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Abstract Background The Covid pandemic and its aftermath have triggered new alarm and social unrest across the Global South over the deepening international debt crisis that now threatens to derail Universal Health Coverage (UHC), other Sustainable Development Goals (SDGs), future pandemic preparedness, and global warming mitigation. The recent Globalization and Health article by Alex Kentikelenis and Thomas Stubbs (May 2024), “Social protection and the International Monetary Fund: promise versus performance”, offers a meticulously quantified rendering of the social costs imposed by the crisis and takes aim at IMF solutions. They advocate for a rejection of IMF austerity programs and offer a valuable prescription for change through the International Labor Organization’s “Universal Social Protection” concept. Main body Similar to the Jubilee movement at the turn of the century, global civil society, humanitarian aid, and health organizations are mobilizing in a variety of global networks to call for debt cancellation and restructuring as well as an end to austerity. These include new debt law campaigns in London and New York to rein in private creditors, calls for new IMF issuance of “Special Drawing Rights”, demands for “Global Public Investment”, and promotion of Universal Social Protection. Conclusion The Universal Social Protection approach described by Kentikelenis and Stubbs provides a focal point for these demands to confront this latest and worst episode of sovereign debt crisis already undermining global health progress.

Abstract Background The Systems Analysis and Improvement Approach (SAIA) is an evidence-based package of systems engineering tools originally designed to improve patient flow through the prevention of Mother-to-Child transmission of HIV (PMTCT) cascade. SAIA is a potentially scalable model for maximizing the benefits of universal antiretroviral therapy (ART) for mothers and their babies. SAIA-SCALE was a stepped wedge trial implemented in Manica Province, Mozambique, to evaluate SAIA’s effectiveness when led by district health managers, rather than by study nurses. We present the results of a qualitative assessment of implementation determinants of the SAIA-SCALE strategy during two intensive and one maintenance phases. Methods We used an extended case study design that embedded the Consolidated Framework for Implementation Research (CFIR) to guide data collection, analysis, and interpretation. From March 2019 to April 2020, we conducted in-depth individual interviews (IDIs) and focus group discussions (FGDs) with district managers, health facility maternal and child health (MCH) managers, and frontline nurses at 21 health facilities and seven districts of Manica Province (Chimoio, Báruè, Gondola, Macate, Manica, Sussundenga, and Vanduzi). Results We included 85 participants: 50 through IDIs and 35 from three FGDs. Most study participants were women (98%), frontline nurses (49.4%), and MCH health facility managers (32.5%). An identified facilitator of successful intervention implementation (regardless of intervention phase) was related to SAIA’s compatibility with organizational structures, processes, and priorities of Mozambique’s health system at the district and health facility levels. Identified barriers to successful implementation included (a) inadequate health facility and road infrastructure preventing mothers from accessing MCH/PMTCT services at study health facilities and preventing nurses from dedicating time to improving service provision, and (b) challenges in managing intervention funds. Conclusions The SAIA-SCALE qualitative evaluation suggests that the scalability of SAIA for PMTCT is enhanced by its fit within organizational structures, processes, and priorities at the primary level of healthcare delivery and health system management in Mozambique. Barriers to implementation that impact the scalability of SAIA include district-level financial management capabilities and lack of infrastructure at the health facility level. SAIA cannot be successfully scaled up to adequately address PMTCT needs without leveraging central-level resources and priorities. Trial registration ClinicalTrials.gov, NCT03425136 . Registered on 02/06/2018.

Abstract Introduction Overall, resilient health systems build upon sufficient, qualified, well-distributed, and motivated health workers; however, this precious resource is limited in numbers to meet people’s demands, particularly in LMICs. Understanding the subnational distribution of health workers from different lens is critical to ensure quality healthcare and improving health outcomes. Methods Using data from Health Personnel Information System, facility-level Service Availability and Readiness Assessment, and other sources, we performed a district-level longitudinal analysis to assess health workforce density and the ratio of male to female health workers between January 2016 and June 2020 across all districts in Mozambique. Results 22 011 health workers were sampled, of whom 10 405 (47.3%) were male. The average age was 35 years (SD: 9.4). Physicians (1025, 4.7%), maternal and child health nurses (4808, 21.8%), and nurses (6402, 29.1%) represented about 55% of the sample. In January 2016, the average district-level workforce density was 75.8 per 100 000 population (95% CI 65.9, 87.1), and was increasing at an annual rate of 8.0% (95% CI 6.00, 9.00) through January 2018. The annual growth rate declined to 3.0% (95% CI 2.00, 4.00) after January 2018. Two provinces, Maputo City and Maputo Province, with 268.3 (95% CI 186.10, 387.00) and 104.6 (95% CI 84.20, 130.00) health workers per 100 000 population, respectively, had the highest workforce density at baseline (2016). There were 3122 community health workers (CHW), of whom 72.8% were male, in January 2016. The average number of CHWs per 10 000 population was 1.33 (95% CI 1.11, 1.59) in 2016 and increased by 18% annually between January 2016 and January 2018. This trend reduced to 11% (95% CI 0.00, 13.00) after January 2018. The sex ratio was twice as high for all provinces in the central and northern regions relative to Maputo Province. Maputo City (OR: 0.34; 95% CI 0.32, 0.34) and Maputo Province (OR: 0.56; 95% CI 0.49, 0.65) reported the lowest sex ratio at the baseline. Encouragingly, important sex ratio improvements were observed after January 2018, particularly in the northern and central regions. Conclusion Mozambique made substantial progress in health workers’ availability during the study period; however, with a critical slowdown after 2018. Despite the progress, meaningful shortages and distribution disparities persist.

BackgroundThere are limited data on home pregnancy test use among women in low-and-middle-income countries. A prior survey found that only 20% of women in western Kenya used a home pregnancy test to confirm their pregnancies before going to antenatal care. This qualitative study aims to understand why women do not use home pregnancy tests in early pregnancy.MethodsFrom April 2021 to July 2021, we interviewed women from four antenatal care clinics in Homa Bay and Siaya counties. We recruited women previously enrolled in the PrEP Implementation for Mothers in Antenatal care (PrIMA) study, a cluster-randomized trial that evaluated the best approaches to implementing PrEP in maternal and child health clinics in Western Kenya (NCT03070600). Interviews were conducted via phone, audio recorded, translated, and transcribed verbatim. We coded and analyzed the transcripts to capture factors influencing women's capability, opportunity, and motivation to use home pregnancy tests.ResultsWe conducted 48 semistructured interviews with women aged 21–42 years. Twenty-seven women did not use a home pregnancy test in their most recent pregnancy. Seventeen of these women reported not using a home pregnancy test before. Lack of knowledge, mistrust in the accuracy of tests, preferring to rely on signs and symptoms of pregnancy or get a test from the health facility, cost, and accessibility were key barriers to home pregnancy test use.ConclusionImproving the uptake of home pregnancy testing during early pregnancy will require efforts to enhance community knowledge of test use and associated benefits and reduce cost burdens by making tests more affordable and accessible.

Abstract Background Across sub-Saharan Africa, evidence-based clinical guidelines to screen and manage hypertension exist; however, country level application is low due to lack of service readiness, uneven health worker motivation, weak accountability of health worker performance, and poor integration of hypertension screening and management with chronic care services. The systems analysis and improvement approach (SAIA) is an evidence-based implementation strategy that combines systems engineering tools into a five-step, facility-level package to improve understanding of gaps (cascade analysis), guide identification and prioritization of low-cost workflow modifications (process mapping), and iteratively test and redesign these modifications (continuous quality improvement). As hypertension screening and management are integrated into chronic care services in sub-Saharan Africa, an opportunity exists to test whether SAIA interventions shown to be effective in improving efficiency and coverage of HIV services can be effective when applied to the non-communicable disease services that leverage the same platform. We hypothesize that SAIA-hypertension (SAIA-HTN) will be effective as an adaptable, scalable model for broad implementation. Methods We will deploy a hybrid type III cluster randomized trial to evaluate the impact of SAIA-HTN on hypertension management in eight intervention and eight control facilities in central Mozambique. Effectiveness outcomes include hypertension cascade flow measures (screening, diagnosis, management, control), as well as hypertension and HIV clinical outcomes among people living with HIV. Cost-effectiveness will be estimated as the incremental costs per additional patient passing through the hypertension cascade steps and the cost per additional disability-adjusted life year averted, from the payer perspective (Ministry of Health). SAIA-HTN implementation fidelity will be measured, and the Consolidated Framework for Implementation Research will guide qualitative evaluation of the implementation process in high- and low-performing facilities to identify determinants of intervention success and failure, and define core and adaptable components of the SAIA-HTN intervention. The Organizational Readiness for Implementing Change scale will measure facility-level readiness for adopting SAIA-HTN. Discussion SAIA packages user-friendly systems engineering tools to guide decision-making by front-line health workers to identify low-cost, contextually appropriate chronic care improvement strategies. By integrating SAIA into routine hypertension screening and management structures, this pragmatic trial is designed to test a model for national scale-up. Trial registration ClinicalTrials.gov NCT04088656 (registered 09/13/2019; https://clinicaltrials.gov/ct2/show/NCT04088656 ).

