaline ethylene glycol pretreatment and two-staged acid hydrolysis. In this study, different instrument characterisations were used to provide the physical and chemical information for the DEFB samples. [Fig. 1](#fig0001){ref-type="fig"} shows the FTIR spectrum of fresh EFB, DEFB and treated DEFB after the alkaline ethylene glycol pretreatment. [Table 1](#tbl0001){ref-type="table"} illustrates the XRD intensity of the similar samples after the alkaline ethylene glycol pretreatment. [Fig. 2](#fig0002){ref-type="fig"} shows their surface morphology while [Table 2](#tbl0002){ref-type="table"} lists the elemental compositions of different EFB samples. The validation of the analysis methods that were used to determine the product compositions of acid hydrolysate is also provided in [Sections 2.3](#sec0005){ref-type="sec"}--[2.5](#sec0007){ref-type="sec"}. Raw data of this article is available in the supplementary files.Fig. 1FTIR Spectrum of (a) treated DEFB after alkaline ethylene glycol pretreatment, (b) untreated DEFB and (c) untreated fresh EFB from 500 cm^−1^ to 4000 cm^−1^.Fig 1Table 1XRD peak intensities at 18.5^∘^ (amorphous region) and at 22.5^∘^ (crystalline region) of cellulose.Table 1EFB samplesIntensity (I.U.)18.5^∘^22.5^∘^Fresh EFB116369Untreated DEFB126340Alkaline ethylene glycol treated DEFB154418Fig. 2Surface morphology of (a) fresh EFB, (b) untreated DEFB, (c) treated DEFB after ethylene glycol pretreatment and (d) treated DEFB after ethylene glycol pretreatment with NaOH under 500  ×  magnification.Fig 2Table 2Elemental composition of fresh EFB, untreated DEFB and treated DEFB after alkaline ethylene glycol pretreatment.Table 2Elemental composition (At%)Fresh EFBDEFBTreated DEFBCarbon64.163.555.3Oxygen34.635.343.1Sodium0.10.11.0Magnesium0.20.30.2Silicon0.70.60.2Phosphorous0.10.10.1Sulfur0.10.10.1Ferum0.10.10.1 2. Experimental design, materials, and methods {#sec0002} ============================================== 2.1. Preparation of DEFB and alkaline organosolv pretreatment of DEFB {#sec0003} --------------------------------------------------------------------- DEFB was collected from a plantation estate which is located at Segamat, Malaysia. Prior to utilisation, DEFB was separated into smaller pieces and dried at 100 °C overnight. The dried DEFB was subsequently grinded into powder form and sieved using the size of 850$\mu$m [@bib0002]. Predetermined amount of powdered DEFB was treated with 50 v/v% ethylene glycol in the presence of 3 v/v% NaOH at 80 °C for 45 min under 10 w/v% mass loading. Treated DEFB was washed thoroughly with distilled water for several times and dried in oven at 100 °C for overnight. Fresh EFB, untreated powdered DEFB and treated DEFB were subjected to characterisation analysis including FTIR, XRD, SEM and EDX as shown in [Fig. 1](#fig0001){ref-type="fig"}, [Table 1](#tbl0001){ref-type="table"}, [Fig. 2](#fig0002){ref-type="fig"} and [Table 2](#tbl0002){ref-type="table"}, respectively. The FTIR spectrums of different EFB samples were obtained with Nicholet IS10 in the range of 500--4000 cm^−1^. XRD patterns were determined by using Shimadzu XRD-6000 from 5^∘^ to 60^∘^ at the speed of 2^∘^/min. The surface morphology with 500  ×  magnification was obtained with Hitachi SEM while elemental composition of sample was determined using Aimtek EDX. 2.2. Component analysis for two-staged acid hydrolysis {#sec0004} ------------------------------------------------------ Treated DEFB was subjected to two-staged acid hydrolysis and the complete methodology can be obtained from our previous work [@bib0001]. 2.3. DNS method for reducing sugars {#sec0005} ----------------------------------- DNS reagent was used to quantify the reducing sugars in acid hydrolysate [@bib0003]. 1 ml of the neutralised sample was added with 2 ml of 3,5-dinitrosalicylic acid (DNS) reagent before immersed in a water bath at 100 °C for 5 min. Afterwards, the solution was added with 7 ml of distilled water and allowed to cool down to room temperature. The solution was detected at 540 nm with UV--vis spectrophotometry (PG Instruments T-60) and glucose was used as the standard solution. The calibration curve of glucose and coefficient of determination (*R*^2^) are shown in [Fig. 3](#fig0003){ref-type="fig"}.Fig. 3Calibration curve of DNS method by using glucose as standard at 540 nm detection.Fig 3 2.4. Folin-Ciocaltues reagent method for phenolic contents {#sec0006} ---------------------------------------------------------- Folin-Ciocalteus reagent was used to determine the phenolic concentration in acid hydrolysate [@bib0004]. A total volume of 9 ml solution which included 8.4 ml distilled water, 0.5 ml Folin-Ciocalteus Reagent and 0.1 ml hydrolysate was prepared. The solution was allowed to react in the dark at room temperature for 1 h. The final solution was then subjected to UV--vis spectrophotometry (PG Instruments T-60) at 750 nm. The standard was prepared by using gallic acid and the calibration curve which recorded a *R*^2^ of 0.9924 is shown in [Fig. 4](#fig0004){ref-type="fig"}.Fig. 4Calibration curve of phenolic compounds by using gallic acid as standard at 750 nm detection.Fig 4 2.5. HPLC method for furfural and HMF {#sec0007} ------------------------------------- The quantifications of furfural and HMF were determined by using a reversed phase HPLC (Shimadzu LC-20AD) which was equipped with a quaternary pump unit, a column oven, auto-sampler and an UV--vis detector. Hypersil Gold C~18~ column (150 mm  ×  4.6 mm, 5 $\mu$m) was used to separate the furfural and HMF which were detected at 280 nm. The mobile phase was composed of 11 v/v% acetonitrile, 88 v/v% deionised water and 1 v/v% acetic acid [@bib0005]. The mobile phase was filtered with the membrane filtration for three times prior to its usage. The analysis was conducted at column temperature of 40 °C with fixed flowrate at 1.0 ml/min. The injection volume of sample was 10 $\mu$l. The HPLC chromatogram of HMF and furfural is shown in [Fig. 5](#fig0005){ref-type="fig"} and [Table 3](#tbl0003){ref-type="table"} shows the intraday and interday parameters of HMF and furfural.Fig. 5HPLC Chromatogram of furans (a) HMF (4.526 min) and (b) furfural (5.851 min) with Hypersil Gold C~18~ with flowrate 1.0 ml/min.Fig 5Table 3Intraday and Interday precision, retention time and limit of detection for HMF and furfural.Table 3ComponentsFurfuralHMFRetention time (min)5.8504.526Intraday precision (%) Retention time0.0200.025 Peak area0.5860.285Interday precision (%) Retention time0.0850.088 Peak area1.0430.312Coefficient of determination (*R*^2^)0.99990.9999 Appendix. Supplementary materials {#sec0009} ================================= Image, application 1Image, application 2 The authors would like to express their gratitude towards financial support provided by Universiti Tunku Abdul Rahman Research Fund, Malaysia (UTARRF/2017-CI/206) and Fundamental Research Grant Scheme (FRGS/1/2018/TK10/UTAR/02/1) from Ministry of Education, Malaysia. Conflict of Interest {#sec0008a} ==================== The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Supplementary material associated with this article can be found, in the online version, at doi:[10.1016/j.dib.2020.105431](https://doi.org/10.1016/j.dib.2020.105431){#interref0002}.

All relevant data are within the paper and its Supporting Information files. Introduction {#sec001} ============ Low temperature is one of the major abiotic stresses that threaten the adaptability of rice and its production. Due to the long growing season and frequent low temperature in North China, Korea, Japan and other countries at high latitudes, the growth duration of cultivars that cannot tolerate low temperature has to be shortened and this usually results in low yields. Cold stresses that often occur during flowering and grain filling all over the world \[[@pone.0120590.ref001]--[@pone.0120590.ref002]\] may also lower grain quality and yield. Cold tolerance of rice is a complex quantitative trait significantly influenced by environment. Thus research on the underlying genetic mechanisms and discovery of genes affecting to cold tolerance in rice will be helpful for developing elite cultivars with strong cold tolerance. Segregating populations are usually used for mapping and cloning genes for cold tolerance. More than 30 quantitative trait loci (QTLs) for cold tolerance have been mapped on 12 chromosomes (according to [www.gramene.com](http://www.gramene.com)). Most of them were for cold tolerance at the germination stage, and a few were cloned. Using backcross inbred lines of Livorno/Haymasari, the QTL *qLTG3--1* was cloned and proven to contribute to the high germinability under low temperatures \[[@pone.0120590.ref003]\]. Mapping QTL for cold tolerance at the booting stage is more difficult because of difficulties in phenotyping and the underlying complex biological and genetic mechanisms involved. Eight QTLs related to spikelet fertility under cold stress were mapped on chromosomes 1, 4, 5, 7, 10 and 11 based on NILs derived from KMXBG as donor and Towada as the recipient \[[@pone.0120590.ref004]--[@pone.0120590.ref005]\]. Saito et al. mapped the QTL *Ctb1* encoding an F-box protein and contributing to normal anther development under cold stress \[[@pone.0120590.ref006]\]. In contrast to traditional QTL mapping, "association mapping" is an alternative approach for studying complex traits. Association mapping, also known as linkage disequilibrium mapping, utilizes allelic variation in natural populations, and is capable of simultaneously identifying many loci for multiple traits \[[@pone.0120590.ref007]\]. Association mapping has been employed in rice to identify QTLs or genes related to yield \[[@pone.0120590.ref008]--[@pone.0120590.ref010]\], heading date \[[@pone.0120590.ref011]\], disease resistance \[[@pone.0120590.ref012]--[@pone.0120590.ref013]\] and tolerance to abiotic stresses \[[@pone.0120590.ref014]\]. Using 174 rice accessions from the mini core collection of cultivated rice and 273 simple-sequence repeat (SSR) markers we detected QTLs associated with cold tolerance during the germination and booting stages. We subsequently screened the accessions with strong cold tolerance and QTL markers showing strong effects, which will be the potential parents and markers in developing cultivars with cold tolerance by molecular marker assisted selection. Materials and Methods {#sec002} ===================== Plant materials {#sec003} --------------- The research material comprised 174 diverse accessions from the mini core collection of cultivated rice \[[@pone.0120590.ref015]\]. Among them, 118 were landraces, 56 were modern improved cultivars and 109 were subspecies *indica*, and 65 were *japonica* ([S1 Table](#pone.0120590.s002){ref-type="supplementary-material"}). Evaluation of cold tolerance at the germination stage {#sec004} ----------------------------------------------------- Fifty seeds of each accession were placed in a drying oven at 50°C for 48 h to break dormancy, and then soaked in distilled water in a 15 cm Petri dish for 24 h. The pre-soaked seeds were germinated in a growth chamber at a constant temperature of 32°C for 36 h. Germinated seeds with 5 mm coleoptiles were stressed at 5°C for 10 days, and then moved to a greenhouse at 20°C for 10 days to allow seedlings to recover and resume normal growth. Seedling survival rates (SSvR) were then scored as a measure of cold tolerance at the germination stage. Evaluation of cold tolerance at the booting stage under naturally low temperature conditions {#sec005} -------------------------------------------------------------------------------------------- One measure of cold tolerance at the booting stage is the seed setting rate under naturally low temperature conditions (SStR-NL). Suitable conditions are available at an experimental farm of Yunnan Academy of Agricultural Sciences, Kunming, where atmospheric temperatures are 15--19°C during the booting stage extending from June to August \[[@pone.0120590.ref016]\]. Seedlings of each cultivar were grown in a nursery seedbed planted 31 March 2006, and transplanted on 9 May with two replications. Twenty plants of each accession were transplanted in a single row with a row × plant distance of 25 × 15 cm. The field was fertilized with N (120 kg/ha^2^) and P~2~O~5~ (80 kg/ha^2^). SStR-NL was scored as the mean seed setting rate of 10 plants from the middle 18 plants in each row. Evaluation of cold tolerance at the booting stage under cold water stress {#sec006} ------------------------------------------------------------------------- The other two measures of cold tolerance at the booting stage were seed setting rate under cold water (SStR-CW) and relative seed setting rate (RSStR-CW, i.e. ratio of seed setting rate under cold water stress to seed setting rate under normal conditions). Cold tolerance under cold water stress was evaluated in Gongzhuling, Jilin province. Seedlings were grown in a nursery seedbed after planting on 15 April 2006, and transplanted on 25 May with two replications. Twelve plants of each accession were transplanted in a single row with a line × plant distance 25 × 15 cm. The field was fertilized with N (120 kg/ha^2^) and P~2~O~5~ (80 kg/ha^2^). On 1 July at panicle initiation, the field was irrigated continuously for 40 days with cold water pumped from a deep well where the water temperature is a constant 19°C. The cold water was maintained at 20 cm depth at the early stage to 30 cm at the late growth stage, such that the young panicles were covered by cold water. Late accessions that could not be harvested from late August to mid-October were enclosed by a plastic greenhouse to avoid the effects of low atmospheric temperatures on grain filling. Another two replications were transplanted and managed under the same conditions, except that they were irrigated with surface water at higher temperatures (daily mean temperature of 22--26°C from 1 July to 10 August). Mean seed setting rates for all accessions under both cold and normal conditions were recorded on the middle 10 plants in each row. SSR polymorphism and genotyping {#sec007} ------------------------------- DNA was extracted from the young leaves using the CTAB method. Two hundred and seventy three simple sequence repeat (SSR) markers randomly distributed across all 12 rice chromosomes ([S2 Table](#pone.0120590.s003){ref-type="supplementary-material"}) were selected for genotyping. The average distance between markers was 835 kb. The minimum distance between adjacent markers was 0.06 kb and the maximum was 4,050 kb; 18% of distances between adjacent markers were less than 150 kb, more than 50% were less than 750 kb, and more than 90% were less than 1,800 kb. PCR conditions, gel electrophoresis of PCR products and genotype scoring methods were as described in Zhang *et al*. \[[@pone.0120590.ref015]\]. Statistical analysis and association mapping {#sec008} -------------------------------------------- STRUCTURE 2.2 \[[@pone.0120590.ref017]\], a model-based program, was utilized to infer population structure with 60 unlinked SSRs from the 273 markers. Ten independent simulations were run for each *K* (the number of clusters, from 1 to 10) using a burn-in length of 10,000, run length of 100,000 and a model for admixture and correlated allelic frequencies. To determine the *K* value, the LnP(D) value in the STRUCTURE output and Evanno's*Δ*K between successive *K* \[[@pone.0120590.ref018]\] were used. Pair-wise linkage disequilibria between markers were evaluated by TASSEL V4.0 (<http://www.maizegenetics.net/tassel>) in a total of 174 accessions and in each of two clusters inferred by STRUCTURE (here, the clusters were subspecies, *indica* and *japonica*). Association analyses between SSR markers and measures of cold tolerance were carried out by TASSEL V4.0. The Q-Q plot of GLM and MLM for four measures of cold tolerance indicated that MLM overestimated the effect of genetic relationship among individuals ([S1 Fig](#pone.0120590.s001){ref-type="supplementary-material"}.). The GLM model was therefore used to analyze the association between SSR markers and four measures of cold tolerance for *indica* and *japonica* separately. To control false discovery in association analyses, the significance level ɑ was adjusted upward by ɑʹ in a rate (1-ɑ)^R^ for R-rejected hypotheses. Here, we set ɑ = 0.05, then ɑʹ = 0.05(1--0.05)^R^. We denoted markers with p-values lower than ɑʹ as significantly associated ones. To provide information about the markers and germplasm resources that may be used in breeding cultivars with strong cold tolerance, we investigated the genotypic effect of each genotype for each QTL. Due to the distinct difference in cold tolerance between the two subspecies, the relative genotypic effect for the i^th^ genotype in the s^th^ subspecies for the corresponding indicator was denoted as $RGE_{\text{si}} = ({\overline{x}}_{si} - {\overline{x}}_{s})/{\overline{x}}_{s}$, where, s represents subspecies (*indica* or *japonica*), ${\overline{x}}_{si}$ is the average phenotype of *i* ^*th*^ genotype in the *s* ^*th*^ subspecies, and ${\overline{x}}_{s}$ is the average phenotype of the *s* ^*th*^ subspecies. Error MS for each marker (MS~E~) estimated during association analysis was used to estimate the error MS of RGE as $MS_{RGE\text{si}} = (MS_{E}/n_{\text{i}})/{\overline{x}}_{s}{}^{2}$. Given that MS~E~ was estimated from a large sample, the z-test was used to test whether the RGE of each genotype was biased from zero. We denoted genotypes significantly larger than zero as positive or cold-tolerant genotypes, and those smaller than zero as negative or cold-sensitive genotypes. Results {#sec009} ======= Population structure and linkage disequilibrium {#sec010} ----------------------------------------------- When we ran the STRUCTURE simulation using 60 SSRs, the LnP(D) value increased with *K* from 1 to 10, but showed an evident knee and there was a sharp peak of Evanno's Δ*K* at *K* = 2 ([Fig. 1](#pone.0120590.g001){ref-type="fig"}). These results indicated that there were two distinctly divergent populations, corresponding to subspecies *indica* and *japonica*. To survey the influence of population structure on LD, we analyzed LD in the whole population and in each of the subgroups (i.e. *indica* and *japonica*) identified by structure analysis ([Fig. 2](#pone.0120590.g002){ref-type="fig"}, [S3 Table](#pone.0120590.s004){ref-type="supplementary-material"}). At the whole population level, the r^2^ within the whole genome was 0.0624±0.0865. For SSR markers with physical distances less than 50 kb, the r^2^ was 0.1443±0.2149; for those between 50 kb and 150 kb, r^2^ was 0.1332±0.1829; for those between 150 kb and 500 kb, r^2^ was 0.0926±0.1353; for those between 500 kb and 1000 kb, r^2^ was 0.0624±0.0765; and for those more than 1,000 kb, r^2^ was 0.0605±0.0818. Thus LD for markers with physical distances shorter than 50 kb was not obviously different from those between 50 kb and 150 kb, but decreased dramatically for those further than 150 kb. Compared to the whole population, LDs within *indica* and *japonica* were obviously lower ([S3 Table](#pone.0120590.s004){ref-type="supplementary-material"} and [Fig. 2](#pone.0120590.g002){ref-type="fig"}). For SSR markers with physical distances shorter than 50 kb, mean r^2^ were 0.1388±0.2135 and 0.1810±0.3203 for *indica* and *japonica*, respectively; for markers between 50 kb and 150 kb, 0.0761±0.0796 and 0.0760±0.0368; for markers between 150 kb and 500 kb, 0.0331±0.0573 and 0.0392±0.0616; for markers between 500 kb and 1000 kb, 0.0155±0.0148 and 0.0336±0.0505; and for markers more than 1000 kb, 0.0134±0.0165 and 0.0201±0.0261. These results indicated that LDs between close markers (such as physical distances less than 50 kb) in both *indica* and *japonica* did not decrease relative to the whole population, however, the LDs between distant markers especially those more than 150 kb decreased dramatically ([S3 Table](#pone.0120590.s004){ref-type="supplementary-material"}). Given the above results, we carried out the association analysis within *indica* and *japonica* independently in order to avoid false positive associations; and given that few non-linked markers showed strong LD, we used the GLM model that controls population structure but not kinship for the association analysis within *indica* and *japonica*. {#pone.0120590.g001} {#pone.0120590.g002} Cold tolerance at the germination and booting stages {#sec011} ---------------------------------------------------- All four measures of cold tolerance showed high variation among accessions at both the germination and booting stages, particularly the latter ([Table 1](#pone.0120590.t001){ref-type="table"}). *Japonica* had apparently strong cold tolerance at the germination stage as measured by seedling survival rate (SSvR = 79.11%) under low temperature; this was more than three-fold stronger than *indica* (23.39%). Both *indica* and *japonica* were sensitive to cold stress at the booting stage, as measured by seed setting rate under natural low temperature conditions in Kunming (SStR-NL), seed setting rate under cold water irrigation in Gongzhuling (SStR-CW), and relative seed setting rate under cold water irrigation in Gongzhuling (RSStR-CW). However, *japonica* was more tolerant to cold stress (SStR-NL and RSStR-CW were higher than 25%) than *indica* (SStR-NL and RSStR-CW were lower than 25%). Correlation analyses among the four measures revealed low correlations between cold tolerance at the germination stage and at the booting stage, and low correlations between cold tolerances in different environments ([Table 2](#pone.0120590.t002){ref-type="table"}). Higher correlations in cold tolerance among different stages and environments occurred in the case of *japonica*. 10.1371/journal.pone.0120590.t001 ###### Phenotypic variation in four cold-tolerant measures in *indica* and *japonica*. {#pone.0120590.t001g} Measure *Indica* *Japonica* ---------- ---------- -------------- ------- -------- ---------------------------------------------- -------------- ------- -------- SSvR 23.39 1.11--100.00 18.03 77.08 79.11[\*\*](#t001fn002){ref-type="table-fn"} 1.11--100.00 28.67 36.23 SStR-NL 18.88 0--74.42 21.33 113.01 29.89[\*\*](#t001fn002){ref-type="table-fn"} 0--80.43 26.41 88.35 SStR-CW 14.71 0--83.49 21.24 144.39 16.87 0--64.31 19.54 115.82 RSStR-CW 14.57 0--89.14 20.53 140.92 25.79[\*](#t001fn001){ref-type="table-fn"} 0--90.47 25.84 100.18 \* Significant at P = 0.05; \*\*: significant at P = 0.01; SSvR, survival at low temperature expressed as percentage (%) of surviving plants; SStR-NL, percentage (%) of filled grains per panicle under natural low temperature conditions in Kunming; SStR-CW, percentage (%) of filled grains per panicle under cold water irrigation in Gongzhuling; RSStR-CW, percentage (%) relative seed setting under cold water irrigation in Gongzhuling. 10.1371/journal.pone.0120590.t002 ###### Correlation coefficients among cold-tolerant measures in *indica* (above the diagonal) and *japonica* (below the diagonal). {#pone.0120590.t002g} Measure SSvR SStR-NL SStR-CW RSStR-CW ---------- ------ --------- --------------------------------------------- ---------------------------------------------- SSvR 1.00 0.16 0.11 0.03 SStR-NL 0.16 1.00 0.00 0.17 SStR-CW 0.17 0.23 1.00 0.98 [\*\*](#t002fn001){ref-type="table-fn"} RSStR-CW 0.27 0.25 0.91[\*\*](#t002fn001){ref-type="table-fn"} 1.00 \*\*: Significant at P = 0.01. QTLs associated with cold tolerance at the germination and booting stages {#sec012} ------------------------------------------------------------------------- Fifty one QTLs related to cold tolerance at the germination and booting stages were detected ([S4 Table](#pone.0120590.s005){ref-type="supplementary-material"}). They were distributed on all 12 chromosomes, with the maximum number on chromosome 1 and only one on chromosome 9 ([Fig. 3](#pone.0120590.g003){ref-type="fig"}). Among them, 22 were detected at the germination stage, 33 at booting, and eight were identified by at least two measures. The genetic contribution of each QTL was 29.20% on average, with a minimum 5.25% (*qLTSSvR3--1* on chromosome 3) and the maximum 59.28% (*qLTSSvR7--1* on chromosome 7). Of the reported QTLs related to cold tolerance in rice ([www.gramene.com](http://www.gramene.com)) more than 54% (21 of 39 QTLs) were detected in the current study ([Fig. 3](#pone.0120590.g003){ref-type="fig"}). We identified four loci associated with cold tolerance at the booting stage that were also detected by Cui *et al*. by association mapping \[[@pone.0120590.ref019]\]. {#pone.0120590.g003} Among the 22 QTLs related to cold tolerance at germination as measured by SSvR, 6 were detected only in *indica* with low average contributions to phenotypic variation (CPV = 12.0%), 15 only in *japonica* with high average CPV (36.2%), and *qCTSSR6--3* in both *indica* and *japonica* with average CPV of 13.5%. The results showing that only one QTL was shared between *indica* and *japonica* and that CPV of QTLs in *japonica* were much higher than those in *indica*, implied that the mechanism of cold tolerance at the germination stage in *japonica* was different from that in *indica*. Of 12 QTLs associated with SStR-NL, five were detected in *indica* with low average CPV (21.8%), and seven in *japonica* with higher average CPV (33.9%) ([Fig. 3](#pone.0120590.g003){ref-type="fig"}, [S4 Table](#pone.0120590.s005){ref-type="supplementary-material"}). Eight of 12 QTLs associated with SStR-CW were identified in *indica* with low average CPV (24.0%), compared with four in *japonica* with high average CPV (35.2%). Six of 15 QTLs associated with RSStR-CW, were in *indica* with low average CPV (24.3%), compared to nine in *japonica* with high average CPV (38.2%). No QTL related to SStR-NL, SStR-CW or RSStR-CW was shared between *indica* and *japonica*. Under the same stress and environmental conditions, three (*qLTRSSR6--1*, *qLTRSSR3--1* and *qLTRSSR1--3*) of eight QTLs identified by SStR-CW were also detected by RSStR-CW in *indica*, and similar findings were observed for two (*qLTRSSR8--1* and *qLTRSSR5--1*) of four QTLs in *japonica*. Although using the same measure (seed setting rate, SStR), only one QTL (*qLTRSSR8--1*) at the booting stage was detected under both naturally low air temperatures (the corresponding measure is SStR-NL) and cold water irrigation (the corresponding measure is SStR-CW) in *japonica* ([Fig. 3](#pone.0120590.g003){ref-type="fig"}). The results indicated that the mechanism of cold tolerance at the booting stage was complex. Moreover, only one QTL (*qLTRSSR8--1*) was detected by the same measure (seed setting rate, SStR) under different kinds of cold stress in *japonica*, i.e. naturally low air temperature in Kunming and cold water irrigation in Gongzhuling. Among QTLs detected at both the germination and booting stages, the same two QTLs (*qLTSSR1--3* and *qCTSSR6--3* with CPV 9.1 and 10.9%, respectively) were detected at the germination and booting stages in *indica*, but there was no similar QTL in *japonica*. That is, *qLTSSR1--3* and *qCTSSR6--3* may provide a common mechanism of cold tolerance at the germination and booting stages to some degree in *indica*, although most of the cold tolerance at both stages may be attributed to different mechanisms. In *japonica* the mechanisms of cold tolerance at the germination and booting stages appear to be more different than in *indica*. Markers and germplasm resources with strong cold tolerance potentially useful for breeding {#sec013} ------------------------------------------------------------------------------------------ According to the relative genotypic effect (RGE) of each genotype at each QTL, we detected 18 positive genotypes and 21 negative genotypes in *indica*, and 19 positive genotypes and 24 negative genotypes in *japonica*. Generally, the negative effects were much stronger than the positive effects in both subspecies ([S5 Table](#pone.0120590.s006){ref-type="supplementary-material"}). Using these genotypes as selection markers we independently screened *indica* and *japonica* accessions containing different numbers of positive genotypes ([Fig. 4](#pone.0120590.g004){ref-type="fig"} and [5](#pone.0120590.g005){ref-type="fig"}). For positive genotypes, the cold tolerance as measured by the four measures increased with the increasing number of positive genotypes in *indica*, but not in *japonica* ([Fig. 4](#pone.0120590.g004){ref-type="fig"}). Likewise, for positive genotypes detected in *japonica*, the cold tolerance as measured by the four measures generally increased in *japonica*, but not in *indica*, apart from two exceptions for measures SStR_NL ([Fig. 5B](#pone.0120590.g005){ref-type="fig"}) and RSStR_CW ([Fig. 5D](#pone.0120590.g005){ref-type="fig"}). We checked the accessions with four genotypes positive for SStR_NL ([Fig. 5B](#pone.0120590.g005){ref-type="fig"}), among which one accession (Ye Li Cang Hua) had quite low seed setting (18%) and contained a strong negative genotype (RM5496_136), which reduced seed setting by up to 60% ([S5 Table](#pone.0120590.s006){ref-type="supplementary-material"}). Similarly, among the accessions with three genotypes positive for RSStR_CW ([Fig. 5D](#pone.0120590.g005){ref-type="fig"}), two cultivars (Ning Hui 21 and Dan Dong Lu Dao) had low seed setting (7% and 13.5%, respectively), and both contained a strong negative genotype (RM6863_155) that reduced seed setting by as much as 67% ([S5 Table](#pone.0120590.s006){ref-type="supplementary-material"}). {#pone.0120590.g004} {#pone.0120590.g005} In addition to screening accessions using positive genotypes we also screened the top five cultivars showing high cold tolerance according to each measure in *indica* and *japonica*, respectively, and subsequently investigated the distribution of the positive and negative genotypes among these accessions in each subspecies ([S5 Table](#pone.0120590.s006){ref-type="supplementary-material"}). There were three obvious characteristics for the distribution of the positive and negative genotypes for each measure within each subspecies. Firstly, the top cultivars contained at least one positive genotype of the corresponding measure in the corresponding subspecies, but not necessarily in the other subspecies, although exceptions existed in *indica* for SStR_NL (where accessions Z102, Z146, Z54 and Z64 had no positive genotypes) and RSStR_CW (where accessions Z126, Z5 and Z60 had no positive genotypes). Secondly, the top accessions excluded most of the negative genotypes, especially those with strong negative effects. Thirdly, both positive and negative genotypes in one subspecies may exist in the other subspecies but with no effect on cold tolerance in that subspecies, implying that *indica* and *japonica* do not share the same set of tolerance genes or alleles at most loci. Discussion {#sec014} ========== QTLs for cold tolerance in *indica* and *japonica* {#sec015} -------------------------------------------------- Association analysis is an effective approach to dissect the genetic bases underlying complex traits in plants \[[@pone.0120590.ref009]--[@pone.0120590.ref010]\], animals \[[@pone.0120590.ref020]\] and humans \[[@pone.0120590.ref021]\]. In the present study we used association analysis to identify 51 QTLs related to cold tolerance at the germination and booting stages in rice. Among them, 46.17% were new QTLs, and 27 corresponded to QTLs reported previously. For example, *qLTSSR4--1* associated with SStR-CW was near the cloned *Ctb1* on chromosome 4; *qLTSSvR7--1* associated with SSvR is 1.4 cM from *qCTB7* on chromosome 7; and *qLTRSSR12--1* associated with RSStR-CW is near *qCTS12*. Four loci identified in the present study were detected at the booting stage by Cui *et al*. \[[@pone.0120590.ref017]\]. Of these, *qLTSSvR6--1* on Chr6 in the current study is near RM528 as reported by Cui *et al*., *qCTSSR9--1* on Chr9 is near RM160, *qCTSSR11--1* on Chr11 is near RM4B, and *qCTSSR12--1* on Chr12 is near RM235. Our results clearly indicated that *japonica* has a higher level of tolerance to cold stress than *indica* at both the germination and booting stages. This agrees with results published by others \[[@pone.0120590.ref022]--[@pone.0120590.ref025]\]. More QTLs were identified in *japonica* (36) than in *indica* (26). Five QTLs were shared among measures in *indica* or *japonica* at the booting stage. Only one QTL (*qCTSSR6--3*) associated with seedling survival rate (SSvR) under low temperatures was shared by *indica* and *japonica*. Reasons for this deserve further discussion. During the thousands of years of cultivation and utilization under diverse environmental conditions tremendous genetic differentiation has occurred in rice produced under various agro-ecosystems. *Indica* is known to be adapted to tropical environments at low latitudes or altitudes typified by warm climatic conditions, whereas *japonica* cultivars were grown in temperate areas at high latitudes or altitudes with relatively cool conditions \[[@pone.0120590.ref026]\], at high altitudes in tropical and sub-tropical areas, and in areas where paddy fields are irrigated by cold water. In such areas, water and soil temperatures at sowing are below 15°C \[[@pone.0120590.ref027]--[@pone.0120590.ref028]\], and thus *japonica* cultivars were preferred due to tolerance to prevailing low temperatures. In double-cropped rice-growing regions, water and soil temperatures at sowing are higher than 15°C but cold air currents from Siberia often occurring in April may cause early rotting of seedlings, resulting in heavy seedling losses and a delayed growing period. *Indica* cultivars grown in such areas are usually characterized by tolerance to short periods of low temperature. Evident correlation has been found between cold stress and altitude or atmospheric temperature. Low temperatures (15--19°C) at booting cause sterile pollen which directly leads to spikelet sterility and ultimately causes serious yield losses \[[@pone.0120590.ref023],[@pone.0120590.ref029]--[@pone.0120590.ref031]\]. In Heilongjiang province an increase of 1°C in negative accumulated temperature GDD17 at the inflorescence differentiation stage can result in a 3.51 kg/ha yield loss, and an increase of 1°C in negative accumulated temperature GDD19 at booting stage can cause a 4.95 kg/ha yield loss \[[@pone.0120590.ref032]\]. *Indica* is adapted to warm areas and is more sensitive to low temperatures. Pollen sterility is observed in *indica* when average temperatures remain below 22°C for three days \[[@pone.0120590.ref033]\]. The seasonal duration for rice gets progressively shorter due to decreased average temperature and accumulated temperature from southern to northern China. *Japonica* cultivars are mainly grown in the area north of the Yellow River, whereas most *indica* cultivars are grown to the south. This distribution implies that *indica*-*japonica* differentiation resulted from adaptation to different climatic and geographic environments. The significant *indica*--*japonica* difference in cold tolerance was probably enhanced by rare genetic exchanges between the two subspecies. During cultivation and improvement over thousands of years, *indica* and *japonica* became genetically separated with adaptation to different agro-ecosystems. Differentiation between the subspecies was further enforced by fertility barriers that prevented genetic exchange between the two subspecies \[[@pone.0120590.ref034]\]. Observations of apparent differences in cold tolerance between *indica* and *japonica* may be partially attributed to the use of SSRs originating from noncoding regions. Only 15% of the SSRs used in the present study were from ESTs: this is higher than the proportion (1.5--7.5%) among all SSRs reported in rice \[[@pone.0120590.ref035]--[@pone.0120590.ref036]\]. Detection of marker-trait associations based on linkage disequilibria (LD) in genetically diverse materials can help to identify QTLs controlling target traits. However, this depends on the chromosome distance between marker and QTL and times between mutational changes in marker and the causal mutation of the target QTL. An association can be detected easily in a population when the mutation of the test marker is near in distance and time to the functional mutation in the target QTL \[[@pone.0120590.ref037]\]. However, 85% of SSRs in the study were nonfunctional markers and there are few genomic exchanges between the two subspecies as mentioned above. This may partially explain why *indica* and *japonica* share so few QTLs of cold tolerance. Both positive and negative genotypes should be considered in breeding by MAS {#sec016} ---------------------------------------------------------------------------- Association mapping is a proven and efficient way to detect natural variation in a diverse population and provides many candidate genes or genomic regions for marker assisted selection in breeding. For example, Huang *et al*. used a worldwide panel of cultivated rice varieties to map 32 new loci associated with flowering time and 10 grain-related traits, allowing identification of candidate genes for 18 associated loci \[[@pone.0120590.ref010]\]. Marker assisted selection is an efficient breeding approach to pyramid elite genes in crop improvement; for example, Narayanan *et al*. developed a line combining blast and blight resistance genes Pi-1, Piz-5 and Xa2 using markers \[[@pone.0120590.ref038]\]. In the present study, we identified 18 and 19 positive genotypes (i.e. cold-tolerant genotypes) in *indica* and *japonica*, respectively, based on 51 QTLs. Our results indicated that the more positive genotypes the selected cultivars contain, the stronger cold tolerance they show. This proved that positive markers can be used to pyramid cold tolerance genes. In addition to those positive genotypes, however, we identified even more negative genotypes (sensitive to cold stress), numbering 21 and 24 in *indica* and *japonica*, respectively. Some cultivars (such as Ning Hui 21 and Dan Dong Lu Dao) are very sensitive to low temperatures due to a single strong negative genotype (such as RM6863_155) even though they may contain up to three positive genotypes. This indicates that certain strong negative genotypes may play the role of the shortest piece of wood as showed in the bucket theory and should be avoided by the breeders lest they cover up the advantage of the positive genotypes. Supporting Information {#sec017} ====================== ###### QQ-plots of GLM and MLM models for four measures of cold tolerance. (DOC) ###### Click here for additional data file. ###### Rice germplasm used in the study. (DOC) ###### Click here for additional data file. ###### SSR markers used in the study. (DOC) ###### Click here for additional data file. ###### LD (r^2^) between markers with different physical distances. (DOC) ###### Click here for additional data file. ###### QTLs based on four measures of cold tolerance. (XLS) ###### Click here for additional data file. ###### Positive and negative genotypes detected in two subspecies and their distribution in the cultivars with top SSvR. (XLS) ###### Click here for additional data file. We thank Professor Robert A McIntosh, University of Sydney, for suggested revisions to the manuscript. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: YHP HLZ ZCL. Performed the experiments: XDM GLC YHP HLZ DLZ. Analyzed the data: YHP HLZ DLZ JJL HYX JPY JLL MARR GGL. Contributed reagents/materials/analysis tools: YHP HLZ DLZ JJL XDM GLC LZH. Wrote the paper: YHP HLZ ZCL MARR.

Introduction {#sec1_1} ============ Mild cognitive impairment (MCI) is defined as a transitional stage between normal aging and dementia and is based on stage 3 of the Global Deterioration Scale (GDS) \[[@B1]\]. In 2004, the Key Symposium in Sweden published consensus criteria for MCI to expand the concept of MCI beyond the memory domain \[[@B2], [@B3]\]. MCI includes impairment in both memory and nonmemory cognitive domains \[[@B2], [@B4]\]. MCI patients could be classified into two groups: amnestic MCI (aMCI) and nonamnestic MCI (naMCI) \[[@B2], [@B3]\]. The classification by subtype could possibly predict the type of dementia that MCI patients may develop \[[@B2]\]. Because of the heterogeneity of the clinical presentation and underlying etiologies within the MCI group, there is no documented treatment for MCI. Furthermore, the clinical course of MCI is more heterogeneous than distinctive. In several previous longitudinal studies, aMCI was closely associated with a high risk of progression to Alzheimer\'s disease (AD) dementia \[[@B5], [@B6], [@B7], [@B8], [@B9]\]. MCI could provide significant information on the risk of becoming demented, and so it is a stage at which intervention could be effective in preventing conversion to dementia \[[@B3]\]. However, not all cases of aMCI progress to AD dementia, and some cases even return to normal \[[@B9]\]. It is conceivable that cognitive deficits can be attributable to a variety of medical and psychiatric illnesses instead of AD. Therefore, identifying MCI patients who will progress to AD dementia simply based on the clinical criteria for MCI may be inadequate. A new set of criteria has been proposed by the National Institute on Aging-Alzheimer\'s Association (NIA-AA) workgroup to further define MCI due to AD \[[@B10]\], or proposed by the International Working Group (IWG-2) to define prodromal AD \[[@B11]\], incorporating pathological biomarkers consistent with AD. In addition, diagnostic research criteria for AD have recommended the use of cerebrospinal fluid (CSF) biomarkers to determine the etiology and prognosis in persons with MCI \[[@B10], [@B11], [@B12], [@B13]\]. These new criteria would certainly improve the predictive value for the diagnosis of MCI. However, amyloid imaging, in particular, is expensive and not easily accessible to most primary care physicians, limiting its applicability. Vandermeeren et al. \[[@B14]\] first demonstrated increased CSF tau concentration in AD. Since then, many reports have demonstrated that CSF biomarkers, in addition to neuropsychological tests or magnetic resonance imaging (MRI), increased the accuracy to predict progression to AD dementia in MCI patients \[[@B15], [@B16], [@B17], [@B18]\]. CSF tau concentration especially may be considered as a potential biomarker for AD or a marker of neurodegeneration in MCI patients. However, the authors considered only conversion of AD dementia from MCI during the follow-up period, but not amyloidosis in MCI individuals at baseline. In addition, none of the previous reports have considered the IWG-2 guidelines in staging the AD spectrum. The aims of this study were to determine the value of CSF biomarkers for differentiating prodromal AD from other MCI types in a group of patients with clinically diagnosed aMCI, and to assess whether they could serve as a gold standard as CSF biomarkers for prodromal AD. Materials and Methods {#sec1_2} ===================== Participants {#sec2_1} ------------ Subjects were prospectively recruited from two centers: Chonnam National University Hospital, and Chosun University Hospital including the National Research Center for Dementia in Gwangju, Korea. We prospectively collected data from 198 patients from September 2015 to August 2017. All subjects were examined by experienced neurologists and received a full dementia screening test, which included past history, neurological examination, laboratory and neuropsychological tests, and brain MRI. In addition, 177 of the 198 subjects underwent brain amyloid positron emission tomography (PET) and CSF analysis within 1 year from the screening test. The exclusion criterion was diagnosis of dementia due to causes not related to AD. Clinical and Neuropsychological Assessments {#sec2_2} ------------------------------------------- Clinical assessments were undertaken according to the recommendations in the European Federation of Neurological Societies guideline \[[@B19]\]. All subjects were examined with a clinical interview including Clinical Dementia Rating \[[@B20], [@B21]\] and GDS. Two activities of daily living (ADL) scales were included in the battery: the basic ADL and the instrumental ADL \[[@B22], [@B23]\]. Demographic data included age, sex, and years of education. Global cognitive function was measured by the Korean version of the Mini-Mental State Examination (K-MMSE) \[[@B24], [@B25]\]. Neuropsychological assessment was conducted using the Seoul Neuropsychological Screening Battery, which measures attention, language and associated functions, visuospatial, memory, and frontal/executive functions \[[@B26]\]. Attention was evaluated using the digit span test (forward and backward). Language was evaluated using the Korean version of the Boston Naming Test. Visuospatial function was tested using the copy score of the Rey-Osterrieth Complex Figure Test. Memory was evaluated using the Seoul Verbal Learning Test (three learning-immediate recall trials of a 12-item list, a 20-min delayed recall trial, and recognition) and Rey-Osterrieth Complex Figure Test immediate recall and a 20-min delayed recall and recognition. Frontal/executive function was evaluated using the go-no-go test/contrasting program test, the Controlled Oral Word Association Test, and Stroop test. Cognitive impairment was defined as a *Z*-score \<--1.5 standard deviation (normed for age and education) on at least one of the neuropsychological tests, which could be a memory, language, visuospatial function, attention, or frontal/executive test. A routine blood test for dementia included complete blood count, biochemistry, thyroid function test, and syphilis serologic tests. For apolipoprotein E (APOE) genotyping, genomic DNA was extracted from 196 of the 198 participants using a commercially available kit (QuickGene DNA Whole Blood Kit, KURABO, catalog No.: DB-S). APOE genotyping was performed by SNaPshot-mediated SNP genotyping assays (Applied Biosystems) at DNALink (Seoul, South Korea). Brain MRI was performed on all patients. The MRI protocol included a three-dimensional volumetric T1-weighted (T1W) sagittal scan, an axial T2W, an axial T2W fluid-attenuated inversion recovery, and a diffusion tensor image with slices angled parallel to the axis through anterior and posterior commissures and taken from the foramen magnum to the vertex. All patients who had structural brain lesions or severe subcortical white matter hyperintensities (WMH \> D3P3; P3 = periventricular WMH\'s cap or band ≥10 mm; D3 = maximum diameter of deep WMH ≥25 mm) based on the Clinical Research Center for Dementia of South Korea WMH visual rating scale \[[@B27]\] were excluded. Classifications by Clinical Diagnosis {#sec2_3} ------------------------------------- We tried to screen 210 individuals for this study and excluded only 12 subjects: 6 who were diagnosed with normal-pressure hydrocephalus, 4 with dementia with Lewy bodies, and 2 with semantic variants of frontotemporal dementia. The 198 subjects included in the study for the clinical diagnosis were classified into three groups: 95 normal elderly people, 67 MCI subjects, and 36 AD dementia patients (Fig. [1](#F1){ref-type="fig"}). Patients with AD dementia fulfilled the clinical criteria of probable AD dementia proposed by the NIA-AA or IWG-2 workgroups \[[@B11], [@B28]\]. The diagnosis of MCI was made according to the MCI criteria proposed by the NIA-AA or IWG-2 on MCI \[[@B2], [@B3], [@B10], [@B11]\]. Brain Florbetaben PET Imaging Acquisition and Analysis {#sec2_4} ------------------------------------------------------ Brain PET images were acquired from participants 90--100 min after intravenous injection of a mean dose of 303 MBq 20% florbetaben, a fluorine-18-labeled stilbene derivative with the trade name of NeuraCeq \[[@B29]\], according to a standardized acquisition and imaging protocol in two hospitals. The florbetaben radiosynthesis, PET imaging scan, and data processing were performed as previously described in detail \[[@B30], [@B31]\]. PET data were acquired with a Discovery STE PET-CT scanner (GE Medical Systems, Milwaukee, WI, USA). The visual assessment of florbetaben PET images was performed in transaxial images using a gray scale by trained readers (J. Kim and H.-C. Song). Each brain region (frontal cortex, lateral temporal cortex, parietal cortex, and posterior cingulate cortex/precuneus) was visually assessed and scored according to the brain beta-amyloid plaque load (BAPL) scoring system for each PET scan. BAPL score: 1 = no beta-amyloid load, 2 = minor beta-amyloid load, 3 = significant beta-amyloid load. BAPL scores of 1 are classified as beta-amyloid-negative PET scan, and BAPL scores of 2 and 3 as beta-amyloid-positive PET scan \[[@B32]\]. Classifications by Amyloid PET {#sec2_5} ------------------------------ Twenty-one of the 198 subjects who were classified by clinical diagnosis were excluded. Of these, 9 subjects had naMCI. The other 12 subjects (8 normal, 2 aMCI, and 2 AD dementia) were not administered amyloid PET tracer. Finally, 177 subjects were selected (87 normal subjects, 56 aMCI patients, and 34 AD dementia patients) (Fig. [1](#F1){ref-type="fig"}; Table [1](#T1){ref-type="table"}). Patients who were diagnosed with prodromal AD in this classification met the IWG-2 guidelines based on amyloid PET results \[[@B11]\]. In this classification, 14 of 87 normal elderly people were amyloid-positive, named "preclinical AD," 27 of 56 aMCI patients, named "prodromal AD," and 31 of 34 AD dementia patients were amyloid-positive (Fig. [1](#F1){ref-type="fig"}; Table [2](#T2){ref-type="table"}). CSF Analysis {#sec2_6} ------------ CSF samples were obtained by lumbar puncture with aseptic technique at the L3--L4 or L4--L5 intervertebral spinous process space, using a 22- or 21-gauge needle, between 8 a.m. and 10 a.m. CSF was collected in Falcon polypropylene tubes (BD Biosciences, Franklin Lakes, NJ, USA), and the first 2--3 mL of CSF were analyzed for routine chemical parameters, including cell count as well as glucose and total protein concentrations. Within 15 min, the remaining CSF samples were centrifuged for 10 min at 2,000 rpm at 4°C to remove cells, aliquoted to 0.45-mL-samples in polypropylene tubes, and then stored at −80°C until analysis. CSF biomarker concentrations of Aβ~1--42~, Aβ~1--40~, total tau protein (t-Tau), and tau protein phosphorylated at threonine 181 (p-Tau^181^) were measured in duplicate using a commercially available double antibody sandwich enzyme-linked immunosorbent assay kit (INNOTEST; Fujirebio, Gent, Belgium) according to the manufacturer\'s protocol. Absorbance was measured at 450 nm using a SpectraMAX M3 microplate reader (Molecular Devices, San Jose, CA, USA). We calculated derived ratios from single biomarkers including Aβ~1--42~/Aβ~1--40~, t-Tau/Aβ~1--42~, and p-Tau^181^/Aβ~1--42~ ratios. The team involved in CSF analyses was blinded to the clinical diagnosis. Statistical Analyses {#sec2_7} -------------------- For the statistical analyses, the IBM SPSS software version 24.0 for Windows (IBM, Armonk, NY, USA) was used. The participants were grouped by the states of the clinical diagnosis, the amyloidosis on amyloid PET, and the combination of them for the statistical analysis. The normality of the continuous variables was tested using the Shapiro-Wilk test. The clinical and demographic characteristics of the subgroups were compared among the clinically defined three groups and between the defined two subgroups using one-way analysis of variance and χ^2^ tests for continuous and categorical variables, respectively. The levels of CSF biomarkers including Aβ~1--42~, Aβ~1--40~, t-Tau, and p-Tau^181^ as well as scores on neuropsychological, clinical, and functional measures were compared between groups using analysis of covariance with post hoc analyses (LSD test). Receiver operating characteristic (ROC) curves were depicted with the true-positive fraction (sensitivity) and the false-positive fraction (1 -- specificity) by SPSS software 24.0. The area under the curve (AUC) was estimated from each ROC curve using an empirical method (confidence interval at 95%) \[[@B33]\], because it does not make the strong normality assumptions and was used as an index of test performance. The specificities and sensitivities of each CSF biomarker level, the ratios of CSF biomarkers, or the combinations of CSF biomarkers for the indicated pairs were optimized using the ROC curve. In addition, the cutoffs for each biomarker were the scores that yielded the highest sum of sensitivity and specificity. The sensitivity and specificity were calculated for each cutoff value. A *p* value \< 0.05 was considered statistically significant in the analysis. Results {#sec1_3} ======= Characteristics and CSF Data of Study Participants with Clinical Diagnosis {#sec2_8} -------------------------------------------------------------------------- The clinical characteristics and CSF data of 177 subjects are presented in Table [1](#T1){ref-type="table"}. There were 87 normal elderly people, 56 aMCI patients, and 34 AD dementia patients. The mean age of the normal, aMCI, and AD dementia subjects was 71.7 ± 5.8, 71.5 ± 8.0, and 69.4 ± 6.0 years, respectively. Age and sex were not statistically different in these three groups (Table [1](#T1){ref-type="table"}). Education level was statistically lowest in AD dementia patients. APOE ε4 allele carrier status was observed in 22 normal subjects (25.3%), in 24 patients with aMCI (42.9%), and in 20 patients with AD dementia (58.8%). The frequency of APOE ε4 allele was significantly higher in AD dementia patients than in normal subjects and in aMCI patients (*p* \< 0.001). The K-MMSE scores were significantly lower in AD dementia patients (18.5 ± 5.4) than in aMCI patients (25.5 ± 3.6) and in normal subjects (26.7 ± 2.6) (*p* \< 0.001). The Clinical Dementia Rating and sum of box scores, GDS, and instrumental ADL were statistically lower in the normal group compared to the aMCI and AD dementia groups (*p* \< 0.001) (Table [1](#T1){ref-type="table"}). The CSF concentrations of Aβ~1--42~ were highest in the normal group (982.4 ± 236.5 pg/mL) compared to the aMCI (771.0 ± 317.0 pg/mL) and AD dementia (559.0 ± 311.0 pg/mL) groups (*p* \< 0.001). CSF Aβ~1--42~ levels can be different in aMCI, AD dementia, or normal subjects. The CSF concentrations of t-Tau were highest in the AD dementia group (487.5 ± 218.6 pg/mL) compared to the normal (247.9 ± 105.2 pg/mL) and aMCI (303.8 ± 229.7 pg/mL) groups (*p* \< 0.001). CSF t-Tau levels can also be different in AD dementia, aMCI, or normal subjects. The CSF concentration of p-Tau^181^ in the AD dementia group was 72.6 ± 28.0 pg/mL, which was statistically higher than in the normal (47.6 ± 17.9 pg/mL) or aMCI (53.1 ± 29.9 pg/mL) groups. CSF p-Tau^181^ levels can also be different in AD dementia compared to normal or aMCI subjects (Table [1](#T1){ref-type="table"}). Characteristics and CSF Data of Study Participants with Amyloid PET {#sec2_9} ------------------------------------------------------------------- On the basis of amyloidosis, 177 subjects were classified into 87 normal elderly people (73 amyloid-negative and 14 amyloid-positive), 56 of the aMCI group (29 amyloid-negative and 27 amyloid-positive), and 34 of the AD dementia group (3 amyloid-negative and 31 amyloid-positive) (Table [2](#T2){ref-type="table"}). APOE ε4 allele carrier status was observed in 8 normal amyloid-positive subjects (preclinical AD, 57.1%), in 18 amyloid-positive aMCI patients (prodromal AD, 66.7%), and in 20 amyloid-positive AD dementia patients (64.5%). Statistically, CSF Aβ~1--42~ protein levels were lower in prodromal AD (548.2 ± 214.9 pg/mL) compared with aMCI amyloid-negative patients (978.4 ± 249.2 pg/mL), whereas CSF p-Tau^181^ levels were higher in prodromal AD (62.4 ± 32.3 pg/mL) compared with amyloid-negative aMCI patients (44.4 ± 24.9 pg/mL). However, CSF Aβ~1--40~ protein and CSF t-Tau levels showed no statistical difference between prodromal AD and amyloid-negative aMCI patients. Preclinical AD patients had statistically lower CSF Aβ~1--42~ (643.0 ± 171.1 pg/mL) and higher t-Tau (300.6 ± 132.0 pg/mL) compared with normal amyloid-negative subjects (1,047.5 ± 186.6 and 237.8 ± 97.2 pg/mL, respectively); however, CSF p-Tau^181^ showed no statistical difference between preclinical AD patients and amyloid-negative normal subjects (Table [2](#T2){ref-type="table"}). All three CSF biomarkers defined and were related to the status of the AD spectrum (from normal to AD dementia) according to the IWG-2, with CSF Aβ~1--42~ protein levels decreasing, and others increasing during AD progression (Fig. [2](#F2){ref-type="fig"}). The analysis revealed that the neuropsychological tests were similar between the amyloid-positive and -negative subjects in each of the three groups (normal, aMCI, AD dementia) (online suppl. Table [1](#S1){ref-type="supplementary-material"}; for all online suppl. material, see [www.karger.com/doi/10.1159/000496920](http://www.karger.com/doi/10.1159/000496920)). CSF Biomarkers between Prodromal AD and Amyloid-Negative aMCI Patients in the aMCI Group, and between Amyloid-Positive AD Dementia Patients and Amyloid-Negative Normal Subjects {#sec2_10} -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- The analysis of CSF biomarkers revealed that they differed significantly between prodromal AD and amyloid-negative aMCI. We generated ROC curves to identify CSF biomarkers that differentiated the two groups. The cutoff values that yielded the best index for prodromal AD were 749.5 pg/mL for Aβ~1--42~, 225.6 pg/mL for t-Tau, 43.5 pg/mL for p-Tau^181^, 0.095 for Aβ~1--42~/Aβ~1--40~, 0.298 for t-Tau/Aβ~1--42~, and 0.059 for p-Tau^181^/Aβ~1--42~. The AUCs, the sensitivity, and the specificity of each CSF biomarker are shown in Table [3](#T3){ref-type="table"}. The ROC curve and AUC for prodromal AD and amyloid-negative aMCI are illustrated in online supplementary Figure [1](#S2){ref-type="supplementary-material"}. The cutoff ratio indicative of prodromal AD from amyloid-negative aMCI was set at Aβ~1--42~/Aβ~1--40~ \< 0.095, t-tau/Aβ~1--42~ \> 0.298, and p-Tau^181^/Aβ~1--42~ \> 0.059 based on the amyloidosis with florbetaben PET (Table [3](#T3){ref-type="table"}). We applied the cutoff values to differentiate preclinical AD patients (*n* = 14), prodromal AD patients (*n* = 27), and amyloid-positive AD dementia patients (*n* = 31) from normal amyloid-negative subjects (*n* = 73). The cutoff values for amyloid-positive AD dementia patients versus amyloid-negative normal subjects were 618.4 pg/mL for Aβ~1--42~, 343.5 pg/mL for t-Tau, 56.2 pg/mL for t-Tau^181^, 0.095 for Aβ~1--42~/Aβ~1--40~, 0.370 for t-Tau/Aβ~1--42~, and 0.075 for p-Tau^181^/Aβ~1--42~. These cutoff values had very good sensitivity, specificity, and AUC in each group comparison (Table [4](#T4){ref-type="table"}). The comparison data of preclinical AD and prodromal AD patients versus normal amyloid-negative subjects are also shown in Table [4](#T4){ref-type="table"}. Discussion {#sec1_4} ========== In this study, we aimed to evaluate the usefulness of CSF biomarkers for differentiating prodromal AD from amyloid-negative aMCI patients in a clinically diagnosed aMCI group, and to determine new cutoff values for CSF biomarkers between the two groups. The new cutoff values for differentiating prodromal AD from amyloid-negative aMCI for Aβ~1--42~ (\< 749.5 pg/mL) and p-Tau^181^ (\> 43.5 pg/mL) showed good sensitivity and specificity; however, the difference in values of t-Tau and Aβ~1--40~ was not significant to enable differentiating between the two groups. The ratios of Aβ~1--42~/Aβ~1--40~ (\< 0.095), t-Tau/Aβ~1--42~ (\> 0.298), and p-Tau^181^/Aβ~1--42~ (\> 0.059) also showed good sensitivity and specificity, and the combination values of Aβ~1--42~ + p-Tau^181^ and p-Tau^181^/Aβ~1--42~ + t-Tau were more accurate than individual CSF biomarkers (Table [3](#T3){ref-type="table"}). CSF biomarkers have become accepted and adopted to varying degrees in clinical trials or the diagnosis of AD dementia worldwide. Previous studies focusing on CSF biomarkers have grouped MCI patients depending on the follow-up results, i.e., whether subjects had converted to dementia or not, instead of depending on amyloidosis at baseline. However, CSF biomarker studies of MCI patients depending on amyloidosis have been rarely reported. To our knowledge, this is the first report that allows differentiating individual with prodromal AD from amyloid-negative aMCI ones. Our findings will be helpful for selecting prodromal AD in clinically diagnosed aMCI groups, which are very heterogeneous. The correct prediction of the MCI outcome is an important point for the prevention of dementia and for opting for appropriate clinical trials that use new disease-modifying agents. The role of CSF biomarkers seems to be crucial in a routine clinical setting for MCI patients, and our results confirm this concept. Our results are expected to become increasingly important in the future. We also determined new cutoff values for CSF biomarkers that differentiate amyloid-positive AD dementia patients (*n* = 31) from amyloid-negative normal subjects (*n* = 73). Our individual cutoff values for t-Tau (\> 343.5 pg/mL) and p-Tau^181^ (\> 56.2 pg/mL) were similar to those reported in previous studies \[[@B34], [@B35], [@B36]\], but that for Aβ~1--42~ (\< 618.4 pg/mL) was higher than that reported in previous studies (\< 481--550 pg/mL) \[[@B34], [@B35], [@B36]\]. These findings may have resulted from the fact that our normal amyloid-negative group did not include amyloid-positive normal individuals that may have been included in previous studies. Many previous studies stated that the CSF biomarkers Aβ~1--42~, t-Tau, and p-Tau could differentiate between AD dementia patients and normal healthy controls as well as between the MCI conversion and MCI stable groups, on the basis of clinical diagnosis instead of the status of amyloidosis. Because of that, our data showed some different cutoff values of CSF biomarkers compared to those of previous studies. We also analyzed CSF biomarkers between normal amyloid-negative subjects (*n* = 73) and preclinical AD patients (*n* = 14). Our results suggested that new cutoff values for CSF Aβ~1--42~ (\< 833.0 pg/mL) and t-Tau (\> 253.5 pg/mL) provided good sensitivity and specificity for differentiating preclinical AD patients from the normal amyloid-negative group. The mean concentration of CSF Aβ~1--42~ (1,047.5 ± 186.6 pg/mL, 643.0 ± 171.1 pg/mL), t-Tau (237.8 ± 97.2 pg/mL, 300.6 ± 132.0 pg/mL), and p-Tau^181^ (46.2 ± 17.1 pg/mL, 55.1 ± 20.9 pg/mL) of amyloid-negative normal subjects and preclinical AD patients in our data (Table [2](#T2){ref-type="table"}) is very similar to the results (Aβ~1--42~: 1,058.7 ± 338.3 vs. 612.5 ± 201.29 pg/mL; t-Tau: 255.4 ± 104.3 vs. 382.3 ± 114.6 pg/mL; p-Tau^181^: 45.1 ± 14.6 vs. 62.3 ± 12.3 pg/mL) of amyloid-negative (*n* = 35) and amyloid-positive (*n* = 16) normal subjects on the status of Aβ deposition reported by Dubois et al. \[[@B37]\]. According to these results, there may be no racial differences of CSF biomarkers in cognitively normal subjects because of amyloidosis. In this study, CSF biomarkers reflect the progression of AD, following each stage of the transition of normal elderly people to AD dementia (Fig. [2](#F2){ref-type="fig"}). The present study showed that the APOE ε4 allele frequency is highest in AD dementia patients among the three clinical diagnostic groups. It is also higher in the MCI group than in normal elderly people (Table [1](#T1){ref-type="table"}). The APOE ε4 frequency in the prodromal AD group is similar to that in the amyloid-positive AD dementia group. On the other hand, the APOE ε4 frequency in amyloid-negative aMCI patients is similar to that in amyloid-negative normal subjects (Table [2](#T2){ref-type="table"}). Previous studies reported that there was a significant association of APOE ε4 allele and late-onset AD, and the APOE ε4 frequency was significantly higher in sporadic AD patients than in controls \[[@B38]\]. APOE ε4 is a very strong risk factor for AD in the cohort study and lowers age at onset of late-onset AD significantly \[[@B39]\]. The APOE ε4 allele is associated with the molecular pathway that promotes late-onset AD \[[@B40]\] and is a risk factor not only for late-onset AD, but also for early-onset AD \[[@B41]\]. Our results showed that the APOE ε4 allele also bears strong association with amyloidosis in preclinical AD and prodromal AD as well as AD dementia, and that it is a strong predictor of amyloidosis in patients with aMCI and preclinical AD. Our findings are important for ongoing and future clinical trials involving participants with prodromal AD. Overall, cognitive performance on all tests did not distinguish prodromal AD from amyloid-negative aMCI in the aMCI group (online suppl. Table [1](#S1){ref-type="supplementary-material"}). This result suggests that Aβ depositions are not severe enough to affect cognitive function or are compensated by brain changes in patients with aMCI. Conclusion {#sec1_5} ========== When considering the sensitivity, specificity, reliability, costs, and invasiveness of different biomarkers, we propose that the described CSF biomarkers be used as a first-line choice for the differentiation of prodromal AD in clinically diagnosed aMCI patients or for selecting very early AD dementia to be treated with the new disease-modifying agents. Accordingly, the introduction of CSF examination as a routine diagnostic procedure for selecting prodromal AD should be considered in the net decisions of diagnostic guidelines for AD dementia. CSF biomarkers, especially Aβ~1--42~ and p-Tau^181^, could be a differentiating marker for the detection of prodromal AD from clinically diagnosed aMCI patients. Statement of Ethics {#sec1_6} =================== Two centers in Gwangju City, Korea (Chonnam National University Hospital and Chosun University Hospital) participated in the study. The study design was approved by the Ethics Committees of Chonnam National University Hospital and Chosun University Hospital, Gwangju, Korea. All participants provided written informed consent at the time of inclusion in the cohort for use of data, samples, and images, and all study procedures were approved by the Institutional Review Boards at the Chonnam National University Hospital and Chosun University Hospital. Disclosure Statement {#sec1_7} ==================== The authors have no conflicts of interest to declare. Author Contributions {#sec1_8} ==================== B.C. Kim and K.H. Lee provided overall project supervision and directed the study. J.E. Park and K.Y. Choi contributed to the analysis and interpretation of data and drafting. S.-M. Choi, M.-K. Song, J.J. Lee, and H.-W. Kim contributed to the clinical data collection. J. Kim, H.-C. Song, and J.-M. Ha contributed to the interpretation of the amyloid PET images. E.H. Seo contributed to the analysis of neuropsychological data. W.K. Song and S.-G. Park contributed to the analysis and interpretation of laboratory data. J.E. Park and J.S. Lee led the analysis and interpretation of CSF assays. All authors edited the manuscript for content and approved its final version. Supplementary Material ====================== ###### Supplementary data ###### Click here for additional data file. ###### Supplementary data ###### Click here for additional data file. This study was supported by the Brain Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (K.H. Lee and B.C. Kim: NRF-2014M3C7A1046041; J.S. Lee: NRF-2016M3C7A1905469; B.C. Kim: NRF-2016M3C7A1905472). {#F1} {#F2} ###### Clinical characteristics and CSF data of subjects with a clinical diagnosis (*n* = 177) Characteristics Total Normal aMCI AD dementia *p* value ------------------------- ------- -------------------- ----------------------------------------------------- ------------------------------------------------------------------------------------ ----------- Subjects, *n* 177 87 56 34 Age, years 177 71.7 (5.8) 71.5 (8.0) 69.4 (6.0) 0.215 Education, years 177 9.6 (5 .0) 9.0 (4 .7) 7.0 (3.9) [^a^](#T1F1){ref-type="table-fn"} 0.026 Female sex 177 46 (52.8%) 26 (46.4%) 19 (55.9%) 0.637 APOE ∊4 carrier status 175 22 (25.3%) 24 (42.9%) 20 (58.8%) [^a^](#T1F1){ref-type="table-fn"}, [^b^](#T1F2){ref-type="table-fn"} 0.002 K-MMSE score, points 177 26.7 (2.6) 25.5 (3 .6) 18.5 (5.4) [^a^](#T1F1){ref-type="table-fn"}, [^b^](#T1F2){ref-type="table-fn"} \<0.001 CDR 177 0.3 (0.2) 0.5 (0.0) [^a^](#T1F1){ref-type="table-fn"} 0.8 (0.4) [^a^](#T1F1){ref-type="table-fn"}, [^b^](#T1F2){ref-type="table-fn"} \<0.001 CDR sum of boxes 177 0.5 (0.6) 1.3 (0.7) [^a^](#T1F1){ref-type="table-fn"} 4.6 (2.6) [^a^](#T1F1){ref-type="table-fn"}, [^b^](#T1F2){ref-type="table-fn"} \<0.001 GDS 177 1.6 (0.5) 2.9 (0.3) [^a^](#T1F1){ref-type="table-fn"} 3.9 (0.9) [^a^](#T1F1){ref-type="table-fn"}, [^b^](#T1F2){ref-type="table-fn"} \<0.001 b-adl 177 20.0 (0.0) 20.0 (0.2) 19.0 (1.7) [^a^](#T1F1){ref-type="table-fn"}, [^b^](#T1F2){ref-type="table-fn"} \<0.001 I-ADL 177 0.04 (0.09) 0.18 (0.16) [^a^](#T1F1){ref-type="table-fn"} 0.60 (0.19) [^a^](#T1F1){ref-type="table-fn"}, [^b^](#T1F2){ref-type="table-fn"} \<0.001 CSF A β~1--42~, pg/mL 177 982.4 (236.5) 771.0 (317.0) [^a^](#T1F1){ref-type="table-fn"} 559.0 (311.0) [^a^](#T1F1){ref-type="table-fn"}, [^b^](#T1F2){ref-type="table-fn"} \<0.001 CSF A β~1\ --40~, pg/mL 177 10,120.1 (4,709.0) 8,120.6 (3,532.1) [^a^](#T1F1){ref-type="table-fn"} 7,311.6 (3,795.3) [^a^](#T1F1){ref-type="table-fn"} 0.001 CSF t-Tau, pg/mL 177 247.9 (105.2) 303.8 (229.7) 487.5 (218.6) [^a^](#T1F1){ref-type="table-fn"}, [^b^](#T1F2){ref-type="table-fn"} \<0.001 CSF p-Tau ^181^, pg/mL 177 47.6 (17.9) 53.1 (29.9) 72.6 (28.1) [^a^](#T1F1){ref-type="table-fn"}, [^b^](#T1F2){ref-type="table-fn"} \<0.001 Data are presented as mean (standard deviation) or *n* (%) unless indicated otherwise. Aβ, beta-amyloid protein; AD, Alzheimer\'s disease; aMCI, amnestic mild cognitive impairment; APOE, apolipoprotein E; B-ADL, basic activities of daily living; CDR, Clinical Dementia Rating; CSF, cerebrospinal fluid; GDS, Global Deterioration Scale; I-ADL, instrumental activities of daily living; K-MMSE, Korean version of the Mini-Mental State Examination; p-Tau^181^, tau protein phosphorylated at threonine 181; t-Tau, total tau protein. Statistically significant difference between the indicated group and the normal group. Statically significant difference between the aMCI and the AD dementia group. ###### Clinical characteristics and CSF data of subjects with amyloid PET (n = 177) Characteristics Total Normal (*n* = 87) aMCI (*n* = 56) AD dementia (*n* = 34) ------------------------------- ------- ------------------------------------------------- ------------------------------------------------- ----------------------------------------------- ------------------------------------------------ ------------------------------------------------- ----------------------------------------------- Subjects, *n* 177 73 14 29 27 3 31 Age, years 177 71.6 (5.9) 72.5 (5.5) 72.4 (7.2) 70.6 (8.9) 71.7 (8.4) 69.2 (5.8) Education, years 177 9.6 (5.0) 9.9 (5.3) 9.8 (4.4) 8.2 (5.0) 9.3 (5.8) 6.8 (3.7) Female sex 177 39 (53.4%\] 7 (50.0%\] 13 (44.8%\] 13 (48.1%) 0 (0.0%\] 19 (61.3%\] APOE *∊* 4 carrier status 175 14 (19.2%) 8 (57.1%) 6 (20.7%) 18 (66.7%) 0 (0.0%) 20 (64.5%) K-MMSE score, points 177 26.6 (2.6) 27.1 (2.9) 25.4 (3.5) 25.5 (3.8) 23.0 (2.6) 18.1 (5.4) CDR 177 0.3 (0.2) 0.3 (0.3) 0.5 (0.0) 0.5 (0.0) 0.5 (0.0)[\*](#T2F1){ref-type="table-fn"} 0.8 (0.4)[\*](#T2F1){ref-type="table-fn"} CDR sum of boxes 177 0.5 (0.7) 0.4 (0.4) 1.2 (0.8) 1.3 (0.6) 1.7 (0.3)[\*\*](#T2F2){ref-type="table-fn"} 4.8 (2.6)[\*\*](#T2F2){ref-type="table-fn"} GDS 177 1.6 (0.5) 1.6 (0.5) 2.9 (0.3) 3.0 (0.2) 3.0 (0.0)[\*](#T2F1){ref-type="table-fn"} 4.0 (0.9)[\*](#T2F1){ref-type="table-fn"} B-ADL 177 20.0 (0.0) 20.0 (0.0) 20.0 (0.2) 20.0 (0.2) 20.0 (0.0) 19.0 (1.7) I-ADL 177 0.05 (0.09) 0.04 (0.07) 0.13 (0.16)[\*\*](#T2F2){ref-type="table-fn"} 0.24 (0.14) [\*\*](#T2F2){ref-type="table-fn"} 0.45 (0.00)[\*](#T2F1){ref-type="table-fn"} 0.62 (0.20)[\*](#T2F1){ref-type="table-fn"} CSFA β~1--42~ levels, pg/mL 177 1,047.5 (186.6)[\*](#T2F1){ref-type="table-fn"} 643.0 (171.1)[\*](#T2F1){ref-type="table-fn"} 978.4 (249.2)[\*](#T2F1){ref-type="table-fn"} 548.2 (214.9)[\*](#T2F1){ref-type="table-fn"} 1,306.8 (269.0)[\*](#T2F1){ref-type="table-fn"} 486.7 (200.7)[\*](#T2F1){ref-type="table-fn"} CSFA β~1--40~ levels, pg/mL 177 10,219.8 (4,668) 9,600.5 (5,066) 8,370.7 (4,132) 7,852.0 (2,802) 10,313.2 (3,939) 7,021.1 (3,717) CSF t-Tau levels, pg/mL 177 237.8 (97.2)[\*\*](#T2F2){ref-type="table-fn"} 300.6 (132.0)[\*\*](#T2F2){ref-type="table-fn"} 250.4 (220.5) 361.3 (229.4) 270.3 (131.5) 508.5 (215.0) CSF p-Tau ^181^ levels, pg/mL 177 46.2 (17.1) 55.1 (20.9) 44.4 (24.9)[\*\*](#T2F2){ref-type="table-fn"} 62.4 (32.3)[\*\*](#T2F2){ref-type="table-fn"} 54.1 (25.2) 74.3 (28.1) Data are presented as mean (standard deviation\] or *n* (%) unless indicated otherwise. A β, beta-amyloid protein; AD, Alzheimer\'s disease; aMCI, amnestic mild cognitive impairment; APOE, apolipoprotein E; B-ADL, basic activities of daily living; CDR, Clinical Dementia Rating; CSF, cerebrospinal fluid; GDS, Global Deterioration Scale; I-ADL, instrumental activities of daily living; K-MMSE, Korean version of the Mini-Mental State Examination; PET, positron emission tomography; p-Tau ^181^, tau protein phosphorylated at threonine 181; t-Tau, total tau protein. *p* \< 0.001, *p* \< 0.05. ###### Sensitivity and specificity of CSF biomarkers in prodromal AD of the aMCI group Characteristics Prodromal AD (*n* = 27) versus amyloid-negative aMCI (*n* = 29) ----------------------------------------------- ----------------------------------------------------------------- ------ ------ ------- ------ ------ --------- CSF A β~1--42~, pg/mL \<749.5 85.2 86.2 0.908 85.2 86.2 \<0.001 CSF t-Tau, pg/mL \>225.6 63.0 65.5 0.692 63.0 65.5 0.087 CSF p-Tau ^181^, pg/mL \>43.5 63.0 69.0 0.678 65.4 66.7 0.036 A β~**1**\ --\ **42**~/A β~**1**\ --\ **40**~ \<0.095 85.2 79.3 0.857 79.3 85.2 0.001 t-Tau/A β~**1**\ --\ **42**~ \>0.298 77.8 82.8 0.853 80.8 80.0 0.004 p-Tau ^181^ /Aβ~1\ --42~ Combination \>0.059 81.5 86.2 0.891 84.6 83.3 0.001 A β~1\ --42~+ p-Tau ^181^ 92.6 89.7 0.935 89.3 92.9 p-Tau ^181^ /Ap~1\ --42~ + t-Tau 92.6 86.2 0.930 81.5 93.1 Aβ, beta-amyloid protein; AD, Alzheimer\'s disease; aMCI, amnestic mild cognitive impairment; AUC, area under the curve; CSF, cerebrospinal fluid; NPV, negative predictive value; PPV, positive predictive value; p-Tau^181^, tau protein phosphorylated at threonine 181; SE, sensitivity; SP, specificity; t-Tau, total tau protein. ###### Diagnostic cutoff values in differentiating patients with preclinical AD (*n* = 14), prodromal AD (*n* = 27), and amyloid-positive AD dementia (*n* = 31) from normal amyloid-negative subjects (*n* = 73) Preclinical AD versus normal Prodromal AD versus normal AD dementia versus normal ---------------------------------- ------------------------------ ---------------------------- --------------------------- ------- --------- ------ ------ ------- --------- ------ ------- ------- CSF Aß~**1**\ --\ **42**~, pg/mL \<833.0 92.9 86.3 0.947 \<752.0 88.9 93.2 0.961 \<618.4 80.6 100.0 0.971 CSF t-Tau, pg/mL \>253.5 71.4 65.8 0.659 \>276.2 59.3 74.0 0.657 \>343.5 74.2 89.0 0.912 CSF p-Tau ^**181**^, pg/mL **A** 71.4 69.9 0.653 \>49.8 55.6 69.9 0.634 \>56.2 71.0 82.2 0.819 A β~**1--42**~/ A β~**1--40**~ \<0.095 64.3 68.5 0.705 \<0.095 85.2 68.5 0.777 \<0.095 71.0 68.5 0.735 t-Tau/Aß~**1--42**~ \>0.295 85.7 82.2 0.871 \>0.295 77.8 82.2 0.864 \>0.370 93.5 93.2 0.988 p-Tau ^**181**^/Aß~**1--\ 42**~ \>0.055 78.6 83.6 0.881 \>0.055 81.5 83.6 0.894 \>0.075 90.3 97.3 0.981 Aβ, beta-amyloid protein; AD, Alzheimer\'s disease; AUC, area under the curve; CSF, cerebrospinal fluid; p-Tau^181^, tau protein phosphorylated at threonine 181; SE, sensitivity; SP, specificity; t-Tau, total tau protein. [^1]: J.E. Park and K.Y. Choi contributed equally to this work.

