Biology subjects

Xu, P.

Publications and source records attributed to Xu, P..

At least 199 records · Page 11Linked to original sources

bioRxiv2020

Non-coding loci without epigenomic signals can be essential for maintaining global chromatin organization and cell viability

The majority of the non-coding regions in the human genome do not harbor any annotated element and are even not marked with any epigenomic signal or protein binding. An understudied aspect of these regions is their possible roles in stabilizing the 3D chromatin organization. To illuminate their "structural importance", we chose to start with the non-coding regions forming many 3D contacts (referred to as hubs) and identified dozens of hubs essential for cell viability. Hi-C and single cell transcriptomic analyses showed that their deletion could significantly alter chromatin organization and impact gene expression located distal in the genome. This study revealed the 3D structural importance of non-coding loci that are not associated with any functional element, providing a new mechanistic understanding of the disease-associated genetic variations (GVs). Furthermore, our analyses also suggested a powerful approach to develop "one-drug-multiple-targets" therapeutics targeting the disease-specific non-coding regions.

genetics↗

bioRxiv2020

Regulatory elements can be essential for maintaining broad chromatin organization and cell viability

Increasing evidence shows that promoters and enhancers could be related to 3D chromatin structure, thus affecting cellular functions. Except for functioning through the canonical chromatin loops formed by promoters and enhancers, their roles in maintaining broad chromatin organization have not been well studied. Here, we focused on the active promoters/enhancers (referred to as hotspots) predicted to form many 3D contacts with other active promoters/enhancers, and identified dozens of loci critical for cell survival. While the essentiality of hotspots is not resulted from their association with essential genes, deletion of an essential hotspot could lead to change of broad chromatin organization and expressions of distal genes. We demonstrated that multiple affected genes that are individually non-essential could have synergistic effects to cause cell death.

bioinformatics↗

bioRxiv2020

GSAP regulates mitochondrial function through the Mitochondria-associated ER membrane in the pathogenesis of Alzheimer's disease

Biochemical, pathogenic and human genetic data confirm that GSAP ({gamma}-secretase activating protein), a selective {gamma}-secretase modulatory protein, plays important roles in Alzheimers disease (AD) and Down syndrome. However, the molecular mechanism(s) underlying GSAP-dependent pathogenesis remains largely elusive. Here, through unbiased proteomics and single-nuclei RNA-seq, we identified that GSAP regulates multiple biological pathways, including protein phosphorylation, trafficking, lipid metabolism, and mitochondrial function. We demonstrated that GSAP physically interacts with Fe65:APP complex to regulate APP trafficking/partitioning. GSAP is enriched in the mitochondria-associated membrane (MAM) and regulates lipid homeostasis through the amyloidogenic processing of APP. GSAP deletion generates a lipid environment unfavorable for AD pathogenesis, leading to improved mitochondrial function and the rescue of cognitive deficits in an AD mouse model. Finally, we identified a novel GSAP single-nucleotide polymorphism that regulates its brain transcript level and is associated with an increased AD risk. Together, our findings indicate that GSAP impairs mitochondrial function through its MAM localization, and lowering GSAP expression reduces pathological effects associated with AD.

neuroscience↗

bioRxiv2020

GSAP Regulates Amyloid Beta Production through Modulation of Amyloid Precursor Protein Trafficking

In addition to participating in {gamma}-secretase activity, presenilin 1 (PS1) regulates trafficking and subcellular localization of {beta}-amyloid precursor protein (APP). We previously showed that gamma-secretase activating protein (GSAP) selectively modulates {gamma}-secretase activity by inducing conformational change in PS1. However, little is known whether and how GSAP might influence APP trafficking and consequent generation of {beta}-amyloid (A{beta}) peptides. Here, to explore whether GSAP has any role in regulating APP trafficking, and to systematically investigate the intracellular trafficking routes of APP, we paired total internal reflection fluorescence microscopy, high-speed line scanning microscopy, and 4D microscopy with comprehensive imaging analysis methodologies to depict the elusive modes of APP trafficking at a single-vesicle level. Mobility and diffusivity changes reveal the existence of two kinetically distinct pathways, classified into mobile and immobile pools, for vesicular APP trafficking, suggesting high association between immobile vesicle pool and amyloidogenic processing. GSAP knockdown significantly lowers immobile pool without overturning APP vesicle diffusivity, suggesting that GSAP affects vesicular APP trafficking by retaining APP in membrane microdomains known to favor amyloidogenic processing. Our study reveals a novel role of GSAP in the regulation of A{beta}-peptide formation that modulates switching of APP vesicles between immobile and mobile pools, which may help identifying new therapeutic strategies to treat Alzheimers disease.

