HIV-infected adults have increased risk for age-related diseases and low cardiorespiratory fitness that can be prevented and improved with exercise. Yet, exercise strategies have not been well studied in older adults with HIV and may require substantial adaptation to this special population.To determine the safety and efficacy of aerobic exercise in older HIV-infected men in a randomized trial comparing different levels of exercise intensity.2peak) measured by treadmill testing. Secondary outcomes were exercise endurance, six-minute walk distance (6-MWD), body composition measured by Dual-energy X-ray absorptiometry (DXA), and fasting plasma levels of lipids and glucose.We conducted a pilot exercise trial in 22 HIV-infected men ≥50 years of age receiving antiretroviral therapy who were randomized 1:1 to moderate-intensity aerobic exercise (Mod-AEX) or high-intensity aerobic exercise (High-AEX) that was performed three times weekly for 16 weeks in a supervised setting. Primary outcome was cardiorespiratory fitness  but not in the Mod-AEX group  with a significant between group difference (p<0.01). Exercise endurance increased in both the High-AEX group  and the Mod-AEX group . The 6-MWD increased in both the High-AEX  and the Mod-AEX group . Changes in VO2peak and 6-MWD were clinically relevant. There were no serious exercise-related adverse events. Dropouts were similar between group  and were related to joint pain.VOThis pilot exercise trial demonstrates that moderate to high-intensity aerobic exercise in older HIV-infected men increases endurance and ambulatory function. However, increased cardiorespiratory fitness was observed only with high-intensity aerobic exercise despite substantial baseline impairment. Future research is needed to determine exercise strategies in older HIV-infected adults that address advanced aging and comorbidity yet are durable and feasible. While the median survival time for HIV-infected adults 50 years of age and older has doubled, it remains lower than the general population. Despite 2peak, oxygen utilization at peak exercise, is an established measure of cardiorespiratory fitness and a physiologic biomarker of aging that declines in sedentary adults 10% per decade [In older adults without HIV the positive impact of exercise on cardiovascular disease, frailty,r decade  and inder decade ,10 Cardir decade ,12 and cr decade  and skelr decade  Results r decade 2peak in younger HIV-infected adults, the effect and safety, in older HIV-infected adults is understudied.[2peak in older HIV-infected men. We hypothesized that aerobic exercise would increase VO2peak in both groups but high-intensity exercise would be poorly tolerated. The aim of the trial was to collect preliminary efficacy and safety data toward the goal of determining the ideal exercise program in HIV-infected adults that targets mechanisms of advanced aging.Although moderate to high-intensity aerobic exercise increases VOrstudied.,17 To ad2peak.Sedentary HIV-infected adults ≥50 years of age were recruited from the Baltimore VA Medical Center (VAMC) HIV Clinic between 2008 and 2011. After completion of screening and baseline testing participants were randomized 1:1 to 16 weeks of moderate-intensity aerobic exercise training (Mod-AEX) or high-intensity aerobic exercise training (High-AEX). The randomization sequence was generated using a computer program by an investigator without participant contact (JS) and distributed using the closed envelope method to the research coordinator. The study was approved by the University of Maryland School of Medicine Institutional Review Board and the Baltimore VAMC Research and Development Committee. Sample size target was based on a randomized trial in 21 younger HIV-infected men that compared the effect of moderate-intensity and high-intensity aerobic training on endurance (time on treadmill) since noEligibility criteria included a stable combination antiretroviral therapy (cART) and no AIDS defining conditions in the prior six months. Participants were excluded for conditions that increase the risk of exercise training per the American College of Sports Medicine (ACSM), which included, but was not limited to, poorly controlled blood pressure, symptoms of angina and claudication, and ambulation with an assistive device. ParticipParticipants attended center-based exercise sessions three times per week at the research exercise training facility located at the Baltimore VA Medical Center. All exercise training was performed under the supervision of research exercise physiologists. Heart rate (HR) was measured continuously during each session using a Polar HR watch and chest strap allowing for assessment and maintenance of treatment fidelity. Exercise logs included details on exercise type, duration, time in target heart rate zone, blood pressure and perceived intensity during each training session. In the High-AEX group, exercise training was performed on a motorized treadmill with occasional substitution with the elliptical machine as needed for joint pain. Target heart rate was based on the baseline treadmill test and was calculated as percentage of the heart rate reserve . Initially, participants trained for 20–30 minutes at 50–60% of HRR. Duration and intensity was increased by 10% weekly so that within 5–7 weeks the aerobic exercise sessions lasted 30–45 minutes at 70–85% of HRR and at the end of the 16 weeks lasted 40–45 minutes at 75–90% of HRR. In the Mod-AEX group, participants performed a self-paced 1-mile walk (3–5 METs) on an indoor track in the same exercise center as the high-AEX group. Initial sessions lasted 20–30 minutes and were increased weekly to 45 minutes in parallel to the duration of the high-AEX group. Participants were encouraged to stay weight neutral and had their weight and diet routinely assessed to meet that goal. Adverse events were evaluated by an investigator blinded to group allocation.2peak) that was measured during a graded exercise test on a treadmill using the modified Bruce protocol as defined by the ACSM.[2peak was calculated as the average of the highest two oxygen utilization values at the end of exercise. Exercise duration was measured as total exercise time on the treadmill.The primary outcome measure of the study was cardiorespiratory fitness .[Dual-energy X-ray absorptiometry (DXA) (Model GE iDXA Scan) was used to measure total and regional lean tissue and fat mass. Computed tomography (CT) scans  at the level of L4-L5 were performed to determine the amount of visceral and subcutaneous fat area of the abdomen. Anthropometric measures included waist and hip circumference. Body mass index (BMI) was calculated as weight/(height)leg fat).Participants had blood drawn after an overnight fast. Plasma glucose was measured by glucose oxidase method  and insulin by double antibody radioimmunoassay . Plasma triglycerides and cholesterol were analyzed by enzymatic methods . HDL-C concentration was measured in the supernatant after precipitation with dextran sulfate. LDL-C concentration was computed using the Friedewald equation.Additional data on clinical characteristics were obtained from the VA electronic medical record (EMR) including medical history, laboratory and pharmacy data, which were confirmed by participant self-report at enrollment. Additional blood testing included CD4 cell count and HIV-1 RNA plasma level . Smoking and injection drug use were assessed by self-report. Diagnosis of hypertension and diabetes was based on clinical history.Descriptive statistics were used to examine data for distribution. Between group differences in characteristics at baseline were evaluated with Student’s t-test, Wilcoxon rank-sum test, and Pearson’s Chi squared test. Within group changes (post-pre intervention) were examined using a paired Student’s t-test or Wilcoxon signed rank test. Between group differences in outcome change were examined using ANCOVA in which the outcome change was the dependent variable in a model adjusted for the baseline value of the outcome variable and group (Mod-AEX vs. High-AEX). Analyses were performed using STATA software .The trial overview is provided in 2peak increased in the High-AEX group  but not in the Mod-AEX group  with a significant between group difference. Time on treadmill significantly increased in both the Mod-AEX (11%) and High-AEX (27%) groups. The six-minute walk distance significantly increased 11% in both the Mod-AEX and High-AEX groups. Weight remained stable (<0.5 kg change) in both groups. Body composition and metabolic parameters did not significantly change in either group. However, the between group difference in change for HDL-C was statistically significant with a clinically relevant increase in the High-AEX group and minor decrease in the Mod-AEX group.Training effects are summarized in 2peak) that was not observed in the moderate intensity group.This pilot randomized exercise trial supports the safety and efficacy of aerobic exercise training in older HIV-infected men. In both the moderate-intensity and high-intensity groups we found clinically significant increases in exercise endurance and six-minute walk distance. Converse to our hypothesis, high-intensity aerobic exercise was well-tolerated and resulted in a clinically and statistically significant increase in cardiorespiratory fitness . In the general geriatric population, an increase of 1 MET over a decade is associated with 15% and 19% lower risk of all-cause and CVD mortality.[2peak after 8–12 weeks of high-intensity aerobic training.