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Nonmuscle-invasive bladder cancer (NMIBC) is a clinically heterogeneous disease, characterized by a high recurrence rate and a progression risk that are challenging to predict. Although traditional risk models based on histopathology and clinical staging are widely used, their ability to inform personalized treatment remains limited. Recent advances in molecular profiling have provided a more comprehensive understanding of NMIBC biology. This article synthesizes current evidence regarding the molecular determinants, highlighting their roles in enhancing prognostication and informing personalized therapy. Integrating these molecular insights into contemporary risk stratification frameworks is enabling treatment strategies that align with tumor biology to optimize patient outcomes.
People with Type 2 diabetes (T2D) are twice as likely to develop cardiovascular disease (CVD), though not all excess risk has been fully elucidated. Plasma metabolomics profiles shared between these conditions may uncover molecular mechanisms linking T2D to CVD. We conducted a cross-sectional case-control analysis, comparing T2D individuals who had prevalent CVD to those without CVD at the time of metabolite measurement. Using untargeted liquid chromatography-mass spectrometry (LC-MS), we collected 522 metabolite abundances measured in 1374 participants with T2D (224 CVD cases) from the Trans-Omics for Precision Medicine (TOPMed) program. We used a mixed effects linear model to assess the association of CVD events with each metabolite abundance, adjusting for key covariates. Metabolites meeting a suggestive significance threshold were examined using metabolite set enrichment analysis and evaluated for replication in an independent cohort Atherosclerosis Risk in Communities (ARIC) (n = 1891; 214 CVD cases). We performed meta-analysis to combine both the discovery and replication associations, and assessed overall significance using an experiment-wide Bonferroni-corrected threshold. Metabolites meeting a suggestive threshold were enriched in metabolite sets linked to obesity and kidney disease. Meta-analysis identified eight metabolites reaching experiment-wide significance, confirming previously established associations of asymmetric dimethylarginine, phosphatidylcholines, and gluconic acid, while additionally identifying specific phosphatidylethanolamine species, N-acetyl-L-methionine, and allantoin associated with prevalent CVD among individuals with T2D. Our results established and replicated metabolite associations with prevalent CVD in people with T2D. These metabolites may help characterize metabolic alterations underlying cardiovascular complications that arise in T2D.
Hubris in contemporary medicine is less an individual failing than a structural condition produced by professional privilege, institutional incentives, and global asymmetries of power. This Perspective argues that modern health systems mistake technical competence for epistemic completeness, fueling iatrogenic harm and algorithmic inequity. We propose disciplined humility and structural reflexivity as operational principles, translated into concrete practices across clinical, global health, and AI contexts.
Ulcerative colitis (UC) is a chronic inflammatory bowel disease marked by relapsing mucosal inflammation. Recent data indicate that subclinical metabolic disturbances persist even in patients who have achieved remission. However, the temporal evolution of these molecular alterations remains poorly defined. This study examined how disease duration shapes systemic metabolic and lipidomic remodeling in UC. Plasma samples from 58 UC patients in clinical remission and 70 age- and sex- matched healthy controls (HC) were analyzed using integrated untargeted gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). Patients were stratified by disease duration (≤ 2 years vs. > 2 years). Multivariate and univariate analyses, including partial least squares discriminant analysis (PLS-DA), variable importance in projection (VIP) scoring, and t-tests or Mann-Whitney U test (p < 0.05), were applied, and pathway enrichment was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. UC patients showed distinct plasma metabolomic and lipidomic signatures compared with HC, with 15 metabolites and 126 lipid species significantly altered. Reduced pyruvic and lactic acids, together with disrupted fatty-acid and amino-acid metabolism, suggested impaired energy and redox homeostasis. Lipidomic profiling revealed broad disturbances across phosphatidylcholines, lysophosphatidylcholines, phosphatidylethanolamines, sphingomyelins, diacylglycerols, and triglycerides, reflecting persistent oxidative and inflammatory imbalance. Stratification by disease duration revealed progressive remodeling, particularly within amino-acid pathways and sphingolipid metabolism. Even during remission, UC exhibits systemic metabolic disequilibrium that evolves with disease chronicity. These time-dependent changes could guide the identification of metabolic biomarkers for disease monitoring and personalized therapeutic strategies.
