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High vaccine wastage can serve as a critical barrier to achieving the gains of vaccination, especially in a country like Nigeria, where data on vaccine wastage are sparsely available. We determined the country-wide vaccine wastage rates and their determinants through a mixed-methods study conducted across 576 health facilities (primary and secondary) in 24 states in Nigeria. We collected facility-based immunization records from June 2018 to May 2019, in addition to healthcare workers' interviews and observations of fixed and outreach sessions. The results show that open-vial wastage ranged from 21.2% (95% CI: 20.2%, 22.2%) for the pentavalent vaccine to 72.6% (95% CI: 71.5%, 73.6%) for BCG. Open-vial wastage rates for BCG, measles, and yellow fever vaccines were higher during outreach sessions, with rates of 76.2%, 64.3%, and 65.2%, respectively. For the outreach and fixed sessions, PCV and Penta had the least wastages of 22.0% for fixed sessions and 20.4% for outreach sessions. This study identified vaccine presentation (liquid vs. lyophilized vaccines), vial size (4 dose vs. 5 dose vs. 10 dose vs. 20 dose), RI service delivery strategies (fixed vs. outreach sessions), number of children vaccinated, and human resources (healthcare workers position/cadre) as key determinants of vaccine wastages in Nigeria.

Background: Sustaining independence is important for older people, but there is insufficient guidance about which community health and care services to implement., Objectives: To synthesise evidence of the effectiveness of community services to sustain independence for older people grouped according to their intervention components, and to examine if frailty moderates the effect., Review Design: Systematic review and network meta-analysis., Eligibility Criteria: Studies: Randomised controlled trials or cluster-randomised controlled trials. Participants: Older people (mean age 65+) living at home. Interventions: community-based complex interventions for sustaining independence. Comparators: usual care, placebo or another complex intervention., Main Outcomes: Living at home, instrumental activities of daily living, personal activities of daily living, care-home placement and service/economic outcomes at 1 year., Data Sources: We searched MEDLINE (1946-), Embase (1947-), CINAHL (1972-), PsycINFO (1806-), CENTRAL and trial registries from inception to August 2021, without restrictions, and scanned reference lists., Review Methods: Interventions were coded, summarised and grouped. Study populations were classified by frailty. A random-effects network meta-analysis was used. We assessed trial-result risk of bias (Cochrane RoB 2), network meta-analysis inconsistency and certainty of evidence (Grading of Recommendations Assessment, Development and Evaluation for network meta-analysis)., Results: We included 129 studies (74,946 participants). Nineteen intervention components, including 'multifactorial-action' (multidomain assessment and management/individualised care planning), were identified in 63 combinations. The following results were of low certainty unless otherwise stated. For living at home, compared to no intervention/placebo, evidence favoured: multifactorial-action and review with medication-review (odds ratio 1.22, 95% confidence interval 0.93 to 1.59; moderate certainty) multifactorial-action with medication-review (odds ratio 2.55, 95% confidence interval 0.61 to 10.60) cognitive training, medication-review, nutrition and exercise (odds ratio 1.93, 95% confidence interval 0.79 to 4.77) and activities of daily living training, nutrition and exercise (odds ratio 1.79, 95% confidence interval 0.67 to 4.76). Four intervention combinations may reduce living at home. For instrumental activities of daily living, evidence favoured multifactorial-action and review with medication-review (standardised mean difference 0.11, 95% confidence interval 0.00 to 0.21; moderate certainty). Two interventions may reduce instrumental activities of daily living. For personal activities of daily living, evidence favoured exercise, multifactorial-action and review with medication-review and self-management (standardised mean difference 0.16, 95% confidence interval -0.51 to 0.82). For homecare recipients, evidence favoured the addition of multifactorial-action and review with medication-review (standardised mean difference 0.60, 95% confidence interval 0.32 to 0.88). Care-home placement and service/economic findings were inconclusive., Limitations: High risk of bias in most results and imprecise estimates meant that most evidence was low or very low certainty. Few studies contributed to each comparison, impeding evaluation of inconsistency and frailty. Studies were diverse; findings may not apply to all contexts., Conclusions: Findings for the many intervention combinations evaluated were largely small and uncertain. However, the combinations most likely to sustain independence include multifactorial-action, medication-review and ongoing review of patients. Some combinations may reduce independence., Future Work: Further research is required to explore mechanisms of action and interaction with context. Different methods for evidence synthesis may illuminate further., Study Registration: This study is registered as PROSPERO CRD42019162195., Funding: This award was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme (NIHR award ref: NIHR128862) and is published in full in Health Technology Assessment ; Vol. 28, No. 48. See the NIHR Funding and Awards website for further award information.

Androgen excess is a hallmark feature of polycystic ovary syndrome (PCOS), the most common form of anovulatory infertility. Clinical and preclinical evidence links developmental or chronic exposure to hyperandrogenism with programming and evoking the reproductive and metabolic traits of PCOS. While critical androgen targets remain to be determined, central GABAergic neurons are postulated to be involved. Here, we tested the hypothesis that androgen signaling in GABAergic neurons is critical in PCOS pathogenesis in 2 well-characterized hyperandrogenic mouse models of PCOS. Using cre-lox transgenics, GABA-specific androgen receptor knockout (GABARKO) mice were generated and exposed to either acute prenatal androgen excess (PNA) or chronic peripubertal androgen excess (PPA). Females were phenotyped for reproductive and metabolic features associated with each model and brains of PNA mice were assessed for elevated GABAergic input to gonadotropin-releasing hormone (GnRH) neurons. Reproductive and metabolic dysfunction induced by PPA, including acyclicity, absence of corpora lutea, obesity, adipocyte hypertrophy, and impaired glucose homeostasis, was not different between GABARKO and wild-type (WT) mice. In PNA mice, acyclicity remained in GABARKO mice while ovarian morphology and luteinizing hormone secretion was not significantly impacted by PNA or genotype. However, PNA predictably increased the density of putative GABAergic synapses to GnRH neurons in adult WT mice, and this PNA-induced plasticity was absent in GABARKO mice. Together, these findings suggest that while direct androgen signaling in GABA neurons is largely not required for the development of PCOS-like traits in androgenized models of PCOS, developmental programming of GnRH neuron innervation is dependent upon androgen signaling in GABA neurons., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society.)

Background: Frailty is a suggested consequence of ageing, but with a variety of different definitions the understanding of what it means to be frail is challenging. There is a common belief that frailty results in a reduction of physical functioning and ability and therefore is likely to significantly affect a person's quality of life. The aim of this study was to explore the understanding of older people about the meaning of frailty and the potential consequences of being classified as frail., Methods: This paper forms a secondary analysis of a process evaluation of a complex intervention that was embedded within the individually randomised Home-based Extended Rehabilitation of Older people (HERO) trial. A maximum variation, purposive sampling strategy sought to recruit participants with a wide range of characteristics. Data collection included observations of the delivery of the intervention, documentary analysis and semi-structured interviews with participants. Thematic analysis was used to make sense of the observational and interview data, adopting both inductive and deductive approaches., Results: Ninety three HERO trial participants were sampled for the process evaluation with a total of 60 observational home visits and 35 interviews were undertaken. There was a wide range in perceptions about what it meant to be classified as frail with no clear understanding from our participants. However, there was a negative attitude towards frailty with it being considered something that needed to be avoided where possible. Frailty was seen as part of a negative decline that people struggled to associate with. There was discussion about frailty being temporary and that it could be reduced or avoided with sufficient physical exercise and activity., Conclusion: Our study provides insight into how older people perceive and understand the concept of frailty. Frailty is a concept that is difficult for patients to understand, with most associating the term with an extreme degree of physical and cognitive decline. Having a label of being "frail" was deemed to be negative and something to be avoided, suggesting the term needs to be used cautiously., Trial Registration: ISRCTN 13927531. Registered on April 19, 2017., (© 2024. The Author(s).)

17α-Estradiol (17αE2), a less-feminising enantiomer of 17β-estradiol, has been shown to prolong lifespan and improve metabolic health in a sex-specific manner in male, but not in female mice. Recent studies have demonstrated the pivotal role of estrogen receptor α (ERα) in mediating the effects of 17αE2 on metabolic health. However, the specific tissues and/or neuronal signalling pathways that 17αE2 acts through remain to be elucidated. ERα expression in glutamatergic and GABAergic neurons (principal excitatory and inhibitory neurons respectively) in the hypothalamus is essential for estradiol signalling. Therefore, we hypothesised that knocking out ERα from one of these neuronal populations would attenuate the established beneficial metabolic effects of 17αE2 in male mice exposed to a high fat diet. To test this hypothesis we used two established brain specific ERα KO models, targeting either glutamatergic or GABAergic neurons (Vglut2/Vgat-ERαKO). We show that both of these ERα KO models exhibit a strong reduction in ERα expression in the arcuate nucleus of the hypothalamus, a control centre for metabolic regulation. Deletion of ERα from GABAergic neurons significantly diminished the effect of 17αE2 on body weight relative to controls, although these animals still show metabolic benefits with 17αE2 treatment. The response to 17αE2 was unaffected by ERα deletion in glutamatergic neurons. Our results support a benefit of 17αE2 treatment in protection against metabolic dysfunction, but these effects do not depend on exclusive ERα expression in glutamatergic and GABAergic neurons and persist when ERα expression is strongly reduced in the arcuate nucleus of the hypothalamus., (© 2024. The Author(s).)

