Molecular Therapy Methods & Clinical Development

{gamma}-secretase is a multi-subunit enzyme complex responsible for cleaving hundreds of substrates in diverse cellular contexts. Variation in subunit composition - including the use of alternate catalytic subunits Presenilin 1 (PSEN1) and Presenilin 2 (PSEN2) - results in diverse {gamma}-secretase complexes. Point mutations in PSEN1 and PSEN2 cause familial forms of Alzheimers disease, while loss-of-function mutations in the {gamma}-secretase subunits PSEN1, PSENEN and NCSTN cause acne inversa. To advance therapeutic strategies targeting {gamma}-secretase in Alzheimers disease, a better understanding of individual {gamma}-secretase complexes is required. In this study, we used CRISPR-Cas9 genome engineering to generate PSEN2-knockout iPSCs in order to compare the consequence of PSEN2 knockout versus PSEN1 knockout in iPSC-derived brain cells. In contrast to PSEN1-knockout, PSEN2-knockout did not alter APP cleavage or A{beta} generation in iPSC-neurons, nor did it disrupt Nicastrin maturation. Similarly, PSEN2-knockout had little impact on TREM2 processing in iPSC-microglia. Instead, our data indicate that loss of PSEN2 primarily impacts the endo-lysosomal system in iPSC-neurons, causing an accumulation of early endosome markers and a reduction in lysosomal markers - phenotypes not observed in PSEN1-knockout neurons. Taken together, these findings highlight distinct and non-redundant functions of PSEN1 and PSEN2 in human brain cells, reinforcing findings in animal models and subcellular localisation studies. This work advances our understanding of distinct {gamma}-secretase complex functions and provides insights that will support future therapeutic efforts to inhibit, modulate or stabilise {gamma}-secretase.

The non-euphorigenic phytocannabinoid cannabidiol (CBD) has demonstrated therapeutic efficacy in childhood-onset epilepsies. Using a songbird preclinical model we have found that CBD promotes recovery of learned vocalizations following focal motor cortical injury. But questions about cellular mechanisms supporting this protection remained. Songbird vocal learning, like human speech, depends on development and maintenance of specialized neural circuits. Partial lesioning (microlesions) of the vocal pre-motor cortical-like song region HVC transiently disrupts song structure and triggers injury-associated cellular stress responses across interconnected song regions. Building on prior findings that CBD reduces neuroinflammation and synaptic loss in zebra finch song circuitry, we investigated potential astrocyte contributions. Here we report that HVC microlesions induce significant cell loss in HVC and its projection targets (vocal motor RA and striatal Area X), with a substantial fraction of apoptotic cells being astrocytes. CBD treatment reduces lesion-induced apoptosis and preserves astrocyte populations, indicating enhanced astrocyte viability as a major factor in CBD-mediated neuroprotection. Microlesions also elevate astrocyte stress, including increased lysosomal burden (LAMP1/LC3 expression) and astrocytic reactivity markers (C3, S100A10, aromatase). CBD attenuates these stress responses while enhancing neuroprotective metabolic and antioxidant mediators (glutamine synthetase [GS], glutamate-cysteine ligase modifier subunit [GCLM]), consistent with improved antioxidant and excitotoxicity resistance. Given that development-dependent sensorimotor skills (e.g. song in songbirds, language and many others in humans) depend on sensitive period establishment and ongoing post-learning maintenance of specialized neural circuits vulnerable to traumatic disruption, the zebra finch model provides a valuable preclinical platform for investigating glial-targeted interventions to promote circuit resilience and functional recovery after TBI.