Abstract In many African countries, hundreds of health-related NGOs are fed by a chaotic tangle of donor funding streams. The case of Mozambique illustrates how this NGO model impedes Universal Health Coverage. In the 1990s, NGOs multiplied across post-war Mozambique: the country’s structural adjustment program constrained public and foreign aid expenditures on the public health system, while donors favored private contractors and NGOs. In the 2000s, funding for HIV/AIDS and other vertical aid from many donors increased dramatically. In 2004, the United States introduced PEPFAR in Mozambique at nearly 500 million USD per year, roughly equivalent to the entire budget of the Ministry of Health. To be sure, PEPFAR funding has helped thousands access antiretroviral treatment, but over 90% of resources flow “off-budget” to NGO “implementing partners,” with little left for the public health system. After a decade of this major donor funding to NGOs, public sector health system coverage had barely changed. In 2014, the workforce/ population ratio was still among the five worst in the world at 71/10000; the health facility/per capita ratio worsened since 2009 to only 1 per 16,795. Achieving UHC will require rejection of austerity constraints on public sector health systems, and rechanneling of aid to public systems building rather than to NGOs.

Abstract Background The introduction of option B+—rapid initiation of lifelong antiretroviral therapy regardless of disease status for HIV-infected pregnant and breastfeeding women—can dramatically reduce HIV transmission during pregnancy, birth, and breastfeeding. Despite significant investments to scale-up Option B+, results have been mixed, with high rates of loss to follow-up, sub-optimal viral suppression, continued pediatric HIV transmission, and HIV-associated maternal morbidity. The Systems Analysis and Improvement Approach (SAIA) cluster randomized trial demonstrated that a package of systems engineering tools improved flow through the prevention of mother-to-child HIV transmission (PMTCT) cascade. This five-step, facility-level intervention is designed to improve understanding of gaps (cascade analysis), guide identification and prioritization of low-cost workflow modifications (process mapping), and iteratively test and redesign these modifications (continuous quality improvement). This protocol describes a novel model for SAIA delivery (SAIA-SCALE) led by district nurse supervisors (rather than research nurses), and evaluation procedures, to serve as a foundation for national scale-up. Methods The SAIA-SCALE stepped wedge trial includes three implementation waves, each 12 months in duration. Districts are the unit of assignment, with four districts randomly assigned per wave, covering all 12 districts in Manica province, Mozambique. In each district, the three highest volume health facilities will receive the SAIA-SCALE intervention (totaling 36 intervention facilities). The RE-AIM framework will guide SAIA-SCALE’s evaluation. Reach describes the proportion of clinics and population in Manica province reached, and sub-groups not reached. Effectiveness assesses impact on PMTCT process measures and patient-level outcomes. Adoption describes the proportion of districts/clinics adopting SAIA-SCALE, and determinants of adoption using the Organizational Readiness for Implementing Change (ORIC) tool. Implementation will identify SAIA-SCALE core elements and determinants of successful implementation using the Consolidated Framework for Implementation Research (CFIR). Maintenance describes the proportion of districts sustaining the intervention. We will also estimate the budget and program impact from the payer perspective for national scale-up. Discussion SAIA packages user-friendly systems engineering tools to guide decision-making by frontline health workers, and to identify low-cost, contextually appropriate PMTCT improvement strategies. By integrating SAIA delivery into routine management structures, this pragmatic trial is designed to test a model for national intervention scale-up. Trial registration ClinicalTrials.gov NCT03425136 (registered 02/06/2018).

Learning climate greatly affects student achievement. This qualitative study aimed to understand community definitions of climate; share lived experiences of students, faculty, and staff; and define priority areas of improvement in the University of Washington School of Public Health (UWSPH). Between March-May 2019, 17 focus group discussions were conducted–stratified by role and self-identified race/ethnicity, gender and sexual orientation–among 28 faculty/staff and 36 students. Topics included: assessing the current climate, recounting experiences related to roles and identities, and recommending improvements. Transcripts were coded using deductive and inductive approaches. Race/ethnicity, gender, and sexual orientation appeared to affect perceptions of the climate, with nearly all respondents from underrepresented or minoritized groups recounting negative experiences related to their identity. Persons of color, women, and other respondents who identified as lesbian, gay, bisexual, transgender, queer/questioning, intersex, and asexual (LGBTQIA) frequently perceived the climate as “uncomfortable.” Most felt that UWSPH operates within a structural hierarchy that perpetuates white, male, and/or class privilege and “protects those in power” while leaving underrepresented or minoritized groups feeling like “the way to move up… is to conform” in order to not be seen as “someone pushing against the system.” Improvement priorities included: increasing community responsiveness to diversity, equity, and inclusion; intentionally diversifying faculty/staff and student populations; designing inclusive curricula; and supporting underrepresented or minoritized groups academically, professionally, and psychologically.

Introduction: Facial diplegia, a rare variant of Guillain-Barré syndrome (GBS), is a challenging diagnosis to make in the emergency department due to its resemblance to neurologic Lyme disease. Case report: We present a case of a 27-year-old previously healthy man who presented with bilateral facial paralysis. Discussion: Despite the variance in presentation, the recommended standard of practice for diagnostics (cerebrospinal fluid albumin-cytological dissociation) and disposition (admission for observation, intravenous immunoglobulin, and serial negative inspiratory force) of facial diplegia are the same as for other presentations of GBS. Conclusion: When presented with bilateral facial palsy emergency providers should consider autoimmune, infectious, idiopathic, metabolic, neoplastic, neurologic, and traumatic etiologies in addition to the much more common neurologic Lyme disease.

Background Vertically oriented global health initiatives (GHIs) addressing the HIV/AIDS epidemic, including the President’s Emergency Plan for AIDS Relief (PEPFAR), have successfully contributed to reducing HIV/AIDS related morbidity and mortality. However, there is still debate about whether these disease-specific programs have improved or harmed health systems overall, especially with respect to non-HIV health needs. Methods As part of a larger evaluation of PEPFAR’s effects on the health system between 2005-2011, we collected qualitative and quantitative data through semi-structured interviews with District Health Officers (DHOs) from all 112 districts in Uganda. We asked DHOs to share their perceptions about the ways in which HIV programs (largely PEPFAR in the Ugandan context) had helped and harmed the health system. We then identified key themes among their responses using qualitative content analysis. Results Ugandan DHOs said PEPFAR had generally helped the health system by improving training, integrating HIV and non-HIV care, and directly providing resources. To a lesser extent, DHOs said PEPFAR caused the health system to focus too narrowly on HIV/AIDS, increased workload for already overburdened staff, and encouraged doctors to leave public sector jobs for higher-paid positions with HIV/AIDS programs. Conclusion Health system leaders in Uganda at the district level were appreciative of resources aimed at HIV they could often apply for broader purposes. As HIV infection becomes a chronic disease requiring strong health systems to manage sustained patient care over time, Uganda’s weak health systems will require broad infrastructure improvements inconsistent with narrow vertical health programming.

