Ovarian clear cell carcinoma (OCCC) is a rare aggressive, and chemo-resistant subtype of epithelial ovarian cancer. Current limitations in precisely characterizing its molecular features have resulted in restricted availability of clinical targeted therapies and significant therapeutic challenges. To address this unmet need, we conducted an integrative multi-omics study of 82 OCCC cases, incorporating whole-exome sequencing (WES), bulk RNA sequencing, and single-cell RNA sequencing (scRNA-seq). Our analysis uncovered recurrent mutations in multiple epigenetic regulators including ARID1A, EP300, and SETD2B, reinforcing chromatin remodeling as a hallmark of OCCC pathogenesis. Strikingly, FOXA2 mutations were absent in early-stage tumors but specifically enriched in advanced-stage cases (19% frequency), with functional validation demonstrating their role in driving malignant progression. Copy number alteration profiling revealed frequent amplifications in chromosomal arms such as 17q, which contains the ERBB2 oncogene that potentially regulates OCCC progression. Chromosomal translocations were detected in 35.59% of cases, including a novel FGFR2/RPAP3 fusion with therapeutic implications. Notably, scRNA-seq delineated immune-rich subsets characterized by abundant cytotoxic T-cell and B-cell infiltration, suggesting immunotherapeutic opportunities in a patient subset. Moreover, molecular subtyping identified ERBB2 amplification/overexpression as a high-risk feature strongly associated with poor survival. Patient-derived xenograft (PDX) models and a retrospective analysis of two clinical cases demonstrated that HER2-targeted antibody-drug conjugates (HER2-ADCs) significantly suppressed tumor growth and progression in OCCC patients with HER2 expression. In summary, our study establishes the comprehensive molecular atlas and a targeted therapeutic subtyping framework, revealing therapeutic vulnerabilities and providing novel insights for advancing precision oncology in OCCC management.

The epidemiology of vancomycin-resistant Enterococcus faecium (VREfm) varies across different countries, with a steady global increase. In Portugal, however, epidemiological data on clinical VREfm have been scarce since the early 2000s. This long-term study investigates VREfm isolates from human infections collected at a Porto hospital between 2010 and 2021. Two hundred VREfm isolates, mostly urinary (39%) were characterized by antimicrobial susceptibility testing to 8 antibiotics, chlorhexidine susceptibility, and PCR-based detection of vancomycin-resistance genes, virulence markers, plasmid replicases, and the bac43/T8 gene. Whole-genome sequencing, by Illumina, was used to assess clonal diversity (MLST, cgMLST, SNP phylogeny) and genomic content of antimicrobial resistance (AMR) genes, bacteriocins (76 genes) and putative virulence markers (35 genes). The plasmidome size and replicase initiation proteins of selected isolates was further improved by incorporating nanopore sequencing which enabled hybrid assemblies. All isolates were multidrug resistant; 98% carried vanA, while two (1%) were resistant to linezolid (G2576T mutation). Chlorhexidine susceptibility remained stably low over time (MICs: 2-4mg/L). The population was polyclonal, with a shift from ST18-like lineages to ST80 and ST117 dominance. While ARGs and virulence markers showed no clear association with clonal waves, bacteriocin profiles did, with bac43 becoming increasingly prevalent. ST117-CT24 emerged as the most persistent clone. The plasmidome comprised stable Rep3-like mobilizable plasmids carrying bacteriocins (bac43, bacAS5), RepA_N mega-plasmids harboring virulence/AMR/bacteriocins, and highly plastic medium-to-large vanA plasmids with diverse replicase initiation proteins. Strikingly, linear vanA plasmids (repUS78_pZY2) appeared in the most recent isolates, paralleling findings in vancomycin-variable E. faecium from the same hospital and VREfm from other countries. Our findings reveal dynamic clonal shifts, novel plasmid architectures, and the key role of bacteriocins in shaping clonal success in a WHO priority pathogen. Furthermore, we highlight the need for AMR surveillance frameworks to consider factors beyond conventional prevalence metrics and core-genome comparisons. Integrating intra-species genomic heterogeneity and non-traditional evolutionary indicators will be essential to more accurately predict, track, and ultimately mitigate the dissemination of multidrug-resistant human pathogens.