Share tables (ST) are tables or stations in school cafeterias where students can return unopened foods and beverages, providing an opportunity to access these items at no cost. Currently, research suggests that milk is among the most wasted items in breakfast and lunch programs in the United States. Share tables present a simple solution for reducing milk waste, but research is needed to understand the microbial spoilage potential of milk in ST. To this end, uninoculated milk cartons and milk cartons inoculated with 2 to 3 log 10 (cfu/mL) Pseudomonas poae, a fast-growing psychrotroph, was exposed to ambient temperature during winter (mean temperature = 20.3°C) and summer (23.1°C) for 125 min, repeated over 5 d (the length of a school week). Microbial counts in the inoculated milk cartons increased linearly, exceeding the spoilage threshold of 6.0 log 10 (cfu/mL) after d 3 and after d 4 in the winter and summer season trials, respectively. In the winter trial, the microbial counts for uninoculated milk cartons never exceeded the lower limit of detection, 2.31 log 10 (cfu/mL), and in the summer trials, microbial counts never reached the spoilage threshold, indicating that initial contamination is a driving factor of microbial milk spoilage. Regardless of sharing status or seasonality, the greatest changes in counts for inoculated milk cartons occurred during overnight refrigeration, ranging from 0.56 to 1.4 log 10 (cfu/mL), while during the share table ranged from no observable change up to 0.29 log 10 (cfu/mL), emphasizing that school nutrition personnel should focus efforts on tightly controlling refrigeration temperatures and returning milk to refrigeration as soon as possible. A previously developed model for school cafeteria share tables was adapted to understand the typical residence time of milk in a simulated cafeteria with an ambient temperature share table for the summer and winter seasons over 1,000 wk. Milk was predicted to have a very short mean residence time (85 min) regardless of sharing status or season, with 99.8% of milk consumed, discarded, or donated within the first 2 d. As a result, only 3 out of 451,410 and 6 out of 451,410 simulated milks spoiled in the winter and summer seasons, respectively. The data generated here can be used to inform science-based decision-making for including milk in share tables, or applied to any system where one might have to accept short-term unrefrigerated storage of milk to meet a waste reduction or food security goal., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Objective: To synthesise evidence of the effectiveness of community based complex interventions, grouped according to their intervention components, to sustain independence for older people., Design: Systematic review and network meta-analysis., Data Sources: Medline, Embase, CINAHL, PsycINFO, CENTRAL, clinicaltrials.gov, and International Clinical Trials Registry Platform from inception to 9 August 2021 and reference lists of included studies., Eligibility Criteria: Randomised controlled trials or cluster randomised controlled trials with ≥24 weeks' follow-up studying community based complex interventions for sustaining independence in older people (mean age ≥65 years) living at home, with usual care, placebo, or another complex intervention as comparators., Main Outcomes: Living at home, activities of daily living (personal/instrumental), care home placement, and service/economic outcomes at 12 months., Data Synthesis: Interventions were grouped according to a specifically developed typology. Random effects network meta-analysis estimated comparative effects; Cochrane's revised tool (RoB 2) structured risk of bias assessment. Grading of recommendations assessment, development and evaluation (GRADE) network meta-analysis structured certainty assessment., Results: The review included 129 studies (74 946 participants). Nineteen intervention components, including "multifactorial action from individualised care planning" (a process of multidomain assessment and management leading to tailored actions), were identified in 63 combinations. For living at home, compared with no intervention/placebo, evidence favoured multifactorial action from individualised care planning including medication review and regular follow-ups (routine review) (odds ratio 1.22, 95% confidence interval 0.93 to 1.59; moderate certainty); multifactorial action from individualised care planning including medication review without regular follow-ups (2.55, 0.61 to 10.60; low certainty); combined cognitive training, medication review, nutritional support, and exercise (1.93, 0.79 to 4.77; low certainty); and combined activities of daily living training, nutritional support, and exercise (1.79, 0.67 to 4.76; low certainty). Risk screening or the addition of education and self-management strategies to multifactorial action from individualised care planning and routine review with medication review may reduce odds of living at home. For instrumental activities of daily living, evidence favoured multifactorial action from individualised care planning and routine review with medication review (standardised mean difference 0.11, 95% confidence interval 0.00 to 0.21; moderate certainty). Two interventions may reduce instrumental activities of daily living: combined activities of daily living training, aids, and exercise; and combined activities of daily living training, aids, education, exercise, and multifactorial action from individualised care planning and routine review with medication review and self-management strategies. For personal activities of daily living, evidence favoured combined exercise, multifactorial action from individualised care planning, and routine review with medication review and self-management strategies (0.16, -0.51 to 0.82; low certainty). For homecare recipients, evidence favoured addition of multifactorial action from individualised care planning and routine review with medication review (0.60, 0.32 to 0.88; low certainty). High risk of bias and imprecise estimates meant that most evidence was low or very low certainty. Few studies contributed to each comparison, impeding evaluation of inconsistency and frailty., Conclusions: The intervention most likely to sustain independence is individualised care planning including medicines optimisation and regular follow-up reviews resulting in multifactorial action. Homecare recipients may particularly benefit from this intervention. Unexpectedly, some combinations may reduce independence. Further research is needed to investigate which combinations of interventions work best for different participants and contexts., Registration: PROSPERO CRD42019162195., Competing Interests: Competing interests: All authors have completed the ICMJE uniform disclosure form at https://www.icmje.org/disclosure-of-interest/ and declare: AC, TC, JE, AF, JGl, MJ, NL, and RRi had financial support from the NIHR Health Technology Assessment Programme for the submitted work; MB had financial support from the PhD Graduate Teaching Fund at the University of Liverpool for the submitted work; DA declares payment made to her employer, University of Leeds Library, from the Academic Unit for Ageing and Stroke Research, Bradford Institute for Health Research, for services that included contributions to the submitted work; TC, AC, and AF received research funding from NIHR Programme Grants for Applied Research; AC and AF also received research funding from NIHR HSDR Programme; AC also received research funding from Health Data Research UK, NIHR ARC Yorkshire and Humber, NIHR Leeds BRC, and Dunhill Medical Trust; AF also declares NIHR Senior Investigator award, National Institute for Health (USA) payment for panel membership in 2021 and 2022, and University of Leeds Governor representative on the Governors Board of Bradford Teaching Hospitals NHS Foundation Trust; MB and MP received NIHR pre-doctoral fellowship funding; RB is supported by matched funding awarded to the NIHR Applied Research Collaboration (West Midlands) and is a member of the data monitoring committee for the Predict and Prevent AECOPD Trial and College of Experts, Versus Arthritis; AC is a member of NIHR HTA Commissioned Research Funding Committee and Dunhill Medical Trust Research Grants Committee; RRi received personal payments for training courses provided in-house to universities (Leeds, Aberdeen, Exeter, LSHTM) and other organisations (Roche), has received personal payments from the BMJ and BMJ Medicine as their statistical editor, is a co-convenor of the Cochrane Prognosis Methods Group and on the Editorial Board of Diagnostic and Prognostic Research, and Research Synthesis Methods, but receives no income for these roles, receives personal payment for being the external examiner of the MSc Medical Statistics, London School of Hygiene and Tropical Medicine, was previously an external examiner for the MSc Medical Statistics at University of Leicester, has written two textbooks for which he receives royalties from sales (Prognosis Research in Healthcare, and Individual Participant Data Meta-analysis), is a lead editor on an upcoming book (Cochrane Handbook for Prognosis Reviews, Wiley, 2025), for which he will receive royalties from sales, has received consulting fees for a training course on IPD meta-analysis from Roche in 2018, the NIHR HTA grant paid for travel to Leeds for one meeting, and is a member of the NIHR Doctoral Research Fellowships grant panel, and a member of the MRC Better Methods Better Research grant panel—for the latter, he receives an attendance fee; MH declares NIHR Academic Clinical Fellowship; OT declares NIHR Academic Clinical Lectureship and Dunhill Medical Trust Doctoral Research Fellowship RTF107/0117; no other relationships or activities that could appear to have influenced the submitted work., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Despite growing efforts to improve access to vaccination, millions of children, especially in developing countries, have not received a single dose of diphtheria, tetanus, and pertussis (DTP) vaccine. Consequently, they are often called zero-dose children (ZDC). With limited health resources, prioritising communities for rapid and mass zero-dose catch-up vaccination in missed communities to avert epidemic outbreaks is complicated by unreliable denominators used to compute vaccination coverages. Incorporating other indicators of access and utilisation of vaccination services can help with identifying and ranking missed communities based on the likelihood of finding ZDC. We described the process of generating a scoring method to rank health areas in Cameroon based on their likelihood of containing ZDC., Methods: We used geospatial analysis to compute and aggregate health area characteristics, including hard-to-reach (HTR) areas (defined as areas of settlement above a one- (for urban areas) or 15-kilometre radius (for rural areas) beyond a vaccinating health facility), amount of area covered by slums and new area settlement, and percentage of children unvaccinated for DTP-1. We attributed a weight based on the ability to limit accessibility or utilisation of vaccination services to each characteristic and computed the score as a weighted average of health area characteristics. The health area score ranged from 0 to 1, with higher scores representing a higher likelihood of containing ZDC. We stratified the analysis by rural and urban health areas., Results: We observed substantial district and regional variations in health area scores, with hotspots health areas (administrative level 4) observed in the Far North (0.83), North (0.81), Adamawa (0.80), East (0.75), and South West (0.67) regions. The Adamawa region had the highest percentage of health areas with the highest score (78%), followed by the East (50%), West (48%), and North (46%) regions. For most regions (Far North, South, South West, Littoral, West, and North West), DTP-1 contributed the most to the score. However, HTR settlement areas within a health area contributed substantially to the overall score in the East, North, and Adamawa regions., Conclusions: We found substantial variations in health area scores with hotspots in the Far North, North, Adamawa, East, and South West regions. Although DTP-1 could be used as an indicator to identify health areas with ZDC for most communities, HTR settlement area was a valuable indicator in ranking priority health areas in the East, North, and Adamawa regions, further emphasising the need to consider other indicators before prioritisation., Competing Interests: Disclosure of interest: The authors completed the ICMJE Disclosure of Interest Form (available upon request from the corresponding author) and disclose no relevant interests., (Copyright © 2023 by the Journal of Global Health. All rights reserved.)