Background: We describe wait and consult times across public-sector clinics and identify health facility determinants of wait and consult times. Design: We observed 8,102 patient arrivals and departures from clinical service areas across 12 public-sector clinics in Sofala and Manica Provinces between January and April 2011. Negative binomial generalized estimating equations were used to model associated health facility factors. Results: Mean wait times (in minutes) were: 26.1 for reception; 43.5 for outpatient consults; 58.8 for antenatal visits; 16.2 for well-child visits; 8.0 for pharmacy; and 15.6 for laboratory. Mean consultation times (in minutes) were: 5.3 for outpatient consults; 9.4 for antenatal visits; and 2.3 for well-child visits. Over 70% (884/1,248) of patients arrived at the clinic to begin queuing for general reception prior to 10:30 am. Facilities with more institutional births had significantly longer wait times for general reception, antenatal visits, and well-child visits. Clinics in rural areas had especially shorter wait times for well-child visits. Outpatient consultations were significantly longer at the smallest health facilities, followed by rural hospitals, tertiary/quaternary facilities, compared with Type 1 rural health centers. Discussion: The average outpatient consult in Central Mozambique lasts 5 min, following over 40 min of waiting, not including time to register at most clinics. Wait times for first antenatal visits are even longer at almost 1 h. Urgent investments in public-sector human resources for health alongside innovative operational research are needed to increase consult times, decrease wait times, and improve health system responsiveness.

Following publication of the original article [1], the authors reported a typo in the title of the article.

In this essay, we seek to understand how the stunning rise of data vacuuming, necessitated by the pretense of ‘partnership’ within global health, has fundamentally altered how routine health data in poor countries is collected, analyzed, prioritized, and used to inform management and policy. Writing as a team of authors with experiences on multiple sides of global health partnerships in the United States, Mozambique, Nepal, Lesotho, Kenya, and Cote d’Ivoire, we argue that solidarity-based partnership between donor and recipient countries is impossible when evidence production and management is effectively outsourced to external organizations to meet the criteria of donor partners. Specifically, to meet the 2030 Sustainable Development Goals, equity-oriented strategies are critically needed to create data collection, analysis, and use activities that are mutually beneficial and sustainable.

Introduction: Time spent viewing visual sexual stimuli (VSS) has the potential to habituate the sexual response and generalize to the partner context. Aim: The aim of this study was to examine whether the time spent viewing VSS is related to sexual responsiveness felt in the laboratory or with a sexual partner. Methods: Nontreatment-seeking men (N = 280) reported their weekly average VSS viewing in hours. VSS hours were examined in relation to the sexual arousal experienced while viewing a standardized sexual film in the laboratory and erectile problems experienced with a sexual partner. Main Outcome Measures: Self-reported sexual arousal in response to sexual films and erectile problems on the International Index of Erectile Function were the main outcome measures. Results: More hours viewing VSS was related to stronger experienced sexual responses to VSS in the laboratory, was unrelated to erectile functioning with a partner, and was related to stronger desire for sex with a partner. Conclusions: VSS use within the range of hours tested is unlikely to negatively impact sexual functioning, given that responses actually were stronger in those who viewed more VSS. Prause N and Pfaus J. Viewing sexual stimuli associated with greater sexual responsiveness, not erectile dysfunction. Sex Med 2015;3:90–98.

Ipomoea carnea is a toxic plant often ingested by livestock in Brazil. Three experiments were conducted to determine if conditioned food aversion was effective in reducing goats' consumption of I. carnea. In the first experiment, 10 mildly intoxicated goats that had been eating I. carnea were averted using LiCl (175 to 200mg kg-1 body weight). These intoxicated goats did not develop an aversion to I. carnea, demonstrating that the technique is not effective in goats that are already accustomed to consuming the plant. In the second experiment, 14 naïve goats were placed in a pasture with I. carnea, and averted after they ingested the plant. In this group the aversion persisted until the end of the experiment, 2 years and 8 months after the initial aversion. In another experiment, 20 goats were placed in a pasture with I. carnea, and after consuming the plant were averted with LiCl. The averted goats were transferred to Marajo Island and periodically observed over a 2 year period at 2-3 month intervals to determine if they were still averted. The averted goats did not ingest the plant while grazing in the pasture, whereas in 6 neighboring goat farms the prevalence of intoxication from I. carnea poisoning was estimated to be about 40%. These results demonstrated the efficacy of conditioned food aversion to avoid ingestion of I. carnea in formerly naïve goats that had only recently begun to ingest the plant.

Quinze ovinos foram adaptados ao consumo de Ipomoea carnea por 36 dias. Posteriormente, foram divididos ao acaso em três grupos de cinco ovinos cada e avertidos com cloreto de lítio (LiCl) (Grupo 1) e Baccharis coridifolia (Grupo 2). O Grupo 3 permaneceu como controle. Os ovinos foram desafiados ao consumo da planta na pastagem e em baias individuais até o 74° dia após o primeiro dia da aversão. Nos dias 14 e 19, o número de ovinos que consumiu I. carnea no Grupo 3 foi significativamente maior que o número de ovinos que consumiu a planta nos Grupos 1 e 2 (P=0,004 e P=0,0004, respectivamente). No dia 24, o número de ovinos que consumiu I. carnea foi significativamente maior no Grupo 3 do que no Grupo 1 (P=0,004). A partir do desafio do dia 29, não houve diferença significativa (P>0,05) no consumo da planta entre os três grupos. A quantidade de I. carnea consumida pelos ovinos do Grupo 3 durante o período do experimento no dia 7 após o primeiro dia da aversão foi significativamente maior do que o consumo das ovelhas dos Grupos 1 e 2 (P=0,0002 e P=0,01, respectivamente). Nos desafios seguintes, não houve diferença na quantidade de I. carnea ingerida pelos ovinos. Na avaliação do comportamento dos ovinos na pastagem infestada por I. carnea, o tempo destinado ao consumo da planta foi de no máximo 2,4%±1,6% do total de tempo de pastejo, não havendo diferença entre os três grupos. I. carnea nunca foi consumida avidamente pelos ovinos experimentais e a aversão incompleta induzida pelos dois agentes utilizados como aversores (LiCl e B. coridifolia) sugere que não seriam eficientes para reduzir ou eliminar o consumo de I. carnea pelos ovinos.

Quinze ovinos foram adaptados ao consumo de Ipomoea carnea por 36 dias. Posteriormente, foram divididos ao acaso em três grupos de cinco ovinos cada e avertidos com cloreto de lítio (LiCl) (Grupo 1) e Baccharis coridifolia (Grupo 2). O Grupo 3 permaneceu como controle. Os ovinos foram desafiados ao consumo da planta na pastagem e em baias individuais até o 74° dia após o primeiro dia da aversão. Nos dias 14 e 19, o número de ovinos que consumiu I. carnea no Grupo 3 foi significativamente maior que o número de ovinos que consumiu a planta nos Grupos 1 e 2 (P=0,004 e P=0,0004, respectivamente). No dia 24, o número de ovinos que consumiu I. carnea foi significativamente maior no Grupo 3 do que no Grupo 1 (P=0,004). A partir do desafio do dia 29, não houve diferença significativa (P>0,05) no consumo da planta entre os três grupos. A quantidade de I. carnea consumida pelos ovinos do Grupo 3 durante o período do experimento no dia 7 após o primeiro dia da aversão foi significativamente maior do que o consumo das ovelhas dos Grupos 1 e 2 (P=0,0002 e P=0,01, respectivamente). Nos desafios seguintes, não houve diferença na quantidade de I. carnea ingerida pelos ovinos. Na avaliação do comportamento dos ovinos na pastagem infestada por I. carnea, o tempo destinado ao consumo da planta foi de no máximo 2,4%±1,6% do total de tempo de pastejo, não havendo diferença entre os três grupos. I. carnea nunca foi consumida avidamente pelos ovinos experimentais e a aversão incompleta induzida pelos dois agentes utilizados como aversores (LiCl e B. coridifolia) sugere que não seriam eficientes para reduzir ou eliminar o consumo de I. carnea pelos ovinos.

