Publications by authors named "Zhi-Qiang Du"

75 Publications

Identification of lncRNAs involved in maternal-to-zygotic transition of in vitro-produced porcine embryos by single-cell RNA-seq.

Reprod Domest Anim 2021 Nov 1. Epub 2021 Nov 1.

College of Animal Science, Yangtze University, Jingzhou, Hubei, China.

Long non-coding RNAs (lncRNAs) function through multiple tiers of molecular circuits and are vital to gamete maturation and early embryo development. However, in pig early embryos, identification and expression dynamics of lncRNAs remain less studied. Here, we systematically analysed the expression dynamics of lncRNAs based on our previously published single-cell RNA-seq data from pig mature oocytes (GSE160334), and single blastomeres biopsied from pig in vitro fertilized (IVF) and early parthenogenetically activated (PA) embryos (1- to 8-cell stages; GSE164812). With the progression of embryo development, the total number of expressed lncRNAs gradually decreased and showed great variation at each developmental stage for both IVF and PA groups. Consecutive stage pairwise comparison of MII oocytes, 1-cell zygotes, 2-cell, 4-cell and 8-cell IVF embryos identified 151, 245, 1119 and 188 differentially expressed (DE) lncRNAs, including 119, 80, 867, 77 up-regulated and 32, 165, 252, 111 down-regulated, while 289, 437, 895 and 495 DE lncRNAs (141, 89, 768, 97 up-regulated and 148, 348, 127, 398 down-regulated) were identified in PA embryos at the same stages. The DE lncRNAs identified within IVF embryos were much different from that identified within PA embryos, showing embryo type-specific manner. Further cross-comparison between PA and IVF embryos identified 184, 656, 2502 and 266 DE lncRNAs for the 1- to 8-cell embryo stages, respectively. Further GO and KEGG enrichment analysis of DE mRNAs targeted by DELs indicated that different signalling pathways were involved in maternal-only and bi-parental embryo development. Collectively, comparative profiling of lncRNA expression dynamics between pig IVF and PA embryos provides a valuable resource, to investigate further regulatory mechanisms of lncRNAs associated with ZGA and maternal RNA decay during early embryo development.
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http://dx.doi.org/10.1111/rda.14034DOI Listing
November 2021

Melatonin mitigates Chloroquine-induced defects in porcine immature Sertoli cells.

Theriogenology 2022 Jan 8;177:1-10. Epub 2021 Oct 8.

College of Animal Science and Technology, Northeast Agricultural University, Harbin, 150030, Heilongjiang, China; College of Animal Science, Yangtze University, Jingzhou, 434025, Hubei, China. Electronic address:

Chloroquine (CQ) could function as a lysosomotropic agent to inhibit the endolysosomal trafficking in the autophagy pathway, and is widely used on malarial, tumor and recently COVID-19. However, the effect of CQ treatment on porcine immature Sertoli cells (iSCs) remains unclear. Here we showed that CQ could reduce iSC viability in a dose-dependent manner. CQ treatment (20 μM) on iSCs for 36h could elevate oxidative stress, damage mitochondrial function and promote apoptosis, which could be partially rescued by melatonin (MT) (10 nM). Transcriptome profiling identified 1611 differentially expressed genes (DEGs) (776 up- and 835 down-regulated) (20 μM CQ vs. DMSO), mainly involved in MAPK cascade, cell proliferation/apoptosis, HIF-1, PI3K-Akt and lysosome signaling pathways. In contrast, only 467 (224 up- and 243 down-regulated) DEGs (CQ + MT vs. DMSO) could be found after MT (10 nM) addition, enriched in cell cycle, regulation of apoptotic process, lysosome and reproduction pathways. Therefore, the partial rescue effects of MT on CQ treatment were confirmed by multiple assays (cell viability, ROS level, mitochondrial function, apoptosis, and mRNA levels of selected genes). Collectively, CQ treatment could impair porcine iSC viability by deranging the signaling pathways related to apoptosis and autophagy, which could be partially rescued by MT supplementation.
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http://dx.doi.org/10.1016/j.theriogenology.2021.10.005DOI Listing
January 2022

Global 3'-untranslated region landscape mediated by alternative polyadenylation during meiotic maturation of pig oocytes.

Reprod Domest Anim 2021 Oct 13. Epub 2021 Oct 13.

College of Animal Science, Yangtze University, Jingzhou, China.