Cooking-related literacy and attitudes may play important roles in preventing and reducing diet-related chronic diseases and nutrition disparities. People living alone are an under-researched but growing population who face above average food insecurity rates. This study's objectives were to 1) test how cooking self-efficacy and attitude are stratified demographically among a sample of people living alone, focusing on variations across gender, age, and food security, and 2) examine how cooking self-efficacy and attitude are associated with two indicators of cooking behavior - cooking frequency and convenience orientation. We draw from a cross-sectional survey analysis of 493 adults living alone in Illinois, USA with validated measures for cooking self-efficacy, attitude, frequency, convenience orientation, and demographic characteristics. Hierarchical linear regression models were used to examine demographic factors explaining variation in self-efficacy and attitude, with attention to interactions between gender, food insecurity, and age. Poisson and OLS linear regression models were used to examine associations between self-efficacy and attitude and cooking frequency and convenience orientation. We find cooking-related self-efficacy and attitude showed strong but distinct associations with cooking frequency and convenience orientation. Overall, food insecure groups had lower self-efficacy than those who were food secure; however, food insecure women had higher self-efficacy than men in similar positions, apart from older-adult women who held particularly low efficacy. Cooking attitudes varied in small ways, notably with food insecure younger and older women possessing more negative cooking attitudes than middle-aged women. This research highlights the importance of understanding the cooking-related orientations of single-living people, while demonstrating that this group's ability to prevent and manage food insecurity is not uniform. These results can inform targeted interventions around food and nutrition insecurity, cooking attitudes, and self-efficacy among single-living populations., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Objectives: To report our experience using version 2 of the Cochrane risk-of-bias tool for randomized trials (RoB 2)., Study Design and Setting: Two reviewers independently applied RoB 2 to results of interest in a large systematic review of complex interventions and reached consensus. We recorded the time taken, and noted and discussed our difficulties using the tool, and the resolutions we adopted. We explored the time taken with regression analysis and summarized our experience of implementing the tool., Results: We assessed risk of bias in 860 results of interest in 113 studies. Staff resource averaged 358 minutes per study (SD 183). Number of results (β = 22) and reports (β = 14) per study and experience of the team (β = -6) significantly affected assessment time. To implement the tool consistently, we developed cut points for missingness and considerations of balance regarding missingness, assumed some concerns with intervention deviations unless otherwise prevented or investigated, some concerns with measurements from unblinded self-reporting participants, and judged low risk of selection for certain dichotomous outcomes despite the absence of an analysis plan., Conclusion: The RoB 2 tool and guidance are useful but resource-intensive and challenging to implement. Critical appraisal tools and reporting guidelines should detail risk of bias implementation. Improved guidance focusing on implementation could assist reviewers., Competing Interests: Declaration of Competing Interest Andrew Clegg declares funding through the NIHR HTA programme, NIHR Programme Grants for Applied Research, NIHR HS&DR programme, NIHR Applied Research Collaboration Yorkshire & Humber, and Health Data Research UK; Anne Forster declares NIHR Senior Investigator Award 2017-present, NIHR Programme Grant 10% of salary, NIHR HS&DR grant 8% of salary, HTA grant 5% of salary, National Institute for Health (USA) payment for panel membership 2021, 2022, participation in Programme Steering Committees for NIHR 202,339 Improving the lives of stroke survivors with data, and NIHR202020 Research Title Personalised Exercise-Rehabilitation FOR people with Multiple long-term conditions (multimorbidity)-The PERFORM trial, University of Leeds Governor representative on the Governors Board of Bradford Teaching Hospitals NHS Foundation Trust, member of HSDR Researcher-Led panel, member of NIHR Doctoral Fellowship Panel member of Policy Research Unit assessment panel. Other authors declare no potential conflicts of interest., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Immunohistochemical visualization of progesterone receptor (PR)-expressing cells in the brain is a powerful technique to investigate the role of progesterone in the neuroendocrine regulation of fertility. A major obstacle to the immunohistochemical visualization of progesterone-sensitive cells in the rodent brain has been the discontinuation of the commercially produced A0098 rabbit polyclonal PR antibody by DAKO. To address the unavailability of this widely used PR antibody, we optimized and evaluated 4 alternative commercial PR antibodies and found that each lacked the specificity and/or sensitivity to immunohistochemically label PR-expressing cells in paraformaldehyde-fixed female mouse brain sections. As a result, we developed and validated a new custom RC269 PR antibody, directed against the same 533-547 amino acid sequence of the human PR as the discontinued A0098 DAKO PR antibody. Immunohistochemical application of the RC269 PR antibody on paraformaldehyde-fixed mouse brain sections resulted in nuclear PR labeling that was highly distinguishable from background, specific to its antigen, highly regulated by estradiol, matched the known distribution of PR protein expression in the female mouse hypothalamus, and nearly identical to that of the discontinued A0098 DAKO PR antibody. In summary, the RC269 PR antibody is a specific and sensitive antibody to immunohistochemically visualize PR-expressing cells in the mouse brain., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society.)

Chemical communication by females remains poorly understood, with most attention focused on female advertisement of sexual receptivity to males or mother-offspring communication. However, in social species, scents are likely to be important for mediating competition and cooperation between females determining individual reproductive success. Here, we explore chemical signaling by female laboratory rats ( Rattus norvegicus ) to test i) whether females target their deployment of scent information differentially according to their sexual receptivity and the genetic identity of both female and male conspecifics signaling in the local environment and ii) whether females are attracted to gain the same or different information from female scents compared to males. Consistent with targeting of scent information to colony members of similar genetic background, female rats increased scent marking in response to scents from females of the same strain. Females also suppressed scent marking in response to male scent from a genetically foreign strain while sexually receptive. Proteomic analysis of female scent deposits revealed a complex protein profile, contributed from several sources but dominated by clitoral gland secretion. In particular, female scent marks contained a series of clitoral-derived hydrolases and proteolytically truncated major urinary proteins (MUPs). Manipulated blends of clitoral secretion and urine from estrus females were strongly attractive to both sexes, while voided urine alone stimulated no interest. Our study reveals that information about female receptive status is shared between females as well as with males, while clitoral secretions containing a complex set of truncated MUPs and other proteins play a key role in female communication.

Introduction: Polycystic ovary syndrome (PCOS) is the most common infertility disorder worldwide, typically characterised by high circulating androgen levels, oligo- or anovulation, and polycystic ovarian morphology. Sexual dysfunction, including decreased sexual desire and increased sexual dissatisfaction, is also reported by women with PCOS. The origins of these sexual difficulties remain largely unidentified. To investigate potential biological origins of sexual dysfunction in PCOS patients, we asked whether the well-characterized, prenatally androgenized (PNA) mouse model of PCOS exhibits modified sex behaviours and whether central brain circuits associated with female sex behaviour are differentially regulated. As a male equivalent of PCOS is reported in the brothers of women with PCOS, we also investigated the impact of maternal androgen excess on the sex behaviour of male siblings., Methods: Adult male and female offspring of dams exposed to dihydrotestosterone (PNAM/PNAF) or an oil vehicle (VEH) from gestational days 16 to 18 were tested for a suite of sex-specific behaviours., Results: PNAM showed a reduction in their mounting capabilities, however, most of PNAM where able to reach ejaculation by the end of the test similar to the VEH control males. In contrast, PNAF exhibited a significant impairment in the female-typical sexual behaviour, lordosis. Interestingly, while neuronal activation was largely similar between PNAF and VEH females, impaired lordosis behaviour in PNAF was unexpectedly associated with decreased neuronal activation in the dorsomedial hypothalamic nucleus (DMH)., Conclusion: Taken together, these data link prenatal androgen exposure that drives a PCOS-like phenotype with altered sexual behaviours in both sexes., Competing Interests: 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., (Copyright © 2023 Donaldson, Prescott, Ruddenklau, Campbell and Desroziers.)

Background: Although there have been recent efforts to improve access to Canadian national parks, many remain not fully accessible to people with disabilities. Winter conditions, in particular, present challenges that limit their participation in outdoor activities., Objective: This study aimed to develop a novel method to assess park access during winter, which will inform recommendations for national park standards to meet the needs of all park visitors (regardless of ability) during winter conditions., Methods: A larger participatory mixed methods research project exploring park access was adapted. A 3-phase approach has already been proposed to achieve the study objectives. In the first phase, a scoping review of the existing accessibility standards will be conducted. In the second phase, objective audits of trails and features in 6 parks, 3 in western Canada and 3 in eastern Canada, will be conducted, as well as mobile interviews with 24 various participants in each region regarding their experiences of and recommendations for improving the park's accessibility. In the final phase, a Delphi participatory consensus development process will be used, based on the data gathered in the first 2 phases, to prioritize recommendations for standards. This paper will focus on the second phase of the study, specifically on whether the in-person winter mobile interviews (ie, walking and wheeling interviews) with people who have a wide range of disabilities while visiting 3 parks in 2 provinces were modified. Changes were made to accommodate the extreme winter weather conditions in Quebec while using safe and informative data collection methods., Results: In Quebec, one park, where winter conditions are safer, has been assessed in person (n=4). Web-based interviews were used to facilitate the assessment of other winter and summer conditions in two other parks (n=8). Winter and web-based interviews were completed in April 2022. Data are currently being collected and analyzed, and results will be completed by December 2022., Conclusions: We expect that adapting the protocol to gather further information on winter conditions and access to parks will provide high-quality and rich data to better inform park access standards. This participatory mixed methods research will inform the development of park standards that consider the accessibility needs of all people., International Registered Report Identifier (irrid): DERR1-10.2196/38715., (©Mike Prescott, Stéphanie Gamache, W Ben Mortenson, Krista L Best, Marie Grandisson, Mir Abolfazl Mostafavi, Delphine Labbé, Ernesto Morales, Atiya Mahmood, Jaimie Borisoff, Bonita Sawatzky, William C Miller, Laura Yvonne Bulk, Julie M Robillard, François Routhier. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 07.10.2022.)

Objective: We conducted an intersectional analysis to explore how demographic characteristics and mobility device use were associated with psychological distress (depression and anxiety) and confidence negotiating physical and social environments., Design: Cross-sectional data were collected using the Hospital Anxiety and Depression Scale, modified Wheelchair Use Confidence Scale, and self-reported functional independence scale., Participants: The sample included 105 participants. Primary mobility devices used included mobility scooters (27%), power wheelchairs (26%), manual wheelchairs (25%), walkers (11%), and cane or crutch (12%). The mean age of participants was 58 yrs, 53% were female, and 52% lived alone and were functionally independent with the use of assistive technology., Results: We were able to explain between 39% and 65% of the variance (adjusted R2) in the dependent variables with parsimonious regression models. Age was an independent predictor of all outcomes. Women were less confident negotiating the physical environment, and walker use was associated with depression and lower confidence negotiating physical environments, but increased confidence negotiating social environments., Conclusions: Age is associated with psychosocial outcomes for assistive device users, and those who use walkers may experience increased challenges with depression and negotiating the physical environment. Pending confirmatory research, the findings may have important implications for targeted interventions related to device provision., Competing Interests: Financial disclosure statements have been obtained, and no conflicts of interest have been reported by the authors or by any individuals in control of the content of this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Lower limb exoskeletons are a recent intervention promoted to improve gait disorders. Available research has focused on clinical outcomes; however, little is known about therapists' experiences using the device in practice., Purpose: We explored the implementation of an exoskeleton at a tertiary rehabilitation center., Method: In this longitudinal qualitative study we conducted semi-structured interviews with 10 therapists. One group of therapists was formally trained using the device, whereas the other group only had clinical exposure to the device. The interviews were transcribed, coded, and analyzed for themes., Results: Three main themes emerged: (1) A steep learning curve described challenges in learning to use the exoskeleton. (2) One step at a time illustrated the complex process of incorporating the exoskeleton in daily work. (3) Not a magic bullet revealed the tensions using this emergent technology in practice., Conclusion: The exoskeleton represents one of the most complicated and labor-intensive interventions provided by therapists. Implementation requires substantial resources, raising questions regarding its efficacy and cost-effectiveness relative to other approaches. Until more evidence becomes available around the use and effectiveness of this rapidly evolving technology, therapists must contend with a high degree of uncertainty.IMPLICATIONS FOR REHABILITATIONUsing a lower limb exoskeleton may reduce physical demands on therapists in high-intensity rehabilitation programs that involve repetitive, effortful movements.However, a number of potential barriers to implementing the exoskeleton into practice need to be taken into consideration, including calibration time, intensive training required to become qualified to administer the intervention, the cost of the device, and comfort and safety of the device affecting user acceptance and uptake.Therapists also need to manage patient expectations related to outcomes related to use of exoskeletons.