Introduction {#S0001} ============ A cutaneous wound is typically protected with a dressing to enhance the rate and quality of healing.[@CIT0001] An ideal wound dressing should possess the capacity to effectively protect the wound from bacterial infection, promote angiogenesis and accelerate the re-epithelialization process.[@CIT0002],[@CIT0003],[@CIT0004] Additionally, a dressing should have a suitable porous structure for water and gas exchange and wound exudate absorption.[@CIT0003],[@CIT0005] However, the demand of such multi-functionality for a wound dressing remains a challenge. Antibiotics such as gentamicin and amoxicillin are generally grafted onto wound dressings to provide antibacterial activity.[@CIT0006],[@CIT0007] However, the increasing emergence of antibiotic-resistant bacteria has become one of the largest threats to public health, as declared by the WHO in 2014.[@CIT0008] Therefore, it is urgent to find alternatives to antibiotics. Functional peptides have been widely used in biomedical sciences, such as cancer therapy and antibacterial therapy in recent years.[@CIT0009],[@CIT0010],[@CIT0011] Particularly, antimicrobial peptides (AMPs) have attracted great attention as an antibacterial agent.[@CIT0011],[@CIT0012],[@CIT0013] As a natural immune defence, AMPs are secreted by animals, plants and even some microorganisms, and they have shown remarkable broad-spectrum antibacterial activity.[@CIT0011],[@CIT0014] It is widely believed that cationic AMPs could target the bacterial surface via the electrostatic attraction between positively charged amino acids and the negatively charged cell surface and destroy bacteria by physically disrupting the bacterial cell membrane.[@CIT0015],[@CIT0016] This particular mechanism of action makes AMPs seldom induce drug resistance. The KR-12 peptide, which is identified as the smallest antibacterial motif (residues 18--29) of human cathelicidin LL-37, displays potent antibacterial activity against both Gram-negative bacteria and Gram-positive bacteria, including methicillin-resistant *Staphylococcus aureus* (MRSA).[@CIT0017],[@CIT0018] Apart from antibacterial activity, KR-12 peptide was reported to modulate inflammation and accelerate the re-epithelialization process via promoting keratinocyte proliferation and migration.[@CIT0019],[@CIT0020] The potential cytotoxicity and hemolysis of free KR-12 and LL-37 narrow their applications, but it has been reported that immobilizing them on materials relieves this concern.[@CIT0018],[@CIT0021] Thus, because of the fascinating advantages, KR-12 peptide is a valuable antibacterial candidate to include in the fabrication of wound dressings. In addition to the inhibition of bacterial infection and promotion of re-epithelialization, angiogenesis is another important issue during the wound healing process because the newly generated blood vessels can transport nutrients and growth factors for tissue formation.[@CIT0004],[@CIT0022] Hyaluronic acid (HA) is a natural linear polysaccharide mainly found in hydrated tissues, such as the eye, cartilage, bone and skin.[@CIT0023],[@CIT0024] It is composed of repeated disaccharides of β-1,4-linked D-glucuronic acid and β-1,3-linked N-acetyl-D-glucosamine and displays unique biological properties, such as biodegradability, biocompatibility, hydrophilicity and water control ability.[@CIT0025],[@CIT0026] As an essential component of the skin extracellular matrix, HA has been demonstrated to be beneficial for rapid wound healing.[@CIT0027],[@CIT0028] More noteworthy, accumulating evidence in recent years has indicated that HA plays an important role in modulating angiogenesis via promoting the proliferation of endothelial cells and secretion of angiogenesis-related cytokines.[@CIT0029],[@CIT0030],[@CIT0031],[@CIT0032] Thus, the immobilization of HA into a wound dressing is helpful for angiogenesis in the wound healing process. The eggshell membrane (ESM) is a fibrous connective tissue between the egg white and eggshell. As a natural biomaterial, ESM is biocompatible with human cells and easy to obtain at low cost.[@CIT0033] ESM possesses a three-dimensional fibrous meshwork structure, and its nano/microfibres are mainly made up of proteins (80--85%).[@CIT0034] The characteristic porous meshwork structure provides ESM with a large surface area for biomolecule deposition and suitable porosity for gas and water exchange, which are essential factors for wound dressings.[@CIT0033],[@CIT0035] Furthermore, ESM has been well demonstrated to be beneficial to fibroblast proliferation and wound healing.[@CIT0036],[@CIT0037] These features make ESM a good scaffold for wound dressings. In this study, a biocompatible composite membrane composed of biomimetic polydopamine-modified ESM nano/microfibres coated with KR-12 peptide and HA (ESM/KR-12) was prepared using a three-step approach ([Figure 1A](#F0001){ref-type="fig"}). First, the ESM was modified with a biomimetic polydopamine, which contains multiple active groups and possesses strong adhesive capacity.[@CIT0038],[@CIT0039] Second, HA was immobilized on the polydopamine coating via chemical/physical interactions. Third, because HA is negatively charged while KR-12 is positively charged, the KR-12 peptide was conjugated onto the HA coating via electrostatic interaction in a facile manner.[@CIT0018],[@CIT0040] The whole fabrication process was carried out in an eco-friendly way at room temperature. We hypothesize that the ESM/KR-12 composite membrane has antibacterial properties, including the capacity to kill multi-drug-resistant bacteria, and can promote angiogenesis and re-epithelialization, leading to rapid wound healing.Figure 1(**A**) Schematic of the preparation and biological effects of ESM/KR-12 membrane. SEM images of (**B**) ESM, (**C**) ESM/DA, (**D**) ESM/HA, (**E**) ESM/KR~300~, (**F**) ESM/KR~600~ and (**G**) ESM/KR~900~ membranes.**Abbreviations:** ESM, eggshell membrane; HA, hyaluronic acid. Materials and methods {#S0002} ===================== Materials and animals {#S0002-S2001} --------------------- Fresh eggs were obtained from a supermarket (Chongqing, People\'s Republic of China). Dopamine (DA) hydrochloride, sodium salt of HA (\~2000 kDa) and 5, 5ʹ-dithiobis-(2-nitrobenzoic acid) (DTNB) were purchased from Solarbio Science & Technology (Beijing, People\'s Republic of China). Analogue-3 of the original KR-12 peptide (CKRIVKRIKKWLR, \>95% purity) was obtained from Sangon (Shanghai, People\'s Republic of China).[@CIT0018],[@CIT0020] *Staphylococcus aureus* (*S. aureus*, ATCC 25923), MRSA (ATCC 43300) and *Escherichia coli* (*E. coli*, ATCC 25922) were obtained from Clinical Microbiology Laboratory, Southwest Hospital (Chongqing, People\'s Republic of China). BALB/c mice (female, \~25 g, 12 weeks old, specific pathogen-free level) were purchased from the Animal Department of Third Military Medical University (TMMU). All animal experimental methods were approved by the ethical committee of TMMU, and all animal tests were performed in accordance with the guidelines of TMMU. Preparation of ESM/KR-12 composite membranes {#S0002-S2002} -------------------------------------------- The ESM was carefully peeled from fresh eggshells using forceps and washed three times with deionized water. Next, the ESM was immersed into a DA solution (2 mg/mL in 10 mM Tris-HCl, pH=8.5) for 16 hrs. After rinsing twice with deionized water, the DA-coated ESM was immersed into a sodium HA aqueous solution (4 mg/mL, pH=6.0) for 4 hrs. Next, the HA was adhered on the DA coating via chemical/physical combinations. After washing twice with 0.15 M NaCl aqueous solution, the HA-treated ESM was dipped into different concentrations of KR-12 solution (300, 600 and 900 μg/mL) in PBS (pH=7.4) for 40 mins to conjugate the peptide via charge attraction. Finally, the membrane was washed with deionized water and harvested for subsequent experiments. The whole process of preparation was conducted at room temperature. Subsequently, the DA-treated ESM, HA-treated ESM and KR-12-treated ESM were designated as ESM/DA, ESM/HA and ESM/KR-12 (ESM/KR~300~, ESM/KR~600~ and ESM/KR~900~) membranes, respectively. Characterization of ESM/KR-12 membranes {#S0002-S2003} --------------------------------------- The membrane morphology was observed under a scanning electron microscope (SEM; Crossbeam 340, Zeiss, Germany). The average porosity and fiber diameter of the membranes was analyzed using ImageJ software based on SEM images. Surface zeta potential measurement was performed by Zetasizer Nano ZSP (Malvern, UK) at room temperature. The water contact angle of the membrane was detected using a contact angle measurement instrument (Theta Lite 101; Biolin Scientific, Sweden). Attenuated total reflectance Fourier transform-infrared (ATR-FTIR) spectra were determined using an ATR-FTIR spectrometer (Nicolet 460, USA). The loading amount of KR-12 was explored using the Ellman's assay, which was based on the absorbance alteration of the solution via the reaction between DTNB and the thiol group of KR-12.[@CIT0020] Briefly, 50 μL of KR-12 solution and 150 μL of DTNB were added into a 96-well plate and incubated in the dark for 15 mins at room temperature. Next, the absorbance value of the solution was measured at 412 nm using a microplate absorbance reader (Thermo Scientific, USA). The loading ratio and loading density were calculated using the following formula: $$\documentclass[12pt]{minimal} \usepackage{wasysym} \usepackage[substack]{amsmath} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage[mathscr]{eucal} \usepackage{mathrsfs} \DeclareFontFamily{T1}{linotext}{} \DeclareFontShape{T1}{linotext}{m}{n} {linotext }{} \DeclareSymbolFont{linotext}{T1}{linotext}{m}{n} \DeclareSymbolFontAlphabet{\mathLINOTEXT}{linotext} \begin{document} $${\rm{Loading\ ratio }}\,\left({\rm{\% }} \right){\rm{ = }}\left({{{\rm{C}}_{\rm{1}}}{\rm{-\, }}{{\rm{C}}_{\rm{2}}}} \right){\rm{/ }}{{\rm{C}}_{\rm{1}}}{\rm{ \times 100\% ,}}$$ \end{document}$$$$\documentclass[12pt]{minimal} \usepackage{wasysym} \usepackage[substack]{amsmath} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage[mathscr]{eucal} \usepackage{mathrsfs} \DeclareFontFamily{T1}{linotext}{} \DeclareFontShape{T1}{linotext}{m}{n} {linotext }{} \DeclareSymbolFont{linotext}{T1}{linotext}{m}{n} \DeclareSymbolFontAlphabet{\mathLINOTEXT}{linotext} \begin{document} $${\rm{Loading\ density = }}\left({{{\rm{C}}_{\rm{1}}}{\rm{-\, }}{{\rm{C}}_{\rm{2}}}} \right){\rm{ \times V/ S,}}$$ \end{document}$$ where C~1~ and C~2~ represent the concentrations of KR-12 solution before and after peptide coating, respectively, V represents the volume of KR-12 solution, and S represents the surface area of the membrane. To measure the swelling percentage, a piece of membrane (10×10 mm) was immersed in PBS (pH=7.4) for various times (0.5--24 hrs) at 37°C, and the membrane was weighed before and after swelling. The swelling percentage (%) of the membrane was calculated using the following formula: $$\documentclass[12pt]{minimal} \usepackage{wasysym} \usepackage[substack]{amsmath} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage[mathscr]{eucal} \usepackage{mathrsfs} \DeclareFontFamily{T1}{linotext}{} \DeclareFontShape{T1}{linotext}{m}{n} {linotext }{} \DeclareSymbolFont{linotext}{T1}{linotext}{m}{n} \DeclareSymbolFontAlphabet{\mathLINOTEXT}{linotext} \begin{document} $${\rm{Swelling\ percentage }}\,\left({\rm{\% }} \right){\rm{ = }}\left({{{\rm{W}}_{\rm{s}}}{\rm{- }}{{\rm{W}}_{\rm{i}}}} \right){\rm{/ }}{{\rm{W}}_{\rm{i}}}{\rm{,}}$$ \end{document}$$ where W~s~ is the weight of membrane after a certain swelling time and W~i~ is the weight of the membrane in the dry state. Antibacterial test {#S0002-S2004} ------------------ The antibacterial activity of the membrane was assessed against Gram-negative *E. coli*, Gram-positive *S. aureus* and MRSA. Briefly, a log-phase bacterial suspension was first diluted with Luria--Bertani (LB) medium to the initial concentration (1×10^6^ colony forming units (CFU)/mL). Next, 100 μL of bacterial suspension was inoculated on the surface of membrane and incubated for 4 hrs at 37°C. After gently washing with PBS for 1 min, the sample was placed in a 24-well plate with 1 mL of PBS, followed by sonication for 10 mins. The solution was then diluted with PBS for 200×, and 25 μL of solution was uniformly spread on an agar plate. After incubation for 18 hrs at 37°C, the number of bacterial colonies was counted, and the bacterial survival rates in different groups were calculated by comparison with the pristine ESM group. To investigate the effect of KR-12-conjugated ESM on biofilm formation, a piece of sample was co-incubated with 300 μL of bacterial suspension (1×10^6^ CFU/mL) in LB medium for 24 hrs at 37°C. Next, the sample was collected and gently rinsed twice with PBS. For SEM analysis, the sample was fixed in 4% formaldehyde, dehydrated with an ethanol solution and sputter-coated with gold. The area of biofilm was then measured using IPP 6.0 software based on SEM images as reported previously.[@CIT0041] For the Live/Dead staining assay, the sample was stained by a Live/Dead staining kit (Invitrogen, USA) in the dark for 15 mins. After rinsing twice with PBS, the biofilm was observed using a fluorescence microscope (Olympus, Japan). The green signal indicated living bacteria, while the red signal indicated dead bacteria. Cell culture and cell proliferation test {#S0002-S2005} ---------------------------------------- Human umbilical vein endothelial cells (HUVECs) and human keratinocytes (HaCaT) were purchased commercially from American Type Culture Collection, and cultured in DMEM supplemented with 10% FBS. Before the cell experiment, the sterilized samples were cut into small disks (diameter: 6 mm) and placed in a 96-well plate. Next, 2,500 HUVECs or HaCaT cells were seeded on each sample and incubated in an incubator at 37°C and 5% CO~2~. To detect the cell proliferation level, the CCK8 assay was conducted at day 1, 3 and 5 post-seeding. Briefly, the culture medium in each well was replaced with 100 μL of fresh medium and 10 μL of CCK8 solution and then was incubated for 2 hrs at 37°C. Next, the optical density of solution at 450 nm was measured using a microplate absorbance reader (Thermo Scientific, USA). Moreover, to observe the cell morphology, the samples after 5 days of incubation were fixed in 4% formaldehyde for 0.5 hr and then were successively stained with phalloidin (100 ng/mL; Thermo Scientific) and DAPI (Thermo Scientific), which labelled F-actin (red) and the nuclei (blue) of cells, respectively. Fluorescence images of HUVECs and HaCaT cells were obtained using a fluorescence microscope (Olympus, Japan). ELISA for vascular endothelial growth factor (VEGF) secretion {#S0002-S2006} ------------------------------------------------------------- To quantify the amount of (VEGF secreted from HUVECs, the cell culture medium of each well was harvested at day 5 post-seeding. Next, the VEGF concentration was detected using an ELISA kit (Dakewe Biotech Co., Ltd, Shenzhen, People\'s Republic of China) according to the manufacturer's instructions. The results were expressed as the mass (pg) of VEGF per milliliter of culture medium. Hemolysis assay {#S0002-S2007} --------------- The hemocompatibility of the membrane was evaluated using a hemolysis assay as described previously.[@CIT0042] Human whole blood (diluted 10-fold with normal saline) was obtained from Southwest Hospital, Chongqing, People\'s Republic of China. All studies involving human whole blood were approved by the ethics committee of first affiliated hospital of TMMU and performed in accordance with the institutional and international guidelines. Written informed consent has been provided by the donors in compliance with the Declaration of Helsinki. Prior to the assay, each sample was cut into a rectangular strip (10×25 mm) and was incubated in 10 mL of normal saline at 37°C for 1 hr. Next, 200 μL of diluted blood was added and incubated for another 1 hr at 37°C. Finally, the solution was centrifuged at 3000 rpm for 5 mins at room temperature, and the absorbance of supernatant fluid at 545 nm was measured using a microplate absorbance reader (Thermo Scientific). Here, the deionized water mixed with blood served as a positive control group, while the normal saline mixed with blood served as a negative control group. The hemolysis ratio (HR) was calculated according to the following formula: $$\documentclass[12pt]{minimal} \usepackage{wasysym} \usepackage[substack]{amsmath} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage[mathscr]{eucal} \usepackage{mathrsfs} \DeclareFontFamily{T1}{linotext}{} \DeclareFontShape{T1}{linotext}{m}{n} {linotext }{} \DeclareSymbolFont{linotext}{T1}{linotext}{m}{n} \DeclareSymbolFontAlphabet{\mathLINOTEXT}{linotext} \begin{document} $${\rm{HR }}\,\left({\rm{\% }} \right){\rm{ = }}\left({{{\rm{A}}_{\rm{M}}}{\rm{- }}{{\rm{A}}_{\rm{N}}}} \right){\rm{/}}\left({{{\rm{A}}_{\rm{P}}}{\rm{-\, }}{{\rm{A}}_{\rm{N}}}} \right){\rm{ \times 100\% ,}}$$ \end{document}$$ where A~M~ represents the absorbance of blood treated with the membrane, A~P~ represents the absorbance of blood treated with deionized water and A~N~ represents the absorbance of blood treated with normal saline. In vivo animal test {#S0002-S2008} ------------------- A murine skin wound model (five mice per group) was conducted for the in vivo test as previously described.[@CIT0043] The mice were anesthetized with 1% pentobarbital via intraperitoneal injection, and the dorsal hairs were shaved. Next, two full-thickness wounds (diameter: 6 mm) were created on either side of the back using a punch. After being photographed by a digital camera, the wounds were immediately covered by ESM, ESM/DA, ESM/HA or ESM/KR~600~ membranes and were kept in place by an adhesive bandage. The wounds that only covered with an adhesive bandage were served as the control group. At days 2, 4 and 8, the wounds were photographed, and the dressings were replaced. Based on the photographs, the wound areas were carefully measured using IPP 6.0 software by two independent researchers. The reserved wound area at a certain time point was calculated using the following formula: $$\documentclass[12pt]{minimal} \usepackage{wasysym} \usepackage[substack]{amsmath} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage[mathscr]{eucal} \usepackage{mathrsfs} \DeclareFontFamily{T1}{linotext}{} \DeclareFontShape{T1}{linotext}{m}{n} {linotext }{} \DeclareSymbolFont{linotext}{T1}{linotext}{m}{n} \DeclareSymbolFontAlphabet{\mathLINOTEXT}{linotext} \begin{document} $${\rm{Percentage\ of\ wound\ area }}\,\left({\rm{\% }} \right){\rm{ = R/ I\times 100\% ,}}$$ \end{document}$$ where R represents the number of pixels of the reserved wound area at a certain time point, while I represents the number of pixels of the initial wound area. Furthermore, to determine the average wound closure times, five additional mice in every group were observed until the wounds were completely closed. Histological examination {#S0002-S2009} ------------------------ The mice were sacrificed at day eight post-surgery, and then the wound tissues with adjacent normal skin (10×10 mm) were carefully harvested and fixed in 4% paraformaldehyde for 24 hrs. Next, the tissues were embedded in paraffin and sliced at a thickness of 5 μm. H&E staining was performed for histological analysis. The number of inflammatory cells in the wound edge, thickness of granulation tissue and length of regenerated epidermis, which was defined as the distance from the border between normal skin and wound region to the anterior edges of newly generated epidermis,[@CIT0043] were quantified using ImageJ software by two independent researchers. Immunohistochemistry analysis {#S0002-S2010} ----------------------------- Immunohistochemistry analysis was conducted as previously described.[@CIT0003] Briefly, wound tissue sections were deparaffinized, rehydrated and boiled in a 100°C citrate buffer bath. Next, the sections were treated with primary antibodies at 4°C overnight. The primary antibodies obtained from Abcam (UK) were as follows: anti-CD31 (cluster of differentiation 31) antibody at 1:250 dilution, ab28364; anti-PCNA (proliferating cell nuclear antigen) antibody at 1:200 dilution, ab15497; and anti-VEGF antibody at 1:200 dilution, ab46154. After incubation with goat-anti-rabbit IgG antibody and avidin-peroxidase reagent, the sections were stained with 3,30-diaminobenzidine tetrahydrochloride (DAB) solution and hematoxylin and then were photographed under a microscope (CTR6000, Leica, Germany). Based on the images, the number of PCNA-positive keratinocytes per field, the number of CD31-positive blood vessels per field and relative intensity of VEGF protein (compared with control group) were counted and analyzed using ImageJ software by two independent researchers. Statistical analysis {#S0002-S2011} -------------------- The data were expressed as the mean±SD, and the results were analyzed by one-way ANOVA. *P*\<0.05 was considered statistically significant. Results and discussion {#S0003} ====================== Characterizations of composite membranes {#S0003-S2001} ---------------------------------------- The composite membranes were first characterized by SEM. As shown in [Figure 1B](#F0001){ref-type="fig"}, pristine ESM exhibited a three-dimensional network structure consisting of interwoven nano/microfibres and hollow spaces. The diameter of fibers ranged from 0.5 to 2.0 μm, and the fiber surface was smooth. After being coated with polydopamine, the ESM/DA fiber was slightly rougher than that of the ESM ([Figure 1C](#F0001){ref-type="fig"}), but no obvious difference was found in the morphology between the ESM and the ESM/DA. [Figure 1D](#F0001){ref-type="fig"} shows the microscopic feature of the ESM coated by HA. The ESM/HA still maintained a fibrous network structure, but a homogeneous layer with aggregations was apparent on the fiber surface. Moreover, compared with the ESM/DA, the average fiber diameter of the ESM/HA measured by ImageJ software was significantly increased from 1.37 to 1.72 μm ([Table 1](#T0001){ref-type="table"}). Correspondingly, the porosity of the ESM/HA was decreased from 44.09% to 42.05%. The overall results suggested that HA was successfully grafted on the polydopamine-modified ESM scaffold. Polydopamine possesses strong adhesive capacity, and its catechol group could react with various biomolecules under physiological conditions; thus, we assumed that HA was immobilized on the polydopamine coating via physical/chemical interactions.[@CIT0038],[@CIT0039] The morphologies of the ESM/KR~300~, the ESM/KR~600~ and the ESM/KR~900~ presented in [Figure 1E](#F0001){ref-type="fig"}-G were similar to that of the ESM/HA, and the cause might be because that the nanosized peptide layer was too thin to distinguish.[@CIT0044]Table 1Porosities and fibre diameters of ESM, ESM/DA, ESM/HA, ESM/KR~300~, ESM/KR~600~ and ESM/KR~900~ membranesESMESM/DAESM/HAESM/KR~300~ESM/KR~600~ESM/KR~900~Porosity (%)44.31±5.9244.09±5.0642.05±4.5741.53±5.8841.25±3.7941.04±4.37Fibre diameter (μm)1.23±0.501.37±0.551.72±0.481.77±0.431.79±0.421.80±0.47[^1] [Figure 2A](#F0002){ref-type="fig"} shows the alteration sign of the zeta potential along with the successive deposition of HA and KR-12 peptide. As previously described, the surface of HA coating exhibited a negative zeta potential.[@CIT0040] However, after treatment with KR-12 solutions, the surface zeta potential became clearly positive, and the charge was enhanced with the concentration increase of KR-12 peptide. This result suggested that positively charged KR-12 was deposited on the negatively charged HA coating via electrostatic attraction.[@CIT0018],[@CIT0040] To determine the loading amount of KR-12 peptide on the ESM scaffold, Ellman's assay was conducted. As shown in [Figure 2B](#F0002){ref-type="fig"}, the loading densities of KR-12 on the ESM/KR~300~, the ESM/KR~600~ and the ESM/KR~900~ membranes were 28.5, 58.8 and 84.4 μg/mL, respectively, which were proportional to the concentration of KR-12 solution in the reaction medium. The loading ratios of the three membranes were all approximately 32.0%, likely because the loading efficacy was not influenced by the concentration of KR-12 peptide.Figure 2Characterizations of the composite membranes (n=3). (**A**) Zeta potentials of ESM coated with HA and ESM coated with different concentrations of KR-12 peptide. (**B**) Loading density and loading ratio of KR-12 peptide on ESM. (**C**) Water contact angles of pristine ESM and composite membranes. (**D**) ATR-FTIR spectra of pristine ESM and composite membranes. (**E**) Swelling percentages of pristine ESM and composite membranes.**Abbreviations:** ESM, eggshell membrane; HA, hyaluronic acid. [Figure 2C](#F0002){ref-type="fig"} shows the water contact angles of the ESM, the ESM/DA, the ESM/HA and the ESM/KR~600~ membranes. After being coated with polydopamine, the contact angles of ESM/DA were significantly decreased from 88.2° to 40.2°, due to the super hydrophilic property of the polydopamine layer.[@CIT0045] Similarly, the subsequent immobilized HA coating kept the membrane surface hydrophilic. The water contact angle of the ESM/KR~600~ was slightly increased to 47.1°, which is likely attributed to the hydrophobic residue of KR-12 present on the membrane surface.[@CIT0017],[@CIT0046] As the surface wettability influenced the biological behaviours of the biomaterial, we speculated that such an improvement in the surface hydrophilicity of the membrane might be beneficial to enhance the cytocompatibility as previously reported.[@CIT0038] [Figure 2D](#F0002){ref-type="fig"} shows the ATR-FTIR spectra of the ESM, the ESM/DA, the ESM/HA and the ESM/KR~600~ membranes. The pristine ESM exhibited characteristic absorption peaks at 1644, 1537 and 1236 cm,^−1^ which represented the amide I (C=O bond), amide II (C−N stretching vibration) and amide III (C−N stretching/N−H bending modes) bands of the proteins, respectively. Moreover, the absorption peak at 3312 cm^−1^ was assigned to the stretching mode of O−H and N−H groups.[@CIT0033] After coating with polydopamine, the absorption peak approximately 3312 cm^−1^ became broader and stronger, due to the phenolic hydroxyl stretching vibration.[@CIT0047] Compared with the ESM/DA, the spectrum of the ESM/HA showed stronger characteristic absorption peaks at 1644 and 1450 cm^−1^ which corresponded to C=O and C=C bonds, respectively, suggesting chemical interactions between HA and polydopamine.[@CIT0048] The spectra of the ESM/HA and the ESM/KR~600~ were similar, suggesting that KR-12 peptide was immobilized on the HA coating via physical combination. [Figure 2E](#F0002){ref-type="fig"} shows the swelling percentages of the ESM, the ESM/DA, the ESM/HA and the ESM/KR~600~ membranes. The ESM and ESM/DA membranes exhibited similar water retention ability, and their swelling percentages were 222.9% and 224.4%, respectively. However, after coating with HA, the swelling percentages of the ESM/HA and ESM/KR~600~ membranes increased to 274.2% and 269.6%, respectively, likely attributed to the visco-elastic property and hydration control ability of HA.[@CIT0024],[@CIT0049] The improvement in the water retention capacity of the ESM/HA and the ESM/KR~600~ suggested that they could effectively absorb excess exudates from the wound, maintaining a suitable moist microenvironment for tissue regeneration.[@CIT0003],[@CIT0050] Thus, these membranes might be applied as wound dressings. Taken together, the results of SEM, zeta potential, water contact angle, ATR-FTIR and swelling percentage indicated that the ESM/KR-12 composite membrane (ESM/KR~600~) was successfully prepared and expected to be used in wound treatment. Antibacterial activities of composite membranes {#S0003-S2002} ----------------------------------------------- The antibacterial activities of composite membranes against Gram-negative *E. coli*, and Gram-positive *S. aureus* and MRSA bacteria are shown in [Figure 3A](#F0003){ref-type="fig"}-[C](#F0003){ref-type="fig"}. Compared with pristine ESM, ESM/DA and ESM/HA, KR-12 peptide-conjugated membranes (ESM/KR~300~, ESM/KR~600~ and ESM/KR~900~) showed significant bactericidal activity against all three bacterial strains in a KR-12-dose-dependent manner. Specifically, the survival rates of the three bacterial strains in ESM/KR~600~ and ESM/KR~900~ groups were all less than 3%, indicating that KR-12 conjugation endowed ESM broad antibacterial activity, including the capacity to kill multi-drug-resistant bacteria. The cause might be that the positively charged KR-12 peptide immobilized on the surface of ESM could target the negatively charged bacteria via electrostatic interaction and damage the bacterial membrane to cause membrane lysis and eventually bacterial death.[@CIT0011],[@CIT0015]Figure 3Antibacterial activity of the composite membranes (n=3). Bacterial viabilities of (**A**) *E. coli*, (**B**) *S. aureus* and (**C**) MRSA in different groups. (**D**) SEM images, (**E**) Live/Dead staining images and (**F**) corresponding areas of MRSA biofilm on ESM, ESM/DA, ESM/HA and ESM/KR~600~ membranes. *\*P*\<0.05, and *\*\*P*\<0.01. **Abbreviations: **ESM, eggshell membrane; HA, hyaluronic acid; MRSA, methicillin-resistant *Staphylococcus aureus.* Bacteria adhered to a biomaterial surface can grow into biofilms, which show high resistance to antibiotics and often cause persistent infection.[@CIT0051],[@CIT0052] Thus, it is very important for a wound dressing to prevent biofilm formation. To assess the effect of as-prepared membranes on biofilm formation, the samples were co-incubated with multi-drug-resistant MRSA bacteria for 24 hrs and then were harvested for SEM analysis and the Live/Dead staining assay. As shown in [Figure 3D](#F0003){ref-type="fig"}, the surfaces of pristine ESM, ESM/DA and ESM/HA were covered by an abundant bacteria, which were grouped in large clusters and tended to grow into biofilms. However, bacteria were hardly detected growing on the surface of the ESM/KR~600~ membrane. The quantitative data also showed that the area covered by bacteria on the surface of ESM/KR~600~ was significantly less than that on the surface of other samples ([Figure 3F](#F0003){ref-type="fig"}). Moreover, Live/Dead staining images clearly revealed that the surfaces of pristine ESM, ESM/DA and ESM/HA were dominant with strong green signals (living bacteria), while only a few green signals and red signals (dead bacteria) could be observed on the surface of ESM/KR~600~ ([Figure 3E](#F0003){ref-type="fig"}), further confirming that ESM/KR~600~ was efficient to prevent the formation of MRSA biofilms. The biofilm inhibition effect of ESM/KR~600~ might be due to the mechanism that KR-12 peptide could fight against bacteria ([Figure 3A](#F0003){ref-type="fig"}-[C](#F0003){ref-type="fig"}), further preventing their adhesion and cloning on the surface of the ESM/KR~600~ membrane.[@CIT0018] Collectively, KR-12 conjugated ESM/KR~600~ showed excellent bactericidal activity and anti-biofilm property and could be used as an antibacterial wound dressing. In vitro effect of composite membranes on HUVECs {#S0003-S2003} ------------------------------------------------ To investigate the effect of composite membranes on the proliferation of HUVECs in vitro, the cell number was first detected using the CCK-8 assay. As shown in [Figure 4A](#F0004){ref-type="fig"}, the proliferation of HUVECs in all groups exhibited an increasing profile after incubation for 1, 3 and 5 days. At day 1 post-seeding, no significant differences were observed among all groups. However, the cell viability in both the ESM/HA and ESM/KR~600~ groups was significantly higher than that of the ESM and ESM/DA groups at days 3 and 5 post-seeding. Moreover, the cells after culture for 5 days were stained with phalloidin and DAPI, and the morphologies were observed under a fluorescence microscope. As shown in [Figure 4C](#F0004){ref-type="fig"}, compared with the ESM and ESM/DA groups, a larger number of cells was clearly found in the ESM/HA and ESM/KR~600~ groups. In line with the above results, the ESM/HA and ESM/KR~600~ membranes were found to increase VEGF secretion ([Figure 4B](#F0004){ref-type="fig"}), which could enhance the proliferation of HUVECs and angiogenesis.[@CIT0053],[@CIT0054] These results suggested that the HA-coated ESM/HA and ESM/KR~600~ membranes could promote angiogenesis. According to previous reports, this might be because that HA bound to its receptor CD44 on the endothelial cell surface, on the one hand, increasing ERK and Akt phosphorylation to promote cell proliferation and VEGF expression and, on the other hand, decreasing cleaved caspase-3 expression to prevent cell apoptosis, resulting in improved HUVEC proliferation and angiogenesis.[@CIT0029],[@CIT0030]Figure 4In vitro effects of the composite membranes on HUVECs (n=3). (**A**) OD values measured by the CCK8 assay at days 1, 3 and 5 post-seeding in different groups. (**B**) Concentration of secreted VEGF and (**C**) fluorescence images of HUVECs at day 5 post-seeding in different groups. *\*\*P*\<0.01. In vitro effect of composite membranes on HaCat cells {#S0003-S2004} ----------------------------------------------------- The effect of composite membranes on the proliferation of HaCaT cells was evaluated using the CCK8 assay and morphology observation. As presented in [Figure 5](#F0005){ref-type="fig"}, both the OD values and fluorescence images revealed that the proliferation level of HaCaT cells in the ESM/KR~600~ group was obviously higher than that in the ESM, ESM/DA and ESM/HA groups at days 3 and 5 post-seeding. Additionally, compared with the ESM and ESM/DA, the ESM/HA promoted the proliferation of keratinocytes at day 5 post-seeding. This result was consistent with previous studies that both HA and KR-12 peptide were helpful for the proliferation of keratinocytes, and we speculated that the ESM/KR~600~ group exhibited the highest level of cell viability likely because ESM/KR~600~ could promote keratinocyte proliferation via the synergistic effects of immobilized HA and KR-12 peptide.[@CIT0019],[@CIT0020],[@CIT0028]Figure 5In vitro effects of the composite membranes on HaCaT cells (n=3). (**A**) OD values measured by the CCK8 assay at days 1, 3 and 5 post-seeding in different groups. (**B**) Fluorescence images of HaCaT cells at day 5 post-seeding in different groups. \*\**P*\<0.01. Hemocompatibility evaluation {#S0003-S2005} ---------------------------- The hemocompatibility of biomaterials such as wound dressings must be carefully evaluated prior to clinical use.[@CIT0055] Therefore, the effect of samples on human whole blood was investigated using the hemolysis assay. As shown in [Figure 6A](#F0006){ref-type="fig"}, compared with the deionized water group, no hemolysis sign was observed in the ESM, ESM/DA, ESM/KR~600~ and ESM/KR~900~ groups. Furthermore, the quantitative data revealed that the hemolysis ratios of all membranes were below the safety threshold of 5% ([Figure 6B](#F0006){ref-type="fig"}).[@CIT0042] This result indicated that KR-12 conjugated membranes had excellent hemocompatibility, which is suitable for use in wound management.Figure 6Blood compatibility of the composite membranes (n=3). (**A**) Images and (**B**) corresponding hemolysis ratios of fresh blood after incubation with saline, ESM, ESM/DA, ESM/HA, ESM/KR~600~, ESM/KR~900~ and deionized water. Abbreviations: ESM, eggshell membrane; HA, hyaluronic acid. In vivo effect of composite membranes on wound healing {#S0003-S2006} ------------------------------------------------------ The wound healing performances of composite membranes were investigated using an in vivo murine wound model. The wounds were covered without a membrane (control group) or with ESM, ESM/DA, ESM/HA and ESM/KR~600~ membranes and were photographed at the determined times. [Figure 7A](#F0007){ref-type="fig"} and [B](#F0007){ref-type="fig"} displayed the macroscopic pictures of wounds and percentages of the wound area, and it was clearly observed that ESM/KR~600~ could significantly promote wound healing compared with other treatments. At day 2 post-surgery, a red and swollen sign was found around the wound in the control, ESM, ESM/DA and ESM/HA groups, but the wound treated by ESM/KR~600~ seemed to be tidy and neat, suggesting that ESM/KR~600~ might reduce the inflammation response. At day 4 and day 8 post-surgery, newly formed epidermis could be apparently observed in the wound treated by ESM/KR~600~, and the quantitative data revealed that the percentage of the wound area in the ESM/KR~600~ group was significantly lower than that in other groups. Particularly, at day 8 post-surgery, the percentage of the wound area in the ESM/KR~600~ group was decreased from 100.0% to 9.0%, while that in the control, ESM, ESM/DA and ESM/HA groups were 24.1%, 23.6%, 23.3% and 16.2%, respectively. ESM/HA also significantly promoted wound closure compared with pristine ESM at day 8 post-surgery, consistent with previous reports that HA contributed to wound healing.[@CIT0027],[@CIT0028] Additionally, [Figure 7C](#F0007){ref-type="fig"} shows that the average wound closure time for the control, ESM, ESM/DA and ESM/HA groups were 10.8, 10.5, 10.7 and 10.2 days, respectively; nevertheless, it only required 8.8 days for the ESM/KR~600~-treated mice, implying that treatment with the ESM/KR~600~ could shorten the wound closure time.Figure 7In vivo effects of the composite membranes on wound healing (n=5). (**A**) Images of wounds and (**B**) the corresponding percentages of wounds at determined time points. (**C**) Average complete healing times in different groups. \**P*\<0.05 represents a significant difference between ESM/KR~600~ and other groups, while ^\#^*P*\<0.05 represents a significant difference between the ESM/HA and ESM groups.**Abbreviations:** ESM, eggshell membrane; HA, hyaluronic acid. To further evaluate the efficacy of ESM/KR~600~ on wound healing, the wound tissues were collected and stained with H&E for histological examination. Re-epithelialization and granulation tissue formation as two important parameters for wound healing were first analyzed. As shown in [Figure 8A](#F0008){ref-type="fig"} and [C](#F0008){ref-type="fig"}, the length of the regenerated epidermis in the ESM/KR~600~ group was obviously longer than that in the control, ESM, ESM/DA and ESM/HA groups, indicating that ESM/KR~600~ was conducive to re-epithelialization during the wound-healing process. On the other hand, the thickness of granulation tissue in both the ESM/KR~600~ and ESM/HA groups was dramatically greater than that in the control, ESM and ESM/DA groups ([Figure 8A](#F0008){ref-type="fig"} and [D](#F0008){ref-type="fig"}), suggesting that ESM/KR~600~ and ESM/HA could promote granulation tissue formation via the beneficial effects of HA coating. Additionally, [Figure 8B](#F0008){ref-type="fig"} and [E](#F0008){ref-type="fig"} reveals that, compared with other groups, treatment with ESM/KR~600~ obviously reduced the number of inflammatory cells infiltrated to the wound edge, suggesting that ESM/KR~600~ might attenuate the inflammation reaction, as observed in the macroscopic pictures of wounds.Figure 8Histological examination of cutaneous wound sections (n=5) at day 8 post-surgery. (**A, B**) Representative H&E staining images of wound sites in different groups. The yellow double arrows indicate the regenerated epidermises, the black double arrows indicate granulation tissues and the black single arrows indicate inflammatory cells. Quantitative determination of (**C**) the length of regenerated epidermis, (**D**) thickness of granulation tissue and (**E**) number of inflammatory cells in different groups. *\*P*\<0.05 and *\*\*P*\<0.01.**Abbreviations:** ESM, eggshell membrane; HA, hyaluronic acid. Thereafter, to explore the underlying mechanism of the observed phenomenon, PCNA, as a marker of cell proliferation, and CD31 and VEGF, as markers of angiogenesis, were detected using immunohistochemistry.[@CIT0003],[@CIT0053] [Figure 9A](#F0009){ref-type="fig"} and [B](#F0009){ref-type="fig"} shows that the number of PCNA-positive keratinocytes per field in the ESM/KR~600~ group was significantly greater than that in other groups, indicating that the ESM/KR~600~ could promote keratinocyte proliferation in vivo and accelerate the re-epithelialization process, which was in line with the in vitro result ([Figure 5](#F0005){ref-type="fig"}) and previous studies that KR-12 peptide contributed to proliferation of epithelial cells.[@CIT0019],[@CIT0020] On the other hand, [Figure 9A](#F0009){ref-type="fig"}, [C](#F0009){ref-type="fig"} and [D](#F0009){ref-type="fig"} shows that, compared with the control, ESM and ESM/DA groups, ESM/KR~600~ dramatically increased both the density of CD31-positive blood vessels and VEGF expression. However, no obvious differences were found between the ESM/KR~600~ and ESM/HA groups. In previous studies, HA had been announced to promote angiogenesis both in vitro and in vivo, through increasing endothelial cell proliferation and decreasing cell apoptosis.[@CIT0029],[@CIT0030] Moreover, Mariarosaria et al reported that systemic administration of HA modulated angiogenesis in diabetic mice by enhancing the expression of angiogenesis-related cytokines such as TGF-β1.[@CIT0031] Combined with similar in vitro results ([Figure 4](#F0004){ref-type="fig"}), we speculated that ESM/KR~600~ might improve angiogenesis mainly via the effect of HA coating, further promoting granulation tissue formation in vivo.Figure 9Immunohistochemical analysis of cutaneous wound sections (n=5) at day 8 post-surgery. (**A**) Representative images of immunohistochemical staining of PCNA, CD31 and VEGF. The black single arrows indicate PCNA-positive keratinocytes and CD-31-positive blood vessels. Quantitative counts of (**B**) PCNA-positive keratinocytes per field, (**C**) CD-31-positive blood vessels per field and (**D**) the relative expression of VEGF. *\*P*\<0.05 and *\*\*P*\<0.01.**Abbreviations:** ESM, eggshell membrane; HA, hyaluronic acid. Taken together, ESM/KR~600~ was efficient to promote wound healing by accelerating angiogenesis and re-epithelialization in vivo, which could be attributed to the synergistic effects of KR-12 peptide and HA. Wound healing is a physiological process and is generally divided into three stages: inflammation, cell proliferation and tissue formation, and remodeling .[@CIT0056] These stages overlap and interact with each other in a complex way that ultimately affects the healing process. Bacterial infection could interfere with the normal healing process and lead to delayed wound closure.[@CIT0057],[@CIT0058] By conjugating with KR-12 peptide, ESM/KR~600~ possessed good antibacterial activity against both Gram-negative and Gram-positive bacteria, including multi-drug-resistant bacteria ([Figure 3](#F0003){ref-type="fig"}), which could effectively protect the wounds from bacterial invasion and cloning, maintaining a natural and non-septic microenvironment for new tissue formation.[@CIT0001] Moreover, KR-12 peptide conjugated on ESM/KR~600~ could promote keratinocyte proliferation in vivo ([Figure 9A](#F0009){ref-type="fig"} and [B](#F0009){ref-type="fig"}) to accelerate the re-epithelialization process and cause rapid wound healing ([Figures 7](#F0007){ref-type="fig"} and [8A](#F0008){ref-type="fig"}). On the other hand, by coating with HA, ESM/KR~600~ could promote sufficient angiogenesis in the wound area ([Figure 9A](#F0009){ref-type="fig"}, [C](#F0009){ref-type="fig"} and [D](#F0009){ref-type="fig"}), increasing the formation of granulation tissue ([Figure 8A](#F0008){ref-type="fig"} and [D](#F0008){ref-type="fig"}). The newly generated granulation tissue could provide support for epithelial growth, further accelerating the re-epithelialization process and promoting wound closure.[@CIT0054] Collectively, ESM/KR~600~ could effectively promote wound healing in vivo and potentially be an ideal wound dressing. Conclusion {#S0004} ========== A biocompatible composite membrane composed of a polydopamine-modified ESM coated with HA and KR-12 peptide was successfully developed. The composite membrane showed excellent antibacterial activity against *E. coli, S. aureus* and multi-drug-resistant MRSA bacteria. More importantly, it could prevent the formation of a MRSA biofilm on the surface. Additionally, the composite membrane promoted the proliferation of keratinocytes and HUVECs. In vivo animal testing showed that the composite membrane accelerated re-epithelialization via promoting keratinocyte proliferation and enhanced granulation tissue formation via promoting angiogenesis, eventually leading to rapid wound healing. Therefore, the prepared composite membrane is a promising dressing for wound treatment. This work was supported by the National Special Scientific Projects of Public Welfare Industry Funding of China (No. 201502015) and State Key Laboratory Funding (SKLZZ201221). Disclosure {#S0005} ========== The authors report no conflicts of interest in this work.  [^1]: **Abbreviations:** ESM,eggshell membrane;HA,hyaluronic acid.

