Biology subjects

Nguyen, D.

Publications and source records attributed to Nguyen, D..

At least 91 records · Page 5Linked to original sources

bioRxiv2020

Sex-specific effects of in vitro fertilization on adult metabolic phenotypes and hepatic transcriptomic and proteomic pathways in mouse.

Although in vitro fertilization (IVF) is associated with adverse perinatal outcomes, an increasing concern is the long-term health implications. We augmented our IVF mouse model to longitudinally investigate cardiometabolic outcomes in offspring from optimal neonatal litter sizes. We found that IVF-conceived females had higher body weight and cholesterol levels compared to naturally-conceived females, whereas IVF-conceived males had higher levels of triglycerides and insulin, and increased body fat composition. Through transcriptomics and proteomics of adult liver, we identified sexually-dimorphic dysregulation of the sterol regulatory element binding protein (SREBP) pathways that are associated with the sex-specfic phenotypes. We also found that global loss of DNA methylation in placenta was linked to higher cholesterol levels in IVF-conceived females. Our findings indicate that IVF procedures have long-lasting sex-specific effects on metabolic health of offspring and lay the foundation to utilize the placenta as a predictor of long-term outcomes.

developmental biology↗

bioRxiv2020

The CD28-transmembrane domain mediates chimeric antigen receptor heterodimerization with CD28

Anti-CD19 chimeric antigen receptor (CD19-CAR)-engineered T cells are approved therapeutics for malignancies. The impact of the hinge (HD) and transmembrane (TMD) domains between the extracellular antigen-targeting and the intracellular signaling modalities of CARs has not been systemically studied. Here, a series of CD19-CARs differing only by their HD (CD8/CD28/IgG4) and TMD (CD8/CD28) was generated. CARs containing a CD28-TMD, but not a CD8-TMD, formed heterodimers with the endogenous CD28 in human T cells, as shown by co-immunoprecipitation and CAR-dependent proliferation to anti-CD28 stimulation. This dimerization depended on polar amino-acids in the CD28-TMD. CD28-CAR heterodimerization was more efficient in CARs containing a CD8-HD or CD28-HD as compared to an IgG4-HD. CD28-CAR heterodimers did not respond to CD80 and CD86 stimulation but led to a significant reduction of CD28 cell-surface expression. These data unveil a new property of the CD28-TMD and suggest that TMDs can modulate CAR T-cell activities by engaging endogenous partners. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/296913v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@bcd17aorg.highwire.dtl.DTLVardef@5cbca9org.highwire.dtl.DTLVardef@660eecorg.highwire.dtl.DTLVardef@5db163_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2020

The importance of charge in perturbing the aromatic glue stabilizing the protein-protein interface of homodimeric tRNA-guanine transglycosylase

Bacterial tRNA-guanine transglycosylase (Tgt) is involved in the biosynthesis of the modified tRNA nucleoside queuosine present in the anticodon wobble position of tRNAs specific for aspartate, asparagine, histidine and tyrosine. Inactivation of the tgt gene leads to decreased pathogenicity of Shigella bacteria. Therefore, Tgt constitutes a putative target for Shigellosis drug therapy. Since only active as homodimer, interference with dimer-interface formation may, in addition to active-site inhibition, provide further means to disable this protein. A cluster of four aromatic residues seems important to stabilize the homodimer. We mutated residues of this aromatic cluster and analyzed each exchange with respect to dimer and thermal stability or enzyme activity applying native mass spectrometry, thermal shift assay, enzyme kinetics, and X-ray crystallography. Our structural studies indicate strong influence of pH on homodimer stability. Obviously, protonation of a histidine within the aromatic cluster promotes the collapse of an essential structural motif within the dimer interface at slightly acidic pH. TOC GraphicFor table of contents use only. O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

molecular biology↗

bioRxiv2020

Learning distance-dependent motif interactions: an interpretable CNN model of genomic events