Polycystic ovary syndrome (PCOS) is associated with androgen excess and, frequently, hyperactive pulsatile luteinizing hormone (LH) secretion. Although the origins of PCOS are unclear, evidence from pre-clinical models implicates androgen signalling in the brain in the development of PCOS pathophysiology. Chronic exposure of female mice to dihydrotestosterone (DHT) from 3 weeks of age drives both reproductive and metabolic impairments that are ameliorated by selective androgen receptor (AR) loss from the brain. This suggests centrally driven mechanisms in hyperandrogen-mediated PCOS-like pathophysiology that remain to be defined. Acute prenatal DHT exposure can also model the hyperandrogenism of PCOS, and this is accompanied by increased LH pulse frequency and increased GABAergic innervation of gonadotrophin-releasing hormone (GnRH) neurons. We aimed to determine the impact of chronic exposure of female mice to DHT, which models the hyperandrogenism of PCOS, on pulsatile LH secretion and putative GABAergic input to GnRH neurons. To do this, GnRH-green fluorescent protein (GFP) female mice received either DHT or blank capsules for 90 days from postnatal day 21 (n = 6 or 7 per group). Serial tail-tip blood sampling was used to measure LH dynamics and perfusion-fixed brains were collected and immunolabelled for vesicular GABA transporter (VGAT) to assess putative GABAergic terminals associated with GFP-labelled GnRH neurons. As expected, chronic DHT resulted in acyclicity and significantly increased body weight. However, no differences in LH pulse frequency or the density of VGAT appositions to GnRH neurons were identified between ovary-intact DHT-treated females and controls. Chronic DHT exposure significantly increased the number of AR expressing cells in the hypothalamus, whereas oestrogen receptor α-expressing neuron number was unchanged. Therefore, although chronic DHT exposure from 3 weeks of age increases AR expressing neurons in the brain, the GnRH neuronal network changes and hyperactive LH secretion associated with prenatal androgen excess are not evident. These findings suggest that unique central mechanisms are involved in the reproductive impairments driven by exposure to androgen excess at different developmental stages., (© 2022 The Authors. Journal of Neuroendocrinology published by John Wiley & Sons Ltd on behalf of British Society for Neuroendocrinology.)

Background: Canada's national parks are world-renowned. However, despite recent attempts to improve access, many are not accessible to people with disabilities. With the advent of provincial and federal legislation, standards are being developed to assist with the design and management of parks., Objective: The overarching objective of this study is to inform accessibility standards for federal parks that meet the needs of all park visitors, regardless of ability. The specific objectives of this study are to identify park accessibility standards that exist internationally, identify the accessibility challenges that people with disabilities face in park environments, and prioritize and recommend accessibility standards for national parks., Methods: A 3-phase approach will be used to achieve the study objectives. In the first phase, a scoping review of the existing accessibility standards will be conducted. The second phase will include objective audits of trails and features in 6 parks, 3 in western Canada and 3 in eastern Canada, as well as mobile interviews with 24 diverse participants in each region regarding their experiences of and recommendations for improving the park's accessibility. In the final phase, a Delphi participatory consensus development process will be used, based on the data gathered in the first 2 phases, to prioritize recommendations for standards., Results: We expect to find gaps in existing standards that do not account for the diverse range of accessibility requirements that people with disabilities have for visiting parks. We also expect to find that existing standards, on their own, may not be enough to ensure equitable access to all the experiences and amenities that parks have to offer. Development of subsequent guidelines and best practices may be necessary to address complex scenarios for which standards may not be the best approach to ensuring accessibility., Conclusions: The participatory and mixed methods approaches used in this study will provide rich insights for developing accessible park standards that consider the diverse needs of people with disabilities. The findings will also support the development or enhancement of park standards at all levels of government., International Registered Report Identifier (irrid): DERR1-10.2196/33611., (©Mike Prescott, François Routhier, Delphine Labbé, Marie Grandisson, Atiya Mahmood, Ernesto Morales, Krista L Best, Mir Abolfazl Mostafavi, Jaimie Borisoff, Stéphanie Gamache, Bonita Sawatzky, William C Miller, Laura Yvonne Bulk, Julie M Robillard, Hailey-Thomas Jenkins, Kishore Seetharaman, W Ben Mortenson. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 31.03.2022.)

Prenatal exposure to excess androgens is associated with the development of polycystic ovary syndrome (PCOS). In prenatally androgenised (PNA) mice, a model of PCOS, progesterone receptor (PR) protein expression is reduced in arcuate nucleus (ARC) GABA neurons. This suggests a mechanism for PCOS-related impaired steroid hormone feedback and implicates androgen excess with respect to inducing transcriptional repression of the PR-encoding gene Pgr in the ARC. However, the androgen sensitivity of ARC neurons and the relative gene expression of PRs over development and following prenatal androgen exposure remain unknown. Here, we used a quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) of microdissected ARC to determine the relative androgen receptor (Ar) and progesterone receptor (Pgr) gene expression in PNA and control mice at five developmental timepoints. In a two-way analysis of variance, none of the genes examined showed expression changes with a statistically significant interaction between treatment and age, although PgrA showed a borderline interaction. For all genes, there was a statistically significant main effect of age on expression levels, reflecting a general increase in expression with increasing age, regardless of treatment. For PgrB and Ar, there was a statistically significant main effect of treatment, indicating a change in expression following PNA (increased for PgrB and decreased for Ar), regardless of age. For PgrA, there was a borderline main effect of treatment, suggesting a possible change in expression following PNA, regardless of age. PgrAB gene expression changes showed no significant main effect of treatment. We additionally examined androgen and progesterone responsiveness specifically in P60 ARC GABA neurons using RNAScope® (Advanced Cell Diagnostics, Inc.) in situ hybridization. This analysis revealed that Pgr and Ar were expressed in the majority of ARC GABA neurons in normal adult females. However, our RNAScope® analysis did not show significant changes in Pgr or Ar expression within ARC GABA neurons following PNA. Lastly, because GABA drive to gonadotropin-releasing hormone neurons is increased in PNA, we hypothesised that PNA mice would show increased expression of glutamic acid decarboxylase (GAD), the rate-limiting enzyme in GABA production. However, the RT-qPCR showed that the expression of GAD encoding genes (Gad1 and Gad2) was unchanged in adult PNA mice compared to controls. Our findings indicate that PNA treatment can impact Pgr and Ar mRNA expression in adulthood. This may reflect altered circulating steroid hormones in PNA mice or PNA-induced epigenetic changes in the regulation of Pgr and Ar gene expression in ARC neurons., (© 2021 British Society for Neuroendocrinology.)

Background: The majority of older people (> 65 years) in hospital have frailty and are at increased risk of readmission or death following discharge home. In the UK, following acute hospitalisation, around one third of older people with frailty are referred on for rehabilitation, termed 'intermediate care' services. Although this rehabilitation can reduce early readmission to hospital (< 30 days), recipients often do not feel ready to leave the service on discharge, suggesting possible incomplete recovery. Limited evidence suggests extended rehabilitation is of benefit in several conditions and there is preliminary evidence that progressive physical exercise can improve mobility and function for older people with frailty, and slow progression to disability. Our aim is to evaluate the effectiveness of the Home-based Older People's Exercise (HOPE) programme as extended rehabilitation for older people with frailty discharged home from hospital or intermediate care services after acute illness or injury., Methods: A multi-centre individually randomised controlled trial, to evaluate the clinical and cost-effectiveness of the HOPE programme. This individualised, graded and progressive 24-week exercise programme is delivered by NHS physiotherapy teams to people aged 65 and older with frailty, identified using the Clinical Frailty Scale, following discharge from acute hospitalisation and linked intermediate care rehabilitation pathways. The primary outcome is physical health-related quality of life, measured using the physical component summary score of the modified Short Form 36- item health questionnaire (SF36) at 12 months. Secondary outcomes include self-reported physical and mental health, functional independence, death, hospitalisations, care home admissions. Plans include health economic analyses and an embedded process evaluation., Discussion: This trial seeks to determine if extended rehabilitation, via the HOPE programme, can improve physical health-related quality of life for older people with frailty following acute hospitalisation. Results will improve awareness of the rehabilitation needs of older people with frailty, and provide evidence on the clinical and cost-effectiveness of the targeted exercise intervention. There is potential for considerable benefit for health and social care services through widespread implementation of trial findings if clinical and cost-effectiveness is demonstrated., Trial Registration: ISRCTN 13927531 . Registered on April 19, 2017., (© 2021. The Author(s).)