neuroscience↗

bioRxiv2020

Cloning and Functional characterization of seed-specific LEC1A promoter from peanut (Arachis hypogaea L.)

LEAFY COTYLEDON1 (LEC1) is a HAP3 subunit of CCAAT-binding transcription factor, which controls several aspects of embryo and postembryo development, including embryo morphogenesis, storage reserve accumulation and skotomorphogenesis. Herein, using the method of chromosomal walking, a 2707bp upstream sequence from the ATG initiation codon site of AhLEC1A which is a homolog of Arabidopsis LEC1 was isolated in peanut. Its transcriptional start site confirmed by 5 RACE was located at 82 nt from 5 upstream of ATG. The bioinformatics analysis revealed that there existed many tissue-specific elements and light responsive motifs in its promoter. To identify the functional region of the AhLEC1A promoter, seven plant expression vectors expressing the GUS ({beta}-glucuronidase) gene, driven by 5 terminal series deleted fragments of AhLEC1A promoter, were constructed and transformed into Arabidopsis. Results of GUS histochemical staining showed that the regulatory region containing 82bp of 5 UTR and 2228bp promoter could facilitate GUS to express preferentially in the embryos at different development periods of Arabidopsis. Taken together, it was inferred that the expression of AhLEC1A during seed development of peanut might be controlled positively by several seed-specific regulatory elements, as well as negatively by some other regulatory elements inhibiting its expression in other organs. Moreover, the GUS expression pattern of transgenic seedlings in darkness and in light was relevant to the light-responsive elements scattered in AhLEC1A promoter segment, implying that these light-responsive elements harbored in the AhLEC1A promoter regulate skotomorphogenesis of peanut seeds, and AhLEC1A expression was inhibited after the germinated seedlings were transferred from darkness to light.

molecular biology↗

bioRxiv2020

SARS-CoV-2 infection causes transient olfactory dysfunction in mice

Olfactory dysfunction caused by SARS-CoV-2 infection represents as one of the most predictive and common symptoms in COVID-19 patients. However, the causal link between SARS-CoV-2 infection and olfactory disorders remains lacking. Herein we demonstrate intranasal inoculation of SARS-CoV-2 induces robust viral replication in the olfactory epithelium (OE), resulting in transient olfactory dysfunction in humanized ACE2 mice. The sustentacular cells and Bowmans gland cells in OE were identified as the major targets of SARS-CoV-2 before the invasion into olfactory sensory neurons. Remarkably, SARS-CoV-2 infection triggers cell death and immune cell infiltration, and impairs the uniformity of OE structure. Combined transcriptomic and proteomic analyses reveal the induction of antiviral and inflammatory responses, as well as the downregulation of olfactory receptors in OE from the infected animals. Overall, our mouse model recapitulates the olfactory dysfunction in COVID-19 patients, and provides critical clues to understand the physiological basis for extrapulmonary manifestations of COVID-19.

microbiology↗

bioRxiv2020

Human Identical Sequences of SARS-CoV-2 Promote Clinical Progression of COVID-19 by Upregulating Hyaluronan via NamiRNA-Enhancer Network

