The FEBS Journal

Purpose: Pathogenic variants in NFIX cause Marshall-Smith syndrome and Malan syndrome (MALNS). We identified a severe subtype of MALNS characterized by adolescent-onset musculoskeletal deterioration and investigated functional consequences of underlying variants. Methods: Clinical data were collected from seven individuals with pathogenic NFIX variants. Wild-type and mutated recombinant NFIX DNA-binding domains (DBDs) were evaluated using biochemical, structural, and DNA-binding assays. Results: Six individuals carrying R116W, R116P, K125E, or G147E NFIX substitutions developed progressive muscle wasting, markedly reduced body mass index, and rapidly progressive scoliosis after the typical childhood features of MALNS; two died from disease-related complications. A seventh individual with R116G did not develop this severe phenotype. Functional studies on recombinant NFIX DBDs showed complete or near-complete loss of DNA-binding activity for R116W, R116P, K125E, and G147E despite preserved protein folding, consistent with disrupted DNA recognition and a potential dominant-negative mechanism. In contrast, R116G exhibited a 7.7{degrees}C decrease in thermal stability, which may support haploinsufficiency mediated by protein degradation. Conclusion: Specific NFIX missense variants define a severe subtype of MALNS associated with progressive musculoskeletal deterioration. In vitro functional studies support variant-specific disruption of DNA binding, providing a mechanistic basis of genotype-phenotype correlations and informing prognosis, clinical surveillance, and therapy development.

Dementia is a progressive condition that impairs cognitive processes such as memory, decision making, and the ability to manage daily activities. Recent estimates suggest that more than half of all dementia cases could be preventable by addressing their risk factors, including disease comorbidities such as diabetes and vision loss. Yet, we lack a comprehensive molecular map of dementia comorbidities. In this work, we analyzed Austrian nationwide hospital claims data, comprising 13 million hospital stays from 2015 to 2019, to systematically assess dementia-related risk across disease comorbidity patterns, covering both their molecular relationships and their epidemiological overrepresentation. We identified disease trajectories occurring before and at the time of dementia diagnosis, revealing both sex-specific and shared comorbidity patterns. Overall, we identified 51 potential risk factors, with a prominent contribution from endocrine and metabolic disorders. While Parkinson's disease emerged as a strong molecularly related driver of dementia, we also identified emerging and previously under chracterized risk factors, including vitamin D deficiency. This integrative framework provides a comprehensive view of dementia associated disease networks and identifies novel, potentially modifiable risk factors. These results offer new opportunities for targeted prevention strategies and advance our understanding of the complex interplay between comorbidities and dementia development.

Prenatal alcohol exposure (PAE) is a major cause of neurodevelopmental disorders, yet most children are diagnosed late or misdiagnosed. Neuroplacentology suggest that placental factors released into maternal and/or umbilical cord blood contribute to fetal brain development. Consistently, a preclinical inter-organ transcriptomic database revealed that PAE disrupts the expression ratio of angiogenic and inflammatory factors suggesting an angio-inflammatory response. This study aimed i) to assay, by multiplex immunoassay, angiogenic and inflammatory factors in maternal and umbilical cord blood from alcohol-consuming women and ii) to perform a maternofetal analysis according to neonatal sex. Afterwards, dysregulated factors from mothers who gave birth to females or males were submitted to STRING and ShinyGO analyses. Results showed that PAE differently altered the distribution profiles of dysregulated angiogenic and inflammatory factors in maternal and umbilical cord blood. Moreover, sex-specific differences were observed, with 36% of dysregulated proteins specific to males, 48% to females, and 16% common to both. STRING analysis revealed robust functional protein-protein interactions linking together inflammatory and angiogenic clusters while the ShinyGO analysis identified enriched pathways related to vascular shear stress. These findings provide the first maternofetal analysis of combined angiogenic and inflammatory factors from alcohol-consuming mothers.