Stroke remains a leading cause of adult disability due to the brains limited regenerative capacity. Although stem cell therapies show favorable safety and feasibility profiles in early clinical trials, poor spatial retention and limited engagement with peri-infarct salvageable tissue constrain efficacy. Here, we engineered human induced pluripotent stem cell-derived neural stem cells (NSC) with a chimeric antigen receptor (CAR)-like architecture to enable targeted recognition of injury-associated cues. Specifically, cells were modified to express a membrane-anchored single chain variable fragment (scFv) targeting integrin v{beta}3, a receptor selectively upregulated in peri-infarct vasculature after stroke. Engineered CAR-NSC retained progenitor identity and selectively bound recombinant integrin v{beta}3 in vitro. Following focal transplantation into a photothrombotic stroke mouse model, CAR-NSC displayed broader dispersion within peri-infarct tissue and covered a greater proportion of the ischemic lesion compared to non-binding control-CAR-NSC. CAR-NSC grafts extended longer neurites that aligned more closely with the lesion border. In addition, CAR-NSC transplantation reduced microglial activation and was associated with increased vascular density and blood-brain barrier integrity in the peri-infarct zone. Together, these findings establish a CAR-like NSC strategy for stroke to direct the spatial distribution and tissue engagement of transplanted cells. Molecular targeting of injury-associated cues may improve the precision and regenerative efficacy of cell-based therapies for stroke and related neurological disorders.

CD28 co-stimulatory blockade is an established therapeutic strategy in autoimmune disease, yet every clinical-stage agent shares a structural limitation: high-affinity, long-lived receptor occupancy that precludes dynamic control of immune suppression. In chronic inflammatory conditions, where prolonged immunosuppression carries infection risk and necessitates treatment interruptions, no existing agent permits rapid restoration of immune function. We report CP8, a disulfide-constrained cyclic peptide antagonist that matches the inhibitory potency of clinical-stage CD28 biologics (FR104, Acazicolcept, and Lulizumab) across primary human immune cells from healthy and ulcerative colitis donors, suppressing IL-2 and IFN-{gamma} production without agonist activity. Unlike these biologics, CP8 enables rapid and near-complete restoration of T-cell function upon compound removal, a property mechanistically inaccessible to antibody-based therapeutics and demonstrated here for the first time for any CD28-targeting agent. In a T-cell transfer colitis model, CP8 maintains efficacy under intermittent dosing and outperforms Acazicolcept, a dual CD28/ICOS inhibitor, under exposure-limited conditions, achieving superior disease suppression, tissue preservation, and cytokine reduction. These results demonstrate that potency and pharmacological persistence are decoupled properties, and reframe cyclic peptides as a superior modality for immune checkpoints where temporal control of signaling is essential to balance efficacy with the risks of chronic immune suppression.

Huntingtons disease (HD) is a progressive neurodegenerative disease caused by a mutation in the exon 1 of the huntingtin (HTT) gene, which leads to an extended polyglutamine (polyQ) tract in the mutant protein. As a result, mutant huntingtin (mHTT) exon 1 fragments aggregate in cells, which disrupts proper neuronal function and eventually induces cell death. The selective reduction of these toxic mHTT fragments without disturbing the wild-type full-length HTT function would be a potential therapeutic strategy to treat and prevent HD. Intracellular antibodies (intrabodies) have emerged as an attractive strategy to specifically target disease-related proteins, with VHH intrabodies being of high interest as they are much smaller than single-chain variable fragments (scFv). Here, we describe the identification and development of VHH 1 as a lead candidate intrabody targeting the first 17 amino acids of the mHTT protein, using a humanized VHH page-display library to screen against mHTT(Q46) exon 1 to identify potential binders. Next, we further optimized VHH 1 into VHH 1a to improve cytoplasmic solubility. Using immortalized mouse striatal cells that express inducible untagged mHTT exon 1 fragments, we investigated the effects of the intrabody on soluble and insoluble mHTT species via microscopy and biochemical assays. We showed that the VHH 1a intrabody reduces the levels of insoluble mHTT species, thereby effectively interrupting the aggregation process. This study highlights the potential for VHH intrabodies to specifically target mHTT fragments, enabling therapeutic strategies to delay and prevent HD pathology. HighlightsO_LIThree binders were down-selected from a phage-display library to bind HTT N17 C_LIO_LIVHH 1a intrabody is the most efficient at reducing mutant HTT exon 1 aggregation C_LIO_LIVHH 1a acts on soluble HTT exon 1 oligomers to block the transition to inclusion body C_LI