In many biological studies, prediction is used primarily to validate the model; the real quest is to understand the underlying phenomenon. Therefore, interpretable deep models for biological studies are required. Here, we propose the Hyper-parameter eXplainable Motif Pair framework (HyperXPair) to model biological motifs and their distance-dependent context through explicitly interpretable parameters. This makes HyperXPair more than a decision-support tool; it is also a hypothesis-generating tool designed to advance knowledge in the field. We demonstrate the utility of our model by learning distance-dependent motif interactions for two biological problems: transcription initiation and RNA splicing.

bioinformatics↗

bioRxiv2020

A Multi-Phenotype System to Discover Therapies for Age-Related Dysregulation of the Immune Response to Viral Infections

Age-related immune dysregulation contributes to increased susceptibility to infection and disease in older adults. We combined high-throughput laboratory automation with machine learning to build a multi-phenotype aging profile that models the dysfunctional immune response to viral infection in older adults. From a single well, our multi-phenotype aging profile can capture changes in cell composition, physical cell-to-cell interaction, organelle structure, cytokines, and other hidden complexities contributing to age-related dysfunction. This system allows for rapid identification of new potential compounds to rejuvenate older adults immune response. We used our technology to screen thousands of compounds for their ability to make old immune cells respond to viral infection like young immune cells. We observed beneficial effects of multiple compounds, of which two of the most promising were disulfiram and triptonide. Our findings indicate that disulfiram could be considered as a treatment for severe coronavirus disease 2019 and other inflammatory infections.

immunology↗

bioRxiv2020

Identification and Characterization of a B-Raf Kinase Alpha Helix Critical for the Activity of MEK Kinase in MAPK Signaling

In the MAPK pathway, an oncogenic V600E mutation in B-Raf kinase causes the enzyme to be constitutively active, leading to aberrantly high phosphorylation levels of its downstream effectors, MEK and ERK kinases. The V600E mutation in B-Raf accounts for more than half of all melanomas and [~]3% of all cancers and many drugs target the ATP-binding site of the enzyme for its inhibition. Since B-Raf can develop resistance against these drugs and such drugs can induce paradoxical activation, drugs that target allosteric sites are needed. To identify other potential drug targets, we generated and kinetically characterized an active form of B-RafV600E expressed using a bacterial expression system. In doing so, we identified an alpha helix on B-Raf, found at the B-Raf-MEK interface, that is critical for their interaction and the oncogenic activity of B-RafV600E. We performed binding experiments between B-Raf mutants and MEK using pull downs and biolayer interferometry, and assessed phosphorylation levels of MEK in vitro and in cells as well as its downstream target ERK to show that mutating certain residues on this alpha helix is detrimental to binding and downstream activity. Our results suggest that this B-Raf alpha helix binding site on MEK could be a site to target for drug development to treat B-RafV600E-induced melanomas.

biochemistry↗

bioRxiv2020

Novel PE and APC Tandems: Additional Bright Near-Infrared Tandem Fluorochromes for Use in Spectral Flow Cytometry

Recent advances in flow cytometry instrumentation and fluorochrome chemistries have greatly increased fluorescent conjugated antibody combinations that can be used reliably and easily in routine experiments. The Cytek Aurora flow cytometer has three excitation lasers (405nm, 488nm, and 640nm) and incorporates the latest Avalanche Photodiode (APD) technology, demonstrating significant improvement in sensitivity for fluorescent emission signals longer than 800nm. However, there are limited commercially available fluorochromes capable of excitation with peak emission signals beyond 800nm. To address this gap, we engineered 6 new fluorochromes: PE-750, PE-800, PE-830 for the 488nm laser and APC-750, APC-800, APC-830 for the 640nm laser. Utilizing the principal of fluorescence resonance energy transfer (FRET), these novel structures were created by covalently linking a protein donor dye with an organic small molecule acceptor dye. Additionally, each of these fluorochrome conjugates were shown to be compatible with Fixation/Permeabilization buffer reagents, and demonstrated acceptable brightness and stability when conjugated to antigen-specific monoclonal antibodies. These six novel fluorochrome reagents can increase the numbers of fluorochromes that can be used on a 3-laser spectral flow cytometer.

immunology↗

bioRxiv2020

Evaluation of Different PCR Assay Formats for Sensitive and Specific Detection of SARS-CoV-2 RNA