Alternative polyadenylation affects the length and composition of 3'-untranslated region (3'-UTR) and regulates mRNA stability or translational activity to affect important biological processes. However, global 3'-UTR landscape and its relationship with gamete maturation remain less studied. Here, we analysed our previously reported single-cell RNA-seq data of germinal vesicle and metaphase II stage oocytes in pigs to systematically catalogue the 3'-UTR dynamics during oocyte maturation. Two softwares (DaPars and APAtrap) were employed and identified 110 and 228 mRNAs with significantly different 3'-UTRs (adjusted p ≤ .05), respectively. Gene enrichment analyses found signalling pathways related with biological processes of female gametophyte production, methyltransferase activity and mRNA surveillance (DaPars) and cell cycle process, regulation of ERK1 and ERK2 cascade, regulation of translation, spindle organization, kinetochore, condensed chromosome and progesterone-mediated oocyte maturation (APAtrap), respectively. Moreover, 18 of 110 mRNAs (|△PDUI| ≥ 0.25 and |log PDUI ratio| ≥ 0.59) and 15 of 228 mRNAs (Perc. diff. ≥ 0.5) were with greater difference of 3'-UTR length or abundance, and integrative genomics viewer analysis further identified 4 (Alg10, Hadhb, Hsd17b4 and Sbds) of 18 mRNAs to be with 3'-UTR length differed ≥150 bp and 6 (Gcc1, Hnrnpa2b1, Lsm6, Prpf18, Sfr1 and Ust) of 15 mRNAs to be with 3'-UTR abundance extremely differed. Furthermore, the location, sequences and number of cis-elements were predicted, which were shown to derange cytoplasmic polyadenylation element, poly(A) site and microRNA binding sites within 3'-UTRs of Alg10, Hadhb, Hsd17b4 and Sbds mRNAs. Taken together, global 3'-UTR landscape changes dynamically with oocyte meiotic maturation, potentially involved in regulating oocyte meiotic process in pigs.
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http://dx.doi.org/10.1111/rda.14026DOI Listing
October 2021

Acute heat stress reduces viability but increases lactate secretion of porcine immature Sertoli cells through transcriptome reprogramming.

Theriogenology 2021 Oct 9;173:183-192. Epub 2021 Aug 9.

College of Animal Science, Yangtze University, Jingzhou, 434025, Hubei, China. Electronic address:

Sertoli cells, important constituents of the somatic niche, supports the growth and development of spermatogonia. Heat stress (HS), among multiple intrinsic and external factors, can induce physiological and biochemical changes in Sertoli cells. However, the underlying molecular mechanism remains largely unclear. Here, we showed that acute heat stress (43 °C, 0.5 h) could reduce cell viability, promote apoptosis, and increase the lactate production of porcine immature Sertoli cells (iSCs) cultured in vitro. Then, transcriptome sequencing identified 126 immediately and 3372 prolonged responded differentially expressed genes (DEGs) after acute heat stress (43 °C, 0.5 h) (HS0.5), and 36 h recovery culture following heat stress (HS0.5-R36), respectively. Enrichment analyses found different signaling pathways: immediate changes including cell response to heat, regulation of cellular response to stress, heat shock protein binding, chaperon-mediated protein folding, and sterol biosynthetic process, but prolonged changes mainly involving cell cycle, regulation of apoptotic process/cell proliferation, reproductive process, P53, PI3K-Akt and Glycolysis/Gluconeogenesis. Furthermore, transcriptional patterns of 9 DEGs (Dnajb1, Traf6, Insig1, Gadd45g, Hdac6, Fkbp4, Serpine1, Pfkp and Galm), and 6 heat shock proteins (HSPs) (Hspa6, Hspb1, Hspd1, HSP90aa1, HSP90ab1 and Hsph1) were validated, as well as the protein pattern of HSP90AA1 via immunostaining and western blot. Taken together, heat stress could initiate immediate changes of heat shock-related genes, and reprogram transcriptome and signaling pathways affecting the viability, apoptosis and metabolite production of pig iSCs.
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http://dx.doi.org/10.1016/j.theriogenology.2021.06.024DOI Listing
October 2021

Single cell RNA-seq reveals genes vital to in vitro fertilized embryos and parthenotes in pigs.

Sci Rep 2021 07 13;11(1):14393. Epub 2021 Jul 13.

College of Animal Science, Yangtze University, Jingzhou, 434025, Hubei, China.