Three experiments were performed to determine the efficacy of various methods of averting naïve cattle to prevent Baccharis coridifolia poisoning: forced oral administration of 0.5g kg-1 body weight of fresh B. coridifolia; forced inhalation of the smoke from burning B. coridifolia and rubbing the plant on the animals' muzzles and mouths; and introducing the animals into paddocks with low invasion by B. coridifolia. Results demonstrated that cattle forced to ingest low doses become strongly averted if introduced into paddocks 23-26 hours after the aversion. In contrast, cattle introduced into the paddocks between 1-10 hours were not fully averted. Inhalation of B. coridifolia smoke, and rubbing the plant on the animals' muzzles and mouths were not efficient to induce an aversion. The introduction of cattle into paddocks with approximately 1% of B. coridifolia was efficient if the animals remained 5 months in the area, but not if they only remained for 60 hours, as cattle required sufficient time to learn to avoid the plant.

Loss to follow-up contributes to the low coverage of HIV care interventions among HIV-exposed infants in Beira, Mozambique. This qualitative study explores the perceptions of HIV-infected women and their health care providers regarding the main obstacles preventing women from attending follow-up visits for HIV care, and factors influencing women’s decisions about newborn care. Fifty-two in-depth interviews and two focus group discussions were conducted; transcripts were coded and analyzed using ATLAS.ti. Interviewees perceived three major barriers to follow-up: food insecurity, difficulties navigating the health system, and women’s familial roles and responsibilities. Our findings unveil the complex context in which HIV-infected women and their children live, and suggest that the structure and function of the HIV care system should be reviewed. Economic empowerment of women is crucial to achieving better compliance with medical care. Integration of mother and child services and more efficient and culturally sensitive medical services may improve follow-up.

BACKGROUND:In 2010, the Uganda Ministry of Health introduced its Safe Male Circumcision (SMC) strategy for HIV prevention with the goal of providing 4.2 million voluntary medical male circumcisions by 2015. Fishing communities, where HIV prevalence is approximately 3-5 times higher than the national average, have been identified as a key population needing targeted HIV prevention services by the National HIV Prevention Strategy. This study aimed to understand perceptions of HIV and identify potential barriers and facilitators to SMC in fishing communities along Lake Victoria. METHODS:We conducted 8 focus group discussions, stratified by sex and age, with 67 purposefully sampled participants in 4 communities in Kalangala District, Uganda. RESULTS:There was universal knowledge of the availability of SMC services, but males reported high uptake in the community while females indicated that it is low. Improved hygiene, disease prevention, and improved sexual performance and desirability were reported facilitators. Barriers included a perceived increase in SMC recipients' physiological libido, post-surgical abstinence, lost income during convalescence, and lengthier recovery due to occupational hazards. Both males and females reported concerns about spousal fidelity during post-SMC abstinence. Reported misconceptions and community-held cultural beliefs include fear that foreskins are sold after their removal, the belief that a SMC recipient's first sexual partner after the procedure should not be his spouse, and the belief that vaginal fluids aid circumcision wound healing. CONCLUSIONS:Previous outreach efforts have effectively reached these remote communities, where availability and health benefits of SMC are widely understood. However, community-specific intervention strategies are needed to address the barriers identified in this study. We recommend the development of targeted counseling, outreach, and communication strategies to address barriers, misconceptions, and community-held beliefs. Interventions should also incorporate female partners into the SMC decision-making process and develop compensation strategies to address lost income during SMC recovery.

Background: Knowledge of the relation between health-system factors and child mortality could help to inform health policy in low-income and middle-income countries. We aimed to quantify modifiable health-system factors and their relation with provincial-level heterogeneity in under-5, infant, and neonatal mortality over time in Mozambique. Methods: Using Demographic and Health Survey (2003 and 2011) and Multiple Indicator Cluster Survey (2008) data, we generated provincial-level time-series of child mortality in under-5 (ages 0–4 years), infant (younger than 1 year), and neonatal (younger than 1 month) age groups for 2000–10. We built negative binomial mixed models to examine health-system factors associated with changes in child mortality. Findings: Under-5 mortality rate was heterogeneous across provinces, with yearly decreases ranging from 11·1% (Nampula) to 1·9% (Maputo Province). Heterogeneity was greater for neonatal mortality rate, with only seven of 11 provinces showing significant yearly decreases, ranging from 13·6% (Nampula) to 4·2% (Zambezia). Health workforce density (adjusted rate ratio 0·94, 95% CI 0·90–0·98) and maternal and child health nurse density (0·96, 0·92–0·99) were both associated with reduced under-5 mortality rate, as were institutional birth coverage (0·94, 0·90–0·98) and government financing per head (0·80, 0·65–0·98). Higher population per health facility was associated with increased under-5 mortality rate (1·14, 1·02–1·28). Neonatal mortality rate was most strongly associated with institutional birth attendance, maternal and child nurse density, and overall health workforce density. Infant mortality rate was most strongly associated with institutional birth attendance and population per health facility. Interpretation: The large decreases in child mortality seen in Mozambique between 2000 and 2010 could have been partly caused by improvements in the public-sector health workforce, institutional birth coverage, and government health financing. Increased attention should be paid to service availability, because population per health facility is increasing across Mozambique and is associated with increased under-5 mortality. Investments in health information systems and new methods to track potentially increasing subnational health disparities are urgently needed. Funding: Doris Duke Charitable Foundation and Mozambican National Institute of Health.

IntroductionTrained human resources are fundamental for well-functioning health systems, and the lack of health workers undermines public sector capacity to meet population health needs. While external brain drain from low and middle-income countries is well described, there is little understanding of the degree of internal brain drain, and how increases in health sector funding through global health initiatives may contribute to the outflow of health workers from the public sector to donor agencies, non-governmental organisations (NGOs), and the private sector.MethodsAn observational study was conducted to estimate the degree of internal and external brain drain among Mozambican nationals qualifying from domestic and foreign medical schools between 1980-2006. Data were collected 26-months apart in 2008 and 2010, and included current employment status, employer, geographic location of employment, and main work duties.ResultsOf 723 qualifying physicians between 1980-2006, 95.9% (693) were working full-time, including 71.1% (493) as clinicians, 20.5% (142) as health system managers, and 6.9% (48) as researchers/professors. 25.5% (181) of the sample had left the public sector, of which 62.4% (113) continued working in-country and 37.6% (68) emigrated from Mozambique. Of those cases of internal migration, 66.4% (75) worked for NGOs, 21.2% (24) for donor agencies, and 12.4% (14) in the private sector. Annual incidence of physician migration was estimated to be 3.7%, predominately to work in the growing NGO sector. An estimated 36.3% (41/113) of internal migration cases had previously held senior-level management positions in the public sector.DiscussionInternal migration is an important contributor to capital flight from the public sector, accounting for more cases of physician loss than external migration in Mozambique. Given the urgent need to strengthen public sector health systems, frank reflection by donors and NGOs is needed to assess how hiring practices may undermine the very systems they seek to strengthen.

Introduction In 2004, Mozambique, supported by large increases in international disease‐specific funding, initiated a national rapid scale‐up of antiretroviral treatment (ART) and HIV care through a vertical “Day Hospital” approach. Though this model showed substantial increases in people receiving treatment, it diverted scarce resources away from the primary health care (PHC) system. In 2005, the Ministry of Health (MOH) began an effort to use HIV/AIDS treatment and care resources as a means to strengthen their PHC system. The MOH worked closely with a number of NGOs to integrate HIV programs more effectively into existing public‐sector PHC services. Case Description In 2005, the Ministry of Health and Health Alliance International initiated an effort in two provinces to integrate ART into the existing primary health care system through health units distributed across 23 districts. Integration included: a) placing ART services in existing units; b) retraining existing workers; c) strengthening laboratories, testing, and referral linkages; e) expanding testing in TB wards; f) integrating HIV and antenatal services; and g) improving district‐level management. Discussion: By 2008, treatment was available in nearly 67 health facilities in 23 districts. Nearly 30,000 adults were on ART. Over 80,000 enrolled in the HIV/AIDS program. Loss to follow‐up from antenatal and TB testing to ART services has declined from 70% to less than 10% in many integrated sites. Average time from HIV testing to ART initiation is significantly faster and adherence to ART is better in smaller peripheral clinics than in vertical day hospitals. Integration has also improved other non‐HIV aspects of primary health care. Conclusion The integration approach enables the public sector PHC system to test more patients for HIV, place more patients on ART more quickly and efficiently, reduce loss‐to‐follow‐up, and achieve greater geographic HIV care coverage compared to the vertical model. Through the integration process, HIV resources have been used to rehabilitate PHC infrastructure (including laboratories and pharmacies), strengthen supervision, fill workforce gaps, and improve patient flow between services and facilities in ways that can benefit all programs. Using aid resources to integrate and better link HIV care with existing services can strengthen wider PHC systems.

