Biology subjects

Cao, X.

Publications and source records attributed to Cao, X..

At least 199 records · Page 11Linked to original sources

bioRxiv2020

IF1 connects obesity and insulin resistance through mitochondrial reprogramming in association with ANT2

IF1 (ATPIF1) is a nuclear DNA-encoded protein with an activity in the inhibition of catalytic activity of F1Fo-ATP synthase (ATPase), an enzyme for ATP synthesis in mitochondria. A role of IF1 remains unknown in the metabolic disorder in obesity. In this study, IF1 was examined in the diet-induced obese (DIO) mice and a decrease in IF1 protein was observed in several tissues including the skeletal muscle, liver and intestine in the absence of mRNA alteration. Significance of the reduction was investigated in the IF1-KO mice, in which insulin sensitivity was improved in the absence of body weight alteration on Chow diet. On a high fat diet (HFD), the IF1-KO mice gain more body weight as a result of enhanced fat tissue growth. The energy expenditure and locomotion activity were decreased in the KO mice without an alteration in food intake. The increase in insulin sensitivity remained in the obese KO mice. The colon tissue exhibited a resistance to the HFD-induced atrophy with less cell apoptosis and more secretion of GLP-1. Mitochondria exhibited an enhanced ATP production and maximal oxygen consumption without an alteration in the respiratory chain proteins. However, the ATP level was reduced in the fasting condition in the muscle as well as the liver. Mitophagy was enhanced with elevated accumulation of PINK1 and Parkin proteins in the mitochondria. The protein abundance of ADP/ATP translocase 2 (ANT2) was decreased in the inner membrane of mitochondria to account for the reduced apoptosis and enhanced mitophagy. The data suggest that the IF1 reduction in obesity leads to reprogramming of mitochondrial metabolism in a compensatory response to maintain the insulin sensitivity through down-regulation of ANT2 protein.

physiology↗

bioRxiv2020

ATPIF1 inactivation promotes antitumor immunity through metabolic reprogramming of CD8+ T cells

Induction of CD8+ T cell activity is a promising strategy in the cancer immunotherapy. In this study, we identified ATP synthase inhibitory factor 1 (ATPIF1) as a potential target in the induction of CD8+ T cell immunity against tumor. Inactivation of ATPIF1 gene in mice promoted the antitumor activity of CD8+ T cells leading to suppression of tumor growth of B16 melanoma and Lewis lung cancer. The phenotype was abolished by deletion of CD8+ T cells in the ATPIF1-KO mice. The tumor infiltrating CD8+ T cells exhibited strong activities in the proliferation, effector and memory as revealed by the single cell RNA sequencing results of CD45+ tumor infiltrating lymphocytes (TILs) isolated from the tumors. The CD8+ T cells expressed more antitumor makers in the tumor microenvironment and in coculture with the tumor cells. The cells had a higher level of glycolysis after the T cell receptor-mediated activation as revealed by the targeted metabolomics assay. The cells exhibited an extra activity of oxidative phosphorylation before the activation as indicated by the oxygen consumption rate. The cells gained capacities in the proliferation, apoptosis resistance and mitophagy in the glucose-limiting environment. These data suggest that inhibition of ATPIF1 activity by gene inactivation rewired the energy metabolism of CD8+ T cells to enhance their immune activities to the tumors. ATPIF1 is a potential molecular target in the induction of antitumor immunity through metabolic reprogramming of CD8+ T cells for the cancer immunotherapy.

immunology↗

bioRxiv2020

Actuation Enhances Patterning in Human Neural Tube Organoids

Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.

