Biology subjects

Cao, Z.

Publications and source records attributed to Cao, Z..

137 records · Page 8Linked to original sources

bioRxiv2019

Circular RNA profiling in the oocyte and cumulus cells reveals that circARMC4 is essential for porcine oocyte maturation

Thousands of circular RNAs (circRNAs) have been recently discovered in cumulus cells and oocytes from several species. However, the expression and function of circRNA during porcine oocyte meiotic maturation have been never examined. Here, we separately identified 7,067 and 637 circRNAs in both the cumulus cell and the oocyte via deep sequencing and bioinformatic analysis. Further analysis revealed that a faction of circRNAs is differentially expressed (DE) in a developmental stage-specific manner. The host genes of DE circRNAs are markedly enriched to multiple signaling pathways associated with cumulus cell function and oocyte maturation. Additionally, most DE circRNAs harbor several miRNA targets, suggesting that these DE circRNAs potentially act as miRNA sponge. Importantly, we found that maternal circARMC4 knockdown by siRNA microinjection caused a severely impaired chromosome alignment, and significantly inhibited first polar body extrusion and early embryo development. Taken together, these results demonstrate for the first time that circRNAs are abundantly and dynamically expressed in a developmental stage-specific manner in cumulus cells and oocytes, and maternally expressed circARMC4 is essential for porcine oocyte meiotic maturation and early embryo development.

developmental biology↗

bioRxiv2019

Heterologous expression of Stlac2, a laccase isozyme of Setosphearia turcica, and the ability of decolorization of malachite green

Laccases can catalyze monoelectronic oxidation and have shown to have an increasing value in industrial application. In this study, as identified by Native-PAGE and ESI-MS/MS, ascomycetous fungus Setosphaeria turcica produced three laccase isozymes: Stlac1, Stlac2, and Stlac6. Stlac2 was heterologously expressed in both eukaryotic and prokaryotic expression systems. The eukaryotic recombinant Stlac2 expressed in Pichia pastoris was inactive, and also showed a higher molecular weight than predicted because of glycosylation. The depression of laccase activity was attributable to the incorrect glycosylation at Asn97. Stlac2 expressed in Escherichia coli and after being renaturated from the inclusion body, the recombinant Stlac2 exhibited activity of 28.23 U/mg with 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate. The highest activity was observed at pH of 4.5 and the temperature of 60 {degrees}C. The activity of recombinant Stlac2 was inhibited by 10 mM Na+, Mg2+, Ca2+, Mn2+, and increased by 10 mM of Fe3+ with a relatively activity of 315% compared with no addition. Cu2+ did not affect enzyme activity. Recombinant Stlac2 was capable of decolorizing 67.08% of 20 mg/L malachite green in 15 min without any mediators. It is suggested that Stlac2 has potential industrial applications. ImportanceSetosphaeria turcica, an ascomycetous fungus causes northern corn leaf blight, product three laccase isozymes identified by Native-PAGE and ESI-MS/MS. The major expression laccase gene StLAC2 was expression in both eukaryotic and prokaryotic expression systems, which found incorrect glycosylation at Asn97 may result in the depression of laccase activity. The heterologous laccase Stlac2 decolorize organic dye malachite green, which had a potential industrial application.

bioengineering↗

bioRxiv2018

Zebrafish DANA retroposon can form large zebrafish sequence in human Hepg2 and 293T cell lines

In this study, we cloned small zebrafish retroposon DANA from zebrafish genome and constructed the lentiviral expression vector pEB-GFP (T2A)PURO. Three human cell lines including 293T, Hepg2 and LO2 were selected as infection targets. After detecting the expression of DANA, we found that the expression of DANA retroposon in three cells had different effects on cell lines through chromosome walking. Among these cells, LO2 showed no DANA retrotrans-position, while 293T and Hepg2 cell lines displayed retrotrans-position with the formation of some zebrafish genome fragments. Thereafter, we constructed a mutant of DANA retroposon and infected it in 293T cells, but no retrotrans-position was found after chromosome walking. Re-sequencing of the two cell lines (293T and Hepg2) showed that a large number of zebrafish genome fragments were found in the genomes of both cell lines, which could be divided into four types. The first type was zebrafish microsatellite sequence, accounting for 79.23% and 74.45% in 293T cell line and Hepg2 cell line, respectively. The second type was the sequence with a small amount of poly A or T, and the third type was the sequence with poly G or C, and the second and third types accounted very low proportion. The fourth type was composed of coding sequence and non-coding sequence, with large difference and very low proportion of common sequences between the two cell lines. Taken together, this study indicated that zebrafish DANA retroposon can result in retrotrans-position using the retrotrans system of human cell lines.