ABSTRACT: The aim of this work was to describe the epidemiological, clinical and pathological aspects of spontaneous poisoning by Merremia macrocalyx in cattle in the Pernambuco state, northeastern Brazil, and to experimentally replicate the poisoning by this plant. To determine the occurrence of poisonings, 30 farms were visited in six municipalities at the Forest Zone of Pernambuco. The plant was found in nine farms, in which history of plant poisoning in cattle, and occasionally in sheep were also reported. Three outbreaks of spontaneous poisonings in cattle were studied. To replicate the disease experimentally, two steers received a single dose of 60g/kg and two steers received 80g/kg of the fresh leaves of M. macrocalyx in the trough for spontaneous ingestion. Two steers were also used as a control group. The main clinical signs observed in spontaneous cases consisted of restlessness, bloat, polyuria, diarrhea, and death within 48 to 72 hours after the onset of clinical signs. Cattle experimentally poisoned presented similar clinical signs to those observed in spontaneous cases. Gross lesions consisted of dryness and impaction of the rumen, omasum and reticulum contents. Abomasal content was fluid, the mucosa was hyperemic, with swollen folds and multiple ulcers. Similar lesions were observed in duodenum mucosae. Histologically, lesions observed in the abomasum and duodenum mucosa consisted of necrosis, hemorrhage and inflammatory infiltration of neutrophils and lymphocytes. The wide distribution and palatability of this plant, associated with the high sensitivity of the bovine species verified in this experiment, highlight the importance of this plant in spontaneous cases of poisoning in cattle.

Increasing evidence shows extracellular vesicles (EVs) are primarily responsible for the beneficial effects of cell-based therapies. EVs derived from mesenchymal stromal cells (MSCs) show promise as a source of EVs for cell-free therapies. The human placental fetal-maternal interface is a rich and abundant source of MSCs from which EVs can be isolated. This study focusses on chorionic MSCs (CMSC) located on the fetal aspect of the interface and decidual MSCs (DMSC) on the maternal aspect. This study used Ligand-based Exosome Affinity Purification (LEAP) chromatography to isolate EVs from well-characterized placental hTERT-transduced CMSC29 and DMSC23 cell lines, which retain many important stem cell-like properties of primary CMSC and DMSC, respectively. After initial biophysical characterization of the EVs isolated from each cell line, the biological activities and the protein, lipid and small RNA contents of CMSC29-EVs and DMSC23-EVs were compared and assessed. LEAP-purified EVs from both sources were validated at the biophysical level by Spectradyne, Cryo-Transmission Electron Microscopy (Cryo-TEM), and Western blot analysis. EVs from each type were labelled with the live cell stain PKH26 and their in vitro uptake and internalization by human dermal fibroblast cells was assessed, as well as their phosphorylation of the protein kinase B/AKT (AKT) pathway. The protein and lipid contents were analyzed by mass spectrometry and the nucleic acid content by RNA sequencing (RNA-seq). Lastly, the biological activities of the EVs were evaluated in a BioMAP® Diversity PLUS® screen system across a panel of 12 human primary cell-based systems and in vitro cell proliferation. EVs isolated from both DMSC23 and CMSC29 significantly increased proliferation of fibroblasts and showed phosphorylation of the AKT pathway. Protein mass spectrometry analysis identified a large number of proteins including cell surface receptors, cytokines, chemokines, matrix molecules and enzymes in both EV types. Lipidomic analysis identified species including phosphatidylcholine, triacylglycerides and diacylglycerides in both DMSC23 and CMSC29-derived EVs. There were some significant differences in identified microRNAs (miRNAs) between the two EV types. The top differentially expressed miRNAs between the two EV types show pathways association with matrix interaction, transcriptional regulation, proliferation, cellular protein modification processes, and vasculogenesis. Differences were also detected between DMSC23- and CMSC29-EVs in the biological activity they displayed in the BioMAP® Diversity PLUS® screen., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Aim: Physical exercise triggers the secretion of small extracellular vesicles (sEVs) into the circulation in humans, enabling signalling crosstalk between tissues. Exercise-derived EVs and their cargo have been proposed to mediate adaptations to exercise; however, our understanding of how exercise-derived EV protein cargo is modulated by factors such as aerobic fitness and age of an individual is currently unknown. Here, we examined the circulating sEV proteome following aerobic exercise in healthy males of different ages and aerobic fitness to understand exercise-induced EV response during the aging process., Methods: Twenty-eight healthy men completed a bout of 20-min cycling exercise at 70% estimated VO 2peak . Small EVs were isolated from blood samples collected before and immediately after exercise, and then quantified using particle analysis and Western blotting. Small EV proteome was examined using quantitative proteomic analysis., Results: We identified a significant increase in 13 proteins in small plasma EVs following moderate-to-vigorous intensity exercise. We observed distinct changes in sEV proteome after exercise in young, mature, unfit, and fit individuals, highlighting the impact of aerobic fitness and age on sEV protein secretion. Functional enrichment and pathway analysis identified that the majority of the significantly altered sEV proteins are associated with the innate immune system, including proteins known to be damage-associated molecular patterns (DAMPs)., Conclusion: Together, our findings suggest that exercise-evoked acute stress can positively challenge the innate immune system through the release of signalling molecules such as DAMPs in sEVs, proposing a novel EV-based mechanism for moderate-to-vigorous intensity exercise in immune surveillance pathways., (© 2024 The Authors. Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

The SLC9C1 gene (which encodes the NHE10 protein) is essential for male fertility in both mice and humans, however the epigenetic mechanisms regulating its testis/sperm-specific gene expression have yet to be studied. Here we identify and characterize DNA regulatory elements of the SLC9C1 gene across three mammalian species: mouse, rat, and human. First, in silico analysis of these mammalian SLC9C1 genes identified a CpG island located upstream of the transcription start site in the same relative position in all three genes. Further analysis reveals that this CpG island behaves differently, with respect to gene regulatory activity, in the mouse SLC9C1 gene than it does in the rat and human SLC9C1 gene. The mouse SLC9C1 CpG island displays strong promoter activity by itself and seems to have a stronger gene regulatory effect than either the rat or human SLC9C1 CpG islands. While the function of the upstream SLC9C1 CpG island may be divergent across the three studied species, it appears that the promoters of these three mammalian SLC9C1 genes share similar DNA methylation-sensitive regulatory mechanisms. All three SLC9C1 promoter regions are differentially methylated in lung and testis, being more hypermethylated in lung relative to the testis, and DNA sequence alignments provide strong evidence of primary sequence conservation. Luciferase assays reveal that in vitro methylation of constructs containing different elements of the three SLC9C1 genes largely exhibit methylation-sensitive promoter activity (reduced promoter activity when methylated) in both HEK 293 and GC-1spg cells. In total, our data suggest that the DNA methylation-sensitive elements of the mouse, rat, and human SLC9C1 promoters are largely conserved, while the upstream SLC9C1 CpG island common to all three species seems to perform a different function in mouse than it does in rat and human. This work provides evidence that while homologous genes can all be regulated by DNA methylation-dependent epigenetic mechanisms, the location of the specific cis-regulatory elements responsible for this regulation can differ across species., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Na + /H + exchangers (NHEs) are known to be important regulators of pH in multiple intracellular compartments of eukaryotic cells. Sperm function is especially dependent on changes in pH and thus it has been postulated that NHEs play important roles in regulating the intracellular pH of these cells. For example, in order to achieve fertilization, mature sperm must maintain a basal pH in the male reproductive tract and then alkalize in response to specific signals in the female reproductive tract during the capacitation process. Eight NHE isoforms are expressed in mammalian testis/sperm: NHE1, NHE3, NHE5, NHE8, NHA1, NHA2, NHE10, and NHE11. These NHE isoforms are expressed at varying times during spermatogenesis and localize to different subcellular structures in developing and mature sperm where they contribute to multiple aspects of sperm physiology and male fertility including proper sperm development/morphogenesis, motility, capacitation, and the acrosome reaction. Previous work has provided evidence for NHE3, NHE8, NHA1, NHA2, and NHE10 being critical for male fertility in mice and NHE10 has recently been shown to be essential for male fertility in humans. In this article we review what is known about each NHE isoform expressed in mammalian sperm and discuss the physiological significance of each NHE isoform with respect to male fertility.

