Differential cell survival outcomes in response to diverse amino acid stress.

Amino acid (AA) detection is fundamental for cellular function, balancing translation demands, biochemical pathways, and signaling networks. Although the GCN2 and mTORC1 pathways are known to regulate AA sensing, the global cellular response to AA deprivation remains poorly understood, particularly in non-transformed cells, which may exhibit distinct adaptive strategies compared with cancer cells. Here, we employed murine pluripotent embryonic stem (ES) cells as a model system to dissect responses to AA stress.

Integration of cell-type resolved spatial proteomics and transcriptomics reveals novel mechanisms in early ovarian cancer.

High-grade serous carcinoma (HGSC) is the most common ovarian cancer subtype, typically diagnosed at late stages with poor prognosis. Understanding early molecular events driving HGSC progression is crucial for timely detection and development of effective treatment strategies. We performed and integrated spatial cell-type resolved proteomics and paired transcriptomics across 25 women with precursor lesions of the fallopian tube and/or HGSC.

Redefining ALS: Large-scale proteomic profiling reveals a prolonged pre-diagnostic phase with immune, muscular, metabolic, and brain involvement.

Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with a largely unknown duration and pathophysiology of the pre-diagnostic phase, especially for the common non-monogenic form.

Methods: We leveraged the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort with up to 30 years of follow-up to identify incident ALS cases across five European countries. Pre-diagnostic plasma samples from initially healthy participants underwent high-throughput proteomic profiling (7,285 protein markers, SomaScan).

Clonal haematopoiesis of indeterminate potential and mortality in coronary artery disease.

Background And Aims: Clonal haematopoiesis of indeterminate potential (CHIP) has been associated with cardiovascular risk, but its prognostic relevance and mechanistic role in coronary artery disease (CAD) remains incompletely understood. This study investigated the association between CHIP and all-cause mortality in CAD and explored the cellular and molecular mechanisms, focusing on TET2 mutations.

Methods: Targeted deep sequencing of 13 CHIP driver genes in 8612 patients with angiographically confirmed CAD was performed.

A novel hybrid high speed mass spectrometer allows rapid translation from biomarker candidates to targeted clinical tests using 15N labeled proteins.

Recent developments in affinity binder or mass spectrometry (MS)-based plasma proteomics are now producing panels of potential biomarker candidates for diagnosis or prognosis. However, clinical validation and implementation of these biomarkers remain limited by the reliance on dated triple quadrupole MS technology. Here, we evaluate a novel hybrid high-speed mass spectrometer, Stellar MS, which integrates the robustness of triple quadrupoles with the enhanced capabilities of an advanced linear ion trap analyzer.

Inflammasome-independent IL-1β activation via staphopain A protease of Staphylococcus aureus.

Interleukin-1β (IL-1β) is a pivotal mediator of innate immunity, essential for orchestrating the acute inflammatory response. While the canonical activation of IL-1β involves cleavage of its inactive precursor (pro-IL-1β) by the inflammatory cysteine protease caspase-1, certain bacterial proteases, such as those secreted by group A Streptococcus and Pseudomonas aeruginosa, can also activate pro-IL-1β. In this study, we demonstrate that infection of human N/TERT-1 immortalized keratinocytes by Staphylococcus aureus induces IL-1β processing independently of the classical inflammasome pathways.

Axonal tau reduction ameliorates tau and amyloid pathology in a mouse model of Alzheimer's disease.

Background: Pathological deposition of hyperphosphorylated tau in the brain closely correlates with the course of Alzheimer's disease (AD). Tau pathology occurs in axons of affected neurons and tau removal from axons might thus be an early intervention strategy.

Methods: We investigated the role of the RNA-binding protein hnRNP R in axonal localization and local translation of Mapt mRNA in neurons cultured from hnRNP R knockout mice.

Proteomic analysis of isolated nerve terminals from NaV1.9 knockout mice reveals pathways relevant for pain perception.

Neuropathic pain substantially affects the mental and physical well-being of patients and magnifies the socio-economic burden on the healthcare system. It is important to understand the molecular mechanisms underlying chronic pain to effectively target it. To investigate peripheral mechanisms relevant to pain signalling, we isolated nerve terminals from mouse footpads of wild type and NaV1.

NNMT inhibition in cancer-associated fibroblasts restores antitumour immunity.

