Biology subjects

Tang, Q.

Publications and source records attributed to Tang, Q..

121 records · Page 7Linked to original sources

bioRxiv2019

Systematical analysis reveals the novel function of Cyp2c29 in liver injury

As a severe lethal cancer, hepatocellular carcinoma (HCC) usually originates from chronic liver injury and inflammation, in which the discovery of key genes is important for HCC prevention. Here, we analyzed the time serial (from 0 week to 30 weeks) transcriptome data of liver injury samples in diethylnitrosamine (DEN)-induced HCC mouse model. Through expression and function analyses, we identified that Cyp2c29 was a key gene continuously downregulated during liver injury. Overexpression of Cyp2c29 suppressed the NF-{kappa}B activation, proinflammatory cytokine production and hepatocyte proliferation by increasing its production 14,15-epoxyeicosatrienoic acid (14,15-EET). Furthermore, in vivo Cyp2c29 protected against liver inflammation in liver injury mice models by reversing the expression on functions of cell proliferation, metabolism and inflammation including suppressing NF-{kappa}B pathway and compensatory proliferation. CYP2C8 and CYP2C9, two human homologs of mouse Cyp2c29, were decreased in human HCC progression and positively correlated with HCC patient survival. Therefore, through systematical analysis and verification, we identified that Cyp2c29 is a novel gene in liver injury and inflammation, which may be a potential biomarker for HCC prevention and prognosis.

bioinformatics↗

bioRxiv2019

Cell-substrate adhesion drives Scar/WAVE activation and phosphorylation, which controls pseudopod lifetime

The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVEs proline-rich domain is polyphosphorylated after the complex is activated. Treatments that stop activation block phosphorylation in both Dictyostelium and mammalian cells. This implies that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether a protrusion is initiated. Unexpectedly, activation-dependent phosphorylation is not promoted by chemotactic signalling, or by signal-dependent kinases such as ERKs, but is greatly stimulated by cell:substrate adhesion. Scar/WAVE that has been mutated to be either unphosphorylatable or phosphomimetic is activated normally, and rescues the phenotype of scar- cells, demonstrating that phosphorylation is dispensible for activation and actin regulation. However, pseudopods and patches of Scar/WAVE complex recruitment last substantially longer in unphosphorylatable mutants, altering cell polarisation and the efficiency of migration. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVEs activity, and that phosphorylation acts as a timer, restricting pseudopod lifetime by promoting Scar/WAVE turnover.

cell biology↗

bioRxiv2019

Mutational mimics of allosteric effectors: a genome editing design to validate allosteric drug targets

Development of drugs that allosterically regulate enzyme functions to treat disease is a costly venture. Screening mutations that mimic allosteric effectors in vitro will identify therapeutic regulatory targets enhancing the likelihood of developing a disease treatment at a reasonable cost. We demonstrate the potential of this approach utilizing human liver pyruvate kinase (hLPYK) as a model. Inhibition of hLPYK was the first desired outcome of our screen. We identified individual point mutations that: 1) mimicked allosteric inhibition by alanine, 2) mimicked inhibition by protein phosphorylation, and 3) prevented binding of fructose-1,6-bisphosphate (Fru-1,6-BP). Our second desired screening outcome was activation of hLPYK. We identified individual point mutations that: 1) prevented hLPYK from binding alanine, the allosteric inhibitor, 2) prevented inhibitory protein phosphorylation, or 3) mimicked allosteric activation by Fru-1,6-BP. Combining the three activating point mutations produced a constitutively activated enzyme that was unresponsive to regulators. Expression of a mutant hLPYK transgene containing these three mutations in a mouse model was not lethal. Thus, mutational mimics of allosteric effectors will be useful to confirm whether allosteric activation of hLPYK will control glycolytic flux in the diabetic liver to reduce hepatic glucose production and, in turn, reduce or prevent hyperglycemia.

biochemistry↗

bioRxiv2019

Do neutral protein positions really exist? A case study with allostery in human liver pyruvate kinase

