Biology subjects

Nguyen, H.

Publications and source records attributed to Nguyen, H..

At least 127 records · Page 7Linked to original sources

bioRxiv2021

REV7/FANCV Binds to CHAMP1 and Promotes Homologous Recombination Repair

A critical determinant of DNA repair pathway choice is the HORMA protein REV7, a small abundant adaptor which binds to various DNA repair proteins through its C-terminal seatbelt domain. The REV7 seatbelt binds to the REV3 polymerase to form the Polymerase {zeta} complex, a positive regulator of translesion synthesis (TLS) repair. Alternatively, the REV7 seatbelt binds to SHLD3 in the Shieldin complex, a positive regulator of NHEJ repair. Recent studies have identified another novel REV7 seatbelt-binding protein, CHAMP1 (Chromosome Alignment-Maintaining Phosphoprotein, though its role in DNA repair is unknown. Here, we show that the REV7-CHAMP1 complex promotes homologous recombination (HR) repair by sequestering REV7 from the Shieldin complex. CHAMP1 competes directly with the SHLD3 subunit of the Shieldin complex for a limited pool of C-REV7, thereby inhibiting the REV7-mediated recruitment of the SHLD2 and SHLD1 effector subunits to DNA double strand breaks. CHAMP1 thereby channels DNA repair away from error-prone NHEJ and towards the competing error-free HR pathway. Similarly, CHAMP1 competes with the REV3 component of the POL{zeta} complex, thereby reducing the level of mutagenic TLS repair. CHAMP1 interacts with POGZ in a heterochromatin complex further promoting HR repair. Importantly, in human tumors, CHAMP1 overexpression promotes HR, confers PARP inhibitor resistance, and correlates with poor prognosis. Thus, by binding to either REV3, SHLD3, or CHAMP1 through its seatbelt, the REV7 protein can promote either TLS repair, NHEJ repair, or HR repair respectively.

molecular biology↗

bioRxiv2021

Utilizing Pine Needles to Temporally and Spatially Profile Per- and Polyfluoroalkyl Substances

As concerns continue to mount over exposure to per- and polyfluoroalkyl substances (PFAS), novel methods of profiling their presence and modifications are greatly needed as some have known toxic and bioaccumulative characteristics while others have unknown effects. This task however is not simple as over 5000 PFAS of interest have been named by the Environmental Protection Agency and this list continues to grow daily. In this work, we utilized widely available archived and field-sampled pine needles and a novel non-targeted analytical method to evaluate the temporal and spatial presence of numerous PFAS. Over 70 PFAS were detected in the pine needles from this study, providing information from the last six decades related to PFAS exposure, contamination, and reduction.

pharmacology and toxicology↗

bioRxiv2021

Efficient inhibition of HIV using CRISPR/Cas13d nuclease system

Recently discovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas13 proteins are programmable RNA-guided ribonucleases that target single-stranded RNA (ssRNA). CRISPR/Cas13 mediated RNA targeting has emerged as a powerful tool for detecting and eliminating RNA viruses. Here, we demonstrate the effectiveness of CRISPR/Cas13d to inhibit HIV-1 replication. We designed guide RNAs (gRNAs) targeting highly conserved regions of HIV-1. RfxCas13d (CasRx) in combination with HIV-specific gRNAs efficiently inhibited HIV-1 replication in cell line models. Furthermore, simultaneous targeting of four distinct sites in the HIV-1 transcript resulted in robust inhibition of HIV-1 replication. We also show the effective HIV-1 inhibition in primary CD4+ T-cells and suppression of HIV-1 reactivated from latently infected cells using the CRISPR/Cas13d system. Our study demonstrates the utility of the CRISPR/Cas13d nuclease system to target acute and latent HIV infection and provides an alternative treatment modality against HIV.

molecular biology↗

bioRxiv2021

Sliding of HIV-1 reverse transcriptase over DNA creates a transient P pocket - Targeting P-pocket by fragment screening

HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We report a crystal structure of RT/dsDNA complex in which RT overstepped the primer 3-end of a dsDNA substrate and created a transient P-pocket at the priming site. We performed a high-throughput screening of 300 drug-like fragments by X-ray crystallography and identified binding of two to P-pocket, which is composed of structural elements from polymerase active site, primer grip, and template-primer that are resilient to drugresistance mutations. Analogs of a fragment were synthesized of which two showed noticeable RT inhibition. An engineered RT/DNA aptamer complex trapped the transient P-pocket in solution. Structures of the RT/DNA complex were determined with a fragment and a synthesized analog bound at P-pocket by single-particle cryo-EM. Identification of P-pocket and the devised structurebased platform provide an opportunity for designing new types of polymerase inhibitors.

