Biology subjects

Belmonte, M.

Publications and source records attributed to Belmonte, M..

6 recordsLinked to original sources

bioRxiv2025

Selection of pre-leukemic hematopoietic stem cells driven by distinct extracellular matrix molecules

Despite rapid advances in mapping genetic drivers and gene expression changes in hematopoietic stem cells (HSCs), there is a relative paucity of studies at the protein level. Here, we perform a deep, multi-omic characterization (epigenome, transcriptome and proteome) of HSCs carrying a loss-of-function mutation in Tet2, a key driver of increased self-renewal in blood cancers. Using state-of-the-art, multiplexed, low-input mass spectrometry (MS)-based proteomics, we profile wildtype (WT) and TET2-deficient (Tet2-/-) HSCs and show that the proteome captures previously unrecognized molecular processes which define the pre-leukemic HSC molecular landscape. Specifically, we obtain more accurate stratification of WT and Tet2-/- HSCs than transcriptomic approaches and identify extracellular matrix (ECM) molecules as novel points of dysregulation upon TET2 loss. HSC expansion assays using ECM-functionalized hydrogels confirm a selective effect on the expansion of Tet2-mutant HSCs. Taken together, our study represents a comprehensive molecular characterization of Tet2-mutant HSCs and identifies a previously unanticipated role of ECM molecules in regulating self-renewal of disease-driving HSCs.

cell biology↗

bioRxiv2021

CHARM: COVID-19 Health Action Response for Marines - association of antigen-specific interferon-gamma and IL2 responses with asymptomatic and symptomatic infections after a positive qPCR SARS-CoV-2 test

SARS-CoV-2 T cell responses are associated with COVID-19 recovery, and Class I- and Class II-restricted epitopes have been identified in the spike (S), nucleocapsid (N) and membrane (M) proteins and others. This prospective COVID-19 Health Action Response for Marines (CHARM) study enabled assessment of T cell responses in symptomatic and asymptomatic SARS-CoV-2 infected participants. At enrollment all participants were negative by qPCR; follow-up occurred biweekly and then bimonthly for the next 6 weeks. Study participants who tested positive by qPCR SARS-CoV-2 test were asked to enroll in an immune response sub-study. FluoroSpot interferon-gamma (IFN-{gamma}) and IL2 responses following qPCR-confirmed infection at enrollment (day 0), day 7 and 14 and more than 28 days later were measured using pools of 17mer peptides covering S, N, and M proteins, or CD4+CD8 peptide pools containing predicted epitopes from multiple SARS-CoV-2 antigens. Among 124 asymptomatic and 105 symptomatic participants, SARS-CoV-2 infection generated IFN-{gamma} responses to the S, N and M proteins that persisted longer in asymptomatic cases. IFN-{gamma} responses were significantly (p=0.001) more frequent to the N pool (51.4%) than the M pool (18.9%) among asymptomatic subjects; however, the difference was not statistically significant (p=0.06) for symptomatic subjects (N pool: 44.4%; M pool: 25.9%). In asymptomatic participants IFN-{gamma} responders to the CD4+CD8 pool responded more frequently to the S pool (55.6%) and N pool (57.1%), than the M pool (7.1%), but symptomatic participants, IFN-{gamma} responses were more frequent to the S pool (75.0%) than N pool (33.3%) and M pool (33.3%). The frequencies of IFN-{gamma} responses to the S and N+M pools peaked 7 days after the positive qPCR test among asymptomatic (S pool: 22.2%; N+M pool: 28.7%) and symptomatic (S pool: 15.3%; N+M pool 21.9%) participants and dropped by >28 days. Magnitudes of post-infection IFN-{gamma} and IL2 responses to the N+M pool were significantly correlated with IFN-{gamma} and IL2 responses to the N and M pools. These data further support the central role of Th1-biased cell mediated immunity IFN-{gamma} and IL2 responses, particularly to the N protein, in controlling COVID-19 symptoms, and justify T cell-based COVID-19 vaccines that include the N and S proteins.

immunology↗

bioRxiv2021

A highly efficient reporter system for identifying and characterizing in vitro expanded hematopoietic stem cells

Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved more than 200-fold expansion of functional HSCs, but their molecular characterization has not been possible due to the substantial majority of cells being non-HSCs and single cell-initiated cultures displaying substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with the EPCR surface marker, we report exclusive identification of HSCs from non-HSCs in expansion cultures. Linking single clone functional transplantation data with single clone gene expression profiling, we show that the molecular profile of expanded HSCs is similar to actively cycling fetal liver HSCs and shares a gene expression signature with functional HSCs from all sources, including Prdm16, Fstl1 and Palld. This new tool can now be applied to a wide-range of functional screening and molecular experiments previously not possible due to limited HSC numbers.

cell biology↗

bioRxiv2021

Genome-wide mutational signatures of immunological diversification in normal lymphocytes

A lymphocyte suffers many threats to its genome, including programmed mutation during differentiation, antigen-driven proliferation and residency in diverse microenvironments. After developing protocols for single-cell lymphocyte expansions, we sequenced whole genomes from 717 normal naive and memory B and T lymphocytes and hematopoietic stem cells. Lymphocytes carried more point mutations and structural variation than stem cells, accruing at higher rates in T than B cells, attributable to both exogenous and endogenous mutational processes. Ultraviolet light exposure and other sporadic mutational processes generated hundreds to thousands of mutations in some memory lymphocytes. Memory B cells acquired, on average, 18 off-target mutations genome-wide for every one on-target IGV mutation during the germinal center reaction. Structural variation was 16-fold higher in lymphocytes than stem cells, with ~15% of deletions being attributable to off-target RAG activity. One Sentence SummaryThe mutational landscape of normal lymphocytes chronicles the off-target effects of programmed genome engineering during immunological diversification and the consequences of differentiation, proliferation and residency in diverse microenvironments.

genomics↗

bioRxiv2019

Investigation of the quorum-sensing regulon of the biocontrol bacterium Pseudomonas chlororaphis strain PA23

Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of protecting canola from stem rot disease caused by the fungal pathogen Sclerotinia sclerotiorum. PA23 produces several of inhibitory compounds that are under control of a complex regulatory network. Included in this cascade is the PhzRI quorum sensing (QS) system, which plays an essential role in PA23 biocontrol. The focus of the current study was to employ RNA sequencing to explore the spectrum of PA23 genes under QS control. Transcriptomic profiling revealed 545 differentially expressed genes (365 downregulated; 180 upregulated) in the phzR mutant and 534 genes (382 downregulated; 152 upregulated) in the AHL-deficient PA23-6863. In both strains, decreased expression of phenazine, pyrrolnitrin, and exoprotease biosynthetic genes was observed. We have previously reported that QS activates expression of these genes and their encoded products. In addition, elevated siderophore and decreased chitinase gene expression was observed in the QS-deficient stains, which was confirmed by phenotypic analysis. Inspection of the promoter regions revealed the presence of "phz-box" sequences in only 58 of the 807 differentially expressed genes, suggesting that much of the QS regulon is indirectly regulated. Consistent with this notion, 41 transcriptional regulators displayed altered expression in one or both of the QS-deficient strains. Collectively, our findings indicate that QS governs expression of approximately 13% of the PA23 genome affecting diverse functions ranging from secondary metabolite production to general metabolism. To the best of our knowledge, this represents the first global transcriptomic analysis of the QS regulon of a biocontrol pseudomonad.

microbiology↗

bioRxiv2019

The CUL5 ubiquitin ligase complex mediates resistance to CDK9 and MCL1 inhibitors in lung cancer cells

Overexpression of anti-apoptotic proteins MCL1 and Bcl-xL is a frequent event in blood and solid cancers. Inhibitors targeting MCL1 are in clinical development, however many cancer models are intrinsically resistant to this approach. To discover mechanisms underlying resistance to MCL1 inhibition, we performed multiple flow-cytometry based genome-wide CRISPR screens that interrogate two drugs directly or indirectly targeting MCL1. Remarkably, both screens identified three components (CUL5, RNF7 and UBE2F) of a cullin-RING ubiquitin ligase complex (CRL5) that resensitized cells to MCL1 inhibition. We find that levels of the BH3-only pro-apoptotic proteins Bim and Noxa are proteasomally regulated by the CRL5 complex. Accumulation of Noxa caused by depletion of CRL5 components particularly skewed the balance in favor of apoptosis when cells were challenged with an MCL1 inhibitor. Discovery of a novel role of CRL5 in apoptosis and resistance to MCL1 inhibitors exposes new drug targets and the potential to improve combination treatments.

cancer biology↗