###### Key questions What is already known about this subject? ========================================= - Resting heart rate (HR) is an independent predictor of cardiovascular and all-cause mortality in men and women with and without diagnosed cardiovascular disease. What does this study add? ========================= - Most previous studies of HR have utilised resting HR. Three previous studies which analysed mean 24 h HR, as in our study, recruited individuals much older than those in our study. Our findings suggest that HR may be a potential marker of elevated cardiovascular risk in asymptomatic individuals, prior to the development of clinical hypertension or cardiovascular disease. How might this impact on clinical practice? =========================================== - Although epidemiological studies have consistently reported that resting HR is an independent predictor of cardiovascular and all-cause mortality, the importance of resting HR as a prognostic factor and potential therapeutic target is not yet generally accepted. Clinical strategies incorporating HR in risk stratification may have possible future utility in identifying asymptomatic individuals to target with individualised preventive therapy. Introduction {#s1} ============ Heart rate (HR) is a key determinant of coronary blood flow, an established marker of sympathetic nervous system (SNS) activity, and an independent predictor of cardiovascular morbidity and mortality in various adult patient populations with and without cardiovascular disease.[@R1] Increased SNS activity increases blood pressure (BP) and contributes to the development and maintenance of hypertension.[@R2] Multiple studies have demonstrated a correlation between increased SNS activity and higher BP elevation in patients with essential hypertension; this association is not present in secondary hypertension.[@R3] Furthermore, elevated HR is present in a significant proportion of adult borderline hypertensives, beginning in childhood.[@R4] These observations suggest that sympathetic activation is a specific feature of essential hypertension and that heightened SNS activity may play a pathogenetic role in hypertension pathophysiology and prognosis.[@R5] It has been hypothesised that increased SNS activity may contribute to the pathogenesis of elevated BP via direct vasoconstrictive effects on blood vessels, via stimulation of release of adipokines and/or via SNS-mediated insulin resistance.[@R6] Up to 70% of newly diagnosed hypertensive cases are attributable to obesity[@R7]; increased kidney and muscle sympathetic nerve activity (SNA) are found in obesity.[@R8] Muscle SNA levels decline with weight loss and increase with weight gain.[@R9] Compared with subcutaneous or lower body fat, visceral abdominal fat, which is the primary source of C reactive protein (CRP) and other proinflammatory adipokines, is more closely associated with increased resting HR and higher SNA in humans.[@R10] Elevated HR has deleterious cardiovascular consequences in addition to elevation of BP. High resting HR may potentially accelerate atherosclerosis via its negative effects on the endothelium.[@R11] There is a close relationship between shear stress and HR[@R1]; accelerated HR may promote endothelial shear stress. To further investigate the potential role of HR in the early pathophysiology of atherosclerosis, we analysed data from a cohort of non-hypertensive young adults. The primary study objective was to evaluate the direct link between ambulatory 24 h HR and ambulatory 24 h BP ([figure 1](#OPENHRT2015000332F1){ref-type="fig"}, pathway \[1\]), accounting for other potential risk factors that may also independently impact BP, including endothelial dysfunction, insulin resistance, inflammatory markers and measures of adiposity ([figure 1](#OPENHRT2015000332F1){ref-type="fig"}, pathways \[2\]--\[5\]). The secondary study objective was to assess potential determinants of HR ([figure 1](#OPENHRT2015000332F1){ref-type="fig"}, pathways \[6\]--\[8\]). {#OPENHRT2015000332F1} Research methods and procedures {#s2} =============================== Participants/study design {#s2a} ------------------------- Data were collected using an observational cross-sectional study design from a cohort of volunteers enrolled in an ongoing investigation of the role of adipokines and insulin resistance in the pathogenesis of hypertension. Participants were recruited from a large urban academic Family Medicine outpatient practice, serving 40 000 individuals in Philadelphia, Pennsylvania, USA. Inclusion criteria were age between 18 and 45 years and absence of hypertension: office systolic BP (SBP) \<140 mm Hg and diastolic BP (DBP) \<90 mm Hg. Exclusion criteria were diabetes, pregnancy, hypertension, coronary or cerebrovascular disease, collagen vascular disease, organ failure (heart, kidney, liver), and statin medication use. The study was approved by the Thomas Jefferson University Institutional Review Board, and written informed consent was obtained from all participants. Measurements and data collection {#s2b} -------------------------------- Information was collected on sociodemographic characteristics and current health behaviours. Height and weight were measured and body mass index (BMI) was calculated as weight divided by height squared (kg/m^2^). ### BP and HR {#s2b1} Resting SBP and DBP and HR were measured at the office with a Dinamap cuff. In addition, the participants were also provided with an oscillometric device (Spacelabs 90217A, Spacelabs) for 24 h ambulatory BP monitoring (ABPM), with readings set at 20 min intervals while awake, and 60 min intervals while asleep. Mean 24 h SBP, DBP and HR were calculated, as well as the means of these measures during the awake and asleep periods. SBP, DBP and HR variability were also computed, as the within-participant SD of the ambulatory measurements. ### Endothelial function {#s2b2} Minimal forearm vascular resistance (MFVR) at maximal vasodilation is considered an integrated measure of vascular resistance in the forearm.[@R12] We assessed forearm vascular resistance and forearm blood flow non-invasively using plethysmography according to the method of Sivertsson.[@R13] First, we measured baseline forearm blood flow (BFBF) at rest and maximum forearm blood flow (MFBF) during postocclusive reactive hyperaemia. We then calculated both baseline forearm vascular resistance (BFVR=baseline mean arterial pressure (MAP) divided by BFBF) and minimum forearm vascular resistance (MFVR=postocclusive MAP divided by MFBF). The main measures used in the analyses were the per cent increase in FBF and the per cent decrease in FVR (computed as (MFBF**−**BFBF)/BFBF×100 and (BFVR**−**MFVR)/BFVR×100, respectively). Several groups have assessed the reproducibility of venous occlusion plethysmography. Roberts et al (1986) demonstrated good within-participant reproducibility for unilateral blood flow measurement, used in this study, with a coefficient of variation of ∼10%. ### Insulin resistance {#s2b3} Fasting plasma samples were obtained following an overnight fast and analysed using a Glucostat analyser (YSI, Model 27). Insulin was assayed using a solid phase radioimmunoassay, 'Coat-A-Count' (Diagnostic Products Corporation). We computed two common indices of insulin sensitivity. The homoeostatic model assessment (HOMA) index was computed as the product of fasting glucose (mM/L) and fasting insulin (uU/mL), divided by 22.5. The quantitative insulin sensitivity check index (QUICKI) was computed as the reciprocal of the sum of the log fasting glucose (mg/dL) and the log fasting insulin (uU/mL). We used QUICKI in the main analyses, as it tends to have a more symmetric (normal) distribution than the HOMA and does not require logarithmic transformation.[@R14] ### Inflammatory markers {#s2b4} We measured five inflammatory markers: plasminogen activator inhibitor-1 (PAI-1), interleukin 6 (IL-6), tumour necrosis factor α (TNFα), adiponectin and high-sensitive CRP. Assays were performed using commercially available ELISA kits, which have coefficients of variation 4--6%. Statistical analyses {#s2c} -------------------- The main study objective was to assess the association of HR and HR variability with BP ([figure 1](#OPENHRT2015000332F1){ref-type="fig"}, pathway \[1\]), controlling for other risk factors, mainly endothelial function and insulin resistance ([figure 1](#OPENHRT2015000332F1){ref-type="fig"}, pathways \[2\] and \[3\]). The main analyses were based on multiple linear regression, with mean 24 h BP (SBP or DBP) as the outcomes, mean 24 h HR or HR variability as the main predictor of interest, and controlling for age, gender, race, endothelial function and insulin sensitivity/resistance. Although endothelial dysfunction and insulin resistance may develop as a consequence of hypertension, they are also possible contributors to the pathogenesis of hypertension. Therefore, we controlled for them in order to isolate the independent association between HR and BP. Additional analyses also controlled for inflammatory markers and measures of adiposity ([figure 1](#OPENHRT2015000332F1){ref-type="fig"}, pathways \[4\] and \[5\]). We also carried out similar analyses with office measurements of BP and HR, instead of the 24 h ambulatory averages. The secondary study objective was to evaluate the extent to which HR is impacted by other atherosclerotic risk factors, specifically measures of adiposity, inflammation and insulin resistance ([figure 1](#OPENHRT2015000332F1){ref-type="fig"}, pathways \[6\] through \[8\]). The main analyses were again based on multiple linear regression, with mean 24 h HR as the outcome, and measures of adiposity, inflammatory markers and insulin sensitivity/resistance as the predictors of interest. Because of their skewed distributions, IL-6, adiponectin, CRP and FVR per cent decrease were log-transformed in all analyses. Statistical analyses were conducted in SAS V.9.4 (SAS Institute; Cary, North Carolina, USA). The study was powered at 80% or higher for detecting outcomes as noted for these analyses. Missing data were not imputed. Results {#s3} ======= The analyses were based on data from 186 participants. Ambulatory BP and HR measurements were based on an average of 42 readings per participant for the awake period and 8 readings per participant for the asleep period (SD=9 and 2, respectively). [Table 1](#OPENHRT2015000332TB1){ref-type="table"} summarises the characteristics of the study participants. No participant was hypertensive, while 44 (24%) were prehypertensive by either their office BP (ie, office SBP 120--139 mm Hg or DBP 80--89 mm Hg) or their 24 h BP (ie, 24 h SBP 130--134 mm Hg or 24 h DBP 80--84 mm Hg). These included 8 (4%) classified as prehypertensive by both office and 24 h BP, 18 (10%) with white coat prehypertension (ie, office but not 24 h) and 18 (10%) with masked prehypertension (ie, 24 h but not office). Finally, 66 participants (35%) were 'SBP non-dippers', that is, their ambulatory mean SBP had dropped less than 10% from their awake to their sleep period. ###### Study participant characteristics (N=186) --------------------------------------------------------- ------------- Age (years), mean (SD) 27 (6) Gender, n (%)  Female 115 (62)  Male 71 (38) Race, n (%)  Caucasian 98 (53)  Black/African-American 32 (17)  Hispanic/Latino 10 (5)  Asian 46 (25) BMI, kg/m^2^,\* mean (SD) 25 (5) Waist circumference (cm),† mean (SD) 83 (12) Sleep quality score (PSQI),‡ mean (SD) 4.8 (3.0) CRP (mg/L), geometric mean (IQR) 1.1 (2.0) PAI-1 (ng/mL), mean (SD) 43 (25) TNFα (pg/mL), mean (SD) 9.3 (2.8) IL-6 (pg/mL), geometric mean (IQR) 1.8 (1.6) Adiponectin (ug/mL), geometric mean (IQR) 8.8 (8.3) HOMA, geometric mean (IQR) 1.2 (0.7) QUICKI, mean (SD) 0.38 (0.03) MFBF (mL/100 mL tissue/min), mean (SD) 27 (9) FBF increase (%), mean (SD) 782 (311) MFVR (mm Hg/mL/100 mL tissue/min), geometric mean (IQR) 3.0 (1.3) FVR decrease (%), geometric mean (IQR) 86 (7) HR 24 h (bpm), mean (SD) 75 (10)  HR awake (bpm), mean (SD) 77 (11)  HR asleep (bpm), mean (SD) 64 (11) HR office (bpm), mean (SD) 69 (11) HR 24 h variability (bpm), mean (SD) 12 (4) SBP 24 h (mm Hg), mean (SD) 117 (9)  SBP awake (bpm), mean (SD) 119 (9)  SBP asleep (bpm), mean (SD) 105 (10) SBP office (mm Hg), mean (SD) 108 (10) DBP 24 h (mm Hg), mean (SD) 72 (6)  DBP awake (bpm), mean (SD) 74 (6)  DBP asleep (bpm), mean (SD) 60 (6) DBP office (mm Hg), mean (SD) 65 (8) Prehypertensive (office or 24 h BP), n (%) 44 (24)  White coat prehypertension (office only), n (%) 18 (10)  Masked prehypertension (24 h only), n (%) 18 (10) 24 h SBP non-dipper, n (%) 66 (35) --------------------------------------------------------- ------------- \*Data available for 175 participants. †Data available for 171 participants. ‡Data available for 117 participants. BMI, body mass index; CRP, C reactive protein; DBP, diastolic blood pressure; HOMA, homoeostatic model assessment; HR, heart rate; IL-6, interleukin-6; MFBF, maximum forearm blood flow; MFVR, minimum forearm vascular resistance; PAI-1, plasminogen activator inhibitor-1; PSQI, Pittsburgh sleep quality index; QUICKI, quantitative insulin sensitivity check index; SBP, systolic blood pressure; TNF, tomour necrosis factor. [Table 2](#OPENHRT2015000332TB2){ref-type="table"} presents the correlations of mean 24 h BP and the various risk factors, including mean 24 h HR and HR variability. HR was a modest predictor of DBP but not SBP. By contrast, endothelial dysfunction, measured as the FBF per cent increase or the FVR per cent decrease, was significantly associated with SBP but not DBP. Insulin sensitivity (QUICKI) was significantly and inversely related with both SBP and DBP. All these unadjusted results were qualitatively similar when office measurements were substituted for mean 24 h values (not shown), as office and mean 24 h measures were highly correlated (correlation=0.67 for SBP, 0.53 for DBP, 0.74 for HR; all p\<0.001). ###### Correlations (p values) of HR and other atherosclerotic risk factors with SBP and DBP (N=186) SBP 24 h DBP 24 h --------------------- --------------- ---------------- HR 24 h 0.04 (0.633) 0.33 (0.001) HR 24 h variability −0.09 (0.236) −0.02 (0.830) FBF increase −0.24 (0.001) −0.09 (0.247) FVR decrease\* −0.22 (0.002) −0.08 (0.260) QUICKI −0.16 (0.025) −0.24 (0.001) CRP\* 0.08 (0.289) 0.13 (0.075) PAI-1 0.22 (0.003) 0.23 (0.002) TNFα −0.05 (0.473) 0.01 (0.879) IL-6\* 0.05 (0.463) 0.07 (0.357) Adiponectin\* −0.17 (0.018) −0.05 (−0.473) BMI 0.23 (0.002) 0.08 (0.319) WC 0.34 (0.001) 0.19 (0.013) \*Log-transformed. BMI, body mass index; CRP, C reactive protein; DBP, diastolic blood pressure; FBF, forearm blood flow; FVR, forearm vascular resistance; HR, heart rate; IL-6, interleukin-6; PAI-1, plasminogen activator inhibitor-1; QUICKI, quantitative insulin sensitivity check index; SBP, systolic blood pressure; TNF, tomour necrosis factor; WC, waist circumference. [Table 3](#OPENHRT2015000332TB3){ref-type="table"} presents the main results regarding the association of 24 h HR with 24 h SBP and DBP ([figure 1](#OPENHRT2015000332F1){ref-type="fig"}, pathway \[2\]), controlling for age, gender, race, endothelial function (FBF % increase from baseline to maximum during postocclusive reactive hyperaemia, [figure 1](#OPENHRT2015000332F1){ref-type="fig"}, pathway \[2\]), and insulin sensitivity (QUICKI, [figure 1](#OPENHRT2015000332F1){ref-type="fig"}, pathway \[3\]). An increment of 10 bpm in the 24 h HR (about 1 SD) was associated with higher 24 h SBP and DBP, by and average of 1.3 mm Hg (p=0.042) and 1.7 mm Hg (p=0.001), respectively. FBF per cent increase was significantly associated with SBP but not DBP (p=0.013 and 0.499, respectively), while QUICKI was not associated with either SBP or DBP (p=0.218 and 0.283, respectively). ###### Multivariable results for HR and other atherosclerotic risk factors as determinants of SBP and DBP (N=186) Increment SBP 24 h DBP 24 h -------------- -------------------- ---------- ---------------- ------- ------ --------------- ------- HR 24 h 10 bpm 1.3 (0.0 to 2.6) 0.042 1.7 (0.8 to 2.5) 0.001 FBF increase 300% −1.4 (−2.5 to −0.3) 0.013 −0.3 (−1.0 to 0.5) 0.499 QUICKI 0.03 −0.8 (−2.1 to 0.5) 0.218 −0.5 (−1.3 to 0.4) 0.283 Age 10 years 2.2 (0.2 to 4.1) 0.033 2.8 (1.5 to 4.1) 0.001 Gender M vs F 9.4 (6.9 to 11.9) 0.001 1.3 (−0.4 to 3.0) 0.123 Race Black vs non-black 0.3 (−3.4 to 4.0) 0.873 −0.4 (−2.9 to 2.0) 0.719 Δ, Mean difference of SBP or DBP (in mm Hg) corresponding to the increment shown for each variable. DBP, diastolic blood pressure; F, female; FBF, forearm blood flow; HR, heart rate; M, male; QUICKI, quantitative insulin sensitivity check index; SBP, systolic blood pressure. In additional analyses that adjusted for inflammatory markers and BMI ([figure 1](#OPENHRT2015000332F1){ref-type="fig"}, pathways \[4\] and \[5\]), the effect of HR on SBP was somewhat attenuated (mean SBP difference=0.6 mm Hg per 10 bpm in HR, p=0.422), but that on DBP remained unchanged (mean DBP difference=1.7 mm Hg per 10 bpm in HR, p=0.001). Furthermore, PAI-1 was also a significant predictor of both SBP (mean difference=1.4 mm Hg per 25 mg/mL PAI-1, p=0.041) and DBP (mean difference=1.1 mm Hg per 25 mg/mL PAI-1, p=0.015). Findings were very similar when waist circumference (WC) was substituted for BMI (results not shown). Awake and asleep HR were both associated with 24 h SBP (p=0.040 and 0.068, respectively) and 24 h DBP (p=0.001 and 0.062). Not surprisingly, the awake HR mean was more strongly associated with the awake SBP and DBP, while the asleep HR mean was more strongly associated with the asleep SBP and DBP. Finally, we did not find any significant association between 24 h HR variability and BP (p=0.443 for SBP and 0.561 for DBP). The main analyses were also repeated using office measurements of HR and BP instead of their 24 h ambulatory averages. The associations were generally similar as those seen with ambulatory measurements, although their strength was attenuated by about one-third to one-half. As a consequence, neither HR nor PAI-1 was significantly associated with BP, although FBF per cent increase was a significant predictor of office SBP (results not shown). [Table 4](#OPENHRT2015000332TB4){ref-type="table"} presents the results regarding the association of BMI, inflammatory markers and insulin sensitivity/resistance with 24 h HR ([figure 1](#OPENHRT2015000332F1){ref-type="fig"}, pathways \[6\]--\[8\]). QUICKI showed a very strong inverse association with HR (p=0.013). Higher CRP was significantly associated with higher HR (p=0.007), while similar associations of PAI-1 and IL-6 with HR were marginally significant (p=0.093 and 0.077, respectively). Unexpectedly, we also found a significant inverse association between TNFα and HR (p=0.015). Neither BMI nor WC (results not shown) was significantly associated with HR. ###### Atherosclerotic risk factors as determinants of HR (N=175) Increment HR 24 h --------------- -------------------- --------- ---------------- ------- Age 10 years −0.6 (−2.8 to 1.7) 0.617 Gender M vs F −6.2 (−9.0 to −3.4) 0.001 Race Black vs non-black 2.7 (−1.7 to 7.1) 0.234 BMI 5 kg/m^2^ 0.4 (−1.3 to 2.2) 0.627 CRP\* Doubling 1.3 (0.4 to 2.3) 0.007 PAI-1 25 ng/Ml 1.2 (−0.2 to 2.7) 0.093 TNFα 3 pg/mL −1.7 (−3.0 to −0.3) 0.015 IL-6\* Doubling 1.1 (−0.1 to 2.4) 0.077 Adiponectin\* Doubling 1.0 (−0.7 to 2.7) 0.270 QUICKI 0.03 −2.8 (−4.4 to −1.1) 0.001 Δ, Mean difference of HR (in bpm) corresponding to the increment shown for each variable. \*Log-transformed. BMI, body mass index; CRP, C reactive protein; F, female; HR, heart rate; IL-6, interleukin-6; M, male; PAI-1, plasminogen activator inhibitor-1; QUICKI, quantitative insulin sensitivity check index; TNF, tumour necrosis factor. Discussion {#s4} ========== Mean 24 h HR was independently associated with mean 24 h SBP and DBP, CRP, and QUICKI. The findings from this study of young non-hypertensive individuals suggest that 24 h HR is associated with known risk factors for atherosclerosis prior to the development of diabetes or sustained hypertension. The magnitude of these associations was modest but still potentially clinically meaningful, particularly given that these young individuals did not exhibit any clinical manifestations of atherosclerosis or cardiovascular disease. Although this study specifically excluded hypertensive individuals, a sizable proportion of participants (35%) were SBP non-dippers, and masked prehypertensives (8%), possibly indicating the presence of early stage cardiovascular pathology already underway. In contrast to HR, HR variability was not significantly associated with BP. Previous studies of HR variability demonstrating an association with BP[@R15] and adipokines[@R16] were conducted in older individuals, suggesting that HR may be an earlier, more sensitive marker of autonomic dysfunction than HR variability. Alternatively, HR variability is typically determined from 24 h ECG recordings. However, HR variability has also previously been calculated as the mean of the SD of HR from ABPM reports as in our study.[@R15] We chose this methodology as we do not have 24 h ECG monitoring equipment. ABPM-calculated HR variability was found in the cited reference to be a significant predictor of carotid intima-media thickness in older hypertensive patients. Our lack of statistically significant findings for HR variability in this study could either be because this alternate methodology may be a less sensitive method for detecting HR variability compared with 24 h ECG monitoring, or due to the inclusion of younger, healthier patients in this study in whom HR variability may not be pathologically altered. Study findings may have been additionally limited by the moderate sample size of 186 participants. Many inter-related factors contribute to raised BP. Among the factors that have been intensively studied are salt intake, obesity and insulin resistance, the renin-angiotensin system, the SNS, genetics, endothelial dysfunction, smoking, alcohol intake, and exercise. We have not included all possible determinants of BP in order to limit the complexity of our analyses in this study; we chose to focus on the variables noted in [figure 1](#OPENHRT2015000332F1){ref-type="fig"}: obesity (BMI), insulin resistance, SNS and endothelial dysfunction. This study presents evidence of an association between elevated SNA, as represented by elevated HR, and atherosclerotic risk factors which have also been implicated in the pathogenesis of elevated BP: endothelial dysfunction, measures of adiposity, insulin resistance and inflammatory cytokines.[@R17] Endothelial dysfunction is central to the initiation and progression of atherosclerosis, since vascular endothelial cells secrete several antiatherogenic substances including the potent vasodilator nitric oxide.[@R18] Overall, research to date suggests that visceral adiposity is a stronger predictor of endothelial activation than overall adiposity. Insulin resistance is associated with systemic coronary artery disease risk factors, including adiposity; increasing evidence suggests that defective insulin signalling in atherosclerotic lesional cells also contributes to disease progression.[@R19] Elevated levels of inflammatory adipokines promote endothelial dysfunction, hypertension and atherosclerosis.[@R17] Both CRP and IL-6 are molecular markers associated with atherosclerosis and its progression, with IL-6 showing more consistent results and stronger independent predictive value than other inflammatory markers.[@R20] Conclusive in vitro and in vivo findings clearly indicate a proatherogenic role of IL-6.[@R21] IL-6 signalling has been shown to contribute to both atherosclerotic plaque development and plaque destabilisation, while elevated PAI-1 levels appear to increase the risk of atherothrombotic events and may also promote the progression of vascular disease.[@R22] We found that measures of adiposity (BMI or WC) were not significantly associated with HR24 ([figure 1](#OPENHRT2015000332F1){ref-type="fig"}, pathway \[6\]), when insulin resistance and inflammatory markers were controlled for. QUICKI ([figure 1](#OPENHRT2015000332F1){ref-type="fig"}, pathway \[8\]), as well as CRP and to some extent PAI-1 ([figure 1](#OPENHRT2015000332F1){ref-type="fig"}, pathway \[7\]) were independently associated with SNS activity. This suggests that the latter factors may be intermediaries in the main causal pathway linking adiposity to SNS activity. However, we also found an unexpected protective effect of TNFα on HR24 ([figure 1](#OPENHRT2015000332F1){ref-type="fig"}, pathway \[7\]). This could be explained by the fact that plasma TNFα values are usually low. Measurement of TNFα receptors, a better marker of TNFα activity, was not part of the study protocol. Despite the evidence presented in these and other studies, the exact mechanism by which the SNS mediates its deleterious effects on BP remains unknown. Possible additional mechanisms that we did not explore in this study include activation of the renin-angiotensin-aldosterone system, impaired baroreceptor reflexes, sleep apnoea-activated chemoreceptor reflexes and adipose tissue-derived leptin-mediated SNS activation.[@R6] Also, although our analyses took into account various links between many known or suspected risk factors of atherosclerosis, our data were cross-sectional and cannot establish temporal relationships or causal pathways. Although multiple epidemiological studies have consistently reported that resting HR is an independent predictor of cardiovascular and all-cause mortality in men and women with and without diagnosed cardiovascular disease, the importance of resting HR as a prognostic factor and potential therapeutic target is not yet generally accepted. Resting HR has been previously found to be a predictor of all-cause mortality in older (aged 45--64 years) prehypertensive older patients.[@R23] HR has also been associated with CRP in elderly men and women (aged 55--75 years),[@R24] middle-aged Israeli men (mean age of about 45 years),[@R25] and with both insulin resistance and CRP in young Japanese men (aged 18--26 years).[@R26] For this study, we specifically recruited young non-hypertensive individuals (including normotensives and prehypertensives) as we aimed to study individuals who have not yet progressed to clinical disease. An additional strength of our study is its diverse population, with substantial fractions of both African-Americans and Asians. African-Americans have not been well studied regarding ambulatory monitoring, although hypertension occurs more commonly and is associated with greater morbidity and mortality in African-Americans compared with Caucasians.[@R27] Most previous studies of HR have utilised resting HR. HR recorded over 24 h has previously been reported to have a better reproducibility (correlation 0.73, with a mean change of only 0.7 bpm over a 3-month interval in 839 hypertensive patients) than office HR (Palatini *et al*, 2000). Twenty-four-hour HR could therefore be a better prognostic indicator than traditional measurement of resting HR. Three previous studies analysed mean 24 h HR in individuals much older than those in our study, one with patients with a mean age of 52,[@R28] and the other among patients aged 55--75 years.[@R29] The large ABP-International study of 7600 Caucasians and Asians reported independent predictive value for night-time HR, but not daytime or 24 h HR, for cardiovascular, but not all-cause mortality.[@R28] Similar findings were reported in a study of 653 elderly Europeans.[@R29] However, ambulatory HR did not add additional prognostic information over office HR in a third study of nearly 5000 elderly hypertensive Europeans.[@R30] In our analyses, associations were markedly stronger for 24 h measurements than for office (resting) measurements, suggesting that the 24 h measures may be more sensitive than their office (resting) counterparts in young non-hypertensive adults. Conclusions {#s5} =========== In conclusion, HR, an established indicator of SNS activity, was associated with elevated BP, CRP and insulin resistance in non-hypertensive individuals. This finding suggests that HR may be a potential marker of elevated cardiovascular risk in asymptomatic individuals, prior to the development of clinical hypertension or cardiovascular disease. Furthermore, our findings suggest that the link of adiposity with HR may be mediated by inflammatory markers and insulin resistance. Prospective longitudinal studies are needed to further elucidate the causal pathways that link adiposity, inflammation, insulin resistance, endothelial function and HR, and the degree to which HR measured in early life can predict the incidence of atherosclerosis and cardiovascular disease. Clinical strategies incorporating HR in risk stratification may have possible future utility in identifying asymptomatic individuals to target with individualised preventive therapy. **Contributors:** Both CC and CD contributed to the work by meeting all four of the following authorship criteria: substantial contributions to the conception or design of the work; also, the acquisition, analysis or interpretation of data for the work; drafting the work or revising it critically for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. **Funding:** Funding for this study was provided by NIH grant HL096593. **Competing interests:** None declared. **Ethics approval:** Thomas Jefferson University Institutional Review Board. **Provenance and peer review:** Not commissioned; externally peer reviewed. **Data sharing statement:** Extra data is available by emailing CC at Cynthia.Cheng\@jefferson.edu.

It is accepted standard that patients undergoing hip joint replacement should receive prophylactic antibiotics to prevent postoperative infections. The type and dose of antibiotics varies between countries, and in Denmark even between hospitals. Cefuroxime has traditionally been the first choice. When cefuroxime and other broad-spectrum antibiotics became associated with Clostridium difficile-associated disease (CDAD) (Baxter et al. [@C2]), guidelines were changed and gentamicin with dicloxacillin was introduced as the standard prophylaxis in many Danish hospitals. However, this new prophylactic regime was suspected of giving a high risk of kidney injury due to the known nephrotoxicity of gentamicin (Quiros et al. [@C10], Wargo and Edwards [@C11]). It is unknown whether exclusion of gentamicin from the antibiotic regime causes noticeable changes in the severity and number of cases of acute kidney injury (AKI). In our department, we noticed a dramatic rise in the number of elective total hip arthroplasty (THA) patients who showed clinical and biochemical symptoms of kidney damage in the years 2010--2013. This rise in cases coincided with the introduction of dicloxacillin/gentamicin prophylaxis. Gentamicin was suspected of being the main cause of this rise, and it was therefore removed from our antibiotic protocol. We investigated whether dicloxacillin alone was associated with fewer and less severe cases of AKI after elective THA compared to dicloxacillin and gentamicin together. Methods {#s0002} ======= We performed a retrospective study involving 136 patients who underwent elective cemented (non- antibiotic-loaded cement) or uncemented total hip replacement. All the patients were treated according to current local guidelines. A list of all patients who underwent THA surgery during that period was retrieved. Using the medical records, we documented the dosage and type of antibiotics used and collected numerical data on pre- and postoperative creatinine levels during the hospital stay. The preoperative creatinine level was measured less than 2 months before the surgery in all cases. The postoperative creatinine level was measured for the first time during the first or second day after surgery in all patients. Acute kidney injury was graded using the classification developed by KDIGO ([@C7]), in which AKI is graded in 3 stages ([Table 1](#TB1){ref-type="table"}). ###### KDIGO classification of acute kidney injury (AKI) AKI staging Serum creatinine ------------- ---------------------------------------------------------------------------------------------------------------------------------- Stage 1 Increase to 150--200% from baseline or increase of ≥26.5 μmol/L (0.3 mg/dL). Stage 2 Increase to 200--300% from baseline. Stage 3 Increase to more than 300% from baseline or increase of ≥354 μmol/L (4.0 mg/dL) with an acute increase of ≥44 μmol/L (0.5 mg/dL) Statistics {#s0003} ---------- The patients were divided into 2 groups, with or without gentamicin, and renal injury status was classified according to KDIGO. The Wilcoxon-White rank-sum test was performed for differences between the 2 study groups, as this was a retrospective study. For differences in treatment regimen, 2-tailed chi-square analysis for significance was performed. An exact 2-tailed Wilcoxon-White rank-sum test was performed for differences in severity between the 2 treatment regimens. A regression analysis was done for relative risk of developing AKI, with the variables sex and age per decade. The level of significance was set at p \< 0.05. Statistical analysis was performed using STATA software version 13.1. Results {#s0004} ======= We included 136 patients who underwent elective total hip arthroplasty during the period 2010--2014: 57 male patients with a median age of 69 (45--88) years and 79 female patients with a median age of 71 (42--88) years. Sex distribution and preoperative creatinine levels were similar between the 2 groups ([Table 2](#TB2){ref-type="table"}). ###### Characteristics of the 2 groups Dicloxacillin/gentamicin Dicloxacillin p-value ------------------------------------------------------- -------------------------- --------------- --------- No. of patients 66 70 -- Sex (men/women) 29/37 28/42 0.6 Mean age, men (SD) 69 (2.2) 70 (1.7) 0.8 Mean age, women (SD) 72 (1.6) 71 (1.7) 0.6 Mean dicloxacillin dose (SD), g 4.92 (1.13) 4.49 (0.85) 0.01 Mean gentamicin dose (SD), mg 174 (58) -- -- Creatinine level[^a^](#TF1){ref-type="table-fn"} (SD)  mean preoperative 79 (27) 77 (21) 0.6  maximum 126 (83) 93 (46) 0.04 μmol/L The first group (74 patients) received dicloxacillin and gentamicin; they were operated on during the period October 10, 2010 to August 31, 2011. 8 patients were excluded because they had received other types of antibiotics or because we lacked information on pre- or postoperative creatinine levels. The 66 patients who were included received prophylaxis consisting of 2--9 g intravenous dicloxacillin and 120--240 mg intravenous gentamicin. The mean dicloxacillin dose was 4,924 mg and the mean gentamicin dose was 174 mg. The second group (79 patients) received dicloxacillin alone; they were operated on during the period August 7, 2014 to October 20, 2014. 9 patients were excluded because they had received other types of antibiotics. The 70 patients who were included received 1--6 g of dicloxacillin intravenously. The mean dicloxacillin dose was 4,485 mg. This group therefore received slightly lower amounts of dicloxacillin than the other group ([Table 2](#TB2){ref-type="table"}). The local guidelines recommend 2 g perioperatively and 1 g × 3 during the first 24 hours postoperatively. The dicloxacillin/gentamicin group had a mean preoperative serum creatinine level of 79 (36--227) μmol/L. The maximum serum creatinine level was 126 (25--422) μmol/L. There were 48 patients with no AKI, 3 with AKI of stage 1, 7 with stage 2, and 8 with stage 3. The dicloxacillin-only group had a mean preoperative serum creatinine level of 77 (38--278) μmol/L and a maximum serum creatinine level of 93 (39--278) μmol/L. There were 61 patients with no AKI, 5 with AKI of stage 1, 3 with stage 2 and 1 with stage 3 ([Table 3](#TB3){ref-type="table"}). In the dicloxacillin group, the creatinine level of 1 of the patients who developed AKI (stage 1) had returned to normal when measured 2 days later. The rest of the patients who developed AKI had elevated serum creatinine levels for more than 24 hours. ###### No. of patients who developed AKI AKI staging Dicloxacillin/gentamicin Dicloxacillin ----------------------- -------------------------- --------------- Stage 1 3 5 Stage 2 7 3 Stage 3 8 1 No AKI 48 61 Total no. of patients 66 70 Overall, the dicloxacillin-only group had less severe cases of AKI (p = 0.02). The overall odds ratio (OR) for developing AKI was 3.2 in the dicloxacillin/gentamicin group (95% Cl: 1.2--8.5; p = 0.02). Male sex had a lower OR of 0.49 (95% CI: 0.19--1.23; p = 0.01). Age group per decade showed no statistically significant additional risk. Discussion {#s0005} ========== We confirmed that prophylaxis with dicloxacillin alone was associated with fewer cases of AKI and also with less severe cases of AKI. The risk of developing AKI was 3 times higher in the dicloxacillin/gentamicin group. According to the Danish Hip Arthroplasty Register, the current rate of THA for arthrosis is 160 per 100,000 inhabitants. This number can be expected to grow in the future as a result of changing demographics. Acute kidney injury is a known but not extensively studied complication of hip replacement surgery (Pulido et al. [@C9]). Perregaard et al. ([@C8]) found that 2.2% of elective hip replacement patients developed AKI postoperatively. There have been a number of studies showing that antibiotic prophylaxis consisting of gentamicin and flucloxacillin is associated with a higher rate of AKI than prophylaxis with cephlosporins (Craxford et al. [@C5], Bailey et al. [@C1], Bell et al. [@C3]). We performed a retrospective study in an effort to investigate the frequency of AKI after removing gentamicin from our antibiotic prophylactic regime of dicloxacillin and gentamincin. With a fair number of patients, we were able to confirm our clinical hypothesis---that the incidence, severity, and relative risk of AKI was lower after we had removed gentamicin from the prophylactic regime. The group with the lower incidence of AKI also received slightly lower doses of dicloxacillin, which may have contributed to decreasing the risk of developing AKI, as shown by Challagundla et al. ([@C4]). The reason for using a lower dose of dicloxacillin in this group was probably that the orthopedics department became more aware of the problem with antibiotics and AKI. Previous studies have shown that hypovolemia, hypotension, and NSAIDs are among the risk factors for development of AKI (Kateros et al. [@C6], Craxford et al. [@C5]). We are aware that our study did not take account of information about other medications, perioperative bleeding, peri- and postoperative fluid administration, or comorbidity---including previous kidney disease. Another limitation of the study was the limited number of patients with previous chronic kidney disease. Only 8 patients in each group had elevated serum creatinine levels preoperatively. This is most probably a result of selection bias, as surgeons would be reluctant to refer patients with previous kidney disease for an operation. Patients with moderate-to-severe chronic kidney disease appear to have a higher risk of developing AKI (Perregaard et al. [@C8]). The patients were drawn from the same population and were operated by the same surgeons, and there was no difference in comorbidity index seen in the Danish Hip Arthroplasty Register, but despite this we cannot be certain that there were no significant differences between the groups regarding these factors. Our department has continued to use dicloxacillin alone as prophylaxis for THA patients, with special care not to exceed the recommended antibiotic regime. As before, we use non-antibiotic-loaded cement. All the postoperative infections have been registered according to Danish regulations. No rise in acute infections has been seen since the change in antibiotic regime. It is not yet possible to make any conclusions about late infections. In conclusion, we found fewer and less severe cases of acute renal injury after we excluded gentamicin from the THA prophylactic regime and became more cautious with the use of dicloxacillin. We recommend that other departments should investigate their prophylactic antibiotic regimens if a rise in acute kidney injury is seen. SJ: data collection, main writer. OMC: statistical analysis, co-writer. AHT: data collection, statistical analysis, co-writer. No competing interests declared.

High blood pressure is a leading risk factor for stroke and coronary heart disease and subsequent disability internationally.^[@R1]^ A 10 mm Hg reduction in blood pressure (BP) is estimated to lead to a 41% reduction in stroke and a 22% reduction in coronary heart disease.^[@R2]^ Lowering blood pressure with pharmacological and nonpharmacological methods is both clinically and cost-effective.^[@R3]^ Ensuring that the correct people receive such treatment maximizes benefit whilst reducing adverse effects. The 2011 National Institute for Health and Care Excellence (NICE) Hypertension Guideline introduced, for the first time in the United Kingdom, recommendations for the diagnosis of hypertension that included the use of out-of-office measurement for confirmation of an initially raised clinic blood pressure.^[@R3]^ The routine use of out-of-office monitoring in the diagnosis of hypertension is now also recommended in Canada, the United States, Japan, and Europe.^[@R4]--[@R7]^ This change was in response to concerns that using clinic blood pressure may result in ≈25% of individuals being misclassified due to white coat hypertension, leading to potential unnecessary costs and adverse events.^[@R8]^ Furthermore, the use of ambulatory blood pressure monitoring (ABPM), and to a lesser extent home blood pressure monitoring, reduces this misclassification^[@R8]^ and is cost-effective compared with clinic monitoring.^[@R9]^ However, these changes were not without controversy. It was suggested that implementation would result in an additional 5000 cardiovascular events each year in England and Wales.^[@R10]^ Others argued that the use of ambulatory monitoring in the diagnosis of hypertension was inappropriate given that most evidence related to the treatment of hypertension is based on clinic blood pressure readings.^[@R11]^ Conversely, at a time of increasing workload and decreasing funding in UK General Practice,^[@R12],[@R13]^ the changes were predicted to reduce clinical workload due to a reduction in the unnecessary treatment of white coat hypertension.^[@R3]^ Sufficient time has now passed since the introduction of the NICE 2011 guideline to assess its impact on the management of hypertension and outcomes. We aimed to use data from the Clinical Practice Research Datalink (CPRD), a large database of routine electronic health records from primary care in England, to examine trends in the incidence of hypertension, use of out-of-office blood pressure monitoring, and cardiovascular morbidity and mortality from 2006 to 2017. We further aimed to test whether the introduction of the revised NICE Hypertension guideline in 2011 was associated with a change in these trends. Methods ======= The data used in this study were obtained under licence from the Medicines and Healthcare products Regulatory Agency, which does not permit data sharing. Equivalent data may be obtained subject to the terms outlined here: <https://www.cprd.com/research-applications>. Study Design ------------ This was a retrospective cohort study of adults (aged 18 years and over) registered at general practices contributing to the CPRD between April 1, 2006 and March 31, 2017. The CPRD is a research database of routinely collected primary care records, drawn from over 600 general practices and 11 million patients who are representative of the UK population.^[@R14]^ Data from CPRD were linked to inpatient Hospital Episodes Statistics (hospital data), Office for National Statistics mortality register data, and Index of Multiple Deprivation data (describing patient socio-economic status). Patients were eligible for inclusion if their records met basic quality measures, such as nontemporary registration and practice registration occurring after birth (termed acceptable records by CPRD) and were eligible for data linkage (which meant patients registered to English practices only). Furthermore, patients had to be registered at practices with continuous data reporting during the study period (termed up-to-standard by CPRD). Patients were excluded if they had a history of hypertension before study entry, because the change in guidance was limited to the use of out-of-office monitoring for diagnosis alone. We hypothesized that the change in guidance would, therefore, have a limited impact on those with a long-standing diagnosis of hypertension. See the extended methods in the [online-only Data Supplement](https://www.ahajournals.org/doi/suppl/10.1161/HYPERTENSIONAHA.119.13926) for further details and sample size calculations.^[@R15],[@R16]^ The study protocol was approved by the Independent Scientific Advisory Committee to CPRD (protocol number 17_239R) and was made available during peer review. Outcomes -------- The primary outcome was incidence of hypertension (defined as the presence of a first diagnostic code in the primary care record; Table S1 in the [online-only Data Supplement](https://www.ahajournals.org/doi/suppl/10.1161/HYPERTENSIONAHA.119.13926)). We considered the incidence of treated hypertension (presence of a clinical diagnostic code with antihypertensive treatment within 30 days) and untreated hypertension (presence of a diagnostic code without treatment). In sensitivity analyses, incidence of hypertension was defined according to (1) a clinical code for hypertension with or without a subsequent record of antihypertensive treatment or (2) 2 raised BP readings (clinic BP ≥140/90 mm Hg or out-of-office BP ≥135/85 mm Hg) followed by a record of antihypertensive treatment. We studied the following prespecified secondary outcomes: rate of new prescriptions of antihypertensive medication, rate of blood pressure monitoring (clinic, ambulatory, home, and overall), incidence of (major) cardiovascular events (cardiovascular death, myocardial infarction, or stroke), cardiovascular mortality, and all-cause mortality. The specific definitions for each outcome are given in the extended methods in the [online-only Data Supplement](https://www.ahajournals.org/doi/suppl/10.1161/HYPERTENSIONAHA.119.13926). Statistical Analysis -------------------- For each outcome, rates per 100 person-years were calculated for each age-sex stratum (male/female and 18--24, 25--44, 45--54, 55--64, 65--74, 75--84, and 85+) in each month. In primary analyses, rates were standardized to the English National Population standard in 2015 to account for changes in the age-sex distribution over time. In sensitivity analyses, rates were standardized according to the age, sex and socio-economic status (Index of Multiple Deprivation) of the cohort in March 2017. We performed prespecified subgroup analysis in those who had/had not developed hypertension before the beginning of each month from April 2007 onwards (allowing 1 year for incident hypertension cases to develop). Standardized rates were examined using interrupted time series analysis.^[@R17]^ Interrupted time series analysis is appropriate for examining the impact of health interventions and policies at a population level, when such interventions have been implemented at a clearly defined point in time.^[@R17]^ We assessed whether the introduction of the NICE Hypertension guideline in 2011 was associated with a step change in rates or a change in trend, by interrupting the time series between April 1, 2011 and March 31, 2012. Since patients could enter and exit the cohort at varying time points across the entire study period, monthly rates were calculated using data from varying person-years of observation. To account for this, analytical weights (equal to the person-years of observation) were used to estimate weighted interrupted time series models. We investigated the presence of autocorrelation and the influence of seasonality by including lag terms (up to order 12) in sensitivity analyses. In post hoc analyses, we examined the incidence of a control condition, asthma. Asthma (like hypertension) is diagnosed, monitored, and managed in primary care but (unlike hypertension) should not be causally related to changes in hypertension guidelines. Inclusion of a negative control helps ensure that any observed changes in outcomes are plausibly related to the exposure of interest rather than secular trends.^[@R18]^ We also examined the incidence of hypertension and major cardiovascular events in practices with high rates of reported out-of-office monitoring (top 20% between 2006 and 2017) to assess the possible impact of coding differences. Data on age and sex were complete. Patients with missing deprivation data (0.08%) were assumed to be in the middle quintile of deprivation. For all outcomes, absence of a relevant diagnostic or medication code was assumed to reflect absence of disease/treatment. All analysis was conducted using Stata version 14.^[@R19]^ Further details of data cleaning and analysis are given in the extended methods section in the [online-only Data Supplement](https://www.ahajournals.org/doi/suppl/10.1161/HYPERTENSIONAHA.119.13926). S.L. Lay-Flurrie had access to the full CPRD database and conducted all data cleaning and analysis. Linked data was provided directly by CPRD. Results ======= In total 3 937 191 patients from the January 2018 CPRD database were eligible for inclusion in the study cohort (Figure [1](#F1){ref-type="fig"}). Mean age at study entry was 39.7 years (SD=17.3), and 49.0% were male. There were 19 088 414 person-years of follow-up in total, and median follow-up was 4.2 years (interquartile range, 1.6--8.0). The characteristics of the cohort at study entry are given in Table [1](#T1){ref-type="table"}. ###### Baseline Study Characteristics  {#F1} Incidence of Hypertension ------------------------- In total, 236 554 (6.0%) patients developed incident hypertension during the study period (crude incidence rate of 1.31 per 100 person-years; Table S2). The age and sex standardized incidence of hypertension decreased over the study period from 2.10 per 100 person-years (95% CI, 1.96--2.25) in April 2006 to 1.39 per 100 person-years (95% CI, 1.28--1.49) in March 2017 (Figure [2](#F2){ref-type="fig"} and Table [2](#T2){ref-type="table"}). The year 2011, when the revised NICE hypertension guideline was introduced, was not associated with a significant change in the incident rate level (change in rate =0.01 \[95 % CI, −0.18--0.20\]). It was, however, associated with a reduction in the yearly downward trend (change in trend =0.093 \[95% CI, 0.035--0.151\]), but a similar reduction was observed in the negative control condition, asthma (Figure S1). The majority of incident hypertension cases were treated, and the same pattern in incidence was observed in treated and untreated groups (Table S3). When considering a more sensitive definition of hypertension, the incidence rate was higher but patterns in adjusted rates were similar (Table S4). ###### Results From Interrupted Time Series Analysis of Age and Sex Standardized Rates of Incident Hypertension Between April 2006 and March 2017, With Interruption Between April 2011 and March 2012  {#F2} Blood Pressure Monitoring ------------------------- Patients were recorded as having home and ambulatory monitoring on 39 260 and 98 071 occasions, respectively (crude rate of 0.2 and 0.5 per 100 person-years, respectively). In comparison, patients had recorded clinic blood pressure on 14 717 205 occasions (crude rate of 77.1 per 100 person-years). The change in guidance in 2011 was associated with a significant change in the age and sex standardized rates of home and ABPM and a significant change in the trend for home monitoring (Table S5 and Figure [3](#F3){ref-type="fig"}). The 2011 guidance was associated with a change in a downwards trend of clinic BP monitoring but not a change in the level. In those with a diagnosis of hypertension, the proportion who had a record of home or ambulatory BP monitoring before their diagnosis increased from 3.7% for those diagnosed in 2006/2007 to 25.6% in 2016/2017. {#F3} Cardiovascular Morbidity, Mortality, and All-Cause Mortality ------------------------------------------------------------ After excluding 63 623 patients with a history of major cardiovascular disease (myocardial infarction or stroke) before study entry, 66 785 patients (1.7%) had an incident MI or stroke during the study period (crude incidence rate of 0.36 per 100 person-years). The standardized incidence rate of cardiovascular disease was unchanged across the study period (Figure [4](#F4){ref-type="fig"}). The introduction of new NICE guidance in 2011 was not associated with changes in the incidence rate or trend in CVD (Table S6). Rates of any CVD and all-cause mortality were unchanged across the study period, but there were small (borderline significant) changes in trend for rates of major CVD mortality and any CVD mortality (5--8/100 000 patient years per year change). {#F4} New Antihypertensive Medication Use ----------------------------------- In total 11% of patients (399008 of 3 643 319) received antihypertensive medications for the first time during the study period (crude incidence rate of 2.5 per 100 person-years). The age-sex standardized rate of new antihypertensive medication use decreased during the study period (Figure S2 and Table S7). The change in guidance in 2011 was not associated with a change in rate (change in rate =−0.105 \[95% CI, −0.406 to 0.196\]) but was associated with a flattening of the previously downward trend (change in trend =0.096 \[95% CI, 0.007--0.185\]). There was no clear change in the pattern of prescribing of at least one medication in each class (Figure S3). Sensitivity and Post Hoc Analyses --------------------------------- There was some evidence of seasonality in rates but accounting for this produced similar results to primary analyses. An exception was for our broad definition of (any) CVD events, where the change in guidance was associated with a small decrease in the incidence rate level and a flattening trend (data not shown). Results for incidence of hypertension were also similar when standardizing according to age, sex, and deprivation in the final month of analysis (data not shown). Results stratified by hypertensive status are given in Figures S4 through S8 and Table S8. Absolute rates of BP monitoring, incident CVD, and incident antihypertensive treatment were higher in patients with hypertension compared with those without. The change in guidance in 2011 was associated with larger absolute changes in the rate of ambulatory blood pressure monitoring and home monitoring in hypertensive compared with normotensive groups but similar relative changes. Yearly trends in the rate of ambulatory and home monitoring remained largely flat across the study period in normotensive patients. Conversely, in hypertensive patients, trends in the rate of ambulatory and home BP monitoring changed significantly (from decreasing to flat and flat to increasing respectively). There was little change in the rate of office BP monitoring which continued its downward trend. For incident major CVD events (myocardial infarction and stroke), rates remained unchanged in both hypertensive and normotensive groups. Finally, the change in guidance was associated with a large increase in the rate of new antihypertensive treatment in patients with hypertension but no change in patients without. In practices with high rates of recorded out-of-office monitoring, the incidence of hypertension and major CVD events were similar to that observed in all practices (Figures S9 and S10). In 2011, there was no change in the incidence rate level of hypertension (change in rate =0.06 \[95% CI, −0.16--0.27) but a leveling off of downward trend (change in yearly trend =0.09 \[95% CI, 0.02--0.15). There was no change in the level or trend for incidence of major CVD events (change in rate =−0.001 \[95% CI, −0.056--0.055\]; change in yearly trend =0.010 \[95% CI, −0.009--0.030\]). Although these practices were selected for having high rates of out-of-office monitoring across the study period, a large increase in the use of out-of-office monitoring was also observed in this subgroup after 2011 (Figure S11). Discussion ========== The introduction of the NICE Hypertension guideline in England in 2011 was associated with a leveling out of downward trends in the incidence of diagnosed hypertension, although this may not be causal. In 2011, there was a step-change in the rate of ambulatory and home BP monitoring, in those with and without hypertension, suggesting changes in BP measurement occurred for diagnosis and monitoring. However, changes were modest in absolute terms implying perhaps issues with guideline implementation or coding of monitoring. By 2017, around a quarter of new diagnoses were accompanied by evidence of out-of-office monitoring. Contrary to previous predictions,^[@R10]^ there was little or no change in the incidence of cardiovascular morbidity or mortality. Strengths and Limitations ------------------------- This was a large scale analysis of high-quality data representative of the UK population.^[@R14]^ The majority of excluded patients were those not registered at contributing practices or within the study age range during the study period (Figure [1](#F1){ref-type="fig"}). Other exclusions were those not eligible for data linkage (largely those registered at practices outside of England) and those without acceptable records for research (primarily those registered as temporary patients with unknown follow-up status). The age and sex profiles of those included/not on this basis were similar (data not shown),and since all people resident in the England are entitled to access national health services, we are confident that selection bias was limited. Our use of standardized rates further increases the applicability of the results to wider populations, even in the presence of differences in age and sex demographics. We used diagnostic codes to define hypertension diagnoses which may have led to an underestimate of incidence.^[@R20]^ However, trends over time were similar when considering a broader definition, and patterns mirrored those observed in the United Kingdom for resistant hypertension.^[@R21]^ We could not use data from the Myocardial Infarction National Audit Project (a comprehensive register of patients experiencing MI) to ascertain fully validated MI outcomes. However, CPRD data is the most complete single source of information and when combined with hospital data as here, 92% of MI cases are identified.^[@R22]^ The effect of any under ascertainment of events in our composite outcomes is, therefore, likely to be limited. Reassuringly, we observed a decline in CVD-related mortality consistent with national statistics.^[@R23]^ Interrupted time series analysis cannot establish causality, as demonstrated by our finding that the incidence rate of asthma changed in 2011, which cannot plausibly be attributed to the hypertension guideline change. Our results may be influenced by coincidental factors including increases in general practitioner workload between 2007 and 2014^[@R12]^ without equivalent increases in staffing levels,^[@R24]^ which may have reduced access for patients, resulting in fewer diagnoses. Conversely, mortality rates declined and healthcare spending increased in the years before 2011, but both have since leveled off.^[@R25]^ Population-level improvements in health in the early years of our study may, therefore, not have been maintained. Nevertheless, our results were similar across analyses of CVD events and mortality regardless of the definition of CVD. Results for our primary outcome were also unaffected when adjusting for deprivation in addition to age and sex or in practices with greater use of out-of-office monitoring. We chose to study major cardiovascular outcomes and mortality, to reflect the overall goal of BP lowering and powered the study to detect small changes in incidence levels and trends in the presence of autocorrelation. Previous studies in UK databases have been able to detect differences in CVD event and mortality rates in smaller numbers of patients with less follow-up.^[@R26]^ We are confident we could have detected changes in these outcomes in the medium term if they truly existed but cannot exclude the possibility that the effects of guideline change may only manifest in the longer term. Such lagged effects could be studied once further time has elapsed. Guideline changes may have affected GP practices differently, depending on uptake. Models including random effects for GP practice were infeasible due to the large volumes of data in this study. This necessitated the use of yearly aggregate data in each age-gender strata and weighted analysis, but contemporary statistical software cannot implement random effects models with analytical weights. However, we were able to repeat analyses in practices with high levels of recorded out-of-office monitoring, and results in this subgroup were similar. Our use of age and sex as standardization variables meant we were unable to study patterns stratified by age or sex. Review evidence suggests that gender, in particular, is associated with the presence of masked and white coat hypertension,^[@R27]^ and therefore, the change in guidance may have had differential effects in men and women. Further work would be required to explore this. Although we standardized our results for deprivation in sensitivity analyses, we did not adjust for other covariates that may influence the risk of hypertension, in particular body mass index. However, trends in body mass index over time are small (0.2 kg/m^2^ between 2007 and 2016),^[@R28]^ and results accounting for deprivation were similar to our main analyses, so further adjustment is unlikely to have altered our results. Comparisons With Existing Literature ------------------------------------ Our finding that the incidence of hypertension has fallen since 2006 is consistent with observed downward trends in blood pressure.^[@R29]^ Considering the aging UK population and that the prevalence of hypertension increases with age, our finding is also consistent with studies showing that the overall prevalence of hypertension is stable.^[@R30]^ The decrease in the incidence of hypertension observed in this study may have been driven by an artificially inflated incidence rate shortly after the implementation of pay-for-performance indicators in 2004 in the United Kingdom. However, such an inflation was not observed when we repeated our analysis using data from 2003 onwards (not shown). Although the change in guidance was associated with a change in the rate of out-of-office monitoring, observed changes were small in absolute terms. We also observed a decline in office BP measurement after 2011, which would be consistent with replacement of at least some of these measurements with out-of-office readings, as recommended. However, our findings suggest that the overwhelming majority of BP measurement is still recorded as performed in the clinic. This may be explained by a failure to implement guideline changes properly or from issues in coding. Previous research examining the impact of other guideline or policy changes have indeed shown limited impact on hypertension-related clinical practice.^[@R16],[@R31]^ However, repeated UK surveys show that the vast majority of UK general practices now have access to home and ambulatory BP monitoring, with increases since 2011.^[@R32]^ Approximately 1 in 5 of all patients are also asked to monitor their BP at home.^[@R33]^ This suggests that the low rate of out-of-office monitoring in this study is more likely to reflect limited use of specific coding, rather than limited uptake of guidance. This appears to be a systematic issue as results were similar in practices where the use of out-of-office BP coding was high. This may be due to difficulty in using codes or the fact that BP-related pay-for-performance measures depend on clinic measurements.^[@R34]^ Implications for Research and Practice -------------------------------------- Our results indicate that concerns that the introduction of the NICE hypertension guideline in 2011 would result in major increases in avoidable cardiovascular events and deaths,^[@R10]^ appear unfounded, at least in the short-medium term. More recent guidelines in North America^[@R35]^ and Europe^[@R36]^ have taken up the UK NICE recommendations. We have found no evidence that such continued guidance would materially affect important patient outcomes. Further studies, in countries which have implemented similar guidance, or restricted to general practices which have fully adopted the guideline recommendations, are warranted to confirm our findings. Our results indicate that the change in guidance was associated with a relative increase in hypertension diagnoses compared with that expected from pre-2011 trends, but further research would be required to understand the mechanism underpinning this. It is possible that the guideline change prompted closer monitoring of patients with borderline clinic BP readings who would have subsequently had hypertension ruled out (indicating white coat effects) or been diagnosed. Perspectives ------------ For the diagnosis of hypertension, out-of-office blood pressure monitoring is more accurate^[@R8]^ and cost-effective^[@R9]^ compared with clinic BP monitoring. The results from this study suggest that the recommendation of out-of-office monitoring for hypertension diagnosis was not associated with observed negative consequences. By the end of the observation period, around a quarter of all new diagnoses were accompanied by recorded out-of-office measurement, but doubt remains regarding whether guidance was appropriately implemented or whether coding issues masked larger increases in the use of out-of-office BP measurement for the diagnosis of hypertension. The wider implementation of out-of-office monitoring, as has been recommended in the recent US, European and 2019 NICE hypertension guidelines, remains appropriate. Acknowledgments =============== The protocol was approved by the Independent Scientific Advisory Committee (ISAC) of the Medicines and Healthcare products Regulatory Agency (ISAC protocol number 17_239R; available from the authors on request). Ethics approval for observational research using the Clinical Practice Research Datalink with approval from ISAC was granted by a National Research Ethics Service committee (Trent Multi Research Ethics Committee, REC reference number 05/MRE04/87). All authors contributed to the study design, funding application, and ethical approval. S.L. Lay-Flurrie conducted data cleaning and analysis with input from R.J. Stevens and J.P. Sheppard. All authors interpreted results. S.L. Lay-Flurrie and R.J. McManus drafted the article which all authors then revised. S.L. Lay-Flurrie is the guarantor. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted. Sources of Funding ================== This study is funded by the National Institute for Health Research (NIHR) School for Primary Care Research (SPCR) (project reference 388). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. J.P. Sheppard is funded by a Wellcome Trust/Royal Society Sir Henry Dale Fellowship (ref: 211182/Z/18/Z) and previously received support from the NIHR Oxford Collaborations for Leadership in Applied Research and Care (CLARHC), and the NIHR School for Primary Care Research. C. Mallen is funded by the NIHR Collaborations for Leadership in Applied Health Research and Care West Midlands and a NIHR Research Professorship in General Practice (NIHR-RP-2014-04-026). F.D.R. Hobbs acknowledges funding from the NIHR School for Primary Care Research, the NIHR CLARHC Oxford, the NIHR Oxford Biomedical Research Centre (BRC, University Hospitals Trust), and the NIHR Oxford Medtech and In-Vitro Diagnostics Co-operative (MIC). R.J. McManus received support from NIHR via Programme Grants for Applied Health research, the SPCR, the Oxford CLAHRC, and a Senior Investigator Award. J. Mant is an NIHR Senior Investigator. The study sponsors were not involved in any aspect of the study including study design, data collection, data analysis, and interpretation of data. Disclosures =========== All authors have completed the International Committee of Medical Journal Editors uniform disclosure form at [www.icmje.org/coi_disclosure.pdf](www.icmje.org/coi_disclosure.pdf) and declare: S.L. Lay-Flurrie reports grants from National Institute for Health Research, School for Primary Care Research, during the conduct of the study; R.J. McManus reports grants from National Institute for Health Research, during the conduct of the study and grants from Omron, outside the submitted work; and being a member of the 2019 National Institute for Health and Care Excellence Hypertension Guideline Committee; J.P. Sheppard reports grants from National Institute for Health Research and Wellcome Trust, during the conduct of the study; C. Heneghan reports grants from National Institute for Health Research and other expenses from World Health Organization, outside the submitted work, and is Editor in chief of the British Medical Journal Evidence-Based Medicine and an National Health Service Urgent Care general practitioner. The other authors report no conflicts. Supplementary Material ====================== The online-only Data Supplement is available with this article at <https://www.ahajournals.org/doi/suppl/10.1161/HYPERTENSIONAHA.119.13926>. Novelty and Significance ======================== What Is New? ------------ - National guidelines in the United Kingdom recommended the use of out-of-office monitoring for hypertension diagnosis in 2011, but it was suggested this may increase rates of cardiovascular disease. This study is the first to formally evaluate the impact of these guideline changes on clinical outcomes at the population level. What is Relevant? ----------------- - Our study shows that changes to guidelines in 2011 were associated with a leveling off of downward trends in the incidence of hypertension and increases in the use of out-of-office monitoring. Changes were not associated with an increase in cardiovascular events, at least in the short-medium term. Summary ------- Based on current evidence, the continued recommendation of out-of-office monitoring for hypertension diagnosis is unlikely to affect hard patient outcomes.

