Biology Open

Fluorescent in situ hybridization (FISH) enables highly sensitive, high-resolution detection of gene transcripts. Moreover, by employing multiple probes, this technique allows for multiplexed, simultaneous detection of distinct gene expression patterns spatiotemporally, making it a valuable spatial transcriptomics approach. Owing to these advantages, FISH techniques are rapidly being adopted across diverse areas of basic biology. However, conventional protocols often rely on volatile, toxic reagents such as formalin or methanol, posing potential health risks to researchers. Here, we present a safer protocol that replaces these chemicals with low-toxicity alternatives, without compromising the high detection sensitivity of FISH. We validated this protocol using both in situ hybridization chain reaction (HCR) and signal amplification by exchange reaction (SABER)-FISH in frozen sections of various model organisms, including mouse (Mus musculus), amphibians (Xenopus laevis and Pleurodeles waltl), and medaka (Oryzias latipes). Our results demonstrate successful multiplexed detection of morphogenetic and cell-type marker genes in these model animals using this safer protocol. The protocol has the additional advantage of requiring no proteolytic enzyme treatment, thus preserving tissue integrity. Furthermore, we show that this protocol is fully compatible with EGFP immunostaining, allowing for the simultaneous detection of mRNAs and reporter proteins in transgenic animals. This protocol retains the benefits of highly sensitive, multiplexed, and multimodal detection afforded by integrating in situ HCR and SABER-FISH with immunohistochemistry, while providing a safer option for researchers, thereby offering a valuable tool for basic biology.

How embryonic cells generate large clones of cells in the adult represents a fundamental question in biology. Here, using melanocyte stem cells (McSCs) in the zebrafish as a model, we explore the function of the master melanocyte transcription factor (MITF) in safeguarding McSCs in embryonic development and their potential to pigment large clones in the adult. MITF is well known is for its role in the specification of melanoblasts from the neural crest (NC) and their differentiation into melanocytes, yet little is known about how this activity shapes the stem cell lineages. Here, we use live imaging coupled with single-cell transcriptomics and lineage tracing to show that MITF (mitfa in zebrafish) protects the melanocyte stem cell (McSC) fate in zebrafish. Utilizing a temperature sensitive mitfavc7 mutant, we show loss of Mitfa leads to a surprising premature and aberrant expansion of McSC progeny at the niche during embryogenesis, coupled with novel emergent transcriptional cell states. Linage tracing of McSCs from the embryonic to juvenile stages reveals Mitfa activity is subsequently required in regeneration by Schwann cell-like and melanocyte stem cell progenitors that serve as a reservoir for fast-responding pigment progenitors. Thus, the impact of Mitfa loss on the melanocyte lineage is cell-state and stage-specific. The emergent cell states upon mitfa loss may have important implications for our understanding the loss of MITF activity in human genetic disease and melanoma.

Altered iron homeostasis has long been implicated in Parkinson's Disease (PD), although the mechanisms have not been clear. Given the critical role of PD-related activating mutations in LRRK2 (leucine-rich repeat protein kinase 2) within membrane trafficking pathways we examined the impact of a homozygous mutant LRRK2G2019S on iron homeostasis within the RAW macrophage cell line with high iron capacity. Proteomics analysis revealed a dysregulation of iron-related proteins in steady state with highly elevated levels of ferritin light chain and a reduction of ferritin heavy chain. LRRK2G2019S mutant cells showed efficient ferritinophagy upon iron chelation, but upon iron overload there was a near complete block in the degradation of the ferritinophagy adaptor NCOA4. These conditions lead to an accumulation of phosphorylated Rab8 at the plasma membrane, which is selectively inhibited by LRRK type II kinase inhibitors. Iron overload then leads to increased oxidative stress and ferroptotic cell death. These data implicate LRRK2 as a key regulator of iron homeostasis and point to the need for an increased focus on the mechanisms of iron dysregulation in PD.