biophysics↗

bioRxiv2021

H3 acetylation selectively promotes basal progenitor proliferation and neocortex expansion by activating TRNP1 expression

Increase in the size of human neocortex, acquired in evolution, accounts for the unique cognitive capacity of humans. This expansion appears to reflect the evolutionarily-enhanced proliferative ability of basal progenitors (BPs) in mammalian cortex, which may have been acquired through epigenetic alterations in BPs. However, whether or how the epigenome in BPs differs across species is not known. Here, we report that histone H3 acetylation is a key epigenetic regulation in BP amplification and cortical expansion. Through epigenetic profiling of sorted BPs, we show that H3K9 acetylation is low in murine BPs and high in human BPs. Elevated H3K9ac preferentially increases BP proliferation, increasing the size and folding of the normally smooth mouse neocortex. Mechanistically, H3K9ac drives BP amplification by increasing expression of the evolutionarily regulated gene, TRNP1, in the developing cortex. Our findings demonstrate a previously unknown mechanism that controls cortical architecture. One Sentence SummaryH3K9ac promotes basal progenitor amplification, neocortex expansion and gyrification by activating TRNP1 expression in evolution.

developmental biology↗

bioRxiv2021

Arkadia-SKI/SnoN signaling cascade differentially regulates TGF-β-induced iTreg and Th17 cell differentiation

TGF-{beta} signaling is fundamental for both Th17 and regulatory T cell (Treg) differentiation. However, these cells differ in requirements for downstream signaling components, such as various SMAD effectors, for their differentiation. To further characterize the mechanisms that distinguish TGF-{beta} signaling requirements for Th17 and Treg cell differentiation, we investigated the role of Arkadia (RNF111), a RING type E3 ubiquitin ligase known to enhance TGF-{beta} signaling during development. We find that Arkadia mediates the differentiation of induced-Treg (iTreg) but not Th17 cells both in vitro and in vivo. Inactivation of Arkadia in CD4+ T cells resulted in impairment of Treg cell differentiation in vitro and loss of ROR{gamma}t+FOXP3+ iTreg cells in the intestinal lamina propria in vivo, which increased susceptibility to microbiota-induced mucosal inflammation. Furthermore, genetic ablation of two substrates of Arkadia, the transcriptional co-repressors SKI and SnoN, rescued in vitro and in vivo iTreg cell-differentiation of Arkadia-deficient cells. These results reveal distinct TGF-{beta} signaling modules that govern Th17 and iTreg cell differentiation programs and that could be exploited therapeutically to selectively modulate T cell subsets in pathological settings such as autoimmunity or cancer.

immunology↗

bioRxiv2021

Understanding LRRK2 kinase activity in preclinical models and human subjects through quantitative analysis of LRRK2 and pRab10

Variants in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with increased risk for familial and sporadic Parkinsons disease (PD). Pathogenic variants in LRRK2, including the common variant G2019S, result in increased LRRK2 kinase activity, supporting the therapeutic potential of LRRK2 kinase inhibitors for PD. To better understand the role of LRRK2 in disease and to support the clinical development of LRRK2 inhibitors, quantitative and high-throughput assays to measure LRRK2 levels and activity are needed. We developed and applied such assays to measure the levels of LRRK2 as well as the phosphorylation of LRRK2 itself or one of its substrates, Rab10 (pT73 Rab10). We observed increased LRRK2 activity in various cellular models of disease, including iPSC-derived microglia, as well as in human subjects carrying disease-linked variant in LRRK2 (G2019S). Capitalizing on the high-throughput and sensitive nature of these assays, we detected a significant reduction in LRRK2 activity in subjects carrying missense variants in LRRK2 associated with reduced disease risk. Finally, we optimized these assays to enable analysis of LRRK2 activity following inhibition in human peripheral blood mononuclear cells (PBMCs) and whole blood, demonstrating their potential utility as biomarkers to assess changes in LRRK2 expression and activity in the clinic.

neuroscience↗

bioRxiv2021

Condensates in RNA Repeat Sequences are Heterogeneously Organized and Exhibit Reptation-like Dynamics