bioengineering↗

bioRxiv2020

RNA LEVER Mediates Long-Range Regulation of ε-globin by Keeping PRC2 in Check

Polycomb Repressive Complex 2 (PRC2) is an epigenetic regulator required for gene silencing during embryonic development. Previous studies have reported that PRC2 interacts with RNA in a promiscuous manner, but the biological functions of such interaction are unknown. Here we present a seesaw mechanism for the regulation of {varepsilon}-globin through inactivating EZH2 by an upstream non-coding RNA (LEVER). We show that LEVER, a non-coding RNA identified by RNA immunoprecipitation sequencing (RIP-seq) of the PRC2 core subunit EZH2 and Nanopore sequencing, binds PRC2 and thereby prevents the accumulation of H3K27 methylation along the genomic region where LEVER RNA is transcribed. The open chromatin within the LEVER locus in turn competes for the chromatin interaction between the {varepsilon}-globin promoter and the Locus Control Region (LCR), working as a negative regulatory element of {varepsilon}-globin expression. Hence, LEVER RNA negatively regulates {varepsilon}-globin by sequestering PRC2 from repressing the LEVER locus, which is a competitor of the {varepsilon}-globin-LCR interaction.

molecular biology↗

bioRxiv2020

Up-Regulation of αCaMKII Impairs Cued Fear Extinction and NMDAR-Dependent LTD in the Lateral Amygdala

Impaired fear extinction is one of the hallmark symptoms of post-traumatic stress disorder (PTSD). The roles of CaMKII have been not extensively studied in fear extinction and LTD. Here, we found PTSD susceptible mice exhibited significant up-regulation of CaMKII in the lateral amygdala (LA). Consistently, increasing CaMKII in LA profoundly not only caused PTSD-like symptoms such as impaired fear extinction and anxiety-like behaviors, but also attenuated NMDAR-dependent LTD at thalamo-LA synapses, reduced GluA1-Ser845/Ser831 dephosphorylation and AMPAR internalization. Suppressing the elevated CaMKII to normal level could completely reverse both PTSD-like symptoms and the impairments in LTD, GluA1-Ser845/Ser831 dephosphorylation and AMPAR internalization. Intriguingly, deficits in AMPAR internalization and GluA1-Ser845/Ser831 dephosphorylation were detected not only after impaired fear extinction, but also after attenuated LTD Our results demonstrate for the first time GluA1-Ser845/Ser831 dephosphorylation and AMPAR internalization are molecular links between LTD and fear extinction, and suggest CaMKII may be a potential molecular determinant of PTSD.

neuroscience↗

bioRxiv2020

Force-dependent stimulation of RNA unwinding by SARS-CoV-2 nsp13 helicase

The superfamily-1 helicase non-structural protein 13 (nsp13) is required for SARS-CoV-2 replication, making it an important antiviral therapeutic target. The mechanism and regulation of nsp13 has not been explored at the single-molecule level. Specifically, force-dependent unwinding experiments have yet to be performed for any coronavirus helicase. Here, using optical tweezers, we find that nsp13 unwinding frequency, processivity, and velocity increase substantially when a destabilizing force is applied to the dsRNA, suggesting a passive unwinding mechanism. These results, along with bulk assays, depict nsp13 as an intrinsically weak helicase that can be potently activated by picoNewton forces. Such force-dependent behavior contrasts the known behavior of other viral monomeric helicases, drawing stronger parallels to ring-shaped helicases. Our findings suggest that mechanoregulation, which may be provided by a directly bound RNA-dependent RNA polymerase, enables on-demand helicase activity on the relevant polynucleotide substrate during viral replication.

biophysics↗

bioRxiv2020

PLEKHA4 Promotes Wnt/β-catenin Signaling-Mediated G1/S Transition and Proliferation in Melanoma

Melanoma patients incur substantial mortality, despite promising recent advances in targeted therapies and immunotherapies. In particular, inhibitors targeting BRAF-mutant melanoma can lead to resistance, and no targeted therapies exist for NRAS-mutant melanoma, motivating the search for additional therapeutic targets and vulnerable pathways. Here, we identify a regulator of Wnt/{beta}-catenin signaling, PLEKHA4, as a factor required for melanoma proliferation and survival. PLEKHA4 knockdown in vitro leads to lower Dishevelled levels, attenuated Wnt/{beta}-catenin signaling, and a block of progression through the G1/S cell cycle transition. In mouse xenograft models, inducible PLEKHA4 knockdown attenuated tumor growth in BRAF- and NRAS-mutant melanomas and synergized with the clinically used inhibitor encorafenib in a BRAF-mutant model. As an E3 ubiquitin ligase regulator with both lipid and protein binding partners, PLEKHA4 presents several opportunities for targeting with small molecules. Our work identifies PLEKHA4 as a promising drug target for melanoma and clarifies a controversial role for Wnt/{beta}-catenin signaling in the control of melanoma proliferation.