Introduction {#Sec1} ============ Rodents, and in particular mice, are increasingly applied to understanding the physiology and neural computations underlying vision. Rodent models of ocular diseases are also an important tool to develop therapies at preclinical level. In this field a great body of work has been dedicated to developing rodent models that capture the critical aspects of human diseases and there are currently \>100 different types of visually impaired mouse^[@CR1]^. A particular focus has been devoted to using these models to develop treatments based on optogenetics, stem cell therapies and gene therapies for such incurable conditions as retinitis pigmentosa and Leber congenital amaurosis^[@CR2]--[@CR4]^. A crucial step in describing degeneration and establishing therapeutic efficacy in such pre-clinical studies is assessment of visual function, commonly by electrophysiological and/or behavioural approaches. Electrical recordings of neural activity in the retina and in the brain can be very useful in revealing physiological responses to visual stimuli^[@CR5],[@CR6]^. However, they don't directly measure integrated visual performance or perceptual components of vision^[@CR7]^. Many commonly used behavioural assays of visual function, such as pupil constriction^[@CR8]^, optokinetic and optomotor reflexes^[@CR9],[@CR10]^, suffer from the same weakness; being sub-conscious, reflex responses whose activity is only indirectly related to perceptual vision. All current tests that allow measurement of perceptual, cortically modulated vision rely on learning^[@CR11]--[@CR13]^. The most commonly used, the visual water task, has been effectively and widely employed to measure spatial acuity and brightness discrimination in mice with intact visual systems. However in animal models of moderate or severe retinal degeneration this task can only be painstakingly learnt through several weeks of intensive training^[@CR7]^. This process is time consuming and can be significantly stressful for the animals^[@CR14]^. Furthermore the visual water task can introduce additional sources of variability including the effects of repeated handling^[@CR15]^ and the learning process itself^[@CR16],[@CR17]^. The aim of this work is to develop a rapid assay of visual stimulus detection, suitable for mice with intact and degenerate retinas, which doesn't rely on training. To meet these requirements we investigate the possibility of using innate behaviours instead of learned tasks. Visually evoked changes in exploratory behaviour have been applied to this problem^[@CR6],[@CR18]^, but such tests are relatively low throughput and we were interested in the possibility of exploiting recent advances in understanding of rapid defensive behaviours as an alternative. Behaviourally salient visual stimuli, such as a looming dark spot^[@CR19],[@CR20]^ or a full field flash of light^[@CR21]^, can reliably trigger different behavioural responses. Several studies aimed at elucidating the circuits involved in controlling these behaviours revealed a substantial involvement of primary visual cortex^[@CR21],[@CR22]^ and first and higher order thalamic nuclei^[@CR23],[@CR24]^ as well as spatial memory^[@CR25]^. This indicates that these behaviours are not simply sub-conscious reflexes and instead rely on processing of higher order image forming pathways and integration with the limbic system. These behaviours are rapidly induced and short lived, meaning that they can, in theory, be evoked multiple times in a single experimental session. However so far such innate responses haven't been used systematically to assay visual ability, nor have they been tested in animals with mild or severe visual impairments. Therefore it is not clear to what extent these responses could be used to discriminate different degrees of retinal function. We show that measuring innate responses to experimentally controlled changes in the visual scene (such as light intensity or spatial frequency) represents an efficient strategy for measuring the limits of visual acuity in mice with an intact visual system. Importantly innate responses are partially maintained in animals with impaired vision and can be used to discriminate different levels of vision loss. Results {#Sec2} ======= Behavioural responses in visually intact mice {#Sec3} --------------------------------------------- We first set out to apply innate responses to measure visual capabilities in mice with an intact visual system. To this end, we developed an algorithm for tracking many landmarks on an animal's body in order to improve our ability to detect and characterise behavioural responses in an open field arena (Fig. [1a,b](#Fig1){ref-type="fig"}, see Methods for details). For each pair of consecutive frames the change in location of landmarks was used to calculate speed of motion (Fig. [1b](#Fig1){ref-type="fig"}, lower panels), and the distribution of velocities across landmarks divided into quantiles (Fig. [1b](#Fig1){ref-type="fig"}, upper panels) and used to generate a multidimensional time series representing the movements of different body parts (Fig. [1c](#Fig1){ref-type="fig"}). By plotting distinct behavioural time series for each quantile we could then detect movie frames in which movement was restricted to parts of the body (e.g. head movements or rearing; Fig. [1b](#Fig1){ref-type="fig"}, middle panel), as well as full body movements (e.g. locomotion; Fig. [1b](#Fig1){ref-type="fig"}, right panel).Figure 1Behavioural arena, data analyses and experimental protocols. (**a**) Schematic of the open field arena used for all tests (dimension: 30 × 30 cm). Power LEDs were used to provide diffuse illumination for the flashes while a rear projection screen was used to deliver the looming stimuli. (**b**) Images of a mouse in the arena (bottom) over successive frames (superimposed pink and green images) during periods of quiescence (left), head movement (middle) and locomotion (right). Landmarks were automatically identified on the mouse's body (blue dots on images at bottom) and the speed of each landmark calculated as proportional to the change in position from previous frame (visualised as yellow lines). Distribution of speed across landmarks was then calculated for each frame (top panels) and 10^th^, 30^th^, 50^th^, 70^th^, 90^th^ speed quantiles (respectively Q10 to Q90) used for subsequent analyses. (**c**) Behavioural time series showing velocity of landmarks (Q10--90) extracted from two cameras (camera 1 and 2, bottom panel; visual stimulus at time 0). Changepoint detection was run independently on each series (single changepoints depicted by black dots) and identified changepoints were then summed for each frame across those series (top panels, "pooled changepoints") and used for changepoint statistics throughout the study. (**d**) Schematic of the two stimulation protocols used for full field flash and pattern detection. In both protocols the order of each stimulus is block randomised (note: each block is represented by the list of all the distinct stimuli for that protocol). We considered two ways of using the tracking data to measure behavioural responses to a visual stimulus. In the simplest, we could use the estimates of landmark speed to identify increases or decreases in movement. In the second, we established changepoint analysis as a method to identify responses, and to quantify the frequency and reliability of visually induced behaviours. Changepoint analysis is used to detect if and when the properties of a time series change. By applying this technique to the behavioural time series, we can identify when a mouse's behaviour changes. We reasoned that if visual stimuli affect behaviour then we would expect higher rates of behavioural changes to occur after the onset of those stimuli (shown in Fig. [1c](#Fig1){ref-type="fig"}). Thus we used Pruned Exact Linear Time algorithm (PELT)^[@CR26]^ to detect changes in speed in our behavioural time series. This method is computationally efficient and can analyse dozens of our time series in less than a second. The PELT algorithm uses a penalty term to prevent overfitting changepoints (see Methods). This penalty term determines how much a changepoint must improve the model fit in order to be included. Small values result in a high rate of changepoints and are suitable to detect small changes in movements while larger values will only detect more substantial changes. In order to identify a suitable value for our data we investigated how the penalty value affects changepoint detection using the CROPS algorithm (Haynes *et al*., 2017) and knee estimation^[@CR27]^ (see Methods for details and Supplementary Fig. [1b,c](#MOESM1){ref-type="media"}). We designed two sets of stimuli aimed at probing different levels of visual capabilities. For the first we presented the animals with sudden changes in ambient light (bursts of 5 × 30 ms flashes over 330 ms, Fig. [1d](#Fig1){ref-type="fig"}, "full field flash") at three different magnitudes (see Table [1](#Tab1){ref-type="table"} for calibrated background and flash intensities). Since the flash stimulus produces a global change in ambient light no spatial discrimination is required. For the second set we generated a modified looming stimulus where the dark enlarging spot was replaced by a static grating whose average intensity was isoluminant with the grey background (Table [2](#Tab2){ref-type="table"}; Spatial Frequency = 0.017, 0.068, 0.272, 0.544, 1.088 cycles/degree; stimulus duration: 660 ms; looming speed = 66 deg/s; Inter-Stimulus-Interval = 73 seconds; Fig. [1d](#Fig1){ref-type="fig"}, "loom + gratings"). By changing the spatial frequency of the grating we could then use this stimulus to probe spatial acuity. As background illumination in both experiments we used low photopic light (see Tables [1](#Tab1){ref-type="table"}, [2](#Tab2){ref-type="table"}).Table 1Background irradiance (log~10~ photon/cm^2^/s).S-cone opsinMelanopsinRhodopsinM-cone opsinFull Field Flash Contrast Response10.055212.76512.834513.0188Gratings Looming for Spatial Acuity10.610613.217113.286813.4712Full Field Flash Contrast Response (RD1 mice)9.1651612.054312.121912.2971Table 2Michelson Contrast (%).Full Field Contrast ResponseS-cone opsinMelanopsinRhodopsinM-cone opsinDIM32.879344.140335.212311.2034MID86.119389.108684.802856.2394BRIGHT99.489199.533499.313197.0982**Gratings Looming for Spatial Acuity** ^**\***^**S-cone opsinMelanopsinRhodopsinM-cone opsin**White vs Grey35.25435.25435.25435.254Black vs Grey−87.113−87.113−87.113−87.113White vs Black93.61793.61793.61793.617**Full Field Flash Contrast Response (RD1 mice)S-cone opsinMelanopsinRhodopsinM-cone opsin**DIM79.177280.233973.714939.9271MID97.965897.675996.643587.1301BRIGHT99.933999.908899.866199.4359^\*^Michelson Contrast measured between white area of the gratings and grey background (White vs Grey), black area of the gratings and the grey background (Black vs Grey), white and black area of the gratings (White vs Black). The mice expressed a variety of responses that differed both quantitatively and qualitatively. The full field stimulus evoked partial body movements, typically head movements or rearing, or full body changes in locomotor activity (respectively Movies [1](#MOESM2){ref-type="media"}, [2](#MOESM3){ref-type="media"}). The looming stimulus instead reduced the animal activity both when engaged in full body movements such as during locomotion and when the animal was performing more stationary exploration (respectively Movies [3](#MOESM4){ref-type="media"}, [4](#MOESM5){ref-type="media"}). Therefore the responses to flashes and looming were qualitatively different and consisted respectively of an increase and a decrease in movement speed. The response to full field stimuli was apparent in analyses based upon speed of movement and changepoint detection. Thus, velocity of movement across landmarks increased following presentation of mid and bright, but not dim, flashes (Fig. [2a](#Fig2){ref-type="fig"}; p = 1, 0.006, 4 \* 10^−7^ respectively dim, mid, bright flash; n = 84 trials from 12 animals, signtest). The average responses to highest intensity were substantially larger than to mid intensity levels (p = 0.013, z = 2.48, n = 84 "mid" & "bri" trials, ranksum test). We also found an increase in the number of changepoints after the stimulus onset compared to baseline activity, and this effect was flash intensity dependent (Fig. [2b](#Fig2){ref-type="fig"}, p \< 0.0001, df = 2, χ^2^ = 27.57, n = 84, kruskalwallis test). The increase in number of changepoints was consistent across individuals for the bright flash (Supplementary Fig. [5a,b](#MOESM1){ref-type="media"}) while the mid flash revealed higher variability across subjects (Supplementary Fig. [5c](#MOESM1){ref-type="media"}). We wondered whether the larger average effects associated with high intensity was also due to an increase in reliability leading to more effective summation across trials. In other words whether part of the higher amplitude response to bright flash could be explained by the fact that dimmer flashes evoke both positive and negative responses that partially cancel out. In order to measure reliability of these responses we compared the rate of changepoints before and after stimulus onset on a single trial basis (calculated over time windows of 0.6 s duration). The rate increased in 74% trials and decreased in 11% trials for the highest intensity while for the mid-level intensity we found increments and decrements respectively in 45% and 27% trials. Therefore since the brightest flash produced a more consistent bias towards positive increments in changepoints we conclude that this stimulus also increased response reliability. The intensity dependent increase in reliability is also clearly apparent by comparing left and right panels in Fig. [2c](#Fig2){ref-type="fig"}, where we reported the changepoints detected for all individual trials for mid (left panel) and bright (right panel) flashes.Figure 2Behavioural responses to full field flashes in visually intact animals. (**a**) Movement responses to dim, mid and bright flashes (respectively left, middle and right panels; visual stimulus at time 0). Each grey line represents a different quantile of the speed distribution (10^th^, 30^th^, 50^th^, 70^th^, 90^th^ quantiles, respectively Q10 to Q90, averaged across the two cameras used to track behaviour, see also Fig. [1a--c](#Fig1){ref-type="fig"} and Methods). (**b**) Difference between changepoint rate after and before stimulus onset (calculated as *Δ\#chp/s* = *\#chp/s (pre) - \#chp/s (post)*, see Methods; the rates were calculated in time windows of 0.6 s). (**c**) Behavioural changepoints for individual trials for mid and bright flashes (respectively left and right panels). For both flashes we collected 84 trials (each trial here reported as row along the y-axis) from 12 animals (7 trials/animal). The flash was delivered at time 0). For a given trial the number of changepoints occurring at the same time frame are colour coded gray-to-black as exemplified in Fig. [1c](#Fig1){ref-type="fig"} ("pooled changepoints"). \*p \< 0.05, \*\*p \< 0.0.01, \*\*\*p \< 0.005, \*\*\*\*p \< 0.001, \*\*\*\*\*p \< 0.0005, \*\*\*\*\*\*p \< 0.0001, ns = not significant. The looming stimuli elicited freezing-like reductions in activity across a wide range of frequencies. This effect could be observed from an average reduction in speed that was significant up to 0.544 cpd but not at 1.088 cycles/degrees (p = 0.021, 5 \* 10^−5^, 5 \* 10^−5^, 0.0063, 0.558 respectively 0.017, 0.068, 0.272, 0.544, 1.088 cycles/degrees; n = 91, 91, 91, 105, 105 trials from 15 animals, signtest; Fig. [3a](#Fig3){ref-type="fig"}). The changepoint analysis replicated the results obtained by measuring movement speed as significant responses could be measured up to 0.544 cycles/degree (Fig. [3b](#Fig3){ref-type="fig"}). The most reliable response occurred with the intermediate spatial frequency gratings 0.068 and 0.272 cycles/degree in which changepoint rate increased respectively in 61% and 65% trials and decreased in 30% and 26%. This effect can be observed in Fig. [3c](#Fig3){ref-type="fig"} where a clear increase in changepoint rate after stimulus onset (at time 0) is apparent at 0.068 and 0.272 cycles/degree (left and middle panel) but not at 1.088 cycles/degree (right panel). The highest reproducibility at 0.272 cycles/degree is consistent with peak in contrast sensitivity recorded with visual water task^[@CR11]^. For spatial frequencies in this high sensitivity range responses were also reliable across individuals (Supplementary Fig. [5d,e](#MOESM1){ref-type="media"}) while, for spatial frequencies around the spatial acuity threshold (0.544--1.088 cycles/degree), responses were more variable across subjects (Supplementary Fig. [5f](#MOESM1){ref-type="media"}). Importantly, the threshold acuity found here (0.544 cpd) is higher than that defined in cortically lesioned animals (\<0.3 cycles/degree with visual water task^[@CR28]^) suggesting that visual cortex is involved in driving these behavioural responses. This possibility is also consistent with the described positive cortical modulation of looming evoked responses in superior colliculus^[@CR22]^. In this study the authors demonstrated that primary visual cortex exerts a positive gain modulation of looming evoked firing rate in superior colliculus. Therefore it is possible that a similar modulation could enhance collicular responses to the highest spatial frequency and increase the probability of a behavioural response. However a conclusive proof of cortical involvement in driving innate freeze-like responses to high spatial frequency would require experimental manipulation of cortical activity. These experiments could be done either by lesioning the visual cortex or by transiently blocking cortical activity (either pharmacologically, or by using optogenetics or chemogenetics in transgenic animals).Figure 3Behavioural responses to looming + gratings in visually intact animals. **(a**) Movement responses (in log scale for 10^th^, 30^th^, 50^th^, 70^th^, 90^th^ quantiles - respectively Q10 to Q90 - averaged across two cameras) to all spatial frequencies tested for the spatial acuity stimulus represented in Fig. [1d](#Fig1){ref-type="fig"} (the spatial frequency for each panel is indicated in cycles/degree in the top left corner of each panel; visual stimulus at time 0). Reduction in movement was significant for all frequencies apart from the highest (1.088 cycles/degree). (**b**) Differences in changepoint rate before and after stimulus onset across all spatial frequencies tested (mean ± sem; spatial frequencies are indicated as cycles/dregrees on the x-axis). (**c**) Behavioural changepoints for individual trials at different spatial frequencies (0.068, 0.272, 1.088 cycles/degree; 91, 91, 105 trials, 7 trials/animal; visual stimulus delivered at time 0). For a given trial the number of changepoints occurring at the same time frame are colour coded gray-to-black as exemplified in Fig. [1c](#Fig1){ref-type="fig"} ("pooled changepoints"). \*p \< 0.05, \*\*p \< 0.01, \*\*\*p \< 0.005, \*\*\*\*p \< 0.001, \*\*\*\*\*p \< 0.0005, \*\*\*\*\*\*p \< 0.0001, ns = not significant. Many of the previous studies on looming evoked defensive responses in mice employed a black looming disc^[@CR19],[@CR20],[@CR24],[@CR25],[@CR29]--[@CR31]^. Therefore we wondered whether the flashes or the loom + gratings evoked responses that were comparable to those expressed by the "standard" black looming stimulus. Since several of those studies focussed on escape responses^[@CR19],[@CR25],[@CR29]^ we performed an additional set of experiments by adding a shelter to provide an escape route for the animal. We found that the bright flash never evoked a clear run to the shelter (n = 0/22 trials, data from 11 animals; we recorded 2 trials per animal) and instead animals expressed diverse increases in movement speed that included enhanced locomotion or rearing (see Movie [9](#MOESM10){ref-type="media"}). Both looming + gratings and "standard" black looming typically evoked transient freeze followed by escape to the shelter (13/22 and 12/22 trials respectively loom + gratings & black looming; see Movie [10](#MOESM11){ref-type="media"}) and less commonly immediate escape (1/22 and 3/22 trials respectively loom + gratings & black looming; see Movie [11](#MOESM12){ref-type="media"}) or freeze (3/22 and 4/22 trials respectively loom + gratings & black looming). The qualitative difference between flash and looming is also apparent from individual trials (see Supplementary Fig. [2a](#MOESM1){ref-type="media"}) where, compared with flashes, black looming and loom + gratings alike elicit both higher and lower peaks speeds (Supplementary Fig. [2b](#MOESM1){ref-type="media"}) associated respectively with escape and freeze. In order to objectively test the possibility that behaviours expressed by flash and looming are largely separable we also performed a principal component analysis on the speed time series. We found that the first component could readily separate flash and looming stimuli on individual trials basis (Supplementary Fig. [2c](#MOESM1){ref-type="media"}; p = 5 \* 10^−6^, 1 \* 10^−6^, respectively for flash vs loom + gratings and flash vs black looming; two sample Kolmogorov-Smirnov goodness-of-fit test) while the score distribution for the first component were largely overlapping for black looming and loom + gratings (p = 0.33 for loom + gratings vs black looming; two sample Kolmogorov-Smirnov goodness-of-fit test). A clear difference between flash and looming evoked behaviours could also be observed from the average speed responses (Supplementary Fig. [2d](#MOESM1){ref-type="media"}). These results indicate that we can effectively use parametrised stimuli (i.e. stimuli in which we can systematically vary relevant visual parameters, such as flash intensity or gratings spatial frequency) to measure different visual abilities, ranging from coarse light detection to noise-limited spatial acuity, in mice with intact retinas. Interestingly reactions to full field flashes are of opposite sign compared with looming gratings and produced qualitatively different behaviours, providing a novel case in which vision guiding of spontaneous behaviour is diverse yet systematic. Behavioural responses in mouse models of retinal degeneration {#Sec4} ------------------------------------------------------------- The use of innate responses as a tool to measure different levels of visual function in mice with intact visual system suggest that these responses could also be used to probe residual visual function in mouse models of retinal degeneration. To investigate this possibility we first repeated our behavioural tests in rd1 mice where a mutation of the Pde6b gene substantially disrupts the rod phototransduction cascade^[@CR32]^. These animals represent a model of severe retinal degeneration characterised by fast onset, in which the rod phototransduction cascade is non-functional from birth and cones undergo rapid progressive degeneration^[@CR33]^. Adult rd1 mice (3--6 months) undergoing intensive training with visual water task (\~3 weeks) can still learn a coarse dark/bright discrimination^[@CR7]^, which is at least partly reliant on inner retina photoreception^[@CR34]^, while detection of spatially structured patterns is abolished^[@CR7]^. Previous work has shown that rd1 animals retain some innate light aversion however this behaviour is expressed only after prolonged light exposure (\~10 minutes;^[@CR35]^). Moreover, open field spontaneous exploratory behaviour is unaffected by exposure to bright light^[@CR5],[@CR6]^. It is currently unknown whether their residual visual functionality can drive more transient behavioural responses resembling those we observed in visually intact animals; and, if that were the case, whether it is possible, based on these responses, to track the progress of visual degeneration. To answer these questions we selected two cohorts of adult rd1 mice associated with different age groups hereinafter defined as "young" and "old" rd1 (n = 8,12 animals; 10.2 ± 0sd and 24 ± 1.75sd weeks). It was previously shown that cone density undergoes substantial changes between these ages^[@CR36]^. The gradual loss of photoreceptor inputs then results in plastic changes and aberrant rhythmic activity at the level of the retinal ganglion cells^[@CR37]^ targeting both visual thalamus and superior colliculus. Therefore behavioural responses, if at all present, would be expected to be more detectable in young animals. As a first test we used the same full field flashes delivered to control mice but we reduced the background light intensity in order to maximise the contrast and effectiveness of the flash stimulus (see Tables [1](#Tab1){ref-type="table"}, [2](#Tab2){ref-type="table"} for calibrated intensities). The highest intensity flash evoked a transient increase in activity in both age groups (Movies [5](#MOESM6){ref-type="media"}, [6](#MOESM7){ref-type="media"}, Fig. [4a](#Fig4){ref-type="fig"}; p = 0.011 and 0.0375, n = 56 and 84 trials, signtest). One potential explanation is that these responses could be driven by residual cones. This possibility is consistent with a previous study that described surviving cone photoreceptors up to 3 months of age^[@CR36]^ and with immunohistochemistry analysis of retinas from animals in the "old" group that also provided positive evidence of residual S-cones (Fig. [4b](#Fig4){ref-type="fig"}**;** see also Supplementary Fig. [7](#MOESM1){ref-type="media"}). Alternatively these responses could be driven by intrinsically photosensitive retinal ganglion cells (ipRGCs), as melanopsin signalling is largely preserved in rd1 mice^[@CR38],[@CR39]^. A further possibility is that both surviving cones and melanopsin contribute to these responses as ipRGCs mediated gain modulation of cone signalling has recently been described^[@CR40]^. Lower intensities did not evoke significant responses ("young": p = 0.894 and 0.504, n = 56 trials; "old": p = 0.230 and 0.063, n = 84 trials; signtest for dim and mid intensity).Figure 4Behavioural responses to full field flashes and looming + gratings in mouse models of retinal degeneration. **(a**) Increase in speed after bright flash for young and old rd1 animals (respectively left and right panels). (**b**) Immunostaining of retinal wholemounts from "old" cohort of *rd1* mice revealed presence of surviving S-cones in peripheral ventral regions. A diagram showing pattern of anti-SWS cone opsin staining in a representative *rd1*retina (left), with an example micrograph of the anti-SWS cone staining we observed in the ventral retina shown on the right. Asterisk indicates location of micrograph in diagram of the retina. An expanded version of this figure is shown in Supplementary Fig. [7](#MOESM1){ref-type="media"}. (**c**) Behavioural changepoint for individual trials under bright flash stimuli for "young" and "old" animals (respectively left and right panels; 56, 84 trials collected from 8, 12 animals; each animals recorded for 7 trials; visual stimulus at time 0). (**d**) Increase in changepoint rate (mean ± sem) as function of flash intensity. (**e**) Looming did not evoke changes in speed in both groups. Speed, represented in log scale, was averaged across the spatial frequencies tested (0.017, 0.068 and 0.272 cycles/degree). (**f**) Changepoint rates did not significantly change after stimulus onset at all spatial frequencies tested (0.017, 0.068, 0.272 cycles/deg; mean ± sem). (**g**) rd12 expressed significant reductions in movement at 0.068 cycles/deg (left panel) and at 0.272 cycles/deg (right panel). (**h**) Behavioural changepoints for individual trials during looming gratings at 0.068 cycles/deg reveals repeatable responses (left). Changepoint rates as function of spatial frequencies (right panel; mean ± sem). \*p \< 0.05, \*\*p \< 0.01, \*\*\*p \< 0.005, \*\*\*\*p \< 0.001, \*\*\*\*\*p \< 0.0005, \*\*\*\*\*\*p \< 0.0001, ns = not significant. The response to the highest intensity flash was significantly larger in the young cohort indicating that this simple test allows to detect differences in coarse light detection among the two groups (p = 0.022, z = 2.3, n = 56 and 84, respectively "young" and "old" trials, ranksum test). Compared with control animals trial-to-trial reproducibility was reduced in both groups as changepoint rate increased in 57% & 52% and decreased in 16% & 23% of the trials (respectively young & old rd1; Fig. [4c](#Fig4){ref-type="fig"}). Individual variability in responses to the bright flash appeared larger that in visually intact animals (compare Supplementary Fig. [6a](#MOESM1){ref-type="media"} with Supplementary Fig. [5b](#MOESM1){ref-type="media"}). Like in control animals increase in changepoint rate was dependent on flash intensity (Fig. [4d](#Fig4){ref-type="fig"}, p = 0.021 and 0.004, df = 2, χ^2^ = 7.70 and 11.047, n = 56 and 84 trials, Kruskal-Wallis test respectively "young" and "old" groups). We next used the looming stimuli in order to assess visual acuity. Since previous studies reported complete lack of spatial discrimination in these animals we focused on a reduced spatial frequency range (0.017, 0.068, 0.272 cycles/degree). Both age groups failed to exhibit significant responses to any of the stimuli presented (Fig. [4e,f](#Fig4){ref-type="fig"}**;** "young": p = 0.689, 0.894, 0.141, n = 56 trials; "old": p = 1, 0.445, 0.586, n = 84 trials; signtest at 0.017, 0.068, 0.272 cycles/degree). These negative results are consistent with data from both visual water task and optomotor assays showing loss of spatial discrimination in this genotype^[@CR7],[@CR10]^. We wondered whether behavioural responses to spatially structured stimuli such as our looming gratings could be observed in animals with less complete retinal degeneration. To investigate this possibility we used rd12 animals, which carry a mutation in the Rpe65 gene that disrupts recycling of the chromophore^[@CR41]^. Previous results based on optokinetic reflex revealed detectable responses below 0.2 cycles/degrees in adult animals aged between 9 and 18 weeks^[@CR42]^. We presented looming grating stimuli over a range of spatial frequencies (0.017--0.272 cpd) to an age-matched cohort (n = 7; 14.4 ± 4.4sd weeks) of rd12 mice. We found significant responses at 0.068 cycles/degrees (Movie [7](#MOESM8){ref-type="media"}, Fig. [4g](#Fig4){ref-type="fig"}, left panel; p = 0.0001, n = 49 trials; signtest) and a similar response at lowest frequency tested that however did not reach significance (Supplementary Fig. [1a](#MOESM1){ref-type="media"}; p = 0.568, signtest, n = 49 trials, signtest; spatial frequency = 0.017 cycles/degree). These results are consistent with previously published measurements of the optokinetic reflex that reported a limit of spatial acuity below 0.2 cycles/degree^[@CR42]^. Additionally we also found significant responses at 0.272 cycles/degree (Movie [8](#MOESM9){ref-type="media"}, Fig. [4g](#Fig4){ref-type="fig"}, right panel; p = 0.009, n = 49 trials, signtest). Responses to all stimuli consisted of a reduction in motor activity similar to the responses observed in control animals. Changepoint results were consistent across stimuli with a larger fraction of trials exhibiting an increase in changepoint rate (51%, 57%, 53% respectively at 0.017, 0.068, 0.272 cycles/degree) compared with trials exhibiting a decrease (34%, 34%, 36%). However reproducibility was lower than in visually intact animals (p = 0.008, permutation test comparing distributions of Δ\#chp/s for visually intact and rd12 animals across all spatial frequencies) and the average increments in changepoints were only significant at 0.068 cycles/degree (Fig. [4h](#Fig4){ref-type="fig"}). Responses were also less reproducible across individuals (compared Supplementary Fig. [6b](#MOESM1){ref-type="media"} with Supplementary Fig. [5e](#MOESM1){ref-type="media"}). Behavioural habituation in mice with intact and degenerate retinas {#Sec5} ------------------------------------------------------------------ As animals are presented with multiple repeats of the same or similar stimuli with no associated reward or punishment the salience of those stimuli is expected to "wash off", reducing the drive for behavioural responses^[@CR43]^. Indeed habituation to looming stimulation has been previously reported^[@CR31]^. As habituation could potentially limit the number of effective trials that can be presented to an individual animal we asked to what extent this phenomenon affected our tests. To quantify habituation we focussed on those stimuli and animal groups associated with clearly detectable behavioural responses as with little (e.g. flash stimuli in "old" rd1, Fig. [4](#Fig4){ref-type="fig"}) or no responses this effect is not measurable (e.g. looming stimuli in rd1, Fig. [4e,f](#Fig4){ref-type="fig"}). Note that in order to pre-emptively mitigate the possibility of habituation by reducing expectation the presentation order of the stimuli throughout our study was block randomised (See Methods). During flash experiments changes in speed in animals with intact vision and in young rd1 were both trending towards lower amplitude in "late" trials (6^th^ and 7^th^ repetitions of the stimulus) compared with those recorded in "early" trials (1^st^ and 2^nd^ repetitions; Fig. [5a](#Fig5){ref-type="fig"}**;** p = 0.049, 0.039, respectively visually intact and young rd1, permutation test). The level of habituation to looming gratings was different in visually intact and rd12 animals. The former did not exhibit significant habituation (Fig. [5c](#Fig5){ref-type="fig"}, left panels; p = 0.482, permutation test) while the latter expressed a substantial reduction in speed changes across trials (Fig. [5c](#Fig5){ref-type="fig"}, right panel; p = 0.001). None of these effects were observed in changepoint rates indicating that while the amplitude of behavioural responses is reduced in "late" trials those responses are still present (Fig. [5b,d](#Fig5){ref-type="fig"}**;** visually intact: p = 0.718, 0.556 respectively flash and looming; rd1: p = 0.283; rd12: p = 0.401; permutation tests). Overall our data indicate that habituation can have measurable short-term effects (on the timescale of minutes) over the course of an experimental session.Figure 5Behavioural habituation to repeated stimulation in visually intact and retinally degenerate mice. **(a)** Average changes in speed after stimulus onset as function of trial order for the highest intensity flash (data shown as mean ± sem). Represented in left and right panels are visually intact and "young" rd1 animals. Both groups exhibit a negative trend (visually intact: p = 0.049, n = 36 trials; young rd1: p = 0.039; n = 24 trials; comparisons between early and late trials, permutation test). **(b)** Difference between changepoint rate after and before stimulus onset (Δ\#chp/s) as function of trial order during highest intensity flash for visually intact and young rd1 animals (respectively left and right panel). Neither group showed a significant trend (visually intact: p = 0.718, n = 36 trials; young rd1: p = 0.283; n = 24 trials; comparisons between early and late trials, ranksum test). (**c**,**d)** Like panels a and b but for looming responses in visually intact and rd12 animals. No significant trend was observed for visually intact animals (panel c: p = 0.482, n = 39 trials; panel d: p = 0.556, n = 39 trials). The rd12 groups exhibited gradual reduction in speed (p = 0.001, n = 21 trials) while no clear trend could be observed in changepoints (p = 0.401, n = 21 trials). (**e)** Alternative protocol based on systematic reduction of spatial frequency from highest to lowest (1.088, 0.544, 0.272, 0.136, 0.068, 0.034, 0.017 cycles/degree). Each spatial frequency was presented only on one trial and repeated three times (each repetition lasting 660 ms as previous tests reported in Fig. [1d](#Fig1){ref-type="fig"}; double arrows represent this time interval) during that trial as in^[@CR19]^. (**f)** Average speed response for all spatial frequencies tested (n = 5 animals, 1 trial/animal). (**g)** Polynomial fit for changes in speed as function of spatial frequency (polynomial degree = 2; R^2^ = 0.263; same dataset shown in from panel f, data shown as mean ± sem). Consistent with results obtained by using the block randomised protocol (Fig. [3a--c](#Fig3){ref-type="fig"}) reduction in speed can be observed up to 0.544 cycles/degrees but not at 1.088 cycles/degrees. In situations where many stimuli have to be presented, an alternative solution to counteract habituation could be to present stimuli in order by starting with those least likely to be detected. To test this possibility we repeated the estimation of spatial acuity in a new cohort of visually intact animals. We selected a wider set of spatial frequencies and presented them in decreasing order (Fig. [5e](#Fig5){ref-type="fig"}). In this way the minimally detectable stimulus (in this case threshold spatial frequency) will be associated with the strongest, non-habituated response, providing the clearest upper bound to estimate visual function. In agreement with the results in Fig. [3a,b](#Fig3){ref-type="fig"} this alternative protocol returned a limiting spatial acuity between 0.5 and 1 cycles/degrees (Fig. [5f,g](#Fig5){ref-type="fig"}). Discussion {#Sec6} ========== Our results provide a proof of principle that systematic assessment of visually guided innate responses, such as freeze or escape, represents a promising tool to assess visual capabilities both in mice with intact visual system and in mice affected by different levels of visual impairment. In the last few years, interest in visually driven innate behaviours has come of age (see e.g.^[@CR29],[@CR30],[@CR44]^). The growth in interest in such behaviours has been attributable primarily to their application in studies of sensorimotor transformations and action selection. As these behaviours rely upon detection of a visual stimulus, and are readily elicited in untrained animals, they also have clear potential as a basis for characterising visual capacity. However several potential barriers stood in the way of realising that potential at the start of this project: 1) the degree to which behaviourally salient stimuli used to evoke innate responses could be parameterised was unknown; in other words, to what extent a systematic variation of a stimulus parameter (such as flash intensity or grating spatial frequency) can induce measurable differences in behavioural responses? 2) reliable, automated and objective methods for measuring innate behavioural responses were not well defined; 3) the intrinsic variability of innate behaviours may make them too unreliable as an indicator of whether a mouse had detected a visual stimulus; 4) innate responses could quickly habituate; 5) the extent to which visually driven behaviours appear in animals with visual impairment was unexplored. Below, by using our methodological and behavioural results as well as other recent studies, we discuss these issues: Parametrising behaviourally salient visual stimuli {#Sec7} -------------------------------------------------- Most studies on visually driven innate responses have focussed on looming stimuli in the form of a dark, enlarging, spot. While variants of this stimulus have been considered (see e.g.^[@CR19]^) the general conclusion that defensive responses are strongly selective might have prevented a full exploration of the space of effective stimuli. Very recently it was shown that escape response to dark looming stimulus exhibits a smooth dependence on the contrast of a looming spot, indicating that negative contrast can be effectively parametrised^[@CR29]^. Here we show that darkening looming stimuli do not represent the only stimulus class able to trigger defensive responses: isoluminant stimuli -- black and white gratings whose average luminance matches a grey background - can reliably evoke freezing (Figs [1d](#Fig1){ref-type="fig"}, [3](#Fig3){ref-type="fig"}). Importantly we have shown that altering the spatial frequency of these gratings induces quantifiable changes in behavioural responses. Therefore those responses can be used to measure mouse spatial acuity. Moreover it is now reasonable to assume that changes in other grating characteristics e.g. contrast, colour etc. could be applied to quantify other visual features. Although mice with advanced degeneration did not respond to gratings (rd1, Fig. [4a--f](#Fig4){ref-type="fig"}), we found that they did respond to another parameterisable stimulus: full field flashes. Responses to full field flashes have been previously shown to evoke behavioural responses whose magnitude depends on flash intensity^[@CR21]^. Unexpectedly the responses we observed, consisting in sharp transient increases in activity, were different and opposite from the behavioural arrests observed by Liang and co-workers^[@CR21]^. We note however that the environment in which those behavioural arrests were recorded, a narrow corridor, was substantially different from the open field arena used for this study. Therefore, a likely explanation for different behavioural outcomes is that different environments trigger diverse behavioural responses. Indeed a similar environmental effect, whereby the presence (or absence) of a shelter triggers escape (or freeze), is well known (compare e.g. results from^[@CR19]^ -- escape to^[@CR30]^ -- freeze). Methods for measuring innate responses {#Sec8} -------------------------------------- In this work we introduce a new method to track multiple body points and establish changepoint analyses to detect innate responses in behavioural time series. These methods allow detection of large changes in behaviours such as sudden locomotion arrests or initiations (see e.g. Movies [2](#MOESM3){ref-type="media"}, [7](#MOESM8){ref-type="media"}) as well as more subtle behavioural responses (see e.g. Movies [1](#MOESM2){ref-type="media"}, [4](#MOESM5){ref-type="media"}). Changepoint analysis is based on PELT algorithm and provides the optimal segmentation under a simple cost function^[@CR26]^. It has only one free parameter, which can be quickly estimated from the data with the CROPS algorithm^[@CR45]^ as shown in Methods, therefore results can be easily reproduced. Changepoint analysis is particularly useful for measuring how reliably behavioural responses occur across trials by simply subtracting the rate of changepoint before and after stimulus onset. The results are robust in respect to the tracking algorithm used and the main results can be also replicated by using the more standard centroid method to track body position (Supplementary Figs [3](#MOESM1){ref-type="media"} and [4](#MOESM1){ref-type="media"}). Therefore, while tracking multiple body parts has the advantage of providing a more graded representation of partial movements (Fig. [1](#Fig1){ref-type="fig"}), a more complete dataset to apply changepoint detection and is robust to occluded views of the animal body (e.g. when a shelter is present, see Movie [12](#MOESM13){ref-type="media"}), any tracking algorithm can potentially be used to detect the range of innate behaviours we describe in this study. Intrinsic variability of innate behaviours {#Sec9} ------------------------------------------ Spontaneous behaviours can vary substantially between individuals. Thus, different animals exposed to the same threatening visual stimulus can employ diverse, and sometimes opposing (e.g. freezing or escape), behavioural strategies. However recent results indicate that different classes of visual stimuli can bias towards different types of innate responses and this bias is shared across individuals. Thus De Franceschi^[@CR20]^ has shown that looming and sweeping dark spots can consistently drive mice to select either escape (for a looming spot) or freezing (for a sweeping spot). In this paper we describe another clear behavioural dichotomy as the same group of animals express sharp increments in speed after a flash presentation and freezing-like behaviours under loom + gratings stimuli (respectively Figs [2a](#Fig2){ref-type="fig"}, [3a](#Fig3){ref-type="fig"}). Innate responses such as freezing and escape have been now described from several laboratories. While details of the motor sequences might vary (for example from Type I to Type II behaviours described in^[@CR31]^) the occurrence of those behaviours is well established. In this work we reinforce this notion by showing that both looming evoked freezing-like behaviours and flash evoked increments in motor activity are maintained across different mouse strains. Moreover changepoint analysis can automatically detect both negative and positive changes in speed and therefore also addresses intrinsic variability of innate responses. For each stimulus in each animal group we also reported the percentage of trials with positive, negative or no response (calculated as difference in number of changepoint before and after presentation of a visual stimulus). These numbers provide a measure of response reliability and can be used in the future to design adequately powered experiments (e.g. by using Experimental Design Assistant^[@CR46]^ or other software) for testing treatments for retinal degeneration. Innate responses could quickly habituate {#Sec10} ---------------------------------------- One obvious potential solution to inherent behavioural variability is to record responses to multiple stimulus presentations. However, when animals are presented with repeated sets of threatening stimuli, that turn out to be inconsequential, behavioural responses will quickly habituate^[@CR43]^. Therefore habituation could represent a barrier to presenting large numbers of similar stimuli to the same animal as a method of addressing response variability and in order to describe responses to quantitative alterations in the stimulus (e.g. when estimating threshold spatial acuity). While we do observe some level of habituation in the amplitude of behavioural responses (Fig. [5a,c](#Fig5){ref-type="fig"}) those responses were still present across multiple repetitions (up to 7) and detectable by changepoint analysis (Fig. [5b,d](#Fig5){ref-type="fig"}). Thus when multiple stimuli must be presented to estimate detection thresholds, we find that habituation can be addressed either by randomising the presentation order of a limited set of stimuli (Fig. [3](#Fig3){ref-type="fig"}) or by presenting a wider set that starts from the least detectable stimulus (Fig. [5e--g](#Fig5){ref-type="fig"}). In this study we did not investigate the potential for long-term habituation to the same set of stimuli presented on different experimental sessions. Others have described this phenomenon and reported it to be stimulus specific^[@CR43]^. Our data are consistent with this view as we were able to record responses to two different sets of stimuli (flash and looming gratings) from the same visually intact animals on different days (Figs [2](#Fig2){ref-type="fig"}, [3](#Fig3){ref-type="fig"}). Thus, while it may not be sensible to re-test the same stimuli on a different occasion, simple modifications of the stimuli (e.g. changing orientation of looming gratings) that have been shown to enhance perception of novelty^[@CR47]^ could allow re-testing. To what extent visually driven behaviours are conserved in animals with different levels of visual impairment? {#Sec11} -------------------------------------------------------------------------------------------------------------- A fundamental concern about the use of visually driven innate responses in describing sight loss is the extent to which they are conserved in animals with impaired visual function. Clearly an all-or-none situation whereby innate behaviours are either reliably expressed or fully abolished would only allow for a binary discrimination between animals with intact or impaired function. Given the wide spectrum of visual impairments occurring in different mouse models of ocular diseases and individual variability in degeneration and treatment efficacy this kind of binary discrimination would be of little use. In this study we used well characterised mouse models of retinal degeneration (see e.g.^[@CR36],[@CR48]^ for rd1 and^[@CR42],[@CR49]^ for rd12) and we showed that visually driven innate responses allow for a finer discrimination across different levels of visual impairments. Thus, quantitative differences (in flash responses between rd1 age groups, Fig. [4a](#Fig4){ref-type="fig"}) as well as qualitative differences (presence or absence of freeze responses to spatial stimuli, Fig. [4e--h](#Fig4){ref-type="fig"}) allowed for discrimination between severity and type of retinal degeneration. Our test is mainly intended to probe retinal function. However since degeneration of retinal output can potentially induce plastic changes in all retinal targets (see^[@CR50]^ for a systematic assessment of retinal projections) it is also possible that those downstream changes also play a role in the suppression of visually evoked behaviours." Conclusions {#Sec12} =========== Our results indicate that systematic assessment of innate responses elicited by parameterisable stimuli represent a viable approach for measuring visual capabilities both in mice with intact visual function and in mouse models of retinal degeneration. Furthermore innate behaviours have several advantages over other established tests of visual function. The tests we propose are quick; can be performed on naïve mice; do not require specialist training for the experimenter; and the analyses are all automated. Compared with tests based on brainstem driven reflexes, such as optokinetic nystagmus or optomotor following, they capture higher limits in spatial acuity that match those obtained with the visual water task. Unlike the visual water task they do not require preliminary training sessions that can last several weeks in animals with limited vision^[@CR7]^, can cause significant distress^[@CR14]^ and introduce further variability in the results due to the learning process itself^[@CR16],[@CR17]^. Finally, the use of innate behaviours allows for recording a wider repertoire of behavioural responses, such as speed increments after full field flashes and freeze after looming, increasing the experimenter's ability to discriminate different levels of visual function. Methods {#Sec13} ======= Ethical statement {#Sec14} ----------------- Experiments were conducted in accordance with the Animals, Scientific Procedures Act of 1986 (United Kingdom) and approved by the University of Manchester ethical review committee. Experimental set-up {#Sec15} ------------------- We used an open field arena and recorded the animals with 2 programmable global shutter cameras (Chamaleon 3 from Point Grey) stably mounted on optical rails (Thorlabs; Fig. [1a](#Fig1){ref-type="fig"}). In order to avoid saturation due to changing light levels in the visual stimuli the camera lenses were covered with infrared cut-on filters (Edmund Optics) and fed with constant infrared light. Visual stimuli were delivered by a projector onto a rear projection screen. The flashes for the full field contrast stimuli were provided by two LEDs mounted inside the arena (LED Engin LZ4-00B208; controlled by T-Cube drivers, Thorlabs). The experiments were controlled by using Psychopy (version 1.82.01)^[@CR51]^. Frame acquisition was synchronized with the projected images and across multiple cameras by a common electrical trigger delivered by an Arduino Uno board (arduino.cc) controlled by Psychopy through a serial interface (pyserial). Triggered acquisition through external TTL from the Arduino board was enabled on Chamaleon 3 cameras through FlyCapture2 software (from Point Grey). All movies were encoded as M-JPEG from RGB 1280 (W) × 1040 (H) images. For tracking RGB images were converted to grayscale. All stimuli were calibrated by using a spectroradiometer (Bentham Instruments) and retinal irradiance values for each photoreceptor were calculated by using Govardovskii templates^[@CR52]^ and lens correction functions^[@CR53]^ as previously described^[@CR54],[@CR55]^. Summary information for all stimuli are provided in Tables [1](#Tab1){ref-type="table"}, [2](#Tab2){ref-type="table"}. For all experiments we delivered 7 blocks of 2--3 different stimuli whose order was independently randomized within each block. We set inter-stimulus interval at 73 seconds. For each trial the video recording started 6 seconds before and ended 7 seconds after the stimulus onset. Framerate was set at 15 Hz and allowed us to collect 200 frames per trials. Animal resources, housing and handling {#Sec16} -------------------------------------- The following animals were used in this study: visually intact C57Bl/6 mice (n = 15, all male), retinally degenerate *rd1* (n = 20 mice, 19 male, 1 female) and *rd12* (n = 7, all male) mice. The *rd12* strain was provided by Prof. Robin Ali at University College London and is on a C57Bl/6 J background. This *rd12* strain originates from Jax Laboratories (JAX stock \#005379). The visually intact C57Bl/6 mice were obtained from the Biological Services facility at University of Manchester, originally from Envigo. The *rd1* mice are on a mixed C57Bl/6 × C3H/HeNCrL background bred at the University of Manchester and were originally obtained from Prof. Mark Hankins at the University of Oxford. All mice were stored in cages of 2--4 individuals and were provided with food and water ad libitum. During transfer between the cage and the behavioural arena we used the tube handling procedure instead of tail picking, as prescribed in^[@CR15]^, in order to minimise stress and reduce variability across animals.Mice were kept on a 12:12 light dark cycle, with behavioural testing conducted during the light phase. Immunohistochemistry and cell imaging {#Sec17} ------------------------------------- Mice were culled by cervical dislocation, enucleated and eyes pierced with 24 gauge needle before being transferred to 4% paraformaldehyde in phosphate buffered saline (PBS) and stored overnight at 4 °C. Retinas were then dissected and permeabilised in PBS with 1% Triton-X for 3 × 10 mins. A background block incubation in PBS with 1% TritonX and 10% Donkey serum was conducted before retinas were incubated in primary antibody solution: PBS with 1% Triton-X with 2.5% donkey serum with 1:250 dilution of rabbit anti-short-wavelength sensitive (SWS) cone opsin antibody (AB5407, Merck Millipore) overnight at 4 °C. This anti-SWS cone opsin antibody is well-characterised for the fluorescent labelling of S-cones in the mouse retina^[@CR56]--[@CR58]^. Retinas were then washed in PBS with 0.2% TritonX for 4 × 1 hour, then incubated in secondary antibody solution: PBS with 1% TritonX and 2.5% Donkey serum with 1:250 dilution of donkey anti-rabbit Alexa 546 (A10040, Thermo Fisher) overnight at 4 °C. Retinas were washed in PBS with 0.2% TritonX for 4 × 1 hour, then washed in distilled water for 10 mins before being mounted ganglion cell-side up on microscope slides using Prolong Gold anti-fade mounting reagent (Thermo Fisher) and left to cure overnight at room temperature. Slides were stored at 4 °C until imaging. Images were acquired using a Leica DM2500 microscope and Leica DFC365 FX camera with CoolLED pE300-white light source. Images were collected using Leica Aplication Suite Advanced Fluorescence6000 (LAS AF6000) software. The filters used were Chroma ET Y3 filter set (excitation = 545 nm, emission = 610 nm). For x20 air objective; exposure time = 100 ms and gain = 2. For x100 oil objective; exposure time = 50 ms and gain = 2. Global enhancements of brightness and contrast were applied equally to all 20× (+40% brightness and +20% contrast) and 100× (+20% brightness and +40% contrast) images using ImageJ in Supplementary Fig. [7](#MOESM1){ref-type="media"}. Mouse tracking {#Sec18} -------------- We first isolated the mouse body by using a static background acquired at the beginning of each experiment before introducing the animal into the arena. The initial subtraction was refined by an opening operation (erosion followed by dilation) that removed mouse excrements that occasionally accumulated on arena floor during the experiment. These operations allowed us to extract a tightly bounded box around the animal body that sped up subsequent calculations. We found that the classic Harris corner detector was effective in detecting most relevant landmarks such as ears, snout tip, paws extremities, tail base and whiskers. However when just applied to the original image it only returned a partial list of features per image. In order to increase the yield of Harris detector we applied it to the associated 6 levels image pyramid representation (sample factor = 1.25 with 13 points Gaussian smoothing, σ = 1.25). This returned a dense labelling of body landmarks as shown in Fig. [1b](#Fig1){ref-type="fig"} (bottom panels). In order to quantify landmark speed the landmarks between two successive images were initially matched by using Pearson's correlation. Typically not all matches were correctly assigned and incorrect assignments could introduce artefactual jumps in the behavioural time series. To address this problem we applied the following algorithm to identify and reject outliers. We estimated a partial Procrustes superimposition (rotation + translation) between matched landmarks coordinates. The estimation was performed by employing the RANSAC algorithm^[@CR59]^ that allowed us to reject outliers given a fixed error threshold (inlier threshold = 10 pixels). While a single linear transformation was effective in capturing movements with consistent direction and amplitude across the whole mouse body (Fig. [1b](#Fig1){ref-type="fig"}, right panel) it failed to capture movements where different body parts where displaced in different directions and/or with different amplitudes. In order to capture the whole gamut of mouse movements we extended our approach by combining clustering and Procrustes analysis. Thus landmark coordinates were clustered via the *kmeans* ++ algorithm^[@CR60]^ by systematically varying K, the number of clusters (K = \[1, 5\]; for each value of K clustering was performed 10 times to avoid suboptimal solutions). Procrustes partial superimposition was then separately estimated for each cluster (Fig. [1b](#Fig1){ref-type="fig"}, centre panel). To identify the optimal number of clusters we then compared the residuals by using as criterion the Minimum Description Length^[@CR61]^ that allowed us to incorporate model complexity (i.e. the number of clusters) and the cost of different number of outliers. The description length of each model was defined as:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$DL={L}_{which\_model}+{L}_{outlier}+{L}_{model}+{L}_{residuals}$$\end{document}$$and$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\begin{array}{ccc}{L}_{which{\rm{\_}}model} & = & Nlo{g}_{2}(K+1)/ln(2)\\ {L}_{outlier} & = & {N}_{outlier}lo{g}_{2}({N}_{x}{N}_{y})/ln(2)\\ {L}_{model} & = & \frac{3}{2\,{\rm{l}}{\rm{n}}(2)}lo{g}_{2}(\prod _{i=1}^{K}{N}_{i})\\ {L}_{residuals} & = & \sum _{i=1}^{K}\{\begin{array}{c}\frac{{N}_{i}}{2}[{\rm{l}}{\rm{n}}(2\pi {\sigma }_{{x}_{i}}^{2})+\,{\rm{l}}{\rm{n}}(2\pi {\sigma }_{{y}_{i}}^{2})]+\sum _{j=1}^{{N}_{i}}\frac{{({x}_{i,j}-{\mu }_{{x}_{i}})}^{2}}{2{\sigma }_{{x}_{i}}^{2}}+\sum _{j=1}^{{N}_{i}}\frac{{({y}_{i,j}-{\mu }_{{y}_{i}})}^{2}}{2{\sigma }_{{y}_{i}}^{2}}\,\end{array}\}\end{array}$$\end{document}$$where *N*~*x*~*N*~*y*~ represents the area of the bounding box around the animal, *N*~*outlier*~ the total number of outliers, *N*~*i*~ the number of inliers for the *i*^*th*^ cluster, *x*~*i*,*j*~ the *j*^*th*^ residual of the *i*^*th*^ cluster on the x direction. The first term of Eq. [1](#Equ1){ref-type=""}, *L*~*which_model*~ accounts for the number of *nats* (natural bits) required to identify each point as outlier or belonging to the *i*^*th*^ cluster. The second term costs the outliers as bounded integers in the area bounding box. The third term penalizes for the number of clusters and the factor 3 corresponds to the number of parameters required to define the partial Procrustes superimposition for each cluster (1 for rotation, 2 for translation). The final term uses the fact that the cost of encoding residuals can be approximated by the negative log likelihood. In order to compare results obtained with this technique with more standard tracking of mouse body centre we also performed additional tracking to evaluate whole body movements. To extract mouse body centre in each frame we first performed background subtraction followed by opening operation as described above. All pixels below a pre-specified threshold were inactivated. The body centre was then calculated as median coordinates of the remaining pixel positions. Generation of movement time series {#Sec19} ---------------------------------- We used the distribution of landmarks speed to generate multidimensional behavioural time series. We first calculated the speed of each landmark between two consecutive frames (Fig. [1b](#Fig1){ref-type="fig"}, bottom panels). From the distribution of landmarks speed we then calculated the 10^th^, 30^th^, 50^th^, 70^th^, 90^th^ speed quantiles for each frame (Fig. [1b](#Fig1){ref-type="fig"}, top panels; respectively Q10 to Q90). Each of these quantiles, estimated separately for each of the two cameras used (Fig. [1a](#Fig1){ref-type="fig"}), defined a dimension of the multivariate time series (Fig. [1c](#Fig1){ref-type="fig"}). For all subsequent analyses the quantiles were then averaged across the cameras (Figs [2a](#Fig2){ref-type="fig"}--[4a,e,g](#Fig4){ref-type="fig"}). Changepoints detection {#Sec20} ---------------------- In order to find the number and the location of the changepoints we used the Pruned Exact Linear Time method (PELT) (Killick *et al*.^[@CR26]^). PELT is designed to minimize a penalised cost function of the following form:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\sum _{i=1}^{m+1}[C({y}_{({\tau }_{i-1}+1):{\tau }_{i}})]+\beta m$$\end{document}$$where *C* is the cost function, *τ* represents the changepoints, *m* is the number of changepoints and β is the penalty for each additional change. This method has two main advantages: it calculates the global optimum of (3) and it does so in a computational cost, under mild conditions, that scales linearly with the number of data points. We define the cost function *C* as follows:$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$C({y}_{({\tau }_{i-1}+1):{\tau }_{i}})=\sum _{{\tau }_{i-1}+1}^{{\tau }_{i}}{({y}_{i}-{y}_{({\tau }_{i-1}+1):{\tau }_{i}})}^{2}$$\end{document}$$where \<y\> represents the average speed between two successive changepoints. The β term in (3) represents a penalty against overfitting. Popular choices for the penalty function include the Schwarz Information Criterion (Schwarz, 1978). Several authors also proposed different solutions to this problem by adaptations of the SIC to the problem of multiple changepoint analyses (Zhang and Siegmund, 2007). As a general consensus lacks about which criterion is more convenient for a given dataset we devised a method to automatically derive a suitable penalty range from the data. Therefore, instead of specifying a penalty value, for each trial we systematically scanned across a wide range of β values by using the Changepoint for a Range Of PenaltieS (CROPS) algorithm (Haynes *et al*., 2017). The CROPS algorithm efficiently finds all the sets of changepoints whose segmentations are optimal under some choice of β within an interval \[βmin, βmax\] by sequentially dividing this interval until guaranteed convergence. We then used the output of this algorithm, the number of changepoints as function of penalty values, to fit a piecewise linear model represented by two lines intersecting at a "knee" point (Supplementary Fig. [1b](#MOESM1){ref-type="media"}). The knee point marks the transition between under and over fitting and thus represents an ideal choice for the penalty. The segmented relationship between the number of changepoints and the penalty value β was modelled following (Muggeo, 2003) as$$\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$E[m]=a\beta +b{(\beta -\psi )}_{+}+c$$\end{document}$$where parameters *a* and *b* represent respectively the slope of the left line segment and the difference in slopes between the left and the right line, $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${(\beta -\psi )}_{+}$$\end{document}$equals 1 for $\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta > \psi $$\end{document}$ and 0 otherwise, and *ψ* represents the knee point. By using the iterative estimation proposed by (Muggeo, 2003) we then obtained a maximum likelihood estimation of all the parameters including the knee point (Supplementary Fig. [1b](#MOESM1){ref-type="media"}). We repeated this procedure across all trials to obtain the distribution of knee values for our datasets that provided a suitable range for choosing the penalty value β (Supplementary Fig. [1c](#MOESM1){ref-type="media"}). Statistics {#Sec21} ---------- Statistics derived from tracking data were applied either to the raw multivariate time series (for full field flash) or to a truncated log transformation of these data (for loom + gratings). The former was positively skewed, the second negatively skewed. Accordingly changepoints applied to the raw data was more biased towards detecting positive changes in movement, such as flights, the second towards negative changes such as freezing. The significance of behavioural responses to individual stimuli was assessed by using the two-tailed signtest (in Figs [2a](#Fig2){ref-type="fig"}--[4a,e,g](#Fig4){ref-type="fig"}). The test statistic was obtained by subtracting the mean landmarks speed after stimulus onset from that recorded before the stimulus onset. Speed values were estimated in time windows of equal duration (0.53 seconds for both looming and flash). Comparison between flash responses was performed on the same speed distributions by using the two tailed Wilcoxon rank-sum test (e.g. between "young" and "old" rd1 mice, Fig. [4a](#Fig4){ref-type="fig"}). Statistical significance for changepoints was calculated by subtracting the rate of change points before - *\#chp/s(pre)* - and after - *\#chp/s(post)* - stimulus onset using the same time windows and applying a two-tailed Wilcoxon rank-sum test. Thus the difference in changepoint rate (Δ\#chp/s in Figs [2b](#Fig2){ref-type="fig"}--[4d,f,h](#Fig4){ref-type="fig"}) was calculated as Δ\#chp/s = *\#chp/s(pre) - \#chp/s(post)*. Statistical significance of intensity dependence for flash responses shown in Figs [2b](#Fig2){ref-type="fig"}, [4d](#Fig4){ref-type="fig"} was calculated from the same data by using Kruskal-Wallis test. In order to test habituation both in speed and changepoint responses we used permutation tests. First the difference in speed responses (or changepoint responses) was calculated between early and late trials (Fig. [5a,b](#Fig5){ref-type="fig"}). Then this difference was recalculated 100000 times by shuffling the order of the stimulus trials and a p-value was determined as the fraction of instances in which these surrogate values were larger than the original statistic. All statistical tests presented in the main and supplementary figures are detailed in Supplementary Tables [1](#MOESM1){ref-type="media"}, [2](#MOESM1){ref-type="media"}. Software resources {#Sec22} ------------------ Changepoint analyses were performed in R^[@CR62]^. The code for changepoint analysis, including the CROPS method, can be found in the 'changepoint' package^[@CR63]^. For knee estimation we used the 'segmented' package^[@CR64]^. The code for mouse tracking was written in MATLAB (Matworks, Natick, Massachusetts, USA). Supplementary information ========================= {#Sec23} Supplementary Information movie 1 movie 2 movie 3 movie 4 movie 5 movie 6 movie 7 movie 8 movie 9 movie 10 movie 11 movie 12 **Publisher's note:** Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Supplementary information ========================= **Supplementary information** accompanies this paper at 10.1038/s41598-019-46836-y. We thank Annette Allen for thorough discussions during the preparation of the manuscript; Sumayyah Zanna Muhammad for helpful feedback during the early phase of the project; and Robin Ali at University College London for supplying us with the rd12 mice. The work was funded by National Centre for Replacement Refinement and Reduction of Animals in Research (NC3Rs) via a David Sainsbury Fellowship (NC/P001505/1) to R.S. and a project grant from the Medical Research Council to R.J.L. (MR/N012992/1). R.S. designed the study; designed and performed the experiments; analysed the data; wrote the manuscript; provided hardware resources; provided computational resources J.R. performed the experiments; analysed the data. M.G. performed the experiments. F.P.M. provided hardware resources. J.W. provided hardware resources. S.R. provided computational resources. C.J.T. provided computational resources. T.F.C. provided computational resources. R.K. provided computational resources; wrote the manuscript. R.J.L. wrote the manuscript. Data will be made available on request. Contact [riccardo.storchi\@manchester.ac.uk]{.ul}. The authors declare no competing interests.