Paroxysmal Nocturnal Hemoglobinuria (PNH) is characterized by hemolysis due to the loss of GPI-anchored complement regulators. While terminal complement inhibitors improve survival, the precise intracellular mechanisms driving the destruction of PNH erythrocytes remain controversial. A recently proposed model suggests PNH cells undergo an inflammatory programmed cell death ("spectosis") driven by an NLRP3-Caspase-8 signaling cascade. Here, we use a whole packed cell lysis approach to map the cytoskeletal degradation of primary erythrocytes across a 22-patient PNH cohort. Our data show that membrane attack complex (MAC) pore formation drives targeted {beta}-spectrin fragmentation, which correlates with rapid intracellular potassium (K+) efflux. Notably, when probing these primary patient samples, we detected a complete absence of the NLRP3 protein and found no functional evidence of Caspase-8 activation during MAC pore formation. Furthermore, caspase inhibition did not alter cytoskeletal degradation or K+ efflux. Instead, our data demonstrate that MAC-induced membrane perforation permits a rapid influx of calcium, which activates calpain, the dominant calcium-dependent protease in erythrocytes. Rather than an inflammatory cascade, this calcium-dependent calpain activity executes the degradation of {beta}-spectrin. These findings challenge current models of PNH hemolysis. We show that the destruction of PNH erythrocytes is a consequence of the MAC-calcium-calpain axis, rather than an inflammatory programmed cell death event. Consequently, therapeutic strategies aimed at targeting the inflammasome or caspase signaling will likely offer no clinical benefit for PNH patients.

Objectives: We tested how multi-level socioeconomic disadvantage relates to biological aging and systemic inflammation in women and men from the population-based Canadian Longitudinal Study on Aging (CLSA). Methods: We examined cross-sectional data from 8,516 CLSA participants with baseline measures on systemic inflammatory biomarkers (C-reactive protein, interleukin-6, and tumoral necrosis factor-) and biological aging (metabolomic and six DNA methylation [DNAm] age estimates). Plasma samples underwent metabolomic profiling by Metabolon, Inc. Metabolomic age was estimated separately in males and females using sex-stratified models based on age-correlated metabolite levels. DNAm data generated using the Illumina Infinium MethylationEPIC v1.0 array were used to estimate DNAm age across six established models, including Horvath, Hannum, PhenoAge, GrimAge, GrimAge2, and DunedinPACE. We used log-transformed metabolite levels to calculate metabolomic age by sex. We linked education, income, material and social deprivation to biomarkers of systemic inflammation and biological aging stratified by sex using generalized linear models. Multivariable models were adjusted by age, major behavioral risk factors, and chronic conditions. Results: Participants were aged on average of 62.6 years of age, and approximately 50% were females. In multivariable linear adjusted models, we found that in comparison to those earning [&ge;]$100K a year, women earning less <$20K were on average 1.14 (95%CI: 0.46, 1.82) year older with respect to metabolomic age; those earning [&ge;]$20K & <$50K were on average 0.90 (95%CI: 0.26, 1.53) years older; and those earning [&ge;]$50K & <$100K were on average 0.70 (95%CI: 0.05, 1.34) years older. We did not observe this dose response among men. A similar dose-response association was observed for interleukin-6 in both men and women. Discussion: These findings suggest that socioeconomic adversity influences not only inflammatory pathways but also distinct biological aging processes, including metabolomic aging.