We aimed to improve understanding of the genetic architecture of type 2 diabetes and glycemic traits by leveraging whole-genome sequencing in diverse populations. Our goal was to identify novel variants, refine known loci, and link genetic signals to regulatory mechanisms through colocalization with expression quantitative trait loci. We discovered novel variants, significantly improved fine-mapping resolution, and identified 80 regulatory colocalization signals in diabetes-relevant tissues. These findings support precision medicine approaches by connecting genetic variation to functional biology in type 2 diabetes.
Epstein-Barr virus (EBV) establishes lifelong latency in humans, yet sporadically reactivates into a productive lytic cycle that enables viral propagation. The lytic phase is critical for sustaining life-long persistence and also contributes to oncogenesis by fostering cellular environments that support EBV-associated malignancies. EBV lytic reactivation has long been understood as a complex process shaped by the integration of multiple, largely host-derived signals. Recent advances in genome-wide CRISPR screening, single-cell transcriptomics, and integrative multi-omic analyses have substantially expanded this framework, adding rich and unexpected molecular detail to how epigenetic repression, transcriptional control, metabolic adaptation, and immune-microenvironmental cues converge to regulate the latent-lytic transition. This review synthesizes recent discoveries that refine our understanding of the molecular logic of EBV reactivation and discusses how these insights are shaping new therapeutic approaches.
The clinical and molecular heterogeneity of diffuse large B cell lymphoma (DLBCL) is incompletely understood. By integrating proteomic, transcriptomic, and genomic data from 478 DLBCL tumors, we identify seven DLBCL proteogenotypes (PGs) reflecting specific pathophysiological features that span known molecular subtypes. PG4 is associated with poor outcome independent of established risk factors such as cell-of-origin, international prognostic index, or genetic features. PG4 contains activated B cell-like and germinal center B cell-like tumors and genetically unclassified cases. It shares a dark-zone-related B cell phenotype and shows enrichment for BTG1 mutations that can activate MYC. Single-cell sequencing and spatial transcriptomics reveal enhanced MYC and TCF3/4 transcriptional activity irrespective of MYC translocations. The PG4 tumor microenvironment is characterized by exhausted CD8+ T cells. Our study identifies common oncogenic themes underlying high-risk DLBCL tumors and provides a proteogenomic framework for future diagnostic and therapeutic approaches.
The development and maintenance of multicellular tissues requires that cell states be closely coupled to their local environment, including geometric and mechanical cues. However, studying this coupling in intact tissues has been challenging, because existing measurement technologies cannot simultaneously assess mechanical properties and molecularly defined cell states. To address this gap, we introduce the Unified Transcriptome and Mechanics Map (UTMM), a method for concurrent measurement of the transcriptome and cytoplasmic stiffness (high-frequency elastic modulus) within intact 3D multicellular structures. UTMM relies on two innovations: (1) a targeted in situ RNA sequencing approach for intact 3D embryos (3DISS), and (2) a strategy that leverages high-frequency intracellular organelle fluctuations to infer cell-level stiffness in vivo. We applied UTMM to mouse embryos from the zygote through the morula stage to characterize how RNA expression and cytoplasmic stiffness become coupled during early lineage specification. Our data reveal that, in the early morula, transcriptional and morphological distinctions emerge between trophectoderm and inner cell mass (ICM) lineages, coinciding with a gradual decrease in cytoplasmic stiffness (softening) across all cells from the 2-cell through morula stages. Furthermore, we observe that early lineage biases align with differential mechanical properties, reflecting distinct emerging developmental programs. When we delayed this softening process via mechanical perturbation, embryonic progression was impeded, highlighting the functional importance of coordinated mechanical and transcriptional changes. Together, these results demonstrate UTMM's ability to bridge molecular and mechanical dynamics in multicellular systems, providing a powerful framework for investigating how biomechanical cues shape cell fate decisions in intact tissues.
