School of Medicine

Emerging studies suggest that antibiotics can disrupt the gut microbiome and alter vaccine-induced immune responses. However, the specific consequences of early-life exposure on neonatal immune development remain poorly understood. Here, we examined how two antibiotics frequently used in perinatal care, broad-spectrum ampicillin (AMP) and the extended-spectrum combination amoxicillin/clavulanate (AMOX/CLAV), administered during gestation and lactation, influence neonatal gut microbiome composition, fecal metabolome profiles, and responses to the 20-valent pneumococcal conjugate vaccine (PCV20). Maternal treatment with AMOX/CLAV, but not AMP, significantly reduced PCV-specific IgG titers at 4 and 6 weeks post-prime immunization compared to untreated controls. Exclusive exposure to AMOX/CLAV also impaired neutrophil-mediated opsonophagocytic killing, indicating reduced antibody functionality. These effects were transient, with immune parameters normalizing by 8 weeks post-prime immunization. Metabolomic and microbiome profiling revealed that maternal AMP and AMOX/CLAV differentially perturbed specific metabolite classes, including bile acids, N-acyl lipids, and indole derivatives. Key commensal taxa, including Bacteroidales and Coriobacteriales were also impacted within the gut microbiota. Together, these findings reveal a previously underappreciated maternal-offspring route of antibiotic influence that is transiently associated with neonatal vaccine responsiveness and microbiome and metabolome alterations. These results highlight maternal antibiotic exposure as a possible modifiable factor shaping early-life immunity.

DNA mismatch repair (MMR) corrects DNA replication errors, some forms of chemical damage to DNA bases, and acts in processes such as heteroduplex rejection, triplet repeat expansion, antibody maturation and cytotoxicity of alkylating agents. During MMR, the newly synthesized DNA strand containing the error must be removed and resynthesized to prevent mutations. In bacteria, this strand is displaced by the UvrD DNA helicase. In eukaryotes, this strand is excised by redundant pathways. In the first excision pathway identified, Saccharomyces cerevisiae Msh2-Msh6 (or Msh2-Msh3) and/or Mlh1-Pms1 (human MLH1-PMS2) recruit Exo1 to 5' nicks, and 5'-to-3' excision by Exo1 excises the daughter strand containing the mispair to generate a single-stranded gap. The Mlh1-Pms1 endonuclease also mediates excision through extensive nicking of the daughter strand to generate single-stranded gaps. In coupled strand-displacement synthesis coordinated by Msh2-Msh6 (or Msh2-Msh3), DNA polymerase delta and Rad27 (human FEN1)-mediated 5' flap cleavage excise the mispair and resynthesize the daughter strand. Resynthesis in the Exo1- and Mlh1-Pms1-mediated pathways can be performed by both DNA polymerase delta and epsilon, and in all excision pathways the nicked product can be sealed by DNA ligase. Genetic analysis in S. cerevisiae suggests that these three pathways correspond to all or at least the major excision pathways; however, other factors such as Fan1, Artemis, Mre11, Dna2, Fun30, and the RSC complex have been implicated in eukaryotic MMR but are less well understood. The roles of excision and its redundant pathways have also been less well characterized for other processes involving MMR proteins.

Cisgender women face an increased risk of HIV acquisition during pregnancy and postpartum. Oral pre-exposure prophylaxis (PrEP) with emtricitabine and tenofovir disoproxil fumarate (F/TDF) is recommended in pregnancy despite limited published pharmacokinetic (PK) and efficacy data during pregnancy. Altered PK during pregnancy may reduce prophylactic efficacy and necessitate higher adherence. Tenofovir alafenamide (TAF) is a newer tenofovir prodrug with higher tenofovir diphosphate (TFV-DP) exposure in peripheral blood mononuclear cells (PBMCs), but the prophylactic efficacy of F/TAF across different reproductive stages in women is understudied. We adapted a multiscale modeling framework to predict the prophylactic efficacy of F/TDF and F/TAF across varying adherence levels and during the second trimester, third trimester, and 6-12 weeks postpartum. Clinical trial simulations were conducted utilizing PBMC TFV-DP and emtricitabine triphosphate concentrations as surrogate markers for F/TAF and F/TDF efficacy. For both F/TDF and F/TAF, prophylactic efficacy was lowest during the second trimester, increased in the third trimester (but remained below nonpregnant levels), and was highest postpartum. At high adherence levels of five or more pills per week, predicted effectiveness remained high across pregnancy and postpartum and effectiveness exceeded 97% for both regimens, with maximum decreases in the second trimester of only 0.9 percent point for F/TDF and 0.3 percent point for F/TAF. These findings suggest that adherence is the primary determinant of PrEP effectiveness during pregnancy and postpartum, while pregnancy-related PK changes have a limited impact on efficacy.

