Metabolic Engineering

Axenic cultivation of novel bacterial lineages, referred to as "gold standard in microbiology", remains challenging for fastidious or uncultured taxa due the challenges of replicating adequate growth conditions. We isolated strain PM69, a representative of the previously undescribed Bacillota class SHA-98, from a phenyl acid degrading, oligotrophic batch culture. By employing a broad spectrum of (anaerobic) culture techniques, biochemical, physiological, and genomic analyses, we characterised the strain as Thermoaminiphila catenidiffluenda, gen. nov., sp. nov., a thermophilic, strictly anaerobic, bacterium fermenting monosaccharides to acetate. Its motility, biofilm forming capacity, and ecological niche in biogas fermenters and hydrocarbon-associated habitats suggest adaptive strategies for harsh environments exhibiting e.g., high concentrations of aromatic compounds. This description of a new bacterial class not only expands the taxonomic diversity of phylum Bacillota but also provides insights into the metabolic versatility of yet uncultured microorganisms, with implications for carbon cycling and biotechnological applications. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/718153v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@109cdd5org.highwire.dtl.DTLVardef@a896faorg.highwire.dtl.DTLVardef@502388org.highwire.dtl.DTLVardef@14ce1fd_HPS_FORMAT_FIGEXP M_FIG C_FIG

Biological nitrification inhibition (BNI) is a plant-mediated process that suppresses nitrification and is widely considered beneficial for reducing nitrous oxide emissions. Here, we show that BNI compounds also inhibit methane oxidation by methanotrophic bacteria, revealing a previously unrecognized trade-off in greenhouse gas regulation. Across soil bioreactor systems and pure cultures of both Type I and Type II methanotrophs, BNI compounds consistently suppressed methane oxidation activity. Kinetic analyses indicated an uncompetitive-like inhibition pattern, characterized by concurrent decreases in Vmax and Km, while reversibility assays showed that inhibition was not associated with loss of cellular viability. Experiments under copper-replete and copper-depleted conditions further showed that inhibition is predominantly associated with particulate methane monooxygenase (pMMO). Transcriptomic analyses demonstrated compound-specific responses, including suppression of methane oxidation pathways and differential regulation of stress-associated genes. These findings suggest that BNI-mediated inhibition of methane oxidation may offset reductions in nitrous oxide emissions, with implications for predicting net greenhouse gas fluxes in agricultural and wetland ecosystems. Incorporating BNI effects into biogeochemical models will be critical for accurately evaluating their role in the global methane budget.

Harpagoside, a high-value anti-inflammatory iridoid compound, is traditionally extracted from the roots of Harpagophytum procumbens (Pedaliaceae, Lamiales), a Southern African desert plant widely used in traditional medicine but currently threatened by overexploitation. Scrophularia nodosa (Scrophulariaceae, Lamiales) is a perennial annual plant widely distributed in Western Europe and accumulates several iridoid compounds with known biological activities, such as catalpol, aucubin, harpagide and particularly harpagoside. We gathered extensive genomic and transcriptomics resources for this species and aimed at deciphering the biosynthetic pathway leading to the most abundant iridoid in S. nodosa, harpagoside. We found that the early iridoid pathway is well conserved with other iridoid-producing plants and validated the enzyme activities by transient co-expression in N. benthamiana. Investigation into the large BAHD family showed subclade 6i expanding in Scrophulariaceae, with an atypical VYPWG motif instead of the canonical DFGWG. In this branch, we discovered and characterized harpagoside synthase, a BAHD-type cinnamoyl transferase enzyme showing unique high specificity to the uncommon cinnamoyl-CoA acyl donor and catalyzing the final step of harpagoside biosynthesis. These results establish S. nodosa as a new model to investigate unexplored branches of the iridoids metabolism, and are a first step towards sustainable harpagoside and high-value cinnamoyl-containing conjugates production.

