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Hearing and balance disorders are among the most prevalent sensory impairments globally, yet their cellular and molecular basis remains poorly understood. This gap stems from the inaccessibility of the inner ear, which is encased in the temporal bone (TB)-the hardest bone in the body-and cannot be biopsied in living patients. Conventional histopathology workflows, particularly the century-old celloidin method, are time-consuming, labor-intensive, and incompatible with modern molecular analyses. We aimed to develop a faster, more versatile histology workflow for human TBs that preserves both morphology and molecular integrity. We developed a reversible polymethyl methacrylate (rPMMA) embedding protocol for formalin-fixed, calcified TBs using low-temperature resin infiltration (-40 to +4 °C). Precision near-serial sections (10-50 µm) were generated via femtosecond laser microtomy or precision diamond wire sawing. Deacrylation was performed to restore tissue accessibility for histological staining, multiplex immunofluorescence, whole-genome sequencing, and in situ RNA detection (RNAscope™). Compared to the celloidin workflow, our method reduced processing time and costs by over 90% while preserving histomorphology of comparable quality (n = 7 human TBs). It maintained antigenicity for multiplexed immunofluorescence, preserved native tissue-implant interfaces in implant-containing specimens, yielded high-quality DNA suitable for whole-genome sequencing (mean coverage 7.4 × in specimens with postmortem intervals < 24 h), and enabled mRNA detection at single-cell resolution. Celloidin-embedded controls consistently failed to support these molecular assays. This rPMMA-based workflow combines gold-standard histomorphology with full compatibility for advanced molecular analyses. With dramatically reduced time and cost, it offers a new benchmark for integrated, spatially resolved studies of human hearing and balance pathologies.
The circulating blood proteome comprises soluble and cellular components that reflect physiological and pathological states across tissues. Advances in mass spectrometry and affinity-based proteomics have improved sensitivity and throughput, enabling the generation of public blood proteomics resources. However, comprehensive assessments of these datasets remain limited. This work reviews the cellular and molecular complexity of publicly available blood proteomics data, recent methodological developments, and the complementarity of diverse data sources across the abundance range, while outlining remaining challenges for translating blood proteomics into personalized medicine.
Species in microbial communities need to stave off competition and capitalize on new resources that become available because of metabolic activities of others. However, intra-cellular molecular changes that underpin these responses are understudied, preventing mechanistic insights into community function and dynamics. Here we analyse proteomic and metabolomic responses in 104 pairwise co-cultures of 15 gut bacteria, spanning a diversity of ecological interactions from competition to mutualism. We find that molecular responses to co-culturing are substantial, with typically 50% of the quantified proteome changing in at least one co-culture, jointly influenced by genome size, species abundance and pH. Even closely related species and orthologue proteins show different expression profiles in response to the same partner, indicating functional diversification at both protein and species level. Small-molecule transport and carbon metabolism are among the most responsive processes, indicating pervasive metabolic interactions. Using metabolomics, we identify likely cross-fed metabolites, emergent polyamine metabolism and niche partitioning in amino acid utilization. Overall, our study uncovers how bacteria respond to the presence of other species through extensive remodelling of their proteome and metabolome.
Different classes of anesthetics induce unconsciousness despite acting through distinct molecular mechanisms, raising the possibility that their convergent effects arise at the level of the dynamics of neural population activity. To explore this, we analyze intracortical electrophysiological recordings during infusions of propofol, ketamine, and dexmedetomidine, applying a rigorous method to estimate the stability of population dynamics during anesthesia. We find that all three anesthetics, despite their molecular differences, similarly affect cortical states by reducing dynamic stability. The estimated destabilization is corroborated by the slower recovery from sensory perturbations and longer stimulus-induced autocorrelation times observed during the anesthetic infusions. Destabilization is most prevalent in the low-frequency band of the population dynamics, linking it to the well-known increase in low-frequency power during anesthesia. Together, these findings suggest that cortical destabilization may be a shared neural correlate of anesthetic-induced unconsciousness and provide a plausible link to the low-frequency oscillations observed during anesthesia.
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.
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.