MOTIVATION: Exome sequencing is widely used for genetic studies; however, accurate detection of copy number variants (CNV) in paralogous genes is challenging due to short-read mapping ambiguity and extensive copy-number variation. The human genome contains several hundred paralogous genes, many of which are known to harbor disease-associated CNVs. Existing exome CNV callers are primarily designed for rare CNV detection in uniquely mappable regions and are not well-suited for paralogous genes. METHODS: We describe a computational method (EdgeCopy) for copy number profiling of paralogous genes using whole-exome sequence data. EdgeCopy aggregates reads mapped to all copies of paralogous genes and relates observed read depth to copy number for multiple exome samples using an approximate composite likelihood function. The likelihood function is optimized using numerical optimization to obtain gene-level fractional copy number estimates that are discretized and refined using a Hidden Markov Model to obtain exon-level copy number estimates. RESULTS: Benchmarking of Edgecopy using experimental copy number data showed high concordance (mean = 0.973) for six disease-associated paralogous genes. We evaluated performance using whole-exome data from approximately 2400 samples across five continental populations from the 1000 Genomes Project. EdgeCopy shows robust concordance with whole-genome sequencing based estimates (0.974-0.982) across populations and 130 paralogous genes spanning a wide range of copy-number variation. In comparison, copy number analysis using a state-of-the-art exome CNV caller failed to estimate copy number for paralogous genes with very high mapping ambiguity and showed much lower concordance (0.565) for CNV events compared to EdgeCopy (0.908). AVAILABILITY: EdgeCopy is freely available at https://github.com/vibansal-lab/edgecopy.

Disruptions in the microtubule cytoskeleton play a role in various neurological diseases that afflict a large fraction of the population. Microtubule function is regulated by post-translational modifications like acetylation, and one consistently identified acetylation site in mammals and Drosophila melanogaster is α-tubulin lysine 394 (K394). Our previous research demonstrated that an acetylation-blocking point mutation-K394R-causes a decrease in microtubule stability in axon terminals at the developing neuromuscular junction. Here, we asked whether K394 acetylation regulates the development of additional neuronal structures. Using the central brain mushroom body as a model, we found that K394R results in β lobe overextension at the midline. The K394R phenotype manifests during metamorphosis and affects β lobe growth in a cell-autonomous manner. Our data suggest that the K394R phenotype may result from changes in Tau, a microtubule-associated protein enriched in the mushroom body and known to play a critical role in regulating neuronal microtubules. Knocking-out tau resulted in defects in midline crossing similar to K394R. However, when the loss of tau was combined with K394R, β lobe extension was normal-indicating that the loss of tau suppresses the K394R phenotype and vice versa. While overexpressing tau also resulted in a midline crossing phenotype, K394R in combination with elevated Tau resulted in a severely malformed mushroom body. Altogether, our work suggests that K394R interacts with tau to regulate axon outgrowth during mushroom body development and raises the potential of manipulating K394 acetylation to ameliorate neurological disease resulting from axonal growth defects and changes in Tau.

Objective: Masculinising chest surgery, also known as top surgery, is the most requested gender-affirming procedure among transgender and gender-diverse (TGD) adolescents, yet research on patient experiences remains limited. This study explored the experiences of TGD adolescents who were seeking or had undergone masculinising chest surgery. Design: Qualitative secondary analysis using existing themes framework and data from the GENDER-Q (GQ) and GENDER-Q Youth (GQY) research programmes, which aim to develop comprehensive patient-reported outcome measures for gender-affirming care. Setting: Participants were sampled from five high-volume gender-affirming care clinics, three in Canada and two in the United States. Interviews were conducted online. Participants: 35 GQ and GQY participants aged 13–18 years who were assigned female at birth, identified as trans men or non-binary, and were pursuing (n=19) or had undergone (n=16) masculinising chest surgery. Results: Three major themes emerged: chest appearance, health-related quality of life (HRQL) and gender practices. Most participants expected a flatter chest that aesthetically aligned with their gender identity. Presurgery participants anticipated that surgery would allow them to engage in previously avoided physical activities and would enhance their relationships. Postoperative participants reported increased physical activity, mental resilience, bodily connection and social comfort. Most reported binder use and related reliance or discomfort as motivators for pursuing surgery. Conclusions: This study highlights the multidimensional experiences surrounding masculinising chest surgery on TGD adolescents with impacts on chest appearance, HRQL and gender practices. Centering adolescents’ perspectives, these findings underscore the importance of accessible, affirming surgical care and provide valuable insights for clinicians, policymakers and future research.