[Several reviews and meta-analyses of randomized exercise trials in younger HIV-infected adults have shown that aerobic exercise increases VOpression.,17,23,24pression. To our kortality. In compatraining. In this training. These fi2peak 0.4 mL/kg/min) despite increased exercise endurance and six-minute walk distance. The 27% increased time on treadmill in the high-intensity group was more than double the 11% increase seen in the moderate-intensity group and is consistent with the higher dose of exercise (intensity x time) received by this group compared to the moderate-intensity group, who exercised for the same duration and frequency. This dose-based increase in time on treadmill by group is similar to results in a 12-week randomized trial of moderate-intensity versus high-intensity aerobic exercise including HIV-infected men who were on average 26 years younger than our participants.[2peak in healthy adults is ≥60% of baseline VO2peak.[2peak in every individual, especially those young and active. However, exercise trials in older adults with low VO2peak and age-related diseases, including heart failure,[2peak with moderate-intensity aerobic training. In our trial, aerobic exercise at an intensity level of only 3 METS should have been sufficient to significantly increase VO2peak in the moderate-intensity group given the low baseline VO2peak (17.8 mL/kg/min).[Surprisingly, we did not find any change in fitness in the moderate aerobic exercise group . Because /kg/min). and The /kg/min). Yet ther/kg/min). Furtherm/kg/min). and loss/kg/min). are impo2peak. Even with adjustment for weight (ml/kg/min), there was no significant change in the VO2peak in the moderate intensity group compared to the substantial change in the high intensity group. Further, we adjusted the between group analyses for baseline differences. We acknowledge the possibility of a type II error  in the moderate-intensity group VO2peak finding. However, it is notable that despite the sample size statistically significant increases were found for both time on treadmill and 6-MWD, which also negates the issue of a learning effect in the high-intensity group who trained on the treadmill.In addition to the study’s small size, specific limitations warrant comment. First, the study population consisted largely of African American men which limits the generalizability of our findings. Second, since there is little information on the safety of exercise interventions in older HIV-infected adults, by design our exclusion criteria were conservative. This process resulted in a high screening to enrollment ratio but minimized the risk of serious adverse events. Therefore, the exercise safety, like the robust exercise training effects, may not be generalizable to the general population of older adults with HIV. Similarly, our attrition rate was lower than the average attrition rate in HIV exercise trials. In this This pilot randomized aerobic exercise trial provides novel findings on the safety and efficacy of moderate to high intensity aerobic exercise in older HIV-infected men. Clinically significant gains in cardiorespiratory fitness, endurance, and ambulatory function were found despite the small sample size. Results also raise the question of an absolute or relative exercise intensity threshold to increase cardiorespiratory fitness. Physical activity recommendations are a critical component of geriatric care and are yet to be formulated as part of the clinical practice of older adults with HIV. Further research is needed to determine effective exercise training strategies in older HIV-infected adults that will promote successful aging with HIV and assure an increased lifespan of quality years.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.


Experience with providers shapes the quality of adolescent health services, including voluntary medical male circumcision (VMMC). This study examined the perceived quality of in-service communication and counseling during adolescent VMMC services.A postprocedure quantitative survey measuring overall satisfaction, comfort, perceived quality of in-service communication and counseling, and perceived quality of facility-level factors was administered across 14 VMMC sites in South Africa, Tanzania, and Zimbabwe. Participants were adolescent male clients aged 10–14 years (n = 836) and 15–19 years (n = 457) and completed the survey 7 to 10 days following VMMC. Adjusted prevalence ratios (aPRs) were estimated by multivariable modified Poisson regression with generalized estimating equations and robust variance estimation to account for site-level clustering.Of 10- to 14-year-olds and 15- to 19-year-olds, 97.7% and 98.7%, respectively, reported they were either satisfied or very satisfied with their VMMC counseling experience. Most were also very likely or somewhat likely (93.6% of 10- to 14-year olds and 94.7% of 15- to 19-year olds) to recommend VMMC to their peers. On a 9-point scale, the median perceived quality of in-service (counselor) communication was 9  among 15- to 19-year-olds and 8  among 10- to 14-year-olds. The 10- to 14-year-olds were more likely than 15- to 19-year-olds to perceive a lower quality of in-service (counselor) communication . Most adolescents were more comfortable with a male rather than female counselor and provider. Adolescents of all ages wanted more discussion about pain, wound care, and healing time.Adolescents perceive the quality of in-service communication as high and recommend VMMC to their peers; however, many adolescents desire more discussion about key topics outlined in World Health Organization guidance. Adolescents’ experiences with a healthcare provider can shape the perceived quality of adolescent sexual and reproductive health (ASRH) services. A systematic literature review focused on ASRH services in sub-Saharan Africa highlights evidence of a disregard for privacy, judgmental attitudes toward adolescents seeking services, and a lack of respect, all of which negatively affect adolescent–provider interaction . InsuffiGlobal guidance on the standards for quality health services for adolescents, as set forth by the World Health Organization (WHO), advocates for competency-based trainings in adolescent health, age-appropriate tools and materials, and ongoing monitoring of the quality of health education and counseling by providers , 4. ThisAdolescent experiences during voluntary medical male circumcision (VMMC), which provides one of the few entry points through which health services can reach male adolescents, have the potential to influence future health-seeking behavior. VMMC services constitute an important opportunity to contribute to structural, policy, and healthcare setting changes aimed at improving the health and well-being of adolescent males . ResearcThe WHO/Joint United Nations Programme on HIV/AIDS Framework for VMMC 2021 includes targets for reaching 90% of males 10–29 years of age by 2021 with VMMC services that include “age-appropriate, comprehensive sexuality and health education” for 10- to 14-year-olds and “detailed sexual health counseling” among other components for 15- to 19-year-olds [The Tanzania National Institute for Medical Research, the Human Sciences Research Council in South Africa, the Medical Research Council of Zimbabwe, and the Johns Hopkins Bloomberg School of Public Health Institutional Review Board all approved the study prior to data collection. Parent permission was obtained for adolescent males <18 years of age, and assent/consent was obtained for all adolescents.Participants were recruited from 14 facilities offering VMMC services to adolescents in South Africa, Tanzania, and Zimbabwe . Data weOverall satisfaction with the VMMC experience and likelihood of recommending VMMC to male peers were measured using ordinal 4-point Likert response scales .Adolescents’ gender preference for providers was measured with a 3-point Likert response scale . Participants were asked about their level of comfort with having a male or female counselor during the individual and group preprocedure counseling sessions. In addition, participants were asked about their level of comfort with having a male or female provider perform the VMMC or check the wound during follow-up.A scale to measure the perceived quality of in-service (counselor) communication, which taps into the quality of patient–counselor interpersonal interactions, was developed using 9 items adapted from the WHO Quality Assessment Guidelines  and the Participants were asked 7 items related to facility-level factors that could influence the perceived quality of their VMMC experience . Responses were coded as 0 (“no/don’t remember”) or 1 (“yes”).To explore participants’ perceptions of the content of counseling, they were asked (1) “What do you wish had been discussed (more) during counseling? and (2) “What do you wish had not been discussed during counseling?” Participants could provide multiple unprompted responses; interviewers had a predetermined list of relevant categories but detailed responses could also be documented. It should be noted that due to the open-ended nature of this question, interviewers did not consistently administer this question to all adolescents. Furthermore, it was not clearly coded if the interviewer skipped the question or the adolescent responded “nothing” to each question. Thus, these findings should be cautiously interpreted.Differences in the distribution of ordinal Likert responses between age-groups 10–14 vs 15–19 years) were assessed using the nonparametric Somers D test with Fisher Z transformation; the Somers D tests accounted for facility-level clustering using the delta-jackknife method and adjusted for country by stratification [–19 yearsP < .15), with the exception of age group and receipt of a postprocedure counseling session, which were included regardless of statistical significance. Only some participants experienced both a preprocedure and postprocedure counseling session, and since the outcome variable did not differentiate between the two, a sensitivity analysis limited to participants who self-reported only receiving preprocedure counseling was conducted. All analyses were among complete cases only.Correlates of a perceived quality of counselor communication score <7/9  were examined. This threshold was selected based on the distribution of scores among the entire sample population . For this analysis, modified Poisson regression models were used with generalized estimating equations and robust variance estimation to account for facility-level clustering. Factors shown to have an association with the outcome after adjustment for country were included in the final multivariable model .A total of 1293 adolescents completed the preprocedure and postprocedure survey, including 836 (64.7%) 10- to 14-year-olds and 457 (35.3%) 15- to 19-year-olds across South Africa, Tanzania, and Zimbabwe. The majority of adolescent clients (97.7% of 10- to 14-year-olds and 98.7% of 15- to 19-year-olds) reported they were either satisfied or very satisfied with their VMMC experience . When asThe overall perceived quality of in-service communication was high; the median perceived quality of in-service communication score was 8  among 10- to 14-year-olds and 9  among 15- to 19-year-olds . Age wasFacility setting was independently associated with a lower perceived quality of in-service communication . When coThese findings were replicated in a sensitivity analysis limited to participants who received only preprocedure counseling  .Adolescents in both age groups tended to feel more comfortable having a male rather than female counselor during both individual and group preprocedure counseling sessions, but this was only significant among 15- to 19-year-olds receiving group counseling after adjustment for country. In comparison to reported comfort among adolescents who had a female provider, both age groups were significantly more comfortable with a male provider performing the procedure and with a male provider being the one checking their wound during the follow-up appointment .P = .005) and were less likely to report feeling embarrassed because others might overhear what was said . Although there were significant differences between the 2 groups (independent of country), the majority had not experienced transport issues or embarrassment during the counseling session.Overall, adolescents had positive perceptions of the facilities where they received VMMC. The majority reported the facilities to be welcoming, they were made to feel comfortable by all staff, wait times were reasonable, and the hours of the facility were convenient . A signiWhen asked what topics they wish had been discussed more during the counseling sessions, both 10- to 14-year-olds and 15- to 19-year-olds reported they wish there had been more discussion about pain , followed by wound care  and healing time  . AdolescDespite reporting high levels of satisfaction with their VMMC service and overall high perceived quality of in-service communication, younger adolescents were significantly more likely to perceive the in-service communication as lower quality compared to older adolescents. Overall, adolescents of all ages found facilities welcoming and reported feeling more comfortable with male counselors and providers compared with female counselors and providers. However, adolescents across all ages reported they would have liked to receive more information about pain, sexual health, wound care, and healing time, and the majority reported not receiving postoperative counseling or a combination of individual and group counseling, as recommended by WHO guidance.The absence of a postprocedure counseling session is concerning since WHO guidance , the US Furthermore, most adolescent clients, regardless of age, did not receive both group and individual preprocedure counseling sessions, although both are clearly defined in the WHO guidelines , 24. TheBeyond worries regarding pain, a main topic also reported by adolescents needing further discussion is sexual health. Younger adolescents aged 10–14 years, reported they would also like more information about condom use. As found in Kenya , provideThis study has limitations. Given the cross-sectional study design, reported associations should be descriptively interpreted, and the measures for perceived quality must be taken in context of this particular study. The study population may contain selection biases, as the study only captured adolescents who underwent VMMC and completed the postprocedure survey. The responses of this study sample may vary from adolescents who did not complete the follow-up survey and adolescents who attended the service provision site but did not ultimately undergo VMMC.While adolescents across the 3 study countries reported high perceived quality of in-service communication and would generally recommend VMMC to their peers, they also reported wanting additional discussion on topics including pain, wound healing, sexual health, and the VMMC procedure itself. As stated by the WHO, adolescents “are not simply older children or younger adults,” but instead are individuals with particular needs who require providers sufficiently competent to respond to those needs and ensure adolescent-responsive care . VMMC prClinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at VanLith_Perceived_SupplementalFile_FinalClick here for additional data file.


Affective disorders impact nearly 10% of the adult population in the United States in a given year. Synaptic dysfunction has recently emerged as a key neurobiological mechanism underlying affective disorders such as anxiety and depression. In this study, we investigate the potential role of two synaptic scaffolding proteins, neurabin and spinophilin, in regulating anxiety- and depression-related behaviors at different ages using genetically deficient mice. Loss of the neurabin gene reduces anxiety-like behavior in the elevated zero maze in young adult mice (3–5 months old), but not in middle aged mice (11–13 months old), whereas loss of spinophilin decreases anxiety in middle-aged mice, but not in young adult mice. Neurabin knockout (KO) mice also show reduced immobility in the repeated force swim test (FST) at 3–5 months, but not 11–3 months, of age, compared to age- and strain-matched wild type (WT) controls. Conversely, spinophilin KO mice display a lower level of this behavioral despair than matched WT controls after repeated FST trials at the middle age (11–13 months) but not the young age (3–5 months). Together, these data indicate that, despite their structural similarities and overlapping function in regulating synaptic cytoskeleton, the two homologs neurabin and spinophilin play important yet distinct roles in the regulation of anxiety- and depression-like behaviors in an age-dependent manner. Our studies provide new insights into the complex neurobiology of affective disorders. Affective disorders, such as anxiety and depression, are a major public health challenge carrying substantial social and economic burdens, impacting nearly 10% of the adult population in the United States in a given year –4. DysreNeurabin and spinophilin, which are also referred as neurabin I and II, are homologous scaffold proteins enriched at the synapse. The two proteins share a similar multi-domain structure which includes actin-binding, G protein-coupled receptor (GPCR)-interacting, protein phosphatase 1 (PP1)-binding, PDZ, and coiled-coil domains , 6. NeurGiven their functions in regulating synaptic morphology and plasticity, we elected to address the potential roles of neurabin and spinophilin in regulating anxiety and depression-related behaviors using neurabin-deficient and spinophilin-deficient mice. Since these behaviors are often affected by age, we tested two age groups, young adult (3–5 months old) and middle aged (11–13 months old), in the open field (OF), elevated zero maze (EZM), and forced swimming test (FST) assays. Our data reveal that, despite the structural similarities between neurabin and spinophilin, and their overlapping function in regulating the synaptic cytoskeleton, inactivation of each gene caused distinct behavioral phenotypes. Furthermore, the phenotypes are age-dependent for both neurabin and spinophilin, although the overall expression levels of these two proteins did not change with age. Our study suggests that neurabin and spinophilin play important, yet distinct, roles in regulating anxiety- and depression-like behaviors in an age-dependent manner.2 inhalation followed by cervical dislocation to ensure the death of the animals. CO2 flow was controlled by a regulator. The procedure was consistent with the recommendations of the AVMA Guidelines for the Euthanasia of Animals.Mice were housed in the Association for Assessment and Accreditation of Laboratory Animal Care-accredited Animal Resources Program at the University of Alabama at Birmingham. Experimental procedures are in accordance with Animal Welfare Act and the 1989 amendments to this Act. All protocols were approved by University of Alabama Institutional Animal Care and Use Committee. Mice were euthanized by CONeurabin-null  and spin2. Total distance traveled and relative time spent in the center of the open field were recorded. EZM was an elevated cycling corridor with two open areas