The COVID-19 pandemic is a widespread and deadly public health crisis. The pathogen SARS-CoV-2 replicates in the lower respiratory tract and causes fatal pneumonia. Although tremendous efforts have been put into investigating the pathogeny of SARS-CoV-2, the underlying mechanism of how SARS-CoV-2 interacts with its host is largely unexplored. Here, by comparing the genomic sequences of SARS-CoV-2 and human, we identified five fully conserved elements in SARS-CoV-2 genome, which were termed as "human identical sequences (HIS)". HIS are also recognized in both SARS-CoV and MERS-CoV genome. Meanwhile, HIS-SARS-CoV-2 are highly conserved in the primate. Mechanically, HIS-SARS-CoV-2, behaving as virus-derived miRNAs, directly target to the human genomic loci and further interact with host enhancers to activate the expression of adjacent and distant genes, including cytokines gene and angiotensin converting enzyme II (ACE2), a well-known cell entry receptor of SARS-CoV-2, and hyaluronan synthase 2 (HAS2), which further increases hyaluronan formation. Noteworthily, hyaluronan level in plasma of COVID-19 patients is tightly correlated with severity and high risk for acute respiratory distress syndrome (ARDS) and may act as a predictor for the progression of COVID-19. HIS antagomirs, which downregulate hyaluronan level effectively, and 4-Methylumbelliferone (MU), an inhibitor of hyaluronan synthesis, are potential drugs to relieve the ARDS related ground-glass pattern in lung for COVID-19 treatment. Our results revealed that unprecedented HIS elements of SARS-CoV-2 contribute to the cytokine storm and ARDS in COVID-19 patients. Thus, blocking HIS-involved activating processes or hyaluronan synthesis directly by 4-MU may be effective strategies to alleviate COVID-19 progression.

molecular biology↗

bioRxiv2020

A triple functional sensing chip for rapid detection of pathogenic Listeria monocytogenes

Here a triple functional sensing chip was created for L. monocytogenes detection by integrating three biomarkers (Listeriolysin O (LLO) at protein level, hly gene at genetic level, and acetoin at metabolic level). Liposome encapsulated catechol was used for LLO detection via LLO pore-forming ability. hly gene was specifically captured by using a thiolated capture probe on nanoporous gold (NPG). As an electroactive label, methylene blue was embedded in double-stranded structures to generate an electrochemical signal for hly detection. Combined with the electrocatalysis of NADH by NPG, the acetoin detection was achieved by measuring the consumption of NADH as a cofactor under acetoin reductase catalysis. Importantly, the L. monocytogenes detection results obtained by detecting three biomarkers using the chip can be mutually verified, which reduces the probability of false positives based on a single marker. Moreover, the detection time was reduced to about 90 min, making it a rapid and reliable tool for L. monocytogenes detection.

microbiology↗

bioRxiv2020

Foxc1 establishes enhancer accessibility for craniofacial cartilage differentiation

The specification of cartilage requires Sox9, a transcription factor with broad roles for organogenesis outside the skeletal system. How Sox9 gains selective access to cartilage-specific cis-regulatory regions during skeletal development had remained unclear. By analyzing chromatin accessibility during the differentiation of neural crest cells into chondrocytes of the zebrafish head, we find that cartilage-associated chromatin accessibility is dynamically established. Cartilage-associated regions that become accessible after neural crest migration are co-enriched for Sox9 and Fox transcription factor binding motifs. In zebrafish lacking Foxc1 paralogs, we find a global decrease in chromatin accessibility in chondrocytes, consistent with a later loss of dorsal facial cartilages. Zebrafish transgenesis assays confirm that many of these Foxc1-dependent elements function as enhancers with region- and stage-specific activity in facial cartilages. We propose that Foxc1-dependent chromatin accessibility helps directs the versatile Sox9 protein to a chondrogenic program in the face. HighlightsO_LIDynamic chromatin accessibility across facial cartilage development C_LIO_LICo-enrichment of Fox- and Sox-binding motifs in accessible regions C_LIO_LIFoxc1 establishes accessibility in a subset of facial cartilage enhancers C_LIO_LIModular activity of Foxc1-dependent cartilage enhancers in zebrafish C_LI

molecular biology↗

bioRxiv2020

Humans surviving cholera develop antibodies against Vibrio cholerae O-specific polysaccharide that inhibit pathogen motility