Background: Caregivers of children with retinoblastoma (RB) face substantial psychological and socioeconomic challenges. However, the factors independently associated with caregiver burden and the distribution of risk across caregiver subgroups remain incompletely characterized. We examined psychosocial and socioeconomic correlates of caregiver burden, identified distinct vulnerability profiles, and evaluated factors associated with high-risk profile membership. Methods: This cross-sectional study enrolled 413 primary caregivers of children with RB at a tertiary ophthalmic oncology center. Participants completed validated measures of caregiver burden (ZBI-22), anxiety (GAD-7), perceived social support (PSSS), family functioning (FAD-GF), and mental and physical quality of life (SF-12 MCS and PCS). Multivariable linear regression identified factors independently associated with caregiver burden and mental quality of life. Mediation analysis evaluated the indirect association between social support and burden through family functioning, and moderation analysis assessed whether household income modified the association between family dysfunction and burden. Latent profile analysis (LPA) identified caregiver risk profiles, and multinomial logistic regression examined factors associated with profile membership. Results: Anxiety showed the strongest independent association with greater caregiver burden (standardized coefficient beta = 0.641, 95% CI [1.46, 1.84], P < 0.001) and poorer mental quality of life (beta = -0.483, 95% CI [-0.12, -0.08], P < 0.001). Family debt was independently associated with greater burden (beta = 0.195, P = 0.040). Family functioning accounted for 32.19% of the total association between social support and burden. Household income modified the association between family dysfunction and burden (interaction B = -0.85, P < 0.001), with a steeper gradient in lower-income households. LPA identified three profiles: severe burden-high vulnerability (n = 82, 19.85%), moderate burden (n = 193, 46.73%), and mild burden-high resilience (n = 138, 33.41%). Low-to-moderate household income was associated with higher odds of severe-profile membership (OR = 31.50, 95% CI [6.56, 151.24], P < 0.001). Conclusions: Caregiver burden in pediatric RB was associated more strongly with psychosocial and socioeconomic factors than with the clinical indicators examined. Family functioning partly accounted for the association between social support and burden, while household income modified the association between family dysfunction and burden. These findings support prospective evaluation of family-centered and financial-support interventions and suggest that profile-based screening may help identify caregivers requiring more intensive support.

Insulin signalling contributes to neurodevelopment and brain function, and insulin resistance (IR)-related traits are associated with cognitive performance. However, the genetic architecture shared across specific cognitive domains and IR-related phenotypes remains insufficiently defined. We analysed large-scale genome-wide association study summary statistics for 11 IR-related traits (N=53,334-933,970) and 10 cognitive measures (N=28,156-436,853) to quantify global and local genetic correlations, fine-map shared association signals, and annotate implicated genes and drug-gene interactions. Pairwise global and local genetic correlations were estimated, and shared high-confidence variants were prioritised using the multivariate Sum of Single Effects model. Positional and expression quantitative trait locus mapping was performed, and implicated genes were examined through functional annotation, tissue enrichment, and drug-gene interaction analyses. Low-to-moderate genetic correlations were observed between six IR-related traits and seven cognitive measures (|rg|=0.08-0.34), with predominantly opposite directions, except for correlations involving visual declarative short-term memory. Local genetic correlations showed mixed effect directions across most trait pairs, and multivariate fine-mapping prioritised 696 shared likely causal variants with high posterior support. Gene annotation indicated enrichment in several pathways, including immune-related, signal transduction, neurogenesis, neurotransmitter metabolism, receptor regulation, and lipid and cholesterol metabolism regulation. Implicated genes were expressed across various brain regions and showed prior associations with neuropsychiatric and cardiometabolic conditions. Several drug-gene interactions were identified, involving immunomodulatory and anti-inflammatory compounds. These findings indicate widespread heterogeneous genetic overlap between IR-related traits, particularly body mass index and waist-to-hip ratio, and cognitive measures of general intelligence, processing speed, and short-term visual declarative memory. The findings prioritise apolipoprotein-related lipid transport and inflammatory and oxidative stress pathways as candidate mechanisms linking cognitive, cardiometabolic, and neuropsychiatric phenotypes.

Red blood cell (RBC) disorders arose as an advantageous evolutionary response to malaria infections. In a heterozygous condition, such as in Southeast Asian Ovalocytosis (SAO), hosts are protected against severe malaria. In malaria-endemic regions, RBC disorders are presumed to be highly prevalent. Tanjung Leidong, a moderately endemic area in North Sumatra (API 1.13 in 2024), lacks comprehensive data on RBC disorder prevalence beyond G6PD deficiency. Therefore, this study aims to characterize the RBC disorders in this region as well as to characterize the anemia status in children living in Tanjung Leidong. Schoolers attending D. I. Panjaitan elementary to high school were recruited and screened for malaria by microscopy and G6PD deficiency using the STANDARD G6PD Assay. The DNA of the participants was also extracted to be genotyped for SAO, Hemoglobin E (HbE), and -thalassemia. Exclusively, G6PD-deficient DNA samples were genotyped further to determine variants. The proportion of G6PD deficiency, SAO, HbE, -thalassemia one-gene deletion, and two-gene deletion were 0.90%, 0.90%, 2.40%, 6.26%, and 0.30%, respectively. Anemia prevalence was approximately 14%, and RBC disorders were observed across children with normal to obese BMI. No malaria infections were detected by microscopy. The predominance of asymptomatic RBC disorders highlights that they are protective against malaria infection, although their protective role against malaria could not be directly assessed in this study. Both nutritional and genetic factors are found to contribute to anemia in this cohort. These findings underscore the importance of integrated screening strategies for RBC disorders and anemia in malaria-endemic settings.