Pseudomonas putida is a gram-negative bacterial species increasingly utilized in biotechnology due to its robust growth, ability to degrade aromatic compounds, solvent tolerance, and genetic tractability. In this study, we report a comprehensive multi-strain analysis of 164 P. putida strains based on the reconstruction of a pan-putida metabolic network and the formulation of strain-specific genome-scale metabolic models (GEMs). We performed whole-genome sequencing and hybrid assembly for 40 strains, contributing a ~8% increase to the available genomic data for P. putida. Furthermore, high-throughput phenotypic profiling using the Biolog phenotype microarray system for 24 strains on 190 unique carbon sources, along with 15 aromatic compounds not present on Biolog plates, yielded 4,920 unique strain-phenotype measurements. These data were leveraged to curate GEMs for 24 representative strains, including a refined model for strain KT2440, which comprised 1,480 genes and 2,191 metabolites, achieving a prediction accuracy of 91.2% in carbon utilization. Systematic comparison of genomes and GEMs revealed both conserved core pathways and significant allelic and functional divergence across strains, highlighting strain-specific variation in aromatic degradation. While pathways for protocatechuate and phenylacetate degradation were widely conserved, metabolic capabilities for compounds such as ferulate, phenol, and cresols varied markedly, suggesting adaptation to distinct ecological niches. Alleleome analysis of enzymes, such as PcaI and PcaJ, revealed distinct, functionally similar clades, indicating possible convergent evolution or horizontal gene transfer. These results provide computable resources and informative models for selecting P. putida strains with desired traits for biomanufacturing and bioremediation and offer insights into the evolution and phylogeny of the P. putida species.IMPORTANCEPseudomonas putida has become an organism of interest for biotechnological applications, but a species-level understanding of its metabolic diversity remains incomplete. In this study, we analyzed 164 P. putida strains using a combination of genome sequencing, phenotypic profiling, and metabolic modeling. Our results indicate that while many metabolic pathways are conserved, notable differences exist across strains, particularly in aromatic compound degradation. These observations may inform future strain selection and engineering strategies tailored to specific industrial or environmental goals. In addition, the genome-scale models and phenotypic data generated here can serve as a foundation for broader studies of metabolism and functional variation within this species.

Kawasaki disease is the most common cause of acquired heart disease in children in the developed world.1 Although Kawasaki disease manifests with acute, transient systemic signs, its long-term morb...

BACKGROUND: Intraosseous venous malformations are rare, benign vascular bone lesions that may mimic malignant sarcomas on imaging. OBSERVATIONS: A 14-year-old male presented with fever and cough. Imaging revealed an incidental ossified rib mass with aggressive features. CT-guided biopsy was nondiagnostic, whereas open biopsy demonstrated a vascular lesion positive for CD31, FLI-1, and ERG. Molecular testing identified an EWSR1-NFATC1 fusion. Gross total resection was achieved, followed by prolonged disease-free survival, and the patient resumed full athletic activity 7 months after surgery. CONCLUSIONS: This case highlights the value of open biopsy and molecular testing to prevent misdiagnosis and overtreatment of intraosseous venous malformations.

BackgroundProteins and RNA circulate in plasma and can offer insights into human physiology. Yet, despite their clinical importance, direct comparisons between these analytes remain unexplored.Methods:Here, we measure and compare plasma cell-free RNA (cfRNA) and protein levels for 263 children diagnosed with inflammatory diseases, specifically either Kawasaki disease (KD) or Multisystem Inflammatory Syndrome in Children (MIS-C), by RNA sequencing (n = 108 KD and n = 47 MIS-C, mean age=4.2 years) and SomaScan proteomics (n = 70 KD and n = 101 MIS-C, mean age=6.8 years).ResultsHere we show that cell-free RNA and protein levels are largely uncorrelated across samples (feature-by-sample correlation coefficient 0.052; median feature-level correlation coefficient 0.009). Nonetheless, machine learning models based on either modality distinguish KD from MIS-C with similar high accuracy (median area under the curve greater than 0.93). Analysis of KD subtypes reveals distinct cell-free RNA and protein signatures, with one group showing molecular similarity to MIS-C.ConclusionsThese findings underscore the complementary nature of cell-free RNA and protein profiling and highlight the utility of integrating multiple plasma analytes to improve disease classification and deepen our understanding of complex inflammatory conditions.