Accurate identification of individuals infected with SARS-CoV-2 is crucial for efforts to control the ongoing COVID-19 pandemic. Polymerase chain reaction (PCR)-based assays are the gold standard for detecting viral RNA in patient samples and are used extensively in clinical settings. Most currently used quantitative PCR (RT-qPCRs) rely upon real-time detection of PCR product using specialized laboratory equipment. To enable the application of PCR in resource-poor or non-specialist laboratories, we have developed and evaluated a nested PCR method for SARS-CoV-2 RNA using simple agarose gel electrophoresis for product detection. Using clinical samples tested by conventional qPCR methods and RNA transcripts of defined RNA copy number, the nested PCR based on the RdRP gene demonstrated high sensitivity and specificity for SARS-CoV-2 RNA detection in clinical samples, but showed variable and transcript length-dependent sensitivity for RNA transcripts. Samples and transcripts were further evaluated in an additional N protein real-time quantitative PCR assay. As determined by 50% endpoint detection, the sensitivities of three RT-qPCRs and nested PCR methods varied substantially depending on the transcript target with no method approaching single copy detection. Overall, these findings highlight the need for assay validation and optimization and demonstrate the inability to precisely compare viral quantification from different PCR methodologies without calibration.

microbiology↗

bioRxiv2020

Association of zinc-finger antiviral protein (ZAP) binding to viral genomic RNA with attenuation of replication of echovirus 7

Previous studies have implicated both zinc finger antiviral protein (ZAP) and oligoadenylate synthetase 3 (OAS3)/RNASeL in the attenuation of RNA viruses with elevated CpG and UpA dinucleotides. Mechanisms and inter-relationships between these two pathways were investigated using an echovirus 7 (E7) replicon with compositionally modified sequences inserted into the 3untranslated region. ZAP and OAS3 immunoprecipitation (IP) assays provided complementary data on dinucleotide composition effects on binding. Elevated frequencies of alternative pyrimidine/purine (CpA and UpG) and reversed (GpC and ApU) dinucleotides showed no attenuating effect nor specific binding to ZAP by IP. However, the bases 3 and 5 to CpG motifs influenced replication and ZAP binding; UCGU enhanced CpG-mediated attenuation and ZAP-binding while A residues shielded CpGs from ZAP recognition. Attenuating effects of elevated frequencies of UpA on replication occurred independently of CpG dinucleotides and bound non-competitively with CpG-enriched RNA consistent with a separate recognition site from CpG. Remarkably, immunoprecipitation with OAS3 antibody reproduced the specific binding to CpG- and UpA-enriched RNA sequences. However, OAS3 and ZAP were coimmunoprecipitated in both ZAP and OAS3 IP, and colocalised with E7 and stress granules (SGs) by confocal microscopy analysis of infected cells. ZAPs association with larger cellular complexes may mediate the recruitment of OAS3/RNAseL, KHNYN and other RNA degradation pathways. ImportanceWe have recently discovered that the OAS3/RNAseL antiviral pathway is essential for restriction of CpG- and UpA-enriched viruses, in addition to the requirement for zinc finger antiviral protein (ZAP). The current study provides evidence for the specific dinucleotide and wider recognition contexts associated with virus recognition and attenuation. It further documents the association of ZAP and OAS3 and association with stress granules and a wider protein interactome that may mediate antiviral effects in different cellular compartments. The study provides a striking re-conceptualisation of the pathways associated with this aspect of antiviral defence.

microbiology↗

bioRxiv2020

Identification of Candidate COVID-19 Therapeutics using hPSC-derived Lung Organoids