cell biology↗

bioRxiv2018

Rearranged zebrafish genomic DNA induces zebrafish mutant after microinjection into fertilized egg and preliminary study of the mechanism

Genomic DNA of zebrafish was first digested incompletely with Msp I, and then the fragments were joined to form rearranged genomic DNA. This rearranged genomic DNA was incompletely digested with EcoR I, and the fragments were linked with a long adaptor. Two primers (Gmprimer1 and Gmprimer2) were designed according to the adaptor sequence for two-step amplification. The Gmprimer1-amplified products were microinjected into fertilized zebrafish eggs after purification and a red flesh mutant was observed among 42 surviving zebrafish. We obtained several introduced sequences by two-step amplification. The second set of Gmprimer2-amplified products were purified and microinjected into fertilized zebrafish eggs; all 37 surviving fish were red flesh mutants. We found that the largest amplified band from the mutant from the first microinjection was also present in the amplified pattern from six mutants from the second microinjection. The length of the sequence was 2,565 bp, but it did not encode any proteins. Microinjecting this sequence into fertilized zebrafish eggs produced the red flesh mutant. The sequences differed slightly among different individuals from the second microinjection. Most regions of these sequences were the same, with the exception of a hypervariable region. We mixed 10 such sequences equally and microinjected them into zebrafish zygotes; the findings showed that most zygotes died and the surviving zebrafish were almost all mutants. By genome walking, we found that the site of insertion of the fragment was the same, beginning at position 41,365,003 of the eighth chromosome, and that downstream of the introduced fragment is a conservative sequence of 6,536 bp (named Cao-sequence), starting from a small reverse repeat sequence, not encoding any gene, nor similar to any known regulatory sequence. It has 322 homologous sequences in the zebrafish genome, which are distributed in all chromosomes. We designed two primers within Cao-sequence and several primers specific for different locations upstream of it. Compared with normal zebrafish, we found that the amplified patterns of all mutants in Cao-sequence regions changed to varying degrees. To further understand the effect of the introduced sequence on the zebrafish genomes, we selected six mutants for whole-genome resequencing. The results showed that numerous Cao-sequences from these six mutants were partially deleted and the lengths of the deletions was mostly approximately 6,100 bp, being located at the 5' end of Cao-sequences. Among them, 43 Cao-sequence loci were commonly deleted from the six mutants (with slightly different locations), and the other deletion sites were not identical. We think that different deletion combinations of Cao-sequence may show different mutation characteristics. The tail part from four red flesh mutants and three individuals of wild type were collected for transcriptome sequencing. TopGO analysis showed that the 4 most significant enrichment nodes were sequence specific DNA binding proteins, sequence specific transcription factors, chromatin proteins and zinc binding proteins. The results of KEGG enrichment analysis showed that the top four affected KEGG-pathways were metabolic pathways, oxidative phosphorylation, citrate cycle and 2-oxocarboxylic acid metabolism.We conclude that deletion of Cao-sequence can affect the expression of a series of transcription regulators and specific DNA binding proteins, then many basic metabolic processes were disturbed which led to mutations.

genetics↗

bioRxiv2018

A membrane-depolarising toxin substrate of the Staphylococcus aureus Type VII protein secretion system targets eukaryotes and bacteria