Successful early embryo development requires the correct reprogramming and configuration of gene networks by the timely and faithful execution of zygotic genome activation (ZGA). However, the regulatory principle of molecular elements and circuits fundamental to embryo development remains largely obscure. Here, we profiled the transcriptomes of single zygotes and blastomeres, obtained from in vitro fertilized (IVF) or parthenogenetically activated (PA) porcine early embryos (1- to 8-cell), focusing on the gene expression dynamics and regulatory networks associated with maternal-to-zygote transition (MZT) (mainly maternal RNA clearance and ZGA). We found that minor and major ZGAs occur at 1-cell and 4-cell stages for both IVF and PA embryos, respectively. Maternal RNAs gradually decay from 1- to 8-cell embryos. Top abundantly expressed genes (CDV3, PCNA, CDR1, YWHAE, DNMT1, IGF2BP3, ARMC1, BTG4, UHRF2 and gametocyte-specific factor 1-like) in both IVF and PA early embryos identified are of vital roles for embryo development. Differentially expressed genes within IVF groups are different from that within PA groups, indicating bi-parental and maternal-only embryos have specific sets of mRNAs distinctly decayed and activated. Pathways enriched from DEGs showed that RNA associated pathways (RNA binding, processing, transport and degradation) could be important. Moreover, mitochondrial RNAs are found to be actively transcribed, showing dynamic expression patterns, and for DNA/H3K4 methylation and transcription factors as well. Taken together, our findings provide an important resource to investigate further the epigenetic and genome regulation of MZT events in early embryos of pigs.
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http://dx.doi.org/10.1038/s41598-021-93904-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8277874PMC
July 2021

Integrated transcriptome and proteome analysis reveals potential mechanisms for differential abdominal fat deposition between divergently selected chicken lines.

J Proteomics 2021 06 23;241:104242. Epub 2021 Apr 23.

Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture and Rural Affairs, Harbin 150030, PR China; Key Laboratory of Animal Genetics, Breeding and Reproduction, Education Department of Heilongjiang Province, Harbin 150030, PR China; College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, PR China. Electronic address:

Genetic selection for meat production performance of broilers concomitantly causes excessive abdominal fat deposition, accompanied by several adverse effects, such as the reduction of feed conversion efficiency and reproduction performance. Our previous studies have identified important genes regulating chicken fat deposition, using the Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF) as an animal model. However, the molecular mechanism underlying fat deposition differences between fat and lean broilers remains largely unknown. Here, we integrated the transcriptome (RNA-Seq) and quantitative proteome (isobaric tags for relative and absolute quantitation, iTRAQ) profiling analyses on abdominal fat tissues from NEAUHLF chicken lines. Differentially expressed genes (2167 DEGs, corrected p-value < 0.01) and differentially abundant proteins (199 DAPs, corrected p-value < 0.05) were identified in lean line compared to fat line. Down-regulated DEGs and DAPs mainly enriched in pathways related to fatty acid metabolism, fatty acid biosynthesis, and PPAR signaling, and interestingly, up-regulated DEGs and DAPs enriched both in lysosome pathway. Moreover, numerous key DEGs and DAPs involved in long-chain fatty acid uptake, in situ lipogenesis (fatty acid and cholesterol synthesis), and lipid droplet accumulation were discovered after integrated transcriptome and proteome analysis. SIGNIFICANCE: Excessive abdominal fat deposition critically affects the health of broilers and causes economic loss to broiler producers, but the molecular mechanism of abdominal fat deposition is still unclear in chicken. We identified key DEGs/DAPs and potential pathways through an integration of chicken abdominal fat tissues transcriptome and proteome analyses. Our findings will facilitate a better revealing the mechanism and provide a novel insight into abdominal fat content discrepancy between the fat and lean chicken lines.
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http://dx.doi.org/10.1016/j.jprot.2021.104242DOI Listing
June 2021

Endoscopic treatment for acute appendicitis with coexistent acute pancreatitis: Two case reports.

World J Clin Cases 2021 Jan;9(1):245-251

Department of Gastroenterology, Jianyang People's Hospital of Sichuan Province, Jianyang 641400, Sichuan Province, China.

Background: Appendectomy is the procedure of choice for the treatment of acute appendicitis. However, surgery may not be appropriate for patients with coexisting severe illness or comorbidities such as acute pancreatitis (AP). Endoscopic retrograde appendicitis treatment (ERAT) may be a novel alternative to surgery for treating such patients where existing medical therapies have failed.

Case Summary: We report 2 cases of moderately severe AP who developed acute uncomplicated appendicitis during their hospital stay and did not respond to traditional medical therapy. One patient had moderately severe AP due to hyperlipidemia, while the other patient had a gallstone induced by moderately severe AP. Neither patient was fit to undergo an appendectomy procedure because of the concurrent AP. Therefore, the alternative and minimally invasive ERAT was considered. After written informed consent was collected from the patients, the ERAT procedure was performed. Both patients exhibited fast postoperative recovery after ERAT with minimal surgical trauma.

Conclusion: ERAT is a safe and effective minimally invasive endoscopic procedure for acute appendicitis in patients with coexistent AP.
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http://dx.doi.org/10.12998/wjcc.v9.i1.245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7809652PMC
January 2021

Single-cell RNA-seq reveals mRNAs and lncRNAs important for oocytes in vitro matured in pigs.