and two closed areas. Time spent in open versus closed areas of the EZM and total distance traveled were documented as endpoints. All data were acquired using the automated EthoVision camera-driven tracker system .OF and EZM tests were performed as we described previously . OF triaFST were performed as previously described  with modMouse brains without the cerebellum were homogenized with lysis buffer containing 75mM Tris, pH6.8, 15% glycerol and 3% SDS. 20μg of each sample was loaded on a 10% polyacrylamide gel and separated by SDS-PAGE. Neurabin, spinophilin and PP1γ were detected by antibodies against neurabin , spinophilin (Upstate) and PP1γ , respectively. Quantitation of Western blots was performed using the LI-COR Odyssey Imaging System.t-tests were used to compare two groups. Two-way ANOVA was used to analyze multiple FST trials with two genotypes and test for trial x genotype interactions in FST. Post hoc Sidak's multiple comparisons were performed following two-way ANOVA to determine the difference between two genotypes in each FST trial.Data were analyzed using GraphPad Prism software. Unpaired Student’s We began by assessing the baseline activity of young adult (3–5 months old) neurabin KO and spinophilin KO mice in the OF assay, comparing with age- and strain-matched WT mice. No difference was observed in either the percentage of time spent in the center of the arena  over the total trial time  or the tWe then examined anxiety-like behavior by EZM tests. Because of the elevated nature of the platform, rodents tend to spend more time in the closed regions than in the open ones. When comparing the effects of genotype, drug administration, etc., any increase in time spent in the open regions indicates decreased anxiety-like behavior, while any increase in time spent in the closed regions indicates increased anxiety-like behavior . As showCenter exploration time in the OF is an additional parameter that can reflect anxiety-related behavior, with increased center time being indicative of an anxiolytic effect . Althougp = 0.05, The FST has been widely used to evaluate behavioral despair , 32, typp < 0.01) for immobility time (p < 0.01) for immobility  for immobility time between WT and neurabin KO mice  for immobility time between WT and spinophilin KO mice , indicative of an anxiolytic effect , but not


Modifiable risk factors such as smoking and sedentary lifestyle adversely affect multiple sclerosis (MS) progression. Few multimodal behavioural interventions have been conducted for people with MS, and follow-up beyond 1 year is rare for lifestyle interventions. This study assessed adoption and adherence to healthy lifestyle behaviours and health outcomes 3 years after a lifestyle modification intervention, using generalized estimating equation models to account for within-participant correlation over time.95 people with MS completed baseline surveys before participating in 5-day MS lifestyle risk-factor modification workshops. 76 and 78 participants completed the 1-year and 3-year follow-up surveys respectively. Mean age at 3-year follow-up was 47 years, 72% were female, most (62.8%) had MS for 5 years or less, and 73% had relapsing remitting MS (RRMS).Compared to baseline, participants reported clinically meaningful increases in physical (mean difference (MD): 8.0, 95% Confidence Interval (CI): 5.2–10.8) and mental health  quality of life (QOL) at 1-year, and physical  and mental health  QOL at 3-year follow-up. There was a small decrease in disability from baseline to 1-year follow-up  and to 3-year follow-up , which was not clinically meaningful. Of those with RRMS, compared to baseline, fewer had a relapse during the year before 1-year follow-up  and 3-year follow-up . Participants’ healthy diet score, the proportion meditating ≥1 hours a week, supplementing with ≥ 5000IU vitamin D daily, and supplementing with omega-3 flaxseed oil increased at 1-year follow-up and was sustained, although slightly lower at 3-year follow-up. However, there was no evidence for a change in physical activity and not enough smokers to make meaningful comparisons. Medication use increased at 1-year follow-up and at 3-year follow-up.The results provide evidence that lifestyle risk factor modification is feasible and sustainable over time, in a small self-selected and motivated sample of people with MS. Furthermore, participation in a lifestyle intervention is not associated with a decrease in MS medication use. The onset of multiple sclerosis (MS), a chronic inflammatory and neurodegenerative disorder, can be explained by an interplay of genetic and environmental risk factors, including modifiable lifestyle factors. The proIn recent years research interest in modifiable lifestyle risk factors for MS risk and progression has increased, and some have postulated that MS may be a lifestyle disease. HoweverMany PwMS are motivated to modify their risk factors for disease progression. A survey showed that 85% of 199 surveyed PwMS in the United States were willing to try a dietary intervention for at least 3 months. FurtherThe aim of this study was to assess adherence to healthy lifestyle recommendations and health outcomes 3 years after a multimodal lifestyle risk-factor intervention in PwMS.This study is part of a larger study evaluating the effects of a lifestyle modification intervention. A subgroup was invited to complete this 3-year follow up. We have previously reported QOL results from a different subgroup using the same intervention, 15. BelThe intervention was advertised on MS websites with a focus on lifestyle, and participants enrolled without needing referral from a clinician. A few weeks before the workshop, all participants were asked to enrol in this study with longitudinal follow up if they were 18 years or older, by signing a consent form and completing an online or hardcopy baseline survey. Participants who had previously participated in the workshops were excluded from the study.The intervention—5-day residential group workshops—promoted a very low saturated fat, plant-based whole food plus seafood diet, omega-3 supplementation, regular exercise, non-smoking, adequate vitamin D levels and stress reduction techniques such as meditation . These wThe facilitators delivered evidence-based information regarding lifestyle-related risk factor modifications associated with improved health outcomes in MS and practical advice on incorporating these into daily life in a face-to-face group setting. Participant fees varied depending on level of accommodation, and also covered food and other expenses, coordinated through a not-for-profit charity Overcoming Multiple Sclerosis. More information regarding the content of the recommendations including the evidence base can be found on their website.Participants were invited by email for follow-up survey completion at one and three years post-intervention, and reminded by phone or follow-up emails. Data were mostly collected using online survey methodology, and some paper surveys sent through mail and entered in manually by the researchers. The survey took approximately 30–40 minutes to complete and participants were able to take breaks and return to the online survey at a later time if needed.Psychometrically sound and previously tested questionnaires were included in the survey, supplemented by researcher-devised items where existing tools were not available. These are summarised and listed below. All measures were self-reported. Participants self-reported the number of doctor-diagnosed relapses experienced in the previous year. For purpose of analysis, participants were categorised as having had a relapse in the previous year, or not. Disability was assessed using the validated and widely used tool Multiple Sclerosis Impact Scale Physical Component MSIS-20), . Health, . ExercisOther recorded variables were age, gender, partner status (partnered vs not), employment status , level of education (university level or below), and current medication use .Written consent was obtained from all participants included in the study at baseline, and if completing paper follow-up surveys. At follow-up a participant information form was attached to the email invitation and a statement on the first page of the online survey reiterated that completion of the online survey was taken as consent to participate. The study was approved by the Health Sciences Human Ethics sub committee of University of Melbourne (1545287).Data were analysed using Stata, V14  and R 3.13 . We report descriptive statistics of demographics, lifestyle behaviours and health outcomes in Of 134 participants who participated for the first time in one of six workshops 39 (29.1%) either did not have a confirmed MS diagnosis at baseline or follow-up , or did not complete a baseline survey, and were therefore excluded from the study. 