Predicting cellular behaviors, a central task in systems biology and metabolic engineering, can be enhanced through integrative modeling of processes such as gene regulation and metabolism. Information flow from gene regulation (modeled via a gene regulatory network) to metabolism (modeled via a genome-scale metabolic model) is well-studied, but the reciprocal regulation of genes by metabolites is less explored. We introduce CausalFlux, a method that models bidirectional feedback between genes and metabolites, in order to predict steady-state reaction fluxes under wild-type (WT) or perturbed (e.g., gene knockout/KO) conditions. CausalFlux does so by iteratively performing causal surgery on a Bayesian gene regulatory network and constraint-based analysis of a coupled metabolic model. CausalFlux enabled us to assess the impact of two-way feedback in several testbed models and real-world biological systems by comparing its predictions to those of TRIMER, a state-of-the-art model of gene-to-metabolite one-way feedback. Incorporating bidirectional feedback, as in CausalFlux, improved the Spearman correlation between actual and predicted fluxes in 92% of the 39 distinct simulation conditions relative to TRIMER. For predicting growth/no-growth phenotype following single-gene KOs in E. coli, CausalFlux achieved a balanced accuracy of 0.79 in identifying essential genes, and TRIMER achieved 0.71 for the same task, again highlighting the importance of modeling two-way feedback. In ablation studies that further dissect the role of specific metabolite[->]gene feedback edges in E. coli, the F1 scores of gene essentiality predictions decreased by 7.5% and 13% upon ablation of feedback edges from any metabolite to the crp gene and the 10 metabolic feedback genes with the highest influence on the KO genes, respectively. Finally, we highlight the application of CausalFlux to predict the essentiality of several hundred genes under different media conditions. Overall, our findings show that CausalFlux can crucially utilize information on feedback metabolites to predict trends in reaction fluxes and qualitative (growth/no-growth) outcomes; thereby encouraging future systems modeling efforts to carefully incorporate not only gene-to-metabolite but also metabolite-to-gene interactions. AvailabilityCode pertaining to the CausalFlux method, and its benchmarking and application is publicly available at: https://github.com/BIRDSgroup/CausalFlux. Author summaryThe myriad processes within a living cell, such as gene regulation or metabolism, are tightly interconnected. Modeling these interconnected processes can offer a deeper mechanistic understanding of cellular behaviors, as well as guide efforts that engineer the metabolic output of a cell. In this work, we develop a novel integrated model of gene regulation and metabolism that incorporates bidirectional feedback between these two processes, via the concept of metabolite-induced causal surgery on a gene regulatory network and gene-induced constraints on the fluxes of metabolic reactions. Our model, which we call CausalFlux, represents an advance over most existing models that capture just the one-way gene-to-metabolism feedback (i.e., genes coding for enzymes that control metabolic reactions). Our CausalFlux methodology opens up an unique opportunity to quantify the impact of two-way feedback in gene-metabolite systems, via comparison of CausalFluxs predictions to those of TRIMER, a published model incorporating one-way feedback alone. For predicting reaction fluxes in testbed models and essential genes in E. coli, quantitative comparison of the performance of CausalFlux vs. TRIMER showed that accounting for two-way feedback leads to more accurate and biologically meaningful predictions. CausalFlux also enabled us to quantify the effect of two-way feedback by comparing prediction performance before and after ablation of certain feedback edges from metabolites to genes. Overall, our findings highlight the importance of modeling gene regulation and metabolism as two-way interconnected systems within a living cell, and encourage future works to incorporate gene{leftrightarrow}metabolite feedback into their analyses.

Fucosylation is a cancer-associated glycosylation change, and core fucosylation of N-glycans catalyzed by the 1,6-fucosyltransferase FUT8, has been closely linked to tumor progression, metastasis, drug resistance, and poor prognosis. However, the core-fucosylated proteins that directly support hepatocarcinogenesis are not fully defined. In a KRAS-G12D-driven mouse liver cancer model, we observed increased fucosylation and FUT8 upregulation, and glycoproteomic analysis of fucose-enriched fractions identified low-density lipoprotein receptor-related protein 1 (LRP1) as a prominent core-fucosylated protein in tumor tissue. In immortalized mouse hepatocytes, genetic or pharmacological inhibition of FUT8 markedly increased Lrp1 mRNA and protein, indicating that loss of core fucosylation is accompanied by robust upregulation of LRP1. In human HepG2 cells, LRP1 knockout suppressed cell proliferation and markedly altered colony morphology, leading to compact rounded clusters instead of the typical polygonal pattern. It also reduced EGFR protein and further inhibited proliferation of HepG2 cell. These findings identify LRP1 as a FUT8-dependent core-fucosylated receptor in experimental hepatocarcinogenesis and suggest that the FUT8-LRP1 axis contributes to the maintenance of proliferative signaling in hepatoma cells.