Although it is known that RNA undergoes liquid-liquid phase separation (LLPS), the interplay between the molecular driving forces and the emergent features of the condensates, such as their morphologies and dynamical properties, is not well understood. We introduce a coarse-grained model to simulate phase separation of trinucleotide repeat RNAs, which are implicated in neurological disorders such as Huntington disease and amyotrophic lateral sclerosis. After establishing that the simulations reproduce key experimental findings (length and concentration dependence of the phase transition in (CAG)n repeats), we show that once recruited inside the liquid droplets, the monomers transition from hairpin-like structures to extended states. Interactions between the monomers in the condensates result in the formation of an intricate and dense intermolecular network, which severely restrains the fluctuations and mobilities of the RNAs inside large droplets. In the largest densely packed high viscosity droplets, the mobility of RNA chains is best characterized by reptation, reminiscent of the dynamics in polymer melts. Our work provides a microscopic framework for understanding liquid-liquid phase separation in RNA, which is not easily discernible in current experiments.

biophysics↗

bioRxiv2021

Post-infection treatment with a protease inhibitor increases survival of mice with a fatal SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a serious global public health threat. The 3C-like protease (3CLpro) is a virus protease encoded by SARS-CoV-2, which is essential for virus replication. We have previously reported a series of small molecule 3CLpro inhibitors effective for inhibiting replication of human coronaviruses including SARS-CoV-2 in cell culture and in animal models. Here we generated a series of deuterated variants of a 3CLpro inhibitor, GC376, and evaluated the antiviral effect against SARS-CoV-2. The deuterated GC376 displayed potent inhibitory activity against SARS-CoV-2 in the enzyme and the cell-based assays. The K18-hACE2 mice develop mild to lethal infection commensurate with SARS-CoV-2 challenge doses and was proposed as a model for efficacy testing of antiviral agents. We treated lethally infected mice with a deuterated derivative of GC376. Treatment of K18-hACE2 mice at 24 hr post infection with a derivative (compound 2) resulted in increased survival of mice compared to vehicle-treated mice. Lung virus titers were decreased, and histopathological changes were ameliorated in compound 2-treated mice compared to vehicle-treated mice. Structural investigation using high-resolution crystallography illuminated binding interactions of 3CLpro of SARS-CoV-2 and SARS-CoV with deuterated variants of GC376. Taken together, deuterated GC376 variants have excellent potential as antiviral agents against SARS-CoV-2.

microbiology↗

bioRxiv2021

Fibrillar Aβ causes profound microglial metabolic perturbations in a novel APP knock-in mouse model

Microglial dysfunction is believed to play a pathogenic role in Alzheimers disease (AD). Here, we characterize the amyloid-{beta} related pathology and microglial responses in an engineered APP knock-in mouse model of familial AD. This model recapitulates key pathological features of AD such as a progressive accumulation of parenchymal amyloid plaques and vascular amyloid deposits, altered glial responses and neurodegeneration. Leveraging multi-omics approaches, we found lipid accumulation and an exacerbated disease-associated transcriptomic response in methoxy-X04-positive, phagocytic microglia. Together, these findings highlight the potential of this novel, open-access mouse model to investigate AD pathogenesis and demonstrate that fibrillar A{beta} triggers lipid dysregulation and immuno-metabolic perturbations in phagocytic microglia. HighlightsO_LINovel open-access APP KI mouse model shows salient AD pathological features C_LIO_LIDeep phenotyping of sorted microglia reveals profound lipidomic perturbations in line with Alois Alzheimers original descriptions of glial adipose inclusions C_LIO_LIImmunometabolic perturbations are exacerbated in microglia accumulating fibrillar A{beta} C_LI

neuroscience↗

bioRxiv2020

BBrowser: Making single-cell data easily accessible

BioTurings BBrowser is a software solution that helps scientists effectively analyze single-cell omics data. It combines big data with big computation and modern data visualization to create a unique platform where scientists can interact and obtain important biological insights from the massive amounts of single-cell data. BBrowser has three main components: a curated single-cell database, a big-data analytics layer, and a data visualization module. BBrowser is available for download at: https://bioturing.com/bbrowser/download.

bioinformatics↗

bioRxiv2020

SLAMF7 engagement super-activates macrophages in acute and chronic inflammation

Macrophages regulate protective immune responses to infectious microbes, but aberrant macrophage activation frequently drives pathological inflammation. To identify regulators of vigorous macrophage activation, we analyzed RNA-seq data from synovial macrophages and identified SLAMF7 as a receptor associated with a super-activated macrophage state in rheumatoid arthritis. We implicated IFN-{gamma} as a key regulator of SLAMF7 expression. Engaging this receptor drove an exuberant wave of inflammatory cytokine expression, and induction of TNF- following SLAMF7 engagement amplified inflammation through an autocrine signaling loop. We observed SLAMF7-induced gene programs not only in macrophages from rheumatoid arthritis patients, but in gut macrophages from active Crohns disease patients and lung macrophages from severe COVID-19 patients. This suggests a central role for SLAMF7 in macrophage super-activation with broad implications in pathology.