Summary ParagraphThe SARS-CoV-2 virus has caused already over 3.5 million COVID-19 cases and 250,000 deaths globally. There is an urgent need to create novel models to study SARS-CoV-2 using human disease-relevant cells to understand key features of virus biology and facilitate drug screening. As primary SARS-CoV-2 infection is respiratory-based, we developed a lung organoid model using human pluripotent stem cells (hPSCs) that could be adapted for drug screens. The lung organoids, particularly aveolar type II cells, express ACE2 and are permissive to SARS-CoV-2 infection. Transcriptomic analysis following SARS-CoV-2 infection revealed a robust induction of chemokines and cytokines with little type I/III interferon signaling, similar to that observed amongst human COVID-19 pulmonary infections. We performed a high throughput screen using hPSC-derived lung organoids and identified FDA-approved drug candidates, including imatinib and mycophenolic acid, as inhibitors of SARS-CoV-2 entry. Pre- or post-treatment with these drugs at physiologically relevant levels decreased SARS-CoV-2 infection of hPSC-derived lung organoids. Together, these data demonstrate that hPSC-derived lung cells infected by SARS-CoV-2 can model human COVID-19 disease and provide a valuable resource to screen for FDA-approved drugs that might be repurposed and should be considered for COVID-19 clinical trials.

microbiology↗

bioRxiv2020

Mechano-bactericidal nanopillars require external forces to effectively kill bacteria

Nanopillars are known to mechanically damage bacteria, suggesting a promising strategy for highly-effective anti-bacterial surfaces. However, the mechanisms underlying this phenomena remain unclear, which ultimately limits translational potential towards real-world applications. Using real-time and end-point analysis techniques, we demonstrate that in contrast to expectations, bacteria on multiple "mechano-bactericidal" surfaces remain viable, unless exposed to a moving air-liquid interface which caused considerable cell death. Reasoning that normal forces arising from surface tension may underlie mechano-bactericidal activity, we developed computational and experimental models to estimate, manipulate, and recreate the impact of these forces. Our experiments together demonstrate that nanopillar surfaces alone do not cause cell death, but require a critical level of external force to deform and rupture bacteria. These studies hence provide fundamental physical insight into the mechanisms by which nanopillar surfaces can serve as effective antibacterial strategies, and describe the use-conditions under which such nanotechnological approaches may provide practical value. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/012153v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@1cfded2org.highwire.dtl.DTLVardef@2dc47dorg.highwire.dtl.DTLVardef@bf002dorg.highwire.dtl.DTLVardef@1a49c2d_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

microbiology↗

bioRxiv2020

Potentially highly potent drugs for 2019-nCoV

The World Health Organization (WHO) has declared the 2019 novel coronavirus (2019-nCoV) infection outbreak a global health emergency. Currently, there is no effective anti-2019-nCoV medication. The sequence identity of the 3CL proteases of 2019-nCoV and SARS is 96%, which provides a sound foundation for structural-based drug repositioning (SBDR). Based on a SARS 3CL protease X-ray crystal structure, we construct a 3D homology structure of 2019-nCoV 3CL protease. Based on this structure and existing experimental datasets for SARS 3CL protease inhibitors, we develop an SBDR model based on machine learning and mathematics to screen 1465 drugs in the DrugBank that have been approved by the U.S. Food and Drug Administration (FDA). We found that many FDA approved drugs are potentially highly potent to 2019-nCoV.

bioinformatics↗

bioRxiv2020

Deep Learning Identifies Cardiomyocyte Nuclei in Murine Tissue with High Precision

Proper identification and annotation of cells in mammalian tissues is of paramount importance to biological research. Various approaches are currently used to identify and label cell types of interest in complex tissues. In this report, we generated an artificial intelligence (AI) deep learning model that uses image segmentation to predict cardiomyocyte nuclei in mouse heart sections without a specific cardiomyocyte nuclear label. This tool can annotate cardiomyocytes highly sensitively and specifically (AUC 0.94) using only cardiomyocyte structural protein immunostaining and a global nuclear stain. We speculate that our method is generalizable to other tissues to annotate specific cell types and organelles in a label-free way.

cell biology↗

bioRxiv2019

Proteomics identifies signal peptide features determining the substrate specificity in human Sec62/Sec63-dependent ER protein import