Background: Kidney transplantation is the preferred treatment strategy for end-stage kidney disease. Deceased donor kidneys usually undergo cold storage until kidney transplantation, leading to cold ischemia injury that may contribute to poor graft outcomes. However, the molecular characterization of potential mechanisms of cold ischemia injury remains incomplete. Results: To bridge this knowledge gap, we leveraged the 10x Visium spatial transcriptomic technology to perform full transcriptome profiling of murine kidneys subject to varying durations of cold ischemia typical in a deceased donor kidney transplant setting. We developed a computational workflow to identify and compare spatiotemporal transcriptomic changes that accompany the injury pathophysiology in a tissue compartment-specific manner. We identified proportional enrichment of oxidative phosphorylation (OXPHOS) genes with increasing duration of cold ischemia injury within the oxygen-lean inner medulla region, suggestive of atypical metabolic presentation. This was distinct in cold ischemia injury tissue compared to warm ischemia-reperfusion kidney injury tissue. Spatiotemporal trends were validated by qPCR and immunofluorescence in a larger cohort of mice. We provide an interactive online browser at https://jef.works/CellCarto-ColdIschemia/ to facilitate exploration of our results by the broader scientific and clinical community. Conclusions: Altogether, our spatiotemporal transcriptomic analysis identified coordinated molecular changes within metabolic pathways such as OXPHOS deep within the cold ischemic kidney, highlighting the need for increased attention to the inner medulla and potential opportunities for new insights beyond those available from superficial biopsy-focused tissue examinations.

Recent elucidation of the bacterial sphingolipid synthesis pathway has revealed that these lipids are produced by a range of taxonomically diverse species. In contrast to the biosynthetic pathways, the mechanism by which sphingolipids are transported from the inner membrane to the cell surface in Gram-negative bacteria remains a mystery. Here, we identify and characterize paralogs of the well-characterized lipopolysaccharide (LPS) inner membrane ABC transporter proteins encoded within the sphingolipid locus. Using Caulobacter crescentus as a model system, we analyzed three putative inner membrane proteins with homology to LptF, LptG, and LptC. Deletion of these genes was lethal, likely due to the accumulation of anionic sphingolipids in the inner membrane. We further show that the LptF and LptG homologues form a complex like their LPS counterparts and discover that they interact with the LPS ATPase LptB. Together, our data suggest that ceramide transport to the outer membrane is facilitated by an ABC transporter consisting of a sphingolipid-specific LptFG homolog coupled to the LPS LptB, supporting a model in which sphingolipid transport partially converges with the LPS transport system. Together, these findings reveal an unexpected evolutionary relationship between sphingolipid and lipopolysaccharide transport.

Epitranscriptomics has recently gained significant momentum due to technological advances and translational applications, however, studies on bacterial RNA modifications remain limited. Bacterial RNA remains notoriously prone to degradation and methodologies to investigate the epitranscriptome are challenging. Prior research has shown RNA modifications modulate antimicrobial resistance, virulence and pathogenicity. This research employed CRISPR interference to knock down five known Escherichia coli rRNA modification genes (rlmF, rlmJ, rluD, rsmF and rsmG) in three E. coli strains. These isolates underwent growth curves, proteome analysis and native RNA sequencing CRISPRi adequately silenced the majority of RNA modification genes in E. coli (>80% reduction). Significant growth delays were associated with rlmF, rsmF and rsmG repression. Unique protein pathways corresponding with RNA modification loss were found for rlmJ (TreB, XylF), rluD (CysH, HycB, PutP, TrpB), rsmF (EvgA) and rsmG (OppC). Known rRNA modification sites for rluD ({Psi}) and rsmG (m7G) were detected from analysis of nanopore electrical signal, however, only a weak signal was apparent for m6A (rlmF, rlmJ) and m5C (rsmF) modifications. The inhibition of rRNA modifications resulted in mRNA modification changes including for genes ompC, cspC, dbhA, dbhB and secY. Our work provides an approach for unravelling the epitranscriptome of E. coli and gain insight into its functional role.

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.

The ability of protoplasts to regenerate into whole plants underpins advances in crop engineering and pluripotency research. However, most protoplasts exhibit poor division and limited shoot regeneration, restricting their broader utility. Here, we present EppTec (Efficient regeneration of transgene-free whole Plants from Protoplasts reprogrammed by Transiently Expressed Combinatorial factors), a transient expression platform using defined combinatorial factors (CFs) to unlock protoplast pluripotency and enable transgene-free whole-plant regeneration. Among 24 CFs tested, co-transfection of SEPW (SCR, ESR1, PSK5, and WOX5) markedly enhanced regeneration across diverse plant species in an evolutionarily conserved manner. We demonstrate that SEPW co-transfection induces epigenomic reprogramming, formation of a distinct cell cluster undergoing reprogramming and cell-cycle re-entry, and long-term reprogramming into pluripotent cells. These findings establish EppTec as a robust strategy to restore the regenerative capacity of plant cells from diverse species. EppTec may serve as a platform to revolutionize regeneration-based plant biotechnologies and conserve endangered plant species.