The anthelmintic praziquantel (PZQ) has been used for decades as the clinical therapy for schistosomiasis, and remains the only available drug. As a cheap and effective drug therapy for all human disease-causing Schistosoma species, usage of PZQ underpins mass drug administration strategies aimed at eliminating schistosomiasis as a public health problem by 2030. Concern over the potential emergence of resistance to PZQ is therefore warranted, as it would constitute a major threat to this approach. In terms of molecular adaptations conferring PZQ resistance, variation in the sequence and/or expression of the drug target is an obvious mechanism and should be a priority for surveillance efforts. The target of PZQ is a transient receptor potential ion channel, TRPMPZQ, which is established as a locus that regulates schistosome sensitivity to PZQ. Here, we describe the establishment of a community resource, 'TRPtracker', which coalesces data on TRPMPZQ natural variants together with measurements of individual TRPMPZQ variant sensitivity to PZQ assessed by profiling TRPMPZQ in a heterologous expression system. A compendium of laboratory-generated mutants in TRPMPZQ is also compiled in the TRPtracker database to delimit regions within TRPMPZQ that are critical for PZQ sensitivity. Aggregation of data from multiple research groups into TRPtracker catalogues which TRPMPZQ variants have been functionally profiled, where geographically these variants have been found, their frequency within populations, and their potential impact on PZQ sensitivity. The overall goal is to facilitate rapid community-wide exchange of data to monitor predicted variants of concern that are likely to be associated with decreased PZQ efficacy.
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.
Biological aging reflects complex cellular and biochemical processes that can be measured across multiple omic layers. Using routine clinical laboratory data from ~31,000 participants in the Mass General Brigham Biobank, we developed EMRAge, a biomarker of mortality risk that can be broadly recapitulated across electronic medical records. Here we show that EMRAge can be modeled using elastic net regression with DNA methylation and multi-omics to generate DNAmEMRAge and OMICmAge, respectively. Both biomarkers are strongly associated with incident and prevalent chronic diseases and mortality, performing comparably or better than current biomarkers across discovery (Massachusetts General Brigham Aging Biobank Cohort, n = 3,451) and validation cohorts (TruDiagnostic, n = 14,213; Generation Scotland, n = 18,672). Importantly, OMICmAge leverages epigenetic biomarker proxies to integrate proteomic, metabolomic and clinical domains while remaining quantifiable from DNA methylation alone. This framework establishes an accessible, scalable measure of biological aging with potential to reveal molecular interconnections that shape healthspan and disease risk.
The gut microbiota plays a pivotal role in bio-transforming dietary components, including tryptophan, an essential amino acid that undergoes microbial metabolism. Microbial metabolism of tryptophan yields indole-3-propionic acid (IPA), an emerging biomarker for gut inflammation. Current IPA detection relies on expensive, time-consuming mass spectrometry. To address this limitation, a fluorescent nanosensor system is presented that uniquely features two optical modalities: one utilizing near-infrared (NIR) emission of a central single-walled carbon nanotube (SWNT), and a separate, visible emission from the corona phase polymer, a cationic conjugated polyelectrolyte (CP3). Selective IPA molecular recognition occurs at the latter, but the binding is optically reported via quenching in both the visible and NIR emission channels. The two modalities provide complementary advantages: CP3-SWNTs' NIR channel enables detection in strongly scattering tissue environments due to reduced Rayleigh scattering at longer wavelengths. Conversely, CP3 visible channel facilitates future rapid, cost-effective point-of-care biological sample screening. Functionality of both modalities is maintained within a gelatin metacrylate hydrogel offering potential for future continuous in vivo monitoring of IPA dynamics. The sensor reveals significant differences in plasma IPA levels between healthy controls and patients with active gut inflammation: ulcerative colitis and Crohn's disease, highlighting its promise in rapid gut health assessment.