In mammalian cells one-third of all polypeptides are integrated into the membrane or translocated into the lumen of the endoplasmic reticulum (ER) via the Sec61-channel. While the Sec61-complex facilitates ER-import of most precursor polypeptides, the Sec61-associated Sec62/Sec63-complex supports ER-import in a substrate-specific manner. So far, mainly posttranslationally imported precursors and the two cotranslationally imported precursors of ERj3 and prion protein were found to depend on the Sec62/Sec63-complex in vitro. Therefore, we determined the rules for engagement of Sec62/Sec63 in ER-import in intact human cells using a recently established unbiased proteomics approach. In addition to confirming ERj3, we identified twenty-two novel Sec62/Sec63-substrates under these in vivo-like conditions. As a common feature, those previously unknown substrates share signal peptides with comparatively longer but less hydrophobic H-region and lower C-region polarity. Further analyses with four substrates, and ERj3 in particular, revealed the combination of a slowly-gating signal peptide and a downstream translocation-disruptive positively charged cluster of amino acid residues as decisive for the Sec62-/Sec63-requirement. In the case of ERj3, these features were found to be responsible for an additional BiP-requirement and to correlate with sensitivity towards the Sec61-channel inhibitor CAM741. Thus, the human Sec62/Sec63-complex may support Sec61-channel opening for precursor polypeptides with slowly-gating signal peptides by direct interaction with the cytosolic amino-terminal peptide of Sec61 or via recruitment of BiP and its interaction with the ER-lumenal loop 7 of Sec61. These novel insights into the mechanism of human ER protein import contribute to our understanding of the etiology of SEC63-linked Polycystic Liver Disease. DatabasesThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (http://www.ebi.ac.uk/pride/archive/projects/Identifiers) with the dataset identifiers: PXD008178, PXD011993, and PXD012078. Supplementary information was deposited at Mendeley Data under the DOI:10.17632/6s5hn73jcv.1 (http://dx.doi.or/10.17632/6s5hn73jcv.1).

biochemistry↗

bioRxiv2019

Loss of CHCHD2 and CHCHD10 activates OMA1 peptidase to disrupt mitochondrial cristae phenocopying patient mutations in vivo

Dominant mutations in the mitochondrial paralogs CHCHD2 (C2) and CHCHD10 (C10) were recently identified as causing Parkinsons disease and ALS/FTD/myopathy, respectively. Disruption of mitochondrial cristae has been observed in mutant C10 patient tissues and animal models, but the mechanism for this disruption remains controversial. Additionally, C10 patient mutant knock-in (KI) mice were recently reported to activate a mitochondrial integrated stress response (mt-ISR) and develop cardiomyopathy not seen in C10 knockout (KO) mice, calling into question whether mutant C10 pathogenesis is related to C2/C10 normal function or purely toxic gain of function. Here, using the first C2/10 double knockout (DKO) mice, we report that C10 pathogenesis and the normal function of C2/10 are intimately linked. Similar to patients with C10 mutations, we found that C2/10 DKO mice (but not either single KO mice) have disrupted mitochondrial cristae, due to cleavage of the mitochondrial shaping protein L-OPA1 by the stress-induced peptidase OMA1. OMA1 was found to be activated similarly in affected tissues of mutant C10 KI mice, demonstrating that L-OPA1 cleavage is a novel mechanism for cristae abnormalities due to both C10 mutation and C2/C10 loss, and that OMA1, a driver of neurodegeneration in other contexts, may be a therapeutic target. Finally, C2/10 DKO mice partially phenocopied mutant C10 KI mice with the development of cardiomyopathy and activation of the mt-ISR in affected tissues, tying mutant C10 pathogenesis to C2/C10 function.

cell biology↗

bioRxiv2019

Phytochromes measure photoperiod in Brachypodium

Daylength is a key seasonal cue for animals and plants. In cereals, photoperiodic responses are a major adaptive trait, and alleles of clock genes such as PHOTOPERIOD DEPENDENT1 (PPD1) and EARLY FLOWERING3 (ELF3) have been selected for in breeding barley and wheat for more northern latitudes (Faure et al., 2012; Turner, Beales, Faure, Dunford, & Laurie, 2005). How monocot plants sense photoperiod and integrate this information into growth and development is not well understood. We show that in Brachypodium distachyon, phytochrome C (phyC) acts as a molecular timer, directly communicating information to the circadian clock protein ELF3. In this way, ELF3 levels integrate night length information. ELF3 is a central regulator of photoperiodism in Brachypodium, and elf3 mutants display a constitutive long day transcriptome. Conversely, conditions that result in higher levels of ELF3 suppress long day responses. We are able to show that these effects are direct, as ELF3 and phyC occur in a common complex, and they associate with the promoters of a number of conserved regulators of photoperiodism, including PPD1. Consistent with observations in barley, we are able to show that PPD1 overexpression accelerates flowering in SD and is necessary for rapid flowering in response to LD. These findings provide a conceptual framework for understanding observations in the photoperiodic responses of key crops, including wheat, barley and rice.