Reprod Domest Anim 2021 Apr 2;56(4):642-657. Epub 2021 Feb 2.

College of Animal Science, Yangtze University, Jingzhou, China.

The faithful execution of molecular programme underlying oocyte maturation and meiosis is vital to generate competent haploid gametes for efficient mammalian reproduction. However, the organization and principle of molecular circuits and modules for oocyte meiosis remain obscure. Here, we employed the recently developed single-cell RNA-seq technique to profile the transcriptomes of germinal vesicle (GV) and metaphase II (MII) oocytes, aiming to discover the dynamic changes of mRNAs and long non-coding RNAs (lncRNAs) during oocyte in vitro meiotic maturation. During the transition from GV to MII, total number of detected RNAs (mRNAs and lncRNAs) in oocytes decreased. Moreover, 1,807 (602 up- and 1,205 down-regulated) mRNAs and 313 (177 up- and 136 down-regulated) lncRNAs were significantly differentially expressed (DE), i.e., more mRNAs down-regulated, but more lncRNAs up-regulated. During maturation of pig oocytes, mitochondrial mRNAs were actively transcribed, eight of which (ND6, ND5, CYTB, ND1, ND2, COX1, COX2 and COX3) were significantly up-regulated. Both DE mRNAs and targets of DE lncRNAs were enriched in multiple biological and signal pathways potentially associated with oocyte meiosis. Highly abundantly expressed mRNAs (including DNMT1, UHRF2, PCNA, ARMC1, BTG4, ASNS and SEP11) and lncRNAs were also discovered. Weighted gene co-expression network analysis (WGCNA) revealed 20 hub mRNAs in three modules to be important for oocyte meiosis and maturation. Taken together, our findings provide insights and resources for further functional investigation of mRNAs/lncRNAs in in vitro meiotic maturation of pig oocytes.
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http://dx.doi.org/10.1111/rda.13901DOI Listing
April 2021

CoQ10 improves meiotic maturation of pig oocytes through enhancing mitochondrial function and suppressing oxidative stress.

Theriogenology 2021 Jan 9;159:77-86. Epub 2020 Oct 9.

College of Animal Science, Yangtze University, Jingzhou, 434025, Hubei, China. Electronic address:

Coenzyme Q10 (CoQ10) is essential to many fundamental biological processes. However, the effect of CoQ10 on meiotic maturation of pig oocytes still remains elusive. In the present study we aimed to understand the effects of CoQ10 on porcine oocyte maturation, by supplementing different concentrations of CoQ10 (25, 50 and 100 μM) into the maturation medium. We showed that CoQ10 at 50 μM had better capacity to promote the nuclear maturation of pig oocytes derived from both small and large antral follicles. Though the cleavage and blastocyst rates of parthenotes stayed stable, 50 μM CoQ10 treatment could accelerate the development of parthenotes to blastocyst stage, and increase the average cell number of blastocyst. For cumulus-oocyte complexes from large antral follicles categorized by the brilliant cresyl blue (BCB) test, 50 μM CoQ10 treatment could specifically promote the nuclear maturation of poor-quality oocytes in the BCB-negative group. Mitochondrial function of oocytes treated by 50 μM CoQ10 could be boosted, through increasing the levels of mitochondrial membrane potential, ATP production and CoQ6, and changing the pattern of mitochondrial distribution as well. Moreover, 50 μM CoQ10 treatment suppressed the level of reactive oxygen species and reduced the percentage of oocytes with early apoptosis signal. Taken together, CoQ10 could improve the meiotic maturation of pig oocytes, especially for poor-quality oocytes, mainly through enhancing mitochondrial function and suppressing oxidative stress to reduce apoptosis.
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http://dx.doi.org/10.1016/j.theriogenology.2020.10.009DOI Listing
January 2021

Ascorbic acid promotes the reproductive function of porcine immature Sertoli cells through transcriptome reprogramming.

Theriogenology 2020 Dec 29;158:309-320. Epub 2020 Sep 29.