While extensive cellular heterogeneity in renal cell carcinomas (RCC) is linked to diverse clinical outcomes, our understanding of this diversity is limited to those driven by clonal patterns or activity of canonical pathways. Here, we present a compendium of over 85,000 single-cell gene expression profiles from primary and metastatic tumors as well as patient-derived models across four RCC subtypes, including the rare clear cell papillary renal cell tumors, which we show are often misclassified and for which we identify CASP14 as a highly sensitive and specific biomarker. We dissect malignant cell variation within and across tumors using a generative modeling framework that accounts for clonal and copy number-driven expression shifts, defining 59 gene expression programs that deconstruct canonical pathways into functional submodules with divergent activity patterns, distinct regulators, and differential association with clinical outcomes. Despite the canonical view that VHL-deficient clear cell RCC exists in a constitutive pseudohypoxic state, we show strong intra-tumor variability of a hypoxia inducible factor 2 (HIF2)-driven program linked to poor outcome. We also identify early, spatially organized activation of a complete epithelial-to-mesenchymal transition (EMT) program, loss of epithelial identity, and upregulation of protein translation programs as key characteristics of metastatic progression. Finally, a metastatic signature capturing cellular de-differentiation and translational activity identifies primary tumors associated with adverse clinical outcomes. Together, this resource establishes a framework for dissecting malignant cell heterogeneity, refines RCC subtype classification, and defines transcriptional programs underlying metastasis progression.

Objective: Photobiomodulation therapy has emerged as a promising modality to facilitate scar healing and pain management in dermatology and plastic surgery. However, its role in postoperative care following spine surgeries remains understudied. This double-blinded, placebo-controlled study aimed to investigate the effects of photobiomodulation in patients with chronic lower back pain undergoing lumbar decompression, with postoperative wound healing as the primary outcome and pain reduction and functional recovery as secondary outcomes. Methods: Patients were randomized to receive either active photobiomodulation braces (N=13) or placebo braces (N=12). Follow-up assessments were performed at 2, 4, 6, 8, and 12 weeks postoperatively. Outcomes included wound healing (Stony Brook Scar Evaluation Scale), back and leg pain (Visual Analog Scale), quality of life (EuroQol 5D), and functional status (Oswestry Disability Index). Results: Compared to the placebo group, the photobiomodulation treatment group had a 4.12-fold cumulative improvement in final scar scores, with significant between-group differences at postoperative weeks 6, 8, and 12 (p = 0.0062, 0.010, 0.042). Among patients with severe preoperative disability, treatment resulted in a 1.89-fold faster improvement in back pain (p=0.025) and a 1.80-fold faster improvement in ODI scores (p=0.025); and superior treatment effect on wound healing were again observed at weeks 6, 8, and 12. Among patients with poor initial scars, treatment led to a significantly better scar outcome than placebo at week 6 and a 1.94-fold faster EQ5D improvement (p=0.052), with significant gains observed as early as two weeks after surgery. There were no adverse events associated with photobiomodulation treatment. Conclusions: Photobiomodulation significantly promoted postoperative wound healing following lumbar decompression surgery, with therapeutic benefits preserved even in patients with poor baseline scar scores and functional impairment. This indicates that the efficacy of photobiomodulation is not limited by the initial scar condition or disability, supporting its broad clinical applicability. Additionally, patients with severe preoperative disability experienced greater benefits from photobiomodulation than placebo, including faster reduction in back pain and more rapid improvement in functional capacity, highlighting its role in postoperative pain management and rehabilitation. These therapeutic effects are likely mediated by photobiomodulation-induced reduction of inflammation and enhancement of tissue repair. Together, this study suggests that photobiomodulation can be a promising adjunct therapy to facilitate postoperative recovery in patients undergoing spine surgery.