Endothelial cells (ECs) orchestrate vascular homeostasis and resilience but can undergo reprogramming into a mesenchymal-like phenotype through an endothelial-to-mesenchymal transition (EndMT). Crucially, EndMT is a linchpin underlying several cardiometabolic diseases, but is almost universally studied as an endpoint. The transcription factor ERG (ETS-related gene) is critical to the maintenance of EC identity and function, yet the dynamic transcriptional and functional consequences of ERG loss on EndMT programs, and whether this can be reversed, has not been explored. We modeled both acute and chronic ERG loss in human aortic ECs using siRNA knockdown and CRISPR/Cas9-mediated ERG deletion. We profiled temporal changes in chromatin accessibility (ATAC-seq), transcriptomic responses (RNA-seq), and endothelial phenotypes, including migration and barrier integrity. The temporal kinetics of ERG loss and restoration was assessed by comparing stable ERG knockout to transient ERG knockdown and recovery over time. The implications to human disease were deciphered by examining ERG gene regulatory networks in human atherosclerosis and linkage with genetic variation associated with human cardiovascular disease. Analysis of gene regulatory networks revealed profound and dynamic rewiring of endothelial and mesenchymal transcriptional programs upon loss of ERG. While endothelial identity was rapidly lost by 24h of ERG knockdown, acquisition of mesenchymal identity, barrier dysfunction, and enhanced cell migration required 72h to manifest. Loss of ERG was accompanied by a rapid reduction in accessibility of ETS motifs and an extensive gain in open chromatin containing AP1 motifs. Disease-relevant endothelial dysfunction programs were associated with dynamically reorganized transcriptional networks. Importantly, restoration of ERG expression reversed EndMT gene regulatory networks and phenotypes. Overall, this study highlights the ETS factor, ERG, as an essential transcriptional safeguard of endothelial identity and function, and demonstrates that ERG loss initiates a progressive, yet reversible, EndMT program with EC identity loss preceding a gain of mesenchymal gene regulatory networks and phenotypes. This study establishes loss of ERG as an early initiating event in EndMT and suggests that ERG-targeted therapies may hold promise for promoting endothelial resilience.

The COVID-19 pandemic triggered rapid, population-wide behavioral and environmental changes, offering a unique natural experiment to study how early-life microbiome development responds to abrupt shifts in social and hygiene-related exposures. Using longitudinal data from 139 infants in the Dutch LucKi Gut study, we compared gut microbiome development in fecal samples collected before and during the pandemic. Whole metagenome sequencing of 808 stool samples was performed across nine time points in the first 14 months of life. An exposure index (EI) capturing variation in household-level pandemic-related behaviors was constructed for the 36 infants with samples collected during the COVID-pandemic to quantify variations in social distancing, lifestyle and hygiene measures. Microbial richness and diversity increased with age, following established developmental trajectories. However, from 6 months onward, the COVID-19 pandemic independently shaped gut microbial composition, explaining up to 2.7% of variation by 11 months of age (Q-value = 0.006). Forty-four species were differentially abundant in pandemic-era samples, including depletion of Gordonibacter pamelaeae and several Actinomyces species. Notably, greater environmental exposure (higher EI scores) was associated with lower abundance of G. pamelaeae, a microbe implicated in bile acid and immunomodulatory metabolism. This is the first longitudinal whole-genome sequencing study to demonstrate that pandemic-related behavioral changes measurably altered infant gut microbiota maturation. These findings highlight the sensitivity of microbiome development to societal-level environmental disruptions and suggest that early-life microbial exposures, modulated by hygiene and social behavior, may carry long-term implications for child health.