cancer biology↗

bioRxiv2020

Polymorphic mobile element insertions contribute to gene expression and alternative splicing in human tissues

BackgroundMobile elements are a major source of human structural variants and some mobile elements can regulate gene expression and alternative splicing. However, the impact of polymorphic mobile element insertions (pMEIs) on gene expression and splicing in diverse human tissues has not been thoroughly studied. The multi-tissue gene expression and whole genome sequencing data generated by the Genotype-Tissue Expression (GTEx) project provide a great opportunity to systematic determine pMEIs role in gene expression regulation in human tissues. ResultsUsing the GTEx whole genome sequencing data, we identified 20,545 high-quality pMEIs from 639 individuals. We then identified pMEI-associated expression quantitative trait loci (eQTLs) and splicing quantitative trait loci (sQTLs) in 48 tissues by joint analysis of variants including pMEIs, single-nucleotide polymorphisms, and insertions/deletions. pMEIs were predicted to be the potential causal variant for 3,522 of the 30,147 significant eQTLs, and 3,717 of the 21,529 significant sQTLs. The pMEIs associated eQTLs and sQTLs show high level of tissue-specificity, and the pMEIs were enriched in the proximity of affected genes and in regulatory elements. Using reporter assays, we confirmed that several pMEIs associated with eQTLs and sQTLs can alter gene expression levels and isoform proportions. ConclusionOverall, our study shows that pMEIs are associated with thousands of gene expression and splicing variations in different tissues, and pMEIs could have a significant role in regulating tissue-specific gene expression/splicing. Detailed mechanisms for pMEIs role in gene regulation in different tissues will be an important direction for future human genomic studies.

genomics↗

bioRxiv2020

End to end differentiable protein structure refinement

Free energy is arguably the most important property of molecular systems. Despite great progress in both its efficient estimation by scoring functions/potentials and more rigorous computation based on extensive sampling, we remain far from accurately predicting and manipulating biomolecular structures and their interactions. There are fundamental limitations, including accuracy of interaction description and difficulty of sampling in high dimensional space, to be tackled. Computational graph underlies major artificial intelligence platforms and is proven to facilitate training, optimization and learning. Combining autodifferentiation, coordinates transformation and generalized solvation free energy theory, we construct a computational graph infrastructure to realize seamless integration of fully trainable local free energy landscape with end to end differentiable iterative free energy optimization. This new framework greatly improves efficiency by replacing local sampling with differentiation. Its specific implementation in protein structure refinement achieves superb efficiency and competitive accuracy when compared with state of the art all-atom mainstream methods.

bioinformatics↗

bioRxiv2020

Reprogramming the endogenous type III-A CRISPR-Cas system for genome editing, RNA interference and CRISPRi screening in Mycobacterium tuberculosis

Mycobacterium tuberculosis (M.tb) causes the current leading infectious disease. Examination of the functional genomics of M.tb and development of drugs and vaccines are hampered by the complicated and time-consuming genetic manipulation techniques for M.tb. Here, we reprogrammed M.tb endogenous type III-A CRISPR-Cas10 system for simple and efficient gene editing, RNA interference and screening via simple delivery of a plasmid harboring a mini-CRISPR array, thereby avoiding the introduction of exogenous proteins and minimizing proteotoxicity. We demonstrated that M.tb genes were efficiently and specifically knocked-in/out by this system, which was confirmed by whole-genome sequencing. This system was further employed for single and simultaneous multiple-gene RNA interference. Moreover, we successfully applied this system for genome-wide CRISPR interference screening to identify the in-vitro and intracellular growth-regulating genes. This system can be extensively used to explore the functional genomics of M.tb and facilitate the development of new anti-Mycobacterial drugs and vaccines. SummaryTuberculosis caused by Mycobacterium tuberculosis (M.tb) is the current leading infectious disease affecting more than ten million people annually. To dissect the functional genomics and understand its virulence, persistence, and antibiotics resistance, a powerful genome editing tool and high-throughput screening methods are desperately wanted. Our study developed an efficient and a robust tool for genome editing and RNA interference in M.tb using its endogenous CRISPR cas10 system. Moreover, the system has been successfully applied for genome-wide CRISPR interference screening. This tool could be employed to explore the functional genomics of M.tb and facilitate the development of anti-M.tb drugs and vaccines.