95 (70.9%) consented and completed the baseline survey, out of those, 76 (80.0%) and 78 (82.1%) completed 1 and 3 year follow-up, respectively, but completion varied per survey item .Improvements in health outcomes were seen 1 year post-intervention, and were mostly sustained 3 years post intervention, with the exception of MSIS at 3 years. All health behaviours improved 1 year post-intervention, and were mostly sustained at 3 years post-intervention, except physical activity, which improved but non-significantly from baseline to 1 and 3 years post-intervention .Patient self-management is recognised as a key to effective and efficient management of chronic disease. In chronic disease, health care professionals ideally assist patients to develop skilled health practices and the patient takes a more active role acquiring and applying relevant knowledge to their own circumstances. Our mulFrom the 1960s onwards, with the rising incidence of chronic disease, a patient’s ability to function (not just the status of their disease) was recognised as an important outcome, and a patient’s attitude towards and perceptions of his or her situation were recognised as determinants of function. Continued engagement with and persistence in efforts to overcome problems related to the disease and positive self-perceptions resulted in both better function and survival. These observations led to the development of the concept of quality of life and the recognition of its importance in outcomes of chronic diseases. Self-reOf those participants with RRMS, the proportion reporting a relapse in the year post-intervention decreased dramatically compared to the year pre-intervention (from 70% to 16%). The proportion experiencing a relapse in the year prior to 3-year follow-up increased slightly to 26% but was still markedly and significantly lower than at baseline. It is possible that participants who had just experienced a relapse enrolled into the intervention for that reason, and this may have inflated the number of relapses reported at baseline, a phenomenon also recognised in another study. This stParticipants reported a small, not clinically meaningful, decrease in disability . DiThe intervention successfully changed behaviours but, more importantly, maintained these healthier behaviours over time although a slight decrease in health behaviours was seen at 3-year follow-up compared to 1 year follow-up. The one exception was physical activity. This may reflect the small proportion of time spent during the intervention on physical activity, and this has since been improved after feedback from participants. The efficacy of physical activity interventions may also differ by MS type, disability status, and weight status .While the general population perceives many barriers to maintaining a healthy lifestyle , common Despite very few multimodal lifestyle interventions for mental and physical health outcomes in PwMS, benefit has also been shown for people with other chronic diseases. In fact, management guidelines of other chronic diseases such as cardiovascular and cerebrovascular disease, and type-2 diabetes now routinely incorporate modification of lifestyle-related risk factors such as smoking cessation, diet, weight and physical activity–39, althWe have used the TIDieR guidelines to describe the intervention , howeverThis study contributes to the literature regarding long-term follow-up after behavioural interventions in PwMS. The results provide evidence that lifestyle risk modification is feasible and sustainable over time, in a small self-selected and motivated sample of PwMS. These findings need to be replicated in larger, controlled studies.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


One of the crucial steps toward understanding the associations among molecular interactions, pathways, and diseases in a cell is to investigate detailed atomic protein-protein interactions (PPIs) in the structural interactome. Despite the availability of large-scale methods for analyzing PPI networks, these methods often focused on PPI networks using genome-scale data and/or known experimental PPIs. However, these methods are unable to provide structurally resolved interaction residues and their conservations in PPI networks.Here, we reconstructed a human three-dimensional (3D) structural PPI network (hDiSNet) with the detailed atomic binding models and disease-associated mutations by enhancing our PPI families and 3D–domain interologs from 60,618 structural complexes and complete genome database with 6,352,363 protein sequences across 2274 species. hDiSNet is a scale-free network (γ = 2.05), which consists of 5177 proteins and 19,239 PPIs with 5843 mutations. These 19,239 structurally resolved PPIs not only expanded the number of PPIs compared to present structural PPI network, but also achieved higher agreement with gene ontology similarities and higher co-expression correlation than the ones of 181,868 experimental PPIs recorded in public databases. Among 5843 mutations, 1653 and 790 mutations involved in interacting domains and contacting residues, respectively, are highly related to diseases. Our hDiSNet can provide detailed atomic interactions of human disease and their associated proteins with mutations. Our results show that the disease-related mutations are often located at the contacting residues forming the hydrogen bonds or conserved in the PPI family. In addition, hDiSNet provides the insights of the FGFR (EGFR)-MAPK pathway for interpreting the mechanisms of breast cancer and ErbB signaling pathway in brain cancer.Our results demonstrate that hDiSNet can explore structural-based interactions insights for understanding the mechanisms of disease-associated proteins and their mutations. We believe that our method is useful to reconstruct structurally resolved PPI networks for interpreting structural genomics and disease associations.The online version of this article (10.1186/s12918-018-0537-2) contains supplementary material, which is available to authorized users. One of the crucial steps toward understanding the associations among molecular interactions, pathways, and diseases in a cell is to investigate detailed atomic protein-protein interactions (PPI) in the structural interactome. Many high-throughput experimental methods, such as high-throughput yeast two-hybrid screening , 2 and cAs the increase in the number of available three-dimensional (3D) structural complexes, there is a new opportunity to develop a fast and accurate computational method for inferring structurally resolved PPIs and constructing structural PPI networks. The structural complexes provide domain-domain interactions and atomic details for thousands of direct physical PPI interactions. Several works have combined protein structures with experimental PPIs to study how mutations affect protein interactions in diseases , 5. For http://gemdock.life.nctu.edu.tw/3d-network.Here, we propose a structural systems biology method for reconstructing human structural interactome (hDiSNet) with physical PPIs by enhancing our previous 3D–domain interologs and scoring functions . We collE-value < 10− 10) from the Integr8 complete genomic database /[0.44/(1–0.44)]).To study the relationship between disease-associated proteins (genes) and their mutations in hDiSNet, we collected the disease-related mutations of these proteins from OMIM database . The datp-value < 0.01, Mann–Whitney U test). We also observed that PPIs derived from human and non-human templates are enriched than IEXP PPIs while the RSS-BP, CC, and MF are higher than 0.9  and all pairs  . In RSS-BP score > 0.9, the ratio of overlapped PPIs (0.50) are similar to hSIN (0.53), and more enriched than HC (0.33), IEXP (0.15) and all pairs (0.03). The results indicate that our reconstructed human structural interactome (hDiSNet) comprehensively includes 14,864 newly discovered PPIs and 4375 overlapped PPIs that enhance the Gene Ontology similarities of PPIs.The interacting proteins are usually involved in similar biological processes and located in similar cellular components. To verify the quality of our human structural interactome (hDiSNet), we calculated the relative specificity similarity (RSS)  of Gene 0.9 Fig. . These r01) Fig.  and c. Tp-value = 0.97). These results show that both of our predicted PPIs derived from human and non-human templates are robust and helpful for investigating the cellular processes.Furthermore, we have found that the reliability of a predicted PPI depends on the evolutionary distance between the target and source species based on our previous studies. Here, we compared the similarities of GO annotations of PPIs derived from human and non-human templates. The average of RSS-BPs of PPIs from human and non-human structural templates is 0.85 and 0.82, respectively. We found that the RSS-BPs of PPIs derived from human templates have no significant difference comparing with the non-human templates  on co-expression of three different gene expression datasets. In addition, the HC PPIs have no difference (mean correlation = 0.07) compared with our inferred PPIs from PPI families , and our predicted PPIs have significant difference with the IEXP PPIs  and significant higher correlation than all protein pairs . Furthermore, the co-expression correlation of 4375 overlapped PPIs from our inferred and IEXP (yellow) has no significant difference compared with that of hSIN , but has significantly higher correlation than that of IEXP  and all protein pairs . These results imply that both of our predicted PPIs derived from human and non-human templates are reliable and they could reflect the specific states of disease in time and space. Moreover, our hDiSNet completely comprises 14,864 newly discovered PPIs and 4375 overlapped PPIs that improve the co-expression correlation of PPIs.To further assess the quality of our hDiSNet, we used three microarray sets, including lung, breast and brain cancer, for comparing co-expression correlation in our predicted PPIs and other PPI sets. , the probability of a node with k links, decreases as the node degree increases on a logarithmic scale plot  with mutations and diseases, we identified two major regions which are signal receiving receptors  and their downstream signal transduction proteins  Fig. . These tAccording to the studies of these three microarray datasets  to regulate key biological processes, such as cell