Background: Noninvasive biomarkers for the detection and monitoring of glioblastoma (GBM) are needed to improve clinical outcomes for patients. The objective of this pilot study was to evaluate the expression of a panel of 48 pre-selected microRNAs (miRNAs) in plasma specimens from GBM patients versus healthy controls to identify candidate miRNA biomarkers for noninvasive diagnosis of GBM. Methods: Selection of candidate miRNA biomarkers was based on a comprehensive literature review and data mining. RNA was extracted from plasma samples obtained prior to resection from patients with GBM (n = 30) and age- and sex-matched healthy controls (n = 30), as well as from matched FFPE GBM tissue samples when available (n = 3). Expression levels of 48 miRNAs were assessed in all samples, and expression data was processed using proprietary software to generate potential biomarkers and train linear classifiers. Results: Overall miRNA expression patterns were similar between matched plasma and FFPE tumor tissues in patients with GBM. miRNA levels were examined in pairs to determine the ratio between two miRNAs, which served to normalize the data. The top five miRNA pairs for distinguishing between GBM and healthy control plasma included miR-17-5p/miR-19b-3p (AUC 0.93, 95% CI = 0.870, 0.970), miR-20a-5p/miR-19b-3p (AUC 0.93, 95% CI = 0.870, 0.970), miR-93-5p/miR-92a-3p (AUC 0.92, 95% CI = 0.875, 0.965), miR-17-5p/miR-92a-3p (AUC 0.91, 95% CI = 0.865, 0.955), and miR-93-5p/miR-19b-3p (AUC 0.90, 95% CI = 0.850, 0.950). For the development of a multi-biomarker combination classifier consisting of up to three miRNA pair biomarkers, miRNA pairs with an AUC ≥ 0.8 were selected to build equal-weight linear classifiers. All possible combinations of three high-performing miRNA pairs were tested across the 60 samples. The top classifier (miR-20a-5p/miR-451a, miR-582-5p/miR-222-3p, and miR-17-5p/miR-222-3p) achieved an AUC value of 0.992, sensitivity of 0.93, specificity of 1, and accuracy of 0.97. Conclusions: These findings support the continued development of a plasma-based miRNA molecular diagnostic approach for the detection of GBM. The strong discriminatory performance observed in this study, including high AUC values, highlights the potential of circulating miRNA signatures as a minimally invasive diagnostic tool. As a pilot analysis, this work establishes a foundation for future prospective studies in larger, independent cohorts-including relevant disease control populations-to further define clinical performance, specificity, and utility in diagnostic and monitoring settings. Collectively, these results represent an important step toward the translation of plasma-based miRNA profiling into clinical application for GBM.
During pregnancy hypoxia leads to complications like fetal growth restriction (FGR). Maternal inhalation hypoxia during late murine pregnancy impairs placental phenotype and fetal development in a severity-dependent manner. To identify the molecular mechanisms and sex-specificity of these effects, placentas from pregnant mice exposed to moderate (13% O₂) or severe (10% O₂) hypoxia were analysed using RNA-sequencing, qPCR, western blotting, histology, and nutrient transport assays. Transcriptomic profiling of male 13% O2 placentas revealed differential gene expression regulating calcium binding, lipid metabolism, and peroxisome proliferator-activated receptor (PPAR) signalling. In both sexes, hypoxia reduced fetal weight and altered placental nutrient transport in a severity-dependent manner. Abundance of PPARα, PPARγ, and associated targets varied with sex and hypoxia severity. Placental calcium deposition was significantly increased by hypoxia irrespective of severity. Human placental datasets revealed that orthologues of key hypoxia-responsive genes in the mouse placenta are also associated with pregnancy outcomes in human pregnancy. These findings implicate placental PPAR signalling and calcium dysregulation as potential mediators of FGR in compromised pregnancies. The online version contains supplementary material available at 10.1007/s00018-026-06110-7.
Sepsis remains a leading cause of pediatric morbidity and mortality, yet its molecular underpinnings are poorly understood. Here, we performed mass spectrometry-based plasma proteomics and cytokine profiling in pediatric sepsis patients at the acute phase (AP) and recovery phase (RP), alongside preoperative surgical controls. In AP vs. control, we identified 41 differentially abundant (DA) proteins, including acute-phase reactants and complement factors, with persistent but attenuated expression in RP. Pathway analysis revealed sustained enrichment in inflammatory and complement activation processes during both AP and RP, with partial restoration of immune surveillance and vascular homeostasis in recovery. Machine learning highlighted complement components (C9, C1R) and LRG1 as candidate AP biomarkers, and S100A9 as an RP-associated marker. Comparative analysis with adult sepsis proteomes uncovered age-specific complement activation patterns: adults displayed higher classical pathway activity, whereas pediatric patients exhibited enhanced alternative pathway activity. Cytokine profiling confirmed sustained immune activation and endothelial perturbation across sepsis phases. We also compared the sepsis cohort with the sterile inflammation (SI) cohort, which revealed distinct adaptive immune enrichment in sepsis while innate immune predominance in SI, enabling the identification of potential sepsis-specific protein signatures. Together, these findings delineate the dynamic immune and vascular proteomic landscape of pediatric sepsis, reveal biomarkers distinguishing sepsis from sterile inflammation, and highlight age-related complement pathway differences with potential therapeutic implications. Trial Registration: ClinicalTrials.gov: NCT04103268, NCT04299828.