BackgroundDental caries and periodontal disease represent the most prevalent global oral health conditions, collectively affecting several billion people. The diagnostic interpretation of dental radiographs, a cornerstone of modern dentistry, is associated with considerable inter-observer variability. In routine clinical practice, clinicians are required to evaluate a high volume of radiographic images daily, a cognitively demanding task in which diagnostic fatigue, time constraints, and the inherent complexity of overlapping anatomical structures can lead to the inadvertent oversight of early-stage pathologies. Artificial intelligence (AI) offers a transformative opportunity to augment clinical decision-making by providing rapid, objective, and consistent radiographic analysis, thereby serving as a tireless adjunct capable of flagging findings that may be missed during routine human inspection. MethodsThis study developed and validated a deep learning system for the automated detection of dental caries and alveolar bone loss using a dataset of 1,063 periapical and bitewing radiographs. Two separate YOLOv8s object detection models were trained and evaluated using a rigorous 5-fold cross-validation methodology. To align with the clinical use-case of a screening tool where high sensitivity is paramount, a custom image-level evaluation criterion was employed: a true positive was recorded if any predicted bounding box had a Jaccard Index (IoU) > 0 with any ground truth annotation. Model performance was systematically evaluated at confidence thresholds of 0.10 and 0.05. ResultsAt a confidence threshold of 0.05, the caries detection model achieved a mean precision of 84.41% ({+/-}0.72%), recall of 85.97% ({+/-}4.72%), and an F1-score of 85.13% ({+/-}2.61%). The alveolar bone loss model demonstrated exceptionally high performance, with a mean precision of 95.47% ({+/-}0.94%), recall of 98.60% ({+/-}0.49%), and an F1-score of 97.00% ({+/-}0.46%). ConclusionThe YOLOv8-based models demonstrated high accuracy and high sensitivity for detecting dental caries and alveolar bone loss on periapical radiographs. The system shows significant potential as a reliable automated assistant for dental practitioners, helping to improve diagnostic consistency, reduce the risk of missed pathology, and ultimately enhance the standard of patient care.

Background and Objectives: We report the first intraoperative deployment of a real-time machine vision system in neurosurgery, derived from our previous anatomical detection work, automatically identifying structures during endoscopic endonasal surgery. Existing systems demonstrate promising performance in offline anatomical recognition, yet so far none have been implemented during live operations. Methods: A real-time anatomy detection model was trained using the YOLOv8 architecture (Ultralytics). Following training completion in the PyTorch environment, the model was exported to ONNX format and further optimized using the NVIDIA TensorRT engine. Deployment was carried out using the NVIDIA Holoscan SDK, the system ran on an NVIDIA Clara AGX developer kit. We used the model for real-time recognition of intraoperative anatomical structures and compared it with the same video labelled manually as reference. Model performance was reported using the average precision at an intersection-over-union threshold of 0.5 (AP50). Furthermore, end-to-end delay from frame acquisition to the display of the annotated output was measured. Results: A mean AP50 of 0.56 was achieved. The model demonstrated reliable detection of the most relevant landmarks in the transsphenoidal corridor. The mean end-to-end latency of the model was 47.81 ms (median 46.57 ms). Conclusion: For the first time, we demonstrate that clinical-grade, real-time machine-vision assistance during neurosurgery is feasible and can provide continuous, automated anatomical guidance from the surgical field. This approach may enhance intraoperative orientation, reduce cognitive load, and offer a powerful tool for surgical training. These findings represent an initial step toward integrating real-time AI support into routine neurosurgical workflows.

BackgroundPeriodontal disease (PD) and diabetes mellitus (DM) have a well-established bidirectional relationship, affecting glycaemic control and chronic disease outcomes. However, the extent to which medical training supports physician awareness of this association remains unclear especially in resource-limited settings. ObjectiveTo assess exposure to oral health education and to identify predictors of awareness of PD-DM association among physicians. MethodsA cross-sectional study was conducted among 146 physicians managing diabetic patients at a tertiary teaching hospital in Ghana. A structured questionnaire assessed exposure to oral health education, periodontal disease knowledge (score range 0-5), and awareness using a 5-item Likert scale (score range 5-25). Multivariable linear regression identified predictors of awareness. ResultsAlthough 62.1% reported exposure to oral health content during undergraduate training, 59.2% rated its quality as poor. Mean awareness score was 20.6 (SD=2.8). Awareness was independently predicted by years of professional experience (p < 0.001) and periodontal disease knowledge (p = 0.008), but not by structured oral health curriculum exposure. ConclusionAwareness of the PD-DM link was high but was not explained by formal educational exposure. Awareness appears to develop through knowledge of PD and professional experience, suggesting a gap between curricular exposure and competency.