Human ATP synthase is a molecular rotary machine bound in inner mitochondrial membranes, built from twenty-eight subunits of seventeen kinds, two encoded in mitochondrial DNA, the remainder in nuclear genes. The machine consists of a rotor and an interacting stator. Turning of the rotor driven by a transmembrane proton motive force effects a cycle of structural changes in the catalytic part of the stator, producing three ATP molecules per rotation. Here, to establish how the stator and rotor are assembled, we deleted subunits and known assembly factors from human cells, purified and accumulated assembly intermediate complexes, and characterized them by gel analysis and mass spectrometry, allowing us to propose pathways of assembly of the rotor and the catalytic F1-module of the stator. These observations provide opportunities for further development by structural analysis of the accumulated intermediates. The compositions of the various assembly intermediates support the view that ATP synthase arose via independent evolution of its three constituent structural components, the catalytic F1-module, the peripheral stalk module, and the membrane-associated Fo-module.
Malaria remains one of the major threats to human health. Breakthrough drugs with high potency and low resistance risk are needed to combat the ever-increasing resistance to currently deployed antimalarials. Here, we explore a series of 4-amino-quinazoline-based sulfonamides, with drug-like physicochemical parameters and a synthetically accessible scaffold. Exemplars exhibit nanomolar potency against blood stage Plasmodium cultures, with up to 300-fold selectivity compared with a mammalian cell line. The compounds are also active against transmissible stages of P. falciparum and are refractory to resistance development. Targeted mass spectrometry reveals that the compounds act as reaction hijacking inhibitors targeting P. falciparum aminoacyl tRNA synthetases (aaRSs). Subtle changes to the chemical structure switch the main target from cytoplasmic tRNA threonine synthetase (PfThrRS) to cytoplasmic asparagine synthetase (PfAsnRS), a change that is associated with increased potency and selectivity. The target preference was confirmed by selective knock-down of different P. falciparum aaRSs and by tolerance selection in a mutator line. Consistent with aaRS targets, exemplar compounds activate the amino acid starvation response. Recombinant enzyme inhibition and thermal stabilisation assays confirm the susceptibility of PfAsnRS to reaction hijacking and show that human AsnRS is less susceptible. A molecular model of Asn-tRNA-bound PfAsnRS reveals that a potent hijacker adopts a pose similar to adenosine 5'-monophosphate (AMP). An AlphaFold model of the native PfAsnRS dimer helps explain the tolerance-conferring effect of a mutation at the dimer interface.
Gene syntax-the order and arrangement of genes and their regulatory elements-shapes the dynamic coordination of both natural and synthetic gene circuits. Transcription at one locus perturbs the transcription of adjacent genes, but the molecular basis of this effect remains poorly understood. In this work, we show that supercoiling-mediated feedback arises from transcription and regulates expression of adjacent genes in a syntax-specific manner. Using a suite of assays, we measured syntax- and induction-dependent formation of chromatin structures in human induced pluripotent stem cells. Applying syntax as a design parameter, we built and improved compact gene circuits, tuning the expression mean, noise, and stoichiometry across delivery methods and cell types. Integrating supercoiling mediated feedback into models of gene regulation will expand our understanding of native and synthetic systems.
The endoplasmic reticulum (ER) requires an oxidative environment to support the efficient maturation of secretory and membrane proteins. This is in part established by glutathione, a redox-active metabolite present in reduced (GSH) and oxidized (GSSG) forms. The ER maintains a higher GSSG:GSH ratio than the cytosol; however, the mechanisms controlling ER redox balance remain poorly understood. To address this, we developed a method for the rapid immunopurification of the ER, enabling comprehensive profiling of its proteome and metabolome. Combining this approach with CRISPR screening, we identified SLC33A1 as the major ER GSSG exporter in mammalian cells. Loss of SLC33A1 led to GSSG accumulation in the ER and a liposome-based assay demonstrated that SLC33A1 directly transports GSSG. Cryogenic electron microscopy structures and molecular dynamics simulations revealed how SLC33A1 binds GSSG and identified residues critical for its transport. Finally, an imbalance in GSSG:GSH ratio induced ER stress and dependency on the ER-associated degradation pathway, driven by a shift in protein disulfide isomerases towards their oxidized forms. Together, our work establishes SLC33A1-mediated GSSG export as a key mechanism for ER redox homeostasis and protein maturation.