College of Animal Science and Technology, Northeast Agricultural University, Harbin, 150030, Heilongjiang, China; College of Animal Science, Yangtze University, Jingzhou, 434025, Hubei, China. Electronic address:

Vitamin C (ascorbic acid, AA) can regulate antioxidation and affect many cellular processes. However, the effect of AA on the reproduction of male animals remains less explored. Here, we showed that by supplementing exogenous AA to porcine immature Sertoli cells (iSCs), AA could promote the proliferation, suppress apoptosis, and decrease the global nucleic acid methylation (5 mC and mA) levels of iSCs. After we profiled mRNA and long non-coding RNA (lncRNA) expression by transcriptome sequencing on iSCs (treated by 250 μM AA for 36 h), 1232 mRNAs and 937 lncRNAs were identified to be differentially expressed (DE). Gene enrichment analysis found multiple significantly enriched biological pathways, including oxidoreductase activity, cell proliferation and apoptosis, regulation of hormone level, regulation of catalytic activity, developmental process, ATP metabolism and reproductive process. Specifically, for the reproductive process, 49 up- and 36 down-regulated DE mRNAs (including highly expressed genes, such as Tfcp2l1, Hmgcs1, Mmp7, Fndc3a, and Zfp36l1) are involved. Moreover, AA supplementation could promote the secretion of anti-müllerian hormone, inhibin B and lactate, and enhance the activity of lactate dehydrogenase as well. Taken together, AA could promote the reproductive function of pig iSCs, potentially through reprogramming the global transcriptome, and elevating hormone secretion and metabolite production.
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http://dx.doi.org/10.1016/j.theriogenology.2020.09.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7524525PMC
December 2020

Single cell RNA-seq reveals molecular pathways altered by 7, 12-dimethylbenz[a]anthracene treatment on pig oocytes.

Theriogenology 2020 Nov 21;157:449-457. Epub 2020 Aug 21.

College of Animal Science, Yangtze University, Jingzhou, 434025, Hubei, China; College of Animal Science and Technology, Northeast Agricultural University, Harbin, 150030, Heilongjiang, China. Electronic address:

Oocytes of better quality and developmental competence are highly demanded, which is affected by many intrinsic and external factors, including environmental pollutants. We have previously demonstrated that 7, 12-dimethylbenz [a]anthracene (DMBA) reduces the developmental competence of porcine oocytes, by desynchronizing nuclear and ooplasmic maturation. However, the underlying molecular mechanism remains obscure. Here we performed single cell RNA-seq to study the transcriptome changes in DMBA-treated porcine MII oocytes, and identified 19 protein-coding genes and 156 novel long non-coding RNAs (lncRNAs) with abundance to be significantly different (P < 0.05), which enriched in signaling pathways such as glycosphingolipid biosynthesis, nicotine addiction, basal transcription factors and nucleotide excision repair. RT-qPCR on oocyte pools confirmed ornithine aminotransferase (Oat) and serine/arginine-rich splicing factor 4 (Srsf4) to be significantly up- and down-regulated, respectively (P < 0.05). Treating porcine COCs with MAPK and PLC pathway inhibitors suppressed DMBA's effects on increasing PB1 extrusion rate. In addition, DMBA co-incubation with 250 μM vitamin C derivative (l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate, AA2P) and 100 μM co-enzyme Q10 (CoQ10) could significantly reduce the DMBA-induced high ROS level, and partially alleviate the DMBA-induced high PB1 rate, whereas the cleavage and blastocyst rates of parthenotes derived from treated mature oocytes remained to be low. Collectively, our findings indicate that single cell RNA-seq can help reveal the dynamics of molecular signaling pathways for porcine oocytes treated by DMBA, and supplement of anti-oxidative reagents could not sufficiently rescue DMBA-induced defects of porcine oocytes.
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http://dx.doi.org/10.1016/j.theriogenology.2020.08.020DOI Listing
November 2020

Molecular characterization and expression analysis of tumor necrosis factor receptor-associated factor 6 (traf6) like gene involved in antibacterial innate immune of fresh water crayfish, Procambarus clarkii.

Fish Shellfish Immunol 2020 Sep 23;104:517-526. Epub 2020 Jun 23.

School of Life Science and Technology, Inner Mongolia University of Science and Technology, Baotou, Inner Mongolia autonomous region, 014010, China. Electronic address:

In invertebrates, innate immunity was the crucial defending pattern against pathogenic microorganisms. For the past few years, Toll or Toll like receptors (TLRs) signaling pathway was studied extensively in crustaceans. Among the components of Toll or Toll like receptors (TLRs) signaling pathway, tumor necrosis factor receptor-associated factor 6 (TRAF6) acted as an important cytoplasmic adaptor, which was conserved from Drosophila to human. In this study, a new traf6 like gene was cloned from hepatopancreas of P. clarkii. After challenged respectively by S. aureus or E. ictaluri, the expression profiles were studied. And the results showed that the mRNA transcript of Pc-traf6 like gene was up-regulated significantly in the hemocytes, hepatopancreas, gills, and intestine of crayfish. After Pc-traf6 like gene was knocked down, the expression levels of transcription factor (Dorsal) and some crucial immunity effectors (ALF 3, Lysozyme 1, Lectin 1, and Crustin 2) in TLRs signaling pathway were dramatically suppressed. Simultaneously, the survival rate of crayfish challenged respectively by S. aureus or E. ictaluri was significantly decreased in RNAi assay. All these results indicated that Pc-traf6 like gene played an important role in regulating the expression of downstream effectors in the TLRs signaling pathway of crayfish.
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http://dx.doi.org/10.1016/j.fsi.2020.06.027DOI Listing
September 2020

Common Gene Modules Identified for Chicken Adiposity by Network Construction and Comparison.