The SLC9C1 gene (which encodes the NHE10 protein) is essential for male fertility in both mice and humans, however the epigenetic mechanisms regulating its testis/sperm-specific gene expression have yet to be studied. Here we identify and characterize DNA regulatory elements of the SLC9C1 gene across three mammalian species: mouse, rat, and human. First, in silico analysis of these mammalian SLC9C1 genes identified a CpG island located upstream of the transcription start site in the same relative position in all three genes. Further analysis reveals that this CpG island behaves differently, with respect to gene regulatory activity, in the mouse SLC9C1 gene than it does in the rat and human SLC9C1 gene. The mouse SLC9C1 CpG island displays strong promoter activity by itself and seems to have a stronger gene regulatory effect than either the rat or human SLC9C1 CpG islands. While the function of the upstream SLC9C1 CpG island may be divergent across the three studied species, it appears that the promoters of these three mammalian SLC9C1 genes share similar DNA methylation-sensitive regulatory mechanisms. All three SLC9C1 promoter regions are differentially methylated in lung and testis, being more hypermethylated in lung relative to the testis, and DNA sequence alignments provide strong evidence of primary sequence conservation. Luciferase assays reveal that in vitro methylation of constructs containing different elements of the three SLC9C1 genes largely exhibit methylation-sensitive promoter activity (reduced promoter activity when methylated) in both HEK 293 and GC-1spg cells. In total, our data suggest that the DNA methylation-sensitive elements of the mouse, rat, and human SLC9C1 promoters are largely conserved, while the upstream SLC9C1 CpG island common to all three species seems to perform a different function in mouse than it does in rat and human. This work provides evidence that while homologous genes can all be regulated by DNA methylation-dependent epigenetic mechanisms, the location of the specific cis-regulatory elements responsible for this regulation can differ across species., Competing Interests: Conflict of Interest The authors declare no conflict of interest.

The release of growth factors, cytokines and extracellular matrix modifiers by activated platelets is an important step in the process of healthy wound healing. Extracellular vesicles (EVs) released by activated platelets carry this bioactive cargo in an enriched form, and may therefore represent a potential therapeutic for the treatment of delayed wound healing, such as chronic wounds. While EVs show great promise in regenerative medicine, their production at clinical scale remains a critical challenge and their tolerability in humans is still to be fully established. In this work, we demonstrate that Ligand-based Exosome Affinity Purification (LEAP) chromatography can successfully isolate platelet EVs (pEVs) of clinical grade from activated platelets, which retain the regenerative properties of the parent cell. LEAP-isolated pEVs display the expected biophysical features of EV populations and transport essential proteins in wound healing processes, including insulin growth factor (IGF) and transforming growth factor beta (TGF-ß). In vitro studies show that pEVs induce proliferation and migration of dermal fibroblasts and increase dermal endothelial cells' angiogenic potential, demonstrating their wound healing potential. pEV treatment activates the ERK and Akt signalling pathways within recipient cells. In a first-in-human, double-blind, placebo-controlled, phase I clinical trial of healthy volunteer adults, designed primarily to assess safety in the context of wound healing, we demonstrate that injections of LEAP-purified pEVs in formulation buffer are safe and well tolerated (Plexoval II study, ACTRN12620000944932). As a secondary objective, biological activity in the context of wound healing rate was assessed. In this cohort of healthy participants, in which the wound bed would not be expected to be deficient in the bioactive cargo that pEVs carry, all wounds healed rapidly and completely and no difference in time to wound closure of the treated and untreated wounds was observed at the single dose tested. The outcomes of this study evidence that pEVs manufactured through the LEAP process can be injected safely in humans as a potential wound healing treatment, and warrant further study in clinical trials designed expressly to assess therapeutic efficacy in patients with delayed or disrupted wound healing., (© 2023 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Na + /H + exchangers (NHEs) are a family of ion transporters that regulate the pH of various cell compartments across an array of cell types. In eukaryotes, NHEs are encoded by the SLC9 gene family comprising 13 genes. SLC9C2, which encodes the NHE11 protein, is the only one of the SLC9 genes that is essentially uncharacterized. Here, we show that SLC9C2 exhibits testis/sperm-restricted expression in rats and humans, akin to its paralog SLC9C1 (NHE10). Similar to NHE10, NHE11 is predicted to contain an NHE domain, a voltage sensing domain, and finally an intracellular cyclic nucleotide binding domain. An immunofluorescence analysis of testis sections reveals that NHE11 localizes with developing acrosomal granules in spermiogenic cells in both rat and human testes. Most interestingly, NHE11 localizes to the sperm head, likely the plasma membrane overlaying the acrosome, in mature sperm from rats and humans. Therefore, NHE11 is the only known NHE to localize to the acrosomal region of the head in mature sperm cells. The physiological role of NHE11 has yet to be demonstrated but its predicted functional domains and unique localization suggests that it could modulate intracellular pH of the sperm head in response to changes in membrane potential and cyclic nucleotide concentrations that are a result of sperm capacitation events. If NHE11 is shown to be important for male fertility, it will be an attractive target for male contraceptive drugs due to its exclusive testis/sperm-specific expression.

Introduction: In preeclampsia (PE), inadequate remodelling of spiral arterioles in the decidua basalis causes oxidative stress and subsequent increased release of antiangiogenic soluble endoglin (sENG) into the maternal circulation. Decidual mesenchymal stem/stromal cells (DMSCs) reside adjacent to endothelial cells in this vascular niche. Surprisingly, DMSCs express membrane-bound ENG (CD105). PE-affected DMSCs (PE-DMSCs) are abnormal and due to reduced extravillous invasion, more of them are present, but the significance of this is not known., Methods: DMSCs were isolated and characterised from normotensive control and severe-PE placentae. Extracellular vesicle (EV) types, shed microvesicles (sMV) and exosomes, were isolated from DMSC conditioned media (DMSC CM ), respectively. Secretion of ENG by DMSCs was assessed by ELISA of DMSC CM , with and without EV depletion. The effects of reducing ENG concentration, by blocking antibody, on human umbilical vein endothelial cell (HUVEC) attachment were assessed by xCELLigence real-time functional assays., Results: ENG was detected in DMSC CM and these levels significantly decreased when depleted of exosomes and sMV. There was no significant difference in the amount of ENG secreted by control DMSCs and PE-DMSCs. Blocking ENG in concentrated DMSC CM , used to treat HUVECs, improved endothelial cell attachment., Discussion: In normotensive pregnancies, DMSC secretion of ENG likely has a beneficial effect on endothelial cells. However, in PE pregnancies, shallow invasion of the spiral arterioles exposes more PE-DMSC derived sources of ENG (soluble and EV). The presence of these PE-DMSCs in the vascular niche contributes to endothelial cell dysfunction., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Aging is associated with a loss of metabolic homeostasis, with cofactors such as nicotinamide adenine dinucleotide (NAD + ) declining over time. The decrease in NAD + production has been linked to the age-related loss of circulating extracellular nicotinamide phosphoribosyltransferase (eNAMPT), the rate-limiting enzyme in the NAD + biosynthetic pathway. eNAMPT is found almost exclusively in extracellular vesicles (EVs), providing a mechanism for the distribution of the enzyme in different tissues. Currently, the physiological cause for the release of eNAMPT is unknown, and how it may be affected by age and physical exercise. Here, we show that release of small EVs into the bloodstream is stimulated following moderate intensity exercise in humans. Exercise also increased the eNAMPT content in EVs, most prominently in young individuals with higher aerobic fitness. Both mature fit and young unfit individuals exhibited a limited increase in EV-eNAMPT release following exercise, indicating that this mechanism is related to both the age and physical fitness of a person. Notably, unfit mature individuals were unable to increase the release of eNAMPT in EVs after exercise, suggesting that lower fitness levels and aging attenuate this important signalling mechanism in the body. EVs isolated from exercising humans containing eNAMPT were able to alter the abundance of NAD + and SIRT1 activity in recipient cells compared to pre-exercise EVs, indicating a pathway for inter-tissue signalling promoted through exercise. Our results suggest a mechanism to limit age-related NAD + decline, through the systemic delivery of eNAMPT via EVs released during exercise., (© 2022 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Background Aims: Extracellular vesicles (EVs) are involved in mediating intercellular communication processes. An important goal within the EV field is the study of the biodistribution of EVs and the identification of their target cells. Considering that EV uptake is assumed to be important for EVs in mediating intercellular communication processes, labeling with fluorescent dyes has emerged as a broadly distributed strategy for the identification of EV target cells and tissues. However, the accuracy and specificity of commonly utilized labeling dyes have not been sufficiently analyzed., Methods: By combining recent advances in imaging flow cytometry for the phenotypic analysis of single EVs and aiming to identify target cells for EVs within therapeutically relevant mesenchymal stromal cell (MSC)-EV preparations, the authors explored the EV labeling efficacy of various fluorescent dyes, specifically carboxyfluorescein diacetate succinimidyl ester, calcein AM, PKH67, BODIPY TR ceramide (Thermo Fisher Scientific, Darmstadt, Germany) and a novel lipid dye called Exoria (Exopharm Limited, Melbourne, Australia)., Results: The authors' analyses qualified Exoria as the only dye that specifically labeled EVs within the MSC-EV preparations. Furthermore, the authors demonstrated that Exoria labeling did not interfere with the immunomodulatory properties of the MSC-EV preparations as tested in a multi-donor mixed lymphocyte reaction assay. Within this assay, labeled EVs were differentially taken up by different immune cell types., Conclusions: Overall, the results qualify Exoria as an appropriate dye for the labeling of EVs derived from the authors' MSC-EV preparations. This study also demonstrates the need for the development of next-generation EV characterization tools that are able to localize and confirm the specificity of EV labeling., Competing Interests: Declaration of Competing Interest BG is a scientific advisory board member of Innovex Therapeutics SL and Mursla Ltd and a founding director of Exosla Ltd. MS and PFJ are employees and shareholders of Exopharm Limited., (Copyright © 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