Electron cryomicroscopy (cryo-EM) allows high spatial resolution visualization of biological specimens; however, it is challenging to chemically identify densities observed in cryo-EM. To overcome this, we combined cryo-EM with chemical imaging using focused ion beam secondary ion mass spectrometry (FIB-SIMS) for integrated spatiochemical analysis of untagged specimens. We show that our correlative workflow permits subcellular localization of molecules inside bacterial cells and is compatible with cryogenic light microscopy and FIB-milled lamellae of eukaryotic specimens. To highlight biological insights enabled by the workflow, we studied the uptake of bisphenol-AF, a widespread chemical pollutant, by environmental bacteria, revealing the storage of these chemicals within cytosolic phase-separated aggregates in pollutant-exposed cells, where they cannot be removed by the bacterial efflux machinery despite its robust upregulation. Cryo-EM-FIB-SIMS therefore represents an effective approach to map elemental and molecular signatures in near-native biological samples.
Posttranslational modifications with ubiquitin-like modifiers (UBLs) are critical for genome maintenance, yet many remain mechanistically uncharacterised. Here, we identify UFM1 as a key regulator of non-homologous end-joining (NHEJ), a major DNA double-strand break repair pathway. Using a structure-guided chemical biology approach, we develop a photo-crosslinkable UFM1 probe and, in combination with NMR, map non-canonical UFM1-binding interfaces in core NHEJ factors, including the disordered XRCC4 tail. Mechanistically, proximity-dependent proteomics and functional assays identify Ku70 as a crucial UFMylation substrate and reveal a UFM1-dependent axis in which XRCC4 engages UFMylated Ku70 to stabilise NHEJ complex assembly on chromatin. Disruption of this molecular mechanism via UFSP2 depletion or a hypomorphic UBA5 variant in patient-derived cells impairs NHEJ function, linking UFMylation defects to compromised genome integrity processes. Our findings define a complete UFM1 signalling module in DNA repair and establish a generalisable framework for dissecting low-affinity UBL networks with broad functional and disease relevance.
Vitiligo is a chronic depigmenting skin disease that can severely affect quality of life. First-line treatments include topical calcineurin inhibitors such as tacrolimus, but their molecular mechanisms of action in vitiligo remain incompletely understood. In this study, we investigated the early proteomic changes in patients with non-segmental vitiligo treated with topical tacrolimus. We included eight patients and collected 2-mm punch biopsies from untreated lesional skin (LS) and nonlesional skin (NLS), as well as from LS skin after 2 and 8 weeks of treatment. Samples were analyzed with mass spectrometry-based proteomics. We used a pairwise comparison between untreated LS and NLS, and linear mixed models to compare untreated LS and tacrolimus-treated LS. In total, 4416 proteins were identified. Comparison between untreated LS and NLS revealed 32 differentially expressed proteins (DEPs; false discovery rate < 0.05), primarily associated with muscle contraction and melanocyte function. After 2 weeks of treatment, LS showed enrichment of 31 DEPs related to acute inflammatory responses and melanocyte adhesion, while LS treated for 8 weeks showed enrichment of 14 DEPs involved in melanogenesis, interferon-induced inflammation, and heat shock responses. This study characterizes the early proteomic response of the skin to tacrolimus treatment and offers insights into disease-associated proteins in vitiligo.