IntroductionComparison of rectal cancer characteristics in Pakistani Americans and native Pakistanis remains poorly investigated, as migrant studies have predominantly concentrated on East and Southeast Asian groups. This research aims to compare clinicopathological characteristics between the two groups. We hypothesize that significant differences will exist between these cohorts, mediated by gene-environment interactions. MethodsThis was a retrospective cohort study utilizing two multi-institutional databases to identify adult patients with rectal cancer: the National Cancer Database in the U.S (2018-2022) and the Rectal Cancer Surgery and Epidemiology Study in Pakistan (2020-2021). Non-Hispanic Whites (NHWs) were included as a reference population for comparative analysis. Clinicopathological characteristics were compared using Wilcoxon rank-sum and chi-square tests. ResultsA total of 523 Pakistani Americans and 608 native Pakistanis were included in the study. The median age at diagnosis was 57 years in Pakistani Americans (IQR 48-68), 42 years (IQR 33-54) in native Pakistanis and 63 years in NHWs (IQR 54-73) (p < 0.001). Native Pakistanis presented with early-stage disease less often than Pakistani Americans and NHWs (5.3%, 25.1%, and 20.5%, respectively; p < 0.001) and had markedly higher rates of signet cell carcinoma (20.1%, 0.6%, and 0.4%, respectively; p < 0.001) and poorly differentiated tumors (29.0%, 10.4%, and 11.4%, respectively; p < 0.001). ConclusionsThis study found that Native Pakistanis with rectal cancer presented at a younger age and with more aggressive tumor characteristics compared to both Pakistani Americans and NHWs. Notably, Pakistani Americans displayed a distinct clinical profile, intermediate between both groups.

Waldenstrom macroglobulinemia (WM) is a rare indolent neoplasm characterized by presence of more than 10% lymphoid cells in BM that exhibit plasmacytoid or plasma cell differentiation that secretes an IgM monoclonal protein. This is a retrospective analysis of 89 patients of WM that describes the clinical and laboratory characteristics, treatment patterns and outcome of patients of WM. The median age of the entire cophort was 66 years with male predominance (67.4%). Most common presentations were symptoms pertaining to anemia (77.5%) and constitutional symptoms (33.7%). Median bone marrow lymphoplasmacytic cells were 41%. Positivity for MYD88 and CXCR4 mutations were seen in 81.8% and 2.4% cases. BR was the most common regimen used (52.8%). Overall response rates were seen at 87.8%. Median overall survival, progression free survival and time to next treatment is 8.49 years, 2.15 years and 3.88 years. BR regimen was associated with highest event free survival.

Objectives: For suspected hip fractures, prehospital protocols directing patients to an orthopaedic centre rather than the nearest emergency department (ED) could reduce time-to-surgery but may impact EMS travel burden. This study evaluates the impact of transfer protocols by quantifying transport to hospitals from long term care (LTC) facilities across Ontario. Methods: A retrospective cross-sectional analysis of all Ontario LTC facilities and hospitals was performed. Two protocols were modeled: standard transfer to the nearest ED with subsequent transfer if required, and selective transfer based on Collingwood Hip Fracture Rule prehospital screening1 directly to the nearest orthopaedic services (orthoED). Median one-way travel distances were calculated from Google Maps. Results: In Ontario, 15.4% of LTC residents require hospital destination decisions because their nearest ED lacks orthopaedic services; for these facilities, median distances were 2.7km to the ED and 36.0km to the orthoED. Among the 52 LTC facilities where selective transfer was distance-optimal, it substantially reduced travel for patients with hip fracture (31.1km vs 49.6km; P<.01) while only modestly increasing travel for patients without hip fracture. Where standard transfer was distance-optimal, little travel difference was noted for patients with hip fracture, however false positive screened patients traveled significantly further to an orthoED. Greatest negative consequences of selective transfer lie in the 1.3% of residents living farthest (>100km) from an orthoED. Conclusions: EMS direct transportation to hospitals with orthopaedics may improve hip fracture care but can increase EMS burden due to patients identified falsely as having a hip fracture, particularly in remote communities.