Fracture nonunion remains a major cause of morbidity, yet patient-specific factors associated with impaired healing remain incompletely characterized. Anemia has been associated with adverse orthopaedic outcomes, but its relationship with fracture nonunion is poorly understood. We examined whether pre-fracture anemia, anemia burden, and clinically relevant anemia subtypes were associated with nonunion following tibial or femoral fractures. Using commercial and Medicare fee-for-service claims from 2016 through 2023, we identified adults aged 19 years or older with a tibial or femoral fracture, continuous enrollment during the preceding year and for at least six months after fracture, and no baseline cancer. Pre-fracture anemia was evaluated as any anemia, the number of distinct anemia diagnoses, and nutritional, hemolytic, aplastic, and other anemia subgroups. Nonunion occurring six to eighteen months after fracture was assessed using incidence rates and multivariable-adjusted hazard models. Among 326,673 adults, 149,704 had pre-fracture anemia and 176,969 did not. The crude incidence of nonunion was 42% higher among individuals with anemia than among those without anemia (incidence rate ratio, 1.42; 95% confidence interval, 1.32 to 1.53) and increased with greater anemia burden. After adjustment for demographic and clinical characteristics, including prior fractures at other anatomical sites, pre-fracture anemia remained associated with nonunion following tibial and femoral fractures, with hazard ratios of 1.83 (95% confidence interval, 1.54 to 2.18) and 1.38 (95% confidence interval, 1.26 to 1.50), respectively. Associations were also observed for nutritional and other anemias, whereas estimates for hemolytic and aplastic anemias were limited by few nonunion events. Within the femur, the association was strongest for distal fractures. These findings demonstrate that pre-fracture anemia is independently associated with nonunion. The increase in risk with greater anemia burden and findings across evaluable subgroups suggest that pre-fracture anemia may help identify patients at increased risk of impaired fracture healing.

Background. Given uncertainty about whether later-life health at similar ages is improving over time, we examined trends across multiple health domains. Methods. We analysed data from community-dwelling adults aged 50 and older in the English Longitudinal Study of Ageing in 2004/05, 2012/13, and 2023/24 (main survey: N=8389, 8549, and 6090, respectively). Outcomes included self-rated health, limiting long-standing illness, pain, mobility limitations, cardiometabolic and chronic conditions, obesity, inflammation, mental health, quality of life and memory. Weighted pooled modified Poisson and linear regressions compared outcomes over time, overall, and by age group and education, with additional adjustment for sex and wealth. Results. Adjusted estimates showed divergent trends. Fair/poor self-rated health increased from 27% to 34%, and any pain from 37% to 47%, whereas mobility impairments declined from 58% to 52%. Self-reported high cholesterol increased from 19% to 39%, while biomarker-defined high cholesterol declined from 78% to 54%; diabetes increased on both measures. Psychiatric problems increased from 6% to 10%, quality of life declined, and memory improved. However, trends differed by age and education, particularly for limiting long-standing illness, mobility limitations, cholesterol biomarkers, and mental health, indicating that aggregate trends masked unevenly distributed changes. Conclusion. Later-life health in England has not improved uniformly. Gains in functioning, biomarkers, and cognition coexist with rising pain and poorer mental health. Trends were also socially and age patterned, producing increasingly multidimensional and socially patterned health outcomes. Multidomain health monitoring is essential for interpreting population health trends and planning healthy ageing, prevention, long-term care, and work policies.