proliferation, survival, migration, and differentiation both during development and in the adult . FGFR2 mTo further investigate the biological meaning of our network (hDiSNet), the Gene Ontology enrichment analysis was carried out. The proteins involved in the similar process and located in similar cellular component would be the neighbors in the PPI network. We identified six cellular components  in the human network based on the CC annotations  can be used to describe the communication between the cellular protein metabolic processes Fig. . The memDisease-related mutations can be roughly classified into two broad categories  . Here, tIn addition, we also assigned the contacting residues of proteins based on the contacting residues of the structural templates. To investigate the relationship between the mutations and contacting residues, we collected 283 proteins which have in-frame mutations on the contacting residues. Based on these 283 proteins, we found that 790 and 1426 mutations are located at the contacting and non-contacting residues, respectively. According to the odds ratio, the disease-related mutations are significantly associated with the contacting residues (2.45) comparing to non-contacting residues     Fig. . The 116st) Fig. . MoreoveBreast cancer is one of the major diseases in women worldwide, with ~ 1.38 million new cases and ~ 458,000 deaths in 2008 alone . MetastaThe AR is a ligand-dependent factor, which transcriptional activity is mediated by interaction with multiple co-activators , and is In comparison with the PPI network derived from experimental PPIs, our hDiSNet provided the clues to reveal that the mutations of target proteins affect the binding between two proteins. According to the structural template (PDB code: 3 h52 ), the coThe grade IV astrocytomas  are categorized as an aggressive class of brain cancer, which involved in hallmark characteristics of proliferation, necrosis, genetic instability, and chemoresistance . In clinAccording to the structural template (PDB code: 1ivo ), the coWe have reconstructed a human structural interactome (hDiSNet) with detailed atomic binding models and disease-associated mutations by enhancing our PPI families and 3D–domain interologs. Our hDiSNet expands the number of PPIs compared to present structural PPI network. The experimental results show that our predicted PPIs have significantly consistent GO annotations and high co-expression correlations. Our hDiSNet provides the insights into human genetic disease and their associated proteins and mutations, such as the structural FGFR (EGFR)-MAPK pathway for interpreting the breast cancer and ErbB sub-network in brain cancer. Our results show that the disease-related mutations are often located at the contacting residues forming the hydrogen bonds and are conserved in the PPI family. Furthermore, the binding affinity analysis indicates our identified contacting residues have high potential as hot spots. We believe that our method is a useful tool to reconstruct structural interactome with detailed atomic interactions to examine the linkages between the diseases and mutations and to interpret structural genomics and disease associations.Additional file 1:Supplementary Material. (DOCX 2898 kb)


SLC2A1 mutation as cause of glucose transporter type 1 deficiency syndrome (GLUT1-DS). This case report presents the first outcome of GPi-DBS in a patient with GLUT1-DS, and suggests that genotype relations may increasingly complement phenotype-based therapy stratification of GPi-DBS in dystonia.Treatment outcomes from pallidal deep brain stimulation are highly heterogeneous reflecting the phenotypic and etiologic spectrum of dystonia. Treatment stratification to neurostimulation therapy primarily relies on the phenotypic motor presentation; however, etiology including genetic factors are increasingly recognized as modifiers of treatment outcomes. Here, we describe a 53 year-old female patient with a progressive generalized dystonia since age 25. The patient underwent deep brain stimulation of the globus pallidus internus (GPi-DBS) at age 44. Since the clinical phenotype included mobile choreo-dystonic features, we expected favorable therapeutic outcome from GPi-DBS. Although mobile dystonia components were slightly improved in the long-term outcome from GPi-DBS the overall therapeutic response 9 years from implantation was limited when comparing “stimulation off” and “stimulation on” despite of proper electrode localization and sufficient stimulation programming. In order to further understand the reason for this limited motor symptom response, we aimed to clarify the etiology of generalized dystonia in this patient. Genetic testing identified a novel heterozygous pathogenic  A 44-year-old female patient with generalized dystonia including mobile components underwent bilateral deep brain stimulation of the globus pallidus internus . When the patient showed limited response to GPi-DBS years from DBS surgery, we performed a detailed diagnostic work-up. This revealed a pathogenic mutation of the glucose transporter 1 deficiency syndrome (GLUT1-DS).The perinatal history was unremarkable, no medical records on motor and speech development was available. She presented with first symptoms in terms of learning disability at age six. Moreover, complex partial seizures occurred in childhood and were successfully treated with carbamazepine. Remission from seizures was reported at age 18. At age 25, both dystonic and choreatic movements as well as action and postural tremor of the right hand appeared. At age 27, cervical dystonia was documented. Finally, at age 37, progression of dystonic symptoms was reported with dystonia affecting the lower limbs resulting in dystonic gait impairment. Family history of the non-consanguineous parents and three siblings was unremarkable with respect to dystonia or other neurologic diseases.Pharmacological treatment did not improve dystonia . The patient did not tolerate tetrabenazine due to depressive symptoms. No premorbid exposure to neuroleptics was documented.In the preoperative state, generalized dystonia including both tonic and mobile choreo-dystonic movement components was the leading motor presentation that prompted DBS therapy  and UDRS . The F-M was similar across conditions . After discontinuing GPi-DBS, no rebound of dystonia was observed. However, in both “stimulation on” and “stimulation off” conditions, cervical dystonia and dystonia of the right upper extremity was less mobile as compared to the preoperative state. Instead, the tonic components of cervical and upper extremity dystonia were more pronounced in the postoperative long-term follow-up as compared to the preoperative state. Moreover, there seemed to be more axial dystonia in both “stimulation off” and “stimulation on” as compared to the preoperative stage including an intermediate increase in torsion of the trunk, pelvis tilt during gait, and slower and bradykinetic gait patterns.Owing to the mild but limited clinical motor effect of GPi-DBS, we reassessed the localization of active electrode contacts, which were in accordance with standards described elsewhere , 2   indicative for GLUT1-DS, which is pathogenic owing to in silico predictions (www.mutationtaster.org). Moreover, other pathogenic mutations were described previously in c.972 position  arises from impaired glucose transport into the brain because of SLC2A1 mutations. The syndrome was first described in 1991  as pathogenic mutation, since both clinical features and disease course are consistent with previously described phenotypes of GLUT1-DS. Moreover, mutations in similar regions of the SLC2A1 gene were reported in other patients with classical GLUT1-DS . Nevertheless, there is indication for a mild but limited DBS effect. For comparison, motor symptoms generally recur within minutes in effective GPi-DBS when DBS is intercepted . MoreoveDYT1 mutations or, vice-versa, near-to-absent response as for example in rapid-onset dystonia parkinsonism (DYT12). In this aspect, we provide first indication that SLC2A1 mutations might associate with a mild but limited therapeutic effect. Future observations are needed to further substantiate this first observation.Together, we initially expected more favorable outcome owing to generalized dystonia with mobile components as leading motor presentation. However, as in other hereditary dystonia syndromes, genotype relations may affect therapeutic outcomes with favorable clinical response such as in SLC2A1 mutation treated with GPi-DBS. Generalized dystonia including mobile components was the leading presentation at the preoperative state. There was indication for initial mild improvement of right arm dystonia and torticollis, however, in the long-term the therapeutic effect was limited. This case report lends further support to the relevance of molecular genetic testing of patients with generalized dystonia in attempt to stratify DBS outcomes. Molecular genetic diagnostics may increasingly add to understand heterogeneity in DBS outcomes in generalized dystonia (In summary, we report the first patient with GLUT1-DS due to a novel heterozygous dystonia , 21.We confirm that the approval of an institutional review board was not required for this work and that we have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines.IH, AG, and DW: design or conceptualization of the study; IH, CK, MS, LR, and DW: analysis or interpretation of the data; IH, CK, MS, LR, YW, RK, CP, AG, and DW: drafting or revising the manuscript for intellectual content.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.