Understanding how each residue position contributes to protein function has been a long-standing goal in protein science. Substitution studies have historically focused on conserved protein positions. However, substitutions of nonconserved positions can also modify function. Indeed, we recently identified nonconserved positions that have large substitution effects in human liver pyruvate kinase (hLPYK), including altered allosteric coupling. To facilitate a comparison of which characteristics determine when a nonconserved position does vs. does not contribute to function, the goal of the current work was to identify neutral positions in hLPYK. However, existing hLPYK data showed that three features commonly associated with neutral positions - high sequence entropy, high surface exposure, and alanine scanning - lacked the sensitivity needed to guide experimental studies. We used multiple evolutionary patterns identified in a sequence alignment of the PYK family to identify which positions were least patterned, reasoning that these were most likely to be neutral. Nine positions were tested with a total of 117 amino acid substitutions. Although exploring all potential functions is not feasible for any protein, five parameters associated with substrate/effector affinities and allosteric coupling were measured for hLPYK variants. For each position, the aggregate functional outcomes of all variants were used to quantify a "neutrality" score. Three positions showed perfect neutral scores for all five parameters. Furthermore, the nine positions showed larger neutral scores than 17 positions located near allosteric binding sites. Thus, our strategy successfully enriched the dataset for positions with neutral and modest substitutions.

biochemistry↗

bioRxiv2019

Mapping Histone Modifications in Low Cell Number and Single Cells Using Antibody-guided Chromatin Tagmentation (ACT-seq)

Modern next-generation sequencing-based methods have empowered researchers to assay the epigenetic states of individual cells. Existing techniques for profiling epigenetic marks in single cells often require the use and optimization of time-intensive procedures such as drop fluidics, chromatin fragmentation, and end repair. Here we describe ACT-seq, a novel and streamlined method for mapping genome-wide distributions of histone tail modifications, histone variants, and chromatin-binding proteins in a small number of or single cells. ACT-seq utilizes a fusion of Tn5 transposase to Protein A that is targeted to chromatin by a specific antibody, allowing chromatin fragmentation and sequence tag insertion specifically at genomic sites presenting the relevant antigen. The Tn5 transposase enables the use of an index multiplexing strategy (iACT-seq), which enables construction of thousands of single-cell libraries in one day by a single researcher without the need for drop-based fluidics or visual sorting. We conclude that ACT-seq present an attractive alternative to existing techniques for mapping epigenetic marks in single cells.

genomics↗

bioRxiv2018

Genomic analyses provide insights into genetic architecture of three-way crossbred pigs

BackgroundCrossbreeding is effective for improving performance in poultry and livestock, which is mainly attributed to heterosis. For pork production, a classic three-way crossbreeding system of Duroc x (Landrace x Yorkshire) (DLY) is widely used to produce terminal crossbred pigs with stable and prominent performance. Nonetheless, studies on the transmission of genetic information and gene expression pattern of DLY have been limited.\n\nFindingsWe analyzed population-scale SNPs based on 30 individuals from these three purebreds and identified 529.93 K SNPs of breed-of-origin of alleles. We also applied whole-genome sequencing of ten individuals from a DLY pig family as well as transcriptome of four representative tissues (adipose, skeletal muscle, heart, and liver) for six DLY individuals. Based on above, we identified a large number of high-confidence ASE genes, among which four ASE genes (KMO, PLIN4, POPDC3 and UGT1A6) were found to be shared over all DLY individuals.\n\nConclusionWe suggest DLY is a more effective strategy of three-way crossbreeding among these three purebreds from genetic aspect. We suppose the numerous breed-of-origin of alleles have close association with improved performance of crossbred individuals. ASE may also play important roles on DLY three-way crossbreeding system. Our findings are valuable for understanding the transmission of genetic information and the gene expression in DLY three-way crossbreeding and may be used to guide breeding and production of pigs in the future.

genomics↗

bioRxiv2018

Generating a sequence map of the pig pan-genome

Pigs (Sus scrofa) exhibit diverse phenotypes in different breeds shaped by the combined effects of various local adaptation and artificial selection. To comprehensively characterize the genetic diversity of pigs, we construct a pig pan-genome by comparing genome assemblies of 11 representative pig breeds with the reference genome (Sscrofa11.1). Approximately 72.5 Mb non-redundant sequences were identified as pan-sequences which were absent from the Sscrofa11.1. On average, 41.7 kb of spurious heterozygous SNPs per individual are removed and 12.9 kb novel SNPs per individual are recovered using pan-genome as the reference for SNP calling, thereby providing enhanced resolution for genetic diversity in pigs. Homolog annotation and analysis using RNA-seq and Hi-C data indicate that these pan-sequences contain protein-coding regions and regulatory elements. These pan-sequences can further improve the interpretation of local 3D structure. The pan-genome as well as the accompanied web-based database will serve as a primary resource for exploration of genetic diversity and promote pig breeding and biomedical research.