Front Genet 2020 29;11:537. Epub 2020 May 29.

College of Animal Science, Yangtze University, Jingzhou, China.

Excessive fat deposition can cause chicken health problem, and affect production efficiency by causing great economic losses to the industry. However, the molecular underpinnings of the complex adiposity trait remain elusive. In the current study, we constructed and compared the gene co-expression networks on four transcriptome profiling datasets, from two chicken lines under divergent selection for abdominal fat contents, in an attempt to dissect network compositions underlying adipose tissue growth and development. After functional enrichment analysis, nine network modules important to adipogenesis were discovered to be involved in lipid metabolism, PPAR and insulin signaling pathways, and contained hub genes related to adipogenesis, cell cycle, inflammation, and protein synthesis. Moreover, after additional functional annotation and network module comparisons, common sub-modules of similar functionality for chicken fat deposition were identified for different chicken lines, apart from modules specific to each chicken line. We further validated the lysosome pathway, and found and its downstream target genes showed similar expression patterns along with chicken preadipocyte differentiation. Our findings could provide novel insights into the genetic basis of complex adiposity traits, as well as human obesity and related metabolic diseases.
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http://dx.doi.org/10.3389/fgene.2020.00537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7272656PMC
May 2020

Rewiring central carbon metabolism for tyrosol and salidroside production in Saccharomyces cerevisiae.

Biotechnol Bioeng 2020 08 15;117(8):2410-2419. Epub 2020 May 15.

State Key Laboratory of Microbial Technology, Shandong University, Qingdao, China.

Metabolic engineering of Saccharomyces cerevisiae for high-level production of aromatic chemicals has received increasing attention in recent years. Tyrosol production from glucose by S. cerevisiae is considered an environmentally sustainable and safe approach. However, the production of tyrosol and salidroside by engineered S. cerevisiae has been reported to be lower than 2 g/L to date. In this study, S. cerevisiae was engineered with a push-pull-restrain strategy to efficiently produce tyrosol and salidroside from glucose. The biosynthetic pathways of ethanol, phenylalanine, and tryptophan were restrained by disrupting PDC1, PHA2, and TRP3. Subsequently, tyrosol biosynthesis was enhanced with a metabolic pull strategy of introducing PcAAS and EcTyrA . Moreover, a metabolic push strategy was implemented with the heterologous expression of phosphoketolase (Xfpk), and then erythrose 4-phosphate was synthesized simultaneously by two pathways, the Xfpk-based pathway and the pentose phosphate pathway, in S. cerevisiae. Furthermore, the heterologous expression of Xfpk alone in S. cerevisiae efficiently improved tyrosol production compared with the coexpression of Xfpk and phosphotransacetylase. Finally, the tyrosol yield increased by approximately 135-folds, compared with that of parent strain. The total amount of tyrosol and salidroside with glucose fed-batch fermentation was over 10 g/L and reached levels suitable for large-scale production.
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http://dx.doi.org/10.1002/bit.27370DOI Listing
August 2020

Long noncoding RNA 2193 regulates meiosis through global epigenetic modification and cytoskeleton organization in pig oocytes.

J Cell Physiol 2020 11 2;235(11):8304-8318. Epub 2020 Apr 2.

College of Animal Science, Yangtze University, Jingzhou, Hubei, China.