Background: Reusable menstrual products provide sustainable and cost effective alternatives to disposable sanitary products; however, their adoption remains limited, even among healthcare professionals. Objectives: To assess awareness, knowledge, perceptions, and utilisation of reusable menstrual products among female medical students and healthcare professionals, and to identify predictors of willingness and use. Design: Cross sectional analytical study. Setting: An online survey was conducted among female medical students and healthcare professionals in Nigeria. Participants: A total of 203 female respondents aged 15 to 55 years. Intervention: Not applicable. Primary Outcome Measures: Utilisation of reusable menstrual products and willingness to adopt their use. Secondary Outcome Measures: Awareness, knowledge, perceptions, and barriers. Methods: Data were collected using a structured questionnaire and analysed using descriptive statistics, chi square tests, and logistic regression. Results: Awareness was high (96.06%), but utilisation was low, with 5.42% ever using and 4.43% currently using reusable products. About 31.53% were willing to use them. Respondent type was not associated with willingness (p = 0.735), although healthcare professionals had higher knowledge (p = 0.024). Positive perception predicted willingness (AOR = 7.58, 95% CI: 3.18 to 18.03, p < 0.001). Good knowledge (AOR = 14.96, p = 0.014) and increasing age (AOR = 1.28, p = 0.004) predicted utilisation. Conclusion: Despite high awareness, utilisation remains low. Perception influences willingness, while knowledge drives use. Targeted behavioural and educational interventions are needed. Keywords: Menstrual hygiene, reusable menstrual products, menstrual cup, sustainability, healthcare professionals

BackgroundPleuroparenchymal fibroelastosis (PPFE) is a rare, fibrotic lung disease with poor prognosis, usually affecting adults which most commonly occurs idiopathically. Biallelic pathogenic variants in DGUOK cause mitochondrial DNA (mtDNA) depletion syndrome, predominantly affecting infants with severe hepatic and neurological symptoms. Detailed description of pulmonary manifestations with late-onset presentation have not been reported. MethodsWe describe nine patients with PPFE and DGUOK-associated mitochondriopathy. Clinical, radiological, histopathological, and genetic data were systematically collected from all patients. Functional studies, single nucleus RNA sequencing (snRNAseq), immunofluorescence staining, transmission electron microscopy and respiratory chain enzyme activity assays were conducted on patient-derived fibroblasts, muscle or lung tissues. mtDNA content quantification was performed on whole genome sequencing (WGS) data. ResultsAll patients (ages 5-36) presented with progressive dyspnea, weight loss and some with spontaneous pneumothoraces. Chest computed tomography and lung biopsies showed features of PPFE. Biallelic pathogenic DGUOK variants were identified in all patients, seven of them carry an unreported intronic variant leading to mtDNA depletion. snRNAseq of lung tissue from four pediatric patients identified Aberrant Basaloid cells and intermediate cells as their precursor localized at the fibrotic edge. Mitochondrial alterations were identified by electron microscopy. ConclusionPPFE in children and young adults is associated with DGUOK-related mitochondriopathy. For the first time, we demonstrate Aberrant Basaloid cells in pediatric fibrotic lung tissue. Since pulmonary involvement may be underrecognized or misinterpreted and the clinical presentation may not always be typical of a mitochondriopathy, we recommend genetic testing in all patients with PPFE of unknown origin.

BackgroundCarrier screening for inherited genetic disorders can reduce the burden of conditions that lead to childhood morbidity and mortality, including thalassaemia, cystic fibrosis, and spinal muscular atrophy. To be successful, national carrier screening programs should aim to maximise uptake, which may depend on population preferences for screening characteristics. In this study, we aimed to determine how expanded carrier screening in Singapore should be designed based on operational factors including suggested copayments, wait times, and disorders included in screening panels. MethodsWe elicited stated preferences for the design of a hypothetical national carrier screening program with seven attributes from 500 Singaporeans of reproductive age (18 to 54). A discrete choice experiment was applied using 30 choice tasks with 3 alternatives per task, divided between 3 blocks. The mixed multinomial logit model was used to estimate willingness-to-pay for each attribute level. Predicted uptake for three plausible screening programs was assessed, with copayment amounts from $0 to $1,200 in increments of $30. Impact on the annual national budget was calculated as a function of 25,000 expected eligible couples per year. All costs were reported in 2026 SGD. ResultsRespondents showed the strongest preferences for cost, followed by the number of diseases included in the panel, then wait times, with limited impact of remaining attributes. With no copayments, predicted uptake ranged from 85% [95% CI: 83% to 87%] to 90% [88% to 92%] for the basic and utility-maximising screening programs, respectively. This declined to 61% [56% to 66%] and 69% [65% to 73%] and, respectively, at a copayment of $1,200 per test. The model predicted higher uptake if a selection of screening alternatives were available, compared to a single program. The budget impact was highly dependent on population eligibility, copayments, and couples decision-making processes, but was unlikely to exceed $22.5m [$19.0m to $26.6m] per year unless expanded beyond married couples. ConclusionsThere was high predicted demand for carrier screening even as copayments increased. Successful strategies to improve uptake may include reducing copays and wait times, increasing the number of screening options available to prospective parents, and increasing program eligibility beyond pre-conception married couples.