INTRODUCTION ============ Morel-Lavallee lesions (MLL) are rare injuries that occur due to a traumatic shearing force or crush injury acting on the skin surface that causes a separation of the skin and subcutaneous tissue from the underlying fascia. This mechanism of the injury is referred to as internal degloving. The traditional and most common location of these injuries is the lateral hip/greater trochanter. Other frequent areas include the pelvis, thigh, and knee.[@b1-wjem-16-438] The separation of the subcutaneous tissue from the fascia in MLLs causes a disruption of the lymphatics and blood vessels in the affected region. This precipitates the accumulation of fluid in this newly formed potential space. Subsequently, the formation of a hematomas or seromas occur.[@b2-wjem-16-438] The inflammatory reaction that ensues if these injuries are not treated in the acute phase can organize granulation tissue into a fibrous capsule.[@b1-wjem-16-438],[@b3-wjem-16-438] This capsule impedes the absorption of the fluid and is thought to be the cause of recurrent fluid collection even after drainage,[@b3-wjem-16-438] MLLs are often not diagnosed initially. Kottmeir et al. reported that they are missed up to 44% of the time.[@b4-wjem-16-438] Early detection and treatment of MLLs is vital to circumvent complications such as re-accumulation of fluid, infection-related morbidity, and chronic pain.[@b5-wjem-16-438],[@b6-wjem-16-438] Recent studies advocate for early treatment via drainage and possible debridement of acute and subacute lesions.[@b2-wjem-16-438],[@b7-wjem-16-438],[@b8-wjem-16-438] We present a patient with a massive MLL of the medial thigh that was initially diagnosed as a quadriceps contusion, which caused delayed treatment. We discuss the pertinent ultrasound and magnetic resonance imaging (MRI) findings as well as address the importance of early identification and proper management of these lesions. CASE ==== The patient was a 22-year-old active duty male in the Navy who presented to the emergency department (ED) with a massively swollen, bruised, and painful right thigh/knee after falling down the stairs onto his knee 11 days prior. Before seeking treatment at the ED he saw his primary care provider and was treated with decadron and toradol injections. He was also given oral nonsteroidal anti-inflammatory drug (NSAIDS), muscle relaxants, an ACE wrap, and crutches. The patient denied fever and chills. His medical history was significant only for hypertension. On physical exam he had a large fluctuant fluid collection along the medial aspect of his right thigh as well as diffuse ecchymosis of the leg centered over the knee. There was no joint line tenderness and no ligamentous laxity of the knee joint. He had full range of movement at the knee, and he was neurovascularly intact distally. All compartments of the leg were soft. Plain films of right knee, femur, tib/fib, were significant for soft tissue swelling and no osseous abnormality. Bedside ultrasound in the ED showed approximately 500mL of subcutaneous, anechoic fluid near the vastus medialis. He was diagnosed with a quadriceps contusion and managed with NSAIDs, compressive dressing, knee immobilizer, and follow up with orthopedics. The patient presented again to the ED approximately three weeks later with worsening pain and swelling of the right thigh and knee. Ultrasound demonstrated a fluid collection measuring 26cm cranio-caudid × 6.2cm AP × 13.8cm transverse ([Figure 1](#f1-wjem-16-438){ref-type="fig"}). The lesion was percutaneously drained and 1900mL of serosanguineous fluid was expressed. Compressive dressing and knee immobilizer were placed and follow up in one week was recommended. One week later the patient had re-accumulation of the fluid and the decision for surgical irrigation and debridement (I and D) with negative pressure wound dressing placement was made. A pre-operative MRI was obtained ([Figure 2a](#f2a-wjem-16-438){ref-type="fig"}--[d](#f2d-wjem-16-438){ref-type="fig"}). The patient eventually underwent one more surgical I and D with delayed primary closure, and at that time the fluid collection had completely resolved. This was more than a month and a half after his initial presentation to the ED. DISCUSSION ========== This case is clinically significant for two primary reasons. First, the lesion was extremely large for its location. There are very few published accounts of massive MLLs occurring in the medial thigh/knee. To our knowledge our patient's lesion may in fact be the largest documented in this region, measuring at 26cm cranio-caudal, 6.2cm AP, and 13.8cm transverse and draining 1900cc. Multiple lesions of this magnitude have been described along the lateral thigh/greater trochanter, but after a thorough literature review only two other lesions that possibly were of similar magnitude in the medial thigh/knee were found. Jones et al. presented a large MLL in a 70-year-old women who had been hit by a car. Her lesion measured 30 × 15cm, but the article did not describe a fluid volume.[@b9-wjem-16-438] The other case is of a 26-year- old male who fell on his anteromedial knee during a soccer match. The case simply describes a medial thigh/knee MLL as massive, but does not make comment of measurements.[@b10-wjem-16-438] Most of the lesions caused by trauma to the knee are significantly smaller than the one described on our case. A 2007 study evaluated 27 cases of MLLs of the knee in the National Football Leauge. The largest suprapatellar and midthigh lesions in this study were up to 300mL in size. The mean amount of fluid that could be aspirated from an area of fluctuance in the knee or thigh was only 46mL with a range of 12--120mL.[@b11-wjem-16-438] The second reason this case is clinically significant is that it demonstrates the importance of early identification and proper treatment of MLLs, which are very often not diagnosed initially.[@b4-wjem-16-438] Our patient's lesion was first thought to be a quadriceps contusion. Complications including recurrence, infection, and chronic pain arise when MLLs are not treated in the acute or subacute window. Although there was evidence present in the history, physical exam, and ultrasound to indicate a MLL, due to its rarity the diagnosis was not made at first. The history detailed a traumatic sheer injury to the soft tissue of the knee. The exam demonstrated diffuse ecchymosis and the hallmark finding of MLLs, a palpable, soft, fluctuant mass over the medial thigh and knee.[@b10-wjem-16-438],[@b11-wjem-16-438] On ultrasound exam the fluid collection was compressible, anechoic and located subcutaneously. In a retrospective study of 21 MLLs of the hip and thigh all demonstrated hypoechoic or anechoic echogencity, were compressible and were located in between the deep subcutaneous fat and the fascia.[@b5-wjem-16-438] Once a fluid collection has been identified as a MLL, research demonstrates that timely intervention via drainage with or without debridement is essential to avoid potential complications. In a 2013 retrospective study of 87 MLLs Nickerson et al. demonstrated that lesions with volumes exceeding 50mL on aspiration were especially prone to reoccur, even after percutaneous aspiration. Specifically, 83% of lesions that drained more than 50ml recurred. This study recommends that lesions with \>50ml aspirated require operative drainage via incision and insertion of suction drain.[@b2-wjem-16-438] In a different study of 19 patients with MLLs the authors used operative percutaneous drainage, irrigation and debridement with drain placement to treat large lesions averaging 30×12cm. The study demonstrated prevention of recurrence in all patients treated, and recommended treatment within 3 days if possible.[@b12-wjem-16-438] A similar study used operative percutaneous drainage, debridement, catheter placement and suction of MLL's at a mean time of 11.9 days from time of injury to intervention. This method was also successful in preventing lesion recurrence in all patients.[@b13-wjem-16-438] Through earlier diagnosis and following the recommendations to operatively drain with or without debridement we propose that our patient may have avoided lesion recurrence and would have healed faster. Although MLLs are admittedly a rare diagnosis, a persistent subcutaneous fluid collection in the setting of trauma should raise clinical suspicion of an underlying MLL. *Section Editor:* Rick A. McPheeters, DO Full text available through open access at <http://escholarship.org/uc/uciem_westjem> *Conflicts of Interest*: By the *West*JEM article submission agreement, all authors are required to disclose all affiliations, funding sources and financial or management relationships that could be perceived as potential sources of bias. The authors disclosed none. {#f1-wjem-16-438} {#f2a-wjem-16-438} {#f2b-wjem-16-438} {#f2c-wjem-16-438} {#f2d-wjem-16-438}

All quantitative data and code will be opely available on the Open Science Framework <https://osf.io/qxt3u/>, DOI: [10.17605/OSF.IO/QXT3U](https://doi.org/10.17605/OSF.IO/QXT3U). Due to ethical constraints, only re-ordered qualitative information can be openly shared (in publicly available data there is no way to link these qualitative responses to any other answers given by participants). This data is sufficient to reproduce all of the results presented in this article. The full raw dataset is stored on the same OSF page but kept private. Authors will provide access for the purpose of validation and fraud detection. Introduction {#sec001} ============ All forms of science communication, including traditional journal articles, involve transforming complicated, often messy data into a coherent narrative form. O'Boyle et al \[[@pone.0200303.ref001]\] likened the process to a Chrysalis effect, turning "ugly initial results into beautiful articles". Repeated failures to reproduce a large proportion of results in the published literature of other disciplines (e.g. \[[@pone.0200303.ref002],[@pone.0200303.ref003]\]) has triggered reflection and meta-research about the ways in which this transformation process is susceptible to confusion and corruption. To date, large scale meta-research and replication projects have not been conducted in ecology and evolution \[[@pone.0200303.ref004],[@pone.0200303.ref005]\]. However, many of the drivers of low reproducibility in other fields, such as publication bias and inflated type I errors, also appear common in ecology and evolution \[[@pone.0200303.ref006]--[@pone.0200303.ref009]\]. For example, Jennions and Møller \[[@pone.0200303.ref010]\] found that 38% of meta-analyses appeared to suffer publication bias, and that adjustments for missing (file drawer) studies changed the statistical conclusion (from statistically significant to non-significant) in 21% of cases. Low statistical power is a long-standing problem in ecology and evolution \[[@pone.0200303.ref011]\], and publishing only statistically significant studies from a pool with low average statistical power selects for inflated effect sizes and type I errors \[[@pone.0200303.ref012]\]. Forstmeier et al \[[@pone.0200303.ref011]\] further explain how, under the conditions of publication bias, Questionable Research Practices like *p* hacking and underpowered research can inflate number of false positive results in the literature. They offer a table of solutions for a range of problematic practices, all specifically relevant to research in ecology and evolution. The majority of advice concerns changes that individual researchers can make to improve the quality of their own research. However, some initiatives look to change the institutions and culture that influence individual behaviour by improving reporting standards in ecology and evolution journals \[[@pone.0200303.ref012],[@pone.0200303.ref013]\] or switching the emphasis of research from p values to effect sizes \[[@pone.0200303.ref014],[@pone.0200303.ref015]\] The widespread prevalence of Questionable Research Practices (QRPs) is now well documented in psychology \[[@pone.0200303.ref016]--[@pone.0200303.ref018]\]. However, this is the first attempt (to the best of our knowledge) to document the prevalence of such practices in ecology and evolution. What are Questionable Research Practices (QRPs)? {#sec002} ------------------------------------------------ QRPs refer to activities such as *p* hacking, cherry picking, and Hypothesizing After Results are Known (HARKing), all of which have been well documented in other fields including psychology and medicine. *Cherry picking* includes failing to report dependent or response variables or relationships that did not reach statistical significance or other threshold and/or failing to report conditions or treatments that did not reach statistical significance or other threshold. *P hacking* refers to a set of activities: checking the statistical significance of results before deciding whether to collect more data; stopping data collection early because results reached statistical significance; deciding whether to exclude data points (e.g., outliers) only after checking the impact on statistical significance and not reporting the impact of the data exclusion; adjusting statistical models, for instance by including or excluding covariates based on the resulting strength of the main effect of interest; and rounding of a *p* value to meet a statistical significance threshold (e.g., presenting 0.053 as *P* \< .05). *HARKing* includes presenting ad hoc and/or unexpected findings as though they had been predicted all along \[[@pone.0200303.ref019]\]; and presenting exploratory work as though it was confirmatory hypothesis testing \[[@pone.0200303.ref020]\]. John et al \[[@pone.0200303.ref017]\] surveyed over 2000 psychological researchers in the US and asked about the prevalence of several questionable practices (we included questions about six of these practices in our survey, see [Table 1](#pone.0200303.t001){ref-type="table"}). Agnoli et al \[[@pone.0200303.ref016]\] repeated John et al's survey with a sample of Italian psychologists, and found strikingly similar results (also shown in [Table 1](#pone.0200303.t001){ref-type="table"}). Failure to report outcome measures and stopping rules has also been documented by LeBel et al \[[@pone.0200303.ref021]\]. O'Boyle et al \[[@pone.0200303.ref001]\] found that in the process of translating PhD theses' results to published articles the proportion of results supporting statistical hypotheses doubled; a change accounted for by the cherry picking of significant results. 10.1371/journal.pone.0200303.t001 ###### Journals used to identify researchers working in ecology and evolution. {#pone.0200303.t001g} Ecology Journals Evolution Journals ------------------------------------------ --------------------------------- Trends in Ecology and Evolution Evolutionary Application Ecology Letters Evolution Annual Review of Ecology and Evolution BMC Evolutionary Biology Frontiers in Ecology and the Environment Evodevo Global Change Biology American Naturalist Ecological Monographs Journal of Evolutionary Biology Methods in Ecology and Evolution Evolutionary Biology Journal of Ecology Evolutionary Ecology Global Ecology and Biogeography Behavioural Ecology ISME Journal of Applied Ecology Publication bias and publish-or-perish research culture {#sec003} ------------------------------------------------------- Publication bias in this context refers to a bias towards publishing statistically significant, 'positive' results and not publishing statistically non-significant ('negative' or null results). The bias exists in many sciences \[[@pone.0200303.ref022]\], has been documented for decades in some disciplines (e.g., in psychology, see Sterling, 1959 \[[@pone.0200303.ref023]\]) and may be getting stronger across science, with a detectable increase in the proportion statistically significant results over the last 25 years \[[@pone.0200303.ref024]\]. The intersection of increasing publication bias and a growing publish-or-perish culture in science may well impact the frequency with which researchers employ QRPs \[[@pone.0200303.ref016],[@pone.0200303.ref025]\]. In a publish-or-perish research culture, studies that were once relegated to a file drawer upon failing to reach statistical significance may now be more likely to be cherry picked, *p* hacked and HARKed back into the literature. In a simulation study, Smaldion & McElreath \[[@pone.0200303.ref026]\] demonstrate how selection for higher output can speed up the dissemination of poor methods within a research community. Simmons et al \[[@pone.0200303.ref027]\] used simulated experimental data to demonstrated how QRPs such as reporting only the subset of dependent/response/outcome variables or experimental conditions that reached statistical significance can inflate the false positive error rate of the research literature. They warned of 'researcher degrees of freedom' in experimental reports, including failing to report the sampling stopping rule. This has been further demonstrated in an ecology and evolution context by Forstmeier et al \[[@pone.0200303.ref011]\]. QRPs, due to their propensity to increase the false positive rate, have been implicated as a contributing factor to the well-publicised reproducibility crisis in psychology and other disciplines \[[@pone.0200303.ref002],[@pone.0200303.ref025],[@pone.0200303.ref028]\]. Aims {#sec004} ---- Publication bias in a publish-or-perish research culture incentivises researchers to engage in QRPs, which inflate the false positive rate leading to a less reproducible research literature. In this sense, QRP rates might be indicators of future reproducibility problems. Arguments about the difficulties in directly evaluating the reproducibility of the ecology and evolution literature have been made elsewhere (e.g., Schnitzer & Carson \[[@pone.0200303.ref029]\] but see Nakagawa & Parker \[[@pone.0200303.ref004]\]). However, the link between QRPs and irreproducibility is rooted in fundamental statistical theory \[[@pone.0200303.ref030]\] and so even in the absence of direct replication measures, a high prevalence of QRPs should alone raise sufficient concern to trigger editorial and institutional action. The specific aims of our research were to: 1. Survey ecology and evolution researchers' own self-reported rate of QRP frequency 2. Survey ecology and evolution researchers' estimated rate of QRP use in their field 3. Compare these rates to those found in other disciplines, particularly psychology, where serious reproducibility problems have been established 4. Explore, through researchers' open-ended comments on each QRP in the survey, attitudes, (mis)understandings, pressures and contexts contributing to QRP use in the discipline Methods {#sec005} ======= Survey participants {#sec006} ------------------- We collected the email addresses of corresponding authors from 11 'ecology' and 9 'evolutionary biology' journals (see [Table 1](#pone.0200303.t001){ref-type="table"}) in line with ethics approval supplied the University of Melbourne Human Research Ethics Committee (Ethics ID: 1646917.1). Journals were chosen from the highest ranking (assessed by 5-year impact factor) journals within the categories defined by the ISI 2013 Journal Citation Reports \[[@pone.0200303.ref031]\]. From the highest impact journals, we selected those that publish a broad range of work and excluded those limited to narrower sub-fields. We extracted authors' email addresses articles published in ecology journal (first 10 ecology journals listed in [Table 1](#pone.0200303.t001){ref-type="table"}) issues between January 2014 and May 2016. We began a trial release of the survey (to check for bugs) on the 5^th^ of December 2016, we had sent the survey to all authors of articles in ecology journals by the 6^th^ of March 2016. Before we looked at the initial data, we decided to expand our sample to include evolutionary biology researchers, and add authors from articles from the Journal of Applied Ecology. We collated email addresses from authors of articles in evolutionary biology journal ([Table 1](#pone.0200303.t001){ref-type="table"}) issues and Journal of Applied Ecology issues between January 2015 and March 2017. We sent the email to these new participants on the 19^th^ of May 2017. We deduplicated our list of email addresses before we sent each survey out to ensure that individual researchers did not receive our survey more than once. We ultimately emailed a total 5386 researchers with a link to our online survey which returned 807 responses (response rate = 15%). Of the 807 responses, 71% (n = 573) were identified through our 'ecology' journal sample and 37% (n = 299) from our 'evolution' journal sample. This imbalance is a product of the number of journals in each sample and the order in which email addresses were collected and deduplicated; we first targeted ecology journals, and then decided to add a second group of evolution journals. Recognising that journal classification is only an approximate guide to disciplinary status, we asked researchers to self-identify their discipline; 411 researchers completed this question. Based on this information we made some adjustments to disciplinary classification as follows. First, we classified responses associated with sub-disciplines including the following terms as being made by evolution researchers: 'evolut\*', 'behav\*', 'reproductive', or 'sexual'. From the remaining set of descriptions, we classified all responses associated including the following terms as being made by ecology researchers: 'plant', '\*population', 'marine biology', 'biodiversity', 'community', 'environment\*', 'conservation', 'ecology', 'botany', 'mycology', or 'zoology'. Researchers who did not use any of these terms and those who did not complete the self-identified sub-discipline question (n = 396) were left in their original journal discipline category as outlined in [Table 1](#pone.0200303.t001){ref-type="table"}. At the end of this reclassification process, the sample (n = 807) consisted of 61% (n = 494) ecology researchers and 39% (n = 313) evolution researchers. Only 69% (558-560/807) of our sample completed the demographic questions at the end of our survey. Of the 560 who completed the gender question, 69% identified as male, 29% as female, 0.2% identified as non-binary and 1% preferred not to say. Of the 558 who completed the career status question, 6% identified as graduate students, 33% as post-doctoral researchers, 24% as midcareer researchers/academics and 37% as senior researchers/academics. The 559 who completed the age question were divided between age categories as follows: under 30 (11.5%), 30--39 (46.7%), 40--49 (25.9%), 50--59 (9.8%), 60--69 (4.8%), and over 70 (1.3%). Survey instrument {#sec007} ----------------- Our research practices survey was administered via Qualtrics (Provo, UT, USA).The survey ([S1 Supplementary Material](#pone.0200303.s001){ref-type="supplementary-material"}) included questions about the following ten research practices: 1. Not reporting studies or variables that failed to reach statistical significance (e.g. p ≤0.05) or some other desired statistical threshold. 2. Not reporting covariates that failed to reach statistical significance (e.g. p ≤0.05) or some other desired statistical threshold. 3. Reporting an unexpected finding or a result from exploratory analysis as having been predicted from the start. 4. Reporting a set of statistical models as the complete tested set when other candidate models were also tested. 5. Rounding-off a p value or other quantity to meet a pre-specified threshold (e.g., reporting p = 0.054 as p = 0.05 or p = 0.013 as p = 0.01). 6. Deciding to exclude data points after first checking the impact on statistical significance (e.g. p ≤ 0.05) or some other desired statistical threshold. 7. Collecting more data for a study after first inspecting whether the results are statistically significant (e.g. p ≤ 0.05). 8. Changing to another type of statistical analysis after the analysis initially chosen failed to reach statistical significance (e.g. p ≤ 0.05) or some other desired statistical threshold. 9. Not disclosing known problems in the method and analysis, or problems with the data quality, that potentially impact conclusions. 10. Filling in missing data points without identifying those data as simulated. Questions 1 to 9 were shown in random order but question 10 was always shown last, because it is particularly controversial and we did not want it to influence the responses to other items. For each of these 10 practices, researchers were asked to: i. estimate the percentage of ecology (evolution) researchers who they believe have engaged in this practice on at least one occasion (0--100%) ii. specify how often they had themselves engaged in the practice (never, once, occasionally, frequently, almost always) iii. specify how often they believe the practice *should* be used (never, rarely, often, almost always) At the end of each QRP, researchers had the opportunity to make additional comments under the open-ended question: 'why do you think this practice should or shouldn't be used?'. At the end of the set of 10 QRP questions, researchers were asked "have you ever had doubts about the scientific integrity of researchers in ecology (evolution)?", and asked to specify the frequency of such doubts, if any, for different sub-groups. Finally, the survey included demographic questions about participants' career stage, gender, age and sub-discipline, discussed above. Data analysis {#sec008} ------------- Analyses were preregistered after data collection had commenced but before the data were viewed \[[@pone.0200303.ref032]\] and performed in R version 3.3.3 \[[@pone.0200303.ref033]\]. The code and data required to reproduce our results are available from <https://osf.io/qxt3u/>. In [Fig 1](#pone.0200303.g001){ref-type="fig"} we plotted the proportion of researchers reporting that they had used each of the 10 QRPs at least once against the researchers' estimates of prevalence in the field, i.e., researchers' responses to question (i) above. For each of the 10 QRPs we also plotted the proportion (with 95% Confidence Intervals, CIs) of researchers in each discipline who stated that they had used the practice 'never', 'once', 'occasionally', 'frequently', and 'almost always' in response to question (ii) above using *ggplot2* \[[@pone.0200303.ref034]\] ([Fig 2](#pone.0200303.g002){ref-type="fig"}). For the QRPs also covered in the John et al \[[@pone.0200303.ref017]\] and Agnoli et al \[[@pone.0200303.ref016]\] surveys, we directly compared proportions of researchers who had engaged in each QRP at least once ([Table 2](#pone.0200303.t002){ref-type="table"}), as this is the primary frequency measure reported in those articles. We examined correlations between how frequently each participant had engaged in a practice and how acceptable they found the practice, and their age and career stage using Kendall's Tau correlation. All 95% CIs are Wilson Score Intervals except for those on Kendall's Tau, which are bootstrapped based on 1000 bootstrapped samples using *NSM3* \[[@pone.0200303.ref035]\]. ![The prevalence of Questionable Research Practices in ecology and evolution.\ Light columns represent the proportion of evolution researchers and dark columns represent the proportion of ecology researchers who reported having used a practice at least once. The dots show researchers' mean estimates of suspected use by colleagues in their field. Dots that are much higher than bars may suggest that the QRP is considered particularly socially unacceptable \[[@pone.0200303.ref017]\]. Error bars are 95% confidence intervals.](pone.0200303.g001){#pone.0200303.g001} {#pone.0200303.g002} 10.1371/journal.pone.0200303.t002 ###### Percentage (with 95% CIs) of researchers in psychology, ecology and evolution who reported having used each Questionable Research Practice at least once. n = 555--626. {#pone.0200303.t002g} ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Questionable Research Practice Psychology\ Psychology USA\ Ecology Evolution Italy\ John et al. \[[@pone.0200303.ref017]\] Agnoli et al. \[[@pone.0200303.ref016]\] ------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------ ---------------------------------------- -------------------- -------------------- Not reporting response (outcome) variables that failed to reach statistical significance[\#](#t002fn001){ref-type="table-fn"} 47.9\ 63.4\ 64.1\ 63.7\ *(41*.*3--54*.*6)* *(59*.*1--67*.*7)* *(59*.*1--68*.*9)* *(57*.*2--69*.*7)* Collecting more data after inspecting whether the results are statistically significant[\#](#t002fn001){ref-type="table-fn"} 53.2\ 55.9\ 36.9\ 50.7\ *(46*.*6--59*.*7)* *(51*.*5--60*.*3)* *(32*.*4--42*.*0)* *(43*.*9--57*.*6)* Rounding-off a p value or other quantity to meet a pre-specified threshold[\#](#t002fn001){ref-type="table-fn"} 22.2\ 22.0\ 27.3\ 17.5\ *(16*.*7--27*.*7)* *(18*.*4--25*.*7)* *(23*.*1--32*.*0)* *(13*.*1--23*.*0)* Deciding to exclude data points after first checking the impact on statistical significance 39.7\ 38.2\ 24.0\ 23.9\ *(33*.*3--46*.*2)* *(33*.*9--42*.*6)* *(19*.*9--28*.*6)* *(18*.*5--30*.*2)* Reporting an unexpected finding as having been predicted from the start[\#](#t002fn001){ref-type="table-fn"} 37.4\ 27.0\ 48.5\ 54.2\ *(31*.*0--43*.*9)* *(23*.*1--30*.*9)* *(43*.*6--53*.*6)* *(47*.*7--60*.*6)* Filling in missing data points without identifying those data as simulated[\*](#t002fn002){ref-type="table-fn"} 2.3\ 0.6\ 4.5\ 2.0\ *(0*.*3--4*.*2)* *(0*.*0--1*.*3)* *(2*.*8--7*.*1)* *(0*.*8--5*.*1)* ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- \#note that these statements began with "in a paper," in John et al. \[[@pone.0200303.ref017]\] and Agnoli et al \[[@pone.0200303.ref016]\]. \*note that this was referred to as "falsifying data" in John et al. \[[@pone.0200303.ref017]\] and Agnoli et al \[[@pone.0200303.ref016]\] which may have influenced the difference in response rates. Results {#sec009} ======= Overall, researchers in ecology and evolution reported high levels of Questionable Research Practices ([Table 2](#pone.0200303.t002){ref-type="table"}, [Fig 1](#pone.0200303.g001){ref-type="fig"}). However, the frequency with which researchers reported using these regularly was much lower ([Fig 2](#pone.0200303.g002){ref-type="fig"}) and qualitative analyses reveals use of these practices in ways that may be less questionable ([S2 Supplementary Material](#pone.0200303.s002){ref-type="supplementary-material"}). Comparing ecology, evolution and psychology researchers {#sec010} ------------------------------------------------------- The responses for ecology and evolution researchers were broadly similar to those from the samples of psychologists studied by John et al. \[[@pone.0200303.ref017]\] and Agnoli et al \[[@pone.0200303.ref016]\] ([Table 2](#pone.0200303.t002){ref-type="table"}). One exception to this is that ecologists were less likely than psychologists or evolution researchers to report 'collecting more data after inspecting whether the results are statistically significant' (see also [Fig 1](#pone.0200303.g001){ref-type="fig"}). Both ecology and evolution researchers were also less likely to report excluding data points after checking significance than psychologists. On the other hand, both ecology and evolution researchers were more likely to acknowledge reporting an unexpected finding as expected than both samples of psychologists. Self-reported QRP use compared to expected QRP use amongst colleagues {#sec011} --------------------------------------------------------------------- Broadly, researchers' self-reported QRP use was closely related to their estimates of prevalence of QRPs in the scientific community ([Fig 1](#pone.0200303.g001){ref-type="fig"}). However, in the case of QRPs 2, 5, 6, 9 and 10, expected prevalence was substantially higher than individual self-reported use, suggesting that these may be considered the least socially acceptable QRPs in the set. Frequency of individual researchers' QRP use {#sec012} -------------------------------------------- It was extremely rare for researchers to report high frequency ('frequently', 'almost always') use of QRPs. Most reported usage was at low frequency ('once', 'occasionally'), with many researchers reporting they had never engaged in these practices ([Fig 2](#pone.0200303.g002){ref-type="fig"}). Age and career stage were not strong predictors of how frequently researchers used Questionable Research Practices (Kendall's Tau of 0.05, 95% CI = 0.001--0.069 and 0.04, 95% CI 0.011--0.058 respectively) but there was a considerable correlation between how often participants thought the practice should be used and how often they used it (Kendall's Tau = 0.6, 95% CI = 0.61--0.65). Those who used practices frequently or almost always were much more likely to indicate that they should be used often. Perceptions of scientific integrity {#sec013} ----------------------------------- Researchers in ecology and evolution expressed considerable doubts about their community's scientific integrity ([Table 3](#pone.0200303.t003){ref-type="table"}), mostly in relation to QRPs rather than scientific misconduct. Concern about the integrity of researchers at their own institution was roughly equal to concern about the integrity of other institutions, nor was there any notable difference in concern about graduate students compared to senior colleagues or collaborators. Our participants expressed least concern about their own integrity, but 44.6% still indicated doubts over their own use of QRPs. 10.1371/journal.pone.0200303.t003 ###### Proportion (with 95% CI) of researchers in ecology and evolution (combined) who reported having doubts about scientific integrity[\*](#t003fn001){ref-type="table-fn"}. {#pone.0200303.t003g}   Questionable Research Practices Scientific Misconduct ----------------------------------------------- --------------------------------- ----------------------- -------------------- -------------------- ------------------ ----- Researchers from other institutions 8.9 56.6 34.5 39.0 55.5 5.5 *(6*.*8--11*.*6)* *(52*.*3--60*.*7)* *(30*.*6--38*.*6)* *(34*.*9--43*.*4)* *(51*.*0--59*.*8)* *(3*.*8--7*.*8)* Research at your institution 27.9 52.2 20.0 69.2 29.1 1.6 *(24*.*2--31*.*8)* *(47*.*9--56*.*4)* *(16*.*8--23*.*6)* *(65*.*0--73*.*1)* *(25*.*3--33*.*3)* *(0*.*8--3*.*1)* Graduate student research at your institution 31.0 48.6 20.4 72.5 25.6 1.8 *(27*.*2--35*.*1)* *(44*.*3--52*.*8)* *(17*.*1--24*.*0)* *(68*.*4--76*.*3)* *(21*.*9--29*.*7)* *(1*.*0--3*.*5)* Senior colleagues or collaborators 31.5 50.8 17.7 73.3 24.7 2.0 *(27*.*6--35*.*5)* *(46*.*6--55*.*1)* *(14*.*7--21*.*2)* *(69*.*2--77*.*0)* *(21*.*1--28*.*7)* *(1*.*1--3*.*7)* Your own research 52.2 44.6 3.2 97.9 2.0 0.0 *(48*.*0--56*.*4)* *(40*.*5--48*.*8)* *(2*.*0--5*.*0)* *(96*.*2--98*.*8)* *(1*.*1--3*.*7)* *(0*.*0--0*.*8)* \*note that not all researchers answered each component of the table above so the total sample size for each of the cells differs slightly, ranging from 488 to 539 samples per cell Qualitative data analysis {#sec014} ------------------------- At the end of each QRP question, researchers had the opportunity to make additional comments on the practice. Overall, we were surprised by the proportion of researchers who made comments. For some QRPs half the researchers left comments, and often substantial ones. Here we have summarised the ecology and evolution groups' comments together, having not detected any major differences between the groups in a qualitative assessment. We interpret the volume of additional comments positively, as evidence of a research community highly engaged with issues of research practice and scientific integrity. The most frequently offered justifications for engaging in QRPs were: publication bias; pressure to publish; and the desire to present a neat, coherent narrative ([Table 4](#pone.0200303.t004){ref-type="table"}). A full description of the qualitative analysis is available in [S2 Supplementary Material](#pone.0200303.s002){ref-type="supplementary-material"}. 10.1371/journal.pone.0200303.t004 ###### Frequently offered arguments against and justifications for various Questionable Research Practices, summarising qualitative comments provided by ecology and evolution researchers. Columns relate to the description of the questionable research practice, complaints respondents made about the practice, indications on why they thought that practice might be tempting, and conditions that respondents identified as justifying the practice. {#pone.0200303.t004g} ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- -------------------------------------------------------------- ------------------------------------------------------------------------ ------------------------------------------------------------------ **Cherry-picking** Description Complaints Temptation Justifications QRP 1: Not reporting studies or variables that failed to reach statistical significance *(n = 408)*\ \- increases false positive rate\ \- hard to publish non-significant results\ \- original method was flawed\ QRP 2: Not reporting covariates that failed to reach statistical significance *(n = 350)*\ - leads to redundant investigation\ - journal word limits\ - analyses were exploratory\ QRP 4: Reporting a subset of statistical models as the complete tested *(n = 386)* - impedes interpretation\ - difficult to create a compelling story with non-significant results\ - results from multiple analyses were the same\ - skews meta-analyses\ - complete report makes boring methods and result sections\ - they were excluded during formal model selection\ - there is important information in non-significant results\ - running extra models improves understanding of the system - variables correlated\ - it is unethical - data did not match model assumptions "Sometimes lots of data are collected and tested. Often non-significant variables are thrown out if they\'re not integral to the story. I think this is okay."\ "Not reporting non-significant results biases the big picture (e.g. meta-analysis), mislead other researchers into thinking that a question is unexplored . . .This publication bias however, is obviously a result of the publication system."\ "If multiple model sets are tested they should all be presented, otherwise we risk presenting misleading results by trying a bunch of stuff until one turns out to be significant" **HARKing** Description Complaints Temptation Justifications QRP 3: Reporting an unexpected finding as having been predicted *(n = 371)* \- it is unethical\ \- makes article sexier\ \- new hypotheses arise from better understanding of the system\ - unexpected results need to be confirmed\ - reviewers ask for this\ - researchers can explain the result\ - increases false positive rate - pressure to publish\ - researchers should have hypothesised something else - not always clear exactly what was hypothesised "well, this is a difficult one---in the statistical sense, this should not happen, but in current times scientists are forced to market their work as best as possible and this is one way to make it more publishable."\ "Encourages, just-so stories, we can always come up with a suitable explanation and prediction. The key point here is to avoid doing so without noticing."\ "I believe it should not be used but editors and reviewers often demand that exploratory results are framed as a priori hypotheses" **P-hacking** Description Complaints Temptation Justifications QRP 5: Rounding- off a p value or other quantity to meet a pre-specified threshold *(n = 409)*\ \- it is unethical\ \- the 0.05 threshold is arbitrary anyway\ \- all results are presented\ QRP 6: Deciding to exclude data points after first checking the impact on statistical significance *(n = 334)*\ - increases false positive rate - hindsight bias\ - process is reported\ QRP 7: Collecting more data for a study after first inspecting whether the results are statistically significant *(n = 364)*\ - pressure to publish\ - decision not based on significance\ QRP 8: Changing to another type of statistical analysis after the analysis initially chosen failed to reach statistical significance *(n = 346)* - reviewers may ask for more data or different analyses - additional data collection already planned\ - original analysis was poorly chosen\ - data didn't meet assumptions of original analysis\ - new analysis better reflects ecological context\ - tests are conducted to test robustness of result "Attempts to conform to strict cut-off significance thresholds demonstrate an adherence to conventional practice over understanding of probability (e.g. the difference between p = 0.013 and 0.010 is and should be viewed as trivial)."\ "This practice leads to statistical significance overshadowing effect sizes and biological significance."\ "Again, one needs to be ethical. Science is about testing hypotheses with experiment, not about publishing p\<0.05 in the sexiest journal possible. A priori and post priori hypotheses are both acceptable, but they need to be labelled as such." ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- -------------------------------------------------------------- ------------------------------------------------------------------------ ------------------------------------------------------------------ Discussion {#sec015} ========== Our results indicate that QRPs are broadly as common in ecology and evolution research as they are in psychology. Of the 807 researchers in our sample, 64% reported cherry picking statistically significant results in at least one publication; 42% reported *p* hacking by collecting more data after first checking the statistical significance of results, and 51% acknowledged reporting an unexpected finding as though it had been hypothesised from the start (HARKing). That these are similar to QRP rates in psychology is hardly surprising given that publication bias and the same publish-or-perish culture persists across disciplines. However, it is important to establish the QRP rate in ecology and evolution, as it provides important evidence on which to base initiatives to improve research practices in these disciplines. Disciplinary differences {#sec016} ------------------------ Our results are most marked by how similar rates of QRPs were across disciplines, but a couple of differences are worth noting. Ecology researchers were less likely to report 'collecting more data after inspecting whether the results are statistically significant' (QRP7) than evolution researchers or psychologists. We suspect this reflects a difference in the constraints of field versus laboratory research, rather than differences in the integrity of the researchers. It is often not physically possible collect more data after the fact in ecology (field sites may be distant, available sites and budgets may be exhausted). This interpretation seems supported by evidence that many ecologists who stated that they had 'never' engaged in this practice indicated that they found it acceptable. The first nine of the QRPs we asked about were certainly controversial practices, generating mixed responses. The tenth is qualitatively different; it essentially asks about data fabrication. The social unacceptability of this practice is well recognised, and we might therefore expect under reporting even in an anonymous survey. The comments volunteered by participants largely reflected this, for example "Is that the science of 'alternative facts'?" and "It is serious scientific misconduct to report results that were not observed". The proportion of researchers admitting to this was relatively high in ecology (4.5%) compared to evolution (2.0%), US psychology (2.3%) and Italian psychology (0.6%). However, it's important to note that our wording of this question was quite different to that in the John et al and Agnoli et al surveys. They asked directly about 'falsifying data' whereas we asked a softer, less direct question about 'filling in missing data points without identifying those data as simulated'. Fiedler et al (2015) found that modified question wording changed QRP reporting rates and we suspect our change to the wording has resulted in an elevated reporting rate. We will not speculate further about ecology researchers reporting a higher rate of this than evolution researchers because the numbers of researchers admitting to this action are very small in both groups and the 95%CIs on these proportions overlap considerably. Novel insights into the usage of QRPs {#sec017} ------------------------------------- Our results contribute to the broader understanding of researchers' practices in two important ways. First, our results on reported frequency provide new insight into the regularity with which researchers engage in these practices; previous surveys in psychology did not elicit this information and asked only if the practice had been used 'at least once'. Information about frequency of use allows us to better estimate the disruption these practices may have had on the published literature. We show that while reports of having engaged in QRPs at least once are alarmingly high, virtually no researchers acknowledge using any of the QRPs more than 'occasionally'. Secondly, our qualitative results offer new understanding of the perceived acceptability of these practices, and common justifications of their use. Our qualitative analysis highlights the perception of a detrimental influence of the current publish-or-perish culture and rigid format currently required in many ecology and evolution journals. Researchers' comments revealed that they feel pressure to present a short, cohesive story with statistically significant results that confirm a priori hypotheses, rather than a full (and likely messy) account of the research as it was conceptualised and conducted. Researchers' qualitative comments also drew attention to grey areas, where the distinction between QRPs and acceptable practice was less clear. For example, in many ecology and evolution articles no hypotheses are overtly stated but the way the background material is described in the introduction can imply that the result was expected; does this constitute HARKing? Similarly, a number of participants answering QRP 6 stated that, although they had technically changed models after investigating statistical significance, their decision to change models was based on finding an error in the original model or discovering that the data did not match the model assumptions. These participants are recorded as using this QRP but whether or not it was 'questionable' in their case is unclear. Social acceptability of QRPs {#sec018} ---------------------------- Discrepancies between individual researchers' self-identified QRP use and their estimates of others' use suggest that certain practices are less socially acceptable. When average estimates of others' use are much higher than average self-report of the practice, it suggests that the practice is particularly socially undesirable and that self-report measures may underestimate prevalence \[[@pone.0200303.ref017]\]. In our results, the greatest discrepancies were observed for QRPs 2, 5, 6, 9, and 10 (see [Fig 2](#pone.0200303.g002){ref-type="fig"}), suggesting that self-reported prevalence may underestimate the true prevalence of these practices. In contrast, where there is little discrepancy between these two measures we can infer that the practice has gained a degree of social acceptability, for example QRPs 1, 4, 7, 8. These may be harder practices to shift, as researchers may not recognise them as problematic. Limitations of the current work {#sec019} ------------------------------- Some key limitations need to be considered when interpreting the results from our study. Firstly, our sample of ecology and evolution researchers might be biased. We contacted only researchers who had published in high impact factor journals, which pre-determined some demographics of our sample. For example, it likely limited the number of graduate students (6%). Our results should be understood as reflecting the practices of post-doctoral, midcareer and senior academic researchers almost exclusively. There is also very likely to be a self-selection bias in our sample of survey respondents. Those who are more confident in their practices--and perhaps more quantitatively confident in general--may have been more likely to respond. If this is the case, then it seems most likely that it would result in an underestimate of QRP rates in the broader ecology and evolution community rather than an overestimate. Another limitation in the data set is that, in order to assure participants of their anonymity, we did not collect any data on their country of origin. Evidence from Agnoli et al \[[@pone.0200303.ref016]\] and John et al. \[[@pone.0200303.ref017]\] suggests that QRPs may be more prevalent among psychology researchers from some countries than others. It seems highly likely that the same is true among ecology and evolution researchers but without data on this we cannot speculate further. Lastly, we collected survey responses between November 2016 and July 2017, it is theoretically possible that the rate of certain QRPs has changed over this time. However, as we ask participants whether they have used any of these practices "never", "once", "occasionally", "frequently", or "almost always", we suspect that any behaviour changes in this time period will not be evident in responses to our surveys. Solutions {#sec020} --------- Our results indicate that there is substantial room to improve research practices in ecology and evolution. However, none of these problems are insurmountable. In fact, the correlation we found between acceptability and prevalence of QRPs and the justifications people provided in text ([S2 Supplementary Material](#pone.0200303.s002){ref-type="supplementary-material"}) suggest that the prevalence of these practices could be reduced by educating researchers about their ramifications. These practices are driven by a publish-or-perish research culture that puts emphasis on producing sexy, novel stories over solid science. The researchers in our sample often commented on this; one researcher commented "the absence of significant result is so hard to publish that people (me included) finally don\'t even try to publish it. The absence of negative result in publications doesn\'t indicate that it wasn\'t tested, only that editors don\'t select them for publication, because they consider them not enough sexy" and another researcher stated "I think there is a problem in ecology where the \'sexy\' findings are promoted, and they often find their way into high ranking journals. Other solid pieces of work often languish in \'specialist\' journals". This culture is perpetuated by science's reliance on publishers which, as private companies, may be more concerned with their reputation and finances than with furthering the scientific endeavour. The open science movement has given rise to a series of solutions that help reduce the temptation for and prevalence of these QRPs \[[@pone.0200303.ref006],[@pone.0200303.ref011],[@pone.0200303.ref013],[@pone.0200303.ref036]\]. A number of these solutions rely on changes in journal practices or institutional policies that may be difficult to implement because these interventions may (at least in the short term) meet resistance from publishing companies. Even though current incentive structures may favour the status quo in scientific publishing, researchers have made inroads through combinations of individual and coordinated action A promising tool for individual researchers to adopt is preregistration. A thorough preregistration specifies researchers' hypotheses, how they will decide on their sample size, data exclusion criteria, and the analyses they will conduct, among other things. This helps researchers think their research through thoroughly, improving its rigor, as well as protecting against HARKing, cherry-picking and p-hacking \[[@pone.0200303.ref037],[@pone.0200303.ref038]\]. Despite the growing use of pre-registration, its widespread adoption remains uncertain in ecology and evolutionary biology. Obstacles include resistance from researchers who mistakenly believe that preregistration limits their creativity and ability to conduct exploratory work \[[@pone.0200303.ref037]\], but also from journals that still preferentially accept manuscript with 'positive' results and clear stories, thus incentivizing HARKing and other QRPs. However, many journals are published by scientific societies and have editorial boards populated by practicing scientists, and these individual scientists can work to promote favourable practices. Changes to reduce QRPs can happen, but this movement is still young. Some editors in ecology and evolutionary biology have also instigated important changes such as requiring data archiving at a handful of prominent journals \[[@pone.0200303.ref039]\]. Although there has been some limited push-back against data archiving \[[@pone.0200303.ref040]\] and compliance falls far short of perfect \[[@pone.0200303.ref041]\], this case demonstrates the potential impact of committed people in influential positions. The archiving movement is now spreading beyond data \[[@pone.0200303.ref042]\], and a small but growing number of journals are starting to use rigorous checklists for authors to encourage more transparent reporting of important aspects of methods and results (e.g. *Conservation Biology*, *Nature*). Conclusion {#sec021} ========== The use of Questionable Research Practices in ecology and evolution research is high enough to be of concern. The rates of QRPs found in our sample of 807 ecologists and evolutionary biologists are similar to those that have been found in psychology, where the reproducibility rates of published research have been systematically studied and found to be low (36--47% depending on the measure \[[@pone.0200303.ref002]\]). Researchers in our survey offered justifications for their practices including: publication bias; pressure to publish; and the desire to present a neat, coherent narrative. We recommend that all journals in ecology and evolution adopt editing and reviewing checklists to ensure more complete and transparent reporting, encourage preregistration and registered reports article formats to minimise HARKing, and encourage open code and data whenever possible. Supporting information {#sec022} ====================== ###### Questionable research practice survey. (PDF) ###### Click here for additional data file. ###### Qualitative data analysis. (DOCX) ###### Click here for additional data file. ###### Disambiguating QRP 1 cherry-picking vs the file drawer. (DOCX) ###### Click here for additional data file. Fiona Fidler is supported by an Australian Research Council Future Fellowship (FT150100297). We would like to thank Felix Singleton Thorn, Franca Agnoli and three reviewers for feedback on the manuscript and Aurora Marquette for her assistance in collecting contact author addresses. [^1]: **Competing Interests:**The authors have declared that no competing interests exist.