Simulated spaceflight perturbs multiple organ systems, yet the integrated impact of spaceflight-relevant stressors on the immune-gut-brain axis remains poorly defined. We used a ground-based model combining hindlimb unloading (HU) with low-dose ionizing radiation (IR; 50 or 100cGy) to quantify neuropathology, peripheral immune phenotypes, intestinal barrier integrity, and behavioral performance in male and female C57BL/6 mice. HU and/or IR induced region-selective neurodegenerative changes consistent with axonal injury across the cortex and major white-matter tracts. In the somatosensory cortex, MAP-2+ neurons were reduced and SMI-312-labeled axonal injury increased, lowering the intact-to-dystrophic axonal area ratio. Long-range fiber pathways (corpus callosum, cingulate gyrus, external capsule) showed robust axonal damage accompanied by gliosis, with elevated Iba-1+ microglia and GFAP+ astrocytes most prominent after HU+IR (100cGy). Peripheral immunophenotyping revealed a sustained, sex-dependent innate inflammatory bias, with expanded CD11b+ myeloid cells and increased TNF-+ myeloid activation after IR and IR+HU, alongside maladaptive T-cell polarization despite largely unchanged total CD8+ and CD4+ frequencies. In parallel, the gut exhibited architectural remodeling and barrier failure, including altered mucin profiles, reduced ZO-1 tight-junction labeling, and increased CD45+ leukocyte infiltration across the jejunum, ileum, and colon. Behavioral assays demonstrated sex-dependent deficits spanning affective, motor, and cognitive domains, including increased anxiety- and depressive-like behaviors, impaired rotarod performance, reduced recognition memory, and less efficient spatial strategies. Overall, these findings identify a sex-dependent immune-gut-brain vulnerability in which combined HU and low-dose IR drive gut barrier breakdown and immune imbalance that coincide with neuroinflammatory axonopathy and measurable neurobehavioral dysfunction.

A complex of the 3'-truncated tRNAArg that lacks 9 nt and tRNase ZL works as a GCCC-recognizing RNA cutter. It recognizes an RNA substrate via four Watson-Crick-Franklin base-pairings with the 3'-truncated tRNAArg. Human SLFN11 and SLFN13 can generate 3'-truncated tRNALeu that lacks 10 nt and 3'-truncated tRNASer that lacks 11 nt, respectively, from their corresponding mature tRNAs. Here, we investigated if these 3'-truncated tRNAs together with tRNase ZL work as sequence-specific RNA cutters. We examined five RNA targets for cleavage by recombinant human tRNase ZL in the presence of the 3'-truncated tRNALeu or tRNASer. We demonstrated that the 3'-truncated tRNALeu and tRNASer together with tRNase ZL indeed work as [~]6-base-recognizing and 7-base-recognizing RNA cutters, respectively.

Background and AimsUnderstanding the etiology of gastrointestinal malfunction requires insights into how heterogeneous gut cell lineages arise during development and the identification of genes controlling these processes. While in utero genetic manipulation has advanced knowledge in other organs, the gut and its intrinsic enteric nervous system (ENS) remain difficult to target. We aimed to establish an approach to resolve precise lineage relationships and achieve conditional gene perturbation of developing gut cell types in vivo. MethodsWe performed ultrasound-guided in utero nano-injection of lentiviral vectors into the mouse amniotic cavity at embryonic day 7.5. By combining lentiviral DNA barcoding with single cell RNA-sequencing, we reconstructed clonal relationships between distinct gut cell types. We used Cre-dependent lentiviral vectors in transgenic mice to achieve cell type-specific expression and tested whether gene overexpression can direct developmental processes. ResultsLentiviral delivery efficiently targeted gut-innervating ganglia and all major gut cell types, including neural, epithelial, immune, and mesenchymal populations. Barcode-based lineage tracing revealed clonal relationships within and across gut cell types. Notably, specialized mesenchymal populations, including pericytes, mesothelial cells, and interstitial cells of Cajal (ICCs) were each linked to different fibroblast subpopulations. Regional analyses demonstrated early establishment of anterior-posterior identity of mesenchyme, whereas ENS and immune cells exhibited long-range clonal dispersion. Cre-dependent targeting enabled selective gene expression in ENS progenitors or neurons, and temporally controlled overexpression of Ascl1 promoted neuronal differentiation. ConclusionsIn utero lentiviral transduction expands experimental access to the developing gastrointestinal tract, enabling lineage-resolved analysis and conditional gene manipulation of gut cell types, including the ENS. This approach enables mechanistic investigation of gut organogenesis and offers a framework to study developmental origins of disease.