Despite recent progress, advanced non-small cell lung cancer (NSCLC) has poor survival outcomes, necessitating the development of novel therapies. TNF-related apoptosis-inducing ligand (TRAIL) selectively induces cancer cell death and can be delivered to tumors by mesenchymal stromal cells (MSCs) due to the cells' migratory properties. This first-in-human phase I trial assessed safety and dose of umbilical cord-derived MSCs expressing TRAIL (UC-MSCTRAIL) alongside standard NSCLC therapy. Participants performance status 0-1 with treatment-naïve, inoperable stage IIIB/IV NSCLC received UC-MSCTRAIL infusions with each cycle of chemotherapy and immunotherapy, up to 3 cycles. A dose de-escalation design was used. Exploratory in vitro and in vivo studies further characterized UC-MSCTRAIL properties. Six participants enrolled; four received 4 × 10⁸ cells/infusion (median 5.4 × 106 cells/kg), and two received 2 × 10⁸ cells/infusion (median 2.4 × 106 cells/kg). Early termination occurred due to asymptomatic pulmonary emboli (N = 5), which included two patients that were anticoagulated as a protocol amendment with prophylactic low molecular weight heparin (enoxaparin 40 mg) and rivaroxaban 20 mg, respectively. Exploratory analyses found no clear pro-coagulant or immunogenic mechanisms, though participants receiving UC-MSCTRAIL had elevated inflammatory markers. This first-in-human study of UC-MSCTRAIL in advanced lung cancer was terminated early due to high prevelance of pulmonary embolism. THough exact mechanism remains unclear, UC-MSCTRAIL may have contributed to a pro-inflammatory environment in participants already at elevated risk of thrombosis due to malignancy. Future MSC-based therapies should incorporate close monitoring for asymptomatic thrombosis and follow a dose escalation design for safety. Safety concerns underscore the need for further research.
Searching and learning from aggregated public metabolomics data spanning thousands of studies remained largely inaccessible. Here we present StructureMASST, a web-based application enabling scalable, structure-centric searches across public metabolomics repositories using molecule names or chemical representations. It queries a precomputed knowledgebase of 2.19 billion spectral matches and 420 million metadata links, supports modification-tolerant and mass-shift searches, and maps chemical structures across taxonomy, biological context and environmental conditions to accelerate discovery.
To develop a sensitive, versatile analytical method capable of simultaneously detecting epigenetically relevant metabolites without chemical derivatization. We also aim to establish a stable isotope tracing methodology to track the biosynthesis of key epigenetic donors, S-adenosylmethionine (SAM) and acetyl-coenzyme A (acetyl-CoA), and demonstrate the method's reproducibility and quantitative accuracy through case-control studies that link metabolism to epigenetics. After a comprehensive literature review, we selected 42 metabolites based on their roles in epigenetic processes such as methylation and acetylation, and devised a targeted metabolomics approach to extract, detect, and quantify these metabolites (Supplementary table 1 and Figure 1). We then optimized ionization parameters and scan rate using pure standards to maximize metabolite coverage in LC-MS/MS. We chose a biphasic extraction method adapted from Lotti et al., using phosphoric acid (15%) and methyl tert-butyl ether (MTBE) for efficient extraction of a wide range of metabolites, including short-chain fatty acids (SCFAs) and formate, without the need for chemical derivatization. The organic phase was analyzed by GC-MS/MS, while the aqueous phase was subjected to LC-MS/MS using a zwitterionic HILIC column with medronic acid to improve peak shape and retention of charged metabolites. To potentially link metabolism and epigenetic modifications, we implemented a stable isotope tracing methodology to track 13C-labeled glucose, glutamine, or serine into SAM and acetyl-CoA. Our method focuses on measuring isotopomers rather than isotopologues, offering a nuanced understanding of labeled carbon atom fate. Our method demonstrated high reproducibility and sensitivity, enabling the quantitative analysis of over 30 epigenetically relevant metabolites, including SCFAs, SAM, and acetyl-CoA, in various biological samples. We successfully quantified these metabolites in three case-control studies: (1) liver and gut content from germ-free and conventional mice, revealing significant differences in SCFA levels and other metabolites linked to one-carbon metabolism and energy production. (2) During OSKM reprogramming of mouse embryonic fibroblasts vitamin B12 supplementation enhances cellular reprogramming. Using 13C-serine as a tracer, we observed a time-dependent increase in SAM enrichment, with additive effects from vitamin B12, primarily due to heightened labeling of the +1 isotopomers formate and methyl group. (3) In an isogenic human glioma cell line with the IDH1 R132H mutation, both wild-type and mutant cells predominantly used glucose carbons for acetyl-CoA synthesis. However, while no significant differences were observed in glucose metabolism between WT and mutant cells, we noted increased glutamine consumption in IDH1-R132H cells, evidenced by higher enrichment of the acetyl group in acetyl-CoA. We present an innovative analytical methodology for the simultaneous detection and quantification of over 30 epigenetically relevant metabolites, including short chain fatty acids. Using stable isotope tracing to track the synthesis of S-adenosylmethionine (SAM) and acetyl-Coenzyme A (acetyl-CoA), our method reveals new insights into metabolism linked to epigenetic modifications, including glycolysis, the pentose phosphate pathway, de novo glycine synthesis, and the folate and methionine cycle. Demonstrating practical utility in case-control studies, this approach supports integrative multi-omics strategies to explore the interplay between metabolism and epigenetics across various biological systems and diseases.