Abstract The ISBAR framework is used to standardize clinical handovers and enhance patient safety. Observational tools based on ISBAR have been developed to assess the completeness of information transfer. However, these instruments have primarily been developed in non-Chinese contexts, and validated Chinese-language observational tools suitable for clinical practice remain limited. In this study, a cross-cultural adaptation and psychometric validation of the ISBAR Structured Handover Observation Tool was conducted, examining its reliability and discriminant validity in Chinese clinical settings. The study was conducted in two phases: cross-cultural adaptation and psychometric evaluation in real-world clinical settings. Content validity was assessed using the Content Validity Index (CVI), and inter-rater reliability was evaluated using the Intraclass Correlation Coefficient (ICC) based on a two-way mixed-effects model with absolute agreement. Discriminant validity was examined using the Mann-Whitney U test to compare scores across nurses with varying levels of clinical experience. A total of 233 handover cases involving patient transfers from the intensive care unit (ICU) to general wards were collected, involving 84 nurses. The scale demonstrated good content validity, with item-level content validity indices (CVI) ranging from 0.88 to 1.00 and a scale-level CVI/Ave of 0.98. The inter-rater reliability, assessed using fifty randomly selected cases, was high, with an intraclass correlation coefficient (ICC) of 0.885 for single-rater assessments and 0.939 for average-rater assessments. Discriminant validity analysis showed that nurses with more clinical experience had significantly higher total scores than those with less experience (Z = -4.772, p < 0.001). The Chinese version of the ISBAR Structured Handover Observation Tool demonstrates good content validity, high inter-rater reliability, and acceptable discriminant validity. This tool provides a standardized and practical method for assessing the completeness of information transfer and is expected to support quality improvement in patient handover from the ICU to general wards in Chinese clinical settings.

BackgroundThe COVID-19 pandemic exposed critical shortages of mechanical ventilators, particularly in low-resource settings. Disruptions in global supply chains and dependence on specialized components highlighted the need for scalable, locally manufacturing alternatives for emergency respiratory support. AimTo describe and evaluate a simplified, supply-chain-independent mechanical ventilator assembled from widely available automotive and simple hardware components, and intended as a last-resort solution. MethodsThe ventilator is based on a reciprocating air pump driven by an automotive windshield wiper motor coupled to parallel shaft bellows and readily assembled passive membrane valves, only requiring materials available from standard hardware retailers, minimal tools, and basic manual skills. Ventilator performance was assessed through bench testing using a patient model simulating severe lung disease in an adult (R=20 cmH2O{middle dot}s/L, C=15 mL/cmH2O) and pediatric (R=50 cmH2O{middle dot}s/L, C=10 mL/cmH2O) patients. Realistic proof of concept was performed in four mechanically ventilated 50-kg pigs. ResultsThe device delivered tidal volumes up to 600 mL and respiratory rates up to 45 breaths/min with PEEP up to 10 cmH2O, covering pediatric and adult ventilation ranges. In vivo testing showed that the ventilator maintained arterial blood gases within the targeted range. Technical details for ventilator construction are provided in an open-source video tutorial. DiscussionThis low-cost ventilator demonstrated adequate performance under demanding conditions. Although not a substitute for commercial intensive care ventilators, its simplicity, autonomy, and independence from fragile supply chains provide a potentially life-saving option in resource-constrained emergency scenarios.