Cancer-associated fibroblasts (CAFs) have a pivotal cancer-supportive role, yet CAF-targeted therapies are lacking. Here, using spatial transcriptomics and single-cell RNA sequencing, we investigate the role of nicotinamide N-methyltransferase (NNMT) in high-grade serous ovarian cancer. Mechanistically, NNMT-induced H3K27me3 hypomethylation drives complement secretion from CAFs, attracting immunosuppressive myeloid-derived suppressor cells (MDSCs) to the tumour.

Privacy-preserving multicenter differential protein abundance analysis with FedProt.

Quantitative mass spectrometry has revolutionized proteomics by enabling simultaneous quantification of thousands of proteins. Pooling patient-derived data from multiple institutions enhances statistical power but raises serious privacy concerns. Here we introduce FedProt, the first privacy-preserving tool for collaborative differential protein abundance analysis of distributed data, which utilizes federated learning and additive secret sharing.

Comparative genomic analysis of clinically relevant human skin-associated fungi.

Fungal skin infections represent a significant global health burden, affecting approximately one billion people annually. Despite their prevalence and major global health impact, the molecular mechanisms underlying pathogenicity remain largely uncharacterized. Here we present high quality genomic datasets for 51 fungal strains, representing highly prevalent and clinically relevant species associated with human skin infections.

Spatial proteo-transcriptomic profiling reveals the molecular landscape of borderline ovarian tumors and their invasive progression.

Epithelial serous borderline tumors (SBT) are non-invasive neoplastic ovarian lesions that may recur as chemo-resistant low-grade serous cancer (LGSC). While genetic alterations suggest a common origin, the transition from SBT to LGSC remains poorly understood. Here, we integrate cell-type resolved spatial proteomics and transcriptomics to elucidate the evolution from SBT to LGSC and its corresponding metastases in both stroma and tumor.

Design of combination therapeutics from protein response to drugs in ovarian cancer cells.

High-grade serous ovarian cancer (HGSOC) remains the most lethal gynecologic malignancy and novel treatment approaches are needed. Here, we used unbiased quantitative protein mass spectrometry to assess the cellular response profile to drug perturbations in ovarian cancer cells for the rational design of potential combination therapies. Analysis of the perturbation profiles revealed proteins responding across several drug perturbations (called frequently responsive below) as well as drug-specific protein responses.

Protocol for spatial proteomic profiling of tonsil cancer microenvironments using multiplexed imaging-powered deep visual proteomics.

Here, we present a protocol for spatial proteomic profiling of the tumor microenvironment in tonsil cancer using multiplexed imaging-powered deep visual proteomics (mipDVP). We describe steps for automated 22-plex immunofluorescence staining and imaging on formalin-fixed paraffin-embedded (FFPE) tissue sections, automated single-cell laser microdissection, and single-cell-type mass spectrometry. This workflow enables the spatially resolved isolation of distinct cell populations for proteomic analysis.

Mast cells modulate macrophage biology through release of prestored CSF1.

Background: Mast cells (MCs) are tissue-resident immune cells present in connective tissues throughout the body. They exert diverse functions in immunity by rapidly releasing a plethora of preformed mediators, including proteoglycans, cytokines, and proteases, which are stored in cytoplasmic granules.

Objective: We sought to systematically and globally identify MC-released protein mediators and elucidate their functions.

DCHS1 Modulates Forebrain Proportions in Modern Humans via a Glycosylation Change.

Comparative anatomical studies of primates and extinct hominins, including Neanderthals, show that the modern human brain is characterised by a disproportionately enlarged neocortex relative to the striatum. To explore the molecular basis of this difference, we screened for missense mutations that are unique to modern humans and occur at high frequency and that alter post-translational sites. One such mutation was identified in a protocadherin family gene, and it was found to disrupt an N-glycosylation site in modern humans.

Ontology-guided clustering enables proteomic analysis of rare pediatric disorders.

The study of rare pediatric disorders is fundamentally limited by small patient numbers, making it challenging to draw meaningful biological conclusions. To address this, we developed a framework integrating clinical ontologies with proteomic profiling, enabling the systematic analysis of rare conditions in aggregate. We applied this approach to urine and plasma samples from 1140 children and adolescents, encompassing 394 distinct disease conditions and healthy controls.

Spatial Proteomic Profiling Reveals Increased Levels of Cholesterol Synthesis Proteins in Psoriasis Vulgaris.

Psoriasis arises from a combination of genetic and environmental factors, triggering inflammatory circuits involving innate and adaptive immune responses. Although the associated histological changes are well-described, the changes on the protein level associated with the inflammation and tissue remodeling have only been characterized to a limited extent and with a low degree of spatial information. Therefore, we aimed to explore skin layer-specific proteomic changes in psoriatic plaques.