Age-related cognitive decline (ARCD) is driven by conserved biological mechanisms of aging, yet no gerotherapeutic directly targets these processes in the brain. Glycyl-L-histidyl-L-lysine complexed with copper (GHK-Cu) is an endogenous peptide with regenerative and anti-inflammatory properties that declines with age. Whether its effects on cognitive aging depend on delivery route or exposure duration remains unclear. Aged C57BL/6J mice (20-21 months) received GHK-Cu (15 mg/kg) via short-term intraperitoneal (IP; 5 days) or longer-term intranasal (IN; 8 weeks) administration. Hippocampal-dependent escape learning was assessed using a spatial navigation task. Molecular effects were evaluated using hippocampal immunohistochemistry and bulk RNA sequencing. Differential gene expression was analyzed using DESeq2 with false discovery rate (FDR) correction, and pathway-level changes were assessed via gene set enrichment analysis (GSEA). IN GHK-Cu improved escape latency across Trials 2-4 in both sexes (P < 0.05), whereas IP dosing produced a transient improvement in males during Trial 2 (P < 0.05) without sustained effects or improvement in females. IN treatment increased synaptophysin in females (P < 0.001) and decreased GFAP in both sexes (P < 0.01), while IP treatment reduced TGF-{beta}, GFAP, and MCP-1 in males (P < 0.05) and decreased p21 in females (P < 0.0001). Transcriptomic analysis revealed distinct molecular programs. IN GHK-Cu induced coordinated suppression of oxidative phosphorylation (male NES -5.44, female NES -4.20; FDR < 0.0001) and MYC target pathways (female NES -4.31, FDR < 0.0001), with additional attenuation of PI3K-AKT-mTOR signaling in females (NES -3.15, FDR = 0.062). In contrast, IP treatment activated oxidative phosphorylation (female NES 4.97, FDR < 0.001), DNA repair (NES 5.58, FDR < 0.001), and MYC targets (NES 4.34, FDR = 0.002), indicating engagement of acute stress-response and repair pathways. GHK-Cu improves hippocampal-dependent learning in aged mice through distinct biological modes: IP exposure activates repair and stress-response pathways, whereas IN delivery induces sustained suppression of growth and mitochondrial metabolic signaling associated with aging biology. These findings demonstrate that functional cognitive improvement can arise from divergent molecular states and identify administrative route and exposure duration as key determinants of gerotherapeutic response.

Enhancing targeted delivery of biomedicines improves efficacy and can reduce off-target effects by lowering the effective dose, but achieving targeting is challenging. Extracellular vesicles (EVs) are promising biological nanovesicles which can be targeted by displaying binding proteins and are being developed as therapeutics. Currently, discovering EV targeting constructs is limited by low throughput and resource-intensive EV production and isolation. To accelerate discovery, we developed a screening pipeline to identify EV targeting constructs without requiring EV production. This approach is premised on the hypothesis that cell-cell interactions may predict some cell-EV interactions. Our cell binding assay (CELLISA) quantifies binding of a cell surface-displayed targeting protein to its cognate receptor on a target cell, employing a microscopy-based analysis pipeline. After validating the premise using existing T cell-targeting reagents, we develop CELLISA for either adherent or suspension EV producer cells. Finally, we use CELLISA to evaluate new binders and validate that hits mediate targeting and/or delivery of genetic cargo to natural killer cells and T cells. CELLISA increased throughput > 6-fold and decreased time by 40% compared to standard EV screens, and it identified a T-cell binder conferring efficient gene delivery. CELLISA is easily adaptable to other laboratories and can accelerate EV research.