Chemical context   {#sec1} ================== Barbiturates play a significant role in biological systems (Hueso-Ureña *et al.*, 2003[@bb8]). Epilepsy (convulsion) is a life-threatening neurological disorder which requires immediate treatment with suitable drugs (Shorvon, 2004[@bb21]). Barbiturates have been proved to be potent drugs for this dreadful disorder (Nadkarni *et al.*, 2005[@bb18]). The iso­quinoline unit also displays a wide spectrum of activity and it is an important component of many biologically active alkaloids (Montalban, 2011[@bb17]). Since 2008, we have been periodically synthesizing new barbiturate derivatives and exploring their anti­convulsant activity (Kalaivani *et al.*, 2008[@bb11]; Kalaivani & Malarvizhi, 2009[@bb10]; Kalaivani & Buvaneswari, 2010[@bb9]; Manickkam & Kalaivani, 2011[@bb14]; Babykala & Kalaivani, 2012[@bb2]; Buvaneswari & Kalaivani, 2013[@bb4]; Vaduganathan & Doraisamyraja, 2014[@bb24]; Gomathi & Kalaivani, 2015[@bb6]). The title mol­ecular salt, which is a new derivative of 1,3-di­methyl­barbituric acid (barbiturate), was recently obtained by our group. Herewith we report its crystal structure. Structural commentary   {#sec2} ======================= In the title compound, (I)[](#scheme1){ref-type="chem"} (Fig. 1[▸](#fig1){ref-type="fig"}), all the bond lengths and bond angles are normal and comparable with those observed in the related barbiturates (Sridevi & Kalaivani, 2012[@bb23]; Gunaseelan & Doraisamyraja, 2014[@bb7]). The plane of the di­nitro­aromatic ring C1--C6 and that of the barbiturate ring C7/C8/N4/C9/N3/C10 form a dihedral angle of 42.78 (9)°. The nitro groups in the 2,4-di­nitro­phenyl fragment attached to the aromatic ring in the *para* and *ortho* positions are twisted from its plane by 3.1 (2) and 45.5 (2)°, respectively. Thus the *para* nitro group is more involved in delocalizing the negative charge than the *ortho* nitro group in the anionic part. This sort of delocalization of the charge over a large area imparts a maroon red colour to the title compound. Supra­molecular features   {#sec3} ========================== The aminium group is involved in formation of an N---H⋯O hydrogen bond (Table 1[▸](#table1){ref-type="table"}) between the isoquinolinium cation (N5---H5*A*) and the deprotonated enol oxygen atom O7. In the crystal, weak C---H⋯O hydrogen bonds (Table 1[▸](#table1){ref-type="table"}) consolidate the crystal packing (Fig. 2[▸](#fig2){ref-type="fig"}). An (6) motif is generated by the C---H groups \[C13---H13 and C20---H20\] of the isoquinolinium cation and oxygen atom O5 of the carbonyl group of the barbiturate ring of the anion. Although there are three rings with cyclically delocalized π electron clouds, no π--π stacking inter­actions are observed between them. 3D Hirshfeld Surface Analysis and 2D Fingerprint Analysis   {#sec4} =========================================================== Hirshfeld surfaces (Spackman & Jayatilaka, 2009[@bb22]) and the associated 2D-fingerprint plots (McKinnon *et al.*, 2007[@bb15]) of the title mol­ecular salt have been generated using *Crystal Explorer 3.1* (Wolff *et al.*, 2013[@bb25]). Hirshfeld surfaces mapped with different properties, *e.g. d* ~e~, *d* ~norm~, *d* ~i~, shapeindex, curvedness, have proven to be a useful visualization tool for the analysis of inter­molecular inter­actions. The 2D-fingerprint plots of Hirshfeld surfaces have been used to pinpoint and scrutinize the percentage of hydrogen-bonding inter­actions present in the crystal structure. The presented graphical plots use the same red-white-blue color scheme, wherein red highlights the shortest inter­molecular atomic contacts (negative *d* ~norm~ values), white is used for contacts around the van der Waals separation, and blue corresponds to longer ones (positive *d* ~norm~ values). Hirshfeld surface analysis of the new barbiturate of present inter­est has *d* ~norm~ values ranging from −0.723 (red) to 1.464 (blue), as specified in Fig. 3[▸](#fig3){ref-type="fig"}. The globularity value (a measure of the degree to which the surface area differs from that of the shape) is less than 1 (0.743), implying a more structured mol­ecular surface and it is an oblate object (asphericity, 0.282). 2D-Fingerprint plots showing contributions from different contacts: (*a*) overall inter­actions (*b*) C⋯H/H⋯C (*c*) C⋯O/O⋯C (*d*) H⋯H (*e*) O⋯H/H⋯O (*f*) N⋯O/O⋯N are depicted in Fig. 4[▸](#fig4){ref-type="fig"}, and Fig. 5[▸](#fig5){ref-type="fig"} (pie chart) clearly demonstrates that the O⋯H/H⋯O inter­actions dominate in the crystal. Pharmacological activity   {#sec5} ========================== Epilepsy affects about 0.5% of the world's population. A seizure is caused by an asynchronous high-frequency discharge of a group of neurons, starting locally and spreading to a varying extent to affect other parts of the brain. 1,3-Di­methyl­barbituric acid is the most significant compound with a heterocyclic structure and exists in two tautomeric forms (keto and enol) due to the mobility of active methyl­ene group hydrogen atoms in its mol­ecule. Barbiturates are drugs that act as central nervous system depressants and can therefore produce a wide spectrum of effects from mild sedation to total anaesthesia. They are also effective as anxiolytics, hypnotics and anti­convulsants. As the mol­ecular salt of the present investigation is a derivative of 1,3-di­methyl­barbituric acid, it has been subjected to the Maximal Electro Shock method to evaluate its anti­convulsant activity (Misra *et al.*,1973[@bb16]; Kulkarni, 1999[@bb12]). It reduces all phases of convulsion (tonic-flexor, tonic-extensor, clonic-convulsion and stupor) even at low dosage (25 mg kg^−1^) and the animals recovered after the experiment. Synthesis and crystallization   {#sec6} =============================== 1-Chloro-2,4-di­nitro­benzene (2.02 g, 0.01 mol) in 40 mL of absolute alcohol was mixed with 1,3-di­methyl­barbituric acid (1.56 g, 0.01 mol) in 30 mL ethanol. To this mixture, 0.02 mol of iso­quinoline was added and the mixture was shaken well for 5 h and kept as such for 24 h. Excess ethanol was removed through evaporation. A maroon-red pasty mass was obtained. This paste was digested with hot ethanol to obtain a maroon-red solid. The solid deposited at the bottom of the flask was filtered, powdered well using an agate mortar, washed again with 20 mL of dry ether and recrystallized from absolute alcohol. Good quality single crystals suitable for X-ray diffraction analysis were obtained by slow evaporation of ethanol at room temperature (yield: 80%; m.p. 413 K). Refinement   {#sec7} ============ Crystal data, data collection and structure refinement details are summarized in Table 2[▸](#table2){ref-type="table"}. The N-bound H atom was located in a difference Fourier map and refined isotropically. C-bound H atoms were positioned geometrically and refined as riding, with C---H = 0.93--0.96 Å and *U* ~iso~(H) = 1.2--1.5 *U* ~eq~(C). Supplementary Material ====================== Crystal structure: contains datablock(s) global, I. DOI: [10.1107/S2056989016005004/cv5504sup1.cif](http://dx.doi.org/10.1107/S2056989016005004/cv5504sup1.cif) Structure factors: contains datablock(s) I. DOI: [10.1107/S2056989016005004/cv5504Isup2.hkl](http://dx.doi.org/10.1107/S2056989016005004/cv5504Isup2.hkl) ###### Click here for additional data file. Supporting information file. DOI: [10.1107/S2056989016005004/cv5504Isup3.cml](http://dx.doi.org/10.1107/S2056989016005004/cv5504Isup3.cml) CCDC reference: [1444879](http://scripts.iucr.org/cgi-bin/cr.cgi?rm=csd&csdid=1444879) Additional supporting information: [crystallographic information](http://scripts.iucr.org/cgi-bin/sendsupfiles?cv5504&file=cv5504sup0.html&mime=text/html); [3D view](http://scripts.iucr.org/cgi-bin/sendcif?cv5504sup1&Qmime=cif); [checkCIF report](http://scripts.iucr.org/cgi-bin/paper?cv5504&checkcif=yes) The authors are thankful to the DST for financial support, the SAIF, IIT Madras, Chennai − 36, for the single crystal XRD data collection and KMCH College of Pharmacy, Coimbatore, for the anti­convulsant activity results. Crystal data {#tablewrapcrystaldatalong} ============ ----------------------------------- --------------------------------------- C~9~H~8~N^+^·C~12~H~9~N~4~O~7~^−^ *D*~x~ = 1.471 Mg m^−3^ *M~r~* = 451.40 Mo *K*α radiation, λ = 0.71073 Å Orthorhombic, *P*2~1~2~1~2~1~ Cell parameters from 9400 reflections *a* = 7.5315 (3) Å θ = 2.3--24.0° *b* = 15.5640 (8) Å µ = 0.11 mm^−1^ *c* = 17.3901 (8) Å *T* = 296 K *V* = 2038.47 (16) Å^3^ Block, brown *Z* = 4 0.35 × 0.30 × 0.25 mm *F*(000) = 936 ----------------------------------- --------------------------------------- Data collection {#tablewrapdatacollectionlong} =============== ------------------------------------------------------------ -------------------------------------- Bruker Kappa APEXII CCD diffractometer 3588 independent reflections Radiation source: fine-focus sealed tube 2797 reflections with *I* \> 2σ(*I*) Graphite monochromator *R*~int~ = 0.038 ω and φ scan θ~max~ = 25.0°, θ~min~ = 2.3° Absorption correction: multi-scan (*SADABS*; Bruker, 2004) *h* = −8→8 *T*~min~ = 0.958, *T*~max~ = 0.984 *k* = −18→18 27633 measured reflections *l* = −20→20 ------------------------------------------------------------ -------------------------------------- Refinement {#tablewraprefinementdatalong} ========== ------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------ Refinement on *F*^2^ Hydrogen site location: mixed Least-squares matrix: full H atoms treated by a mixture of independent and constrained refinement *R*\[*F*^2^ \> 2σ(*F*^2^)\] = 0.055 *w* = 1/\[σ^2^(*F*~o~^2^) + (0.0596*P*)^2^ + 2.2423*P*\] where *P* = (*F*~o~^2^ + 2*F*~c~^2^)/3 *wR*(*F*^2^) = 0.164 (Δ/σ)~max~ \< 0.001 *S* = 1.11 Δρ~max~ = 0.29 e Å^−3^ 3588 reflections Δρ~min~ = −0.26 e Å^−3^ 302 parameters Absolute structure: Flack *x* determined using 1033 quotients \[(*I*^+^)-(*I*^-^)\]/\[(*I*^+^)+(*I*^-^)\] (Parsons *et al.*, 2013) 0 restraints Absolute structure parameter: 0.5 (4) ------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------ Special details {#specialdetails} =============== ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes. Refinement. The elements in the sample do not have sufficient anomalous scattering power for Mo(kα) radiation. Hence the Flack parameter and its standard deviation obtained from refinement have no physical significance. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å^2^) {#tablewrapcoords} ================================================================================================== ------ ------------- ------------- ------------ -------------------- -- *x* *y* *z* *U*~iso~\*/*U*~eq~ C1 1.0128 (8) 0.1849 (4) 0.6047 (3) 0.0420 (13) C2 0.9775 (8) 0.1140 (3) 0.5603 (3) 0.0430 (13) H2 0.9489 0.0617 0.5830 0.052\* C3 0.9850 (7) 0.1217 (3) 0.4818 (3) 0.0380 (12) C4 1.0322 (7) 0.1979 (3) 0.4434 (3) 0.0360 (11) C5 1.0660 (8) 0.2679 (3) 0.4922 (3) 0.0443 (13) H5 1.0970 0.3204 0.4705 0.053\* C6 1.0549 (8) 0.2615 (4) 0.5715 (3) 0.0495 (14) H6 1.0761 0.3094 0.6021 0.059\* C7 1.0594 (7) 0.2047 (3) 0.3605 (3) 0.0337 (11) C8 1.1510 (7) 0.1402 (3) 0.3218 (3) 0.0357 (11) C9 1.1333 (8) 0.2196 (3) 0.2030 (3) 0.0421 (13) C10 1.0069 (7) 0.2807 (3) 0.3207 (3) 0.0388 (12) C11 0.9855 (12) 0.3574 (4) 0.1982 (4) 0.072 (2) H11A 0.9241 0.3963 0.2317 0.108\* H11B 1.0872 0.3856 0.1766 0.108\* H11C 0.9074 0.3396 0.1576 0.108\* C12 1.2891 (9) 0.0820 (4) 0.2065 (3) 0.0569 (16) H12A 1.3142 0.0369 0.2426 0.085\* H12B 1.2236 0.0590 0.1639 0.085\* H12C 1.3985 0.1062 0.1884 0.085\* C13 1.4554 (8) 0.0032 (4) 0.5236 (3) 0.0486 (14) H13 1.4114 0.0499 0.5511 0.058\* C14 1.5694 (7) −0.0536 (3) 0.5585 (3) 0.0404 (12) C15 1.6326 (7) −0.1244 (4) 0.5152 (3) 0.0449 (13) C16 1.5786 (8) −0.1322 (4) 0.4383 (3) 0.0522 (15) H16 1.6195 −0.1776 0.4084 0.063\* C17 1.4675 (8) −0.0738 (4) 0.4083 (3) 0.0546 (15) H17 1.4313 −0.0788 0.3573 0.065\* C18 1.6259 (8) −0.0443 (4) 0.6357 (3) 0.0554 (15) H18 1.5888 0.0026 0.6647 0.066\* C19 1.7348 (9) −0.1043 (6) 0.6669 (4) 0.069 (2) H19 1.7689 −0.0991 0.7181 0.083\* C20 1.7960 (10) −0.1728 (5) 0.6242 (4) 0.074 (2) H20 1.8716 −0.2126 0.6471 0.088\* C21 1.7487 (9) −0.1835 (4) 0.5502 (4) 0.0642 (18) H21 1.7926 −0.2298 0.5223 0.077\* N1 1.0035 (8) 0.1760 (4) 0.6875 (3) 0.0628 (15) N2 0.9243 (7) 0.0456 (3) 0.4385 (3) 0.0496 (12) N3 1.0427 (7) 0.2826 (3) 0.2415 (2) 0.0435 (11) N4 1.1845 (6) 0.1485 (3) 0.2441 (2) 0.0389 (10) N5 1.4081 (7) −0.0079 (3) 0.4514 (3) 0.0508 (13) O1 1.0316 (9) 0.2406 (4) 0.7261 (3) 0.0887 (18) O2 0.9721 (10) 0.1063 (4) 0.7153 (3) 0.099 (2) O3 0.8197 (6) 0.0553 (3) 0.3865 (3) 0.0632 (12) O4 0.9781 (7) −0.0246 (3) 0.4618 (3) 0.0690 (14) O5 0.9298 (6) 0.3428 (3) 0.3499 (2) 0.0578 (11) O6 1.1665 (7) 0.2254 (3) 0.1336 (2) 0.0643 (12) O7 1.2084 (6) 0.0724 (2) 0.3535 (2) 0.0534 (11) H5A 1.330 (8) 0.032 (4) 0.431 (3) 0.043 (15)\* ------ ------------- ------------- ------------ -------------------- -- Atomic displacement parameters (Å^2^) {#tablewrapadps} ===================================== ----- ----------- ----------- ----------- ------------ ------------- ------------- *U*^11^ *U*^22^ *U*^33^ *U*^12^ *U*^13^ *U*^23^ C1 0.050 (3) 0.052 (3) 0.025 (2) 0.002 (3) 0.002 (2) −0.005 (2) C2 0.053 (3) 0.039 (3) 0.037 (3) −0.001 (3) −0.001 (3) 0.005 (2) C3 0.042 (3) 0.037 (3) 0.035 (3) −0.002 (2) 0.000 (2) −0.002 (2) C4 0.036 (3) 0.039 (3) 0.034 (3) 0.002 (2) −0.005 (2) −0.003 (2) C5 0.050 (3) 0.040 (3) 0.044 (3) 0.000 (3) −0.004 (3) 0.002 (2) C6 0.054 (3) 0.051 (3) 0.043 (3) 0.002 (3) −0.010 (3) −0.018 (3) C7 0.041 (3) 0.030 (2) 0.030 (3) −0.002 (2) −0.004 (2) 0.000 (2) C8 0.035 (3) 0.039 (3) 0.033 (3) 0.000 (2) −0.004 (2) 0.002 (2) C9 0.047 (3) 0.042 (3) 0.038 (3) −0.013 (3) 0.001 (2) 0.006 (2) C10 0.042 (3) 0.032 (3) 0.043 (3) −0.004 (2) −0.001 (2) 0.002 (2) C11 0.108 (6) 0.053 (4) 0.054 (4) 0.004 (4) −0.006 (4) 0.015 (3) C12 0.059 (4) 0.065 (4) 0.047 (3) 0.008 (3) 0.012 (3) 0.000 (3) C13 0.048 (3) 0.042 (3) 0.055 (4) 0.004 (3) 0.003 (3) −0.004 (3) C14 0.037 (3) 0.039 (3) 0.045 (3) −0.002 (2) 0.002 (2) 0.004 (2) C15 0.039 (3) 0.046 (3) 0.049 (3) 0.005 (3) 0.002 (3) 0.008 (3) C16 0.051 (3) 0.052 (3) 0.054 (4) 0.007 (3) 0.002 (3) −0.011 (3) C17 0.055 (4) 0.064 (4) 0.045 (3) −0.002 (3) −0.006 (3) 0.004 (3) C18 0.054 (4) 0.063 (4) 0.049 (4) −0.006 (3) −0.001 (3) −0.004 (3) C19 0.053 (4) 0.105 (6) 0.049 (4) −0.014 (4) −0.013 (3) 0.012 (4) C20 0.066 (4) 0.082 (5) 0.073 (5) 0.019 (4) −0.013 (4) 0.021 (4) C21 0.062 (4) 0.057 (4) 0.074 (5) 0.020 (3) −0.007 (4) 0.008 (3) N1 0.070 (4) 0.081 (4) 0.037 (3) 0.018 (3) −0.002 (3) −0.004 (3) N2 0.057 (3) 0.041 (3) 0.051 (3) −0.010 (2) 0.009 (3) −0.007 (2) N3 0.062 (3) 0.033 (2) 0.036 (2) 0.002 (2) −0.002 (2) 0.0105 (19) N4 0.040 (2) 0.042 (2) 0.035 (2) 0.003 (2) 0.0034 (19) 0.0010 (19) N5 0.051 (3) 0.048 (3) 0.054 (3) 0.008 (2) −0.008 (2) 0.008 (3) O1 0.122 (5) 0.099 (4) 0.045 (3) 0.009 (4) −0.011 (3) −0.026 (3) O2 0.158 (6) 0.094 (4) 0.046 (3) −0.004 (4) 0.003 (4) 0.015 (3) O3 0.066 (3) 0.065 (3) 0.058 (3) −0.014 (2) −0.012 (2) −0.016 (2) O4 0.099 (4) 0.040 (2) 0.068 (3) −0.005 (2) 0.012 (3) −0.001 (2) O5 0.076 (3) 0.044 (2) 0.054 (2) 0.010 (2) −0.002 (2) 0.006 (2) O6 0.086 (3) 0.063 (3) 0.044 (3) −0.011 (3) 0.009 (2) 0.013 (2) O7 0.060 (3) 0.055 (3) 0.045 (2) 0.017 (2) 0.003 (2) 0.007 (2) ----- ----------- ----------- ----------- ------------ ------------- ------------- Geometric parameters (Å, º) {#tablewrapgeomlong} =========================== ----------------------- ------------ ----------------------- ------------ C1---C6 1.362 (8) C12---H12A 0.9600 C1---C2 1.372 (7) C12---H12B 0.9600 C1---N1 1.448 (7) C12---H12C 0.9600 C2---C3 1.372 (7) C13---N5 1.316 (7) C2---H2 0.9300 C13---C14 1.374 (8) C3---C4 1.406 (7) C13---H13 0.9300 C3---N2 1.476 (7) C14---C18 1.415 (8) C4---C5 1.404 (7) C14---C15 1.418 (7) C4---C7 1.460 (7) C15---C16 1.402 (8) C5---C6 1.385 (8) C15---C21 1.408 (8) C5---H5 0.9300 C16---C17 1.342 (8) C6---H6 0.9300 C16---H16 0.9300 C7---C8 1.391 (7) C17---N5 1.346 (8) C7---C10 1.425 (7) C17---H17 0.9300 C8---O7 1.267 (6) C18---C19 1.357 (10) C8---N4 1.381 (6) C18---H18 0.9300 C9---O6 1.235 (6) C19---C20 1.379 (10) C9---N3 1.369 (7) C19---H19 0.9300 C9---N4 1.373 (7) C20---C21 1.346 (10) C10---O5 1.236 (6) C20---H20 0.9300 C10---N3 1.404 (7) C21---H21 0.9300 C11---N3 1.452 (7) N1---O2 1.211 (7) C11---H11A 0.9600 N1---O1 1.228 (8) C11---H11B 0.9600 N2---O3 1.209 (6) C11---H11C 0.9600 N2---O4 1.234 (6) C12---N4 1.455 (7) N5---H5A 0.93 (6) C6---C1---C2 120.6 (5) N5---C13---C14 120.4 (5) C6---C1---N1 121.1 (5) N5---C13---H13 119.8 C2---C1---N1 118.3 (5) C14---C13---H13 119.8 C3---C2---C1 118.9 (5) C13---C14---C18 122.8 (6) C3---C2---H2 120.6 C13---C14---C15 118.3 (5) C1---C2---H2 120.6 C18---C14---C15 118.9 (5) C2---C3---C4 123.8 (5) C16---C15---C21 122.4 (6) C2---C3---N2 115.1 (5) C16---C15---C14 118.5 (5) C4---C3---N2 120.9 (5) C21---C15---C14 119.1 (6) C5---C4---C3 114.5 (5) C17---C16---C15 119.5 (6) C5---C4---C7 121.0 (5) C17---C16---H16 120.2 C3---C4---C7 124.4 (4) C15---C16---H16 120.2 C6---C5---C4 122.3 (5) C16---C17---N5 120.5 (6) C6---C5---H5 118.9 C16---C17---H17 119.8 C4---C5---H5 118.9 N5---C17---H17 119.8 C1---C6---C5 120.0 (5) C19---C18---C14 119.5 (6) C1---C6---H6 120.0 C19---C18---H18 120.3 C5---C6---H6 120.0 C14---C18---H18 120.3 C8---C7---C10 120.1 (4) C18---C19---C20 121.2 (6) C8---C7---C4 119.6 (4) C18---C19---H19 119.4 C10---C7---C4 120.0 (4) C20---C19---H19 119.4 O7---C8---N4 116.1 (4) C21---C20---C19 121.5 (7) O7---C8---C7 124.1 (4) C21---C20---H20 119.3 N4---C8---C7 119.8 (4) C19---C20---H20 119.3 O6---C9---N3 121.8 (5) C20---C21---C15 119.9 (7) O6---C9---N4 120.8 (5) C20---C21---H21 120.1 N3---C9---N4 117.5 (4) C15---C21---H21 120.1 O5---C10---N3 118.4 (5) O2---N1---O1 123.3 (6) O5---C10---C7 125.4 (5) O2---N1---C1 119.5 (6) N3---C10---C7 116.1 (5) O1---N1---C1 117.2 (6) N3---C11---H11A 109.5 O3---N2---O4 124.8 (5) N3---C11---H11B 109.5 O3---N2---C3 118.9 (5) H11A---C11---H11B 109.5 O4---N2---C3 116.2 (5) N3---C11---H11C 109.5 C9---N3---C10 124.1 (4) H11A---C11---H11C 109.5 C9---N3---C11 117.9 (5) H11B---C11---H11C 109.5 C10---N3---C11 118.0 (5) N4---C12---H12A 109.5 C9---N4---C8 122.3 (4) N4---C12---H12B 109.5 C9---N4---C12 119.4 (4) H12A---C12---H12B 109.5 C8---N4---C12 118.2 (4) N4---C12---H12C 109.5 C13---N5---C17 122.8 (5) H12A---C12---H12C 109.5 C13---N5---H5A 117 (3) H12B---C12---H12C 109.5 C17---N5---H5A 121 (3) C6---C1---C2---C3 −0.1 (9) C13---C14---C18---C19 178.2 (6) N1---C1---C2---C3 179.7 (5) C15---C14---C18---C19 −1.8 (9) C1---C2---C3---C4 1.9 (9) C14---C18---C19---C20 2.1 (10) C1---C2---C3---N2 −172.9 (5) C18---C19---C20---C21 −0.8 (12) C2---C3---C4---C5 −2.1 (8) C19---C20---C21---C15 −0.8 (11) N2---C3---C4---C5 172.5 (5) C16---C15---C21---C20 −180.0 (7) C2---C3---C4---C7 173.1 (5) C14---C15---C21---C20 0.9 (9) N2---C3---C4---C7 −12.4 (8) C6---C1---N1---O2 −177.3 (7) C3---C4---C5---C6 0.5 (8) C2---C1---N1---O2 2.9 (10) C7---C4---C5---C6 −174.8 (6) C6---C1---N1---O1 1.3 (9) C2---C1---C6---C5 −1.4 (9) C2---C1---N1---O1 −178.4 (6) N1---C1---C6---C5 178.9 (5) C2---C3---N2---O3 131.3 (5) C4---C5---C6---C1 1.2 (9) C4---C3---N2---O3 −43.7 (8) C5---C4---C7---C8 132.9 (5) C2---C3---N2---O4 −44.6 (7) C3---C4---C7---C8 −41.9 (8) C4---C3---N2---O4 140.4 (5) C5---C4---C7---C10 −41.3 (7) O6---C9---N3---C10 −177.7 (5) C3---C4---C7---C10 143.8 (5) N4---C9---N3---C10 3.2 (8) C10---C7---C8---O7 177.1 (5) O6---C9---N3---C11 0.2 (9) C4---C7---C8---O7 2.9 (8) N4---C9---N3---C11 −179.0 (6) C10---C7---C8---N4 −2.6 (7) O5---C10---N3---C9 177.3 (5) C4---C7---C8---N4 −176.8 (5) C7---C10---N3---C9 −4.4 (8) C8---C7---C10---O5 −177.8 (5) O5---C10---N3---C11 −0.5 (8) C4---C7---C10---O5 −3.6 (8) C7---C10---N3---C11 177.7 (5) C8---C7---C10---N3 4.0 (7) O6---C9---N4---C8 179.4 (5) C4---C7---C10---N3 178.2 (5) N3---C9---N4---C8 −1.4 (8) N5---C13---C14---C18 179.4 (6) O6---C9---N4---C12 4.0 (8) N5---C13---C14---C15 −0.6 (8) N3---C9---N4---C12 −176.8 (5) C13---C14---C15---C16 1.2 (8) O7---C8---N4---C9 −178.5 (5) C18---C14---C15---C16 −178.8 (5) C7---C8---N4---C9 1.2 (7) C13---C14---C15---C21 −179.7 (5) O7---C8---N4---C12 −3.1 (7) C18---C14---C15---C21 0.4 (8) C7---C8---N4---C12 176.7 (5) C21---C15---C16---C17 179.9 (6) C14---C13---N5---C17 −0.3 (9) C14---C15---C16---C17 −0.9 (9) C16---C17---N5---C13 0.6 (9) C15---C16---C17---N5 0.1 (9) ----------------------- ------------ ----------------------- ------------ Hydrogen-bond geometry (Å, º) {#tablewraphbondslong} ============================= --------------------- ---------- ---------- ----------- --------------- *D*---H···*A* *D*---H H···*A* *D*···*A* *D*---H···*A* N5---H5*A*···O7 0.93 (6) 1.74 (6) 2.592 (6) 150 (5) C13---H13···O5^i^ 0.93 2.40 3.260 (7) 153 C16---H16···O6^ii^ 0.93 2.33 3.187 (7) 154 C17---H17···O2^iii^ 0.93 2.61 3.424 (8) 146 --------------------- ---------- ---------- ----------- --------------- Symmetry codes: (i) *x*+1/2, −*y*+1/2, −*z*+1; (ii) −*x*+3, *y*−1/2, −*z*+1/2; (iii) −*x*+5/2, −*y*, *z*−1/2. {ref-type="chem"} showing the atom numbering and 40% probability displacement ellipsoids. The doubled-dashed line denotes the N---H⋯O hydrogen bond between the cation and anion.](e-72-00570-fig1){#fig1} {ref-type="chem"} viewed approximately down the *a* axis. Hydrogen bonds are shown as purple dotted lines.](e-72-00570-fig2){#fig2} {ref-type="chem"} mapped over (*a*) *d* ~norm~ ranging from −0.723 (red) to 1.464 (blue); (*b*) *d* ~e~; (*c*) *d* ~i~; (*d*) curvedness; (*e*) shapeindex.](e-72-00570-fig3){#fig3} {#fig4} {ref-type="chem"}.](e-72-00570-fig5){#fig5} ###### Hydrogen-bond geometry (Å, °) *D*---H⋯*A* *D*---H H⋯*A* *D*⋯*A* *D*---H⋯*A* ------------------- ---------- ---------- ----------- ------------- N5---H5*A*⋯O7 0.93 (6) 1.74 (6) 2.592 (6) 150 (5) C13---H13⋯O5^i^ 0.93 2.40 3.260 (7) 153 C16---H16⋯O6^ii^ 0.93 2.33 3.187 (7) 154 C17---H17⋯O2^iii^ 0.93 2.61 3.424 (8) 146 Symmetry codes: (i) ; (ii) ; (iii) . ###### Experimental details -------------------------------------------------------------------------- --------------------------------------------------------------------------------------------------------------------------- Crystal data Chemical formula C~9~H~8~N^+^·C~12~H~9~N~4~O~7~ ^−^ *M* ~r~ 451.40 Crystal system, space group Orthorhombic, *P*2~1~2~1~2~1~ Temperature (K) 296 *a*, *b*, *c* (Å) 7.5315 (3), 15.5640 (8), 17.3901 (8) *V* (Å^3^) 2038.47 (16) *Z* 4 Radiation type Mo *K*α μ (mm^−1^) 0.11 Crystal size (mm) 0.35 × 0.30 × 0.25   Data collection Diffractometer Bruker Kappa APEXII CCD Absorption correction Multi-scan (*SADABS*; Bruker, 2004[@bb3]) *T* ~min~, *T* ~max~ 0.958, 0.984 No. of measured, independent and observed \[*I* \> 2σ(*I*)\] reflections 27633, 3588, 2797 *R* ~int~ 0.038 (sin θ/λ)~max~ (Å^−1^) 0.594   Refinement *R*\[*F* ^2^ \> 2σ(*F* ^2^)\], *wR*(*F* ^2^), *S* 0.055, 0.164, 1.11 No. of reflections 3588 No. of parameters 302 H-atom treatment H atoms treated by a mixture of independent and constrained refinement Δρ~max~, Δρ~min~ (e Å^−3^) 0.29, −0.26 Absolute structure Flack *x* determined using 1033 quotients \[(*I* ^+^)−(*I* ^−^)\]/\[(*I* ^+^)+(*I* ^−^)\] (Parsons *et al.*, 2013[@bb19]) Absolute structure parameter 0.5 (4) -------------------------------------------------------------------------- --------------------------------------------------------------------------------------------------------------------------- Computer programs: *APEX2*, *SAINT* and *XPREP* (Bruker, 2004[@bb3]), *SIR92* (Altomare *et al.*, 1993[@bb1]), *SHELXL2014* (Sheldrick, 2015[@bb20]), *ORTEP-3 for Windows* (Farrugia, 2012[@bb5]) and *Mercury* (Macrae *et al.*, 2008[@bb13]).

1. Introduction {#sec1-sensors-16-01535} =============== Structural Health Monitoring (SHM) systems are automated tools, aimed at rapidly identifying the onset of structural damage and at tracking the condition of structures during forced or natural excitation \[[@B1-sensors-16-01535]\]. Current SHM systems almost adopt Non-Destructive Testing (NDT) techniques, which can be divided into two types: on-demand type and in situ. The on-demand NDT techniques are based on instrumentation brought to the measurement site on-demand, including X-ray, infrared thermography, laser scanning and microwave radar \[[@B2-sensors-16-01535],[@B3-sensors-16-01535],[@B4-sensors-16-01535],[@B5-sensors-16-01535]\]. However, these techniques cannot measure the physical quantities of concrete directly and the measured results are not real-time. The second family is based on the deployment of several local sensors permanently coupled with structural elements. The in situ techniques can also be divided into two groups, according to the use of wired or wireless technology. Among the wired ones \[[@B6-sensors-16-01535],[@B7-sensors-16-01535]\], high measuring precision, great resolution, and real time monitoring can be obtained, however they are quite time-consuming and expensive because of the complicated manufacturing as well as the extensive signal processing. Thanks to fast and easy installation, wireless sensors are gaining a growing interest. Most of the wireless sensors for SHM are based on Wireless Sensor Networks (WSN), which are battery-powered and permit long-range operating \[[@B8-sensors-16-01535],[@B9-sensors-16-01535]\]. However, once these wireless sensors are immersed in concrete, their batteries cannot be replaced again and their lifespan are limited to several years. Furthermore, their cost and complexity are still relatively high. Recently, with the rapid development of Radio Frequency Identification (RFID) technology, the wireless sensors based on passive RFID technology have aroused great interest \[[@B10-sensors-16-01535]\]. The passive tag is able to passively communicate with the interrogator on a zero-powered backscatter mechanism, resulting in simple architecture, low power, and low cost. The passive RFID sensors are excellent choices for long-term and low-cost SHM \[[@B11-sensors-16-01535],[@B12-sensors-16-01535]\]. Humidity is one of the parameters considered as a critical factor in the progress of cement maturity by influencing the stability, the transformation of hydrates, and strength development \[[@B13-sensors-16-01535]\]. The result of this process is an increased strength and decreased permeability. Uncontrolled moisture diffusion during the setting and curing process can cause a number of detrimental effects. For example, fast moisture diffusion resulting from high temperature during curing, may lead to higher shrinkage stresses and lower long-term concrete strength \[[@B14-sensors-16-01535],[@B15-sensors-16-01535]\]. An insufficient water supply due to this diffusion and the evaporation of the internal moisture will hinder the hydration of cement \[[@B16-sensors-16-01535]\]. Besides, during the process of casting reinforced concrete, the corrosion of steel reinforcing bars due to high humidity will decrease the service life of reinforced concrete structures \[[@B17-sensors-16-01535]\]. Therefore, humidity monitoring is significant for concrete. There are various kinds of RFID-based humidity sensors. The simplest kind is the chipless RFID temperature sensor, which requires no Integrated Circuits (ICs) and transmits sensor data by changing the radar cross section of the RFID antenna \[[@B18-sensors-16-01535]\]. The characters of transmitting analog sensor data and no digital blocks lower its performances and constrain its applications mainly in low-cost fields. In situations where there are high accuracy requirements or a large number of sensors in close proximity, chip-based RFID sensors are employed to incorporate digital modulation. A humidity-to-frequency sensor in CMOS technology with wireless readout is presented in \[[@B19-sensors-16-01535]\], however this wireless humidity sensor only achieves a working range of 30 mm because of 13.56 MHz operating frequency. A wireless temperature and humidity sensors based on Ultra-High Frequency (UHF) is proposed in \[[@B20-sensors-16-01535]\]. Because of the complex architecture and off-chip humidity sensor, the wireless humidity sensing range in air is only 2.7 m, which cannot meet the tough requirements of concrete monitoring. This paper presents a novel wireless humidity sensor for SHM. To our knowledge, this is the first work to measure the concrete humidity based on UHF RFID technology. The rest of the paper is organized as follows. [Section 2](#sec2-sensors-16-01535){ref-type="sec"} analyzes electromagnetic wave transmission in concrete and proposes a novel architecture of wireless humidity sensor for ultra-low power application. [Section 3](#sec3-sensors-16-01535){ref-type="sec"} presents the detailed design of key blocks. [Section 4](#sec4-sensors-16-01535){ref-type="sec"} illustrates the measurement results and compared it with the results of traditional measurement. The conclusion is made in [Section 5](#sec5-sensors-16-01535){ref-type="sec"}. 2. System Design of Proposed Wireless Humidity Sensor {#sec2-sensors-16-01535} ===================================================== Since concrete has distinct electromagnetic properties at different humidity conditions, the total power loss for electromagnetic waves penetrating concrete are analyzed through the 1 MHz to 1 GHz frequency range for various humidity conditions (at depth of 0.2 m), i.e., 0.5%, 2.5%, 5.5%, and 13%. The matlab simulation results are shown in [Figure 1](#sensors-16-01535-f001){ref-type="fig"}, which are calculated from the ratio of water to the volume of the specimen \[[@B21-sensors-16-01535],[@B22-sensors-16-01535],[@B23-sensors-16-01535]\]. As expected, due to the reverse variations of the transmission and propagation losses, an optimum frequency range exists, within which there is significantly smaller power loss. For example, the total loss in 20--80 MHz frequency range for wet concrete (13% humidity) is about 5 to 10 dB less than the total loss at either 1 MHz or 1 GHz. The proposed wireless sensor works at 915 MHz, the total loss is about 10 dB at 915 MHz, which is acceptable for concrete monitoring. The aim of the proposed RFID sensor is to perform humidity measurement inside the concrete, thus the type of the antenna should be taken into consideration. [Figure 2](#sensors-16-01535-f002){ref-type="fig"} compares the return loss performances of the same RFID tag respectively equipped with two typical types of antenna (dipole and patch) in wet concrete and air. The results is based on the extended Debye model of concrete \[[@B24-sensors-16-01535]\] and simulated by HFSS. The two pairs of RFID tags, one residing in air and another one residing inside the concrete slab at 8 cm depth, are designed to resonate at 915 MHz. From [Figure 2](#sensors-16-01535-f002){ref-type="fig"}a we can see the performance of dipole is dramatically affected when it is embedded in the concrete, the bandwidth is wider and the transmission loss increases obviously. However seen from [Figure 2](#sensors-16-01535-f002){ref-type="fig"}b, the performance of patch antenna is less sensitive to the humidity change of concrete. Hence, the patch antenna is more suitable for concrete monitoring. The theoretical practicable operating power of an RFID tag *P~t~* is calculated from the Friis transmission equation \[[@B25-sensors-16-01535]\]: $$P_{t} = E_{r} \cdot G_{a} \cdot \eta_{r} \cdot \left( \frac{\lambda}{4\pi d} \right)^{2}$$ where *E~r~* is the effective isotropic radiation power of a reader, *G~a~* is the tag antenna gain, *η~r~* is the RF-to-DC power conversion efficiency of the rectifier, *λ* is the wavelength of the electromagnetic wave, and *d* is the communication distance. From Equation (1), the communication distance *d* can be expressed as: $$d = \frac{\lambda}{4\pi}\sqrt{\frac{E_{r} \cdot G_{a} \cdot \eta_{r}}{P_{t}}}$$ Hence, in order to achieve longer communication distance, lower *P~t~* and higher *η~r~* are critical for the design of UHF RFID tag, for *E~r~* is limited by regional regulations (4 W is the maximum transmitted power) and *G~a~* is roughly determined by the allowable antenna area (1.64 for the λ/2 dipole antenna). Because concrete is a high-loss material for electromagnetic wave transmission, this paper proposes a novel architecture to lower the overall power dissipation of wireless sensor for longer operating distance. [Figure 3](#sensors-16-01535-f003){ref-type="fig"} shows the architecture of the proposed wireless humidity sensor based on UHF RFID technology. The blocks, except for the antenna and matching network, are integrated on a single chip. The sensor antenna, which is matched with the sensor chip through the matching network, receives the electromagnetic waves from the RFID reader. The rectifier multiplies and transfers the received RF signal to a DC supply voltage *V~R~* for the subsequent circuitry. Once the output of the rectifier reaches the operating voltage, the Power-On-Reset (POR) block generates a reset signal *R~st~* for the temperature sensor. The clock generator provides a reference clock CLK for the sensor interface. Because the sensor interface and the clock generator should operate under a stable supply voltage *V~DD~*, a regulator block is employed to stabilize the output voltage of the rectifier. This architecture does not include the demodulator and the baseband blocks in the traditional RFID tag, which means that this wireless humidity sensor will operate without any addressing as long as the sensor receives enough energy from the RFID reader \[[@B26-sensors-16-01535]\]. 3. Key Blocks Design {#sec3-sensors-16-01535} ==================== Inspired by the reported CMOS humidity sensor designs \[[@B27-sensors-16-01535],[@B28-sensors-16-01535],[@B29-sensors-16-01535],[@B30-sensors-16-01535]\], interdigitated top metal fingers with polyimide filled into the finger gaps, can be utilized for capacitive humidity sensing. [Figure 4](#sensors-16-01535-f004){ref-type="fig"}a illustrates the structure of the proposed capacitive humidity sensor. The proposed humidity sensor was fabricated in the TSMC 0.18 μm 1P6M CMOS process. The top metal layer (Metal 6) was deposited and patterned with standard optical lithography and wet etching over the isolation layer to form the interdigitated structure. The sensing capacitor was then covered with a humidity-sensitive polyimide layer. The sensor is fabricated in a standard CMOS process without any post-processing. As seen from [Figure 4](#sensors-16-01535-f004){ref-type="fig"}b, *L* is the length of metal electrodes, *S* is the width of each electrode, and *W* is the distance between adjacent electrodes. The thickness of the polyimide layer is *H*, which is generally larger than metal thickness *h*. For a *N* finger array sensor, the total sensor capacitance *C~h~* can be expressed as \[[@B31-sensors-16-01535]\]: $$C_{h} = N\mathsf{\varepsilon}_{wet}\frac{Lh}{W}$$ where *L* is the length of metal electrodes, *h* is the thickness of metal layer, *W* is the distance between adjacent electrodes, and *ε~wet~* represents the dielectric constant of the polyimide film with absorbed water. Considering the factors including sensor capacitance, chip area sensitivity, etc., this work chooses *N* = 40, *L* = 200 µm, *W* = 2.5 µm, *S* = 2.5 µm, *h* = 1 µm, and *H* = 2 µm. The capacitive humidity sensor acts as a capacitor when it works with the sensor interface. The traditional interface starts with a capacitance-to-voltage converter, which is then followed by a voltage-to-digital converter \[[@B32-sensors-16-01535],[@B33-sensors-16-01535]\]. This technique can achieve high speed and high resolution performances. However, due to the use of an operational amplifier, this technique consumes too much power. [Figure 5](#sensors-16-01535-f005){ref-type="fig"}a shows the architecture of proposed sensor interface, which converts the sensor signal in frequency domain for low-power application. The N-stage ring oscillator is employed to generate a controlled frequency *f~osc~* which is then countered by a 10-bit counter to output the corresponding digital output *B~o~*. For a standard ring oscillator, assuming an equal rise and fall time for the different stages respectively, the oscillating frequency of the ring oscillator *f~osc~* can be expressed as follows: $$f_{osc} = \frac{1}{t_{d}} = \frac{I_{l}}{V_{m}C_{l}}$$ where *t~d~* is the delay time of the loop, *I~l~* is the current flowing through the inverter, *C~l~* is the equivalent load capacitance of the loop, and *V~m~* is the swing range of the output voltage which mostly equals the supply voltage *V~DD~*. Because *C~l~* is mainly determined by the capacitance *C~h~* of humidity sensor, *f~osc~* is a sensor-controlled frequency and *B~o~* is the corresponding digital output of humidity sensor. The simulation results of the proposed frequency *f~osc~* versus temperature on different process corners are shown in [Figure 5](#sensors-16-01535-f005){ref-type="fig"}b. The TT, FF, and SS corners correspond to "nominal", "fast" and "slow" MOSFET devices, respectively. The *f~osc~* achieves good linearity with temperature and the simulated worst case variation across corners is around ±9%. A popular performance metric of a rectifier is its power conversion efficiency which is defined as: $$\eta_{r} = \frac{P_{out}}{P_{in}}$$ where *P~out~* is the average DC output power generated at the output of the rectifier and *P~in~* is the average RF power available at the input of the rectifier. From a circuit-level point of view, *η~r~* is mainly degraded due to the forward drop of the switch diodes or transistors \[[@B34-sensors-16-01535]\]. Differed from the reported rectifier structure \[[@B35-sensors-16-01535],[@B36-sensors-16-01535],[@B37-sensors-16-01535]\], [Figure 6](#sensors-16-01535-f006){ref-type="fig"} shows the schematic of the proposed rectifier, which consists of two identical stages. The NMOS transistors M~N11--22~ and PMOS transistors M~P11--22~ form the differential-drive switch. In order to achieve a flat *η~r~* curve, the bias-voltage V~N1,2~ and V~P1,2~ are employed to boost the gate-source voltage of NMOS switch and PMOS switch, respectively \[[@B31-sensors-16-01535]\]. The large resistor *R~S~* is added to block the AC component of V~N1,2~ and V~P1,2~. In CMOS process, *R~S~* could be replaced by a PMOS transistor that operates in the cut-off region to avoid the large silicon area. [Figure 7](#sensors-16-01535-f007){ref-type="fig"} depicts in more details how the proposed scheme sets the DC level of PMOS and NMOS gate voltages. The RF intermediate voltage (M~U~,~L~) is superimposed on the bias voltages Bias~N2~ and Bias~P2~ to provide the shifted voltages Gate~N2~ and Gate~P2~ to the gate of NMOS and PMOS switches, respectively. Due to the large variation of the output voltage of rectifier, a reference circuit with small supply voltage coefficient is necessary to generate a stable reference voltage. The traditional way to generate stable 1.25 V reference voltage is using a bandgap reference circuit \[[@B38-sensors-16-01535]\]. However, it cannot meet the low-voltage requirement of low-power application. [Figure 8](#sensors-16-01535-f008){ref-type="fig"} shows the schematic of the proposed voltage regulator. It is implemented in standard CMOS process without a bandgap reference and consists of three parts. The transisitors M~P0~--M~P1~ and M~N0~--M~N2~ compose a start-up block, which is added as a precautionary measure to ensure bias in the desired state. The transistors M~P2~--M~P4~ and M~N3~--M~N6~ compose a reference generator block to provide a reference voltage *V~REF~* of 0.26 V which is compensated by temperature and supply voltage. The transistors M~P5~--M~P10~ and M~N7~--M~N9~ compose a regulator to generate a stable voltage *V~DD~* of 1.0 V for other circuits. [Figure 9](#sensors-16-01535-f009){ref-type="fig"} shows the *V~DD~* − *V~out~* characteristic at room temperature of 25 °C. The circuit starts working properly with *V~DD~* = 0.47 V. In the supply voltage range from 0.47 V to 2 V, an average reference voltage of 266.5 mV is generated. In this measured *V~DD~* range, the output voltage changes at most by 1.8 mV, thus leading to a line sensitivity of 0.441%/V. The clock generator always employs ring oscillator architecture based on current-starved inverters. The output voltage of traditional current-starved inverters is close to the supply voltage and ground, which improves the output range while increasing the power consumption. As is shown in [Figure 10](#sensors-16-01535-f010){ref-type="fig"}, in order to achieve the balance between output range and power consumption, this paper proposed a novel structure of ring oscillator for the clock generator's design. The PTAT oscillator discussed above in [Figure 3](#sensors-16-01535-f003){ref-type="fig"} also adopts the same architecture. The transistors M~1~--M~6~ consist of an *N* stage current-starved inverter, the transistor M~7~--M~9~ and M~10~--M~12~ consist the current mirrors of inverter. Compared with the conventional structure, the extra transistors M~H1~, M~H2~, M~L1~, and M~L2~ are employed to reduce the voltage swing \[[@B39-sensors-16-01535]\]. Microstrip antenna is designed by including a ground plane structure, which has a natural anti-metal advantage compared to the dipole antenna. The length of rectangular microstrip patch antenna length is approximately equal to half of its electrical length, which is not suitable for the miniaturization of tags. As shown in [Figure 11](#sensors-16-01535-f011){ref-type="fig"}a, we presented a miniaturized microstrip antenna using the embedded short stub and U-type slot \[[@B40-sensors-16-01535]\]. The impedance of chip was 19-j172 Ω at 915 MHz. The copper traces with the thickness of 0.035 mm were printed on a FR4 substrate, whose permittivity is 4.4 and thickness is 2 mm. The current in the short circuited stub of this antenna is the strongest, so the impedance of the antenna can be adjusted easily by adjusting the value of Lf1 and Lf2. The short stub embedded in radiation patch internal can lead to the reduction of the antenna volume. Furthermore, because the current can flow around the slot, it results in the increasing of the length of current path and further achieving the miniaturization purpose. The return-loss plot of this antenna is shown in [Figure 11](#sensors-16-01535-f011){ref-type="fig"}b. The return-loss at 915 MHz frequency is −29 dB and the bandwidth below −10 dB is from 889 MHz to 923 MHz. The gain of the proposed patch antenna is −3.2 dB and the maximum radiation direction is right above the antenna. The detailed parameters of the proposed patch antenna is shown in [Table 1](#sensors-16-01535-t001){ref-type="table"}. A protection device is necessary before the sensor tag is embedded into concrete, so that the sensor tag is not impaired by hydration heat arising from grouted concrete and not soaked directly in water of concrete. The transmission signal of sensor tag is sent out by electromagnetic wave, hence it is important to pay attention to wireless transmission impedance matching. Based on civil engineering techniques, this study proposes a protective high permeability acrylic package box. In order to keep the sensor in contact with the environment fully, 16 holes were punched on the side and 20 holes were punched at the bottom of the proposed package box. [Figure 12](#sensors-16-01535-f012){ref-type="fig"} shows the top view of protection device. The package box covers an area of 4 × 6.5 mm^2^ and the diameter of the hole is 0.2 cm. 4. Experimental Characterization {#sec4-sensors-16-01535} ================================ [Figure 13](#sensors-16-01535-f013){ref-type="fig"}a shows the proposed wireless humidity sensor, which was fabricated in the TSMC 0.18 μm CMOS process. The sensor tag chip covers around 8 × 3.5 mm^2^ and was equipped with this antenna on FR4 substrate by using flip chip. Then the sensor tag was packaged with the package box for concrete monitoring as shown in [Figure 13](#sensors-16-01535-f013){ref-type="fig"}b. [Figure 14](#sensors-16-01535-f014){ref-type="fig"} shows the wireless testing environment for the proposed humidity sensor. The VISN-R1200 is a special RFID tester from VI Service Network, which can process, analyze, and display the testing signals simultaneously. The sensor performance was measured in a temperature and humidity chamber of Votsch VCL4003. The temperature range and resolution of VCL4003 are −40--180 °C and 0.1 °C respectively, the humidity range and resolution of VCL4003 are 10%--98% RH and 1% RH, respectively. The measured *η~r~* curve of the proposed rectifier is shown in [Figure 15](#sensors-16-01535-f015){ref-type="fig"}a. As compared to the conventional differential-drive rectifier \[[@B36-sensors-16-01535]\], the proposed rectifier shows a flat *η~r~* curve. When input power is 13 dBm, the two curves both reach the optimal point 69%. As for the input power range whose *η~r~* is above 60%, the proposed rectifier and the conventional rectifier achieve −19 dBm and −7 dBm range, respectively. [Figure 15](#sensors-16-01535-f015){ref-type="fig"}b illustrates that the output node of the clock generator swings between the supply voltage 1.0 V and the ground, while the internal nodes of the clock generator vary from 0.3 V to 0.7 V. The reduction of the voltage swing to nearly 50% of its nominal value lowers the dynamic power consumption of the internal nodes by 75% and the overall power consumption by much more than 25%. [Figure 16](#sensors-16-01535-f016){ref-type="fig"}a shows the twice measured results of the sensor tag at 20 °C, 35 °C, and 60 °C respectively within the relative humidity (RH) range from 10% to 90%. The sensor tag achieves high linearity and shows a maximum error of 12% for 40 °C offset. The temperature dependence of the dielectric constant of the polyimide film *ε~wet~* results in this error. [Figure 16](#sensors-16-01535-f016){ref-type="fig"}b shows the measured results of six test chips at 20 °C, 35 °C, and 60 °C within the range from 10% RH to 90% RH. Due to the process variation, the chips exhibit different digital outputs for a given RH value but all the results show an excellent linearity. The average sensitivity is 0.55% RH. The hysteresis performance of the sensor at 20 °C is shown in [Figure 16](#sensors-16-01535-f016){ref-type="fig"}c. The maximum difference between the moisture adsorption and desorption at the point 80% RH does not exceed 10%. [Table 2](#sensors-16-01535-t002){ref-type="table"} compares the performances of the proposed RFID humidity sensor with previous RFID humidity sensor. The chipless RFID humidity sensor \[[@B18-sensors-16-01535]\] is specially designed for ultra-low cost application, however it has the lowest accuracy because there is no digital block in it. The wireless humidity sensor with 13.56 MHz operating frequency \[[@B19-sensors-16-01535]\] can obtain a high power conversion efficiency, however its operating distance is very limited. A RFID-based temperature and humidity sensor \[[@B20-sensors-16-01535]\] can achieve complex function, however its operating distance is only 2.7 m. The proposed wireless humidity sensor achieves a minimum power dissipation of 5.7 μW, resulting in a maximum operating distance of 17 m in free space. Our design is especially for the tough sensing application in concrete. [Figure 17](#sensors-16-01535-f017){ref-type="fig"}a shows the experimental scheme of the proposed wireless sensor in concrete. The depth of the wireless humidity embedded in concrete is *d*, the distance of the RFID reader placed above the concrete is *H*, the incidence angle between the reader and the sensor is *θ*. The photo of the test site is shown in [Figure 17](#sensors-16-01535-f017){ref-type="fig"}b. We embedded the sensor into the concrete at a depth of 8 cm and then investigated the influence of incidence angle on power transmission. The relationship between the incidence angle *θ* and power transmission is shown in [Figure 18](#sensors-16-01535-f018){ref-type="fig"}a. From that, we can see the minimum power loss is achieved when *θ* is 90°, the power transmission efficiency is 85%. When *θ* is larger than 75°, the power transmission efficiency is nearly 0%. Hence, in order to achieve the best test results, the incidence angle should be as vertical as possible. When we set *θ* = 90° and *d* = 8 cm, we measure the maximum communication distance of RFID reader. A calibrated humidity sensor SHT75 is embedded together with the proposed wireless humidity sensor to monitor the concrete humidity. For each measurement, the reader was instructed to perform 1000 attempts to read the sensor data from the wireless humidity sensor. When the success ratio remains above 80%, the communication distance is considered to be reliable. As shown in [Figure 18](#sensors-16-01535-f018){ref-type="fig"}b, the measured maximum communication distance decreases when the concrete humidity increases. The maximum communication distance is 0.52 m or 0.11 m respectively when the concrete humidity is 0% or 30%. This work employs concrete made of normal aggregate (1 h pre-wetting, moisture content of 6.6%) as the sample. The proposed wireless humidity sensor is embedded at a depth of 8 cm inside the concrete sample with the calibrated wired humidity sensor SHT75. The results measured by these two sensors compared with results of the moisture probe are plotted in [Figure 19](#sensors-16-01535-f019){ref-type="fig"}. According to \[[@B41-sensors-16-01535]\], during the early age of concrete maturing, the traditional wired humidity sensor can get more accurate results in the first 14 days resulting from the exchange of air when measuring by moisture probe. Due to the long time embedded in wet concrete, zero drift of the traditional wired humidity sensor will decrease the accuracy of the measurement results. Thus, after the 14th day the moisture probe achieves higher measurement accuracy. From [Figure 18](#sensors-16-01535-f018){ref-type="fig"} we can see in the first 14 days the proposed wireless sensor achieves nearly the same results with the traditional humidity sensor. After that, the measurement results of the proposed sensor are close to the results measured by the moisture probe. Hence the proposed wireless humidity sensor achieves better performances during the entire measurement. 5. Conclusions {#sec5-sensors-16-01535} ============== Humidity monitoring plays an important role in concrete measurement, and thus this work presents a wireless humidity sensor based on UHF RFID technology. Considering the high losses of electromagnetic waves in concrete, a patch antenna is proposed to ensure the sensor tag can work inside of the concrete. The wireless humidity sensor employs a novel architecture and is carefully designed for low power application. The measured results show the proposed sensor tag achieves high humidity linearity with a normalized sensitivity of 0.55% %RH at 20 °C. The maximum operating distance is 0.52 m when the proposed wireless sensor is embedded into the concrete at a depth of 8 cm. The measured results are highly consistent with the results measured by traditional methods. The whole experiment demonstrates that the proposed wireless humidity sensor can provide reliable performance. This work was supported by National Key Research and Development Program (2016YFC0700802), National Natural Science Foundation (61501162), China Postdoctoral Science Foundation (2015M571926), and Science and Technology Support Project of Jiangxi Province (20161BBE50076). Shuangxi Zhou is in charge of this work and Fangming Deng provides the instructions for the design of wireless humidity sensors. Xiang Wu and Baiqiang Yin provide the helps in designing the RFID sensor tag. Bing Li and Lehua Yu provide the helps in measurement. The authors declare no conflict of interest. {#sensors-16-01535-f001} {#sensors-16-01535-f002} {#sensors-16-01535-f003} {#sensors-16-01535-f004} {#sensors-16-01535-f005} {#sensors-16-01535-f006} {#sensors-16-01535-f007} {#sensors-16-01535-f008} {#sensors-16-01535-f009} {#sensors-16-01535-f010} {#sensors-16-01535-f011} {#sensors-16-01535-f012} {#sensors-16-01535-f013} {#sensors-16-01535-f014} {#sensors-16-01535-f015} {#sensors-16-01535-f016} {#sensors-16-01535-f017} {#sensors-16-01535-f018} {#sensors-16-01535-f019} sensors-16-01535-t001_Table 1 ###### Design parameters of the patch antenna. Wp Lp Ls1 Ls2 Ws1 Ws2 Ws3 Lf1 Lf2 S ------- ------- ------- ------- ------- --------- -------- ------- ------ -------- 42 mm 53 mm 23 mm 35 mm 32 mm 29.5 mm 1.7 mm 15 mm 3 mm 2.5 mm sensors-16-01535-t002_Table 2 ###### Performances comparison of various type RFID humidity sensor. Design Frequency Normalized Sensitivity Inaccuracy Distance Humidity Range Cost ----------------------------- ----------- ------------------------ ------------ ---------- ---------------- ----------- \[[@B18-sensors-16-01535]\] 900 MHz 1.1% 9.2% No 20%--70% Ultra low \[[@B19-sensors-16-01535]\] 13.56 MHz 0.8% 4.7% 0.03 m 15%--85% Low \[[@B20-sensors-16-01535]\] 900 MHz 0.7% 4.5% 2.7 m 20%--80% Low This work 915 MHz 0.55% 3.8% 17 m 10%--90% Low

1. Introduction {#sec0001} =============== Autophagy is an intracellular degradation pathway identified more than 50 years ago. The 2016 Nobel prize award to Pr Ohsumi for his work on autophagy renewed interest for the involvement of this process in various fields and autophagy is now recognized as a critical process in bone homeostasis [@bib0001]. Although autophagy\'s role in cancer is complex and context-dependent, pharmacological modulation of this process is emerging in clinical trials. However, understanding the role of autophagy within the tumor and its microenvironment is an essential prerequisite. Osteosarcoma is an aggressive cancer mainly occurring in children and young adults. Osteosarcoma treatment relies on chemotherapy and surgery, leading to a 70% 5-year survival in patients with a non-metastatic disease. Nevertheless, the 5-year survival of patients with a metastatic disease is around 20%, emphasizing the importance to develop new therapeutic strategies. As autophagy modulation could be part of these new options, the aim of the present review is to summarize the major findings regarding the role of autophagy in osteosarcoma. 2. Autophagy, a major degradative pathway {#sec0002} ========================================= Autophagy is an intracellular degradation process which eliminates and recycles damaged proteins and organelles [@bib0002], [@bib0003]. An isolation membrane, called a phagophore, is formed in the cytosol to sequester the damaged material. After elongation and closure of the phagophore, a double-membrane vesicle called an autophagosome is generated. Autophagosomes then fuse with lysosomes to generate autolysosomes, in which the content is degraded and recycled. Basal autophagy exists in all eukaryotic cells and exerts a quality control function. Autophagy can also be stimulated by various stresses such as starvation or hypoxia to sustain cell survival [@bib0002], [@bib0003]. Although autophagy is essentially a prosurvival mechanism, overactivated autophagy can lead to cell death\[4\]. In addition to its classical degradation role, autophagy was recently shown to participate in some secretion processes [@bib0005]. Autophagy is a complex process regulated by the coordinated action of more than 30 autophagy-related proteins (ATG) ([Fig. 1](#fig0001){ref-type="fig"}). Autophagy initiation is mediated by the UNC-51-like kinase (ULK1) complex which is negatively regulated by the mammalian target of rapamycin (mTOR) in nutrient-rich conditions [@bib0006]. Upon starvation, mTOR is inactivated, leading to ULK-1 activation and autophagy induction [@bib0007]. The Class III PI3K complex I is then recruited to initiate autophagosome formation. Elongation and closure of the phagophore requires two complexes containing ubiquitin-like proteins and their respective conjugation machineries [@bib0008]. The first complex mediates the covalent conjugation of ATG12 to ATG5 due to the action of ATG7 and ATG10 enzymes. The ATG12-ATG5 conjugate then binds to ATG16 to form the ATG12-ATG5/ATG16 complex which is essential for autophagosome biogenesis. The second ubiquitin-like conjugation system allows the conjugation of phosphatidylethanolamine (PE) to the microtubule-associated light chain 3 (LC3 or ATG8). Nascent LC3 is first processed by the protease ATG4, activated by the ATG7 enzyme, transferred to the ATG3 conjugating enzyme, and then conjugated with PE [@bib0009]. LC3-PE (LC3-II) is an integral membrane protein present in autophagosomes and is used as an autophagosome marker.Fig. 1Autophagy mechanism. The autophagy mechanism and the different molecular complexes involved in the process are presented. In response to different stimuli such as mTORC1 inactivation, autophagy is initiated through the action of the ULK1 complex and the class III PI3K complex. A phagophore is generated in the cytosol to isolate damaged organelles, aggregates and proteins. The ATG12 conjugation system and the LC3 conjugation system are then involved in the elongation and closure of the phagophore, leading to autophagosome formation. Finally, the autophagosome fuses with lysosomes to degrade the material which will then be recycled.Fig. 1 The cellular material targeted to degradation is sequestered by autophagosomes through the action of selective autophagy receptors such as SQSTM1/p62, NBR1, NDP52 or Optineurin. Due to a specific amino acid sequence binding to ATG8 protein family members (LC3-interacting region or LIR motif), these receptors mediate the cargo delivery to autophagosomes [@bib0010], [@bib0011]. 3. Autophagy in bone {#sec0003} ==================== Bone is a complex organ in which several cell types act in a coordinated manner. Bone remodeling starts by the resorption of old mineralized bone matrix by osteoclasts (OC) followed by de novo bone formation by osteoblasts (OB). Osteocytes (OST), the multifunctional mechanosensing cells, which are embedded within the bone matrix, orchestrate this remodeling process. Autophagy is involved in OB differentiation [@bib0012], [@bib0013], survival [@bib0014], [@bib0015] and function, i.e. bone matrix mineralization [@bib0013], [@bib0016]. In OC, autophagy is also required for differentiation [@bib0017], [@bib0018], [@bib0019] and several autophagic proteins are involved in bone resorption [@bib0020]. OST, which are long-lived key regulators of bone remodelling, are also highly dependent on autophagy for their survival and function [@bib0021], [@bib0022]. Finally, several lines of evidence suggest that an autophagy defect could be related to some bone pathologies such as osteoporosis [@bib0023], Paget\'s disease of bone [@bib0024], or osteopetrosis [@bib0025]. 4. Autophagy in cancer {#sec0004} ====================== In the context of cancer, numerous studies have demonstrated that autophagy is used by tumor cells as a highly dynamic mechanism to repress initial steps in carcinogenesis and/or support the survival and growth of established tumors [@bib0026], [@bib0027] ([Fig. 2](#fig0002){ref-type="fig"}). In the early stages of tumorigenesis, autophagy may be tumor suppressive by (i) limiting chromosomal instability, (ii) restricting oxidative stress which can act as an oncogenic stimulus, (iii) preventing intratumoral necrosis and local inflammation. In advanced cancers, tumor-promoting functions of autophagy rely on survival in response to stresses due to cancer progression, metastasis and treatments [@bib0027]. Tumor cells use autophagy to maintain mitochondrial function and homeostasis required by their high energy demand of unrestrained proliferation. In addition, autophagy-mediated secretion can play a role in tumor microenvironment modification [@bib0028]. Autophagy has also been involved in the generation of a dormancy state which protects cells from chemotherapeutic stress [@bib0029]. Moreover, autophagy was shown to be required in the maintenance of stem cell properties, particularly in cancer stem cells (CSC) [@bib0030]. Autophagy is also required for the motility and invasion of metastatic tumor cells by the promotion of focal adhesion disassembly in an autophagic dependent manner via paxilline degradation [@bib0031]. Lastly, autophagy can also be used by cancer cells to modify host antitumor immunosurveillance and to inhibit adaptative and innate immune responses [@bib0032].Fig. 2Autophagy role in cancer. In early stages of tumor development, autophagy can exert tumor suppressive functions by DNA damage limitation through reactive oxygen species (ROS) elimination, inflammation limitation and cell death induction. In established tumors, autophagy can support tumor growth through various mechanisms such as nutrient generation, microenvironment modification, drug resistance, CSC maintenance promotion and immune response inhibition.Fig. 2 Hence, autophagy exerts context-dependent roles in cancer and most clinical trials, which target established tumors, were performed to inhibit autophagy [@bib0033]. These trials, associating autophagy inhibitors with conventional treatments, led to encouraging results but further research is needed to define the safety and utility of this approach in each cancer type [@bib0033]. In osteosarcoma, a clinical trial combining the autophagy inhibitor hydroxychloroquine with gemcitabine and docetaxel is presently in progress [@bib0034]. 5. Autophagy in osteosarcoma {#sec0005} ============================ 5.1. Autophagy is deregulated in osteosarcoma {#sec0006} --------------------------------------------- Numerous genomic and epigenetic studies have revealed a striking genomic complexity and heterogeneity in osteosarcoma tumors. Several heritable genetic predisposition syndromes with germline mutations in the tumor suppressor genes such as TP53 and RB1 or in RecQL4 gene are associated with osteosarcoma [@bib0035], [@bib0036]. In addition, altered candidate driver genes with biological evidence for a role in osteosarcoma development have been identified in osteosarcoma somatic genomes [@bib0035]. Among the tumor suppressors frequently inactivated in osteosarcoma, several ones have been demonstrated to be involved in autophagy regulation [@bib0037], [@bib0038] ([Fig. 3](#fig0003){ref-type="fig"}). Indeed, RB1, ARF, WIF1, PTEN and TSSC3 were shown to trigger autophagy through various mechanisms [@bib0039], [@bib0040], [@bib0041], [@bib0042], [@bib0043]. TP53 exhibit a dual role in autophagy regulation, nuclear TP53 inducing autophagy through transcriptional regulation and cytoplasmic TP53 acting as a master autophagy repressor [@bib0038]. RECQL4 was also recently shown to exert a dual regulation of autophagy [@bib0044]. In addition, several oncogenes activated in osteosarcoma such as IGF2, H19, COPS3 and RUNX2 also regulate autophagy [@bib0029], [@bib0045], [@bib0046], [@bib0047]. Finally, different studies evidenced that the mTOR pathway was overactivated in osteosarcoma, suggesting autophagy inhibition [@bib0048], [@bib0049]. Taken together, these data indicate that numerous autophagy regulators are affected in osteosarcoma, suggesting that this critical process is deregulated. Nevertheless, autophagy can be induced in OS and the molecular mechanisms involved in autophagy triggering have been reviewed elsewhere [@bib0050].Fig. 3Potential autophagy deregulation in osteosarcoma. Effect on autophagy of tumor suppressors frequently inactivated and oncogenes frequently activated in osteosarcoma. Green arrow: autophagy stimulation; Orange arrow: dual effect on autophagy. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)Fig. 3 The present review aims at updating our current knowledge regarding the complex pro and antitumoral role of autophagy in OS. Indeed, autophagy can either promote cell survival [@bib0029], [@bib0046], [@bib0051], [@bib0052], [@bib0053] or contributes to cell death [@bib0054]. Moreover, autophagy can be involved in chemosensitivity or chemoresistance during osteosarcoma therapy [@bib0054]. In the next paragraphs, we will review several studies illustrating both aspects of autophagy role in osteosarcoma. 5.2. Autophagy as a protumoral process in osteosarcoma {#sec0007} ------------------------------------------------------ In normal conditions, autophagy is a prosurvival pathway that preserves organelle function, prevents cellular waste product toxicity, and produces energy and substrates for survival. In this context, tumors can use autophagy to survive in a hostile microenvironment and to increase growth and aggressiveness. ### 5.2.1. Autophagy role in OS tumor cells {#sec0008} The protumoral role of autophagy has been demonstrated in several osteosarcoma cell lines by the knockdown of key autophagy genes. It has been shown that ATG4B, the cysteine proteinase activating LC3, is crucial for osteosarcoma development in the Saos-2 cell model [@bib0051]. Indeed, treatment with an ATG4B chemical inhibitor results in autophagy deficiency and leads to a decreased proliferation in vitro and tumor growth in vivo [@bib0051]. Similarly, using a siRNA approach in three osteosarcoma cell lines (HOS, MG-63, and U2OS), Zhang et al. have shown that BECN1 knockdown induces a decrease in cell growth and invasion in vitro and reduces metastasis development in vivo [@bib0052]. ### 5.2.2. Autophagy role in treatment resistance {#sec0009} In addition to the role of autophagy in osteosarcoma survival and progression, numerous studies have demonstrated its role in treatment resistance [@bib0054], [@bib0055]. First, several studies demonstrated that chemotherapy-resistant OS cells exhibit increased autophagy [@bib0056], [@bib0057], [@bib0058]. Then, different factors known to mediate drug resistance such as HMGB1, HSP90AA1 or GFRA1, exert their effects through autophagy induction [@bib0059], [@bib0060], [@bib0061]. Moreover, several microRNAs (miRNAs) inhibiting autophagy are downregulated in OS, participating in treatment resistance. miRNAs are small noncoding RNAs that regulate gene expression by binding to the 3′ untranslated region of their target mRNAs, leading to their translational suppression or degradation. MiRNAs are now recognized as major contributors in cancer development and treatment resistance, including in OS [@bib0062]. In addition, miRNAs were shown to regulate autophagy [@bib0063], and several miRNAs target autophagy genes such as Atg5 [@bib0064] or Atg16L1 [@bib0065]. MiR-410, which directly decreases the autophagy gene ATG16L1 expression, is markedly downregulated in human osteosarcoma tissues [@bib0065]. MiR-30a, which targets BECN1, is significantly reduced in doxorubicin-resistant OS cells [@bib0066]. Chemoresistance of osteosarcoma tumor cells to doxorubicin is also associated with the downregulation of miR-143 expression, leading to autophagy activation with upregulation of ATG2B, Bcl-2 and LC3-II protein levels [@bib0067]. Similarly, miR-140-5p, which suppresses autophagy through HMGN5, is downregulated in OS and associated with patient\'s chemoresistance [@bib0068]. More recently, it has been shown that some long non-coding RNAs are involved in autophagy activation in OS through miRNA targeting [@bib0064], [@bib0069]. Finally, autophagy inhibition through BECN1 or Barkor/ATG14 knockdown results in an increased sensitivity of OS cells to chemotherapy [@bib0052], [@bib0070]. Autophagy involvement in treatment resistance can be mediated through various mechanisms. The protective effect can be mediated by ROS reduction, as it was described for radioresistance of OS cells [@bib0071]. Treatment resistance can also involve the degradation of apoptosis-promoting proteins. This is the case in cisplatin-resistant OS cell lines exhibiting autophagic degradation of FOXO3A, which leads to decreased PUMA expression and apoptosis inhibition [@bib0056]. Autophagy was also shown to participate in the maintenance of a dormancy-like state protecting OS cells from chemotherapeutic drugs [@bib0029]. Finally, Ma et al. have shown in osteosarcoma tissues that a weak level of the autophagy substrate p62, which correlates with an active autophagic flux, may be associated with higher metastatic and chemoresistant rates in osteosarcoma patients, suggesting a protumoral effect of autophagy [@bib0072]. Considering the role of autophagy in OS treatment resistance, many studies combined the use of anti-cancer agents with autophagy inhibitors such as chloroquine, 3-methyladenine or spautin-1 (specific and potent autophagy inhibitor-1). These autophagy inhibitors enhance OS cell death induced by cisplatin [@bib0073], doxorubicin [@bib0074], bone-targeted gallium compound KP46 [@bib0075], and increase in vivo tumor regression following photodynamic therapy [@bib0076] or mTOR inhibition [@bib0077]. ### 5.2.3. Autophagy role in OS cancer stem cells {#sec0010} Autophagy was also shown to participate in the maintenance of cancer stem cell (CSC) properties [@bib0078]. Regarding OS, very few studies investigated the role of autophagy in CSC. Zhang et al. have used the CD271 marker to isolate CSC from two human OS cell lines. CD271+ OS cells showed a higher autophagy activity than CD271- OS cells under hypoxia and low nutrient condition. Autophagy deficiency in the CD271+ cells decreased the stemness marker expression, restored chemotherapeutics sensitivity and restricted the advantage of CD271+ OS cells in terms of tumorigenesis in vivo [@bib0079]. More recently, calpain-6 was demonstrated to control OS CSC fate by promoting autophagy and preventing senescence [@bib0080]. 5.3. Autophagy as an antitumoral process in osteosarcoma {#sec0011} -------------------------------------------------------- While the cytoprotective role of autophagy is well described, particularly in response to cancer treatment, its role in cell death has long been controversial. However, several evidences now demonstrate autophagy implication in cell death, including in osteosarcoma. Indeed, autophagy can result in an autophagic cell death, defined as an autophagy-mediated cell death that can be suppressed by the inhibition of the autophagic pathway [@bib0004], [@bib0081]. Such an autophagic cell death was observed in vitro in WT and doxorubicin-resistant U2OS cells after treatment with the bisindolic alkaloid voacamine [@bib0082]. Voacamine induced an apoptosis-independent cell death associated with autophagy stimulation, and knockdown of key autophagy genes resulted in decreased cell death [@bib0082]. In addition, autophagy can also promote apoptosis or necroptosis through various mechanisms [@bib0081]. This can be achieved, for example, through the degradation of anti-apoptotic and cell survival factors [@bib0081]. In this context, treatment of U2OS or Saos-2 osteosarcoma cell lines with Riccardin (RD), a naturally occurring macrocyclic bisbibenzyl, triggers both autophagy and apoptosis and pharmacological or genetic autophagy inhibition decreased RD-mediated cell death [@bib0083]. Similarly, celastrol, a triterpene from traditional chinese medicine induces JNK activation and ROS generation, resulting in apoptosis and autophagy in MG-63 osteosarcoma cells [@bib0084]. Pharmacological autophagy inhibition diminished caspase-3 and PARP cleavage, suggesting that autophagy promoted apoptosis. Although a large number of studies demonstrate that chemotherapy triggers a cytoprotective autophagy, some of them indicate that autophagy can sensitize OS cells to chemotherapy. Inhibition of camptothecin-induced autophagy was shown to decrease cytotoxicity in DLM8 OS cells and to increase cytotoxicity in K7M3 OS cells [@bib0085]. Similarly, autophagy inhibition led to opposite effect in LM7 and CCH---OS-D or K7M3 OS cells [@bib0050]. In this last report, the authors showed that the expression of phosphorylated heat shock protein 27 (HSP27) after chemotherapy, predicts the effect of autophagy inhibition on OS cell survival or death. Interestingly, a recent study of Livingston et al. demonstrated that the presence of LC3B+ puncta, an autophagosome marker, is an independent prognostic biomarker of improved survival following neoadjuvant chemotherapy [@bib0086]. This work, which was performed in tumor specimens isolated from 260 osteosarcoma patients, suggests that autophagy could be beneficial in this context. 6. Conclusion {#sec0012} ============= Autophagy is a prosurvival pathway used by OS tumor cells to increase their proliferation and development, resist to cancer treatments and preserve a pool of CSC within the tumor. Nevertheless, autophagy can also be antitumoral in OS and lead to cell death but several general questions remains to be answered: which signal turns prosurvival autophagy into a death process ? Is there an autophagy threshold level required for cell death induction ? We only start to address these issues which are essential to understand the role of autophagy in OS and to use this pathway as a potential weapon against this cancer. Conflicts of interest {#sec0013} ===================== The authors declare no conflicts of interest. The MATOs team is supported by grants from the France Cancer association, Fondation ARC pour la recherche sur le cancer and Cancéropôle PACA. We apologize to the authors that we were unable to cite due to space limitations.