The type VII protein secretion system (T7SS) is conserved across Staphylococcus aureus strains and plays important roles in virulence and interbacterial competition. To date only one T7SS substrate protein, encoded in a subset of S. aureus genomes, has been functionally characterized. Here, using an unbiased proteomic approach, we identify TspA as a further T7SS substrate. TspA is encoded distantly from the T7SS gene cluster and is found across all S. aureus strains as well as in Listeria and Enterococci. Heterologous expression of TspA from S. aureus strain RN6390 indicates its C-terminal domain is toxic when targeted to the Escherichia coli periplasm and that it depolarizes the cytoplasmic membrane. The membrane depolarizing activity is alleviated by co-production of the membrane-bound TsaI immunity protein, which is encoded adjacent to tspA on the S. aureus chromosome. Using a zebrafish hindbrain ventricle infection model, we demonstrate that the T7SS of strain RN6390 promotes bacterial replication in vivo, and deletion of tspA leads to increased bacterial clearance. The toxin domain of TspA is highly polymorphic and S. aureus strains encode multiple tsaI homologues at the tspA locus, suggestive of additional roles in intra-species competition. In agreement, we demonstrate TspA-dependent growth inhibition of RN6390 by strain COL in the zebrafish infection model that is alleviated by the presence of TsaI homologues. Significance statementStaphylococcus aureus, a human commensal organism that asymptomatically colonizes the nares, is capable of causing serious disease following breach of the mucosal barrier. S. aureus strains encode a Type VII secretion system (T7SS) that is required for virulence in mouse infection models, and some strains also secrete a nuclease toxin by this route that has antibacterial activity. Here we identify TspA, widely found in Staphylococci and other pathogenic bacteria, as a T7 substrate. We show that TspA has membrane-depolarizing activity and that S. aureus uses TspA to inhibit the growth of a bacterial competitor in vivo.

microbiology↗

bioRxiv2018

Dietary fatty acids promote lipid droplet diversity through seipin enrichment in an ER subdomain

Exogenous metabolites from microbial and dietary origins have profound effects on host metabolism. Here, we report that a sub-population of lipid droplets (LDs), which are conserved organelles for fat storage, is defined by metabolites-driven targeting of the C. elegans seipin ortholog, SEIP-1. Loss of SEIP-1 function reduced the size of a subset of LDs while over-expression of SEIP-1 had the opposite effect. Ultrastructural analysis revealed SEIP-1 enrichment in an endoplasmic reticulum (ER) subdomain, which co-purified with LDs. Analyses of C. elegans and bacterial genetic mutants indicated a requirement of polyunsaturated fatty acids (PUFAs) and microbial cyclopropane fatty acids (CFAs) for SEIP-1 enrichment, as confirmed by dietary supplementation experiments. In mammalian cells, heterologous expression of SEIP-1 promoted lipid droplet expansion from ER subdomains in a conserved manner. Our results suggest that microbial and polyunsaturated fatty acids serve unexpected roles in regulating cellular fat storage by enforcing LD diversity.

cell biology↗

bioRxiv2018

Bidirectional Control of Coronary Vascular Resistance by Eicosanoids via a Novel GPCR

Arachidonic acid metabolites epoxyeicosatrienoates (EETs) and hydroxyeicosatetraenoates (HETEs) are important regulators of myocardial blood flow and coronary vascular resistance (CVR), but their mechanisms of action are not fully understood. We identified G protein-coupled receptor 39 (GPR39) as a microvascular smooth muscle cell (mVSMC) receptor antagonistically regulated by two endogenous eicosanoids: 15-HETE, which stimulates GPR39 to increase mVSMC intracellular calcium and augment microvascular CVR, and 14,15-EET, which inhibits these actions. Furthermore, zinc ion acts as an allosteric modulator of GPR39 to potentiate the efficacy of the two ligands. Our findings will have a major impact on understanding the roles of eicosanoids in cardiovascular physiology and disease, and provide an opportunity for the development of novel GPR39-targeting therapies for cardiovascular disease.\n\nOne Sentence SummaryGPR39 is a microvascular smooth muscle cell receptor regulated by two vasoactive eicosanoids with opposing actions.