Long noncoding RNAs (lncRNAs) regulate a variety of physiological and pathological processes. However, the biological function of lncRNAs in mammalian germ cells remains largely unexplored. Here we identified one novel lncRNA (lncRNA2193) from single-cell RNA sequencing performed on porcine oocytes and investigated its function in oocyte meiosis. During in vitro maturation (IVM), from germinal vesicle (GV, 0 hr), GV breakdown (GVBD, 24 hr), to metaphase II stage (MII, 44 hr), the transcriptional abundance of lncRNA2193 remained stable and high. LncRNA2193 interference by small interfering RNA microinjection into porcine GV oocytes could significantly inhibit rates of GVBD and the first polar body extrusion, but enhance the rates of oocytes with a nuclear abnormality. Moreover, lncRNA2193 knockdown disturbed cytoskeletal organization (F-actin and spindle), and decreased DNA 5-methylcytosine (5mC) and histone trimethylation (H3K4me3, H3K9me3, H3K27me3, and H3K36me3) levels. The lncRNA2193 downregulation induced a decrease of 5mC level could be partially due to the reduction of DNA methyltransferase 3A and 3B, and the elevation of 5mC-hydroxylase ten-11 translocation 2 (TET2). After parthenogenetic activation of MII oocytes, parthenotes exhibited higher fragmentation but lower cleavage rates in the lncRNA2193 downregulated group. However, lncRNA2193 interference performed on mature MII oocytes and parthenotes at 1-cell stage did not affect the cleavage and blasctocyst rates of pathenotes. Taken together, lncRNA2193 plays an important role in porcine oocyte maturation, providing more insights for relevant investigations on mammalian germ cells.
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http://dx.doi.org/10.1002/jcp.29675DOI Listing
November 2020

Bouveret syndrome: A case report.

World J Clin Cases 2019 Dec;7(23):4144-4149

Department of Gastroenterology, People's Hospital of Jianyang City, Jianyang 641400, Sichuan Province, China.

Background: Bouveret syndrome is a rare complication of cholelithiasis, with only 315 cases reported in the literature between 1967 and 2016. Delay in diagnosis is associated with a high mortality rate. Diagnosis is based upon clinical manifestations, gastroscopy, and imaging studies such as abdominal computed tomography and magnetic resonance cholan-giopancreatography. Endoscopic stone extraction or lithotripsy is the preferred choice for treatment as it is safe and minimally invasive with few complications. However, if endoscopy fails, surgery is required.

Case Summary: A 61-year-old female patient presented with recurrent epigastric pain for more than 6 mo. On endoscopy, a large amount of food residue was present in the stomach with multiple stones and ulcers in the antro-pyloric region. Based on these findings, a diagnosis of gastrolithiasis was made. However, computed tomography of the abdomen revealed the correct diagnosis of Bouveret syndrome. Initially, endoscopic treatment was attempted but it failed. Later, she was successfully managed by cholecystectomy with duodenal stone extraction and fistula repair (one-step method). At the last follow-up 6 mo after surgery, the patient was symptom-free.

Conclusion: Bouveret syndrome is a rare complication of gallstones that requires prompt endoscopic or surgical treatment to prevent mortality.
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http://dx.doi.org/10.12998/wjcc.v7.i23.4144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6906555PMC
December 2019

A new antimicrobial peptide isoform, Pc-crustin 4 involved in antibacterial innate immune response in fresh water crayfish, Procambarus clarkii.

Fish Shellfish Immunol 2019 Nov 1;94:861-870. Epub 2019 Oct 1.

Key Laboratory of Inshore Resources Biotechnology (Quanzhou Normal University) Fujian Province University, Quanzhou, 362000, ‬China; College of Marine and Food Sciences, ‬Quanzhou Normal University, ‬Quanzhou, 362000, ‬China. Electronic address:

The main advantage of antimicrobial peptides (AMPs) used as the effectors in the innate immunity system of invertebrates is that the high specificity is not indispensable. And they play important roles in the systemic defenses against microbial invasion. In this study, a new full-length cDNA of the crustins molecule was identified in red swamp crayfish, P. clarkii (named Pc-crustin 4). The ORF of Pc-crustin 4 contained 369 bp which encoded a protein of 122 amino acids, with a 20-amino-acid signal peptide sequence. On the base of the classification method established by Smith et al., Pc-crustin 4 belonged to Type Ⅰ crustin molecule. The Pc-crustin 4 transcripts were expressed in hemocytes at relatively high level, and relatively low level in hepatopancreas, gills, and intestine in normal crayfish. After respectively challenged with S. aureus or E. ictaluri, the expression levels of Pc-crustin 4 showed up-regulation trends at different degrees in the hemocytes, hepatopancreas, gills, and intestine tissues. Besides, the results of liquid antibacterial assay showed that rPc-crustin 4 inhibited obviously the growth of S. aureus and E. ictaluri. The results of bacteria binding assay showed that rPc-crustin 4 could bind strongly to S. aureus and E. ictaluri. Finally, RNAi assay was performed to study the immunity roles of Pc-crustin 4 in crayfish in vivo. Taken together, Pc-crustin 4 is an important immunity effector molecule, which plays crucial roles in defending against bacterial infection in crayfish.
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http://dx.doi.org/10.1016/j.fsi.2019.10.003DOI Listing
November 2019

Lysosomal dysfunction disturbs porcine oocyte maturation and developmental capacity by disorganizing chromosome/cytoskeleton and activating autophagy/apoptosis.

Theriogenology 2019 Dec 13;140:44-51. Epub 2019 Aug 13.