Background: Access to mental health services continues to be a systemic problem in the United States and around the world owing to a variety of barriers including the limited availability of skilled providers and lack of mental health literacy among patients. Individuals seeking mental health treatment may not be aware of the multiple modalities of digital mental health care available to address their problems (eg, self-guided and group modalities, or one-to-one care with a provider). In fact, one-to-one, in-person treatment is the dominant care model with a masters- or doctoral-level trained mental health provider, and it may or may not be the appropriate or preferred level of care for an individual. Technology-enabled mental health platforms may be one way to improve access to mental health care by offering stepped care, but more research is needed to understand the care modality preferences of digital mental health care seekers because additional modalities become increasingly validated as effective treatment options., Objective: The purpose of this study was to describe and evaluate the predictors of care modality preferences among individuals enrolled in a technology-enabled stepped mental health care platform., Methods: This exploratory, cross-sectional study used employee data from the 2021 Modern Health database, an employer-sponsored mental health benefit that uses a technology-enabled platform to optimize digital mental health care delivery. Chi-square tests and one-way analysis of variance (ANOVA) were conducted to evaluate associations among the categorical and continuous factors of interest and the preferred care modality. Bivariate logistic regression models were constructed to estimate the odds ratios (ORs) of preferring a one-on-one versus self-guided group, or no preference for digital mental health care modalities., Results: Data were analyzed for 3661 employees. The most common modality preference was one-on-one care (1613/3661, 44.06%). Approximately one-fourth of the digital mental health care seekers (881/3661, 24.06%) expressed a preference for pursuing self-guided care, and others (294/3661, 8.03%) expressed a preference for group care. The ORs indicated that individuals aged 45 years and above were significantly more likely to express a preference for self-guided care compared to individuals aged between 18 and 24 years (OR 2.47, 95% CI 1.70-3.59; P<.001). Individuals screening positive for anxiety (OR 0.73, 95% CI 0.62-0.86; P<.001) or depression (OR 0.79, 95% CI 0.66-0.95; P=.02) were more likely to prefer one-on-one care., Conclusions: Our findings elucidated that care modality preferences vary and are related to clinical severity factors and demographic variables among individuals seeking digital mental health care., (©Elissa Kozlov, Meghan McDarby, Maximo Prescott, Myra Altman. Originally published in JMIR Formative Research (https://formative.jmir.org), 15.09.2021.)

Although polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility worldwide, the aetiology of the disorder remains poorly defined. Animal-based evidence highlights the brain as a prime suspect in both the development and maintenance of PCOS. Prenatally androgenised (PNA) models of PCOS exhibit excessive GABAergic wiring associated with PCOS-like reproductive deficits in adulthood, with aberrant brain wiring detected as early as postnatal day (P) 25, prior to disease onset, in the PNA mouse. The mechanisms underlying this aberrant brain wiring remain unknown. Microglia, the immune cells of the brain, are regulators of neuronal wiring across development, mediating both the formation and removal of neuronal inputs. Here, we tested the hypothesis that microglia play a role in the excessive GABAergic wiring that leads to PCOS-like features in the PNA brain. Using specific immunolabelling, microglia number and morphology associated with activation states were analysed in PNA and vehicle-treated controls across developmental timepoints, including embryonic day 17.5, P0, P25 and P60 (n = 7-14 per group), and in two regions of the hypothalamus implicated in fertility regulation. At P0, fewer amoeboid microglia were observed in the rostral preoptic area (rPOA) of PNA mice. However, the greatest changes were observed at P25, with PNA mice exhibiting fewer total microglia, and specifically fewer "sculpting" microglia, in the rPOA. Based on these findings, we assessed microglia-mediated refinement of GABAergic synaptic terminals at two developmental stages of peak synaptic refinement: P7 and P15 (n = 7 per group). PNA mice showed a reduction in the uptake of GABAergic synaptic material at P15. These findings reveal time-specific changes in the microglia population and refinement of GABAergic inputs in a mouse model of PCOS driven by prenatal androgen excess and suggest a role for microglia in shaping the atypical brain wiring associated with the development of PCOS features., (© 2021 British Society for Neuroendocrinology.)

Chronic stress exerts multiple negative effects on the physiology and health of an individual. In the present study, we examined hypothalamic, pituitary and endocrine responses to 14 days of chronic variable stress (CVS) in male and female C57BL/6J mice. In both sexes, CVS induced a significant decrease in body weight and enhanced the acute corticosterone stress response, which was accompanied by a reduction in thymus weight only in females. However, single-point blood measurements of basal prolactin, thyroid-stimulating hormone, luteinising hormone, growth hormone and corticosterone levels taken at the end of the CVS were not different from those of controls. Similarly, pituitary mRNA expression of Fshb, Lhb, Prl and Gh was unchanged by CVS, although Pomc and Tsh were significantly elevated. Within the adrenal medulla, mRNA for Th, Vip and Gal were elevated following CVS. Avp transcript levels within the paraventricular nucleus of the hypothalamus were increased by CVS; however, levels of Gnrh1, Crh, Oxt, Sst, Trh, Ghrh, Th and Kiss1 remained unchanged. Oestrous cycles were lengthened slightly by CVS and ovarian histology revealed a reduction in the number of preovulatory follicles and corpora lutea. Taken together, these observations indicate that 14 days of CVS induces an up-regulation of the neuroendocrine stress axis and creates a mild disruption of female reproductive function. However, the lack of changes in other neuroendocrine axes controlling anterior and posterior pituitary secretion suggest that most neuroendocrine axes are relatively resilient to CVS., (© 2021 British Society for Neuroendocrinology.)

The aim of this study was to understand the mobility experiences of mobility device users regarding the environmental and social barriers and facilitators in their community and to discuss the benefits and challenges of using photovoice, as a participatory methodology, to increase social participation for people with disabilities. Photovoice was used with mobility device users (n = 70) in two different locations in Canada. The participants took pictures for two weeks and then participated in individual interviews and focus group sessions to discuss their photographs. The participants took over 1,000 pictures that were grouped into five themes around social participation and accessibility. They chose the most illustrative pictures to share in an exhibit to create a dialogue with different stakeholders. Using photovoice offered many benefits such as allowing the participants to be equal partners of the research and made their voices heard, but also presented disability and study-related challenges., (© 2020 Wiley Periodicals, Inc.)

Professional development has been identified as a critical component for school nutrition professionals (SNPs) to successfully implement school meal standards in the United States. However, training needs may vary based on different factors. This study examined (1) the topics of highest priority for SNPs; (2) preferred learning methods; (3) where and when trainings should be conducted; and (4) whether responses differ according to important factors including position type, school locale (urban vs. rural), or job experience. Participants completed surveys that included questions on demographics and preferences for learning methods and training topics ( n = 492). Descriptive statistics characterized survey responses. Chi square tests assessed differences in learning method and training topic preferences by participant role, locale, and job experience; Cramer's V assessed the strength of association for each chi square result. Qualitative responses to open-ended questions were analyzed using an inductive thematic analysis method. Nearly all training topic preferences were significantly different ( p < 0.001 using Bonferroni method) when stratified by role. Significant differences were also observed for school locale and years of experience, but to a lesser degree. There was less variation in learning method preferences across staff role. Qualitative results ( n = 93) identified three key themes related to training needs: role-specific trainings, innovative learning methods, and geographic access. The combination of quantitative and qualitative analysis indicate that professional development for SNPs should mostly be conducted in-person, be easily accessible, and include hands-on activities. Further, training should be tailored by job role and address situational barriers unique to the geographic area.

The mechanism and function of autophagy as a highly-conserved bulk degradation pathway are well studied, but the physiological role of autophagy remains poorly understood. We show that autophagy is involved in the adaptation of Saccharomyces cerevisiae to respiratory growth through its recycling of serine. On respiratory media, growth onset, mitochondrial initiator tRNA modification and mitochondrial protein expression are delayed in autophagy defective cells, suggesting that mitochondrial one-carbon metabolism is perturbed in these cells. The supplementation of serine, which is a key one-carbon metabolite, is able to restore mitochondrial protein expression and alleviate delayed respiratory growth. These results indicate that autophagy-derived serine feeds into mitochondrial one-carbon metabolism, supporting the initiation of mitochondrial protein synthesis and allowing rapid adaptation to respiratory growth.

Polycystic ovary syndrome (PCOS), the most common form of anovulatory infertility, is associated with altered signaling within the hormone-sensitive neuronal network that regulates gonadotropin-releasing hormone (GnRH) neurons, leading to a pathological increase in GnRH secretion. Circuit remodeling is evident between GABAergic neurons in the arcuate nucleus (ARN) and GnRH neurons in a murine model of PCOS. One-third of ARN GABA neurons co-express neuropeptide Y (NPY), which has a known yet complex role in regulating GnRH neurons and reproductive function. Here, we investigated whether the NPY-expressing subpopulation (NPY ARN ) of ARN GABA neurons (GABA ARN ) is also affected in prenatally androgenized (PNA) PCOS-like NPY ARN reporter mice [Agouti-related protein (AgRP)-Cre;τGFP]. PCOS-like mice and controls were generated by exposure to di-hydrotestosterone or vehicle (VEH) in late gestation. τGFP-expressing NPY ARN neuron fiber appositions with GnRH neurons and gonadal steroid hormone receptor expression in τGFP-expressing NPY ARN neurons were assessed using confocal microscopy. Although GnRH neurons received abundant close contacts from τGFP-expressing NPY ARN neuron fibers, the number and density of putative inputs was not affected by prenatal androgen excess. NPY ARN neurons did not co-express progesterone receptor or estrogen receptor α in either PNA or VEH mice. However, the proportion of NPY ARN neurons co-expressing the androgen receptor was significantly elevated in PNA mice. Therefore, NPY ARN neurons are not remodeled by prenatal androgen excess like the wider GABA ARN population, indicating GABA-to-GnRH neuron circuit remodeling occurs in a presently unidentified non-NPY/AgRP population of GABA ARN neurons. NPY ARN neurons do, however, show independent changes in the form of elevated androgen sensitivity., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society.)

The ability to move about the community safely and easily plays a key role in the lives of people who use mobility devices. This study explored how personal characteristics, mobility device use, transportation, and environmental factors affected the size of their activity spaces (i.e., daily path area). Seventy-eight people who used manual wheelchairs, power wheelchairs, scooters, walkers, canes, and/or crutches tracked their movements in the community using a geographical positioning system device over a 7-day period. Participating in productive activities, walkability, the interaction between access to a vehicle and walkability, seasonality, and location helped to explain 43% of the variability in daily path area. The type of mobility device used and years using a device were associated with activity space size in bivariate analysis but not significantly significant in the regression model. The findings underscore the complex challenges that people who use mobility devices face moving about the community. Implications for researchers and transportation planners are discussed., (Copyright © 2020. Published by Elsevier Ltd.)