Blood banks represent highly controlled healthcare environments where microbiological surveillance has traditionally focused on blood products rather than environmental microbial reservoirs. Despite their critical role in transfusion safety, the ecology of surface-associated microorganisms and the persistence traits that enable their long-term survival remain poorly understood. Here, we combined scanning electron microscopy, culture-based microbiology, phenotypic characterization, MALDI-TOF mass spectrometry, and whole-genome sequencing to investigate whether surfaces within a public blood bank facility constitute reservoirs of environmentally derived bacteria with enhanced persistence potential. Samples collected from a public blood bank in Tucuman, Argentina yielded 37 culturable bacterial isolates, predominantly Gram-positive environmental taxa together with a limited number of opportunistic Gram-negative species. More than 30% of the isolates exhibited multidrug resistance, while several strains displayed strong biofilm formation, amyloid-like fiber production, motility, and hemolytic activity, indicating multiple phenotypic strategies associated with long-term surface persistence. Whole-genome sequencing of six representative isolates confirmed species identity, identified genes related to antimicrobial resistance, adhesion, biofilm formation, stress adaptation, and cytotoxicity, and revealed frequent genotype-phenotype discordance, highlighting the importance of integrating genomic and phenotypic analyses. Notably, one isolate exhibited less than 92% average nucleotide identity with publicly available genomes, suggesting the presence of a previously undescribed environmental species. Thus, blood bank surfaces function as selective ecological niches favoring bacteria with persistence-associated traits rather than simply reflecting contamination from blood products. These microorganisms may constitute latent biosafety hazards if environmental barriers fail, particularly in facilities handling biological materials intended for vulnerable patients. Our results support the incorporation of integrated bioimaging, phenotypic characterization, and genome-resolved environmental surveillance into infection prevention strategies and transfusion biosafety programs within a One Health framework.

Background Coccidioidomycosis is an emerging fungal disease across the arid Americas and a frequent cause of community-acquired pneumonia. Understanding where Coccidioides populations originate, how they move across space, and whether they are expanding is important for interpreting changing patterns of Valley fever and anticipating future infection risk. Methods We prospectively collected and whole-genome sequenced 186 Coccidioides-positive clinical isolates submitted to a national diagnostic laboratory, and included 126 previously sequenced genomes. We applied genomic clustering, time-calibrated phylogenetic reconstruction, ancestral area reconstruction, mating-type assignment, and demographic inference to identify major populations, infer dispersal patterns, assess evidence for recombination and clonality, and reconstruct historical population dynamics. Findings We analyzed 312 genomes (139 C. immitis; 173 C. posadasii) and identified three major genetic populations within each species. C. immitis included two California-centered populations and one Pacific Northwest population, whereas C. posadasii included two Arizona-centered populations and one Texas-centered population. The most recent common ancestor was estimated at approximately 127,000 years for C. immitis and 234,000 years for C. posadasii. Most populations were not fully monophyletic, consistent with retained ancestral variation and/or ongoing gene flow. Inferred dispersal was largely asymmetric, with most movement originating from California in C. immitis and from Arizona and Texas in C. posadasii. Most populations contained both mating types, but one C. immitis population and a Brazilian subgroup of C. posadasii were clonal. All populations showed recent demographic expansion. Interpretation The evolutionary history of Coccidioides is characterized by strong geographic structure, ongoing gene flow, and recent demographic expansion. These processes are likely to influence future patterns of Valley fever endemicity and supports the use of genomic surveillance to detect shifts in disease risk as environmental conditions change.

CRISPR-Cas12a nucleases are classically activated through CRISPR RNA (crRNA) guided and PAM-dependent target recognition, which together establish a canonical heteroduplex associated with nuclease activation. Here we identify a crRNA- and PAM-independent activation pathway for Cas12a that reveals previously unrecognized conformational plasticity within its nucleic acid recognition interface. We show that short RNAs can directly occupy the canonical crRNA-binding channel and trigger a catalytically competent trans cleavage state in the absence of PAM recognition or canonical R-loop formation. Biochemical assays indicate that short RNAs bind the crRNA-binding channel and are competitively displaced by cognate crRNA, consistent with binding at a conserved nucleic acid-binding interface. Cryo-electron microscopy (cryo-EM) further reveals that Cas12a maintains its global catalytic architecture while exhibiting loss of canonical PAM-dependent stabilization and increased flexibility of the RuvC lid, alongside accommodation of a noncanonical RNA-DNA hybrid with inverted polarity relative to the crRNA-target duplex. This crRNA-independent activation pathway enables programmable, amplification-free detection of DNA and RNA targets independent of canonical guide-mediated recognition. Together, these findings define an alternative activation geometry for Cas12a and expand models of Class 2 CRISPR-Cas effector activation beyond crRNA- and PAM-directed recognition.

