Biology subjects

Allard, B.

Publications and source records attributed to Allard, B..

9 recordsLinked to original sources

bioRxiv2026

CACNA2D4 variants are associated with exertional heat stroke susceptibility

Exertional Heat Stroke (EHS) is a life-threatening disease defined by severe hyperthermia and sudden neurological dysfunction in healthy individuals exposed to intense physical exercise. As is the case with some forms of malignant hyperthermia, mutations in genes involved in skeletal muscle contraction could predispose some patients to EHS. However, the genetic basis of EHS remains poorly documented. Here we identify EHS-associated variants in the CACNA2D4 gene encoding the Cav Ca2+ channel subunit, 2{delta}4, and describe their impact in heterologous expression systems and a knock-in mutant mouse strain heterozygous for the S299R variant (S299R+/-). In transfected cells, each CACNA2D4 variant, including S299R, affected the trafficking and function of 2{delta}4 and its regulation of the Cav1.1 channel. S299R+/-mice showed EHS-like crisis with signs of rhabdomyolysis and an elevation in core body temperature when submitted to an intense exercise protocol. We show that a CACNA2D4 transcript is expressed in mouse skeletal muscle and that the presence of the S299R variant in 2{delta}4 induces a modification in the calcium flux triggered by muscle cell depolarization. Altogether, our data reveal an unexpected connection between CACNA2D4 gene variants and pathophysiological alterations in skeletal muscle Ca2+ signaling that may increase EHS susceptibility.

genetics↗

bioRxiv2025

Aerosolized ApoA1 Nanoparticles Synthesized by Microfluidics Cross the Lung Barrier and Modulate Inflammation

High-density lipoproteins exert vasculoprotective effects, mainly through apolipoprotein A1, which has led to the development of treatments based on apolipoprotein A1 nanoparticles (A1NPs) administered intravenously, mainly for the treatment of cardiovascular diseases. However, their potential as therapy for lung pathologies has not yet been explored. In this work, we produced A1NPs using microfluidics and characterized their therapeutic potential for lung delivery. Their morphology was characterized by dynamic light scattering and transmission electron microscopy. A1NPs toxicity and cellular uptake were performed on both endothelial (HMEC-1) and epithelial (A549) cells and their anti-inflammatory activity was evaluated on TNF--stimulated HMEC-1. A1NPs biodistribution was explored in lung mice after aerosolization and their transcytosis was further investigated using A549 air-liquid interface model. Our results demonstrate that the microfluidic synthesis of A1NPs was reproducible and yielded discoidal particles with sizes ranging from 7-12 nm. A1NPs were internalized by both cells without being cytotoxic and significantly reduced IL-6 expression. Aerosolization resulted in homogeneous distribution in lungs, without causing an immunogenic response. A fraction of A1NPs crossed alveolar epithelial cells both in vitro and in vivo, paving the way for future therapeutic strategies targeting not only the lungs, but also other peripheral organs. These results are promising for the use of A1NPs as vectors for therapeutic molecules, which could exert synergistic protective effects with Apolipoprotein A1. This is the first study to show the non-invasive administration of A1NPs by aerosolization, which may improve their bioavailability in lungs and appears to be a promising approach for treating lung diseases. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/663869v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@2f3436org.highwire.dtl.DTLVardef@10cd3aeorg.highwire.dtl.DTLVardef@11d7542org.highwire.dtl.DTLVardef@fd627a_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Integrator complex subunit 12 knockout overcomes a transcriptional block to HIV latency reversal

The latent HIV reservoir is a major barrier to HIV cure. Combining latency reversal agents (LRAs) with differing mechanisms of action such as AZD5582, a non-canonical NF-kB activator, and I-BET151, a bromodomain inhibitor is appealing towards inducing HIV-1 reactivation. However, even this LRA combination needs improvement as it is inefficient at activating proviruses in cells from people living with HIV (PLWH). We performed a CRISPR screen in conjunction with AZD5582 & I-BET151 and identified a member of the Integrator complex as a target to improve this LRA combination, specifically Integrator complex subunit 12 (INTS12). Integrator functions as a genome-wide attenuator of transcription that acts on elongation through its RNA cleavage and phosphatase modules. Knockout of INTS12 improved latency reactivation at the transcriptional level and is more specific to the HIV-1 provirus than AZD5582 & I-BET151 treatment alone. We found that INTS12 is present on chromatin at the promoter of HIV and therefore its effect on HIV may be direct. Additionally, we observed more RNAPII in the gene body of HIV only with the combination of INTS12 knockout with AZD5582 & I-BET151, indicating that INTS12 induces a transcriptional elongation block to viral reactivation. Moreover, knockout of INTS12 increased HIV-1 reactivation in CD4 T cells from virally suppressed PLWH ex vivo, and we detected viral RNA in the supernatant from CD4 T cells of all three virally suppressed PLWH tested upon INTS12 knockout suggesting that INTS12 prevents full-length HIV RNA production in primary T cells. Finally, we found that INTS12 more generally limits the efficacy of a variety of LRAs with different mechanisms of action.