The use of porous 3D scaffolds for the repair of bone nonunion and osteoporotic bone is currently an area of great interest. Using a combination of thermally-induced phase separation (TIPS) and 3D-plotting (3DP), we have generated hierarchical 3DP/TIPS scaffolds made of poly(lactic-co-glycolic acid) (PLGA) and nanohydroxyapatite (nHA). A full factorial design of experiments was conducted, in which the PLGA and nHA compositions were varied between 6-12% w/v and 10-40% w/w, respectively, totaling 16 scaffold formulations with an overall porosity ranging between 87%-93%. These formulations included an optimal scaffold design identified in our previous study. The internal structures of the scaffolds were examined using scanning electron microscopy and microcomputed tomography. Our optimal scaffold was seeded with MC3T3-E1 murine preosteoblastic cells and subjected to cell culture inside a tissue culture dish and a perfusion bioreactor. The results were compared to those of a commercial CellCeram™ scaffold with a composition of 40% β-tricalcium phosphate and 60% hydroxyapatite (β-TCP/HA). Media flow within the macrochannels of 3DP/TIPS scaffolds was modeled in COMSOL software in order to fine tune the wall shear stress. CyQUANT DNA assay was performed to assess cell proliferation. The normalized number of cells for the optimal scaffold was more than twofold that of CellCeram™ scaffold after two weeks of culture inside the bioreactor. Despite the substantial variability in the results, the observed improvement in cell proliferation upon culture inside the perfusion bioreactor (vs. static culture) demonstrated the role of macrochannels in making the 3DP/TIPS scaffolds a promising candidate for scaffold-based tissue engineering., Competing Interests: Declarations Conflicts of interest/Competing interests: The authors declare that there are no conflicts of interest or competing interests.

Objective: Our study aimed to quantify structural changes in relation to metabolic abnormalities in the cerebellum, thalamus, and parietal cortex of patients with late-onset GM2-gangliosidosis (LOGG), which encompasses late-onset Tay-Sachs disease (LOTS) and Sandhoff disease (LOSD)., Methods: We enrolled 10 patients with LOGG (7 LOTS, 3 LOSD) who underwent a neurological assessment battery and 7 age-matched controls. Structural MRI and MRS were performed on a 3 T scanner. Structural volumes were obtained from FreeSurfer and normalized by total intracranial volume. Quantified metabolites included N-acetylaspartate (NAA), choline (Cho), myo-inositol (mI), creatine (Cr), and combined glutamate-glutamine (Glx). Metabolic concentrations were corrected for partial volume effects., Results: Structural analyses revealed significant cerebellar atrophy in the LOGG cohort, which was primarily driven by LOTS patients. NAA was lower and mI higher in LOGG, but this was also significantly driven by the LOTS patients. Clinical ataxia deficits (via the Scale for the Assessment and Rating of Ataxia) were associated with neuronal injury (via NAA), neuroinflammation (via mI), and volumetric atrophy in the cerebellum., Interpretation: The decrease of NAA in the cerebellum suggests that, in addition to cerebellar atrophy, there is ongoing impaired neuronal function and/or loss, while an increase in mI indicates possible neuroinflammation in LOGG (more so within the LOTS subvariant). Quantifying cerebellar atrophy in relation to neurometabolic differences in LOGG may lead to improvements in assessing disease severity, progression, and pharmacological efficacy. Lastly, additional neuroimaging studies in LOGG are required to contrast LOTS and LOSD more accurately., Competing Interests: Declaration of Competing Interest O.E. Rowe, D. Rangaprakash, A. Weerasekera, N. Godbole, E. Haxton, P.F. James, C.D. Stephen, R.L. Barry, F.S. Eichler, and E-M. Ratai report no conflicts of interest related to the manuscript., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Gait and balance difficulties are among the most common clinical manifestations in adults with X-linked adrenoleukodystrophy, but little is known about the contributions of sensory loss, motor dysfunction, and postural control to gait dysfunction and fall risk. Objective: To quantify gait and balance deficits in both males and females with adrenoleukodystrophy and evaluate how environmental perturbations (moving surfaces and visual surrounds) affect balance and fall risk. Methods: We assessed sensory and motor contributions to gait and postural instability in 44 adult patients with adrenoleukodystrophy and 17 healthy controls using three different functional gait assessments (25 Foot Walk test, Timed Up and Go, and 6 Minute Walk test) and computerized dynamic posturography. Results: The median Expanded Disability Status Scale score for the patient cohort was 3.0 (range 0.0-6.5). Both males and females with adrenoleukodystrophy showed impairments on all three functional gait assessments relative to controls ( P < 0.001). Performance on walking tests and Expanded Disability Status Scale scores correlated with incidence of falls on computerized dynamic posturography, with the 25 Foot Walk being a moderately reliable predictor of fall risk (area under the ROC curve = 0.7675, P = 0.0038). Conclusion: We demonstrate that gait difficulties and postural control deficits occur in patients with adrenoleukodystrophy, albeit at an older age in females. Postural deficits were aggravated by eyes closed and dynamic conditions that rely on vestibular input, revealing challenges to the interplay of motor, sensory and vestibular circuitry in adrenoleukodystrophy., Competing Interests: FE reports sponsored research contracts with Minoryx Therapeutics and consultancies with Autobahn Therapeutics, Minoryx Therapeutics, and SwanBio Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Godbole, Sadjadi, DeBono, Grant, Kelly, James, Stephen, Balkwill, Lewis and Eichler.)

