Biology subjects

Clair, G. C.

Publications and source records attributed to Clair, G. C..

12 recordsLinked to original sources

bioRxiv2026

Reproducible-by-design: Romics Processor, a FAIR ecosystem for multi-omics and spatial-omics analysis

Multi-omics and spatial-omics technologies are exploding in use, producing increasingly complex datasets. Existing bioinformatics tools are developing rapidly but fail to fully enforce the FAIR principles, leaving the field vulnerable to escalating issues in computational reproducibility. Here, we introduce a reproducible-by-design paradigm represented in an omics data processing package, RomicsProcessor. At its core, the "Romics_object", which is a self-contained digital artifact that encapsulates the full history of the data from the original data to the fully processed state, capturing the details of the transformative steps and the required dependencies. This architecture ensures that computational workflows are fully portable and reproducible. In this manuscript, we demonstrate RomicProcessors computational capabilities and scalability on diverse datasets, including bulk proteomics, large-scale multiplexed immunofluorescence, and multi-batch mass spectrometry imaging. Providing a robust framework for truly FAIR Data Principles-based analysis, RomicsProcessor is a blueprint for the next generation of reproducible bioinformatics tools that can dramatically accelerate discovery in multi-omics biology in the era of artificial intelligence.

bioinformatics↗

bioRxiv2026

Carbamylome analysis reveals regulatory roles for lysine carbamylation in β-cell glycolytic and insulin-processing enzymes

In type 1 diabetes (T1D), insulin-producing {beta} cells are destroyed by an autoimmune response driven by pro-inflammatory cytokines, including interferons. {beta}-cell cytokine signaling is mediated in part by post-translational modifications, such as phosphorylation and acetylation. However, the role of other post-translational modifications in {beta}-cell cytokine signaling represents an important knowledge gap. In the context of autoimmune diseases, lysine carbamylation has gained attention for its role in pathogenesis. Here, we investigate the role of carbamylation in T1D. We found that pancreatic islet cells from the T1D model, non-obese diabetic (NOD) mice, exhibit 11% carbamylation-positive cells, whereas non-diabetic CD1 mice have only 5%. Proteomics analysis of the MIN6 insulin-producing cell line treated with a cocktail of three pro-inflammatory cytokines IFN{gamma} + IL-1{beta} + TNF identified 284 carbamylated peptides from 222 proteins impacted by the cytokine treatment. Integration of carbamylation and acetylation provided a deep view of the cytokine-regulated PTMs and potential points of interplay. A functional-enrichment analysis revealed that carbamylation was enriched in pathways related to autoimmune diseases, metabolism, DNA replication, and protein translation. Moreover, functional testing demonstrated that carbamylation inhibits the glycolytic enzyme aldolase A and the insulin-processing enzyme carboxypeptidase E, identifying a possible role for cytokine-induced {beta}-cell dysfunction. In summary, protein carbamylation is elevated in islets from NOD mice, and pro-inflammatory cytokine treatment regulates protein carbamylation in MIN6 cells. These data identify carbamylation as a potential regulatory mechanism for {beta}-cell metabolism and insulin production in the context of islet inflammation.

biochemistry↗

bioRxiv2026

CD248 activates TGF-β receptor I to promote vascular remodeling in pulmonary arterial hypertension