The mechanism of protection against cholera afforded by previous illness or vaccination is currently unknown. We have recently shown that antibodies targeting O-specific polysaccharide (OSP) of Vibrio cholerae correlate highly with protection against cholera. V. cholerae is highly motile and possesses a flagellum sheathed in O-specific polysaccharide (OSP), and motility of V. cholerae correlates with virulence. Using high speed video microscopy, and building upon previous animal-related work, we demonstrate that sera, polyclonal antibody fractions, and OSP-specific monoclonal antibodies recovered from humans surviving cholera block V. cholerae motility at both subagglutinating and agglutinating concentrations. This anti-motility effect is reversed by pre-adsorbing sera and polyclonal antibody fractions with purified OSP; and is associated with OSP-specific but not flagellin-specific monoclonal antibodies. F[ab] fragments of OSP-specific polyclonal antibodies do not inhibit motility, suggesting a requirement for antibody-mediated crosslinking in motility inhibition. We show that OSP-specific antibodies do not directly affect V. cholerae viability, but that OSP-specific monoclonal antibody highly protects against death in the murine cholera model. We used in vivo competitive index studies to demonstrate that OSP-specific antibodies impede colonization and survival of V. cholerae in intestinal tissues, and that this impact is motility-dependent. Our findings suggest that the impedance of motility by antibodies targeting V. cholerae OSP contributes to protection against cholera. IMPORTANCECholera is a severe dehydrating illness of humans caused by Vibrio cholerae. V. cholerae is a highly motile bacterium that has a single flagellum covered in lipopolysaccharide (LPS) displaying O-specific polysaccharide (OSP), and V. cholerae motility correlates with its ability to cause disease. The mechanisms of protection against cholera are not well understood; however, since V. cholerae is a non-invasive intestinal pathogen, it is likely that antibodies that bind the pathogen or its products in the intestinal lumen contribute to protection from infection. Here, we demonstrate that OSP-specific antibodies isolated from humans surviving cholera in Bangladesh inhibit V. cholerae motility and are associated with protection against challenge in a motility-dependent manner.

microbiology↗

bioRxiv2020

Implantation and Gastrulation Abnormalities Characterize Early Embryonic Lethal Mouse Lines

The period of development between the zygote and embryonic day 9.5 in mice includes multiple developmental milestones essential for embryogenesis. The preeminence of this period of development has been illustrated in loss of function studies conducted by the International Mouse Phenotyping Consortium (IMPC) which have shown that close to one third of all mouse genes are essential for survival to weaning age and a significant number of mutations cause embryo lethality before E9.5. Here we report a systematic analysis of 21 pre-E9.5 lethal lines generated by the IMPC. Analysis of pre- and post-implantation embryos revealed that the majority of the lines exhibit mutant phenotypes that fall within a window of development between implantation and gastrulation with few pre-implantation and no post-gastrulation phenotypes. Our study provides multiple genetic inroads into the molecular mechanisms that control early mammalian development and the etiology of human disease, in particular, the genetic bases of infertility and pregnancy loss. We propose a strategy for an efficient assessment of early embryonic lethal mutations that can be used to assign phenotypes to developmental milestones and outline the time of lethality.

developmental biology↗

bioRxiv2020

Droplet microfluidic sequencing of HIV genomes and integration sites

Sequencing individual HIV-proviruses and their adjacent cellular junctions can elucidate mechanisms of infected cell persistence in vivo. Here, we present a high throughput microfluidic method to sequence entire proviruses in their native integration site context. We used the method to analyze infected cells from people with HIV on suppressive antiretroviral therapy, demonstrating >90% capture and sequencing of paired proviral genomes and integration sites. This method should enable comprehensive genetic analysis of persistent HIV-infected cell reservoirs, providing important insights into the barriers to HIV cure.

genomics↗

bioRxiv2020

Chromosome-level genome of Poropuntius huangchuchieni provides a diploid progenitor-like reference genome for the allotetraploid Cyprinus carpio