This paper reports on the hybrid process we have used for producing hierarchical scaffolds made of poly(lactic-co-glycolic) acid (PLGA) and nanohydroxyapatite (nHA), analyzes their internal structures via scanning electron microscopy, and presents the results of our in vitro proliferation of MC3T3-E1 cells and alkaline phosphatase activity (ALP) for 0 and 21 days. These scaffolds were produced by combining additive manufacturing (AM) and thermally induced phase separation (TIPS) techniques. Slow cooling at a rate of 1.5 °C/min during the TIPS process was used to enable a uniform temperature throughout the scaffolds, and therefore, a relatively uniform pore size range. We produced ten different scaffold compositions and topologies in this study. These scaffolds had macrochannels with diameters of ∼300 µm, ∼380 µm, and ∼460 µm, generated by the extraction of embedded porous 3D-plotted polyethylene glycol (PEG) matrices. The other experimental factors included different TIPS temperatures (-20 °C, -10 °C, and 0 °C), as well as varying PLGA concentrations (8%, 10%, and 12% w/v) and nHA content (0%, 10%, and 20% w/w). Our results indicated that almost all these macro/microporous scaffolds supported cell growth over the period of 21 days. Nevertheless, significant differences were observed among some scaffolds in terms of their support of cell proliferation and differentiation. This paper presents the results of our in vitro cell culture for 0 and 21 days. Our optimal scaffold with a porosity of ∼90%, a modulus of ∼5.2 MPa, and a nHA content of 20% showed a cell adhesion of ∼29% on day 0 and maintained cell proliferation and ALP activity over the 21-day in vitro culture. Hence, the use of additive manufacturing and designed experiments to optimize the scaffold fabrication parameters resulted in superior mechanical properties that most other studies using TIPS.

Ageing and parturition share common pathways, but their relationship remains poorly understood. Decidual cells undergo ageing as parturition approaches term, and these age-related changes may trigger labour. Mesenchymal stem/stromal cells (MSCs) are the predominant stem cell type in the decidua. Stem cell exhaustion is a hallmark of ageing, and thus ageing of decidual MSCs (DMSCs) may contribute to the functional changes in decidual tissue required for term spontaneous labour. Here, we determine whether DMSCs from patients undergoing spontaneous onset of labour (SOL-DMSCs) show evidence of ageing-related functional changes compared with those from patients not in labour (NIL-DMSCs), undergoing Caesarean section. Placentae were collected from term (37-40 weeks of gestation), SOL (n = 18) and NIL (n = 17) healthy patients. DMSCs were isolated from the decidua basalis that remained attached to the placenta after delivery. DMSCs displayed stem cell-like properties and were of maternal origin. Important cell properties and lipid profiles were assessed and compared between SOL- and NIL-DMSCs. SOL-DMSCs showed reduced proliferation and increased lipid peroxidation, migration, necrosis, mitochondrial apoptosis, IL-6 production and p38 MAPK levels compared with NIL-DMSCs (P < 0.05). SOL- and NIL-DMSCs also showed significant differences in lipid profiles in various phospholipids (phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine), sphingolipids (ceramide, sphingomyelin), triglycerides and acyl carnitine (P < 0.05). Overall, SOL-DMSCs had altered lipid profiles compared with NIL-DMSCs. In conclusion, SOL-DMSCs showed evidence of ageing-related reduced functionality, accumulation of cellular damage and changes in lipid profiles compared with NIL-DMSCs. These changes may be associated with term spontaneous labour., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In bone tissue engineering, 3D scaffolds are often designed to have adequate modulus while taking into consideration the requirement for a highly porous network for cell seeding and tissue growth. This paper presents the design optimization of 3D scaffolds made of poly(lactic-co-glycolic) acid (PLGA) and nanohydroxyapatite (nHA), produced by thermally induced phase separation (TIPS). Slow cooling at a rate of 1°C/min enabled a uniform temperature and produced porous scaffolds with a relatively uniform pore size. An I-optimal design of experiments (DoE) with 18 experimental runs was used to relate four responses (scaffold thickness, density, porosity, and modulus) to three experimental factors, namely the TIPS temperature (-20°C, -10°C, and 0°C), PLGA concentration (7%, 10%, and 13% w/v), and nHA content (0%, 15%, and 30% w/w). The response surface analysis using JMP ® software predicted a temperature of -18.3°C, a PLGA concentration of 10.3% w/v, and a nHA content of 30% w/w to achieve a thickness of 3 mm, a porosity of 83%, and a modulus of ~4 MPa. The set of validation scaffolds prepared using the predicted factor levels had a thickness of 3.05 ± 0.37 mm, a porosity of 86.8 ± 0.9 %, and a modulus of 3.57 ± 2.28 MPa.

The limitations in the transport of oxygen, nutrients, and metabolic waste products pose a challenge to the development of bioengineered bone of clinically relevant size. This paper reports the design and characterization of hierarchical macro/microporous scaffolds made of poly(lactic-co-glycolic) acid and nanohydroxyapatite (PLGA/nHA). These scaffolds were produced by combining additive manufacturing (AM) and thermally induced phase separation (TIPS) techniques. Macrochannels with diameters of ~300 μm, ~380 μm, and ~460 μm were generated by embedding porous 3D-plotted polyethylene glycol (PEG) inside PLGA/nHA/1,4-dioxane or PLGA/1,4-dioxane solutions, followed by PEG extraction using deionized (DI) water. We have used an I-optimal design of experiments (DoE) and the response surface analysis (JMP® software) to relate three responses (scaffold thickness, porosity, and modulus) to the four experimental factors affecting the scaffold macro/microstructures ( e.g ., PEG strand diameter, PLGA concentration, nHA content, and TIPS temperature). Our results indicated that a PEG strand diameter of ~380 μm, a PLGA concentration of ~10% w/v, a nHA content of ~10% w/w, and a TIPS temperature around -10°C could generate scaffolds with a porosity of ~90% and a modulus exceeding 4 MPa. This paper presents the steps for the I-optimal design of these scaffolds and reports on their macro/microstructures, characterized using scanning electron microscopy (SEM) and micro-computed tomography (micro-CT)., Competing Interests: Conflict of Interest Disclosure The authors declare no competing financial interest.

The α4 Na,K-ATPase is a sperm-specific protein essential for sperm motility and fertility yet little is known about the mechanisms that regulate its expression in germ cells. Here, the potential involvement of DNA methylation in regulating the expression of this sperm-specific protein is explored. A single, intragenic CpG island (Mα4-CGI) was identified in the gene encoding the mouse α4 Na,K-ATPase (Atp1a4), which displayed reduced methylation in mouse sperm (cells that contain α4) compared to mouse kidney (tissue that lacks α4 expression). Unlike the intragenic CGI, the putative promoter (the -700 to +200 region relative to the transcriptional start site) of Atp1a4 did not show differential methylation between kidney and sperm nevertheless it did drive methylation-dependent reporter gene expression in the male germ cell line GC-1spg. Furthermore, treatment of GC-1spg cells with 5-aza2-deoxycytidine led to upregulation of the α4 transcript and decreased methylation of both the Atp1a4 promoter and the Mα4-CGI. In addition, Atp1a4 expression in mouse embryonic stem cells deficient in DNA methyltransferases suggests that both maintenance and de novo methylation are involved in regulating its expression. In an attempt to define the regulatory function of the Mα4-CGI, possible roles of the Mα4-CGI in regulating Atp1a4 expression via methylation-dependent transcriptional elongation inhibition in somatic cells and via its ability to repress promoter activity in germ cells were uncovered. In all, our data suggests that both the promoter and the intragenic CGI could combine to provide multiple modes of regulation for optimizing the Atp1a4 expression level in a cell type-specific manner., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Mesenchymal stem cells (MSCs) have been the subject of many studies in recent years, ranging from basic science that looks into MSCs properties to studies that aim for developing bioengineered tissues and organs. Adult bone marrow-derived mesenchymal stem cells (BM-MSCs) have been the focus of most studies due to the inherent potential of these cells to differentiate into various cell types. Although, the discovery of induced pluripotent stem cells (iPSCs) represents a paradigm shift in our understanding of cellular differentiation. These cells are another attractive stem cell source because of their ability to be reprogramed, allowing the generation of multiple cell types from a single cell. This paper briefly covers various types of stem cell sources that have been used for tissue engineering applications, with a focus on bone regeneration. Then, an overview of some recent studies making use of MSC-seeded 3D scaffold systems for bone tissue engineering has been presented. The emphasis has been placed on the reported scaffold properties that tend to improve MSCs adhesion, proliferation, and osteogenic differentiation outcomes.