genomics↗

bioRxiv2018

Penton-dodecahedron of fowl adenovirus serotype 4 as a vaccine candidate for the control of related diseases

In some serotypes of adenovirus (Ad), the penton base protein and attached trimeric fiber assemble into dodecameric virus-like particles called penton-dodecahedron (Pt-Dd), which can internalize into cells and can be used to deliver the vaccine antigen and drugs. Fowl adenovirus serotype 4 (FAdV-4) is an important poultry pathogens and causing seriously economic lost to poultry industry in China and several other counties. The produce of Pt-Dd in FAdV-4 infected cells as well as in those infected with the recombinant human Ad expressing fiber-1, fiber-2, and penton base was determine by Transmission electron microscopy (TEM). For the First time, we proved that FAdV-4 produced Pt-Dd in infected cells, which can also be assembled by the overexpressed recombinant proteins fiber-1, fiber-2, and penton base. Pt-Dd, as well as the recombinant proteins fiber-1, fiber-2, and penton base, were then used to immunize chickens. The humoral immune response, cell mediate immune response (CMI), and challenge results were used to evaluate the immune efficacy of the vaccine candidates. Pt-Dd induced the highest level of enzyme-linked immunosorbent assay antibodies and high levels of CMI, showing a significantly (p < 0.05) high level of interferon {gamma}, interleukin-4, and major histocompatibility complex II expressions in peripheral blood mononuclear cells at 48 h post-infection. The challenge results showed that Pt-Dd, inactivated FAdV-4 vaccine, as well as fiber-1 induced the best protection (100%), followed by fiber-2 (80%) and penton (67%). The present study showed that FAdV-4-Pt-Dd and recombinant fiber-1 are good FAdV-4 vaccine candidates and could be used to replace the tissue-sourced inactivated FAdV-4 vaccine.\n\nImportanceFAdV-4-Pt-Dds were discovered in FAdV-4 infected cells, and which were also assembled in cells transduced with recombinant human adenovirus expressing fiber-1, fiber-2, and penton base. FAdV-4-Pt-Dds internalize into cells with high efficiency, so that it can be used for delivery vaccine antigen or drugs. Immunization chickens with Pt-Dd and fiber-1 obtained by transduction HEK-293T cells induced significant high level humoral and cellular mediated immune responses, and also 100% challenge protection in chickens indicating that they are good FAdV-4 vaccine candidates. What more, the Pt-Dd obtained by transduction HEK-293T cell would have no DNA and adenovirus contamination as adenovirus could not package in HEK-293T cells.

immunology↗

bioRxiv2018

ROCK1/Drp1-mediated aberrant mitochondrial fission is crucial for dopaminergic nerve cell apoptosis

Dopamine deficiency caused by apoptosis of the dopaminergic nerve cells in the midbrain substantia nigra is the main pathological basis of Parkinson's disease (PD). Recent research has shown that dynamin-related protein 1 (Drp1)-mediated aberrant mitochondrial fission plays an important role in dopaminergic nerve cell apoptosis. However, the upstream regulatory mechanism remains unclear. Our study shows that knockdown of Drp1 blocked aberrant mitochondrial fission and dopaminergic nerve cell apoptosis. Importantly, we found that ROCK1 was activated in an MPP+-induced PD cell model and that ROCK1 knockdown and the specific ROCK1 activation inhibitor Y-27632 blocked Drp1-mediated aberrant mitochondrial fission and apoptosis of dopaminergic nerve cell through suppression of Drp1 dephosphorylation/activation. Our in vivo study confirmed that Y-27632 significantly improved symptoms of a PD mouse model through inhibition of Drp1-mediated aberrant mitochondrial fission and apoptosis of dopaminergic nerve cell. Collectively, Our study suggests an important molecular mechanism of PD pathogenesis involving ROCK1-regulated dopaminergic nerve cell apoptosis via activation of Drp1-induced aberrant mitochondrial fission.

neuroscience↗

bioRxiv2018

Overexpressed microRNA-141-3p enhance proliferation via targeting PLAG1 in non-diabetic macrosomia