microbiology↗

bioRxiv2020

Transcriptome analysis of the Nematode Caenorhabditis elegans in acidic stress environments

Ocean acidification and acid rain, caused by modern industrial fossil fuels burning, lead to decrease of living environmental pH, which results in a series of negative effects on many organisms. However, the underlying mechanisms of animals’response to acidic pH stress are largely unknown. In this study, we used the nematode Caenorhabditis elegans as an animal model to explore the regulatory mechanisms of organisms’response to pH decline. Two major stress-responsive pathways were found through transcriptome analysis in acidic stress environments. Firstly, when the pH dropped from 6.33 to 4.33, the worms responded to the pH stress by up-regulation of the col, nas and dpy genes, which are required for cuticle synthesis and structure integrity. Secondly, when the pH continued to decrease from 4.33, the metabolism of xenobiotics by cytochrome P450 pathway genes (cyp, gst, ugt, and ABC transporters) played a major role in protecting the nematodes from the toxic substances probably produced by the more acidic environment. At the same time, cuticle synthesis slowed down might due to its insufficient protective ability. Moreover, the systematic regulation pattern we found in nematodes, might also be applied to other invertebrate and vertebrate animals to survive in the changing pH environments. Thus, our data might lay the foundation to identify the master gene(s) responding and adaptation to acidic pH stress in further studies, and might also provide new solutions to improve assessment and monitoring of ecological restoration outcomes, or generate novel genotypes via genome editing for restoring in challenging environments especially in the context of acidic stress through global climate change.View Full Text

zoology↗

bioRxiv2020

Establishment of a marine nematode model for animal functional genomics, environmental adaptation and developmental evolution

Nematodes play key roles in marine ecosystem. Although oceans cover 71% of the Earths surface, none of marine model nematode has been reported. Here, we constructed the first inbred line of free-living marine nematode Litoditis marina, sequenced and assembled its genome. Furthermore, we successfully applied CRISPR/Cas9 genome editing in L. marina. Comparative genomics revealed that immunity and oxygen regulation genes are expanded, which is probably central to its sediment adaptation. While L. marina exhibits massive gene contractions in NHRs, chemoreceptors, xenobiotics detoxification and core histones, which could explain the more defined marine environment. Our experiments showed that dozens of H4 genes in Caenorhabditis elegans might contribute to its adaptation to the complex terrestrial environments, while two H4 genes in L. marina are involved in salinity stress adaptation. Additionally, ninety-two conserved genes appear to be positively selected in L. marina, which may underpin its osmotic, neuronal and epigenetic changes in the sea. With short generation time, highly inbred lines, and genomic resources, our report brings L. marina a promising marine animal model, and a unique satellite marine model to the well-known biomedical model nematode C. elegans. This study will underpin ongoing work on animal functional genomics, environmental adaptation and developmental evolution.

genomics↗

bioRxiv2020

Discovery and Predictive Modeling of Urine Microbiome, Metabolite and Cytokine Biomarkers in Hospitalized Patients with Community Acquired Pneumonia