Maintenance of connective tissue integrity is fundamental to sustain function, requiring protein turnover to repair damaged tissue. However, connective tissue proteome dynamics remain largely undefined, as do differences in turnover rates of individual proteins in the collagen and glycoprotein phases of connective tissue extracellular matrix (ECM). Here, we investigate proteome dynamics in the collagen and glycoprotein phases of connective tissues by exploiting the spatially distinct fascicular (collagen-rich) and interfascicular (glycoprotein-rich) ECM phases of tendon. Using isotope labelling, mass spectrometry and bioinformatics, we calculate turnover rates of individual proteins within rat Achilles tendon and its ECM phases. Our results demonstrate complex proteome dynamics in tendon, with ~1000 fold differences in protein turnover rates, and overall faster protein turnover within the glycoprotein-rich interfascicular matrix compared to the collagen-rich fascicular matrix. These data provide insights into the complexity of proteome dynamics in tendon, likely required to maintain tissue homeostasis., Competing Interests: HC, DS, DW, MP, AP, JD, PC, PP, CT No competing interests declared, (© 2020, Choi et al.)

Background: Ecological momentary assessment (EMA) is a promising data collection tool for mobile health interventions targeting episodic health behaviors. For substance-using men who have sex with men (SUMSM), EMA is becoming more widely utilized in efforts to characterize substance use and sexual risk factors for HIV transmission. However, recent literature demonstrates emerging concerns over compliance and lower EMA engagement and data concordance among racial and ethnic minority SUMSM., Objective: This study aimed to provide a qualitative evaluation of the barriers and facilitators of EMA as a data collection tool among racial and ethnic minority SUMSM., Methods: Between October and November 2017, 45 racial and ethnic minority SUMSM were recruited from a list of prior research participants at the San Francisco Department of Public Health to participate in daily EMA surveys on their substance use and sexual health behaviors for 1 week, followed by in-person focus groups (FGs). A total of 4 FGs explored the participants' experiences with the surveys, issues regarding privacy and confidentiality, and suggestions for improvement. Qualitative analysis was performed using content analysis. Descriptive statistics and Fisher exact tests were used to assess the associations between demographics or substance use behaviors and EMA completion., Results: Overall, 93.9% (295/314) of all delivered surveys were initiated, and of those, 98.0% (289/295) were completed. Neither participant demographics, including race (P=.65) or age (P=.43), nor substance use behaviors, including the frequency of alcohol (P=.40) or methamphetamine (P=.91) use or any cocaine (P=.28), crack (P=.99), or polysubstance use (P=.24), were found to be associated with survey completion. Overall, participants were receptive to the text message-based EMA surveys. Facilitators included survey timing, user-friendly survey design, survey-stimulated self-reflection, coding of sensitive phrases, and other privacy benefits of a mobile survey. Barriers included an inability to correct texting errors and participants' perception of judgment or stigmatization related to questions about condomless sex. To improve EMA compliance and uptake, participants suggested adding response confirmations, clarifying survey language, and continuing to diversify the study audience., Conclusions: EMA appears to be feasible and acceptable among this sample of racial and ethnic minority SUMSM. Close attention to EMA study design and the development of nonjudgmental, contextualized questions regarding stigmatized health behaviors may be critical to further improve EMA compliance., (©Elaine Hsiang, Claudine Offer, Maximo Prescott, Amy Rodriguez, Emily Behar, Tim Matheson, Diane Santa Maria, Glenn-Milo Santos. Originally published in JMIR mHealth and uHealth (http://mhealth.jmir.org), 29.04.2020.)

Two-component signal transduction systems (TCSTS) are abundant among prokaryotes and regulate important functions, including drug resistance and virulence. The Gram-negative bacterium Burkholderia pseudomallei, which causes the severe infectious disease melioidosis, encodes 136 putative TCSTS components. In silico analyses of these TCSTS indicated that the predicted BbeR-BbeS system (BPSL1036-BPSL1037) displayed significant amino acid sequence similarity to the Shigella flexneri virulence-associated OmpR-EnvZ osmoregulator. To assess the function of the B. pseudomallei BbeR-BbeS system, we constructed by allelic exchange a ΔbbeRS double mutant strain lacking both genes, and single ΔbbeR and ΔbbeS mutants. All three mutant strains caused disease in the BALB/c acute melioidosis model at the same rate as the wild-type strain, displayed unchanged swarming motility on semi-solid medium, and were unaffected for viability on high-osmolarity media. However, when cultured at 37 °C for at least 14 days, ΔbbeS and ΔbbeR colonies developed a distinct, hypermucoid morphology absent in similarly-cultured wild-type colonies. At both 30 °C and 37 °C, these hypermucoid strains produced wild-type levels of type I capsule but released increased quantities of extracellular DNA (eDNA). Upon static growth in liquid medium, all B. pseudomallei strains produced pellicle biofilms that contained DNA in close association with bacterial cells; however, the ΔbbeS and ΔbbeR strains produced increased biofilms with altered microscopic architecture compared to the wild-type. Unusually, while the ΔbbeS and ΔbbeR single-deletion mutants displayed clear phenotypes, the ΔbbeRS double-deletion mutant was indistinguishable from the wild-type strain. We propose that BbeR-BbeS indirectly affects eDNA secretion and biofilm formation through cross-talk with one or more other TCSTS., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Introduction: The central regulation of fertility is carefully coordinated with energy homeostasis, and infertility is frequently the outcome of energy imbalance. Neurons in the hypothalamus expressing neuropeptide Y and agouti-related peptide (NPY/AgRP neurons) are strongly implicated in linking metabolic cues with fertility regulation., Objective: We aimed here to determine the impact of selectively activating NPY/AgRP neurons, critical regulators of metabolism, on the activity of luteinizing hormone (LH) pulse generation., Methods: We employed a suite of in vivo optogenetic and chemogenetic approaches with serial measurements of LH to determine the impact of selectively activating NPY/AgRP neurons on dynamic LH secretion. In addition, electrophysiological studies in ex vivo brain slices were employed to ascertain the functional impact of activating NPY/AgRP neurons on gonadotropin-releasing hormone (GnRH) neurons., Results: Selective activation of NPY/AgRP neurons significantly decreased post-castration LH secretion. This was observed in males and females, as well as in prenatally androgenized females that recapitulate the persistently elevated LH pulse frequency characteristic of polycystic ovary syndrome (PCOS). Reduced LH pulse frequency was also observed when optogenetic stimulation was restricted to NPY/AgRP fiber projections surrounding GnRH neuron cell bodies in the rostral preoptic area. However, electrophysiological studies in ex vivo brain slices indicated these effects were likely to be indirect., Conclusions: These data demonstrate the ability of NPY/AgRP neuronal signaling to modulate and, specifically, reduce GnRH/LH pulse generation. The findings suggest a mechanism by which increased activity of this hunger circuit, in response to negative energy balance, mediates impaired fertility in otherwise reproductively fit states, and highlight a potential mechanism to slow LH pulsatility in female infertility disorders, such as PCOS, that are associated with hyperactive LH secretion., (© 2019 S. Karger AG, Basel.)

Prenatal androgen excess is suspected to contribute to the development of polycystic ovary syndrome (PCOS) in women. Evidence from preclinical female animal models links maternal androgen excess with the development of PCOS-like features and associated alterations in the neuronal network regulating the reproductive axis. There is some evidence suggesting that maternal androgen excess leads to similar reproductive axis disruptions in men, despite the critical role that androgens play in normal sexual differentiation. Here, the specific impact of maternal androgen excess on the male hypothalamic-pituitary-gonadal axis was investigated using a prenatal androgenization protocol in mice shown to model PCOS-like features in females. Reproductive phenotyping of prenatally androgenised male (PNAM) mice revealed no discernible impact of maternal androgen excess at any level of the reproductive axis. Luteinising hormone pulse characteristics, daily sperm production, plasma testosterone and anti-Müllerian hormone levels were not different in the male offspring of dams administered dihydrotestosterone (DHT) during late gestation compared to controls. Androgen receptor expression was quantified through the hypothalamus and identified as unchanged. Confocal imaging of gonadotropin-releasing hormone (GnRH) neurons revealed that in contrast with prenatally androgenised female mice, PNAM mice exhibited no differences in the density of putative GABAergic innervation compared to controls. These data indicate that a maternal androgen environment capable of inducing reproductive dysfunction in female offspring has no evident impact on the reproductive axis of male littermates in adulthood.

Filarial nematodes possess glutathione transferases (GSTs), ubiquitous enzymes with the potential to detoxify xenobiotic and endogenous substrates, and modulate the host immune system, which may aid worm infection establishment, maintenance and survival in the host. Here we have identified and characterized a σ class glycosylated GST (OoGST1), from the cattle-infective filarial nematode Onchocerca ochengi, which is homologous (99% amino acid identity) with an immunodominant GST and potential vaccine candidate from the human parasite, O. volvulus, (OvGST1b). Onchocerca ochengi native GSTs were purified using a two-step affinity chromatography approach, resolved by 2D and 1D SDS-PAGE and subjected to enzymic deglycosylation revealing the existence of at least four glycoforms. A combination of lectin-blotting and mass spectrometry (MS) analyses of the released N-glycans indicated that OoGST1 contained mainly oligomannose Man5GlcNAc2 structure, but also hybrid- and larger oligommanose-type glycans in a lower proportion. Furthermore, purified OoGST1 showed prostaglandin synthase activity as confirmed by Liquid Chromatography (LC)/MS following a coupled-enzyme assay. This is only the second reported and characterized glycosylated GST and our study highlights its potential role in host-parasite interactions and use in the study of human onchocerciasis.

The aim of this study was to examine the histopathological and immunohistochemical changes caused by natural and experimentally-induced Ornithobacterium rhinotracheale infection in the respiratory system of chickens. To this end, three different studies were carried out. The first was a retrospective study of 82 field cases with respiratory disorders compatible with O. rhinotracheale infection. The bacterium was immunohistochemically detected in the lungs in 48 of 82 field cases, and 50 β-haemolytic (BH) and non-haemolytic (NH) strains were isolated. In the second study, an experimental model of the disease was created using 3-week-old broiler chickens, to identify possible differences of pathogenicity between the BH and NH isolates by the intravenous (IV) and intratracheal (IT) inoculation routes (IR). The group challenged with the NH isolate showed more severe lung lesions than the group challenged with the BH isolate at 7-days postinoculation (p.i.). The 14-day p.i. groups challenged with either the BH or NH isolates by the IT or IV IR had a higher histologic grade of pulmonary and hepatic lesions and a higher total histologic grade of lesions suggesting more severe pathology with longer time of exposure. A direct association between the inoculation routes and the organs affected was shown. Finally, a slaughterhouse study was carried out from October 2014 to May 2015, in which the histologic grade of lesions was significantly higher in immunohistochemically positive for O. rhinotracheale lungs of dead-on-arrival chickens.