immunology↗

bioRxiv2020

The soybean (Glycine max L.) cytokinin oxidase/dehydrogenase multigene family; identification of natural variations for altered cytokinin content and seed yield

Cytokinins (CKs) play a fundamental role in regulating dynamics of organ source/sink relationships during plant development, including flowering and seed formation stages. As a result, CKs are key drivers of seed yield. The cytokinin oxidase/dehydrogenase (CKX) is one of the critical enzymes responsible for regulating plant CK levels by causing their irreversible degradation. Variation of CKX gene activity is significantly correlated with seed yield in many crop species while in soybean (Glycine max L.), the possible associations between CKX gene family members (GFMs) and yield parameters have not yet been assessed. In this study, seventeen GmCKX GFMs were identified, and natural variations among GmCKX genes were probed among soybean cultivars with varying yield characteristics. The key CKX genes responsible for regulating CK content during seed filling stages of reproductive development were highlighted using comparative phylogenetics, gene expression analysis and CK metabolite profiling. Five of the seventeen identified GmCKX GFMs, showed natural variations in the form of single nucleotide polymorphisms (SNPs). The gene GmCKX14, with high expression during critical seed filling stages, was found to have a non-synonymous mutation (H105Q), on one of the active site residues, Histidine 105, previously reported to be essential for co-factor binding to maintain structural integrity of the enzyme. Soybean lines with this mutation had higher CK content and desired yield characteristics. The potential for marker-assisted selection based on the identified natural variation within GmCKX14, is discussed in the context of hormonal control that can result in higher soybean yield. Key MessageNatural variations in soybean cytokinin oxidase/dehydrogenase gene, GmCKX14, with high expression during seed development, were linked to increased sink strength via altered cytokinin profiles in high yielding cultivars.

plant biology↗

bioRxiv2020

Subtype Heterogeneity and Epigenetic Convergence in Neuroendocrine Prostate Cancer

Neuroendocrine carcinomas (NEC) are tumors expressing markers of neuronal differentiation that can arise at different anatomic sites but have strong histological and clinical similarities. Here we report the chromatin landscapes of a range of human NECs and show convergence to the activation of a common epigenetic program. With a particular focus on treatment emergent neuroendocrine prostate cancer (NEPC), we analyzed cell lines, patient-derived xenograft (PDX) models and human clinical samples to show the existence of two distinct NEPC subtypes based on the expression of the neuronal transcription factors ASCL1 and NEUROD1. While in cell lines and PDX models these subtypes are mutually exclusive, single cell analysis of human clinical samples exhibit a more complex tumor structure with subtypes coexisting as separate sub-populations within the same tumor. These tumor sub-populations differ genetically and epigenetically contributing to intra- and inter-tumoral heterogeneity in human metastases. Overall our results provide a deeper understanding of the shared clinicopathological characteristics shown by NECs. Furthermore, the intratumoral heterogeneity of human NEPCs suggests the requirement of simultaneous targeting of coexisting tumor populations as a therapeutic strategy.

cancer biology↗

bioRxiv2020

Quantification of cell behaviours and computational modelling show that cell directional behaviours drive zebrafish pectoral fin morphogenesis

MotivationUnderstanding the mechanisms by which the zebrafish pectoral fin develops is expected to produce insights on how vertebrate limbs grow from a 2D cell layer to a 3D structure. Two mechanisms have been proposed to drive limb morphogenesis in tetrapods: a growth-based morphogenesis with a higher proliferation rate at the distal tip of the limb bud than at the proximal side, and directed cell behaviors that include elongation, division and migration in a nonrandom manner. Based on quantitative experimental biological data at the level of individual cells in the whole developing organ, we test the conditions for the dynamics of pectoral fin early morphogenesis. ResultsWe found that during the development of the zebrafish pectoral fin, cells have a preferential elongation axis that gradually aligns along the proximodistal axis (PD) of the organ. Based on these quantitative observations, we build a center-based cell model enhanced with a polarity term and cell proliferation to simulate fin growth. Our simulations resulted in 3D fins similar in shape to the observed ones, suggesting that the existence of a preferential axis of cell polarization is essential to drive fin morphogenesis in zebrafish, as observed in the development of limbs in the mouse, but distal tip-based expansion is not. AvailabilityUpon publication, biological data will be available at http://bioemergences.eu/modelingFin, and code source at https://github.com/guijoe/MaSoFin. Contactjoel.dokmegang-kassap@stu.mmu.ac.uk, nadine.peyrieras@cnrs.fr or rene.doursat@iscpif.fr Supplementary informationSupplementary data are included in this manuscript.