Oncolytic virotherapy represents a promising yet under-explored approach for precision cancer treatment, particularly when tailored to tumor-specific molecular profiles. Patients with high-grade isocitrate dehydrogenase (IDH) mutant astrocytomas have limited treatment options and poor prognoses. Here, we investigate the therapeutic efficacy of rQNestin34.5 v.2 (CAN-3110), an engineered oncolytic herpes simplex virus 1 (oHSV-1), in IDH1-R132H-mutant diffuse gliomas. We demonstrate that the IDH1-R132H mutation enhances glioma susceptibility to viral infection through upregulation of Nectin-1, the main HSV-1 entry receptor. Concurrently, IDH1-R132H-driven DNA hypermethylation suppresses interferon (IFN) signaling, creating a permissive microenvironment that facilitates viral replication and tumor cell apoptosis. In immunocompetent murine glioma models, intratumoral administration of rQNestin34.5 v.2 induces robust antitumor immune activation, including increased immune infiltration and systemic IFN-γ release. However, elevated expression of poliovirus receptor (PVR) and the immune checkpoint T-cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) on tumor-infiltrating leukocytes suggests a potential resistance mechanism to virotherapy. Combining rQNestin34.5 v.2 with TIGIT blockade enhances therapeutic efficacy compared to monotherapy, identifying IDH1-R132H as a potential predictive biomarker for oncolytic virotherapy response.
Eosinophilic esophagitis (EoE) is a type 2 inflammatory disease of the esophagus, characterized by eosinophilic inflammation and an altered esophageal transcriptome. Dupilumab, a fully human mAb that blocks IL-4 and IL-13 signaling, is approved for use in multiple type 2 inflammatory diseases, including EoE, where it produces histologic, symptomatic, and endoscopic improvements in pediatric, adolescent, and adult patients. We sought to investigate the effect of dupilumab on the dysregulated esophageal transcriptome in pediatric, adolescent, and adult patients with EoE. ClinicalTrials.gov Identifiers: NCT02379052, NCT03633617, and NCT04394351. Changes in the esophageal transcriptome were analyzed from pretreatment and posttreatment esophageal biopsy specimens provided by pediatric, adolescent, and adult patients with EoE who participated in 1 of 3 placebo-controlled clinical trials of dupilumab. Dupilumab treatment resulted in marked normalization of the EoE disease transcriptome, including gene signatures associated with eosinophils as well as other aspects of type 2 inflammation and esophageal dysfunction including mast cells, fibrosis, barrier function, and other cell functions and pathways. Overall, gene ontology enrichment analysis identified 41 biological processes dysregulated in EoE that were normalized with dupilumab treatment, including processes likely to be eosinophil independent. Transcriptome changes were comparable and sustained through week 52 across pediatric, adolescent, and adult patients with EoE. IL-4 and IL-13 are the critical drivers of the molecular differences in the esophageal mucosa of patients with EoE compared with control subjects, consistent with the clinical benefit of dupilumab treatment.
Maternal diabetes confers two opposing risks to fetal growth, resulting in macrosomia in mild cases and intrauterine growth restriction (IUGR) in severe cases. The mechanisms governing these divergent responses are poorly understood, given the intimate regulation of insulin by glucose and insulin's fetal growth-promoting effects. We hypothesised that the degree of maternal hyperglycaemia dictates a bimodal pattern of fetal insulin secretion that determines fetal growth, and that use of a ketogenic diet (KD) as a nutritional intervention could modify this outcome. We used the Insulin-rtTA;TET-DTA mouse model to induce preconception diabetes. Dams were stratified based on maternal blood glucose, namely non-diabetes (glucose <9.6 mmol/l), mild diabetes (glucose range 9.6-16.7 mmol/l) or severe diabetes (glucose >16.7 mmol/l), and maintained on either a normal diet or a KD. We assessed fetal growth and plasma C-peptide, performed islet functional assays ex vivo, and characterised changes in plasma metabolites. Fetal pancreases were analysed by immunohistochemistry for beta cell area, proliferation, maturation and mechanistic target of rapamycin complex 1 (mTORC1) activity. Mild maternal diabetes induced fetal macrosomia, driven by beta cell hyperplasia, hyperinsulinaemia and premature beta cell functional maturation, as reflected by glucose-stimulated insulin secretion and upregulated MafA expression. This was associated with strong activation of the mTORC1 pathway. In contrast, severe diabetes caused IUGR associated with reduced beta cell mass and profound functional impairment. The KD had divergent effects: it normalised fetal growth in the mild diabetes group by preventing beta cell proliferation and premature maturation, thereby reducing insulin secretion, but failed to rescue IUGR in the severe diabetes group, despite partially restoring beta cell function. Notably, the KD uncoupled the positive correlation between fetal insulin and body weight, revealing a primary, insulin-independent, growth-restrictive effect. Fetal growth in a mouse model of diabetes in pregnancy is governed by a bimodal beta cell response to the maternal glycaemic environment, orchestrated at the molecular level by the mTORC1 pathway. A KD can prevent diabetes-derived macrosomia by reducing beta cell stimulation and through insulin-independent mechanisms, but cannot reverse IUGR, warranting further studies of its role in diabetes during pregnancy.