Single cell spatial proteomics maps human liver zonation patterns and their vulnerability to fibrosis.

Understanding protein distribution patterns across tissue architecture is crucial for deciphering organ function in health and disease. Here, we applied single-cell Deep Visual Proteomics to perform spatially-resolved proteome analysis of individual cells in native tissue. We combined this with a novel strategic cell selection pipeline and a continuous protein gradient mapping framework to investigate larger clinical cohorts.

Altering metabolism programs cell identity via NAD-dependent deacetylation.

Cells change their metabolic profiles in response to underlying gene regulatory networks, but how can alterations in metabolism encode specific transcriptional instructions? Here, we show that forcing a metabolic change in embryonic stem cells (ESCs) promotes a developmental identity that better approximates the inner cell mass (ICM) of the early mammalian blastocyst in cultures. This shift in cellular identity depends on the inhibition of glycolysis and stimulation of oxidative phosphorylation (OXPHOS) triggered by the replacement of D-glucose by D-galactose in ESC media. Enhanced OXPHOS in turn activates NAD + -dependent deacetylases of the Sirtuin family, resulting in the deacetylation of histones and key transcription factors to focus enhancer activity while reducing transcriptional noise, which results in a robustly enhanced ESC phenotype.

Deep Immune Phenotyping Reveals Distinct Immunopathogenesis in Checkpoint Inhibitor-Induced Colitis Compared with Ulcerative Colitis.

Although initially assumed to be similar, immune checkpoint inhibitor (ICI)-induced autoimmunity can differ from spontaneous autoimmune disease in regard to clinical presentation, pathogenesis, and therapy. Despite limited pathogenetic understanding, ICI-induced colitis (irColitis), a common adverse event during ICI therapy, is treated analogously to its autoimmune counterpart, ulcerative colitis (UC). Thus, there is a tremendous need to characterize immunophenotypes in both forms of colonic inflammation to ultimately identify specific therapeutic strategies.

Deep Visual Proteomics maps proteotoxicity in a genetic liver disease.

Protein misfolding diseases, including α1-antitrypsin deficiency (AATD), pose substantial health challenges, with their cellular progression still poorly understood. We use spatial proteomics by mass spectrometry and machine learning to map AATD in human liver tissue. Combining Deep Visual Proteomics (DVP) with single-cell analysis, we probe intact patient biopsies to resolve molecular events during hepatocyte stress in pseudotime across fibrosis stages.

Cell-intrinsic insulin signaling defects in human iPS cell-derived hepatocytes in type 2 diabetes.

Hepatic insulin resistance is central to type 2 diabetes (T2D) and metabolic syndrome, but defining the molecular basis of this defect in humans is challenging because of limited tissue access. Utilizing inducible pluripotent stem cells differentiated into hepatocytes from control individuals and patients with T2D and liquid chromatography with tandem mass spectrometry-based (LC-MS/MS-based) phosphoproteomics analysis, we identified a large network of cell-intrinsic alterations in signaling in T2D. Over 300 phosphosites showed impaired or reduced insulin signaling, including losses in the classical insulin-stimulated PI3K/AKT cascade and their downstream targets.

A Proteogenomic View of Synchronous Endometrioid Endometrial and Ovarian Cancer.

Purpose: Increasing genomics-based evidence suggests that synchronous endometrial and ovarian cancer (SEOC) represents clonally related primary and metastatic tumors. A systematic analysis of the global protein landscape of SEOCs, heretofore lacking, could reveal functional and disease-specific consequences of known genetic alterations, the directionality of metastasis, and accurate histologic markers to distinguish SEOCs from single-site tumors.

Experimental Design: We performed a systematic proteogenomic analysis of 29 patients diagnosed with SEOC at three international gynecologic oncology treatment centers (Chicago, Vancouver, and Tübingen).

Regulatory T cells in the mouse hypothalamus control immune activation and ameliorate metabolic impairments in high-calorie environments.

The hypothalamus in the central nervous system (CNS) has important functions in controlling systemic metabolism. A calorie-rich diet triggers CNS immune activation, impairing metabolic control and promoting obesity and Type 2 Diabetes (T2D), but the mechanisms driving hypothalamic immune activation remain unclear. Here we identify regulatory T cells (Tregs) as key modulators of hypothalamic immune responses.