Pneumonia is the leading cause of infectious related death costing 12 billion dollars annually in the United States alone. Despite improvements in clinical care, total mortality remains around 4%, with inpatient mortality reaching 5-10%. For unknown reasons, mortality risk remains high even after hospital discharge and there is a need to identify those patients most at risk. Also of importance, clinical symptoms alone do not distinguish viral from bacterial infection which may delay appropriate treatment and may contribute to short-term and long-term mortality. Biomarkers have the potential to provide point of care diagnosis, identify high-risk patients, and increase our understanding of the biology of disease. However, there have been mixed results on the diagnostic performance of many of the analytes tested to date. Urine represents a largely untapped source for biomarker discovery and is highly accessible. To test this hypothesis, we collected urine from hospitalized patients with community-acquired pneumonia (CAP) and performed a comprehensive screen for urinary tract microbiota signatures, metabolite, and cytokine profiles. CAP patients were diagnosed with influenza or bacterial (S. aureus and S. pneumoniae) etiologies and compared with healthy volunteers. Microbiome signatures showed marked shifts in taxonomic levels in patients with bacterial etiology versus influenza and CAP versus normal. Predictive modeling of 291 microbial and metabolite values achieved a +90% accuracy with LASSO in predicting specific pneumonia etiology. This study demonstrates that urine from patients hospitalized with pneumonia may serve as a reliable and accessible sample to evaluate biomarkers that may diagnose etiology and predict clinical outcomes. Author SummaryUrine has been classically considered sterile since most microorganisms are not readily culturable under healthy circumstances. Further, many pneumonia patients are immediately placed on antibiotics rendering culture-based techniques useless. However, the advent of next generation sequencing has enabled unprecedented analysis of the microbial communities - living or detected as free DNA - found in many niches of the human body. Here, we describe a urine microbiome as well as metabolites and cytokines measured in patients newly admitted to the hospital diagnosed with influenza or bacterial (S. aureus and S. pneumoniae) infection pneumonia, compared with healthy controls. Using these parameters alone, we were able to achieve high success in predicting patient pneumonia. This study provides a proof of concept that urine samples, which are easily accessible in outpatient and inpatient settings, could provide additional diagnostic insights to patient infectious status and future risk factor for complication.

systems biology↗

bioRxiv2020

Alt-RPL36 downregulates the PI3K-AKT-mTOR signaling pathway by interacting with TMEM24

Thousands of previously unannotated small and alternative open reading frames (alt-ORFs) have recently been revealed in the human genome, and hundreds are now known to be required for cell proliferation. Many alt-ORFs are co-encoded with proteins of known function in multicistronic human genes, but the functions of only a handful are currently known in molecular detail. Using a proteomic strategy for discovery of unannotated short open reading frames in human cells, we report the detection of alt-RPL36, a 148-amino acid protein co-encoded with and overlapping human RPL36 (ribosomal protein L36). Alt-RPL36 partially localizes to the endoplasmic reticulum, where it interacts with TMEM24, which transports the phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] precursor phosphatidylinositol from the endoplasmic reticulum to the plasma membrane. Knock-out of alt-RPL36 in HEK 293T cells increases PI(4,5)P2 levels in the plasma membrane, upregulates the PI3K-AKT-mTOR signaling pathway, and increases cell size. Four serine residues of alt-RPL36 are phosphorylated, and mutation of these four serines to alanine abolishes interaction with TMEM24 and, consequently, abolishes alt-RPL36 effects on PI3K signaling and cell size. These results implicate alt-RPL36 as a novel regulator of PI(4,5)P2 synthesis upstream of the PI3K-AKT-mTOR signaling pathway. More broadly, these results show that the alt-RPL36 transcript can express two sequence-independent polypeptides from overlapping ORFs that regulate the same process - protein synthesis - via different molecular mechanisms (PI3K signaling and ribosome composition), expanding our knowledge of the mechanisms by which multicistronic human genes function.

genomics↗

bioRxiv2020

Branched chain amino acid aminotransferase 2 Regulates Ferroptotic Cell Death in Cancer Cells