Importance Pharmacogenomic (PGx) guidelines can improve medication efficacy and reduce toxicity, but their application in pediatric intensive care units (PICUs) remains largely unexplored. Objective To determine the frequency of medications with established PGx guidelines administered in the PICU and assess the capacity of exome sequencing to capture PGx phenotypes for these medications. Design Retrospective cohort study integrating electronic medical record and exome sequencing data. Setting Morgan Stanley Children's Hospital of NewYork-Presbyterian, a single center tertiary care children's hospital. Participants A total of 4,939 children admitted to the PICU (2020 - 2024), and 192 children admitted to the PICU who underwent exome sequencing for research purposes (2015 - 2023). Exposure Critical illness requiring PICU admission. Main Outcomes and Measures Frequencies of administration of medications with established PGx guidelines in the PICU and the proportion of individuals with exome sequencing with identifiable PGx phenotypes. Results Among 4,939 PICU patients, 37.2% (n=1,837) received at least one medication with established PGx guidelines and 14.4% (n=712) received two or more such medications. Twenty PGx genes were implicated; CYP2C9 was most common (17.3%, n=853). An estimated 8.2% of patients received medications for which PGx-guided recommendations would have altered clinical management. Among 192 patients who underwent exome sequencing, at least one metabolizer phenotype was identified in 62% (n=119). Conclusions and Relevance Many critically ill children receive medications with established PGx guidelines. This study highlights an opportunity for more personalized medicine for critically ill children admitted to a tertiary care hospital and assesses the strengths and weaknesses of exome sequencing to uncover pertinent PGx phenotypes.

Background. Post acute sequelae of COVID 19 (PASC) is clinically heterogeneous and mechanistically unresolved, and single-analyte studies have struggled to explain it. Methods. We profiled matched plasma proteomics, metabolomics and whole-blood transcriptomics at acute infection and convalescence (mean 86 days later) in a Belgian cohort, using linear mixed models, multiomic gene-set enrichment, and a degree-matched differential-correlation approach to quantify how each node's interactions were rewired between patients who developed PASC and those who recovered; seven axis proteins were additionally quantified by multiplex immunoassay as orthogonal validation. Findings. Single omic testing yielded few FDR significant features, yet multi-omic enrichment showed sustained complement cascade involvement from acute illness to follow-up in PASC. Correlation networks re-organised topologically toward C3 and lost the immunoglobulin V gene coexpression seen in recovery. The most rewired nodes, heparin cofactor II (SERPIND1), alpha 1 antitrypsin (SERPINA1), complement factor H related 5 (CFHR5), prothrombin/thrombin (F2) and immunoglobulin V gene transcripts (notably IGLV3 21), changed in their co-expression structure rather than in abundance. In multiplex validation, acute CRP was elevated in patients who developed PASC (FDR = 0.012), whereas the directly measured abundances of the network-nominated proteins were unchanged. Interpretation. These trajectory aware, cross omic networks nominate a thrombo inflammatory axis in which complement and coagulation regulation remain dysregulated in PASC at the level of wiring rather than abundance, providing a systems framework for validation and for exploring interventions at the complement coagulation platelet interface.

Background: Monoclonal antibodies have emerged as a prophylactic strategy to prevent symptomatic SARS-CoV-2 infection in immunocompromised individuals. However, the evolutionary and clinical implications of breakthrough infections under this regime remain unclear. Methods: A male in their 80s with a haematological/oncological diagnosis received a 2000 mg intravenous infusion of sotrovimab in March 2023 and was diagnosed with COVID-19 by RT-qPCR from a nasopharyngeal swab in August 2023. Weekly samples (n=24) were collected through February 2024 (171 days). All samples underwent whole-genome sequencing, with select mutations subjected to functional assessment. Findings: Sequencing identified the GE.1 lineage at all timepoints. An intra-host recombination event in ORF1ab (positions 8942-12458) was detected prior to 23 weeks post-detection, followed by a 14-fold increase in viral load (7.42e+06 to 1.00e+08 RNA copies/mL) and a marked shift in the viral population. E340D, a sotrovimab resistance mutation, was detected at low abundance (46%) within the first week post-infection, fluctuated over time, and was nearly fixed by week 15 (107 days) post-detection. We assessed five spike mutations - V36M, S98F, and V213G in the N-terminal domain, Y505P in the receptor-binding domain, and P681Q near the S1/S2 cleavage site - and additionally evaluated the impact of E340D. V36M conferred the highest infectivity across all cell lines, with the most significant effect in low-TMPRSS2 cells. While all mutations showed enhanced infectivity with the addition of E340D, the effect was most pronounced in mutations with lower baseline infectivity. The addition of E340D significantly decreased relative neutralizing titres for V36M, S98F, and V213G, enabling escape from neutralizing antibodies in XBB-responsive individuals, illustrating an enhanced phenotypic advantage. Patient neutralizing activity was absent pre-sotrovimab, and sotrovimab-induced neutralization was further compromised by selection of E340D. Interpretation: Sotrovimab pre-exposure prophylaxis in an immunocompromised patient did not prevent SARS-CoV-2 infection, and selected for resistant mutation E340D, with unexpected fitness consequences across non-receptor binding domain spike regions.