Recombinant peptide production was pioneered in the 1970s for the generation of therapeutic peptides, with notable examples including insulin and somatostatin. These early methods required the use of cyanogen bromide (BrCN) for cleavage of the native peptide sequence from a fusion protein. Since that time, while numerous BrCN-dependent peptide methods continue to be reported, the accessibility and cost of site-specific proteases have improved dramatically. These developments have enabled alternative approaches to recombinant peptide generation that obviate the need for BrCN, an environmentally destructive toxin. We recently created an immobilized SUMO protease that can replace BrCN usage in recombinant peptide production workflows by releasing native peptides expressed as part of a SUMO-peptide fusion protein. We have used this approach to generate P113 peptide, the minimal active fragment of the antifungal peptide Histatin 5. In this report, we describe the creation and characterization of this immobilized SUMO protease and its application in the production of experimentally viable quantities of active P113 peptide.

The human leukocyte antigen (HLA) system is the primary determinant of donor selection in allogeneic hematopoietic cell transplantation (HCT) and plays a central role in solid organ transplantation, immune-mediated disease studies, evolutionary population genetics, and immunotherapy. Large-scale sampling of registry participants reflecting major US ancestry groups allows for characterization of the complex landscape of HLA haplotype diversity for the classical HLA class I (HLA-A, HLA-B, HLA-C) and HLA class II (HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DQA1, HLA-DQB1, HLA-DPA1, and HLA-DPB1) genes. Here we present nine-locus classical HLA allele and haplotype frequency estimates for five broad (Black, White, Asian or Pacific Islander, Hispanic and Native American) and 21 detailed US populations based on 9,671,082 donors with targeted genotyping by DNA-based methods. Frequency estimation used an expectation-maximization (EM) framework specifically adapted to handle mixed-resolution and ambiguous HLA genotyping data. Advancements in next-generation sequencing provide extensive HLA genotyping, offering new insights into the haplotype structure and diversity of the human MHC complex, expanding knowledge especially for HLA class II haplotypes. Population analyses reveal that the most common high-resolution haplotypes are predominantly population-specific, with only three haplotypes shared across the top-100 lists of all five broad population groups, and that Black populations exhibit the greatest nine-locus haplotypic diversity, a pattern that persists after controlling for differences in registry sample size. These frequencies, derived from the largest US cohort to date, support clinical decision-making and research in histocompatibility, immunogenetics, and transplantation and are publicly available at https://zenodo.org/records/17966993.

BackgroundMajor depressive disorder (MDD), a leading cause of disability worldwide, exhibits substantial heterogeneity in treatment outcomes. Patients who do not respond to standard antidepressant therapy account for the majority of MDDs disease burden. Risk factors have been implicated in treatment response, including genes impacting on how antidepressants are metabolised. Yet, despite its clinical importance, risk factors for treatment-resistant depression (TRD) remain unexplored in low- and middle-income countries (LMIC). We used data from the DIVERGE study on MDD to investigate the risk factors of TRD in Pakistan. MethodsDIVERGE is a genetic epidemiological study that recruited adult MDD patients ([&ge;]18 years) between Sep 27,2021 to Jun 30, 2025, from psychiatric care facilities across Pakistan. Detailed phenotypic information was collected by trained interviewers and blood samples taken. Infinium Global Diversity Array with Enhanced PGx-8 from Illumina was used for genotyping followed by DRAGEN calling to infer metaboliser phenotypes for Cytochrome P450 (CYP) enzyme genes. We defined TRD as minimal to no improvement after [&ge;]12 weeks of adherent antidepressant therapy. We conducted multi-level logistic regression to test the association of demographic, clinical and pharmacogenetic variables with TRD. FindingsAmong 3,677 eligible patients, polypharmacy was rampant; 86% were prescribed another psychotropic drug along with an antidepressant. Psychological therapies were uncommon (6%) while 49% of patients had previously visited to a religious leader/faith healer in relation to their mental health problems. TRD was experienced by 34% (95%CI: 32-36%) patients. The TRD group was characterised by more psychotic symptoms and suicidal behaviour (OR=1.39, 95%CI=1.04-1.84, p=0.02; OR=1.03, 95%CI=1.01-1.05, p=0.005). Social support (OR=0.55, 95%CI=0.44-0.69, p=1.4x10-7) and parents being first cousins (OR=0.81, 95%CI=0.69-0.96, p=0.01) were associated with lower odds of TRD. In 1,085 patients with CYP enzyme data, poor (OR=1.85, 95%CI=1.11-3.07, p=0.01) and ultra-rapid (OR=3.11, 95%CI=1.59-6.12, p=0.0009) metabolizers for CYP2C19 had increased risk of TRD compared with normal metabolisers. InterpretationThere was an excessive use of polypharmacy in the treatment of depression while psychological therapies were uncommon highlighting the need for more evidence-based practice. This first large study of MDD from Pakistan uncovered the importance of culture-specific forms of social support in preventing TRD, highlighting opportunities for interventions in low-income settings. Pharmacogenetic markers can be leveraged to predict TRD.