Ferroptosis has been implicated as a tumor-suppressor function for cancer therapy. Recently the sensitivity to ferroptosis was tightly linked to numerous biological processes, including metabolism of amino acid. Here, using a high-throughput CRISPR/Cas9 based genetic screen in HepG2 cells to search for metabolic proteins inhibiting ferroptosis, we identified branched chain amino acid aminotransferase 2 (BCAT2) as a novel suppressor of ferroptosis. Mechanistically, ferroptosis inducers (erastin, sorafenib and sulfasalazine) activated AMPK/SREBP1 signaling pathway through ferritinophagy, which in turn inhibited BCAT2 transcription. We further confirmed that BCAT2 mediating the metabolism of sulfur amino acid, regulated intracellular glutamate level, whose activation by ectopic expression specifically antagonize system Xc- inhibition and protected liver and pancreatic cancer cells from ferroptosis in vitro and in vivo. Finally, our results demonstrate the synergistic effect of sorafenib and sulfasalazine in downregulating BCAT2 expression and dictating ferroptotic death, where BCAT2 can also be used to predict the responsiveness of cancer cells to ferroptosis-inducing therapies. Collectively, these findings identify a novel role of BCAT2 in ferroptosis, suggesting a potential therapeutic strategy for overcoming sorafenib resistance.

cancer biology↗

bioRxiv2020

A synergistic action of sirtuins directly recognizes DNA breaks and potentiates DNA damage response and repair

The DNA damage response (DDR) is a highly orchestrated process but how double-strand DNA breaks (DSBs) are initially recognized is unclear. Here, we show that polymerized SIRT6 deacetylase recognizes DSBs and potentiates the DDR. First, SIRT1 deacetylates SIRT6 at residue K33, which is important for SIRT6 polymerization and mobilization toward DSBs. Then, K33-deacetylated SIRT6 anchors to {gamma}H2AX, allowing its retention on and subsequent remodeling of local chromatin. We show that a K33R mutation that mimics hypoacetylated SIRT6 can rescue defective DNA repair as a result of SIRT1 deficiency in cultured cells. These data highlight the synergistic action between SIRTs in the spatiotemporal regulation of the DDR and DNA repair.

biochemistry↗

bioRxiv2019

Whole Genome Sequencing and Assembly of the Asian Honey Bee Apis dorsata

The Asian honey bee (Apis dorsata) is distinct from its more widely distributed cousin A. mellifera by a few key characteristics. Most prominently, A. dorsata, nest in the open by forming a colony clustered around the honeycomb, while A. mellifera nest in concealed cavities. Additionally, the worker and reproductive castes are all of the same size in A. dorsata. In order to investigate these differences, we performed whole genome sequencing of A. dorsata using a hybrid Oxford Nanopore and Illumina approach. The 223MB genome has an N50 of 35kb with the largest scaffold of 302kb. We have found that there are many genes in the dorsata genome that are distinct from other hymenoptera and also large amounts of transposable elements, and we suggest some candidate genes for A. dorsatas exceptional level of defensive aggression.

genomics↗

bioRxiv2019

RNA G-quadruplex structures exist and function in vivo

Guanine-rich sequences are able to form complex RNA structures termed RNA G-quadruplexes in vitro. Because of their high stability, RNA G-quadruplexes are proposed to exist in vivo and are suggested to be associated with important biological relevance. However, there is a lack of direct evidence for RNA G-quadruplex formation in living cells. Therefore, it is unclear whether any purported functions are associated with the specific sequence content or the formation of an RNA G-quadruplex structure. Here, we profiled the landscape of those guanine-rich regions with the in vitro folding potential in the Arabidopsis transcriptome. We found a global enrichment of RNA G-quadruplexes with two G-quartets whereby the folding potential is strongly influenced by RNA secondary structures. Using in vitro and in vivo RNA chemical structure profiling, we determined that hundreds of RNA G-quadruplex structures are strongly folded in both Arabidopsis and rice, providing direct evidence of RNA G-quadruplex formation in living eukaryotic cells. Subsequent genetic and biochemical analysis showed that RNA G-quadruplex folding was sufficient to regulate translation and modulate plant growth. Our study reveals the existence of RNA G-quadruplex in vivo, and indicates that RNA G-quadruplex structures act as important regulators of plant development and growth.

plant biology↗