I.BackgroundPulmonary arterial hypertension (PAH) is a debilitating cardiopulmonary disease characterized by progressive remodeling of the pulmonary vasculature. Pathologic transforming growth factor-{beta} (TGF-{beta}) signaling is an essential driver of vascular remodeling in PAH. While global inhibitors of TGF-{beta} exist, their clinical application is limited by systemic adverse effects. Therefore, a critically unmet need in PAH is to identify pulmonary vascular-specific regulators of the TGF-{beta} axis, which would selectively enhance clinical efficacy while minimizing adverse effects. As the clinical care of PAH largely promotes vasodilation, and only one FDA-approved agent targets vascular remodeling, this study aimed to identify selective, therapeutically targetable regulators of the TGF-{beta} axis in the PAH pulmonary vasculature. MethodsCD248 was identified via liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics in human lungs. CD248 levels were assessed across human, rat, and mouse lung tissues using western blotting, RTqPCR, and/or immunofluorescence techniques. CD248-null (CD248-/-) mice were used to study the contribution of CD248 to hypoxia-sugen (H/S)-induced PAH. The mechanistic role of CD248 in PAH vascular remodeling and TGF-{beta} signaling was assessed by genetic (siRNA knockdown; overexpression) and pharmacologic (Ontuxizumab) manipulation of primary human pulmonary vascular cells. ResultsLC-MS/MS proteomics coupled with pathway enrichment analysis of human lung tissue identified CD248 as a putative mediator of vascular remodeling that is elevated in PAH lungs. CD248 was elevated in PAH pulmonary artery smooth muscle cells (PASMCs) across human, rat, and mouse lung tissue. CD248-/- mice were protected from H/S-induced elevations in right ventricular (RV) systolic pressure (RVSP), RV hypertrophy, and pulmonary artery muscularization. CD248 knock-down reduced cell proliferation and migration of primary PAH PASMCs. CD248 was essential for phospho-activation of TGF-{beta} receptor I (T{beta}RI) at S165 and canonical phosphorylation of SMAD3 at S423/425. CD248 loss blunted TGF-{beta}-induced gene expression (FN1, Col11, -SMA) and activated expression of the vasoprotective matrix metalloprotease, MMP-8. Mechanistically, CD248 interacted with and enhanced de novo phosphorylation and stability of T{beta}RI, blocking its ubiquitin-mediated proteasomal degradation. Ontuxizumab promoted T{beta}RI instability and attenuated the production of FN1, Col11, and -SMA in primary PAH PASMCs. ConclusionsThis work identifies CD248 as a previously unrecognized co-activator of T{beta}RI in PAH. As CD248 is largely quiescent in most adult tissues yet pathologically upregulated in the PAH pulmonary vasculature, this study supports the potential of anti-CD248 therapy as a novel pulmonary vascular-specific alternative to systemic TGF-{beta} inhibition.

molecular biology↗

bioRxiv2026

Unbiased proteomics following inflammasome activation identifies caspase targets in primary intestinal epithelial cells

Inflammasomes are cytosolic innate immune sensors that, once activated by a pathogenic threat, lead to activation of the inflammatory Caspase-1. Inflammasome activation and its consequences have been studied extensively in myeloid cells and in overexpression systems. Recent studies have identified cell type specific effects that are not fully explained by the known cleavage targets of Caspase-1. Here, we identified targets of caspase cleavage using mass spectrometry in primary intestinal epithelial cells by specifically activating the NAIP-NLRC4 inflammasome. We have taken an unbiased approach and developed a novel method for analyzing mass spectrometry data for evidence of caspase activity. Our approach can also be applied to existing proteomic datasets to establish the presence of caspase activity under various biological conditions. These results lay the groundwork for future studies on mechanisms of caspase-induced processes such as intestinal epithelial cell extrusion.

immunology↗

bioRxiv2025

Sexually distinct multi omic responses to progressive endurance exercise training in the rat lung. Findings from MoTrPAC

Despite the lungs being essential for ventilation and aerobic exercise capacity, conventionally the lungs are not thought to adapt to exercise training. Endurance exercise is key to pulmonary rehabilitation programs, which also displays sex-specific differences in therapeutic efficacy. Given the molecular underpinnings of sex-specific lung adaptations to endurance exercise are uncharacterized, we used a multi-omics approach to study sex differences in the lungs of 6-month-old Fischer 344 rats in response to an 8 week progressive endurance treadmill training protocol. This was accomplished by reannotating publicly accessible data from the Molecular Transducers of Physical Activity Consortium (MoTrPAC) and integrating newly-analyzed acetylome data to assess multi-omic sex differences in sedentary and progressively trained states. Female rats displayed enrichment in immune-related features and pathways at the transcriptome and proteome level that were maintained with training. Conversely, in the male rat lung there was an overall decrease in immune pathways following 8 weeks of training. Sexually conserved responses to training included increased enrichment in transcriptomic pathways related to type I alveoli and proteomic pathways related to cilia, and decreased mitochondrial protein acetylation. In both sexes, features known to be enriched in lung diseases were attenuated with training. Together our findings provide novel insight into sex specific responses to endurance exercise training in the rat lung and may offer translational insight into sex-specific differences in lung disease pathogenesis and treatment.

physiology↗

bioRxiv2024

Integrating N-glycan and CODEX imaging reveal cell-specific protein glycosylation in healthy human lung