systems biology↗

bioRxiv2020

Delineation of a molecularly distinct terminally differentiated memory CD8 T cell population

Memory CD8 T cells provide durable protection against diverse intracellular pathogens and can be broadly segregated into distinct circulating and tissue-resident populations. Paradigmatic studies have demonstrated circulating memory cells can be further divided into effector memory (TO_SCPLOWEMC_SCPLOW) and central memory (TO_SCPLOWCMC_SCPLOW) populations based on discrete functional characteristics. Following resolution of infection, we identified a persisting antigen-specific CD8 T cell population that was simultaneously terminally-fated with potent effector function but maintained memory T cell qualities and conferred robust protection against reinfection. Notably, this terminally-differentiated effector memory CD8 T cell population (terminal-TO_SCPLOWEMC_SCPLOW) was conflated within the conventional TO_SCPLOWEMC_SCPLOW population, prompting redefinition of the classical characteristics of TO_SCPLOWEMC_SCPLOW cells. Murine terminal-TO_SCPLOWEMC_SCPLOW were transcriptionally, functionally, and developmentally unique compared to TO_SCPLOWEMC_SCPLOW cells. Through mass cytometry and single-cell RNAseq analyses of human peripheral blood from healthy individuals, we also identified an analogous terminal-TO_SCPLOWEMC_SCPLOW population of CD8 T cells that was transcriptionally distinct from TO_SCPLOWEMC_SCPLOW and TO_SCPLOWCMC_SCPLOW. A key finding of this study was that parsing of terminal-TO_SCPLOWEMC_SCPLOW from conventionally defined TO_SCPLOWEMC_SCPLOW challenges classical characteristics of TO_SCPLOWEMC_SCPLOW biology, including enhanced presence in lymphoid tissues, robust IL-2 production and recall potential, greater than expected homeostatic fitness, refined transcription factor dependencies, and a distinct molecular phenotype. Classification of terminal-TO_SCPLOWEMC_SCPLOW and clarification of TO_SCPLOWEMC_SCPLOW biology hold broad implications for understanding the molecular regulation of memory cell states and harnessing immunological memory to improve immunotherapies.

immunology↗

bioRxiv2020

Phase separation enables heterochromatin domains to do mechanical work

The condensation of proteins and nucleic acids underlies the formation of membraneless organelles, which have emerged as major drivers of cellular organization. It remains largely unexplored, however, whether these condensates can impart mechanical function(s) to the cell. The heterochromatin protein HP1- (Swi6 in S. pombe) crosslinks histone H3K9 methylated nucleosomes and has been proposed to undergo condensation to drive the liquid-like clustering of heterochromatin domains. Here we leverage the genetically tractable S. pombe model and a separation-of-function Swi6 allele to elucidate a mechanical function imparted by its condensation. Using a combination of single-molecule imaging, force spectroscopy on individual nuclei, and high-resolution live-cell imaging, we show that Swi6 is critical for nuclear resistance to external force. Strikingly, it is this condensed yet dynamic pool of Swi6, rather than the chromatin-bound molecules, that is essential to imparting mechanical stiffness. Our findings suggest that Swi6 condensates embedded in the chromatin meshwork establish the emergent mechanical behavior of the nucleus as a whole, revealing that biomolecular condensation can influence organelle and cell mechanics.

cell biology↗

bioRxiv2020

Development of mPing-based Activation Tags for Crop Insertional Mutagenesis

Modern plant breeding increasingly relies on genomic information to guide crop improvement. Although some genes are characterized, additional tools are needed to effectively identify and characterize genes associated with crop traits. To address this need, the mPing element from rice was modified to serve as an activation tag to induce expression of nearby genes. Embedding promoter sequences in mPing resulted in a decrease in overall transposition rate; however, this effect was negated by using a hyperactive version of mPing called mmPing20. Transgenic soybean events carrying mPing-based activation tags and the appropriate transposase expression cassettes showed evidence of transposition. Expression analysis of a line that contained a heritable insertion of the mmPing20F activation tag indicated that the activation tag induced overexpression of the nearby soybean genes. This represents a significant advance in gene discovery technology as activation tags have the potential to induce more phenotypes than the original mPing element, improving the overall effectiveness of the mutagenesis system.

plant biology↗