Heart failure with preserved ejection fraction (HFpEF) is a poorly understood, multisystem disease with high morbidity and mortality. To improve understanding of its pathobiology, we analyzed single-nucleus RNA sequencing in human HFpEF myocardium versus controls. Septal myocardial biopsies from 19 HFpEF and 24 nonfailing controls were analyzed using the 10× Genomics Chromium platform, with nuclei isolated from combined samples (6 patients/pool). Genotype-based demultiplexing was performed with souporcell, and gene expression was quantified with CellRanger and CellBender. After quality control, nuclei were annotated by cell types, and differential expression was performed between HFpEF versus controls using limma-voom. Functional analysis was performed using Gene Set Enrichment Analysis. Data were compared with prior single-nucleus RNA sequencing in dilated cardiomyopathy versus controls. We successfully demultiplexed pooled myocardial biopsies, assigning >70% of nuclei to individuals. After quality control, we recovered 48 886 nuclei and identified 14 cell types. Many differentially expressed genes across cell types were detected in HFpEF versus controls (fibroblasts, 5905; cardiomyocytes, 5159; endothelial cells, 2143; pericytes, 1812; and macrophages, 1405). Enriched pathways common to multiple cell types included immune activation, transcription/translation, metabolism, and protein quality control. They were particularly shared between cardiomyocytes and fibroblasts. Vascular smooth muscle cells had a more synthetic, proliferative phenotype. Immune cell analyses suggested enhanced T-cell activation and reduced macrophage clearance programs. Comparative analysis between HFpEF and dilated cardiomyopathy identified transcriptional differences primarily in cardiomyocytes. Two of 3 cardiomyocyte differential expression genes unique to HFpEF were validated to have concordant protein expression changes in HFpEF (MAP2K6 and PLPP3). Our findings reveal a distinct, cell-type-specific transcriptomic landscape in the human HFpEF myocardium. While HFpEF and dilated cardiomyopathy share significant molecular pathways across most cell types, the profound divergence within cardiomyocytes suggests a unique pathological driver for HFpEF. These signatures may provide a high-resolution roadmap for identifying precision therapeutic targets in HFpEF.
Rare coding alleles have crucial roles in the molecular diagnosis of genetic diseases. However, the systematic identification of these alleles has been challenging due to their scarcity in the general population. Here we discovered and characterized rare coding alleles contributing to genetic dyslipidemia, a principal risk for coronary artery disease (CAD), among 1,158,017 multi-ancestral individuals. Testing 2,997,401 rare coding variants, we identified 800 exome-wide significant associations (176 predicted loss of function (pLoF) and 624 missense variants). Associated alleles are enriched in functional variant classes, show significant additive and recessive associations, exhibit similar effects across populations and resolve pathogenicity for variants of unknown significance. Furthermore, we identified five lipid-associated genes associated with CAD. Among them, silencing RORC represents a potential therapeutic target for lowering low-density lipoprotein cholesterol. This study provides resources and insights for understanding causal mechanisms, quantifying the expressivity of rare coding alleles and identifying new drug targets for dyslipidemia across diverse populations.