Several studies have shown microRNAs (miRNAs) could regulate the placental development, yet the role and mechanism of miRNAs in the development of non-diabetic macrosomia (NDFMS) remains unclear. The key miRNA that abnormal expressed in NDFMS placentas was screened out by miRNA microarray and verified using qRT-PCR in 91 subjects. The effects of the key miRNA were verified by proliferation assay and invasion assay in HTR-8/SVneo cell, and also in pregnant C57BL/6J mice. miR-141-3p was determined as the key miRNA with the most significant difference, which could promote the proliferation and invasion by regulating the expression of target gene PLAG1. Overexpression of PLAG1 could reverse the effect of cell proliferation and invasion ability caused by miR-141-3p overexpression. Significant difference in fetal birth weight was observed between the control group and treated group with miR-141-3p agomir in late pregnancy, but not in early pregnancy. This study revealed miR-141-3p could increase the proliferation of placenta to participate in the occurrence and development of NDFMS through regulating PLAG1 expression.

developmental biology↗

bioRxiv2018

The holdase function of Escherichia coli Hsp70 (DnaK) chaperone

In Escherichia coli, the DnaK/DnaJ/GrpE system plays a critical role in mediating protein refolding and buffering against protein aggregation due to environmental stress. The underlying mechanism remains unclear. In this work, we probe the activity of DnaK/DnaJ/GrpE system with single-molecule protein refolding assay using tandem repeats of titin immunoglobulin 27 (I27)8. We provide direct evidence that DnaK in apo- and ADP-bound state is predominantly a holdase, which kinetically stabilizes the polyprotein in its unfolded form. Binding of ATP relieves DnaKs holding, allowing protein refolding. The presence of co-chaperone DnaJ and GrpE modulates this holding-release switching, possibly by altering DnaKs nucleotide state. Our findings thus provide important insights to the molecular mechanism of DnaK/DnaJ/GrpE system.

molecular biology↗

bioRxiv2018

Structure of the receptor-activated human TRPC6 ion channel

TRPC6 is a receptor-activated nonselective cation channel that belongs to the family of canonical transient receptor potential (TRPC) channels. It is activated by diacylglycerol, a lipid second messenger. TRPC6 is involved in many physiological processes and implicated in human genetic diseases. Here we present the structure of human TRPC6 homotetramer in complex with a newly identified high affinity inhibitor BTDM solved by single-particle cryo-electron microscopy to 3.8 [A] resolution. The structure shows a two-layer architecture, in which the bell-shaped cytosolic layer holds the transmembrane layer. Extensive inter-subunit interactions of cytosolic domain, including N terminal ankyrin repeats and C terminal coiled-coil, contribute to the tetramer assembly. The high affinity inhibitor BTDM wedges between S5-S6 pore domain and voltage sensor-like domain to inhibit channel opening. Our structure uncovers the molecular architecture of TRPC channels and provides a structural basis for understanding the mechanism of these channels.

biophysics↗

bioRxiv2017

Estrogen represses Tgfbr1 and Bmpr1a expression via estrogen receptor beta in MC3T3-E1 cells

MC3T3-E1 is a clonal pre-osteoblastic cell line derived from newborn mouse calvaria, which is commonly used in osteoblast studies. To investigate the effects of estrogen on osteoblasts, we treated MC3T3-E1 cells with various concentrations of estrogen and assessed their proliferation. Next, we performed RNA deep sequencing to investigate the effects on estrogen target genes. Bmpr1a and Tgfbr1, important participants in the TGF-beta signaling pathway, were down-regulated in our deep sequencing results. Bioinformatics analysis revealed that estrogen receptor response elements (EREs) were present in the Bmpr1a and Tgfbr1 promoters. Culturing the cells with the estrogen receptor (ER) alpha or beta antagonists 1,3-bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H-pyrazole dihydrochloride (MPP) or 4-[2-phenyl-5,7-bis(trifluoromethyl) pyrazolo[1,5-alpha]pyrimidin-3-yl] phenol (PTHPP), respectively, demonstrated that ER beta is involved in the estrogen-mediated repression of Tgfbr1 and Bmpr1a.The chromatin immunoprecipitation (ChIP) results were consistent with the conclusion that E2 increased the binding of ER beta at the EREs located in the Tgfbr1 and Bmpr1a promoters. Our research provides new insight into the role of estrogen in bone metabolisms.

biochemistry↗