The diploid Poropuntius huangchuchieni in the cyprinid family, which is distributed in the Mekong and Red River basins, is one of the most closely related diploid progenitor-like species of allotetraploid common carp. Therefore, the P. huangchuchieni genome is essential for polyploidy evolution studies in Cyprinidae. Here, we report a chromosome-level genome assembly of P. huangchuchieni by integrating Oxford Nanopore and Hi-C technology. The assembled genome size was 1021.38 Mb, 895.66 Mb of which was anchored onto 25 chromosomes with a N50 of 32.93 Mb. The genome contained 486.28 Mb repetitive elements and 24,099 protein-coding genes. Approximately 95.9% of the complete BUSCOs were detected, suggesting a high completeness of the genome. Evolutionary analysis revealed that P. huangchuchieni diverged from Cyprinus carpio at approximately 12 Mya. Genome comparison between P. huangchuchieni and the B subgenome of C. carpio provided insights into chromosomal rearrangements during the allotetraploid speciation. With the complete gene set, 17,474 orthologous genes were identified between P. huangchuchieni and C. carpio, providing a broad view of the gene component in the allotetraploid genome, which is critical for future genomic and genetic analyses. The high-quality genomic dataset created for P. huangchuchieni provides a diploid progenitor-like reference for the evolution and adaptation of allotetraploid carps.

genomics↗

bioRxiv2020

Introduction of a precocious metamorphosis BmNPV infection in the latter half of the fifth instar silkworm larvae

The silkworm, Bombyx mori, is a complete metamorphosis insect, the model to study insect physiology and biochemistry. Bombyx mori nucleopolyhedrovirus (BmNPV) is a principal pathogen of the silkworm and its host range is restricted to silkworm larvae, requiring interaction with larvae to accomplish virus replication. Prothoracic glands (PGs) are a model for synthetic ecdysone with regulating insect growth and development. This study performed a transcriptome analysis of silkworm PGs after BmNPV infection. Transcriptome data were annotated with KEGG, GO, and shown to be of high quality by RT-qPCR. The spatial expression profiles of BmJing and BmAryl indicate that they may be specifically expressed in silkworm PGs. The RT-qPCR results of the DEGs in the PGs of BmNPV-infected larvae at 24, 48, and 72 h and at the developmental stages of days-6 and 7, comparing to day-3, reveal that the DEGs may be related to the BmNPV infection via promoting early maturation in the latter half of the silkworm fifth instar. This study is the first report on the identification of possible genes in PGs correlating with the precocious molting and metamorphosis of silkworm larvae under BmNPV infection in the latter half of the fifth instar. Our findings will help to address the interactions between BmNPV infection and host developmental response. This work provides a new perspective on BmNPV infection and host developmental response, as well as suggesting candidate genes for further research.

molecular biology↗

bioRxiv2020

TIN2 facilitates TRF1-mediated trans- and cis-interactions on physiologically relevant long telomeric DNA

The shelterin complex consisting of TRF1, TRF2, RAP1, TIN2, TPP1, and POT1, functions to prevent false recognition of telomeres as double-strand DNA breaks, and to regulate telomerase and DNA repair protein access. TIN2 is a core component linking double-stranded telomeric DNA binding proteins (TRF1 and TRF2) and proteins at the 3 overhang (TPP1-POT1). Since knockdown of TIN2 also removes TRF1 and TRF2 from telomeres, determining TIN2s unique mechanistic function has been elusive. Here, we investigated DNA molecular structures promoted by TRF1-TIN2 using complementary single-molecule imaging platforms, including atomic force microscopy (AFM), total internal reflection fluorescence microscopy (TIRFM), and the DNA tightrope assay. We demonstrate that TIN2S and TIN2L isoforms facilitate TRF1-mediated DNA compaction (cis-interactions) and DNA-DNA bridging (trans-interactions) in a telomeric sequence- and length-dependent manner. On the short telomeric DNA substrate (6 TTAGGG repeats), the majority of TRF1 mediated telomeric DNA-DNA bridging events are transient with a lifetime of ~1.95 s. On longer DNA substrates (270 TTAGGG), TIN2 forms multi-protein complexes with TRF1 and stabilizes TRF1-mediated DNA-DNA bridging events that last for at least minutes. Preincubation of TRF1 with its regulator protein Tankyrase 1 significantly reduces TRF1-TIN2 mediated DNA-DNA bridging, whereas TIN2 protects the disassembly of TRF1-TIN2 mediated DNA-DNA bridging upon Tankyrase 1 addition. Our study provides evidence that TIN2 functions to promote TRF1 mediated trans-interactions of telomeric DNA, leading to new mechanistic insight into sister telomere cohesion.