Controlled human infection models (CHIMs) are increasingly used in infectious disease research to study pathogen dynamics and evaluate interventions under controlled conditions. However, these studies are resource-intensive and involve ethical and safety constraints, making efficient study design critical. Dose-finding is a key early component in CHIMs, where the aim is to identify a challenge dose that achieves a target infection probability. Traditional rule-based designs are commonly used but can be inefficient, motivating the use of model-based adaptive approaches such as the Bayesian Continual Reassessment Method (CRM). Although CRM has been extensively studied and widely adopted in Phase I oncology trials for identifying the maximum tolerated dose of therapeutics, its application in CHIM settings remains limited, particularly when the endpoint of interest is infection. This tutorial provides step-by-step guidance for implementing a Bayesian CRM in dose-finding CHIMs, using an oropharyngeal Neisseria gonorrhoeae challenge as a motivating case study. The framework outlines key design components, including dose-grid specification, dose-response model, prior elicitation, Bayesian updating, decision rules, and stopping criteria, with particular emphasis on a clinically interpretable parameterisation. Trial operating characteristics are evaluated through simulation studies under multiple dose-response scenarios and prior-predictive analyses, and compared with a commonly used '3+3' type rule-based design. This work highlights the advantages of Bayesian model-based designs for dose-finding in CHIMs over classic rule-based designs and provides a structured, reproducible framework for implementing CRM, supporting their application in future CHIM studies.

Background and Aims The optimal treatment for first episodes and first recurrences of Clostridioides difficile infections (CDI) is unknown and there is emerging evidence for pulse and taper (P-T) regimens. Therefore, we sought to estimate the relative efficacy of treatment options. Methods MEDLINE and CENTRAL were searched from database inception to May 21, 2025 and unpublished conference abstracts were searched from recent infectious disease conferences. RCTs on the treatment of first episodes or first recurrences of CDI comparing fixed-dose or P-T regimens of fidaxomicin or vancomycin were included. The primary and secondary outcomes were 40- and 56-day CDI recurrence, respectively. A random-effects network meta-analysis on the risk ratio (RR) scale was conducted using a standard regimen (10-14 days) of vancomycin as the comparator. Treatments were ranked using the surface under the cumulative ranking curve (SUCRA). Results 8 RCTs were included comprising a total of 2181 patients. For 40-day recurrence, fidaxomicin P-T had the highest probability of ranking best (RR=0.10, 95%Confidence Interval [95%CI]=0.10-0.49, SUCRA=1.00), followed by vancomycin P-T (RR=0.49, 95%CI=0.32-0.76, SUCRA=0.61), fixed-dose fidaxomicin (RR=0.61, 95%CI=0.49-0.76, SUCRA=0.39), and, finally, fixed-dose of vancomycin (SUCRA=0.00). The treatments ranked in the same order for 56-day recurrence, though only 3 RCTs reported on this timepoint. Conclusion Vancomycin P-T, fidaxomicin P-T, and fixed-dose fidaxomicin were all superior to a fixed-dose vancomycin. Head-to-head comparative effectiveness RCTs are needed to quantify their relative effect sizes of and impact on long-term prevention of recurrent CDI.