BackgroundIn the absence of structured donor registries, social media platforms have become a dominant mechanism for blood donor recruitment in many low-resource settings. However, the implications of this shift for transfusion timeliness and system reliability remain unclear. ObjectiveTo evaluate the impact of social media-sourced donors on transfusion delay, donor reliability, and hemovigilance-related outcomes compared with conventional donor pathways. MethodsThis prospective analytical study included 400 transfusion episodes across tertiary hospitals in Bangladesh. Donor sources were categorized as social media (SM) or conventional (CON). The primary outcome was delay-to-transfusion. Secondary outcomes included donor-related irregularities, documentation completeness, near-miss events, and acute transfusion reactions. Multivariable logistic regression identified predictors of delay [&ge;]4 hours. ResultsSocial media-sourced donors were associated with significantly longer transfusion delays (5.98 vs 2.97 hours; p<0.001). Delay [&ge;]4 hours occurred in 83.6% of SM cases versus 17.6% of CON cases (OR 23.78). Donor-related irregularities were observed in 85% of SM episodes and absent in CON donors. Safety outcomes did not differ significantly between groups. Social media donor sourcing remained the strongest independent predictor of delay (adjusted OR 18.09). ConclusionUnregulated social media-based donor recruitment introduces substantial delays and undermines system reliability without improving access. Integration of digital tools into regulated donor systems is essential to strengthen transfusion timeliness and hemovigilance in resource-limited settings.

ObjectivesEpidural spinal cord stimulation (SCS) is an emerging therapy for motor rehabilitation following spinal cord injury (SCI) and other motor disorders. Conventionally, SCS leads are placed along the dorsal spinal cord (SCSD), where stimulation activates large diameter afferent fibers, which indirectly activate motoneurons through reflex pathways. This leads to broad activation of flexor and extensor muscles and limited fine-tuned control of motor output. Targeting the ventral spinal cord (SCSV) may enable more direct activation of motoneuron pools, potentially improving the specificity of muscle activation; however, there is currently no established method to place leads ventrally. To address this, we evaluated the feasibility of four modified percutaneous implantation techniques to target the ventrolateral thoracolumbar spinal cord. Materials and methodsPercutaneous SCSV implantation was performed in three human cadaver torso specimens under fluoroscopic guidance. The following approaches were evaluated: sacral hiatus, transforaminal, interlaminar contralateral, and interlaminar ipsilateral. The leads in the latter 3 approaches were inserted between L1 and L5. Eighteen implants were attempted, with nine leads retained for analysis. Lead and electrode position were assessed using computed tomography (CT) with three-dimensional reconstruction, along with anatomical dissection to verify lead and electrode placement within the epidural space. ResultsSuccessful ventral epidural lead placement was achieved using all four implantation approaches. The sacral hiatus (16/16 electrodes) and transforaminal (8/8 electrodes) approaches resulted in exclusively ventrolateral placement. The interlaminar contralateral approach led to 27/32 electrodes positioned ventrolaterally and 5/32 dorsally. The interlaminar ipsilateral implantation approach led to 14/32 electrodes positioned ventrolaterally and 18/32 positioned ventromedially. ConclusionsThese findings demonstrate that ventral epidural SCS lead placement can be achieved using modified percutaneous implant techniques. The four approaches outlined here provide a clinically feasible pathway to SCSV and establishes a foundation for future clinical studies investigating SCSV for motor rehabilitation following SCI.

Social stigma surrounding chronic skin Ulcer leads patients to hide their wounds or delay seeking medical care. The aim of this study was to explore the types and causes of chronic skin ulcers among patients seen in the dermatology departments of two university hospitals in Burkina Faso. This was a cross-sectional, retrospective study covering an 11-year period, from 2013 to 2023. A review of consultation records allowed for the collection of sociodemographic and clinical data from 104 patients who were seen for chronic skin ulcers over the 11-year period, averaging 9 patients per year. The patients were primarily adults (n=60) and older adults (n=21). Leg ulcers were the condition observed in most patients (n=59). Eight cases of Buruli ulcer (7.69%) were identified among the 104 patients. Five of the eight cases, or 62.50%, were aged between 0 and 19 years. Half of the eight patients resided in Ouagadougou. These results highlight low utilization of dermatology services for chronic skin ulcers. Furthermore, indigenous cases of Buruli ulcer have been identified in Burkina Faso. Consequently, our findings call for the implementation of strategies focused on addressing social perceptions of these ulcers and on the screening and management of this disease.