plant biology↗

bioRxiv2019

Myf6/MRF4 is a Myogenic Niche Regulator Required for the Maintenance of the Muscle Stem Cell Pool

In metazoans, skeletal muscle evolved to contract and produce force. However, recent experimental evidence suggests that skeletal muscle has also acquired endocrine functions and produces a vast array of myokines. Using ChIP-Seq and gene expression analyses of myogenic factors, we show that Myf6/MRF4 transcriptionally regulates a broad spectrum of myokines and muscle-secreted proteins, including ligands for downstream activation of key signaling pathways such as EGFR, STAT3 and VEGFR. Homozygous deletion of Myf6 causes a significant reduction in the ability of muscle to produce key myokines such as EGF, VEGFA and LIF. Consequently, although Myf6 knockout mice are born with a normal muscle stem cell compartment, they undergo progressive reduction in their stem cell pool during postnatal life. Mechanistically, muscle stem cells from the Myf6 knockout animals show defects in activation of EGFR and STAT3 signaling, upregulate the p38 MAP kinase pathway and spontaneously break from quiescence. Exogenous application of recombinant EGF and LIF rescue the defects in the muscle stem cell pool of Myf6 knockout animals. Finally, skeletal muscles of mice lacking Myf6 have a significantly reduced ability to sustain donor-engrafted muscle stem cells. Taken together, our data uncovers a novel role for Myf6 in regulating the expression of niche factors and myokines to maintain the skeletal muscle stem cell pool in adult mice.

genetics↗

bioRxiv2019

Host-Microbial Interactions in Systemic Lupus Erythematosus and Periodontitis

Systemic lupus erythematosus (SLE) is a potentially fatal complex autoimmune disease, that is characterized by widespread inflammation manifesting tissue damage and comorbidities across the human body including heart, blood vessels, joints, skin, liver, kidneys, and periodontal tissues. The etiology of SLE is partially attributed to a deregulated inflammatory response to microbial dysbiosis and environmental changes. In the mouth, periodontal environment provides an optimal niche to assay local dynamic microbial ecological changes in health and disease important to systemic inflammation in SLE subjects. Our aim was to evaluate the reciprocal impact of periodontal subgingival microbiota on SLE systemic inflammation. Ninety-one female subjects were recruited, including healthy (n=31), SLE-inactive (n=29), and SLE-active (n=31). Patients were screened for probing depth (PD), bleeding on probing (BOP), clinical attachment level (CAL), and classified with or without periodontal dysbiosis, periodontitis. Serum inflammatory cytokines were measured by human cytokine panel and subgingival biofilm was examined by DNA-DNA checkerboard. The results showed significant upregulation of proinflammatory cytokines in individuals with SLE when compared to controls. Stratification of subjects into SLE-inactive (I) and SLE-active (A) phenotypes or periodontitis and non-periodontitis groups provided new insights into SLE pathophysiology. While low-grade inflammation was found in SLE-I subjects, a potent anti-inflammatory cytokine, IL-10 was found to control clinical phenotypes. Out of twenty-four significant differential oral microbial abundances found in SLE, fourteen unique subgingival bacteria profiles were found to be elevated in SLE. Pathogens from periodontal disease sites (Treponema denticola and Tannerella forsythia) showed increase abundance in SLE-A subjects when compared to controls. Cytokine-microbial correlations showed that periodontal pathogens dominating the environment increased proinflammatory cytokines systemically. Deeper clinical attachment loss and periodontal pathogens were found in SLE subjects, especially on SLE-I, likely due to long-term chronic and low-grade inflammation. Altogether, local periodontal pathogen enrichment was positively associated with high systemic inflammatory profiles, relevant to the overall health and SLE disease pathogenesis.

immunology↗