N-linked glycosylation, the major post-translational modification of cellular proteins, is important for proper lung functioning, serving to fold, traffic, and stabilize protein structures and to mediate various cell-cell recognition events. Identifying cell-specific N-glycan structures in human lungs is critical for understanding the chemistry and mechanisms that guide cell-cell and cell-matrix interactions and determining nuanced functions of specific N-glycosylation. Our study, which used matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) combined with co-detection by indexing (CODEX) to reveal the cellular origin of N-glycans, is a significant step in this direction. This innovative technological combination enabled us to detect and differentiate N-glycans located in the vicinity of cells surrounding airways and blood vessels, parenchyma, submucosal glands, cartilage, and smooth muscles. The potential impact of our findings on future research is immense. For instance, our algorithm for grouping N-glycans based on their functional chemical features, combined with identifying group niches, paves the way for targeted studies. We found that fucosylated N-glycans are dominant around immune cells, tetra antennary N-glycans in the cartilage, high-mannose N-glycans surrounding the bronchus originate from associated collagenous structures, complex fucosylated-tetra antennary-polylactosamine N-glycans are spread over smooth muscle structures and in epithelial cells surrounding arteries, and N-glycans with Hex:6 HexNAc:6 compositions, which, according to our algorithm, can be ascribed to either tetra antennary or bisecting N-glycan, are highly abundant in the parenchyma. The findings suggest cell or region-specific functions for these localized glycan structures.

biochemistry↗

bioRxiv2024

Alterations of the skeletal muscle nuclear proteome after acute exercise reveals a post-transcriptional influence.

Exercise is firmly established as a key contributor to overall well-being and is frequently employed as a therapeutic approach to mitigate various health conditions. One pivotal aspect of the impact of exercise lies in the systemic transcriptional response, which underpins its beneficial adaptations. While extensive research has been devoted to understanding the transcriptional response to exercise, our knowledge of the protein constituents of nuclear processes that accompany gene expression in skeletal muscle remains largely elusive. We hypothesize that alterations in the nuclear proteome following exercise hold vital clues for comprehending the transcriptional regulation and other related nuclear functions. We isolated skeletal muscle nuclei from C57BL/6 mice both sedentary control and one-hour post 30-minute treadmill running, to gain insights into the nuclear proteome after exercise. A substantial number of the 2,323 proteins identified, were related to nuclear functions. For instance, we found 59 proteins linked to nucleocytoplasmic transport were higher in sedentary mice compared to exercise, hinting at an exercise-induced modulation to nuclear trafficking. Furthermore, 135 proteins exhibited increased abundance after exercise (FDR < 0.1) while 89 proteins decreased, with the most prominent changes in proteins linked to mRNA processing and splicing. Super resolution microscopy further highlights potential localization change in mRNA processing proteins post-exercise, further suggesting changes in nuclear transport dynamics. Nonetheless, our data provide important considerations for the study of the nuclear proteome and supports a paradigm through which exercise downregulated mRNA processing and splicing, offering valuable insights into the broader landscape of the impact from acute exercise. New & NoteworthyExercise plays a crucial role in promoting muscle health, but our understanding of nuclear proteins orchestrating exercise responses is limited. Isolation of skeletal muscle nuclei coupled with mass spectrometry enhanced the identification of nuclear proteins. This approach was used to investigate the effects of acute exercise, revealing changes in the muscle nuclear proteome 1-hour post-exercise, including proteins linked to post-transcriptional processing and splicing. Our findings offer insights into the exercise-induced changes within muscle nuclear proteins.

physiology↗

bioRxiv2021

LungMAP Portal Ecosystem: Systems-Level Exploration of the Lung

An improved understanding of the human lung necessitates advanced systems models informed by an ever-increasing repertoire of molecular omics, cellular, imaging and pathological datasets. To centralize and standardize information across broad lung research efforts we expanded the LungMAP.net website into a gateway portal. This portal connects a broad-spectrum of research networks, bulk and single-cell multi-omics data and a diverse collection of image data that span mammalian lung development and disease. The data are standardized across species and technologies using harmonized data and metadata models that leverage recent advances including those from the Human Cell Atlas, diverse ontologies, and the LungMAP CellCards initiative. To cultivate future discoveries, we have aggregated a diverse collection of single-cell atlases for multiple species (human, rhesus, mouse), to enable consistent queries across technologies, cohorts, age, disease and drug treatment. These atlases are provided as independent and integrated queriable datasets, with an emphasis on dynamic visualization, figure generation and reference-based classification of user-provided datasets (Azimuth). As this resource grows, we intend to increase the breadth of available interactive interfaces, data portals and datasets from LungMAP and external research efforts.