Importance: Suicide is a leading cause of death in the United States with risk strongly influenced by Interpersonal trauma, contributing to treatment resistance and clinical complexity. Objective: To assess clinical and genetic factors in individuals who died from suicide, with and without interpersonal trauma exposure. Design: Individuals who died from suicide with and without trauma were compared in a retrospective case-case design. Prevalence of 19 broad clinical categories was assessed between groups. Results directed selection of 42 clinical subcategories, and 40 polygenic scores (PGS) for further assessment. Multivariable logistic regression models, adjusted for critical covariates and multiple tests, were formulated. Models were also stratified by age group (<26yo and >=26yo), sex, and age/sex. Setting: A population-based evaluation of comorbidity and polygenic scoring in two suicide death subgroups. Participants: A total of 8 738 Utah Suicide Mortality Research Study individuals (23.9% female, average age = 42.6 yo) who died from suicide were evaluated, divided into trauma (N = 1 091) and non-trauma exposed (N = 7 647) individuals. A subset of unrelated European genotyped individuals was also assessed in PGS analyses (Trauma N = 491; Non-trauma N = 3 233). Exposures: Trauma is here defined as interpersonal trauma exposure, including abuse, assault, and neglect from International Classification of Disease coding. Main Outcomes and Measures: Prevalence of comorbid clinical sub/categories and PGS enrichment in trauma versus non-trauma exposed suicide deaths. Results: Overall, trauma-exposed individuals died from suicide earlier (mean age of 38.1 yo versus 43.3 yo; P <0.0001) and were disproportionately female (38% versus 21%, OR = 3.3, CI = 2.9-3.8). Prevalence of asphyxiation and overdose methods, prior suicidality, psychiatric diagnoses, and substance use (OR range = 1.3-3.7) were elevated in trauma exposed individuals who died from suicide. Genetic PGS were also elevated in trauma-exposed individuals who died from suicide for depression, bipolar disorder, cannabis use, PTSD, insomnia, and schizophrenia (OR range = 1.1-1.4) with ADHD and opioid use showing uniquely elevated PGS in trauma exposed males (OR range = 1.2-1.4). Conclusions and Relevance: Results demonstrated multiple convergent lines of age- and sex-specific evidence differentiating trauma-exposed from non-trauma exposed suicide death. Such findings suggest unique biological backgrounds and may refine identification and treatment of this high-risk group.

In the context of global health, the ability of frontline primary health providers to identify potential Drug-Drug Interactions (DDIs) is a critical component of patient safety. This is particularly true in settings like Tanzania, where drug dispensers often serve as the primary point of contact for healthcare. In this study, we establish a baseline for drug decision-making capabilities across multiple cadres of healthcare providers in Kibaha, Tanzania. We specifically distinguish between the ability to recognize safe drug combinations versus harmful ones. The findings reveal a critical asymmetry in provider performance: while professional training improves the recognition of safe combinations, it provides no advantage over lay intuition (and in some cases, a significant disadvantage) in detecting potentially harmful interactions.