Study of epigenetics is currently a high-impact research topic. Multi stage methylation is also an area of high-dimensional prospect. In this article, we provide a new study (intra and inter-species study) on brain tissue between human and rhesus on two methylation cytosine variants based data-profiles  and 5-methylcytosine (5mC) samples) through TF-miRNA-gene network based module detection.First of all, we determine differentially 5hmC methylated genes for human as well as rhesus for intra-species analysis, and differentially multi-stage methylated genes for inter-species analysis. Thereafter, we utilize weighted topological overlap matrix (TOM) measure and average linkage clustering consecutively on these genesets for intra- and inter-species study.We identify co-methylated and multi-stage co-methylated gene modules by using dynamic tree cut, for intra-and inter-species cases, respectively. Each module is represented by individual color in the dendrogram. Gene Ontology and KEGG pathway based analysis are then performed to identify biological functionalities of the identified modules. Finally, top ten regulator TFs and targeter miRNAs that are associated with the maximum number of gene modules, are determined for both intra-and inter-species analysis.The novel TFs and miRNAs obtained from the analysis are: MYST3 and ZNF771 as TFs , BAZ2B, RCOR3 and ATF1 as TFs , and mml-miR-768-3p and mml-miR-561 as miRs ; and MYST3 and ZNF771 as miRs(for inter-species study). Furthermore, the genes/TFs/miRNAs that are already found to be liable for several brain-related dreadful diseases as well as rare neglected diseases  mediated enchaphilits, Parkinsons Disease, Bipolar disorder and Schizophenia etc.) are mentioned.The online version of this article (doi:10.1186/s12863-017-0574-7) contains supplementary material, which is available to authorized users. DNA methylation placed at the 5th position of cytosine  –3 is oneOxidative product of 5mC is 5-hydroxymethylcytosine (5hmC). The 5hmC mark was discovered approximately six decades ago in T-even bacteriophage . Later iDifferential methylation is stated to have significant differences in methylation values of a gene across the group of cancer  samples and the group of normal (control) samples. Statistical test is a technique of statistical inference which is applied for measuring the differential methylation between two groups (populations). Differential methylation analysis is carried out for determining whether methylation levels of the genes change significantly in terms of mean and/or standard deviation across samples of experimental and control groups. Co-methylation is another important term that basically signifies that the methylation levels of these genes might increase or decrease together in between a sub-range of time-series rather than the overall time-line. Statistical test is important to detect differential methylation samples. Co-methylation provides an insight that differential methylation on one gene sample can become a cause for differential methylation of other gene samples. It can help to understand unusual behavior of certain path having these gene memebers.Many studies have already been performed on 5-methylcytosine (5mC) for finding disease related information. Recently, researchers have changed their focus from only considering 5mC to including another methylation mark 5-hydroxymethylcytosine (5hmC) with it for observing the variation of regulation due to higher-dimensional methylation marks. Xu et al. 2011) [11 [20] pIn this section, a proposed framework for intra-species study on multiple cytosine based methylation data (having 5hmC and 5hmC samples) for human as well as rhesus through TF-miRNA-gene network based module identification is presented. For intra-species, we first identify differentially 5hmC-methylated genes, and then compute traditional (weighted) topological overlap measure (TOM) on each pair of the resulting genes. Subsequently, we apply average linkage hierarchical clustering technique using the corresponding dissimilarity values of the TOM scores, and generate a dendrogram of genes. Thereafter, intra-species co-methylated gene-modules are identified from the dendogram, by performing dynamic tree cutter algorithm. An intra-species co-methylated gene-module consists of the genes which are not only correlated in 5mC samples but also in the 5hmC samples for the same category or species (e.g. human or rhesus). Moreover, we carry out literature search as well as Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and gene-ontology analysis using David database for biological verification of the genes representing the modules for each case. The transcription factors (TFs) that regulate the genes belonging to the different intra-species co-methylated gene-modules, are collected from Transfac and ITFP databases . The miRThe sets of 5-hydroxymethylcytosine (5hmC) samples and 5-methylcytosine (5mC) samples for the brain tissue for human as well as rhesus are first collected7. For each case of intra-species analyses , 5mC samples are considered as controlled samples whereas 5hmC samples are utilized as experimental samples.FCintra) is defined as the fraction of the mean of the 5hmC samples  to the mean of the 5mC samples (control group) for each species. Since we deal with very small number of samples for intra-species study, therefore simple fold-change method  is applii, and i. Notably, we set a common threshold i.e., 0.25 for both the human and rhesus for the intra-species study.where Thus, for each species (human and rhesus), we find different genesets having the differentially 5hmC-methylated genes. For this case, the collective list of the differentially 5hmC-methylated genes for human is symbolized as http://hms-dbmi.github.io/scw/WGCNA.html) as a weighted connectivity measure for the geneset i and j for each intra-species study ) can be stated as follows. In order to measure the correlation in terms of methylation levels for intra-species study, we initially perform Pearson’s correlation between pairwise genes belonging to the geneset A denotes weighted adjacency matrix of the nodes having Pearson’s correlation values as the weights for each intra-species study.where TOMintra values (similarity scores). We thereafter compute the corresponding dissimilarity scores of dissimintra scores that is represented as: Average linkage hierarchical clustering  is then where q and r are two clusters made for each intra-species, Da stands for distance between a point of first cluster and a point of second cluster for each intra-species, and Lintra denotes the distance between two clusters using average linkage clustering for each intra-species. Then the corresponding dendrogram of the clusters are found.Here Dynamic tree cutter  is then We carry out KEGG pathway and gene-ontology analyses individually using DAVID database on the genes of each module of atabases  that regatabases  which tamentclass2pt{minimIn a similar way, KEGG pathway and gene-ontology analyses are performed separately on the genes of each gene-module of atabases ; whereasatabases . ThereafIn this section, we elaborate the proposed framework for inter-species study on multiple cytosine based methylation data (having 5mC and 5hmC samples) between human and rhesus through TF-miRNA-gene network based module identification. The steps of the proposed inter-species framework are described as follows:In case of inter-species study, we consider both the 5mC and 5hmC samples for the same tissue utilized in “Since it is already mentioned in “FCinter) is defined as the fraction of the mean of all the samples of rhesus (including both the 5hmC and 5hmC samples) to the mean of all the samples of human (including both the 5mC and 5hmC samples). Fold change for each gene for inter-species study ) can be stated as follows. The mentioned weighted topological matrix score between two vertices Ad denotes weighted adjacency matrix of the nodes having Pearson’s correlation values as the weights for the inter-species study.where TOMinter values (similarity scores). We thereafter compute the corresponding dissimilarity scores of dissiminter scores that is demonstrated as follows. Average linkage clustering  is then where s and t are two clusters made for inter-species, De stands for distance between a point of first cluster and a point of second cluster for inter-species, and Linter denotes the distance between two clusters using average linkage clustering for inter-species. Then we get corresponding dendrogram of the clusters. Dynamic tree cut [GM2humanrhesus.Here, tree cut  is then GM2humanrhesus for the purpose of verifying them with existing pathway evidences. Additionally, we determine TFs using ITFP and Transfac databases [DM2humanrhesus. We also determine miRs using miRTarBase and PITA databases [DM2humanrhesus.We individually perform KEGG pathway and gene-ontology analyses using DAVID database on the genes of each module belonging to atabases  that regatabases  which taThe top ten regulator TFs that associate with maximum gene-modules, are identified; whereas top ten targeter miRNAs that link with maximum gene-modules, are determined. It is noted that when more than one TF/miRNA are connected to same number of gene-modules, then these TFs/miRNAs are further ranked in descending order of the maximum number of participating regulated/targeted genes from the different modules. However, these top ten TFs/miRNAs are then verified by literature search for recognizing how many of them have an association with the corresponding diseases related to the sample tissue. The framework of gene-module detection for the inter-species study between the rhesus and human is represented in Fig. For this study, we utilize a methylation dataset (NCBI ref. id.: GSE49177)  having bIn this section, initially we brief about the experimental dataset, and thereafter list up the outcomes after following the proposed frameworks highlighted in Figs. TOM is computed for each gene-pair belonging to the gene-list TOM values. Using these dissimilarity values, we perform average linkage clustering and extract the corresponding dendrogram. Thereafter, dynamic tree cut algorithm is applied to identify the color specific highly correlated modules with highly correlated genes.As mentioned in “MYST3 and ZNF771). Similarly, among the top ten miRNAs, all are known for human brain tissue  among them remain novel . In case of human, for example, according to [We recognize some top TFs/miRNAs/gene-modules that are responsible for some known diseases as well as several types of