Introduction {#s1} ============ Lung cancer remains the leading cause of death in the world, and small-cell lung cancer (SCLC) accounts for 15--25% of cases [@pone.0091388-Crivellari1]. Clinically, SCLC is distinguished from non-small cell lung cancer (NSCLC) by rapid tumor growth and the early onset of metastases. Unlike in most Western countries, SCLC rates are still increasing in China, where smoking prevalence also continues to increase. Although SCLC is considered highly responsive to chemotherapy and radiotherapy, relapse often occurs within two years and the overall survival (OS) beyond five years is approximately 3%--8%. To improve clinical outcomes, new drugs have been developed, but it is discouraging that the survival for both localized and advanced SCLC has plateaued in the past 20 years. SCLC is thought to carry a variety of molecular abnormalities that are quite different from NSCLC. For example, TP53 mutations, Rb inactivation and c-Myc amplification are common in SCLC compared to NSCLC [@pone.0091388-Salgia1], [@pone.0091388-WistubaGazdar1]. In contrast, epidermal growth factor receptor (EGFR) mutations, K-ras mutations and P16 inactivation are frequently found in NSCLC, but not in SCLC [@pone.0091388-Salgia1]--[@pone.0091388-Shiao1]. However, the underlying mechanisms by which SCLC rapidly progresses remain to be defined, and the identification of these mechanisms could promote the development of novel therapeutic agents and improve the management of this challenging disease. MicroRNAs (miRNAs) are a class of noncoding RNAs that are approximately 22 nucleotides long and are post-transcriptional regulators of gene expression. Because of their fundamental role in development and differentiation, involvement in the biological mechanisms underlying tumorigenesis and progression, as well as low complexity, stability, and easy detection, miRNAs represent a promising class of tissue- and blood-based biomarkers of cancer. A number of researchers have demonstrated that aberrant miRNA expression is closely related to the development and progression of many cancers, behaving as tumor suppressors or oncogenes [@pone.0091388-Zhao1]--[@pone.0091388-Calin1]. Recent data from multiple studies strongly support the potential of microRNAs as biomarkers for NSCLC [@pone.0091388-Takamizawa1]--[@pone.0091388-Hayashita1]. In addition, altered microRNA expression is also associated with tumor progression and survival in NSCLC cancer patients. A global miRNA expression pattern assessed using microarray based analysis has been established, and several NSCLC "miRNA signatures" have been proposed for improved molecular staging and classification of NSCLC [@pone.0091388-Eder1]--[@pone.0091388-Yu1]. However, the clinical significance of miRNAs in SCLC has not been well established. To investigate the role of aberrant miRNA expression profiles in SCLC, we first surveyed 924 known miRNAs in formalin-fixed paraffin-embedded (FFPE) specimens from 42 patients and identified a dual-miRNA signature to predict the clinical outcome of early stage SCLC patients treated with surgical resection followed by adjuvant chemotherapy. This association was further validated with quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis of 40 additional patient specimens, and the prognostic value of the signature appears to be independent of age, gender and smoking status in multivariate Cox regression analysis. Materials and Methods {#s2} ===================== Ethics Statement {#s2a} ---------------- This retrospective study was approved by the Institutional Review Board of the Cancer Hospital and Institute of Chinese Academy of Medical Sciences and Peking Union Medical College. All of the patient records used in this study were made anonymous and de-identified before analysis. Patients and samples {#s2b} -------------------- We retrospectively studied formalin-fixed paraffin-embedded (FFPE) specimens from patients who were diagnosed with resectable limited SCLC and underwent surgery followed by adjuvant chemotherapy (platinum-based regimens such as cisplatin/etoposide and carboplatin/etoposide) at the Cancer Hospital and Institute of Chinese Academy of Medical Sciences (Beijing, China) between January 2000 and December 2005. All of the patients had an ECOG performance status of 0--1, and patients with mixed small-cell/large-cell carcinoma or combined small cell carcinoma were excluded. In total, 82 SCLC patient samples were analyzed, among which 42 samples were used as a screening set to identify miRNAs associated with survival using a miRNA microarray. The selected miRNAs were then validated in the remaining 40 patients using real-time PCR. Hematoxylin and eosin (H&E) sections of all of the samples were reviewed by a pathologist to confirm the diagnosis. Three normal lung tissue (NL) samples were obtained from macroscopically uninvolved areas 2--3 cm away from benign nodules of patients who underwent surgical resection. All of the NL tissues were histopathologically assessed and were morphologically normal. RNA extraction {#s2c} -------------- Low-molecular-weight RNA was isolated from total RNA, which was extracted with TRIZOL, using a PEG solution precipitation method [@pone.0091388-Watanabe1]. In brief, to obtain low molecular weight RNA, total RNA was precipitated with equal amounts of PEG solution, and the supernatant was precipitated using isopropanol. All of the RNA extracts were assessed and showed no signs of RNA degradation. miRNA microarray analysis {#s2d} ------------------------- Microarray analysis was performed as previously described [@pone.0091388-Cao1]. Briefly, RNA was labeled using the T4 RNA ligase labeling method. Hybridization was performed using a custom microRNA microarray panel (CapitalBio, Beijing, China), which included probes in triplicate for 924 mature human and mouse miRNA sequences and eight short oligonucleotides that possessed no homology to any known RNA sequence as external controls. The hybridized arrays were scanned with a LuxScan 10K-A laser confocal scanner, and the images obtained were then analyzed using the LuxScan 3.0 software (CapitalBio, Beijing, China) [@pone.0091388-Guo1]. For data processing, only data for the human miRNAs (546 of 924) was examined. First, the raw data were pre-processed to filter out miRNAs with maximum intensities less than 300 to reduce bias in the normalization step. After filtration, 456 (83.5%) of 546 miRNAs were included for further analysis. Then, the average values of the replicate spots for each miRNA were background-subtracted, and the signals were normalized using the median center tool for genes with Cluster 3.0 software. Hazard ratios (HR) from univariate Cox regression analysis, a standard method in biostatistics for examining survival data that minimizes bias by simply removing censored patients, were used to identify which miRNAs were correlated with the OS of the patients [@pone.0091388-Yu1]. We used a P-value of 0.1 as the cutoff for gene selection. A risk score for the miRNA signature survival prognosis was calculated according to a combination of the expression level of the miRNA weighted by the regression coefficient derived by univariate Cox regression analysis [@pone.0091388-Yu1], [@pone.0091388-Lossos1], [@pone.0091388-Liu1]. A class comparison with the nonparametric Mann-Whitney U test was also performed to identify differently expressed miRNAs between the 42 SCLC samples and the three NL samples. All of the raw data were deposited in a MIAME compliant database (ArrayExpress, GEO: GSM678225 - GSM678266). miRNA quantitative real-time RT-PCR {#s2e} ----------------------------------- cDNA was prepared from 100 ng of total RNA with specific primers for miR-886-3p, miR-150 or U6 as the internal control. For the detection of mature miRNAs, quantitative miRNA RT-PCR was performed using the stem-loop qRT-PCR method [@pone.0091388-Chen1]. The oligos used included the miRNA common reverse primer, GTGCAGGGTCCGAGGT; miR-150, TCTCCCAACCCTTGTACCAGTG; miR-150 RT primer, GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCACTGG; miR-150 forward primer, GTCTCCCAACCCTTGTACCA; miR-886-3p, CGCGGGTGCTTACTGACCCTT; miR-886-3p RT primer, GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAGGGT; miR-886-3p forward primer, CACGCGGGTGCTTACTGAC; U6 forward primer, CTCGCTTCGGCAGCACA; and U6 reverse primer, AACGCTTCACGAATTTGCGT. qRT-PCR was performed using an ABI Prizm 7300 Sequence Detection System with LightCycler DNA Master SYBR Green I mix (Roche Molecular Biochemicals, Mannheim, Germany) following the manufacturer\'s instructions. The level of miRNAs was determined using the 2^--ΔΔCt^ method with SDS 1.3 software, normalized to the level of the internal control, U6, and plotted for inter-cellular comparison. Endpoints {#s2f} --------- The primary endpoint of this study was OS. Secondary endpoints included progression-free survival (PFS), local-regional control (LRC), and distant metastasis-free survival (DMFS). OS was calculated from the date of diagnosis to death from any cause or the last known date that the patient was alive. PFS was defined as the duration from the date of diagnosis to the date of failure either in the thorax or at distant sites, the date of death from any cause, or the date of the last patient follow-up. LRC was calculated from the date of diagnosis to the first local-regional (thorax) recurrence, and DMFS was calculated from the date of diagnosis to the first distant metastasis, the date of death from any cause, or the date of the last patient follow-up. Statistical analysis {#s2g} -------------------- The clinical characteristics of different groups were compared using the independent student\'s t test, χ^2^ test, or Fisher\'s exact test. The correlation between the microarray data and the qRT-PCR data was analyzed with the Spearman correlation. OS, PFS, LRC and DMFS were estimated with the Kaplan-Meier method and log-rank test. HRs were calculated with Cox regression analysis. A multivariate Cox regression model was used to test if the miRNA signature was an independent prognostic factor when adjusted for age, sex, and smoking status. All of the analyses were performed using the SPSS software package (version 11.5, SPSS Inc., Chicago, IL). A two-sided p-value of less than 0.05 was considered to be statistically significant. Results {#s3} ======= The clinical characteristics of the 82 SCLC patients are listed in [Table 1](#pone-0091388-t001){ref-type="table"}. The median follow-up time was 57.2 months. Forty-four patients are still alive. Forty-two patients had recurrent disease, and the median time to a diagnosis of relapse was 12.3 months. 10.1371/journal.pone.0091388.t001 ###### Clinical characteristics of the LD-SCLC patients. {#pone-0091388-t001-1} Characteristic Training set Testing set Total --------------------------------------- -------------- ------------- ------------ **Age, years** Median 60.1 55.0 57 Range 33.0--77.0 33.0--73.0 33.0--77.0 **Sex** Male (%) 30 (71.4) 32 (80.0) 62 (75.6) Female (%) 12 (28.6) 8 (20.0) 20 (24.4) **Smoking status** Current/former smokers (%) 30 (71.4) 29 (72.5) 59 (72.0) Never smokers (%) 12 (28.6) 11 (27.5) 23 (28.0) **No. of ChT cycle (Platinum-based)** 2--4 24 19 33 4--6 17 13 30 \>6 1 8 9 **Median OS, months** 56.0 67.4 67.4 **Median PFS, months** NR 44.6 47.7 **Median LRC, months** NR NR NR **Median DMFS, months** NR 60.0 60.0 LD, limited stage disease; SCLC, small cell lung cancer; No, number; ChT, chemotherapy; OS, overall survival; PFS, progression-free survival; LRC, local-regional control; DMFS, distant metastasis-free survival; NR, not reached. In the training set, we identified two miRNAs, miR-150 and miR-886-3p, that were associated with poor OS using the Cox proportional hazard regression model. Both of them were protective ([Table 2](#pone-0091388-t002){ref-type="table"}). The comparison between NL and SCLC tissues also verified that miR-150 and miR-886-3p expression levels in SCLC tumors were much lower than in NL samples ([Fig 1](#pone-0091388-g001){ref-type="fig"}). {#pone-0091388-g001} 10.1371/journal.pone.0091388.t002 ###### microRNAs associated with overall survival in SCLC. {#pone-0091388-t002-2} miRNA Hazard ratio p value Location Gene references into functions ---------------- -------------- --------- ---------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **miR-886-3p** 0.54 0.045 5q31.1 Predicting outcome in AML [@pone.0091388-Treppendahl1]; regulating cell proliferation and migration in familial non-medullary thyroid cancer [@pone.0091388-Xiong1]. **miR-150** 0.26 0.049 19q13.33 Down-regulated in leukemia (1), colorectal cancer [@pone.0091388-Pizzini1] and malignant pancreatic tissues [@pone.0091388-Srivastava1]; down-regulated associated with shorter survival and a worse response to adjuvant chemotherapy in colorectal cancer [@pone.0091388-Ma1]. We then derived a formula to calculate the risk score for every patient from the expression level of the two miRNAs associated with OS, weighted by Cox regression coefficients: Risk Score  = (0.545 × expression level of miR-150) + (0.617 ×expression level of miR-886-3p). Patients in the training set were divided into high-risk or low-risk groups using the median miRNA signature risk score as the cutoff. Patients with higher risk scores are expected to have poor OS. Compared to patients with a low-risk miRNA signature, patients with a high-risk signature had significantly shorter median OS (12.6 months compared with not reached, p = 0.02) ([Fig. 2A](#pone-0091388-g002){ref-type="fig"}). Likewise, patients in the high-risk group had shorter PFS and DMFS times than those in the low-risk group (Both p-values were 0.045) ([Fig. 2B-C](#pone-0091388-g002){ref-type="fig"}). However, LRCs were similar between the two groups (p = 0.36) ([Fig. 2D](#pone-0091388-g002){ref-type="fig"}). {#pone-0091388-g002} To validate the microarray hybridization expression data, the expression levels of the two miRNAs associated with OS were quantified using qRT-PCR in 10 SCLC tissues randomly chosen from the training set. Significant correlation was observed between log 2-transformed array signal intensities and qRT-PCR Ct values for both miR-150 (r = 0.94, p\<0.001) and miR-886-3p (r = 0.75, p = 0.01) ([Fig. 3](#pone-0091388-g003){ref-type="fig"}). {#pone-0091388-g003} Next, we used the same risk score formula obtained from the training set to calculate the miRNA signature risk score for 40 patients in the testing set, for which the miRNA levels in FFPE specimens were determined by qRT-PCR. Again, the high miRNA signature risk score patients exhibited shorter survival than in the low-risk group (median OS 18.9 months compared with not reached, p = 0.005) ([Fig. 4A](#pone-0091388-g004){ref-type="fig"}). PFS, DMFS, and LRC were also significantly shorter in the high than the in the low risk group (PFS p = 0.017, DMFS p = 0.019 and LRC p = 0.031, [Fig. 4B, 4C, and 4D](#pone-0091388-g004){ref-type="fig"}). The multivariate Cox-regression analysis was performed to further evaluate whether the dual-miRNA signature is an independent prognostic factor associated with survival in this testing set. Our results showed that the miRNA signature remained an independent prognostic factor for OS (HR: 0.26, 95%: CI 0.10--0.69, p = 0.007), PFS (HR: 0.36, 95%: CI 0.15--0.86, p = 0.02), DMFS (HR: 0.34, 95%: CI 0.13--0.86, p = 0.02), and LRC (HR: 0.26, 95%: CI 0.07--0.99, p = 0.05) after adjustment for age, gender and smoking status ([Table 3](#pone-0091388-t003){ref-type="table"}). {#pone-0091388-g004} 10.1371/journal.pone.0091388.t003 ###### Multivariate Cox regression analysis of miRNA signature and survival in the testing set (N = 40). {#pone-0091388-t003-3} Variable Hazard Ratio 95%CI p value -------------------------------------- -------------- ------------- ---------- **Overall survival** miRNA signature 0.26 0.10--0.69 0.007 Age 1.96 0.77--5.02 0.16 Gender 1.12 0.11--11.71 0.92 Smoking status 0.57 0.07--4.49 0.60 **Progression-free survival** miRNA signature 0.36 0.15--0.86 **0.02** Age 1.45 0.60--3.52 0.41 Gender 1.15 0.11--11.73 0.91 Smoking status 0.45 0.06--3.46 0.44 **Distant metastasis-free survival** miRNA signature 0.34 0.13--0.86 **0.02** Age 1.48 0.57--3.84 0.42 Gender 0.84 0.07--9.83 0.89 Smoking status 0.50 0.07--3.89 0.51 **Local-regional control** miRNA signature 0.26 0.07--0.99 **0.05** Age 2.38 0.66--8.54 0.19 Gender 0.52 0.04--6.90 0.62 Smoking status 1.34 0.15--11.67 0.79 Discussion {#s4} ========== In this study, we initiated a discovery phase exercise to identify key miRNAs by surveying the expression of 924 known miRNAs in FFPE specimens from 42 SCLC cases. We discovered that a dual-miRNA signature, miR-150 and miR-886-3p, was associated with OS and PFS. These findings were subsequently confirmed in an independent cohort of 40 SCLC patients using the qRT-PCR method, and the concordance between these two platforms was high. Applying an optimal normalization method is particularly important for miRNA profiling to minimize technical variations and to get meaningful biological changes. However there is no consensus normalization method and recommendations at present for either microarray or qRT-PCR methods in terms of miRNA profiling. In this study, we used a global normalization method in the training set, which is considered more suitable for large-scale miRNA-profiling data sets [@pone.0091388-Pritchard1]. In the testing set, we used U6 RNA for normalizing data because it is the most frequently reported reference gene for miRNA expression studies in lung cancer in the literature [@pone.0091388-Yu1], [@pone.0091388-Lee1]--[@pone.0091388-Lebanony1]. We also correlated the expressions of the two miRNAs on qRT-PCR with array expressions, and confirmed that different miRNA-profiling methods did not influence the results of miR-150 and miR-886-3p expressions ([Fig.3A-B](#pone-0091388-g003){ref-type="fig"}). In addition, the correlation of U6 expression on qRT-PCR with global mean array expression was observed (r = 0.71, p\<0.001; [Fig. 3C](#pone-0091388-g003){ref-type="fig"}). U6 RNA showed absolute fold changes lower than 1.2-times and had no significant differences between the NL and SCLC samples (p = 0.96, [Fig. 3D](#pone-0091388-g003){ref-type="fig"}). Currently, there are only two published studies examining miRNA expression in human SCLC samples [@pone.0091388-Lee2], [@pone.0091388-Ranade1]. Lee et al. investigated the expression of a panel of seven miRNAs (miR-21, miR-29b, miR-34a/b/c, miR-155, and let-7a) in 31 SCLC tumors [@pone.0091388-Lee2]. They reported that the expression of these seven miRNAs was unrelated to the clinical characteristics of SCLC patients and was not prognostic in terms of OS or PFS, nor was the expression level predictive of treatment response. These data were consistent with our findings. Ranade and colleagues evaluated the expression of 880 validated human mature miRNAs with an additional 473 validated human pre-miRNAs in 34 formalin-fixed, paraffin-embedded SCLC tumor specimens and found that miR-92a-2\* was independently associated with survival. Although this study showed for the first time that miRNAs can be prognostic for SCLC, it included cases with both limited stage (12.1%) and extensive stage (87.9%) cancer at diagnosis, and patients with extensive-stage disease have cancers that are considered more heterogeneous. Furthermore, in this study, the tumor tissues were collected not only from primary tumors but also from lymph nodes and distant metastases, which would confound the evaluation results because miRNA expression is considered to be tissue specific. In addition, only half of the patients in this study received radiation during first-line therapy. This is important because radiation might significantly affect survival in both limited-stage [@pone.0091388-Simon1] and extensive-stage SCLC [@pone.0091388-Zhu1]. To date, our study is the first showing that in early stage SCLC patients treated with surgery and adjuvant chemotherapy, a miR-150/miR-886-3p signature predicts OS and PFS in both training and testing sets. This association was independent of age, gender and smoking status. These findings indicate that the miR-150/miR-886-3p signature might be a good candidate as a molecular prognostic marker and would potentially help doctors to identify patients at high-risk who may benefit from more extensive adjuvant therapy. We also found that both miR-150 and miR-886-3p were negatively associated with OS. Comparing the miRNA levels between NL and SCLC tissues also verified that miR-150 and miR-886-3p expression levels in SCLC were much lower than in NL samples. Because of the small NL sample size, we were unable to identify a pattern of persistently small p values. However, these data consistently suggested that miR-150 and miR-886-3p might serve as important tumor suppressors in the development and progression of SCLC and might function with potential anticancer targets. Our previous experimental study has demonstrated a novel miR-886-3p-mediated mechanism underlying the aberrant expression of in polo-like kinase 1 (PLK1) and transforming growth factor beta 1 (TGF-β1) in SCLC [@pone.0091388-Cao1]. The 3′ untranslated regions of PLK1 and TGF-β1 contain highly conserved miR-886-3p binding motifs, and direct interactions with miR-886 down-regulates endogenous PLK1 and TGF-β1 protein levels. We also showed that miR-886-3p over-expression inhibited proliferation, migration and invasion in SCLC cells, as well as suppressing the growth and metastasis of SCLC xenografts in nude mice. Finally, we revealed that the mechanism behind the down-regulated expression of miR-886-3p in SCLC was mediated by DNA hypermethylation of its promoter. Altogether, these findings establish a miR-886-3p-PLK1/TGF-b1 nexus as a novel regulator and promising therapeutic target for SCLC. The observation of miR-150 as a tumor suppressor in SCLC is consistent with similar observations in pancreatic cancer [@pone.0091388-Srivastava1], liver cancer [@pone.0091388-Zhang1], colorectal cancer [@pone.0091388-Ma1], NSCLC [@pone.0091388-Li1], malignant lymphoma [@pone.0091388-Watanabe2] and acute leukemia [@pone.0091388-Jiang1]. However, its involvement in SCLC has not been reported, and the precise molecular mechanisms behind its altered expression are unclear. Jiang et al. recently found that miR-150 maturation is repressed by MYC in MLL-associated leukemia. In addition, miR-150 is reported to be a protective miRNA that directly targets c-Myb and other factors [@pone.0091388-Zhang1], [@pone.0091388-Jiang1]. miR-150 interacts with the 3′UTR of c-Myb mRNA, and over-expression of miR-150 down-regulates c-Myb protein levels [@pone.0091388-Zhang1]. Interestingly, both MYC family members and the c-Myb gene are two well-known and dominant proto-oncogenes that are frequently found to be amplified or over-expressed in SCLC [@pone.0091388-Cook1]. Taken together, these recently published data together with ours, suggests that miR-150 has a tumor suppressor effect in SCLC because low levels of miR-150 expression was correlated with poor survival. However, additional studies are needed to elucidate the molecular mechanisms of both the cause and effect of altered miR-150 expression in the development and progression of SCLC. Limitations of this study include its relatively small sample size, a necessity because only a small number of patients (less than 5%) with early peripheral disease [@pone.0091388-IgnatiusOu1] are surgical candidates. More than eight hundred SCLC patients were treated with chemotherapy at our hospital during the same time period. As a result, biomarker analyses from SCLC patients are reported much less frequently compared with NSCLC patients. Additionally, because of this fact, multi-test corrections were not applied in this study because these corrections prevent type I errors at the cost of a rise in type II errors. Nevertheless, the correlation between the miRNA signature and the clinical outcomes were independently confirmed in a second patient set. Larger-scale studies are needed to confirm our findings. Another limitation is the retrospective design. However, all measurements were performed in a blind manner. Finally, the potential interaction between miR-150 and miR-886-3p should be further investigated to elucidate the mechanism behind the prognostic role of this dual-miRNA signature. In conclusion, our preliminary results suggested that the miR-150/miR-886-3p signature might predict cancer progression and survival in early stage SCLC patients and might be a promising prognostic biomarker and potential therapeutic targets for SCLC patients. This study was accepted for oral presentation at the 15th World Conference on Lung Cancer, Sydney, Australia, October 27--October 31, 2013. [^1]: **Competing Interests:**The authors have declared that no competing interests exist. [^2]: Conceived and designed the experiments: NB JC Y. Song QZ LW. Performed the experiments: NB JC Y. Song JS WL JF. Analyzed the data: NB JC Y. Song LW. Contributed reagents/materials/analysis tools: JC JH Y. Shi XZ NL. Wrote the paper: NB Y. Song LW.

Introduction {#Sec1} ============ Progress in the development of newer antigenic candidates for diagnosis as well as vaccination in visceral leishmaniasis (VL) is largely hampered due to our limited and insufficient knowledge of complex immune responses. Several *Leishmania* proteins have been identified for diagnostic tests. Selection of proteins that have good immunogenicity for B lymphocytes could be an advantage in the diagnosis of VL. Earlier, several vector host and parasite proteins have been identified and evaluated for antibody response within the human host. Heat shock protein (HSP) 83, rGRP78 and HSP70 are some of the most conserved evolutionary proteins identified for serodiagnosis of VL^[@CR1]^. Leishmanial histone proteins such as H2A, H2B, H3, and H4 have also been identified and tested^[@CR2]^. Protein A2 from amastigotes has been investigated for antibody response against *L*. *donovani* infection in India, Sudan and America with variable sensitivities^[@CR3],[@CR4]^. Several other nuclear proteins such as rlepp2 and rpaplee22^[@CR5],[@CR6]^, ribosomal proteins rLiP2a, rLiP2b and rP20^[@CR7],[@CR8]^, enzymes rCysteine proteinase and rSignal peptidases^[@CR9],[@CR10]^, and other antigens such as rORFF and Q protein have been studied for diagnostic purposes^[@CR11],[@CR12]^. Antigens with molecular masses of 116 kDa, 72 kDa, 66 kDa and 36 kDa have been used as the biomarker for VL in many earlier studies^[@CR13],[@CR14]^. Most of the proteins identified as diagnostic candidates have been screened for serodiagnosis and limited to laboratory scale validation only. Genome sequence accessibility of *L*. *donovani* has helped in the study of the expression of genes and proteins by multiple immunoproteomic approaches such as 2D-gel electrophoresis, mass spectrometry and B cell epitope mapping. Immunoproteomics permit the researchers to determine parasite-specific proteins, their interactions with host cells and then specific immune responses during infection. For serological diagnosis of VL, *L*. *chagasi* derived recombinant kinesin-related antigen, rK39 is widely used commercially. However, rK39 antigen often shows cross-reactivity with endemic healthy controls^[@CR15]^. This antigen has better sensitivity and specificity in the Indian subcontinent as compared to the East African countries and South America^[@CR16]^. In the last decade, several newer antigens have been identified and characterized for serological diagnosis of VL. The immunodominant domain of kinesin antigen rKE16 has been cloned from an Indian *L*. *donovani* clinical isolate. 100% sensitivity and specificity have been reported with this antigen in Old World VL countries such as India, Pakistan, China, and Turkey^[@CR17]^. In a further study rKE16 showed comparable sensitivity (96.6%) and specificity (96.2%) with rK39 antigen in India. However, the performance was weaker compared to rK39 in Sudan and France^[@CR18]^. A fusion protein, rK28 has been generated from three *L*. *donovani* proteins having homology with K39, K26 and K9 of *L*. *infantum*^[@CR19]^. In this study rK28 RDT showed better sensitivity (94.5%) and specificity (97.6%) with Sudanese serum samples^[@CR20]^. Another kinesin antigen rKLO8 has been identified and expressed from autochthonous *L*. *donovani* strain in Sudan^[@CR21]^. The sensitivities, 98%, 96.2% and 100%, and specificities, 100%, 96.06% and 81.85% for rKLO8 have been reported in Sudan, India, and France, respectively^[@CR18]^. rKRP42 is another kinesin-related protein that has been reported for diagnostic purpose^[@CR22]^. Development of novel antigen targets for non-invasive diagnosis of VL is still lacking. In some studies, however, antigens which had been developed for serodiagnosis have also been illustrated for urine reactivity. In one such study in Bangladesh rK28 antigen showed 95.4% sensitivity and 98.3% specificity through ELISA with urine samples^[@CR23]^. In recent years, alternatively, with the help of bioinformatic tools analysis of even unknown putative protein sequences, their role in infection and B cell epitopes have been predicted and subsequently synthesized for diagnostic tests^[@CR24]^. Earlier, we have reported the diagnostic ability of leishmanial membrane antigens (LAg) isolated from promastigote form of *L*. *donovani* strain AG83 (ATCC^®^ PRA-413^™^). Reactivity of this crude membrane antigen with urine antibodies paved the way for non-invasive diagnosis of VL^[@CR25]^. In this study, by means of immunoproteomic approach seeking more defined antigens we identified several urine reactive components of LAg through electrophoresis, immunoblot and mass spectrometry. The study further sought B cell epitope mapping of selected antigens and their corresponding peptides were synthesized and evaluated for VL diagnosis. Results {#Sec2} ======= SDS PAGE of *Leishmania* membrane antigens LAg {#Sec3} ---------------------------------------------- Earlier we have reported the diagnostic potential of *L*. *donovani* promastigote membrane antigens (LAg) in ELISA (97.94% sensitive and 100% specific) and dipstick (100% sensitive and 100% specific) systems with urine samples^[@CR25]^. Despite a crude mixture of antigens the sensitivity and specificity of LAg were found to be excellent. Here, we have separated the different protein constituents present in LAg through SDS-PAGE and visualized by Coomassie blue staining. LAg comprises of approximately 15--20 membrane residing proteins ranging in molecular masses from 25--280 kDa. Some of the LAg proteins have good band intensity while others possess comparatively lesser intensity. The major LAg bands visualized with Coomassie were 28, 31, 34, 36, 45, 51, 55, 63, 72, 91, 97, 120, 145, 200 and 280 kDa proteins (Fig. [1](#Fig1){ref-type="fig"}).Figure 1Proteins of different molecular masses separated from *Leishmania* membrane antigens (LAg) on 10% SDS-PAGE. Lane M, molecular weight markers (26--315 kDa); Lane LAg, separated LAg proteins. Both the lanes were part of a single gel. Immunoblot assay of urine with LAg proteins {#Sec4} ------------------------------------------- We further evaluated the reactivity of different LAg proteins with urine samples through immunoblotting. A total of 47 samples of *L*. *donovani* infected patients were used to study the anti-leishmanial antibodies in their urine against different LAg proteins. Out of the fourteen proteins of LAg observed in immunoblot, six proteins corresponding to relative molecular masses 34, 51, 55, 63, 72 and 120 kDa showed 100% reactivity with all active VL urine (Fig. [2A](#Fig2){ref-type="fig"}). Other frequently recognized proteins were of molecular masses 28, 31, 36, 45, 91, 97, 145 and 200 kDa. The data indicated that some of these proteins are more immunogenic than others and can be selected for utilization as future selective diagnostic candidates. The percent recognition of each protein tested with 47 urine VL cases is tabulated in Table [1](#Tab1){ref-type="table"}. Urine reactivity of LAg was also tested with non-leishmanial urine samples for cross-reactivity. Eighteen urine samples from five endemic and non-endemic healthy controls and two each for malaria, viral fever, tuberculosis, and typhoid were tested in the immune blot assay. Antibodies specific to the proteins 28, 31, 34, 36, 45, 51, 55, 63, 72, 91, 97, 120, 145 and 200 kDa, which were present in infected urine were completely absent in healthy controls and other diseases (Fig. [2B](#Fig2){ref-type="fig"}). However, cross-reactive bands at 26 and 102 kDa were present in some controls.Figure 2Immunoblot assay of *Leishmania* membrane proteins, LAg with **(A)** 9 infected urine samples (VL1-VL9) and **(B)** 8 non-VL urine samples including non-endemic healthy controls (C1&C2), endemic healthy controls (C3&C4), malaria (M1), viral fever (V1), tuberculosis (TB1) and typhoid (T1) with an infected VL urine sample (VL). Molecular weight marker lane, M, is marked in the left in kilodaltons. Corresponding blots in part A have been cut, treated and arranged from a single gel. Similarly blots in part B belong to a single gel. Images of reactive *Leishmania* membrane proteins to infected **(C)** and healthy **(D)** urine samples were obtained through Image Lab software and urine reactive proteins were marked as peaks in kDa.Table 1Percent recognition of LAg proteins against VL urine samples.Molecular mass of LAg proteins (kDa)2001451209791726355514536343128Recognition with *Leishmania* infected urine samples (%)31.948.910096.598.210010010010072.395.71005963.8 Immunoreactive protein band analysis {#Sec5} ------------------------------------ To estimate the accurate position of the immunoreactive LAg proteins against *Leishmania*-infected urine, blots were analyzed with the Image Lab software in gel doc Instrument. The software showed the intensity of each band in the blot with relative molecular mass, relative front (R~f~) value, band percentage and the lane percentage occupied by each band. With a typical VL urine sample, eleven proteins of molecular masses 31, 34, 36, 45, 51, 55, 63, 72, 91, 97 and 120 kDa were identified (Fig. [2C](#Fig2){ref-type="fig"}) with the software. Bands of 51, 55 and 63 kDa showed the highest intensity by volume with approximately 20, 28 and 20% contribution in the whole antigen, respectively (Supplementary Table [S1](#MOESM1){ref-type="media"}). Cross-reactivity of LAg with healthy urine sample was also analyzed. None of the components of LAg were detected by the software in healthy urine sample (Fig. [2D](#Fig2){ref-type="fig"}). Urine reactivity of LAg after cure {#Sec6} ---------------------------------- Immunoblot assay with 54 paired urine samples from 18 *Leishmania* infected patients before treatment started (day 0), 18 cured samples after one month treatment (day 30) and six months (day \> 180) follow-up were tested to study the antigenic reactivity of each polypeptide of LAg protein with therapy. In the active phase of infection before the treatment started, all the urine samples were reactive with 31, 34, 36, 45, 51, 55, 63, 72, 91, 97 and 120 kDa proteins. After completing treatment ten urine samples showed a decline in protein reactivity in 30 days (Fig. [3B](#Fig3){ref-type="fig"}). In these cases, antibodies reactive to band 31, 34, 36, 45, 51, 91, 97 and 120 kDa completely declined after 30 days of treatment but 55, 63 and 72 kDa proteins remain detectable. However, urine samples of eight cured VL cases showed almost equal reactivity and band intensity at day 30 similar to the active *Leishmania* infection before treatment (Fig. [3A,C](#Fig3){ref-type="fig"}). Samples after six months of treatment initiation illustrated disappearance of all the reactive bands completely except in two VL cases where bands of 31, 34, 45, and 72 kDa were found to be present (Fig. [3C](#Fig3){ref-type="fig"}). Thus following six months of treatment 16 out of 18 samples showed complete fall in antibody titer against polypeptides of LAg.Figure 3*Leishmania* membrane proteins immunoblotted with paired urine samples before treatment started (day 0), after one month (day 30) and after six months (day \> 180) of treatment. Samples were collected from a longitudinal study of 18 VL patients at three time points. Figure shows a representative pattern of reactivity during treatment. **(A)** Urine antibodies did not fall after one month but completely disappeared after six months. **(B)** Urine antibodies fell after one month and completely disappeared after 6 months. **(C)** Urine antibodies remain positive after six months. M, molecular weight markers in kDa. The blots of figures A, B and C were cropped from three separate gels. We thus observed three types of protein reactivity with urine samples obtained from *Leishmania*-infected patients in the course of treatment. In the majority of the patients, the antibody level did not fall just after therapy but took at least six months for the full disappearance. In some VL cases, urine antibody titer significantly fell during the course of treatment and could not be detected post-treatment. In two cases antibodies were present in the urine even after six months. This could be a signal for relapse or future development of PKDL. This needs more studies for validation.Table 2Identified proteins of LAg in MALDI ms/ms analysis.Protein BandProtein NameAccession No.Protein Score63Glycoprotein 63gi\|2195446414355α-tubulingi\|7184035320651elongation factor 1αgi\|14608315370 Mass spectrometry of urine reactive *Leishmania* proteins {#Sec7} --------------------------------------------------------- After analysis of each component of LAg for its diagnostic potential and ability to monitor treatment response, we selected three immunodominant antigenic proteins of LAg for identification through mass spectrometry. Urine antibodies against 51, 55 and 63 kDa proteins were found prominently in all VL cases with 100% recognition by urine samples from *Leishmania*-infected patients. Since antibodies against these proteins disappeared completely after six months of treatment, they best suited as candidate biomarkers. In order to identify these three proteins, bands were excised from Coomassie stained SDS-PAGE 2D-gel and subjected to in-gel tryptic digestion (Fig. [4A](#Fig4){ref-type="fig"}). The digested proteins were analyzed by MALDI-TOF mass spectrometry and the MS and MS/MS spectra were obtained through MASCOT search against *Leishmania* sequences in MSDB and NCBI databases. Three *Leishmania* proteins which were identified unambiguously from 51, 55 and 63 kDa bands were elongation factor 1α of *L*. *infantum*, α-tubulin of *L*. *donovani*, and glycoprotein or leishmanolysin of *L*. *donovani*, respectively (Supplementary Figs [S1](#MOESM1){ref-type="media"}--[S3](#MOESM1){ref-type="media"}). Protein bands identified with their accession no. in database and protein score obtained in MALDI are listed in Table [2](#Tab2){ref-type="table"}.Figure 4**(A) **Two-dimensional gel electrophoresis of *Leishmania* membrane proteins (LAg). 51, 55 and 63 kDa spots were picked and subjected to tryptic digestion. **(B)** 63, 55 and 51 kDa proteins of LAg were electroeluted and subjected to SDS-PAGE. Corresponding lanes in part B was part of a single gel. RNA sequence expression profiling of selected LAg proteins {#Sec8} ---------------------------------------------------------- In *Leishmania* infection parasite enters into the human bloodstream by the bites of sandflies, the carriers of the promastigote form. These then transform into the amastigote in the peripheral macrophages^[@CR16]^. Several studies have shown the increased level of antibodies in human sera against both promastigote and amastigote antigens^[@CR26]^. Presence of promastigote antigen-specific antibodies in the active phase of *L*. *donovani* infection and even after treatment is possible if these antigens are common in both the forms of the parasite^[@CR27]^. We have shown the reactivity of several *L*. *donovani* promastigote antigens (LAg) with VL urine antibodies. We next investigated the presence of LAg proteins, elongation factor 1α (EF1-α), α-tubulin, and glycoprotein 63 (GP63) in the amastigote stage of the parasite in both virulent and avirulent forms. Intracellular amastigotes from infected peritoneal macrophages of mice after 12 h post-infection were used for RNAseq expression profiling of antigens. All three urine reactive LAg proteins, EF1-α, α-tubulin and GP63 which were identified in promastigotes were also found in the amastigote form of *L*. *donovani* through RNAseq profiling. Moreover, the GP63 protein was up-regulated and EF1-α and α-tubulin were down-regulated in virulent amastigotes as compared to the avirulent ones (Table [3](#Tab3){ref-type="table"}).Table 3RNA Sequence profiling of three LAg proteins in *L*. *donovani* amastigotes.Proteins nameGP63, leishmanolysinAlpha tubulinElongation factor 1alphaGene namesLDBPK_100510LDBPK_130330LDBPK_170170Base mean avirulent206.5517918.2321886.93Base mean virulent296.6514252.9720677.37Fold change1.430.790.94RegulationupdowndownFunctionsMetallo endopeptidase activityGTPase activity; structural constituent of cytoskeletonGTP binding; translation elongation factor activity Electroelution and urine reactivity of proteins through ELISA {#Sec9} ------------------------------------------------------------- The candidate diagnostic proteins verified by mass spectrometry and RNA sequence profiling, were further evaluated using quantitative ELISA assay. EF1-α, α-tubulin and GP63 proteins were electroeluted from SDS-PAGE gel (Fig. [4B](#Fig4){ref-type="fig"}) and subjected to antibody capture ELISA in their native state. Urine antibody response against antigen EF1-α was comparable with LAg reactivity in differentiating active VL cases from other diseases, endemic and non-endemic healthy controls with p values \< 0.0001, \<0.0001 and 0.0001, respectively (Fig. [5A](#Fig5){ref-type="fig"}). However, cross-reactivity with non-VL healthy and other disease controls was found with α-tubulin and GP63. Therefore, out of the three electroeluted native antigens, EF1-α appears to have better diagnostic ability using urine samples.Figure 5**(A)** Reactivity of three electroeluted antigens GP63, α-tubulin and EF1-α along with crude antigens, LAg, were tested against urine samples of kala-azar infected individuals (VL, n = 20), non-endemic healthy controls (NEHC, n = 10), endemic healthy controls (EHC, n = 10), and other diseases (OD, n = 10) including two of each malaria, viral fever, tuberculosis, typhoid, and pneumonia. **(B)** The whole protein sequence of glycoprotein 63, α-tubulin and elongation factor 1α with their mapped epitopes (red block). **(C)** In comparison to crude membrane antigens, LAg, reactivity of three synthetic peptides P1, P2 and P3 were tested against urine samples of kala-azar infected individuals (VL, n = 40), non-endemic healthy controls (NEHC, n = 20), endemic healthy controls (EHC, n = 10) and other diseases (OD, n = 18) including 6 malaria, 4 viral fever, 4 tuberculosis, 2 typhoid and 2 pneumonia urine samples. B cell epitope mapping of selected *Leishmania* antigens {#Sec10} -------------------------------------------------------- Since most of the antigens are conserved throughout evolution, crude and native antigens often have a chance of cross-reaction with other symptomatically similar infections. To select specific sequences that are different from sequences of other closely related infective organisms as well as the parasite hosts B cell epitope sequences were derived from the whole antigen. B cell epitopes may recognize definite antibodies thus allowing diagnosis with more specificity. In this study, selected proteins of *L*. *donovani*, EF1-α, α-tubulin, and GP63 were scanned separately for their potential B cell epitope identification. Epitopes were predicted from the whole protein sequence of antigens in NCBI database search through accession no. that were obtained by mass spectrometry MS/MS. The sequence of B cell epitopes for each antigen was acquired in BepiPred program which had the highest score. Epitopes *VCGNSKNDPPKEAAD* from EF1-α, *IQPDGSMPSD* from α-tubulin, and *IEDQGGAGSAGS* from GP63 protein were obtained and selected from the software. Subsequently, each B cell peptide was allowed for the determination of possible hydropathy of epitopes through Expasy software (Supplementary Fig. [S4](#MOESM1){ref-type="media"}). All epitopes were shown as a surface region of the globular protein. Fig. [5B](#Fig5){ref-type="fig"} shows the B cell epitopes of EF1-α, α-tubulin, and GP63 in their whole protein sequences. Immunoreactivity of synthetic peptides of B cell epitopes {#Sec11} --------------------------------------------------------- To select potentially specific target antigens for non-invasive diagnosis of VL we synthesized three B cell epitopes obtained from the above study. B cell epitope of antigens EF1-α, α-tubulin and GP63 were named Peptide1, Peptide 2 and Peptide 3, respectively. Synthesized B cell linear peptides are unfolded structures and might be useful for possible antibody binding in immunodiagnosis. These peptides were tested against urine samples from VL patients, healthy controls, and other disease controls. Amongst all synthetic peptides, peptide P1 that was mapped from protein EF1-α showed significantly higher reactivity with *Leishmania* infected urine as compared to the non-VL control urine samples, similar to LAg. The p values for antibody titer in VL urine against Peptide P1 were found to be 0.0009, 0.0005 and 0.0007 in comparison to endemic healthy controls, non-endemic healthy controls and other disease urine samples, respectively. Peptides P2 and P3, however, showed lower significant reactivity (p values 0.03, 0.02, 0.04 & 0.07, 0.01, 0.02) with VL urine as compared to endemic healthy controls, non-endemic healthy controls and other disease controls, respectively (Fig. [5C](#Fig5){ref-type="fig"}). Discussion {#Sec12} ========== Antibody detection methods have provided great ease and simplicity in VL diagnostics in comparison to the gold standard tissue aspiration test. Use of leishmanial antigens for diagnostic purposes has been found effective with almost comparable and even better sensitivity and specificity than tissue aspiration. Most of the antigens discovered for VL diagnosis are based on serological assays. However, these antigens are often not well characterized and are either crude or semi-purified such that they often show false positive results or cross-reactivity with other similar infections. Parameters such as sensitivity, specificity, stability, and reproducibility might influence the performance of the antigen to be a good diagnostic candidate. In VL, the commonly used antigen for rapid diagnosis is rK39, a single cloned antigen isolated from *L*. *chagasi* parasite and expressed in a bacterial expression system. However, the performance of this recombinant antigen is variable in different endemic regions and often shows cross-reactivity with endemic healthy controls^[@CR15],[@CR16]^. Availability of newer methodologies such as protein profiling through 2D gel electrophoresis, protein identification and sequence analysis by mass spectrometry and other immunoproteomic approaches of reactive epitope prediction for synthetic peptides have boosted up the strategies for development of novel diagnostic antigens. Therefore, a well defined new generation antigen based on recombinant proteins or peptides is the need for non-invasive diagnosis of VL. Unlike sera, very few studies have been performed to determine the reactivity of leishmanial antigens against urine antibodies. Nevertheless, most of the antigens that were studied with urine samples have been initially developed for serum-based assays. To the best of our information, this is the first study where the immunoproteomic approach was used to identify and evaluate the defined antigens specific to urine antibodies of VL. In this study, we have identified fourteen proteins of *L*. *donovani* membrane antigens which demonstrate immunoreactivity with urine antibodies of VL patients. In immunoblot assay and subsequent analysis with Image Lab software six peptides of molecular masses 34, 51, 55, 63, 72, and 120 kDa showed 100% reactivity with VL urine samples. After one month of treatment 31, 34, 36, 45, 51, 91, 97 and 120 kDa proteins showed recognition only in 44% of the samples. However, after six months of cure recognition goes down to 12% for 31, 34, 45 and 72 kDa proteins whereas reactivity with 36, 51, 55, 63 and 120 kDa proteins was not observed. Therefore, proteins with molecular masses of 51, 55 and 63 were selected for further studies. These proteins can be useful not only in the specific diagnosis of VL using urine samples but also for monitoring treatment response. Through mass spectrometry 51, 55 and 63 kDa bands were identified as elongation factor 1α of *L*. *infantum*, α-tubulin of *L*. *donovani*, and glycoprotein of *L*. *donovani*, respectively. Glycoprotein 63 (GP63) is the major zinc metalloprotease of *L*. *donovani* and is conserved in all *Leishmania* species^[@CR28]^. GP63 promotes infection and survival in the host cells. It has been shown that *Leishmania* alters host cell signaling through GP63 interaction which is involved in the uptake of the parasites by the host macrophages^[@CR29],[@CR30]^. Persistence of GP63 specific antibodies in the serum of VL patients after the cure has been reported which suggests its role in life-long immunity in cured individuals^[@CR31],[@CR32]^. We have shown the presence of GP63 specific antibodies in the urine samples of all kala-azar patients. Anti-GP63 antibodies were found in most VL cases after treatment which completely disappeared after six months. Protein α-tubulin is one of the constituents of microtubule networks present in *Leishmania*. It is associated with cell shape, locomotion, and division of the parasite. In several studies, α-tubulin has been shown to be immunogenic in VL patients' sera and implicated as a vaccine candidate for kala-azar^[@CR33]^. A high intensity of anti-β tubulin antibodies was observed in urine of VL infected patients through immunoblot which disappears after six months of treatment. EF1-α plays a major role in protein synthesis and their assembly. Belonging to GTP-binding protein, it is highly conserved, and engages in protein translation. EF1-α has also been reported as a virulence factor in *Leishmania* where it diffuses into the infected host macrophages and deactivates several pathways^[@CR34],[@CR35]^. EF1-α has been found to be involved in the humoral immune response by inducing antibodies against *Leishmania*^[@CR36]^. We have observed the presence of EF1-α specific antibodies in all the urine samples from VL patients. These antibodies persist in most VL cases just after treatment but fall significantly and become negative after six months. We have always been eager to know why promastigote specific antibodies reside in the human host for so long even after treatment while within 24 h of infection promastigotes transform into amastigotes. This is possible only when amastigotes also share these antigens, thus promoting antibody production throughout infection. We have confirmed this by transcriptome analysis of virulent and avirulent amastigote proteins. We found all the three proteins of promastigote, EF1-α, α-tubulin, and GP63, are present in the amastigote stage of parasite too. GP63 protein was found up-regulated in the virulent form of amastigotes. This suggests a direct role of these proteins in parasite infection and progression. EF1-α and α-tubulin proteins were found to be down-regulated in the virulent stage of amastigotes, suggesting its role in parasite survival and normal growth maintenance in adverse conditions. Therefore, the function of these proteins in *Leishmania* infection supports them as diagnostic biomarkers. To further investigate the quantitative reactivity of EF1-α, α-tubulin, and GP63 proteins were electroeluted and subjected to antibody capture ELISA. All the three chosen proteins helped distinguish active VL from other diseases and healthy controls. However, EF1-α showed better specificity than α-tubulin and GP63 in their native state. Native proteins either purified or recombinant ones may carry amino acid sequences that can cross-react with other infections. B cell epitope prediction for pathogenic organisms has widened the variety of antigenic peptides and has helped to improve the antigenic immunogenicity. In the current study we have predicted and synthesized three epitopic peptides P1, P2 and P3 from *L*. *donovani* antigens EF1-α, α-tubulin and GP63, respectively. Synthetic peptide sequences were evaluated for antigenicity and diagnosis of VL with urine samples. Peptide P1 demonstrates best sensitivity and specificity among the three synthetic peptides and comparable to LAg. Results with synthetic peptides might be improved further with combinations of two and more peptides and can be used in a point of care test in future studies (already started). Synthetic peptides in VL diagnosis could circumvent the dependency of native antigens which often differ in protein content during batch to batch production and also in reactivity in different endemic regions. However, synthetic peptides are rather simple and do not show much variation in the assay. In conclusion, in this study, we identified and evaluated urine antibody specific defined peptides that can be used for the development of a non-invasive diagnosis of VL. Methods {#Sec13} ======= Sample collection {#Sec14} ----------------- Urine samples for this study were obtained from School of Tropical Medicine (STM), Kolkata and Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna. Random urine samples were collected from parasitologically confirmed kala-azar patients before the actual treatment started (47 patients at day 0), after completion of treatment (18 patients at day 30) and after at least six months of treatment commencement (18 patients at day more than 180). Urine samples were also collected from 20 non-endemic and 10 endemic healthy controls each along with 20 other diseases samples including 6 malaria, 4 viral fever, 4 tuberculosis, 4 typhoid and 2 pneumonia. After collection, 0.1% sodium azide (NaN~3~) was added to each sample as a preservative and stored at 4 °C until the study was done. Ethical approval {#Sec15} ---------------- Ethical approval for the human study was acquired from the Ethical Committee of CSIR-Indian Institute of Chemical Biology, Kolkata, India along with STM, Kolkata and RMRIMS, Patna. The experiments conducted for the study were carried out with the regulations and guidelines of the committee. Written informed consent in prescribed format was received from all the participants before sample collection. The aim of the study and privacy of participants were mentioned in the consent form. Ethical approval was also taken from the Institute's animal ethical committee (IICB, Kolkata). SDS-PAGE {#Sec16} -------- Leishmanial antigen, LAg was isolated from strain AG83 (ATCC^®^ PRA-413™) of *L*. *donovani* promastigotes as described elsewhere^[@CR37],[@CR38]^. Antigen LAg (10 μg/lane) was resolved in 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a Mini-Protein II apparatus (BioRad Laboratories, USA). Briefly, prior to loading protein was denatured in sample buffer (2% SDS, 10% glycerol, and 0.025% bromophenol blue in 60 mM Tris-HCl, pH 6.8. 5% 2-mercaptoethanol) in a boiling water bath for 5 min. 10% acrylamide for separating and 4% acrylamide for stacking were used with 1 mm gel thickenings. Finally, proteins were run at constant voltage (60 V). Markers (BioRad Laboratories, USA) of known molecular weights were used for comparison. Protein bands were visualized by Coomassie brilliant blue. Molecular mass of proteins presented in LAg were determined by the R~f~ values obtained with standard marker proteins. Additionally, protein gel was also analyzed with Image Lab software to automatically define the molecular weights in reference to the standard proteins. 2D-gel electrophoresis {#Sec17} ---------------------- LAg proteins were also separated in two dimensional (2D) gel electrophoresis. The first dimension of isoelectric focusing (IEF) was carried out with 18 cm strip, pH 4--7 (BioRad Laboratories, USA). The strip was rehydrated (4% CHAPS, 2% ampholyte, 8 M urea, 30 mM DTT and bromophenol blue) overnight at room temperature followed by IEF with Mutiphor II and a DryStrip kit (GE Healthcare, Germany). The strip was then equilibrated for 15 min in buffer containing 6 M urea, 2% SDS, 20% glycerol, 1% DTT, and 5 mM Tris-HCl, pH 8.8. This time the strip was equilibrated with 2.5% iodoacetamide. For the second dimension, 1.5 mm thick 12% SDS-PAGE was run and protein spots were visualized with Coomassie brilliant blue. Immunoblot assay {#Sec18} ---------------- The PAGE-separated proteins were electrophoretically transferred to nitrocellulose membranes at 85 V/cm for 75 minutes in a transblot apparatus (BioRad Laboratories, USA). Transferred LAg proteins on nitrocellulose membrane were confirmed and visualized with Ponceau dye and different lanes were cut into strips and marked for reaction with different urine samples. Each nitrocellulose membrane strip was then blocked overnight in 5% BSA at 4 °C in 100 mM TBS containing 0.1% Tween 20. The next day strips were washed once with washing buffer (100 mM TBS & 0.05% Tween-20) and then incubated with diluted urine samples (1:5 in washing buffer) for 2 hr at room temperature with constant stirring. After incubation with urine, strips were then washed two times for five minutes in wash buffer and dipped in diluted (1:2000) peroxidase-conjugated goat anti-human IgG for 90 min at room temperature. Strips were then washed two more times with the same wash buffer and the third wash was with 100 mM TBS only (without Tween-20). After washing the strips were spread with Luminol and H~2~O~2~ and pictures were taken in ChemiDoc chemiluminescent apparatus (BioRad Laboratories, USA). In another set of an experiment after the last wash strips were incubated for 5 min in freshly prepared substrate solution of 0.05% of DAB + 0.05% of H~2~O~2~ in 100 mM TBS. Finally, the strips were rinsed with distilled water and dried. Image of the membrane blots was captured and analyzed in Image Lab software (version 54.2.1). Mass spectrometry {#Sec19} ----------------- LAg proteins were separated by 2D gel electrophoresis and stained with Coomassie blue dye. Proteins of molecular mass 51, 55 and 63 kDa were excised from the gel through pipette tip as 1 mm^[@CR3]^ diameter pieces. The in-gel tryptic digestion of proteins was carried out according to the manufacturer's manual (Pierce, Rockford). Briefly, gel pieces were destained completely in ammonium bicarbonate and acetonitrile solution followed by alkylation and reduction by tris, 2-carboxyethyl phosphine (TCEP) and iodoacetamide, respectively. The gel pieces were digested in 10 ng/µl concentration of enzyme trypsin in ammonium bicarbonate solution and incubated overnight at 37 °C. The peptides (0.5 µl) were co-crystallized with matrix (5 mg/ml CHCA, 5% ACN and 0.1% TFA) in the metal plate well of ABSciex 4800 mass spectrometer (Applied Biosystems). Both mass spectrometry (MS) and MS/MS spectra were obtained by matrix-assisted laser desorption ionization--time of flight (MALDI-TOF/TOF) mass Spectrometer. Database searching for protein identification was performed with mass spectrometry data using GPS Explorer (Applied Biosystems) software with MASCOT (Matrix Science) search engine. Electroelution of proteins {#Sec20} -------------------------- The LAg proteins of molecular masses 51, 55 and 63 kDa were excised from 10% SDS-PAGE gel stained with Coomassie blue. Excised protein bands were eluted using electrophoresis apparatus (BioRad, Model-422) under running buffer (1% SDS, 0.192 M glycine and 0.025 M Tris) for 4 h at 10 mA. Subsequently, eluted proteins were dialyzed and resuspended in PBS. Finally, proteins were quantified by Lowry's method and position of the proteins was reconfirmed in SDS-PAGE. RNA sequence profiling {#Sec21} ---------------------- Peritoneal macrophages were isolated from healthy BALB/c mice. 3^rd^ to 5^th^ passage of promastigote culture was used for virulent *Leishmania* infection and 25^th^ passage culture was used for avirulent infection in peritoneal macrophages. After approximately 12 h of infection total RNA was isolated and RNAseq profiling was done to compare the expressed genes of virulent and avirulent parasites. Promastigote specific proteins corresponding to their RNAseq and genes were compared against amastigote and database using BLASTp search. B cell epitope mapping {#Sec22} ---------------------- The sequence of three *L*. *donovani* proteins, EF1-α, α-tubulin, and GP63 were obtained from NCBI database. All the sequences were mapped by using BepiPred software program with a threshold of 0.35 (<http://www.cbs.dtu.dk/services/BepiPred>). B cell epitopes predicted for each protein were analyzed further with Expasy for possible hydropathy of the epitopes (<http://web.expasy.org/protscale>). Selected epitopes were blast searched in NCBI database for similarity analysis with other organisms. B cell epitopic peptide sequences matched by the two programs were selected and synthesized at the Biotech Desk peptide synthesis facility. One peptide was synthesized for each of the three proteins with 96% purity in HPLC. Antibody capture ELISA {#Sec23} ---------------------- Antibody capture ELISA with electroeluted proteins and synthetic peptides was performed using 96-well flat bottom ELISA plates (Nunc Maxisorp, Denmark). Briefly, 1 µg/well of electroeluted antigens, EF1-α, α-tubulin and GP63, as well as the crude antigen, LAg were coated in 0.02 M phosphate buffer, pH 7.4 (100 µl/well) and kept overnight at 37 °C. In another set of ELISA 10 µg/well of synthetic peptides P1, P2 and P3 along with antigen LAg were used. Plates were blocked with 1% of bovine serum albumin (BSA) (Sigma, USA) in 200 µl/well of 0.02 M phosphate buffer saline (PBS, pH 7.4) for 2 h at 37 °C. Subsequently, 100 µl/well of urine samples (1:10 dilution in blocking) followed by HRP-conjugated anti-human IgG (Bangalore GeNei, India) (1:4000 dilution) were applied and incubated at 37°C for 1 h. ELISA plates were washed three times with PBS containing Tween-20 in each step. Finally, 0.05% (w/v) o-phenylenediamine dihydrochloride (OPD) (Sigma, USA) as substrate was added in 50 mM phosphate-citrate buffer (100 µl/well) with 0.05% H~2~O~2~ (Merck, Germany). Subsequently, 2 N sulfuric acid (50 µl/well) was used to stop the reaction and the optical density was determined at 492 nm in an ELISA reader (RS232C, Thermo Scientific, MA, USA). Statistics {#Sec24} ---------- Statistical analyses in this study were performed with the software Graph Pad Prism version 5.0. Two-tailed, Mann-Whitney *U* test, unpaired t-tests were used for comparison of ELISA values of different groups and considered statistically significant if the *P* values \< 0.05. Electronic supplementary material ================================= {#Sec25} 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/s41598-018-30546-y. The authors want to acknowledge Mr. Naresh Kumar Sinha and Mr. Ritesh Mishra of RMIRMS, Patna and Dr. Yogiraj Roy of STM, Kolkata for their assistance in urine collection. S.A.E. and N.A. conceived and designed this study. S.A.E. carried out the experiments and wrote the manuscript. A.B., S.T.C., S.G. and A.S. provided experimental support, analyzed results and reviewed the manuscript. K.P., V.N.R.D., P.D., M.R., and R.P.G. contributed in clinical sample collection, conducted confirmatory tests and provided ethical clearance from the hospitals. All data generated or analyzed during this study are included in this published article (and its Supplementary Information files). Competing Interests {#FPar1} =================== This work has been supported by Raja Ramanna Fellowship, Department of Atomic Energy and J. C. Bose National Fellowship, Science and Engineering Research Board, India.