Background: Preterm births contribute to approximately 35% of neonatal deaths globally, with an estimated 13.4 million infants born prematurely each year. Despite this substantial burden, limited evidence exists on time to discharge and its determinants among preterm neonates admitted to Neonatal Intensive Care Units (NICUs), particularly in rural Ugandan settings. This study aimed to investigate time to discharge and associated factors among preterm neonates admitted to Kiwoko Hospital in Nakaseke District, Uganda. Methods: A retrospective cohort study was conducted using secondary data from Kiwoko Hospital on preterm neonates admitted to the Neonatal Intensive Care Unit (NICU) between 2020 and 2021 (n = 847). The cumulative incidence function was used to estimate the probability of discharge within 28 days of admission, accounting for competing events. A Fine and Gray sub-distribution hazard regression model was fitted to identify factors associated with time to discharge. Results: Of the 847 preterm admissions, 70.1% were discharged alive within 28 days. The median time to discharge was 14 days. The cumulative incidence of discharge by 28 days was 68%, accounting for competing events. During follow-up, 165 neonates did not complete the 28-day period, including 88 deaths. Factors significantly associated with time to discharge included place of delivery (SHR: 0.62; 95% CI: 0.53-0.73; p<0.001), maternal residence in other districts (SHR: 0.69; 95% CI: 0.48-0.99; p=0.044), extreme preterm (SHR: 0.05; 95% CI: 0.03-0.09; p<0.001), very preterm (SHR: 0.18; 95% CI: 0.14-0.25; p<0.001), moderate preterm (SHR: 0.59; 95% CI: 0.46-0.76; p<0.001), triplet births (SHR: 0.40; 95% CI: 0.23-0.68; p=0.001), 2-4 ANC visits (SHR: 0.70; 95% CI: 0.56-0.87; p=0.002), <=1 ANC visit (SHR: 0.64; 95% CI: 0.49-0.85; p=0.002), respiratory distress syndrome (SHR: 0.64; 95% CI: 0.48-0.74; p<0.001), and birth trauma (SHR: 2.62; 95% CI: 1.60-4.29; p<0.001). Conclusions: Respiratory distress syndrome, fewer antenatal care visits, out-of-district residence, and higher degrees of prematurity were associated with prolonged time to discharge among preterm neonates. Strengthening antenatal care utilization and improving access to quality neonatal care in underserved areas may enhance discharge outcomes.

Objectives: Acute gastroenteritis is a leading cause of pediatric emergency department (ED) visits. While ondansetron reduces vomiting, intravenous rehydration, and hospital admissions, its efficacy when initiated at triage remains unclear. We aimed to evaluate whether triage nurse-initiated administration of ondansetron in children with suspected gastroenteritis reduces the proportion of patients requiring observation following initial physician assessment. Methods: We conducted a randomized, double-blind, placebo-controlled trial in a tertiary pediatric ED in Canada. Children aged 6 months to 17 years presenting with morae than 3 episodes of vomiting in the preceding 24 hours (including 1 within 2 hours of arrival), were eligible. At triage, we randomized participants to receive liquid ondansetron or a color- and taste-matched placebo. The primary outcome was the proportion of patients requiring observation after the first physician evaluation. Secondary outcomes included post-intervention vomiting, ED length of stay, patient comfort, and 48-hour return visits. The trial was registered at ClinicalTrials.gov (NCT03052361). Results: Recruitment was stopped prematurely due to the COVID-19 pandemic. Ninety-one participants were randomized to ondansetron (n= 44) or placebo (n= 47). Overall, 40 patients (45%) were discharged immediately after the initial physician assessment, with no difference between the ondansetron and placebo groups (44% vs. 45%; absolute difference -1%, 95% CI: -20% to 19%). No significant differences were observed in all secondary outcomes. Conclusion: In this trial, triage nurse-initiated ondansetron administration did not reduce the need for ED observation in children with presumed gastroenteritis. While being underpowered, this study could inform researchers planning larger clinical trials.