systems biology↗

bioRxiv2021

High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip

Global quantification of protein abundances in single cells would provide more direct information on cellular function phenotypes and complement transcriptomics measurements. However, single-cell proteomics (scProteomics) is still immature and confronts technical challenges, including limited proteome coverage, poor reproducibility, as well as low throughput. Here we describe a nested nanoPOTS (N2) chip to dramatically improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow. The N2 chip allows reducing cell digestion volume to <30 nL and increasing processing capacity to > 240 single cells in one microchip. In the analysis of [~]100 individual cells from three different cell lines, we demonstrate the N2 chip-based scProteomics platform can robustly quantify [~]1500 proteins and reveal functional differences. Our analysis also reveals low protein abundance variations (median CVs < 16.3%), highlighting the utility of such measurements, and also suggesting the single-cell proteome is highly stable for the cells cultured under identical conditions.

systems biology↗

bioRxiv2021

Robust, sensitive, and quantitative single-cell proteomics based on ion mobility filtering

Unbiased single-cell proteomics (scProteomics) promises to advance our understanding of cell functions within complex biological systems. However, a major challenge for current methods is their ability to identify and provide accurate quantitative information for low abundance proteins. Herein, we describe an ion mobility-enhanced mass spectrometry acquisition and peptide identification method, TIFF (Transferring Identification based on FAIMS Filtering), designed to improve the sensitivity and accuracy of label-free scProteomics. TIFF significantly extends the ion accumulation times for peptide ions by filtering out singly charged background ions. The peptide identities are then assigned by a 3-dimensional MS1 feature matching approach (retention time, accurate mass, and FAIMS compensation voltage). The TIFF method enabled unbiased proteome analysis to a depth of >1,700 proteins in single HeLa cells with >1,100 proteins consistently quantified. As a demonstration, we applied the TIFF method to obtain temporal proteome profiles of >150 single murine macrophage cells during a lipopolysaccharide stimulation experiment and identified time-dependent proteome profiles.

systems biology↗

bioRxiv2020

A lipid metabolic map of the pathogenic fungus Histoplasma capsulatum

Lipids play a fundamental role in fungal cell biology, being essential cell membrane components and major targets of antifungal drugs. A deeper knowledge of lipid metabolism is key for developing new drugs and a better understanding of fungal pathogenesis. Here we built a comprehensive map of the Histoplasma capsulatum lipid metabolic pathway by incorporating proteomic and lipidomic analyses. We performed genetic complementation and overexpression of H. capsulatum genes in Saccharomyces cerevisiae to validate reactions identified in the map and to determine enzymes responsible for catalyzing orphan reactions. The map led to the identification of both the fatty acid desaturation and the sphingolipid biosynthesis pathways as targets for drug development. We found that the sphingolipid biosynthesis inhibitor myriocin, the fatty acid desaturase inhibitor thiocarlide and the fatty acid analog 10-thiastearic acid inhibit H. capsulatum growth in nanomolar to low micromolar concentrations. These compounds also reduced the intracellular infection in an alveolar macrophage cell line. Overall, this lipid metabolic map revealed pathways that can be targeted for drug development.

microbiology↗

bioRxiv2019

Candida auris: multi-omics signature of an emerging and multidrug-resistant pathogen

Candida auris is a recently described pathogenic fungus that is causing invasive outbreaks on all continents. The fungus is of high concern given the numbers of multidrug-resistant strains that have been isolated in distinct sites across the globe. The fact that its diagnosis is still problematic suggests that the spreading of the pathogen remains underestimated. Notably, the molecular mechanisms of virulence and antifungal resistance employed by this new species are largely unknown. In the present work, we compared two clinical isolates of C. auris with distinct drug susceptibility profiles and a Candida albicans reference strain using a multi-omics approach. Our results show that, despite the distinct drug-resistance profile, both C. auris strains appear to be very similar, albeit with a few notable differences. However, when compared to C. albicans both C. auris strains have major differences regarding their carbon utilization and downstream lipid and protein content, suggesting a multi-factorial mechanism of drug resistance. The molecular profile displayed by C. auris helps to explain the antifungal resistance and virulence phenotypes of this new emerging pathogen. ImportanceCandida auris was firstly described in Japan in 2009 and has now been the cause of significant outbreaks across the globe. The high number of isolates that are resistant to one or more antifungals, as well as the high mortality rates from patients with bloodstream infections, has caught the attention of the medical mycology, infectious disease and public health communities to this pathogenic fungus. In the current work, we performed a broad multi-omics approach on two clinical isolates isolated in New York, the most affected area in the USA and found that the omic profile of C. auris differs significantly from C. albicans. Besides our insights into C. auris carbon utilization and lipid and protein content, we believe that the availability of these data will enhance our ability to combat this rapidly emerging pathogenic yeast.

microbiology↗