physiology↗

bioRxiv2018

Niacin fine-tunes energy homeostasis through canonical GPR109A signaling

Niacin has long been considered as a high-potency drug for beneficially treating lipid abnormalities, however, its anti-atherosclerotic effects have been challenged by recent studies. Here, we demonstrated that oral supplementation of niacin resulted in a significant reduction in body weight and fat mass without affecting food intake in high-fat diet-fed wild-type mice, but not in GPR109A-defeicient mice. Further investigation showed that niacin challenge led to a remarkable inhibition of hepatic lipogenesis via a GPR109A-dependent ERK1/2/AMPK pathway. Additionally, we demonstrated that niacin treatment stimulated thermogenesis in brown adipose tissue by induction of thermogenic genes via GPR109A. Moreover, we observed that mice exposed to niacin exhibited a dramatic decrease in intestinal absorption of fatty acids. Together, our data demonstrate that acting on GPR109A, niacin shows the potential to maintain energy homeostasis by fine-tuning hepatic lipogenesis, BAT/beige thermogenesis and intestinal fat absorption, representing a potential approach to the treatment of lipid abnormalities.

cell biology↗

bioRxiv2018

Changes in the urinary proteome in a Patient-Derived Xenograft model

In this report, the urinary proteome from a patient-derived xenograft (PDX) model was compared at the peptide level to study the origins of urinary proteins in tumor-bearing nude mice. Urine was collected from the PDX mice before and after tumor implantation. A total of 515 mouse proteins were identified, of which 8 were differential proteins. Seventy-eight unambiguous human peptides from 42 human proteins were identified in the tumor-bearing group. Compared with the differential urinary proteins from the tumor-bearing immuno-competent rats, the differential proteins in the urine from the PDX model had no host immune response proteins in the very early stage urine in the tumor-bearing immuno-competent rat model.

biochemistry↗

bioRxiv2018

5C-ID: Increased resolution Chromosome-Conformation-Capture-Carbon-Copy with in situ 3C and double alternating primer design

Mammalian genomes are folded in a hierarchy of compartments, topologically associating domains (TADs), subTADs and looping interactions. Currently, there is a great need to evaluate the link between chromatin topology and genome function across many biological conditions and genetic perturbations. Hi-C generates high quality, high resolution maps of looping interactions genome-wide, but is intractable for high-throughput screening of loops across conditions due to the requirement of an enormous number of reads (>6 Billion) per library. Here, we describe 5C-ID, an updated version of Chromosome-Conformation-Capture-Carbon-Copy (5C) with restriction digest and ligation performed in the nucleus (in situ Chromosome-Conformation-Capture (3C)) and ligation-mediated amplification performed with a new double alternating design. 5C-ID reduces spatial noise and enables higher resolution 3D genome folding maps than canonical 5C, allowing for a marked improvement in sensitivity and specificity of loop detection. 5C-ID enables the creation of high-resolution, high-coverage maps of chromatin loops in up to a 30 Megabase subset of the genome at a fraction of the cost of Hi-C.

genomics↗

bioRxiv2017

gkm-DNN: efficient prediction using gapped k-mer features and deep neural networks

How to extract informative features from genome sequence is a challenging issue. Gapped k-mers frequency vectors (gkm-fv) has been presented as a new type of features in the last few years. Coupled with support vector machine (gkm-SVM), gkm-fvs have been used to achieve effective sequence-based predictions. However, the huge computation of a large kernel matrix prevents it from using large amount of data. And it is unclear how to combine gkm-fvs with other data sources in the context of string kernel. On the other hand, the high dimensionality, colinearity and sparsity of gkm-fvs hinder the use of many traditional machine learning methods without a kernel trick. Therefore, we proposed a flexible and scalable framework gkm-DNN to achieve feature representation from high-dimensional gkm-fvs using deep neural networks (DNN). We first proposed a more concise version of gkm-fvs which significantly reduce the dimension of gkm-fvs. Then we implemented an efficient method to calculate the gkm-fv of a given sequence at the first time. Finally, we adopted a DNN model with gkm-fvs as inputs to achieve efficient feature representation and a prediction task. Here, we took the transcription factor binding site prediction as an illustrative application. We applied gkm-DNN onto 467 small and 69 big human ENCODE ChIP-seq datasets to demonstrate its performance and compared it with the state-of-the-art method gkm-SVM. We demonstrated that gkm-DNN can not only improve the limitations of high dimensionality, colinearity and sparsity of gkm-fvs, but also make comparable overall performance compared with gkm-SVM using the same gkm-fvs. In addition, we used gkm-DNN to explore the representation power of gkm-fvs and provided more explanation on how gkm-fvs work.

bioinformatics↗