Introduction. Empirical evidence linking specific national structural policies to the provision of key HIV services in low- and middle-income settings remains scarce. This study addresses the research gap by quantifying the within-country relationships between six national structural policy indicators and the presence of the HIV prevention service component targeted at sex workers in Southeast Asia. Methods. We constructed a balanced panel dataset covering eight Southeast Asian countries from 2018 to 2025 from the UNAIDS Global AIDS Monitoring (GAM) framework. We used Fixed-Effects (FE) and Random-Effects (RE) models to analyse the relationships, with the FE model selected as the more statistically appropriate estimator. We enhanced robustness by using clustered standard errors and one-period lagged explanatory variables. Results. The primary finding from the FE model indicated a statistically significant and positive contemporaneous association between the existence of legal or administrative barriers to social protection (barriers_spi,t) and the presence of HIV prevention services for sex workers ({beta} = 0.8531; p < 0.001). However, the robustness check revealed a statistically significant negative association between the two when using the lagged barrier variable (barriers_spi,t-1), suggesting a decline in HIV prevention service availability over time ({beta} = -0.3540; p < 0.05). We did not find any other policy variable's coefficient to be statistically significant in the FE models. Conclusions. While the immediate recognition (contemporaneous effect) of structural barriers to access social protection may occur alongside prioritised HIV prevention service provision, the sustained presence of these impediments acts as a long-term constraint that undermines the effectiveness and sustainability of targeted HIV programmes. National HIV programmes must urgently prioritise the removal of structural barriers to ensure long-term service stability for key populations.

ObjectiveTo evaluate the real-world effectiveness of Intact Fish Skin Graft (IFSG) compared with standard of care (SOC) in the treatment of Stage 3-4 pressure ulcers, using clinically meaningful outcomes including wound healing rate and percent area reduction (PAR). Materials and MethodsA retrospective matched cohort study was conducted using deidentified electronic health record (EHR) data from the U.S. Wound Registry. Patients with Stage 3-4 pressure ulcers treated with IFSG (n=40) were compared to a matched SOC control group (n=40). 1:1 covariate matching was performed to reduce confounding across key patient and wound characteristics, including age, mobility status, comorbidities (e.g., diabetes, peripheral artery disease), and wound features (age, size, location, and depth). Outcomes included healed status, healed or improved rate, and percent area reduction (PAR). ResultsThe study population represented a high-risk, real-world cohort (n=40 per group), with only 37.5% ambulatory patients and a high prevalence of multiple concurrent wounds. IFSG treatment demonstrated superior clinical outcomes compared to SOC: O_LIHealed or improved: 67.5% (IFSG) vs 55.0% (SOC) (p=0.0379) C_LIO_LIHealed: 45.5% (IFSG) vs 33.3% (SOC) C_LIO_LIPercent area reduction (PAR): 49% (IFSG) vs 34% (SOC) (p=0.0028) C_LI These findings indicate statistically significant improvements in percent area reduction and in the proportion of wounds that were healed or improved with IFSG. The proportion achieving complete healing was numerically higher with IFSG than with SOC, but this difference did not reach statistical significance. ConclusionIn this real-world matched cohort analysis, Intact Fish Skin Graft demonstrated superior effectiveness compared to standard of care in the management of Stage 3-4 pressure ulcers, with improvements in healing-related outcomes and percent area reduction. These results support the use of IFSG as an effective advanced therapy for hard-to-heal pressure ulcers.

In this article we argue that intersectoral collaboration is ultimately manifested at the neighbourhood level, where professionals from diverse sectors engage in a joint network to improve population health outcomes. To strengthen intersectoral collaboration in neighbourhoods with low SES, it is crucial to include the community voice, representing diverse citizens who must be heard and engaged in decision-making processes. This study aims to contribute to the literature of intersectoral collaboration by exploring how networks emerge and evolve over time. We focus on the development of the roles of citizens in the professional network and diverse sectoral involvement within a local network of the team called The Connectors, in a neighbourhood with low socioeconomic scores (SESs). Methodologically, we use a combination of social network analysis (SNA) and action research. Results show that the network expanded significantly over time, both in terms of the number of actors and the diversity of sectors involved. At both measurement points T1 and T2, the majority of collaborations occurred across sectoral boundaries. By the second measurement, the proportion of intersectoral relationships had increased. This indicates that as the network expanded, new collaborations were not confined to existing sectoral clusters but increasingly bridged different sectors. The dual role that citizens have taken on during the development of the network, serving both as community voice representatives and professionals, can be empowering, offering pathways for personal growth and career advancement. However, it also introduces complexity, as these individuals may experience tensions between personal commitments and professional responsibilities. To enable network development, policies should allow room beyond standard protocols and organizational silos, as well as provide sufficient time for relationships and structures to mature. Although network building is a gradual and complex process, once established, these networks can play a pivotal role in delivering integrated and responsive care.