Spiroplasma are wall-less helical bacteria that swim by switching the handedness of their helices. In most bacteria, MreB, a bacterial actin, forms relatively static filaments beneath the membrane to organize cell wall synthesis. In contrast, Spiroplasma eriocheiris encodes five MreB isoforms (SpeMreBs), and swimming requires a pair of isoforms, SpeMreB5 together with SpeMreB4 (or the related isoform SpeMreB1) (1) Yet how these MreBs generate force and membrane deformation remains unclear. To examine the membrane-deforming activities of SpeMreBs, we demonstrated a simple reconstitution system using the non-motile synthetic bacterium JCVI-syn3B and purified SpeMreBs expressed in Escherichia coli. Lysates of syn3B expressing SpeMreB5 deformed liposomes in a concentration-dependent manner. In contrast, lysates co-expressing both SpeMreB5 and SpeMreB4 showed a plateau in the frequency of deformation, suggesting that SpeMreB4 suppresses membrane deformation driven by SpeMreB5. Deformed liposomes exhibited either fluctuating or stable behaviors. ATP depletion changed both the frequency and behavior of deformation, indicating that membrane remodeling depends on the nucleotide state of SpeMreBs. Reconstitution with purified SpeMreBs from E. coli confirmed that SpeMreB5 alone deforms membranes, whereas SpeMreB1, a member of the same class as SpeMreB4, suppresses deformation. These results suggest that membrane shape in Spiroplasma is dynamically regulated by antagonistic interactions among isoforms of SpeMreBs isoforms and their nucleotide-dependent assembly states.

Antimicrobial resistance (AMR) has resulted in the need for the development of alternative strategies for combating pathogens and growth promotion of poultry, including the use of plant-derived compounds such as hop (Humulus lupulus) isolates. The present study evaluates the correlation patterns of the biological activity of five major hop isolates (humulone, lupulone, isohumulone, xanthohumol, and isoxanthohumol) against Bacillus subtilis, Micrococcus luteus, and a chicken cell line UMNSAH/DF-1 using a two-dimensional checkerboard assay. Fractional inhibitory concentrations ({sum}FIC) were used to classify interactions as additive, synergistic, or antagonistic, and selectivity indices assessed antibacterial versus cytotoxic effects. On B. subtilis, combinations were predominantly additive, whereas M. luteus, in contrast, showed variable interactions, including also synergistic (humulone + lupulone) and antagonistic combinations (isohumulone + isoxanthohumol), demonstrating the impact of the metabolic resilience of the target organism. Cytotoxicity in UMNSAH/DF-1 cells was largely additive, with synergistic effects observed only for isomerized compounds. Selectivity analysis highlighted humulone-lupulone combinations as most favorable, offering high antibacterial activity with minimal cytotoxicity. These results provide novel insights for selecting hop isolate combinations for the development of phytogenic feed additives (PFAs), emphasizing that both compound composition and target organism physiology critically shape efficacy and safety outcomes.

Fusarium head blight (FHB) is a major threat to global wheat production and food safety due to contamination with mycotoxins, such as deoxynivalenol (DON). The emergence of new mycotoxin chemotypes, including 7--hydroxy,15-deacetylcalonectrin (3ANX), presents an evolving challenge for disease management and resistance breeding. Here, we performed a field-based, systems-level proteome analysis of wheat infected with Fusarium graminearum strains belonging to the common 15ADON and recently emerged 15ADON/3ANX chemotypes. Across host and pathogen, we quantified more than 9,200 proteins, providing extensive coverage of infection-associated molecular responses. Infection with 15ADON/3ANX strains suppressed canonical wheat detoxification pathways while promoting structural and oxidative defence responses. Concurrently, the fungal proteome of 15ADON/3ANX-producing strains indicated altered mitochondrial ribosome function and alternative virulence strategies. Further investigation of the host-pathogen interface defined hub protein networks negatively regulating classical detoxification markers, suggesting coordinated regulation of host defence responses regardless of chemotype. Molecular responses were linked to field phenotypes by quantification of DON-3-glucoside/DON ratios and disease severity, defining positive correlations in 15ADON infections, which were abolished upon 15ADON/3ANX infection, indicating chemotype-specific evasion or suppression of host defenses. These findings demonstrate reprogramming of host-pathogen interaction networks and reveal molecular targets that may inform chemotype-aware breeding strategies to enhance crop resilience.