Major urinary proteins (MUP) are the major component of the urinary protein fraction in house mice (Mus spp.) and rats (Rattus spp.). The structure, polymorphism and functions of these lipocalins have been well described in the western European house mouse (Mus musculus domesticus), clarifying their role in semiochemical communication. The complexity of these roles in the mouse raises the question of similar functions in other rodents, including the Norway rat, Rattus norvegicus. Norway rats express MUPs in urine but information about specific MUP isoform sequences and functions is limited. In this study, we present a detailed molecular characterization of the MUP proteoforms expressed in the urine of two laboratory strains, Wistar Han and Brown Norway, and wild caught animals, using a combination of manual gene annotation, intact protein mass spectrometry and bottom-up mass spectrometry-based proteomic approaches. Cluster analysis shows the existence of only 10 predicted mup genes. Further, detailed sequencing of the urinary MUP isoforms reveals a less complex pattern of primary sequence polymorphism in the rat than the mouse. However, unlike the mouse, rat MUPs exhibit added complexity in the form of post-translational modifications, including the phosphorylation of Ser4 in some isoforms, and exoproteolytic trimming of specific isoforms. Our results raise the possibility that urinary MUPs may have different roles in rat chemical communication than those they play in the house mouse. Shotgun proteomics data are available via ProteomExchange with identifier PXD013986.

The purpose of this study was to determine if the associations between eating competence (EC) and eating behaviors that were found in a USA sample of predominantly Hispanic parents of 4th grade youth could be replicated in a USA sample of predominantly non-Hispanic white parents of 4th graders. Baseline responses from parents ( n = 424; 94% white) of youth participating in a year-long educational intervention were collected using an online survey. Validated measures included the Satter Eating Competence Inventory (ecSI 2.0 TM ), in-home fruit/vegetable (FV) availability, healthful eating behavior modeling, and FV self-efficacy/outcome expectancies (SE/OE). Data were analyzed with general linear modeling and cluster analyses. The findings replicated those from the primarily Hispanic sample. Of the 408 completing all ecSI 2.0 TM items, 86% were female, 65% had a 4-year degree or higher, and 53% were EC (ecSI 2.0 TM score ≥ 32). Compared with non-EC parents, EC modeled more healthful eating, higher FV SE/OE, and more in-home FV availability. Behaviors clustered into those striving toward more healthful practices ( strivers ; n = 151) and those achieving them ( thrivers ; n = 255). Striver ecSI 2.0 TM scores were lower than those of thrivers (29.6 ± 7.8 vs. 33.7 ± 7.6; p < 0.001). More EC parents demonstrated eating behaviors associated with childhood obesity prevention than non-EC parents, encouraging education that fosters parent EC, especially in tandem with youth nutrition education.

Background: Enhanced GABA activity in the brain and a hyperactive hypothalamic-pituitary-gonadal axis are associated with polycystic ovary syndrome (PCOS), the most common form of anovulatory infertility. Women with PCOS exhibit elevated cerebrospinal fluid GABA levels and preclinical models of PCOS exhibit increased GABAergic input to GnRH neurons, the central regulators of reproduction. The arcuate nucleus (ARN) is postulated as the anatomical origin of elevated GABAergic innervation; however, the functional role of this circuit is undefined., Methods: We employed a combination of targeted optogenetic and chemogenetic approaches to assess the impact of acute and chronic ARN GABA neuron activation. Selective acute activation of ARN GABA neurons and their fiber projections was coupled with serial blood sampling for luteinizing hormone secretion in anesthetized male, female and prenatally androgenised (PNA) mice modelling PCOS. In addition, GnRH neuron responses to ARN GABA fiber stimulation were recorded in ex vivo brain slices. Chronic activation of ARN GABA neurons in healthy female mice was coupled with reproductive phenotyping for PCOS-like features., Findings: Acute stimulation of ARN GABA fibers adjacent to GnRH neurons resulted in a significant and long-lasting increase in LH secretion in male and female mice. The amplitude of this response was blunted in PNA mice, which also exhibited a blunted LH response to GnRH administration. Infrequent and variable GABA A -dependent changes in GnRH neuron firing were observed in brain slices. Chronic activation of ARN GABA neurons in healthy females impaired estrous cyclicity, decreased corpora lutea number and increased circulating testosterone levels., Interpretation: ARN GABA neurons can stimulate the hypothalamic-pituitary axis and chronic activation of ARN GABA neurons can mimic the reproductive deficits of PCOS in healthy females. Unexpectedly blunted HPG axis responses in PNA mice may reflect a history of high frequency GnRH/LH secretion and reduced LH stores, but also raise questions about impaired function within the ARN GABA population and the involvement of other circuits., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Exhaled breath condensate (EBC) analysis is a noninvasive method to assess the lower respiratory tract. In human subjects, EBC hydrogen peroxide (H 2 O 2 ), pH and leukotriene B 4 (LTB 4 ) are useful for detection and monitoring of inflammatory lung diseases, including asthma., Objectives: To determine associations between EBC biomarkers and cytological and endoscopic definitions of lower airway inflammation (LAI) while controlling for sampling and environmental variables., Study Design: Prospective, cross-sectional study., Methods: Clinical, endoscopic and airway cytological findings from 47 horses were compared with EBC pH and concentrations of H 2 O 2 and LTB 4 by univariate and multivariable analyses. Dichotomous (presence/absence of airway inflammation) and continuous outcome variables (differential cell counts in tracheal aspirate and bronchoalveolar lavage fluid, BALF) were evaluated and potential effects of collection and methodological factors were included., Results: EBC pH and H 2 O 2 concentrations were higher in horses with LAI and both were positively associated with the percentage of neutrophils in BALF (P<0.05). Mast cell percentage in BALF was negatively associated with EBC pH, and BALF eosinophil percentage was positively associated with EBC LTB 4 (P<0.05). Ambient temperature, relative humidity and assay methodology significantly impacted some analytes., Main Limitations: LAI is challenging to categorise due to a variety of clinical and cytological phenotypes. Although the study was designed to overcome this limitation, numbers of horses were small in some categories., Conclusions: EBC pH and H 2 O 2 concentrations are altered by airway inflammation, suggesting a role for these biomarkers in the diagnosis and monitoring of airway disease. Environmental and methodological factors can influence these biomarkers and should be considered in the interpretation of results., (© 2018 EVJ Ltd.)

Gonadotrophin-releasing hormone (GnRH) neurone function is dependent upon gonadal steroid hormone feedback, which is communicated in large part through an afferent neuronal network. The classical neurotransmitters GABA and glutamate are important regulators of GnRH neurone activity and are implicated in mediating feedback signals. In the present study, we aimed to determine whether GABAergic or glutamatergic input to GnRH neurones differs between males and females and/or exhibits morphological plasticity in response to steroid hormone feedback in females. Tissue collected from GnRH-green fluorescent protein (GFP) male and female mice in dioestrus underwent immunofluorescence labelling of GFP and either the vesicular GABA transporter (VGAT) or the vesicular glutamate transporter 2 (VGLUT2). No differences in the densities or absolute numbers of VGAT-immunoreactive (-IR) or VGLUT2-IR puncta apposed to GnRH neurones were identified between males and females. The most significant input from either neurotransmitter was to the proximal dendritic region and 80% of VGAT-IR puncta apposed to GnRH neurones co-localised with synaptophysin. Putative inputs were also assessed in ovariectomised (OVX) female mice treated with negative (OVX+E) or positive (OVX+E+E) feedback levels of oestrogen, and OVX+E+E mice were killed during the expected GnRH/luteinising hormone surge. No differences in VGLUT2-IR contacts to GnRH neurones were identified between animals under the negative-feedback influence of oestrogen (OVX+E) or the positive influence of oestrogen (OVX+E+E), regardless of cFos activation status. By contrast, a significant elevation in putative GABAergic inputs to GnRH neurones at the time of the preovulatory surge was found in the cFos-negative subset of GnRH neurones, both at the level of the soma and at the proximal dendrite. Taken together, these data suggest that, although GABAergic and glutamatergic innervation of GnRH neurones is not sexually differentiated, cyclic fluctuations in steroid hormone feedback over the female oestrous cycle result in plastic changes in GABAergic inputs to a subpopulation of GnRH neurones., (© 2018 British Society for Neuroendocrinology.)

GnRH neuron cell bodies are scattered throughout the basal forebrain but funnel their projections to the median eminence to release GnRH into the pituitary portal system to control fertility. Prior studies have shown that GnRH neurons located in the anterior hypothalamus send projections to the median eminence that have characteristics of both dendrites and axons. These unusual structures have been termed "dendrons." To address whether the dendron is unique to anterior hypothalamic GnRH neurons or is also a characteristic of more rostral GnRH neurons, we used viral vector‒mediated GnRH neuron‒specific tract-tracing coupled with CLARITY optical clearing. Individual rostral preoptic area GnRH neurons in female mice were identified to elaborate processes up to 4 mm in length that exhibited spines and projected all the way to the median eminence before branching into multiple short axons. The synaptic innervation patterns of distal GnRH neuron dendrons and their short axons in the vicinity of the median eminence were examined using electron microscopy. This revealed the presence of a high density of synaptic inputs to distal dendrons at the border of the median eminence. In contrast, no synapses were detected on any GnRH neuron axons. These studies demonstrate that GnRH neurons in the rostral preoptic area project dendrons to the edge of the median eminence, whereupon they branch into multiple short axons responsible for GnRH secretion. The dense synaptic innervation of these distal dendrons likely represents an efficient mechanism for controlling GnRH secretion required for fertility.