Naive human pluripotent stem cells (hPSCs) represent a pre-implantation epiblast state able to efficiently differentiate into embryonic and extraembryonic pre-implantation lineages and to self-organise in vitro into blastocyst-like structures called blastoids. Naive hPSC maintenance routinely relies on co-culture with mouse embryonic fibroblast (MEFs) as feeder cells, a method prone to variability and analytical confounders. Here, we describe a feeder-free culture system based on serum coating that supports long-term maintenance of naive hPSCs. Across five laboratories, 30 serum batches were evaluated for the expansion of eight naive hPSCs lines for up to 25 passages. Mass spectrometry analysis identified fibronectin and collagens as extracellular matrix proteins consistently present in serum coating. Cells cultured on serum coating displayed growth kinetics, clonogenic capacity, mutation rates, and global gene expression profiles comparable to MEF-based cultures. Importantly, serum-cultured naive hPSCs efficiently underwent germ layer specification, retained trophectoderm competence, and generated blastoids with efficiency similar to MEF-based cultures. Collectively, serum coating provides a scalable, cost-effective, and robust alternative to feeder-based systems, preserving genomic stability and developmental potential while eliminating MEF-associated disadvantages and variability. This platform facilitates large-scale applications of naive hPSCs and enables more reproducible mechanistic studies.
Despite being heavily infiltrated by immune cells, tuberculosis (TB) granulomas often subvert the host response to Mycobacterium tuberculosis (Mtb) infection and support bacterial persistence. Human TB granulomas are enriched for immunosuppressive factors typically associated with tumor-immune evasion, raising the possibility that they promote tolerance to infection. Here we identify candidate drivers for establishing this tolerogenic niche and show that the magnitude of this response correlates with bacterial persistence. We conducted a multimodal spatial analysis of 52 granulomas from 16 nonhuman primates infected with low-dose Mtb for 9-12 weeks. Each granuloma's bacterial burden was quantified individually, enabling us to assess how granuloma spatial structure and function relate to infection control. We found that a universal feature of TB granulomas is partitioning of the myeloid core into two distinct metabolic environments, one of which is hypoxic. This hypoxic environment is associated with pathological immune cell states, dysfunctional cellular organization of the granuloma, and a near-complete blockade of lymphocyte infiltration that would be required for a successful host response. The extent of these hypoxia-associated features correlates with higher bacterial burden. We conclude that hypoxia correlates with immune cell state and organization within granulomas and might subvert immunity to TB.
Roughly 20% of circulating B cells react with self-antigens. An open question is whether germline autoreactivities expand when immune tolerance is breached. To test this, we supplied the 564Igi mouse model of spontaneous autoreactive germinal centers (GCs) with naive B cells that were polyclonal with human-like CDRH3 diversity but were genetically constrained to one of two alleles of the human antibody VH gene IGHV1-69. This polymorphism is skewed across global ethnicities, encoding either F54 or L54 in the CDRH2 loop, with L54 endowing autoreactive B cell receptors (BCRs). L54 B cells were selectively retained within 564Igi mice, leading to their incorporation into autoimmune GCs. This advantage was lost within wild-type C57Bl/6. We also demonstrate human-like L54 IGHV1-69 usage within the geographic variation of ancestral Neanderthals and Denisovans. Collectively, our results suggest that the self-reactive B cell pool is ancestral and is positioned for expansion by autoimmune environments.