While the need for continuous blood pressure (BP) monitoring in Japan is high, there are no commercially available cuffless devices for personal daily monitoring use. Fingertip-based sensors are a promising alternative as they eliminate the discomfort of repeated cuff inflation. However, their reliability during winter has been a major technical limitation due to cold-induced peripheral vasoconstriction. This study aimed to address this issue by validating a novel fingertip-based continuous BP monitor used by exercising adults during summer and winter. Eleven community-dwelling older adults (mean age, 73.1 {+/-} 8.8 years) were included in this seasonal comparative study. During exercise, we compared a personal fingertip-based continuous monitor (ArteVu) with a standard oscillometric cuff device (Omron) in summer (mean, 26.5{degrees}C) and winter (mean, 7.4{degrees}C). The study also evaluated the device's accuracy during exercise-induced BP fluctuations and seasonal environmental changes. Awareness of the participants regarding BP management was also assessed using questionnaires. There were strong correlations for systolic BP (SBP) between summer and winter (r = 0.93 in summer; r = 0.88 in winter). Although the mean difference for the SBP was higher in winter than in summer (3.1 {+/-} 11.2 mmHg vs. 0.2 {+/-} 9.4 mmHg), the values remained within a clinically acceptable range for personal monitoring. Notably, 72.7% of participants reported that the ease of using the fingertip-based device significantly increased their awareness and motivation for daily BP management. This study confirms the feasibility of cuffless fingertip-based continuous BP monitoring across different seasons, including in winter. By overcoming the seasonal limitations, this device fills a critical gap in the Japanese health-monitoring market. Our findings support the development of smaller and more portable models, representing a shift from traditional "snapshot" cuff measurements to continuous and integrated lifestyle monitoring for older adults.

Irritable Bowel Syndrome (IBS) affects a substantial proportion of university students, yet its factors remain incompletely characterised in South Asian populations. We reanalysed a publicly available dataset of 550 Bangladeshi students from Hasan et al. (2025), conducting a data audit that identified implausible records, including males reporting menstrual symptoms, and reduced the analytic sample to 506 observations. Using Explainable Boosting Machines (EBMs), which capture non-linear effects and pairwise interactions without sacrificing interpretability, we found that psychological distress, elevated BMI and academic dissatisfaction were the strongest predictors of IBS (mean AUC = 0.852 across 100 stratified train-test splits). Critically, several findings diverged from the original logistic regression analysis. Physical activity showed a non-linear risk pattern only at high intensity, the association with gender was substantially weaker when we accounted for metabolic and psychological factors as well and malnourishment does not have a strong an impact as in the original study. These divergences likely arise because the machine-learning model captures non-linear effects and interactions that were not represented in the original regression specification. Our findings underscore the value of reanalysing existing datasets with methods suited to capturing complexity and highlight data quality verification as a necessary step in the secondary analysis.

BackgroundPatent ductus arteriosus (PDA) is a common and potentially serious cardiovascular condition in preterm infants, particularly those with low gestational age and birth weight. Its management remains controversial due to variability in screening, diagnostic criteria, and treatment strategies. This study aimed to evaluate risk factors, outcomes, and management strategies for PDA in preterm infants, and to identify predictors of clinical and echocardiographic response to therapy. MethodsWe conducted a retrospective cohort study over a 4-year period (2016-2019) in the neonatal intensive care unit (NICU) of a tertiary care center. All consecutive preterm infants admitted during the study period were eligible. Infants with echocardiographically confirmed PDA who received pharmacological treatment with intravenous paracetamol or ibuprofen were included in the analysis. Missing data were minimal and handled using available-case analysis. Statistical analyses included descriptive statistics, Pearsons chi-square test, and multivariable logistic regression. ResultsAmong 2154 preterm infants admitted to the NICU, 60 were diagnosed with PDA (incidence : 2.8%). The mean gestational age was 29 {+/-} 2.6 weeks, and the median birth weight was 1200 g. Respiratory distress occurred in 95% of cases, mainly due to hyaline membrane disease (86.7%). PDA was symptomatic in 80% of infants. First-line treatment resulted in clinical improvement in 77% and ductal closure in 83.3% of cases, most within 3 days. Predictors of successful closure included gestational age [&ge;] 28 weeks (OR = 5.9; 95% CI : 1.7-20.2) and antenatal corticosteroid exposure (OR = 1.2; 95% CI : 1.0-1.6). Overall mortality was 35% and was significantly higher in infants < 28 weeks (OR = 5.0; 95% CI : 2.4-10.3). Clinical improvement (OR = 3.7) and echocardiographic closure (OR = 4.5) after first-line treatment were associated with reduced mortality. ConclusionsPDA in preterm infants is associated with substantial morbidity and mortality, particularly in those born before 28 weeks of gestation. Early diagnosis, antenatal corticosteroid exposure, and timely pharmacological treatment may improve outcomes. Systematic echocardiographic screening in high-risk neonates should be considered.