biophysics↗

bioRxiv2020

Ribonucleotide incorporation characteristics around yeast autonomously replicating sequences reveal the labor division of replicative DNA polymerases

Ribonucleoside monophosphate (rNMP) incorporation in DNA is a natural and prominent phenomenon resulting in DNA structural change and genome instability. While DNA polymerases have different rNMP incorporation rates, little is known whether these enzymes incorporate rNMPs following specific sequence patterns. In this study, we analyzed a series of rNMP incorporation datasets, generated from three rNMP mapping techniques, and obtained from Saccharomyces cerevisiae cells expressing wild-type or mutant replicative DNA polymerase and ribonuclease H2 genes. We performed computational analyses of rNMP sites around early and late firing autonomously replicating sequences (ARSs) of the yeast genome, from which bidirectional, leading and lagging DNA synthesis starts. We find the preference of rNMP incorporation on the leading strand in wild-type DNA polymerase yeast cells. The leading/lagging-strand ratio of rNMP incorporation changes dramatically within 500 nt from ARSs, highlighting the Pol {delta} - Pol {varepsilon} handoff during early leading-strand synthesis. Furthermore, the pattern of rNMP incorporation is markedly distinct between the leading the lagging strand. Overall, our results show the different counts and patterns of rNMP incorporation during DNA replication from ARS, which reflects the different labor of division and rNMP incorporation pattern of Pol {delta} and Pol {varepsilon}.

bioinformatics↗

bioRxiv2020

Hypoxia-induced miR-92a regulates p53 signalling pathway and apoptosis by targeting calcium-sensing receptor in Genetically Improved Farmed Tilapia (Oreochromis niloticus)

miR-92a miRNAs are immune molecules that regulate apoptosis (programmed cell death) during the immune response. Apoptosis helps to maintain the dynamic balance in tissues of fish under hypoxia stress. The aim of this study was to explore the role and potential mechanisms of miR-92a in the liver of tilapia under hypoxia stress. We first confirmed that CaSR (encoding a calcium-sensing receptor) is a target gene of miR-92a in genetically improved farmed tilapia (GIFT) using luciferase reporter gene assays. In GIFT under hypoxia stress, miR-92a was up-regulated and CaSR was down-regulated in a time-dependent manner. Knocked-down CaSR expression led to inhibited expression of p53, TP53INP1 and caspase-3/8, reduced the proportion of apoptotic hepatocytes, and decreased the activity of calcium ions induced by hypoxia in hepatocytes. GIFT injected in the tail vein with an miR-92a agomir showed up-regulation of miR-92a and down-regulation of CaSR, p53, TP53INP1, and caspase-3/8 genes in the liver, resulting in lower serum alanine aminotransferase and aspartate aminotransferase activities under hypoxia stress. These findings suggest that stimulation of miR-92a interferes with hypoxia-induced apoptosis in hepatocytes of GIFT by targeting CaSR, thereby alleviating liver damage. These results provide new insights into the adaptation mechanisms of GIFT to hypoxia stress.

molecular biology↗

bioRxiv2020

Disproportionate presence of adenosine in mitochondrial and chloroplast DNA of Chlamydomonas reinhardtii

Ribonucleoside monophosphates (rNMPs) represent the most common non-standard nucleotides found in the genomic DNA of cells. The distribution of rNMPs in DNA has been studied only in limited genomes, such as yeast nuclear and mitochondrial DNA, as well as human mitochondrial DNA. In this study, we used the ribose-seq protocol and the Ribose-Map bioinformatics toolkit to reveal the distribution of rNMPs incorporated into the whole genome of a photosynthetic unicellular green alga, Chlamydomonas reinhardtii. The study presents the discovery of a disproportionate incorporation of adenosine in the mitochondrial and chloroplast DNA, in contrast to the nuclear DNA, relative to the nucleotide content of these C. reinhardtii genomes. Our results demonstrate that the rNMP content in the DNA of the algal organelles reflects an elevated ATP level present in the algal cells. In addition, we reveal specific rNMP biases and patterns in the mitochondrial, chloroplast and nuclear DNA of C. reinhardtii.

molecular biology↗