INTRODUCTION ============ Vitamin D receptors (VDR) are expressed in the skeletal system and in various extraskeletal tissues and cells. Furthermore, certain tissues and cells express the enzyme that converts the primary inactive form of vitamin D and 25-hydroxyvitamin D (25(OH)D) to the active form, 1,25-hydroxyvitamin D (1,25(OH)~2~D). A number of studies have investigated on the effect of vitamin D on metabolism in cancer, cardiovascular diseases, infectious diseases and immune disorders \[[@B1][@B2][@B3][@B4][@B5]\]. Vitamin D exerts pleiotropic effects through VDRs that form vitamin D-responsive elements in the nucleus to produce traditional genomic effects. VDRs are also present at the cell membrane and exert rapid nongenomic responses through second messengers. VDRs at the cell membrane mediate effects on the pancreas, vascular smooth muscle, monocytes, and immune cells. To the best of our knowledge, the expression of VDR on nasal mucosa is has not been extensively investigated. Serum 25(OH)D is the most abundant and stable form of vitamin D in human systemic circulation, and is typically considered to directly represent the serum vitamin D level. The serum 25(OH)D concentration is affected by age, season, diet, and latitude. The associations between low levels of 25(OH)D and an increased risk of asthma, atopic dermatitis, and allergic rhinitis (AR) are controversial \[[@B6][@B7]\], and the effect of vitamin D on allergic diseases remains unclear. The current study aimed to examine the association between serum vitamin D levels and persistent AR. MATERIALS AND METHODS ===================== Study subjects -------------- The present study included 32 patients with persistent AR (AR group) and 25 nonallergic subjects (control group). All the participants underwent septoplasty surgery at the Affiliated Jiangyin Hospital, Medical College of Southeast University (Jiangsu, China) between February 2014 and January 2015. The patients with persistent AR were diagnosed clinically according to the Allergic Rhinitis and its Impact on Asthma guidelines \[[@B8]\]. The subjects had symptoms and signs of AR with positive skin-prick tests and/or were positive for serum specific IgE against common aeroallergens, without other nasal disorders, and allergic diseases (such as chronic rhinosinusitis, asthma, and eczema/atopic dermatitis). The subjects had not received any vitamin D supplementation within the 3 months prior to blood testing. This study was approved by the Institutional Review Board of the Affiliated Jiangyin Hospital, Medical School of Southeast University (approval number: 2013-118). Informed consent was obtained from all participants or their guardian. Allergy testing --------------- Skin-prick tests were performed with a standard panel of common aeroallergens, including house dust mites, animal dander, cockroaches, fungi, and pollens. Positive (histamine) and negative (normal saline) controls were also used. A mean wheal diameter of 3 mm or more 20 minutes after application was considered a positive skin reaction. Serum total and specific IgE levels were detected using an Allergen Screen Test kit (MEDIWISS Analytic GmbH, Moers, Germany). Specific IgE levels equal to or greater than 0.7 kU/L were considered positive. Blood eosinophil count and its proportion were performed using Sysmex XE-2100 Automated Hematology system (TOA Medical Electronics, Kobe, Japan). The normal range for eosinophil count in peripheral blood was 0.02--0.5×10^9^/L, and the normal proportion in white blood cell was 0.5%--5%. Serum 25(OH)D measurement ------------------------- Serum 25(OH)D was assayed by an enz yme-linked immunosorbent assay (ELISA; Cloud-Clone Corp., Katy, TX, USA) according to the manufacturer\'s instructions. The insufficient value was defined as 21--30 ng/mL, the deficient value was \<21 ng/mL, and the normal value was\>30 ng/mL \[[@B9]\]. For seasonal blood analysis, the seasons were defined as follows: spring (March, April, and May); summer (June, July, and August); autumn (September, October, and November) and winter (December, January, and February). VDR expression on nasal mucosa ------------------------------ VDR expression on nasal mucosa was measured by immunohistochemical staining. Inferior turbinate mucosa tissues were collected from septoplasty surgical samples, fixed in 10% formaldehyde solution, embedded in paraffin blocks and cut into 3-µm-thick sections. Standard immunohistochemical stains for VDR (rabbit polyclonal antibody; Bioss Antibodies Inc., Woburn, MA, USA) were performed \[[@B10]\]. Accordingly, paraffin sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval was performed by heated citrate buffer in a pressure cooker. Endogenous peroxidases were blocked by treating the sections with 0.3% hydrogen peroxide for 30 minutes, followed by washing 3 times with phosphate buffered saline (PBS). Nonspecific binding was blocked by incubating sections with 10% goat serum for 20 minutes at room temperature. The rabbit anti-VDR primary polyclonal antibody was applied in a moist chamber overnight at 4℃. Following rewarming to room temperature for 15 minutes, the slides were washed 3 times for 5 minutes in PBS. Then, the sections were incubated with secondary antibody, (DAKO; Agilent Technologies Inc., Santa Clara, CA, USA) for 25 minutes at room temperature. Af ter 3 additional rinsing steps with PBS for 5 minutes, diaminobenzidine was applied to the sections. This was followed by rinsing of the slides in deionized water. Counter staining with hematoxylin was performed prior to dehydration and clearing. Cover slips were then added to the sections. Control staining with no primary antibody incubation was also performed. The expression of VDR was assessed by a pathologist according to a semiquantitative reference scale, accounting for the intensity and proportion of immunoreactive cells in individual slides of nasal mucosa. The staining was scored as follows: No staining; weak (\<50% cells stained light brown in color); moderate (between weak and strong), and strong (\>70% cells obviously stained deep brown in color). A cell was considered 'stained' if there was a discrete brown color associated with the cell, and the color was different from the cells stained only with hematoxylin or above any background color. Statistical analysis -------------------- Data were evaluated using SPSS ver. 16.0 (SPSS Inc., Chicago, IL, USA). Differences in the distribution of characteristics, including age, sex, body mass index (BMI), the season of blood sampling, serum 25(OH)D levels, and the VDR expression between the patients with AR and the control subjects were analyzed using independent-samples t test or contingency table chi-square tests. The value of serum 25(OH)D were converted into binary variable, and multiple regression models were used to estimate the effect of mean serum 25(OH)D level and demographic characteristics on blood eosinophil count and its proportion. *p* \< 0.05 was considered to indicate a statistically significant difference. RESULTS ======= General characteristics ----------------------- A total of 57 participants were included in the study: 32 in the AR group and 25 in the control group. The general characteristics of the subjects of the study are presented in [Table 1](#T1){ref-type="table"}. The majority of the participants were male (92%). There were no significant differences in age, sex, BMI, and the season of blood sampling between the 2 groups (all *p* \> 0.05). Serum 25(OH)D levels, blood eosinophils, and AR ----------------------------------------------- The difference in serum 25(OH)D levels between the AR group and the control group was not significant (*t* = -0.903, *p* = 0.371) ([Table 2](#T2){ref-type="table"}). There was also no significant difference in the proportion of participants with insufficient and deficient 25(OH) D levels (χ^2^ = 0.101, *p* = 0.751) ([Table 2](#T2){ref-type="table"}). Additionally, when female participants were excluded from the analysis, the difference in 25(OH)D levels between the 2 groups remained nonsignificant (*t* = -0.355, *p* = 0.724). Furthermore, there was no significant association between serum 25(OH)D levels and AR (odds ratio, 1.01; 95% confidence interval, 0.994--1.027; *p* = 0.235) when adjusting for age, sex, BMI, and the season of blood sampling as potential confounders. The eosinophil count and its proportion in white blood cell samples from patients with AR were significantly higher than those of the control group (*t* = 3.081, *p* = 0.003, and *t* = 3.729, *p* \< 0.0001, respectively) ([Table 2](#T2){ref-type="table"}). Interestingly, in AR group, the serum 25(OH)D levels were negatively correlated with blood eosinophil count and its proportion (*p* = 0.019 and *p* = 0.010, respectively) even when adjusting confounding factors including age, sex, BMI, and the season of blood sampling. However, the correlation was not significant in the control group (both *p* \> 0.05). VDR expression on nasal mucosa ------------------------------ In the majority of nasal mucosa samples, VDR expression was observed as moderate-to-strong staining. There was no significant difference in the expression level of VDR between the AR group and the control group (χ^2^ = 1.436, *p* = 0.231). Meanwhile, VDR expression was predominantly detected on the plasma membrane of epithelial cells in most samples, rather than in the cell nucleus and matrix ([Fig. 1](#F1){ref-type="fig"}). DISCUSSION ========== Vitamin D was proposed to play a protective role on immune system and respiratory infections \[[@B11][@B12][@B13][@B14]\]. A number of studies have previously investigated the immunoregulatory mechanisms of vitamin D \[[@B5][@B14]\]. Vitamin D functions in immune responses via VDR in the majority of immune cell types, including B cells, T cells, macrophages, dendritic cells, neutrophils, and eosinophils \[[@B12][@B15]\]. However, vitamin D insufficiency and deficiency appear to be very common in general population without signs of nutritional rickets \[[@B16]\]. The present study identified that patients with persistent AR exhibited a lower serum level of 25(OH)D compared with the level in nonallergic subjects. The blood eosinophil count of the AR group was significantly higher than the control group. Meanwhile, the serum 25(OH)D levels of AR patients were inversely associated with the blood eosinophil count and its proportion in the regression models when adjusting for confounding factors (age, sex, BMI, and the season of blood sampling). However, the differences of serum 25(OH)D level between the 2 groups did not arrive statistical significance. This result might be due to a relatively higher mean serum 25(OH)D level and high standard deviation in control group. In addition, the expression of VDR on nasal mucosa in the 2 groups was similar. Vitamin D impacts both innate and adaptive immune according to recent data. For the innate immune system, it stimulates the production of cathelicidin, which is an antimicrobial peptide \[[@B11]\]. As to adaptive immune, vitamin D reduces the production of T-helper type 1 (Th1) cells through suppressing IL-12 \[[@B12]\]. Meanwhile, different studies found vitamin D upregulates or deregulates expressions of interleukin (IL)-4 in different immune time. *In vitro* studies have demonstrated that vitamin D directly reduces IgE produced by peripheral human B cells \[[@B17]\]. However, certain studies failed to detect an inverse association between vitamin D and IgE levels \[[@B7]\]. Additionally, *in vitro* studies demonstrated that vitamin D may be involved in eosinophil recruitment to noninflammatory sites via regulation of C-X-C chemokine receptor type 4 expression in eosinophils \[[@B15]\]. These findings suggest that vitamin D may not be a direct factor in the pathogenesis of AR. In the current study, serum 25(OH) D levels were observed to be inversely associated with blood eosinophil count and its proportion in patients with persistent AR. A previous *in vitro* study reported results consistent with the findings of the present study. The previous study reported that vitamin D deficiency in neonatal mice contributed to the severity of airway hyper-responsiveness with increased eosinophilic inflammation; however, supplementation of vitamin D reduced pulmonary eosinophilia \[[@B18]\]. It is well established that 1,25(OH)~2~D, the activated form of 25(OH)D, causes genomic responses through interaction with the nuclear VDR; however, 1,25(OH)~2~D is also involved in rapid responses in a variety of cell types, including keratinocytes, chondrocytes and muscle cells \[[@B19]\]. The rapid responses take place within seconds to minutes inside the cells, resulting in the activation of various signaling pathways. To date, 2 receptors for 1,25(OH)~2~D have been identified to mediate these rapid responses: the VDR present on the plasma membrane, and membrane-associated protein disulfide isomerase family A member 3 \[[@B20]\]. These 2 receptors, when activated by 1,25(OH)~2~D, signal via different signaling pathways and initiate downstream cellular effects. Additionally, 1,25(OH)~2~D has been demonstrated to be capable of generating differently shaped ligands for the VDR, which allows it to select for different response arms: genomic or rapid responses \[[@B21]\]. Recent studies observed the pro- and anti-inflammatory properties of vitamin D in epithelial cells. Paplińska-Goryca et al. \[[@B22]\] found 1,25(OH)~2~D decreased the levels of IL-33 and IL-25 in cultured nasal epithelial cells. In inflammatory bowel disease, vitamin D also aids in sustaining the structural integrity of intestine epithelial cells while enhancing innate immune responses in the mucosa \[[@B23]\]. In the current study, VDR expression was predominantly detected on the plasma membrane in nasal epithelial cells. This might indicate that vitamin D plays a part role in intracellular signaling on nasal mucosa. Nevertheless, it is still unclear whether this nongenomic action of vitamin D regulated the immune responses of nasal mucosa \[[@B24]\]. The findings regarding the association between vitamin D levels and AR are inconclusive \[[@B14]\]. A study in Korean adults demonstrated that the serum 25(OH)D level in patients with AR was lower than the non-AR group, and the prevalence of AR steadily decreased in the patients with higher 25(OH)D \[[@B25]\]. Another study in Qatari children below the age of 16 identified that there was a higher prevalence of vitamin D deficiency (25(OH)D level \< 10 ng/mL) in children with AR (18.5%) and asthma (17%) compared with in normal children (10.5%). Vitamin D deficiency was strongly associated with the allergic diseases \[[@B26]\]. However, a similar investigation in Korean adults reported that individuals with vitamin D insufficiency have an increased likelihood of having atopic dermatitis, but not allergic airway diseases, including AR and asthma \[[@B27]\]. A German study did not detect a decreased risk of AR and asthma with higher 25(OH) D levels in 10-year-old children; however, eczema and allergen-specific IgE levels were significantly correlated with those of vitamin D \[[@B28]\]. The results of the present study demonstrated that the serum 25(OH)D levels in the AR group were lower, but not significantly different to the control group. In addition, certain reports demonstrated that serum 25(OH)D were associated with AR in men and not in women \[[@B29]\], whereas others reported that male and female patients with AR had lower vitamin D levels \[[@B30]\]. The majority of subjects involved in the current study were male (92.0% and 84.4% in AR and control groups, respectively). If excluding female participants, the results remained statistically insignificant. Furthermore, our previous study investigating the polymorphisms of VDR observed that age and gender may have an impact on the genetic association of VDR polymorphisms with the risk of mite-sensitized persistent AR \[[@B31]\]. Wegienka et al. \[[@B32]\] reported that the association between vitamin D levels and allergy-associated outcomes, including eczema, skin-prick tests, and specific IgE levels, varied between different races: the results differed between Caucasian and black children. Therefore, the factors that influence vitamin D levels in patients with AR require further analysis in larger randomized studies, including identifying differences in sex, age, and race. As for the effect of vitamin D supplementation on AR, studies are less and also inconclusive. A Danish cohort study involving midpregnancy (25 weeks) vitamin D intake from food and supplementation suggested no association with children AR at 18 months and 7 years but there was an inverse relationship between high total vitamin D intake and asthma at 7 years \[[@B33]\]. Another Finland birth cohort study found maternal vitamin D intake self-reported 5.1 ± 2.6 mg/day from food might be negatively associated with risk of asthma and AR at children by 5 years, but no association was found between vitamin D supplements alone and AR \[[@B34]\]. Bunyavanich et al. \[[@B35]\] also found food containing vitamin D in maternal diets rather than vitamin D supplements was associated with reduced odds of AR at school age. Regardless of the vitamin D supplementation, a recent study found vitamin D binding protein polymorphisms might lead to a racial difference of 25(OH)D level. Mean 25(OH)D levels of African American participants (77% with 25(OH)D \< 20 ng/mL) were significantly lower than that of White Americans. However, African American have consistently higher bone mineral density and serum calcium than White Americans \[[@B36]\]. So whether or how 25(OH)D supplementation affecting the immunological disease are needed to be answered. There are some limitations in our study. In the procedures of immunohistochemistry, we did not operate the intracellular staining, thus the staining might have been main observed in the cell membrane. In addition, the number of subjects in this study was small. These limitations need to be made up in further studies. In conclusion, the present study suggests that serum 25(OH)D might be inversely associated with blood eosinophils in patients with persistent AR. However, the relationship between vitamin D and AR still requires further clarification. This work was supported by grants from the National Natural Science Foundation (81300834), the Priority Academic Program Development of Jiangsu Higher Education Institutions (JX10231801), and the Health Promotion Project of Jiangsu Province (RC2011071), the People\'s Republic of China. We thank Assistant Professor Yong-Ke Cao at the School of Foreign Languages of Nanjing Medical University for kind advice in the manuscript preparation. {#F1} ###### General characteristics of the total study population  AR, allergic rhinitis; SD, standard deviation. ###### Serum 25(OH)D levels and blood eosinophils in patients with AR and the controls  25(OH)D, 25-hydroxyvitamin D; AR, allergic rhinitis; SD, standard deviation.

Neutrophils are key immune cells that participate in host defense through a variety of mechanisms including phagocytosis, the generation of reactive oxygen species (ROS), release of granular contents, and the formation of neutrophil extracellular traps (NETs). The capacity of neutrophils to orchestrate inflammatory and immune responses is dependent on their release of neutrophil-derived molecules, including cytokines, alarmins, and NETs, as well as their capacity to interact with and direct other innate and adaptive immune cells. Over the past decade, the field of neutrophil biology has exploded, with remarkable discoveries highlighting neutrophils as indispensable players in immune regulation. While neutrophils are recognized to be prominent phagocytes involved in the clearance of pathogens and cell debris, they are beginning to emerge as essential communicators crucial in shaping immune response. In this Research Topic, a series of articles provides comprehensive insights into the current view of neutrophil biology, highlighting neutrophil interactions with platelets, infectious agents, and tumor cells, as well as neutrophil generation of inflammatory mediators such as NETs. In the review by [Wirestam et al.](https://doi.org/10.3389/fimmu.2019.02734), we are reminded of the devastating role of neutrophils in rheumatic diseases, including systemic lupus erythematosus (SLE) and anti-phospholipid syndrome (APS) wherein neutrophils recognize circulating immune complexes promoting ROS-dependent NET formation. Intriguingly, SLE patients have impaired NOX2, relying on mitochondrial ROS for NET formation. This results in concomitant mitochondrial extrusion and a qualitatively distinct NETs with inflammatory oxidized mitochondrial DNA as an important cargo. [Takeda et al.](https://doi.org/10.3389/fimmu.2019.01334) highlighting the important role for immune complexes, namely bactericidal/permeability-increasing protein (BPI) immune complexes, in mediating NET formation in systemic anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis. The regulation and role of the main immune complex receptor, Fc gamma R, in steady state and inflammatory conditions, is comprehensively covered in an excellent review by Wang and Jönsson. Beyond immune complexes, large aggregates, including monosodium urate (MSU) crystals are known to activate macrophages and neutrophils, though the underlying signaling events have not been well-characterized. In an elegant study by [Tatsiy et al.](https://doi.org/10.3389/fimmu.2019.02996), performing transcriptomic analyses, the authors demonstrate the novel finding that neutrophils, upon engulfment of MSU crystals, are programmed to attract monocytes in an NF-κB- and CCL4-dependent manner. This observation is particularly interesting as neutrophils are essential in licensing macrophages for cytokine production in atherosclerosis, indicating a pathophysiological role for their crosstalk ([@B1]). Furthermore, the authors find that TAK1 and Syk are early mediators, upstream of MAPK and Akt, partaking in NET formation, and as such, representing a potential therapeutic target for gouty arthritis. A similar transcriptomic approach was undertaken by [Miralda et al.](https://doi.org/10.3389/fimmu.2020.00497) to assess neutrophil signaling pathways regulated by the bacteria *Filifactor alocis*. Among the many pathways being regulated by *F. alocis*, the authors made the intriguing finding that the bacteria blunted TNF-induced MAPK activation in neutrophils, suggesting that this may be a bacterial strategy to subvert innate immunity. Another study investigating the underlying requirements for NET formation ([Gößwein et al.](https://doi.org/10.3389/fimmu.2019.02481)), made the novel observation of a crosstalk between a protease, calpain, and peptidylarginine deiminase 4 (PAD4) in nuclear decondensation, with PAD4-mediated citrullination facilitating proteolytic cleavage of nuclear proteins. This observation markedly advances our understanding of the molecular events during NET formation and may allow for additional therapeutic strategies to prevent exacerbated NETosis. In the final NET-related paper included in this Research Topic, [Agarwal et al.](https://doi.org/10.3389/fimmu.2019.02148) demonstrate that NETs are not only a product of neutrophil activation, but also a potent stimulus for secondary NETosis, inducing NET formation through TLR9-dependent mechanisms. This mechanism may be particularly important in post-traumatic inflammation, when NETs disrupted by mechanical strain may amplify inflammation and prevent physiological wound healing. Two interesting papers in this collection suggest a critical role for platelets as orchestrators of neutrophil recruitment to diseased organs. Psoriasis is a systemic inflammatory disease characterized by intense leukocyte infiltration into the skin. [Herster et al.](https://doi.org/10.3389/fimmu.2019.01867) undertook an unbiased screen of neutrophil surface markers in individuals with psoriasis and found upregulation of CD41 and CD61 (two classic platelet glycoproteins). These surface markers were attributable to increased platelet-neutrophil aggregates in the blood of patients with psoriasis ([Herster et al.](https://doi.org/10.3389/fimmu.2019.01867)). The aggregates also appeared to infiltrate the skin, where platelets could be found in close proximity to both intact neutrophils and neutrophil remnants suggestive of NETs ([Herster et al.](https://doi.org/10.3389/fimmu.2019.01867)). Intriguingly, platelet depletion was protective against neutrophil infiltration and overall skin inflammation in a mouse model of psoriasis ([Herster et al.](https://doi.org/10.3389/fimmu.2019.01867)). Systemic inflammatory response syndrome (SIRS) puts many organ systems at risk including the lungs, where it may lead to acute respiratory distress syndrome. [Hook et al.](https://doi.org/10.3389/fimmu.2019.01472) characterized a model of SIRS triggered by administration of zymosan. This model has previously been shown to be exacerbated by NADPH oxidase 2 (Nox2) deficiency. In this new study ([Hook et al.](https://doi.org/10.3389/fimmu.2019.01472)), Nox2-deficient mice demonstrated upregulation of platelet-derived chemokines such as CXCL4 and CXCL7 in the bronchoalveolar fluid. Neutrophils strongly expressing P-selectin glycoprotein ligand-1 were found at increased levels in the same fluid ([Hook et al.](https://doi.org/10.3389/fimmu.2019.01472)). While Nox2 deficiency may protect against NETosis in some contexts, that was not the case in this model where exaggerated NETosis appeared to be driven by platelets in a PAD4-dependent fashion ([Hook et al.](https://doi.org/10.3389/fimmu.2019.01472)). In summary, these studies shine a light on the role of platelets in recruiting neutrophils into inflamed tissues, and suggest the potential for anti-platelet agents to have efficacy in neutrophil-dependent diseases. A further group of articles have focused on some of the functional crosstalk that neutrophils are known to engage in with other leukocytes. Firstly, [Bernson et al.](https://doi.org/10.3389/fimmu.2019.02444) extended our knowledge on the molecular basis underlying the capacity of natural killer (NK) cells to trigger neutrophil apoptosis under inflammatory conditions, by performing a comprehensive characterization of neutrophil expression of ligands for NK cell receptors. Ultimately, the authors demonstrated that inflammatory neutrophils, including either blood neutrophils stimulated *in vitro* or *in vivo*-transmigrated neutrophils, become more susceptible to NK cell-mediated apoptosis than resting neutrophils. Furthermore, their enhanced sensitivity is associated with a pronounced downregulation of HLA class I expression, which is known to release activating signaling by NK cells ([Bernson et al.](https://doi.org/10.3389/fimmu.2019.02444)). Secondly, [Aarts et al.](https://doi.org/10.3389/fimmu.2019.02144) compared the immunosuppressive capacity of circulating neutrophils from healthy donors (HDs) with those displayed by autologous bone marrow (BM) myeloid cell fractions at various differentiation stages. They show that, unlike "early mature" BM neutrophils, immature BM neutrophils, *per se*, are not efficient in suppressing T cells when activated with physiological stimuli ([Aarts et al.](https://doi.org/10.3389/fimmu.2019.02144)). The authors therefore speculate that, for instance in cancer patients, functional granulocyte-myeloid-derived suppressor cells (g-MDSCs) require neutrophil differentiation from immature populations in the bloodstream, which are unable to perform any immunosuppressive activity in their own right ([Aarts et al.](https://doi.org/10.3389/fimmu.2019.02144)). In another article, [Oberg et al.](https://doi.org/10.3389/fimmu.2019.01690), first, exhaustively review current knowledge on the reciprocal interactions between neutrophils, tumor cells, and particularly gamma/delta T cells. They then provide data on the modulation of anti-tumor cytotoxicity of short-term expanded human gamma/delta T cell lines by autologous freshly isolated neutrophils. Their results demonstrate that, under certain circumstances, the presence of neutrophils can enhance, rather than inhibit, the killing capacity of gamma/delta T cells, by increasing their release of cytotoxic mediators ([Oberg et al.](https://doi.org/10.3389/fimmu.2019.01690)). Finally, the role of neutrophils in malignancy was extensively reviewed by [Lecot et al.](https://doi.org/10.3389/fimmu.2019.02155), highlighting the heterogeneity of neutrophils, and their effector functions contributing to cancer suppression as well as progression. The authors also reviewed different strategies aimed at targeting neutrophils in malignancy, ranging from neutrophil depletion to succinct targeting of recruitment or other key effector functions. In conclusion, neutrophils are heterogeneous cells with a huge range of effector functions partaking in shaping our immune system. The current papers included in this Research Topic highlights the emerging role of neutrophils as communicators in health and disease with promises of future therapies targeting these pathways. Author Contributions {#s1} ==================== All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. Conflict of Interest {#s2} ==================== 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 authors are grateful for the high quality of the authors and reviewers involved in this Research Topic. **Funding.** This study was funded by Lupus Research Alliance (519414) to CL, Associazione Italiana per la Ricerca sul Cancro (AIRC, IG-20339), Ministero dell\'Istruzione, dell\'Università e della Ricerca (MIUR-PRIN 2015YYKPNN and 20177J4E75_004) to MC. RJ was supported by grant 1126403 from National Health and Medical Research Council Australia. JK was supported by grants from NIH (R01HL115138), Lupus Research Alliance, and Burroughs Wellcome Fund. [^1]: Edited and reviewed by: Francesca Granucci, University of Milano-Bicocca, Italy [^2]: This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology

Introduction ============ Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract.[@b1-ppa-6-253] GIST most commonly occur in the stomach (60%--70%) or small intestine (20%--25%) at a rate of 20 cases per million persons per year.[@b2-ppa-6-253],[@b3-ppa-6-253] The introduction of the tyrosine kinase inhibitor (TKI) imatinib (Glivec^®^/Gleevec^®^, Novartis AG, Basel, Switzerland) has improved the treatment of KIT^+^ GIST. Clinical results have shown improvements in response rates and progression-free survival for patients with unresectable and/or metastatic KIT^+^ GIST treated with imatinib at doses between 400 mg/day and 800 mg/day.[@b4-ppa-6-253],[@b5-ppa-6-253] Interruption of imatinib after 1, 3, or 5 years has been associated with increased risk of relapse, even in patients who achieve complete response, suggesting that continuous therapy may be necessary until disease progression or intolerance occurs.[@b6-ppa-6-253],[@b7-ppa-6-253] As such, adherence to self-administered imatinib therapy is an essential component of the daily routine of these patients, and adherence evaluation is a key component of disease management for health care providers (HCPs). The clinical benefit of imatinib as adjuvant therapy, given for 3 years after resection in patients with KIT^+^ GIST, has been validated in the randomized Phase III Scandinavian Sarcoma Group (SSG)/Sarcoma Group of the Arbeitsgemeinschaft Internistische Onkologie (AIO; SSGXVIII/AIO) trial.[@b8-ppa-6-253] Although surgery is considered the only curative treatment for primary resectable GIST, even after complete resection, almost 50% of patients experience disease recurrence within 5 years.[@b9-ppa-6-253] As a result, imatinib is indicated for adjuvant treatment of adult patients following resection of GIST expressing KIT (CD117),[@b10-ppa-6-253]--[@b12-ppa-6-253] and the National Comprehensive Cancer Network (NCCN) guidelines suggest a duration of at least 36 months of imatinib in patients at high risk of recurrence.[@b11-ppa-6-253] Targeted, oral medications are being developed increasingly for cancer treatment. In spite of the severity of many cancers, adherence to these self-administered medications can be a challenge. One study showed that patients taking oral cancer medications may overestimate their adherence to therapy by a factor of 2.[@b13-ppa-6-253] Results from another study of patients with KIT^+^ GIST or Philadelphia chromosome-positive chronic myeloid leukemia on imatinib therapy showed that only half of the patients achieved 100% compliance over a 24-month period.[@b14-ppa-6-253] Moreover, a comprehensive patient record review of patients with GIST (adjuvant: 147 records; metastatic: 225 records) who were treated with imatinib showed that less than half of the patients were perfectly compliant over their last four prescription refills (all refills exactly on time; data not published). In addition, approximately 10%--14% of GIST patients missed 10% or more of their prescribed daily dose during the last four refills in the study. Effective management of adverse events (AEs) in patients with GIST can facilitate continued treatment and adherence to TKI treatment. Although imatinib is generally well tolerated, virtually all patients with metastatic GIST (\>95%) who have been treated with imatinib experienced at least one AE of any grade.[@b4-ppa-6-253],[@b15-ppa-6-253]--[@b18-ppa-6-253] However, many of the AEs occurring in imatinibtreated patients with metastatic GIST may be related to surgical procedure, underlying malignancy, or other illnesses that may occur during prolonged imatinib administration. Significantly longer progression-free survival has been observed in patients with metastatic disease on continuous imatinib therapy compared with patients whose therapy was stopped after 1, 3, or 5 years.[@b6-ppa-6-253],[@b7-ppa-6-253],[@b18-ppa-6-253] Even for patients who have achieved complete response, discontinuation can result in loss of remission and rapid tumor progression.[@b6-ppa-6-253] Moreover, interruption of continuously scheduled treatment due to nonadherence or other reasons can lead to low imatinib plasma concentrations, which have been reported to be associated with a short median time to progression for patients with advanced GIST, confirming the importance of maintaining optimal drug levels through adherence.[@b19-ppa-6-253] Dose escalation of imatinib may be considered in patients showing objective signs of disease progression at a lower dose and in the absence of severe adverse drug reactions.[@b11-ppa-6-253] In cases where maximum supportive care with imatinib is not effective, or where imatinib results in life-threatening side-effects, sunitinib is recommended as second-line therapy.[@b11-ppa-6-253] The treatment of GIST involves not only an understanding of medical therapy regimens and their associated AEs, but also an understanding of the patient emotional journey through diagnosis and treatment. The current study explored the GIST patient journey from diagnosis through the start of TKI treatment, initial response, if any, to treatment, loss of response in a subset of patients deriving clinical benefit from TKI therapy, and subsequent switching to second-line or investigational therapies. Ethnographic tools to examine patients' experience with issues such as adherence, disease knowledge, disease management, and relationship with HCPs were used. Methods ======= Study design ------------ Patients were recruited from individual clinical practice centers. Exclusion criteria included: patients working in advertising; marketing/market research; public relations; any media (eg, TV, radio); the pharmaceutical industry; the health care industry as physicians, pharmacists, mental health professionals, or registered/licensed nurses; and patients aged younger than 21 years or older than 80 years. The ethnographic approach sought to engage patients with GIST in their own environments to obtain open feedback on their everyday experiences. Multiple, in-context techniques, including individual and group interviews, life histories, and participant observation, were used. Together, these methods served to elicit patients' perspectives on their disease and treatment. Researchers documented all interactions and observations with participants by taking field notes; these notes were then examined to address key topic areas (see 'Content and questions' section). Patient perspectives were then compared, and any emerging concepts or themes were assembled. The main approach included a 2.5-hour, in-home interview with patients who either had resected or metastatic GIST, and their immediate family members. The patient sample comprised North American (United States and Canada) and global patients (Brazil, France, Germany, Russia, and Spain). After the interview, patients from North America, Brazil, and France completed a 7-day photo journal followed by a 45-minute telephone interview. All patients provided written consent to participate in the investigation. Patient privacy is maintained based on a signed consent agreement between the researchers and the patient participants and their families. Per the consent document, we maintain GIST patient anonymity but have permission to use patient quotes. Content and questions --------------------- The ethnographic interview protocol included seven sections with suggested discussion time limits to provide a general structure to the interview ([Table 1](#t1-ppa-6-253){ref-type="table"}). These seven sections addressed issues relating to daily life, management techniques for adherence and TKI-related side-effects, their relationship with their HCP, and how these patients perceived their disease and associated treatment options. Questions were developed by the investigators with input from specialists as well as psychosocial scientists trained in evaluating patient experiences. The interviews were audiotaped. National data were analyzed by researchers at each site, and the composite national results for the global dataset were then analyzed by two independent researchers. Results ======= Participants and settings ------------------------- Fifty patients with GIST were interviewed and included in the study ([Table 2](#t2-ppa-6-253){ref-type="table"}). A total of five patients from each country outside North America with either (a) resected primary GIST who received adjuvant imatinb therapy (n = 2), or (b) metastatic GIST who received TKI treatment (n = 3), were interviewed between February 15, 2010, and May 31, 2010. The North American country samples consisted of ten patients from the United States (adjuvant) and 15 from Canada (n = 5, adjuvant; n = 10, metastatic) who were interviewed between January 19, 2010, and April 19, 2010. Overview of the stages of the GIST patient journey -------------------------------------------------- The GIST patient journey included stages of: *crisis*, *hope*, *adaptation*, *new normal*, and *uncertainty* ([Figure 1](#f1-ppa-6-253){ref-type="fig"}). Patients experienced *crisis* when they were given the diagnosis of GIST and were faced with the ensuing treatment decisions that spark worry about the road ahead and mortality. The *crisis* dissipated as patients saw a therapeutic path forward, and *hope* emerged with drug treatment, and in those patients who had an early response, followed by disease regression or stability. Patients then entered a period of *adaptation*, where a prolonged period of adjustment required changes in behavior, outlook, and lifestyle. This period culminated in altered expectations and acceptance of a new outlook on life. Most patients eventually "moved on with life" in the stage of *new normalcy*. The *new normal* for these patients was markedly different, both mentally and physically, from their life before receiving a GIST diagnosis. The stage of *uncertainty* included fear of and/or experience of treatment failure that precipitated anxiety about the disease. Progression triggered *crisis*, and during this stage patients commonly searched for information on GIST and new treatment options that would bring the disease back under control. Patients progressed through these stages over time, and the stages were abbreviated or prolonged depending on the degree of each individual's disease (eg, primary resected or metastatic, comorbidities), therapy response, and circumstances. The patient journey has changed with the availability of new clinical treatment options, information resources, and disease awareness among patients and physicians. The success of imatinib has altered how patients think about GIST; patients noted that they considered TKI treatment to be "easy" compared with how they perceived other cancer treatments are (ie, chemotherapy), and the proven outcomes of imatinib gave physicians confidence and gave patients optimism. The evolution of cancer treatment paradigms with new therapies provides imatinib-resistant or imatinib-intolerant patients with additional treatment options. These additional options along with improved outcomes meant that the *uncertainty* was not always a result of drug resistance or disease progression, but could also result from anxiety surrounding disease monitoring, treatment side-effects, or dose changes. The GIST patient journey has been extended with the advent of second-line treatment with the multitargeted TKI sunitinib (Sutent^®^; Pfizer, New York, NY) and other investigational therapies currently in development. The NCCN guidelines recommend sunitinib 37.5 mg once daily without interruption or sunitinib 50 mg once daily on a schedule of 4 weeks on followed by 2 weeks off.[@b11-ppa-6-253] When a patient can no longer tolerate, or becomes resistant to, imatinib, second-line and investigational therapies can offer hope, but these therapies can also cause patients to revisit earlier journey stages. Patients reported returning to a period of *crisis* with disease recurrence (adjuvant patients) or progression (metastatic patients). With the assistance of their HCPs and support network, patients may then readapt to the new stage of their journey followed by a different *new normal* period. Crisis ------ For patients with GIST, diagnosis of the disease began the tumultuous journey of discovery about GIST and its associated prognoses, treatment options, and paradigms. Regardless of the time to the diagnosis of GIST, the stage of *crisis* inevitably ensued. In the case of protracted diagnoses, many patients had longstanding indeterminate symptoms, or the sense of knowing that something was "not right." Some patients went through months, even years, of anxiety, generalized symptoms, and declining health before receiving the diagnosis of GIST. One patient said, for example: > "It took months. I went to a doctor, and he didn't know what was wrong. They did one gastroscopy and colonoscopy after another until they finally did a \[computed tomography scan\] -- then they knew it." Some patients received the diagnosis quickly and incidentally, or after an acute event. One patient explained, for example: > "\[In a routine procedure\] they found a bump on my rectum, but I thought it was no big deal. The shock was a week later when the doctor called and said it was a very rare disease called GIST." Patients reported that shock is a central part of *crisis*, which results from diagnosis, because GIST is relatively unknown to most patients, who have never heard of this disease. The feeling of fear was heightened in patients who were told by their HCPs that they had a "rare" disease. At diagnosis, patients learned that they had a tumor, but most patients reported that the word "cancer" was not part of the conversation. Few patients reported hearing this word at diagnosis. For example: > "The diagnosis was evasive. The radiologist said 'GIST', but at the time GIST didn't mean cancer. But \[my wife\] checked out the Internet and saw it was cancer." Hope ---- For patients, *hope* came from the knowledge that there is an existing treatment with proven efficacy in GIST, so that they could see a path forward. Some patients felt psychologically reassured and physically improved when they began treatment. As one patient explained: > "They said \[the therapy\] might shrink the tumor. I felt better quickly, fantastic, in 2 weeks. It was an amazing difference. I learned later that shrinkage meant it would liquefy \[ie, cystification\] inside." *Hope* also fueled patients' search for current information about their disease and its associated treatment options. Many patients reported encountering out-of-date, contradictory, or incorrect information, which was confusing at times, as well as anxiety provoking. For example: > "I checked Google with "stromal tumors" or "GIST." Google seemed to show older results, dating back to 2002 or 2003, and that was worrisome." Although patients uniformly grasped the idea that imatinib is a treatment and not a cure for metastatic GIST, differences in the levels of confusion arising from undefined treatment durations were observed for patients with different treatment modalities (ie, therapy in the adjuvant or metastatic and/or unresectable disease settings). Patients with metastatic GIST understood that they would most likely be on therapy for life. However, many patients who underwent surgery for primary GIST were unsure how long they would receive adjuvant treatment, because their HCPs were themselves unsure. The optimal duration of adjuvant imatinib treatment is currently unknown. As a result, treatment duration for these patients is commonly not specified by physicians upfront, and patients reported that imatinib therapy came with an indeterminate timeline they found to be unsettling. As one patient explained: > "With metastases, you will be treated with \[imatinib\] automatically, but clinical data is not clear for adjuvant. Everybody has his own opinion here." Patients also expressed frustration at the lack of communication and coordination among the physicians responsible for their treatment. Patients reported that surgeons failed to set postsurgical expectations, while oncologists were focused on treating the cancer but provided little guidance on postsurgical lifestyle management. Adaptation ---------- As patients with GIST settled into medical therapy, they struggled with profound changes. Patients progressed through a period of *adaptation*, whereby they came to terms with the course of events: a cancer and/or rare-disease diagnosis, surgery, medications, and a harsh confrontation with their own mortality. During this transitory period, many patients reported suffering from symptoms of depression, anxiety, and insomnia. *Adaptation* also brought the physical challenges of postsurgery functionality, including changes in diet brought about by altered digestion, and more broad-spectrum lifestyle changes arising from increased fatigue. For example: > "Anyone who goes through a gastrectomy knows that things don't work the same anymore. I used to wonder if my symptoms were from the drug, but now I know that these things are due to my surgery. They took out a lot of parts!" *Adaptation* meant an altered sense of the patient's own body. Patients expressed a myriad of feelings about having cancer in their bodies, referring to it as an "invader" or "alien." During this stage, patients with metastatic GIST adjusted to the continuous presence of cancer, while patients in the adjuvant setting adjusted to the potential risk for recurrence. Although few understood the cause of the cancer, many reported that GIST was a "wake-up call." Many became focused on the "important" things in life, such as families, relationships with others, and spirituality during this stage. Over time, patients with GIST developed methods for managing their disease and treatment (ie, self-care). A key element of developing self-care involved coming to terms with the necessity of drug adherence, either in the adjuvant or metastatic disease setting. Most patients were motivated to take their medication, as it gave them an active role in their long-term health. Many patients acknowledged that it took time to develop steady, consistent, drug-taking routines, and that they needed to adapt and learn to manage treatment side-effects. During *adaptation,* patients also developed a sense of personal advocacy, whereby they fostered and strengthened new relationships with their HCPs and other members of the GIST community. Having recognized that GIST is a disease that few people know about or understand, many patients felt "singled out" and were obliged to become their own best advocates. As a means of personal advocacy, patients sought out GIST specialists and often sought guidance from other patients dealing with similar experiences by participating in online communities. This personal momentum may help contribute to patients returning to a new sense of *normalcy.* New normal ---------- For patients with GIST, the *new normal* stage meant achieving a semblance of life pre-GIST diagnosis. *New normal* came as patients moved beyond merely adapting to their new circumstances, but started participating in activities within their new circumstances; for many, it was a time of returning to activities they used to do. Of special note, all participating patients claimed that the need to adhere to TKI therapy was a prevailing thought during this stage, and most claimed that they had established routines to try to remember to take their medications. For example, many patients stored their drugs in the kitchen; some also reported filling pill boxes at a given time each week. Other strategies patients used to remain adherent included: having a family member remind them to take their medication, setting reminder mechanisms, and keeping a calendar. In addition, some patients took their medication at regularly spaced periods during the day, while others took it all at once at a specific time. Patients also viewed TKI therapy as an "insurance policy" against progression (those with metastatic and/or unresectable disease) and recurrence (those receiving adjuvant therapy). For example: > "When I walk my dog, I take a golf club, just in case the coyotes return. I consider my \[imatinib\] to be like a golf club, something there to protect me just in case the cancer returns." For patients with GIST, *normal* life was punctuated by moments of fragility. All patients experienced increased anxiety leading up to their quarterly or biannual checkups, which included surveillance imaging scans; this increased stress is referred to, in the GIST community, as "scanxiety." In response to being informed of an "all-clear" scan (ie, no detected progression or recurrence), patients felt a palpable sense of relief, and some reported feeling they now had "time to breathe." > "When my doctor tells me that I am still cancer free, I feel so many emotions. ... I pretty much live from scan to scan." Most patients with GIST in this study became "hyper-surveillant" with their health. This attitude was evident regardless of treatment setting (metastatic or adjuvant), or time since diagnosis. Patients were sensitive to any physical changes that could signal progression (for metastatic disease) or recurrence (for resected GIST). Although patients in this study began to wonder what long-term TKI therapy may be doing to their bodies, few expressed strong concerns about potential side-effects arising from the long-term therapy. For most patients, their desire to continue treatment outweighs the potential risks. The *new normal* did not mean complete resolution of all patients' emotional issues and concerns. Patients had trouble conceptualizing the details of their cancer, and perceived their disease in absolute dichotomous terms (ie, they either had GIST or were disease-free). The degree of tumor burden was irrelevant to patients, and all wanted their tumor "gone." Patients with metastatic GIST were clearly aware that their cancer was ever-present. Patients treated in the adjuvant setting worried that their current state -- "no evidence of disease" -- was tenuous or temporary. Many patients believed that tumor biopsies at diagnosis may have seeded cancer in their bodies, or that surgeons may not have achieved clear margins during resection. These preconceptions lead to a relative degree of *uncertainty* about the nature of their illness and its associated treatments. Uncertainty ----------- Patients experienced periodic episodes of *uncertainty* for various reasons along their disease journey and at various times. Clinical changes, uncertain test results, and new disease symptoms or treatment side-effects triggered *uncertainty*, whereby patients regressed to previous emotional patterns. Patients in both the adjuvant and metastatic settings in this study tended to cycle back through all the stages (*crisis*, *hope*, *adaptation*, *new normal*) during episodes of *uncertaint*y; adjuvant patients responded to potential events or symptoms with emotions similar to metastatic patients, despite their lack of evidence of disease. Many patients with GIST felt the need to stay abreast of new information. However, new scientific data, especially for adjuvant therapy, could trigger *uncertainty*. For example, reading new GIST studies or new clinical trial data led to anxiety as patients interpreted or misinterpreted results. Patients on adjuvant therapy carefully followed changing information about duration of treatment and were continuously concerned with whether stopping adjuvant therapy would allow for disease recurrence. Switching therapy due to TKI resistance, disease recurrence, or progression was a key trigger for *uncertainty* and often precipitated a step back in the emotional journey ( [Figure 2](#f2-ppa-6-253){ref-type="fig"}). Patients believed that this meant that the worst outcome had occurred, and many patients reverted to *crisis*. Most patients were devastated by TKI failure and their unknown future path. Switching therapies also revived information-seeking behavior as patients re-energized around the need for personal advocacy. For example, one patient questioned: > "What will happen now? Will this new drug work as well as \[the last drug\]?" Switching drugs due to intolerance often brought relief instead of *crisis*. Metastatic or adjuvant patients who suffered intolerance to a TKI often readapted to the new therapy with relative ease, and their physical and emotional outlooks improved considerably after switching. Eventually these patients settled back to the normalcy of tolerable side-effects and good response. Conversely, emergence of new side-effects after switching therapy could lead affected patients to experience *crisis*, as one patient explained: > "When I went on the \[new\] med, it was really tough. I had a hard time on it, because of the side-effects. Compared to what I am enduring now, \[the prior therapy\] was a cakewalk." Discussion ========== This global ethnographic study explored the GIST patient journey from diagnosis, through initiation of treatment, response to treatment in most patients, loss of response, and switching to second-line or investigational therapies. The study revealed that patients with GIST progressed through periods of *crisis*, *hope*, *adaptation*, *new normal*, and *uncertainty*. For patients with metastatic GIST, persistent *uncertainty* represented the overriding reality ([Figure 3](#f3-ppa-6-253){ref-type="fig"}). The impact of the disease was very high, and each stage of the journey was difficult. *Crisis* involved making high-stakes decisions within a vacuum of knowledge and misunderstanding. *Hope* for treatment success was tempered by the reality of a limited prognosis. *Adaptation* was prolonged and painful, and coming to terms with the presence of cancer was daunting. The *new normal* was a fragile time -- physically sustainable but emotionally tumultuous. *Uncertainty* was a frequent occurrence even with stable disease, coinciding with anxiety experienced in anticipation of HCP visits and scans. The adjuvant GIST patient journey displayed many similarities to the journey of the metastatic patient ( [Figure 4](#f4-ppa-6-253){ref-type="fig"}). However, the adjuvant patient experienced *hope* in treatment success tempered by ongoing or recent medical intervention (eg, surgery). The *adaptation* stage for these patients was also difficult. Shortly after resections and despite no evidence of disease, physical changes arising from surgery were profound, and emotions were raw. Patients' *new normal* was also imperfect, as they were hypervigilant about physical changes associated with surgery and treatment. In addition, patients with GIST in both the adjuvant and metastatic settings experienced "scanxiety" prior to follow-up examinations (ie, *uncertainty* about the constant threat of disease recurrence/progression that heightened during their follow-up periods). The patients with GIST in this study were highly self-directed, but they encountered challenges finding accurate, relevant, and available information about GIST. Although a strong sense of personal advocacy drove the search for information, what patients learned was not always accurate, or they misinterpreted results from clinical trials. Thus, each interaction between a patient and an HCP offers a critical opportunity to clarify and reinforce accurate and relevant information. Physicians also could reinforce and support their search and ensure that the information patients receive is appropriate by directing them to medically accurate GIST support materials and communities. The provision of accurate information may lead to a greater sense of independence and fewer touch points with HCPs. For patients transitioning from *crisis* to *hope*, HCPs can help set expectations around drug efficacy and differentiate among treatments. HCPs can also help patients understand the real risk of disease progression and recurrence by educating them about the effectiveness of TKI therapy and providing a roadmap to those with GIST in the adjuvant setting to help them understand issues surrounding duration of treatment. Additionally, to reduce the risk of relapse or recurrence, the importance of adherence to therapy should be emphasized to patients. As patients moved through *adaptation* to *new normal*, physicians had the chance to remind and reassure patients of the safety of drug and disease monitoring. Patients with either metastatic or primary GIST were hypervigilant about physical changes. Regular checkups during this stage offered physicians the opportunity to remind patients of the need for adherence to therapy, to reinforce to patients the favorable tolerability of TKIs, and to ensure the best possible longterm care. Most patients with GIST undergo surgery at some point in their patient journey, whether to remove the primary tumor or the metastases. Coordination and consistent communication are important between the different disciplines (ie, oncologists and surgeons), as well as with the patients. This also presents an opportunity for physicians to help patients make distinctions between side-effects associated with TKI therapy and those occurring for other reasons (eg, invasive surgical procedures, illnesses unrelated to GIST) so patients do not mistakenly attribute discomfort or side-effects to their medicine with potentially consequent-reduced adherence. Limitations =========== Inclusion in this voluntary study involved patients who were actively engaged and aware of their disease, and many patients participating in this study were driven to gather information; this may reflect a potential selection bias. Multiple data collection techniques were used in this study to lessen these effects. Furthermore, it is important to note that the degree of personal advocacy demonstrated by interviewed patients in this study may not be representative of the GIST patient population as a whole. Nonetheless, the general GIST patient population may require more HCP and staff guidance than the patients participating in this study. Although care was taken to accommodate cultural and health care system differences across the participating countries, these factors may also influence patient perceptions. Conclusion ========== The GIST patient journey is evolving as more information becomes available and patients become further educated about the disease. Most patients with GIST are highly self-directed and understand the importance of adherence to TKI therapy, while acknowledging that therapy may have an impact on their daily lives. HCPs can optimize adherence by managing patient expectations about therapy duration and dissociating medication side-effects from postsurgical events where appropriate. Each phase of the patient journey offers opportunities for physicians and staff, and drug manufacturers to provide support to patients. The authors acknowledge Robert Gillespie, PhD, of Chameleon Communications International, who provided medical writing services with funding from Novartis Pharmaceuticals. **Disclosure** This study was sponsored by Novartis Pharmaceuticals and conducted by N Macdonald and A Shapiro while employed by Hall & Partners Group. C Bender, M Paolantonio, and J Coombs are shareholders and employees of Novartis Pharmaceuticals. {#f1-ppa-6-253} {#f2-ppa-6-253} {#f3-ppa-6-253} {#f4-ppa-6-253} ###### Interview protocol Section Main topic Subtopics ---------------- -------------------------------- --------------------------------------------------------------------------------------------------------------------------------------------------- 1 (10 minutes) Personal/family context BackgroundFamilyEmployment 2 (45 minutes) Illness and treatment history Medical history (timeline)DiagnosisTreatment historyExperience with imatinibRole imatinib plays in your lifeDiagnostics and switching medications 3 (20 minutes) Relationship with medical team Physicians and facilitiesProvider selection processFrequency of visitsQuality of relationshipSource of education about disease and treatments 4 (20 minutes) Current management of illness Logistical and financial concernsEducational resources for disease treatmentsRoutines to help manage diseaseLifestyle modifications 5 (15 minutes) Support Family supportProvider supportOther resources 6 (20 minutes) Impact of cancer on life Emotional impact 7 (20 minutes) Medicine cabinet tour Locations of medicationsMedication adherence routines ###### GIST treatment setting and patient nationality Country Adjuvant patients Metastatic patients --------------- ------------------- --------------------- Brazil 2 3 Canada 5 10 France 2 3 Germany 2 3 Russia 2 3 Spain 2 3 United States 10 0 **Abbreviation:** GIST, gastrointestinal stromal tumors.