ImportanceGuideline-concordant care for young children with attention-deficit/hyperactivity disorder (ADHD) includes recommending parent training in behavior management (PTBM) as first-line treatment. However, assessing guideline adherence through manual chart review is time-consuming and costly, limiting scalable and timely quality-of-care measurement. ObjectiveTo evaluate the accuracy and explainability of large language models (LLMs) in identifying PTBM recommendations in pediatric electronic health record (EHR) notes as a scalable alternative to manual chart review. Design, Setting, and ParticipantsThis retrospective cohort study was conducted in a community-based pediatric healthcare network in California consisting of 27 primary care clinics. The study cohort included children aged 4-6 years with [&ge;] 2 primary care visits between 2020-2024 and ICD-10 diagnoses of ADHD or ADHD symptoms (n=542 patients). Clinical notes from the first ADHD-related visit were included. A stratified subset of 122 notes, including all cases with model disagreement, was manually annotated to assess model performance in identifying PTBM recommendations and rank model explanations. ExposuresAssessment and plan sections of clinical notes were analyzed using three generative large language models (Claude-3.5, GPT-4o, and LLaMA-3.3-70B) to identify the presence of PTBM recommendations and generate explanatory rationales and documentation evidence. Main Outcomes and MeasuresModel performance in identifying PTBM recommendations (measured by sensitivity, positive predictive value (PPV), and F1-score) and qualitative explainability ratings of model-generated rationales (based on the QUEST framework). ResultsAll three models demonstrated high performance compared to expert chart review. Claude-3.5 showed balanced performance (sensitivity=0.89, PPV=0.95, and F1-score=0.92) and ranked highest in explainability. LLaMA3.3-70B achieved sensitivity=0.91, PPV=0.89, and F1-score=0.90, ranking second for explainability. GPT-4o had the highest PPV [0.97] but lowest sensitivity [0.82], with an F1-score of 0.89 and the lowest explainability ranking. Based on classifications from the best-performing model, Claude-3.5, 26.4% (143/542) of patients had documented PTBM recommendations at their first ADHD-related visit. Conclusions and RelevanceLLMs can accurately extract guideline-concordant clinician recommendations for non-pharmacological ADHD treatment from unstructured clinical notes while providing clear explanations and supporting evidence. Evaluating model explainability as part of LLM implementation for medical chart review tasks can promote transparent and scalable solutions for quality-of-care measurement.

Objective Cerebral palsy (CP) affects approximately 1 million Americans and 18 million individuals worldwide, yet contemporary US epidemiologic data remains limited. We aimed to use Cerebral Palsy Research Network (CPRN) clinical registry to describe demographics and clinical characteristics of individuals with CP across the US and determine associations with gross motor function and genetic etiology. Methods Registry subjects were included if they had clinician-confirmed CP and prospectively entered data for Gross Motor Function Classification System (GMFCS) Level, gestational age, genetic etiology, CP distribution, and tone/movement types. Logistic regression was used to determine which of these variables plus race, sex, ethnicity, and age were associated with GMFCS level and genetic etiology. Results A total of 9,756 children and adults with CP from 22 CPRN sites met inclusion criteria. Participants were predominantly White (73.0%), male (57.3%), non-Hispanic (87.8%), and younger than 18 years (73.7%). Most were classified as GMFCS levels I-III (55.6%), born preterm (52.8%), had spasticity (83.8%), and had quadriplegia (41.9%); 12.2% were identified as having a genetic etiology. Tone/movement types, CP distribution, and gestational age were significantly associated with both GMFCS level and genetic etiology (p<0.001). Compared to White individuals, Black individuals were more likely to have greater gross motor impairment (p<0.001). Conclusion In this large US cohort, clinical and demographic factors, including race, were associated with gross motor function and genetic etiology in CP. These findings highlight persistent disparities and demonstrate the value of a national clinical registry for informing prognostication, quality improvement efforts, and targeted genetic testing strategies.

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: 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.