microbiology↗

bioRxiv2024

BET Degraders Reveal BRD4 Disruption of 7SK and P-TEFb is Critical for Effective Reactivation of Latent HIV in CD4+ T-cells

HIV cure strategies that aim to induce viral reactivation for immune clearance leverage latency reversal agents to modulate host pathways which directly or indirectly facilitate viral reactivation. Inhibition of BET (bromo and extra-terminal domain) family member BRD4 reverses HIV latency, but enthusiasm for the use of BET inhibitors in HIV cure studies is tempered by concerns over inhibition of other BET family members and dose-limiting toxicities in oncology trials. Here we evaluated the potential for bivalent chemical degraders targeted to the BET family as alternative latency reversal agents. We observed that despite highly potent and selective BRD4 degradation in primary CD4+ T-cells from ART-suppressed donors, BRD4 degraders failed to induce latency reversal as compared to BET inhibitors. Further, BRD4 degraders failed to mimic previously observed synergistic HIV reactivation between BET inhibitors and an activator of the non-canonical NF-{kappa}B pathway. Mechanistic investigation of this discrepancy revealed that latency reversal by BET inhibitors is not related to the abatement of competition between Tat and BRD4 for P-TEFb, but rather the ability of BRD4 to disrupt 7SK and increase the levels of free P-TEFb. This activity is dependent on the shift of BRD4 from chromatin-bound to soluble and retargeting of P-TEFb to chromatin which is dependent on intact BRD4 but independent of the bromodomains.

molecular biology↗

bioRxiv2022

The CD73 immune checkpoint promotes tumor cell metabolic fitness

CD73 is an ectonucleotidase overexpressed on tumor cells that suppresses anti-tumor immunity. Accordingly, several CD73 inhibitors are currently being evaluated in the clinic, including in large randomized clinical trials. Yet, the tumor cell-intrinsic impact of CD73 remain largely uncharacterized. Using metabolomics, we discovered that CD73 significantly enhances tumor cell mitochondrial respiration and aspartate biosynthesis. Importantly, rescuing aspartate biosynthesis was sufficient to restore proliferation of CD73-deficient tumors in immune deficient mice. Seahorse analysis of a large panel of mouse and human tumor cells demonstrated that CD73 enhanced oxidative phosphorylation (OXPHOS) and glycolytic reserve. Targeting CD73 decreased tumor cell metabolic fitness, increased genomic instability and suppressed poly ADP ribose polymerase (PARP) activity. Our study thus uncovered an important immune-independent function for CD73 in promoting tumor cell metabolism, and provides the rationale for previously unforeseen combination therapies incorporating CD73 inhibition.

cancer biology↗

bioRxiv2021

The myotendinous junction marker collagen XXII enables zebrafish postural control learning and optimal swimming performance through its force transmission activity

Although the myotendinous junction (MTJ) is essential for skeletal muscle integrity, its contribution to skeletal muscle function remains largely unknown. Here, we show that CRISPR-Cas9-mediated gene ablation of the MTJ marker col22a1 in zebrafish identifies two distinctive phenotypic classes: class 1 individuals reach adulthood with no overt muscle phenotype while class 2 display severe movement impairment and eventually dye before metamorphosis. Yet mutants that are unequally affected are all found to display defective force transmission attributed to a loss of ultrastructural integrity of the MTJ and myosepta, though with distinct degrees of severity. The behavior-related consequences of the resulting muscle weakness similarly reveal variable phenotypic expressivity. Movement impairment at the critical stage of swimming postural learning eventually causes class 2 larval death by compromising food intake while intensive exercise is required to uncover a decline in muscle performance in class 1 adults. By confronting MTJ gene expression compensation and structural, functional and behavioral insights of MTJ dysfunction, our work unravels variable expressivity of col22a1 mutant phenotype. This study also underscores COL22A1 as a candidate gene for myopathies associated with dysfunctional force transmission and anticipates a phenotypically heterogeneous disease.

developmental biology↗

bioRxiv2020

Sequence evaluation and comparative analysis of novel assays for intact proviral HIV-1 DNA