rare rding to , the toprding to . Interesrding to , TF EPASrding to , Parkinsrding to , Oxidatirding to  and Alzhrding to , respectrding to , the toprding to . Furtherrding to , Bipolarrding to , Moleculrding to , Neuroderding to , and Psyrding to , respectIn case of rhesus, for example, the TFs namely E2F4, ZBTB20, FOXM1, SSRP1, TEDP1, ZNF207 and MYBL2 are connected with the diseases namely SIV encephalitis , Age assAccording to Table p-value= 3.6E-2) in which several genes of the module are significantly involved. Notably, Liu et al. [Table u et al.  supportsp-value = 4.0E-2). Notably, Wei et al. [p-value = 1.0E-2), and GO:MFs of motor activity  and transcription factor activity . Here, GO:MF of motor activity is involved with motor neuron degeneration [p-value = 1.7E-3), GO:MFs of DNA binding , and Transcription factor activity . In case of yellow module, we identify an important pathway viz., mTOR signaling pathway .Table i et al.  supportsneration , whereasneration . For GreDM2humanrhesus genes for both human and rhesus species. Thereafter, Pearson’s correlation score is calculated for each gene-pair belonging to DM2humanrhesus. Weighted TOM is then performed as a weighted connectivity measure where the aforementioned Pearson’s correlation scores are utilized as weighted values of the adjacency matrix. The corresponding dissimilarity values are then computed from the Weighted TOM (similarity) values. Using those dissimilarity values, a dendrogram is constructed by average linkage clustering. Dynamic tree cutter method is applied with color thresholding. As a result, we identify eighteen inter-species multi-stage co-methylated modules (denoted as GM2humanrhesus). See Fig. As mentioned in “After obtaining these color modules, we use different databases to determine TFs and miRNAs (miRs) that regulate and target genes from different color modules respectively. We utilize Transfac and ITFP databases  to identp-value = 8.41E-13) and Neuro-Trophin Signaling pathway . Several gene-ontology terms are also associated with different genes of the Black module viz., GO:CC of Postsynaptic density ; GO:MFs of Zinc ion binding , and cytokine binding . For Blue module, 83 miRNAs target different genes from this module, whereas 71 TFs regulate different genes from this module. For this module, we identify two important KEGG pathways Viz., Tight junction , and phatidylinositol signaling system . Like the previous module, different genes have been associated with several gene ontology terms Viz., GO:BPs of Intracellular signaling transduction , and cell morphogenesis ; GO:CCs of Synaptosome , and histone methyl transferase complex . For Brown module, 53 miRNAs target different genes of the module, whereas 59 TFs regulate different genes of the module. For this module, the related significant gene-ontology terms for the different genes of the Brown module are GO:BPs of Neuron development , and cell morphogenesis ; GO:CC of intracellular organelle lumen ; GO:MFs of Protein kinase activity , Protein serine/threonine kinase activity , MAP kinase tyrosine/serine/threonine phosphatase activity , Protein tyrosine kinase activity . For Cyan module, 5 miRNAs target different genes of this module, whereas 4 TFs regulate different genes of this module. In case of Green module, 27 miRNAs target different genes of the module, whereas 9 TFs regulate different genes from this module. We identify gene-ontology terms that are associated with the different genes of this module Viz., GO:CCs of plasma membrane part , and golgi apparatus part . For Greenyellow module, 6 miRNAs target different genes of the module, whereas 4 TFs regulate different genes of the module. Grey60 module is connected with 17 targeter miRNAs and 3 regulator TFs. However, 14 miRNAs target different genes of Lightcyan module, whereas one TF regulates different genes of the Lightcyan module. The associated gene-ontology terms with this module are GO:BPs of branching morphogenesis of an epithelial tube , and angiogenesis . For Lightyellow module, 26 miRNAs target different genes of the module, whereas one TF regulates different genes of the module. In this case, two important KEGG pathways are identified i.e., B-cell receptor signaling pathway , and T-cell receptor signaling pathway . Different genes of Lightyellow associate with some gene-ontology terms viz., GO:BPs of negative regulation of transport , and regulation of cellular localization . Different genes of Magenta module are targeted by 3 miRNAs and regulated by 6 TFs. The related GO:CCs for this module is synapse . Thereafter, one miRNA targets different genes of Midnightblue module, whereas one TF regulates different genes of the Midnightblue module. In case of Pink module, three miRNAs target different genes of the pink module. We have identified related GO:BP of response to organic cycle substance . In case of Purple module, five TFs regulate different genes of the module. In case of Red module, 80 miRNAs target different genes of the module, whereas 11 TFs regulate different genes of the module. For Salmon module, we have identified 44 miRNAs targeting different genes of this module and two TFs regulating different genes of this module. After that, 250 miRNAs target different genes of Turquoise module, whereas 130 TFs regulate different genes of that module. Here, we find one significant related KEGG pathway i.e., adherens junction . The related significant gene-ontology terms associated with this module are GO:BPs of negative regulation of signal transduction , negative regulation of cell communication ; GO:CC of Neuron projection ; GO:MFs of protein complex binding , SMAD binding , DNA binding , enzyme binding , and ion binding . Finally, 62 miRNAs target different genes of Yellow module, whereas 82 TFs are identified that regulate different genes of the Yellow module. We get an important KEGG pathway i.e., MAPK signaling pathway . Besides this pathway, several gene-ontology terms are also associated with different genes of the Yellow module. These are GO:BPs of hindbrain morphogenesis , hindbrain development , and Ras protein signal transduction ; GO:CCs of Cell junction , Synapse , Postsynaptic density ; GO:MFs of GTPase regulator activity , transcription factor binding .Table ZNF771 and MYST3) are novel (unknown) in both human as well as rhesus; whereas four  are known , and remaining four TFs  are partially known . Notably, according to [Table rding to , ZNF286Arding to , ZNF286Arding to  in humanrding to  in rhesurding to  in humanrding to  in rhesurding to  in humanrding to  in rhesurding to  in case rding to  in humanrding to  in humanSimilarly, Table In terms of intra species analysis, number of color modules is lower than inter species analysis. However, number of modules in human is higher than rhesus. It is proper evidence to comment on methylation level variety of rhesus and human. On other hand, in mammals brain tissue specific methylation study gives a proper insight on differential methylation and diseases. The brain specific diseases, incorporated with unusual methylation level and hydroxy methylation level, has already been mentioned. The effect of miRNA degradation influences methylation and hydroxymethylation for cancers. From the study, it can be observed that brain related diseases are associated with miRNA and methylation. There is a chance to observe similar type of functionality in case of brain related diseases. So from these extensive study, an insight to therapeutic aspects can be discussed over the whole disease signaling pathway depending on controlling unusual methylation and hydroxy methylation rate.In this article, we provide a novel study  between human and rhesus on the brain tissue of 5mC and 5hmC data-profiles through TF-miRNA-gene network based gene module identification. The gene-modules are the verified by KEGG pathway and gene-ontology is analyzed by DAVID database on the gene representing the individual module. Moreover, top ten regulator TFs and targeter miRNAs that connect the maximum number of gene-modules for the intra-species study as well as the inter-species analysis, are determined. During intra-species analysis, for human, two regulator TFs  whereas three regulator for rhesus TFs , and two targeter miRNAs  are identified novel. For the inter-species study, we have identified two novel TFs  of which the related information regarding both human and rhesus are unknown in literature. These TFs/miRNAs might be highly responsible for any brain related disease. We have highlighted the participating genes of each intra-species and the inter-species module along with the regulator TFs or targeter miRNAs that are connected with several brain-related dreadful diseases as well as rare neglected diseases . For example, a TF namely ZNF423 is connected with Joubart’s Syndrome in human, and TF namely SP1 is associated with “Disrupt in early Huntington’s disease” in human, whereas hsa-miR-34c-5p is responsible for Bipolar disorder and Schizophenia in human, and miR-524-5p is associated with Amyotrophic lateral sclerosis in human. From the rate of diversity in gene methylation profiling, the variety in tissue specific gene samples can be understood. In terms of number of color modules, rate of diversity in human is greater than that in rhesus. Finally, as discussed in resulting section, there is chance to derive some therapeutic aspects by studying tissue specific signaling pathways at epigenetic level.Additional file 1Complete list of MicroRNA targeters and TF regulators of color modules from Human Intra-Species analysis. A table of all MicroRNA targeters and TF regulators of color modules from Human Intra-species analysis. (XLSX 32 kb)Additional file 2Color modules for rhesus and corresponding TF regulator and MicroRNA targeter where 5hmC is controlled and 5mC is diseased. A table of generated color modules for rhesus samples where 5hmC is controlled and 5mC is diseased and corresponding predicted TF regulators and miRNAs are also mentioned. (XLSX 12 kb)Additional file 3Color modules for Human and corresponding TF regulator and MicroRNA targeter where 5hmC is controlledand 5mC is diseased. A table for Generated color modules for human samples where 5hmC is controlled and 5mC is diseased and corresponding predicted TF regulators and miRNAs are also mentioned. (XLSX 13 kb)Additional file 4Complete list of MicroRNA targeters and TF regulators of color modules from Rhesus monkey Intra-Species analysis. A table of all MicroRNA targeters and TF regulators of color modules from Rhesus Intra-species analysis. (XLSX 28 kb)