Background: Large language models (LLMs) are increasingly used in mental health contexts, yet their detection of suicidal ideation is inconsistent, raising patient safety concerns. Objective: To evaluate whether an independent safety monitoring system improves detection of suicide risk compared with native LLM safeguards. Methods: We conducted a cross-sectional evaluation using 224 paired suicide-related clinical vignettes presented in a single-turn format under two conditions (with and without structured clinical information). Native LLM safeguard responses were compared with an independent supervisory safety architecture with asynchronous monitoring. The primary outcome was detection of suicide risk requiring intervention. Results: The supervisory system detected suicide risk in 205 of 224 evaluations (91.5%) versus 41 of 224 (18.3%) for native LLM safeguards. Among 168 discordant evaluations, 166 favored the supervisory system and 2 favored the LLM (matched odds ratio {approx}83.0). Both systems detected risk in 39 evaluations, and neither in 17. Detection was highest in scenarios with explicit suicidal ideation and lower in more ambiguous presentations. Conclusions: Native LLM safeguards frequently failed to detect suicide risk in this structured evaluation. An independent monitoring approach substantially improved detection, supporting the role of external safety systems in high-risk mental health applications of LLMs.

BackgroundProspective studies of pregnant adolescents are essencial to effectively address this global health priority. They help answer vital questions about their health, but such studies are uncommon due to the difficulty in retaining adolescents. This paper describes the successes and challenges of the research strategies used to ensure sufficient recruitment and retention of pregnant adolescents in a longitudinal study about adolescent childbearing in an under-resourced setting. MethodsThe Adolescence and Motherhood Research project was conducted in a rural region of Northeast Brazil in 2017-2019 and assessed 50 primigravids between 13-18 years (adolescents) and 50 primigravids between 23-28 years (young adults) during the first 16 weeks of pregnancy with two follow-ups (third trimester of pregnancy, and 4-6 weeks postpartum). Recruitment strategies involved engagement of health sector and community, as well as referrals from health care professionals and dissemination of the project in different locations. Retention strategies included maintaining contact with the participants between assessments and providing transportation for them to attend the follow-up procedures. ResultsRecruitment took 10 months to complete. A total of 78% of the participants were recruited from the primary health care units, mainly after referral from a health care provider. Retention reached 95% of the sample throughout the study (90%: adolescents; 98%: adults). ConclusionA combination of approaches is necessary to successfully recruit and retain youth in longitudinal studies and engaging local stakeholders may help to increase community-perceived legitimacy of the research. Working closely with front-line staff is essential when conducting research in rural low-income communities.

Background: HIV/AIDS remains a major public health challenge in Tanzania, where viral load suppression among adults on ART stands at 78% and HVL testing uptake among eligible patients is approximately 22%. Since the introduction of the National HVL Testing Guideline in 2015, little has been done to systematically evaluate its implementation. Objective: To evaluate adherence to the National HVL Testing Guideline across CTC clinics in Dar es Salaam Region, covering ART monitoring, documentation, turnaround time, and factors affecting implementation. Methods: A cross-sectional study was conducted in 2021 across 15 public health facilities with CTC clinics in all five Dar es Salaam districts. A total of 330 PLHIV on ART for more than six months were selected through systematic random sampling with proportional to size allocation, and 45 healthcare providers through convenient sampling. Data were collected via abstraction forms and self-administered questionnaires, and analysed using SPSS Version 23 with descriptive statistics, bivariate analysis, and binary logistic regression. Results: Only 25.1% of patients had their first HVL sample taken at six months as per guideline, with 68.8% delayed beyond six months. Second and third samples were similarly delayed. MoHCDGEC sample tracking forms were absent in 96.7% of facilities and incomplete in 99.1%, and no facility captured specimen acceptance or rejection as site feedback. Turnaround time exceeded the 14-day guideline threshold in 64.5%, 66.7%, and 69.4% of first, second, and third results respectively. Patient negligence (AOR=9.84; 95% CI: 1.83-52.77) and storage (AOR=5.72; 95% CI: 0.94-35.0) were independently associated with guideline adherence. Conclusion: Adherence to the National HVL Testing Guideline in Dar es Salaam is suboptimal across testing timelines, documentation, and turnaround time, with patient negligence and storage capacity as significant determinants. Targeted interventions are needed to strengthen patient education, improve storage infrastructure, enhance documentation systems, and support providers in adhering to guideline-specified timelines.