The HIV proviral reservoir is the major barrier to cure. The predominantly replication-defective proviral landscape makes the measurement of virus that is likely to cause rebound upon ART-cessation challenging. To address this issue, novel assays to measure intact HIV proviruses have been developed. The Intact Proviral DNA Assay (IPDA) is a high-throughput assay that uses two probes to exclude the majority of defective proviruses and determine the frequency of intact proviruses, albeit without sequence confirmation. Quadruplex PCR with four probes (Q4PCR), is a lower-throughput assay that uses limiting dilution long distance PCR amplification followed by qPCR and near-full length genome sequencing (nFGS) to estimate the frequency of sequence-confirmed intact proviruses and provide insight into their clonal composition. To explore the advantages and limitations of these assays, we compared IPDA and Q4PCR measurements from 39 ART-suppressed people living with HIV. We found that IPDA and Q4PCR measurements correlated with one another but frequencies of intact proviral DNA differed by approximately 19-fold. This difference may be in part due to inefficiencies in long distance PCR amplification of proviruses in Q4PCR, leading to underestimates of intact proviral frequencies. In addition, nFGS analysis within Q4PCR explained that some of this difference is explained by proviruses that are classified as intact by IPDA but carry defects elsewhere in the genome. Taken together, this head-to-head comparison of novel intact proviral DNA assays provides important context for their interpretation in studies to deplete the HIV reservoir and shows that together the assays bracket true reservoir size. ImportanceThe Intact Proviral DNA Assay (IPDA) and Quadruplex PCR (Q4PCR) represent major advances in accurately quantifying and characterizing the replication competent HIV reservoir. This study compares the two novel approaches for measuring intact HIV proviral DNA in samples from 39 ART-suppressed people living with HIV, thereby informing ongoing efforts to deplete the HIV reservoir in cure-related trials.

microbiology↗

bioRxiv2020

1-Methylnicotinamide is an immune regulatory metabolite in human ovarian cancer

Immune regulatory metabolites are key features of the tumor microenvironment (TME), yet with a few notable exceptions, their identities remain largely unknown. We uncovered the immune regulatory metabolic states and metabolomes of sorted tumor and stromal, CD4+, and CD8+ cells from the tumor and ascites of patients with high-grade serous ovarian cancer (HGSC) using high-dimensional flow cytometry and metabolomics supplemented with single cell RNA sequencing. Flow cytometry revealed that tumor cells show a consistently greater uptake of glucose than T cells, but similar mitochondrial activity. Cells within the ascites and tumor had pervasive metabolite differences, with a striking enrichment in 1-methylnicotinamide (MNA) in T cells infiltrating the tumor compared to ascites. Despite the elevated levels of MNA in T cells, the expression of nicotinamide N-methyltransferase, the gene encoding the enzyme that catalyses the transfer of a methyl group from S-adenosylmethionine to nicotinamide, was restricted to fibroblasts and tumor cells. Treatment of T cells with MNA resulted in an increase in T cell-mediated secretion of the tumor promoting cytokine tumor necrosis factor alpha. Thus, the TME-derived metabolite MNA contributes to an alternative and non-cell autonomous mechanism of immune modulation of T cells in HGSC. Collectively, uncovering the tumor-T cell metabolome may reveal metabolic vulnerabilities that can be exploited using T cell-based immunotherapies to treat human cancer.

immunology↗

bioRxiv2020

Intraflagellar transport-A deficiency ameliorates ADPKD renal cystogenesis in a renal tubular- and maturation-dependent manner

Primary cilia are sensory organelles built and maintained by intraflagellar transport (IFT) multi-protein complexes. Deletion of different IFT-B genes attenuates polycystic kidney disease (PKD) severity in juvenile and adult Autosomal Dominant (AD) PKD mouse models, while deletion of an IFT-A adaptor, Tulp3, attenuates PKD severity in adult mice only. These studies indicate that dysfunction of specific cilia components has potential therapeutic value. To broaden our understanding of cilia dysfunction and its therapeutic potential, here we investigate the impact of global deletion of an IFT-A gene, Thm1, in juvenile and adult ADPKD mouse models. Both juvenile and adult models exhibited increased kidney weight:body weight (KW/BW) ratios, renal cysts, inflammation, lengthened renal cilia, and increased levels of the nutrient sensor, O-linked {beta}-N-acetylglucosamine (O-GlcNAc). Thm1 deletion in juvenile ADPKD mice reduced KW/BW ratios and cortical collecting duct cystogenesis, but increased proximal tubular and glomerular dilations and did not reduce inflammation, cilia lengths, and O-GlcNAc signaling. In contrast, Thm1 deletion in adult ADPKD mice markedly attenuated renal cystogenesis, inflammation, cilia lengths, and O-GlcNAc. Thus, unlike IFT-B genes, the role of Thm1 deletion in ADPKD mouse models is development-specific. Unlike an IFT-A adaptor gene, deleting Thm1 in juvenile ADPKD mice is partially ameliorative. Our studies suggest that different microenvironmental factors found in distinct nephron segments and between developing and mature kidneys modify ciliary homeostasis and ADPKD pathobiology. Further, elevated levels of O-GlcNAc, which regulates cellular metabolism and ciliogenesis, may be a novel feature and critical regulator of certain key ADPKD pathological processes.

genetics↗