Objectives: To describe heavy alcohol use patterns and correlates in a diverse sample of MSM., Methods: We used respondent-driven sampling (RDS) to enroll 252 alcohol-using MSM in San Francisco from March 2015-July 2017. We examined heavy alcohol use patterns and conducted RDS-adjusted multivariable analyses to characterize correlates of hazardous alcohol consumption and binge drinking., Results: RDS-adjusted prevalence of weekly and at least weekly binge drinking was 24.9% and 19.3%, respectively. Hazardous consumption was common; prevalence of mid- and high-levels of hazardous drinking was 11.4% and 29.9%, respectively. In multivariable analyses, identifying as Hispanic/Latino or mixed/other race; being moderately or extremely interested in reducing alcohol use; ever receiving alcohol treatment; using ecstasy; reporting syphilis diagnosis; and having more than 5 male partners were independently associated with hazardous alcohol consumption. Less hazardous consumption was associated with having a bachelor's degree or completing post-graduate studies; and not being in a relationship. Reporting chlamydia infection; being somewhat, moderately or extremely interested in reducing alcohol use; and having multiple male sex partners were associated with higher odds of at least weekly binge drinking. Lower odds of binge drinking were associated with completing post-graduate studies. Moreover, for the outcomes of hazardous alcohol consumption and binge-drinking, we observed significant interaction effects between race/ethnicity and interest in reducing alcohol, past receipt of alcohol treatment, use of ecstasy, syphilis diagnosis, and number of male partners., Conclusion: Among alcohol-using MSM in San Francisco, heavy drinking patterns were common and independently associated with greater number of male sexual partners and sexually transmitted infections (STI). Moreover, significant racial/ethnic and socioeconomic disparities related to heavy alcohol use were observed and race/ethnicity modified the effect of the risk factors associated with these outcomes. These findings underscore the need to develop more MSM-specific interventions that jointly address heavy alcohol use and HIV/STI risk, as well as culturally-tailored and targeted strategies to alleviate health disparities., Competing Interests: The authors of this article declare no conflicts. Co-author Phillip Coffin has directed NIH-funded studies that have received donated medications from Alkermes (2013-2015) to treat methamphetamine dependence and Gilead (2016-2017) to treat Hepatitis C infections. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Objective: To identify why the National Clinical Guideline recommendation of 45 minutes of each appropriate therapy daily is not met in many English stroke units., Design: Mixed-methods case-study evaluation, including modified process mapping, non-participant observations of service organisation and therapy delivery, documentary analysis and semi-structured interviews., Setting: Eight stroke units in four English regions., Subjects: Seventy-seven patients with stroke, 53 carers and 197 stroke unit staff were observed; 49 patients, 50 carers and 131 staff participants were interviewed., Results: Over 1000 hours of non-participant observations and 433 patient-specific therapy observations were undertaken. The most significant factor influencing amount and frequency of therapy provided was the time therapists routinely spent, individually and collectively, in information exchange. Patient factors, including fatigue and tolerance influenced therapists' decisions about frequency and intensity, typically resulting in adaptation of therapy rather than no provision. Limited use of individual patient therapy timetables was evident. Therapist staffing levels were associated with differences in therapy provision but were not the main determinant of intensity and frequency. Few therapists demonstrated understanding of the evidence underpinning recommendations for increased therapy frequency and intensity. Units delivering more therapy had undertaken patient-focused reorganisation of therapists' working practices, enabling them to provide therapy consistent with guideline recommendations., Conclusion: Time spent in information exchange impacted on therapy provision in stroke units. Reorganisation of therapists' work improved alignment with guidelines.

Introduction: Several models of differentiated care for stable HIV patients on antiretroviral therapy (ART) in Malawi have been introduced to ensure that care is efficient and patient-centered. Three models have been prioritized by the government for a deeper and broader understanding: adjusted appointment spacing through multi-month scripting (MMS); fast-track drug refills (FTRs) on alternating visits; and community ART groups (CAGs) where rotating group members collect medications at the facility for all members. This qualitative study aimed to understand the challenges and successes of implementing these models of care and of the process of patient differentiation., Methods: A qualitative study was conducted as a part of a broader process evaluation in 30 purposefully selected ART facilities between February and May 2016. Semi-structured, in-depth interviews with 32 health workers that managed and coordinated ART clinics and 30 focus groups were held with 216 ART patients. Interviews and focus groups were audio recorded, transcribed, and coded thematically., Results: Participants reported that the models of differentiated care have yielded key benefits, including: reduced patients' travel and visit time, decongestion of facilities, and enhanced social support. Participants suggested that these benefits could lead to improved HIV treatment outcomes for patients. At the same time, some challenges were reported, such as inconsistent stocks of drugs, which can inhibit implementation of MMS. For CAGs, the group-based nature of the model presented some unique problems, such as conflicts within groups or concerns about privacy. Health workers also described some of the reasons why eligible patients may not receive the models or conversely why ineligible patients sometimes get the models., Conclusions: Documenting patient and health worker perspectives on models of differentiated care is critical to understanding and improving these models. While these models can offer important benefits, the models may not be appropriate for all sites or patients, and patient status and needs may change over time. Key challenges should be recognized and addressed for optimal utilization of the models., Competing Interests: The authors have declared that no competing interests exist.

Androgen excess is a hallmark of polycystic ovary syndrome (PCOS), a prevalent yet poorly understood endocrine disorder. Evidence from women and preclinical animal models suggests that elevated perinatal androgens can elicit PCOS onset in adulthood, implying androgen actions in both PCOS ontogeny and adult pathophysiology. Prenatally androgenized (PNA) mice exhibit a robust increase of progesterone-sensitive GABAergic inputs to gonadotropin-releasing hormone (GnRH) neurons implicated in the pathogenesis of PCOS. It is unclear when altered GABAergic wiring develops in the brain, and whether these central abnormalities are dependent upon adult androgen excess. Using GnRH-GFP-transgenic mice, we determined that increased GABA input to GnRH neurons occurs prior to androgen excess and the manifestation of reproductive impairments in PNA mice. These data suggest that brain circuit abnormalities precede the postpubertal development of PCOS traits. Despite the apparent developmental programming of circuit abnormalities, long-term blockade of androgen receptor signaling from early adulthood rescued normal GABAergic wiring onto GnRH neurons, improved ovarian morphology, and restored reproductive cycles in PNA mice. Therefore, androgen excess maintains changes in female brain wiring linked to PCOS features and the blockade of androgen receptor signaling reverses both the central and peripheral PNA-induced PCOS phenotype.

Sacubitril/valsartan (LCZ696) is indicated for the treatment of patients with heart failure and reduced ejection fraction (HFrEF). Since patients with HFrEF may receive sacubitril/valsartan and sildenafil, both increasing cyclic guanosine monophosphate, the present study evaluated the pharmacokinetic and pharmacodynamic drug interaction potential between sacubitril/valsartan and sildenafil. In this open-label, three-period, single sequence study, patients with mild-to-moderate hypertension (153.8 ± 8.2 mmHg mean systolic blood pressure (SBP)) received a single dose of sildenafil 50 mg, sacubitril/valsartan 400 mg once daily for 5 days, and sacubitril/valsartan and sildenafil coadministration. When coadministered with sildenafil, the AUC and C max of valsartan decreased by 29% and 39%, respectively. Coadministration of sacubitril/valsartan and sildenafil resulted in a greater decrease in BP (-5/-4/-4 mmHg mean ambulatory SBP/DBP/MAP (mean arterial pressure)) than with sacubitril/valsartan alone. Both treatments were generally safe and well tolerated in this study; however, the additional BP reduction suggests that sildenafil should be administered cautiously in patients receiving sacubitril/valsartan. Unique identifier: NCT01601470., (© 2017 The Authors Clinical Pharmacology & Therapeutics published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.)

The presence of ultrasound-induced cavitation in sonodynamic therapy (SDT) treatments has previously enhanced the activity and delivery of certain sonosensitisers in biological systems. The purpose of this work was to investigate the potential for two novel anti-cancer agents from natural derivatives, sanguinarine and ginger root extract (GRE), as sonosensitisers in an SDT treatment with in vitro PANC-1 cells. Both anti-cancer compounds had a dose-dependent cytotoxicity in the presence of PANC-1 cells. A range of six discreet ultrasound power-frequency configurations were tested and it was found that the cell death caused directly by ultrasound was likely due to the sonomechanical effects of cavitation. Combined treatment used dosages of 100μM sanguinarine or 1mM of GRE with 15s sonication at 500kHz and 10W. The sanguinarine-SDT and GRE-SDT treatments showed a 6% and 17% synergistic increase in observed cell death, respectively. Therefore both sanguinarine and GRE were found to be effective sonosensitisers and warrant further development for SDT, with a view to maximising the magnitude of synergistic increase in toxicity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Human Respiratory Syncytial Virus (HRSV) is a leading cause of bronchopneumonia in infants and the elderly. To date, knowledge of viral and host protein interactions within HRSV is limited and are critical areas of research. Here, we show that HRSV Matrix (M) protein interacts with the cellular adaptor protein complex 3 specifically via its medium subunit (AP-3Mu3A). This novel protein-protein interaction was first detected via yeast-two hybrid screen and was further confirmed in a mammalian system by immunofluorescence colocalization and co-immunoprecipitation. This novel interaction is further substantiated by the presence of a known tyrosine-based adaptor protein MU subunit sorting signal sequence, YXXФ: where Ф is a bulky hydrophobic residue, which is conserved across the related RSV M proteins. Analysis of point-mutated HRSV M derivatives indicated that AP-3Mu3A- mediated trafficking is contingent on the presence of the tyrosine residue within the YXXL sorting sequence at amino acids 197-200 of the M protein. AP-3Mu3A is up regulated at 24 hours post-infection in infected cells versus mock-infected HEp2 cells. Together, our data suggests that the AP-3 complex plays a critical role in the trafficking of HRSV proteins specifically matrix in epithelial cells. The results of this study add new insights and targets that may lead to the development of potential antivirals and attenuating mutations suitable for candidate vaccines in the future.

Heparin and heparan sulfate (HS) by nature contain multiple isomeric structures, which are fundamental for the regulation of biological processes. Here we report the use of a porous graphitized carbon (PGC) LC-MS method with effective separation and sensitivity to separate mixtures of digested HS oligosaccharides. Application of this method allowed the separation of oligosaccharide mixtures with various degree of polymerization (dp) ranging from dp4 to dp8, two dp4 isomers that were baseline resolved, four dp6 isomers, and the observation of a dp3 oligosaccharide. PGC LC-MS of complex mixtures demonstrated that compounds eluted from the column in decreasing order of hydrophilicity, with the more highly sulfated structures eluting first. Our data indicate that sulfation levels, chain length, and conformation all effect elution order. We found that PGC's resolving capabilities for the dp4 and dp6 isomeric structures makes this methodology particularly useful for the sequencing of HS saccharides, because the lack of contaminating isomeric structures provides unambiguous structural assignments from the MS/MS data. Collectively this work demonstrates that PGC column-based methods are powerful tools for enhanced separation and analysis of heterogeneous mixtures of HS saccharide species.