Key Laboratory of Animal Cellular and Genetic Engineering of Heilongjiang Province, Northeast Agricultural University, Harbin, 150030, Heilongjiang, China; College of Animal Science and Technology, Northeast Agricultural University, Harbin, 150030, Heilongjiang, China. Electronic address:

Lysosome, an important organelle in eukaryotes, can sequester macromolecules submitted by the endocytosis and autophagy pathways for degradation and recycling. Massive macromolecular turnover is also vital to the growth and development of mammalian oocytes. However, the functional role of lysosomes in the meiotic maturation of mammalian oocytes remains largely unexplored. Here, by treating in vitro matured porcine cumulus-oocyte complexes (COCs) with chloroquine (CQ), a lysosome inhibitor, we showed that regardless of CQ concentration, lysosomal inhibition affected neither the extrusion of the first polar body (PB1), nor the ROS levels. However, CQ treatment dramatically decreased the rates of oocytes with normal chromosome alignment and cytoskeleton organization (P < 0.05), but boosted the rates of oocytes with apoptosis (P < 0.05). Subsequently, after pathenogenetic activation or in vitro fertilization, the death or fragmentation rates of oocytes treated by CQ (both 35 μM and 45 μM) were significantly higher (P < 0.05), whereas the rates of embryo cleavage, embryos developed to blastocysts, and average blastomere number per blastocyst, were all significantly lower (P < 0.05), respectively. Furthermore, CQ (35 μM) treatment activated the autophagy pathway by elevating the LC3 II/I ratio. Taken together, lysosomes could affect porcine oocyte maturation and subsequent developmental capacity partially through the chromosome organization/cytoskeleton assembly and autophagy/apoptosis pathways.
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http://dx.doi.org/10.1016/j.theriogenology.2019.08.019DOI Listing
December 2019

Molecular characterization and expression analysis of dopa decarboxylase involved in the antibacterial innate immunity of the freshwater crayfish, Procambarus clarkii.

Fish Shellfish Immunol 2019 Aug 8;91:19-28. Epub 2019 May 8.

School of Life Science and Technology, Inner Mongolia University of Science and Technology, Baotou, Inner Mongolia Autonomous Region, 014010, China. Electronic address:

Dopa decarboxylase (DDC) is responsible for the synthesis of dopamine, which acts as an important modulator in the nervous systems of vertebrates and invertebrates. Recent studies have indicated that DDC also plays crucial roles in the insect innate immune system. However, the functions of DDC in immunomodulation in crustaceans have not been thoroughly elucidated to date. In this study, a new full-length cDNA of the DDC protein was identified from red swamp crayfish, Procambarus clarkii (named Pc-ddc). The ORF of Pc-ddc encoded 474 amino acids, which possessed a 377-amino-acid domain. Pc-ddc was expressed at a relatively high level in the hemocytes and gills of crayfish. This protein was expressed at a relatively low level in the hepatopancreas and intestine. The expression level of Pc-ddc was clearly upregulated in hemocytes, hepatopancreas, gills, and intestine tissues after challenge with S. aureus or E. ictaluri. The results of the enzyme catalysis assay showed that the enzyme catalysis activity of rPc-DDC was 35 ± 2.8 ng h mg (n = 3). In addition, the results of the mimetic crayfish hemocytes encapsulation assay showed that the encapsulation rate of beads coated with rPc-DDC was clearly increased. The results of the bacterial binding assay showed that rPc-DDC strongly binds to S. aureus and E. ictaluri. Finally, when Pc-ddc was knocked down, the number of surviving crayfish clearly decreased after S. aureus or E. ictaluri was injected. All of these results indicate that Pc-DDC is an important immunomodulating enzyme in the neuroendocrine-immune (NEI) system of crayfish.
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http://dx.doi.org/10.1016/j.fsi.2019.05.013DOI Listing
August 2019

A new crustin homologue (SpCrus6) involved in the antimicrobial and antiviral innate immunity in mud crab, Scylla paramamosain.

Fish Shellfish Immunol 2019 Jan 28;84:733-743. Epub 2018 Oct 28.

East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai, 200090, China; Key Laboratory of East China Sea Fishery Resources Exploitation, Ministry of Agriculture, Shanghai, 200090, China. Electronic address:

Crustins play important roles in defending against bacteria in the innate immunity system of crustaceans. In present study, we identified a crustin gene in Scylla paramamosain, which was named as SpCrus6. The ORF of SpCrus6 possessed a signal peptide sequence (SPS) at the N-terminus and a WAP domain at the C-terminus. And there were 5 Proline residues, 5 Glycine and 4 Cysteine residues between SPS and WAP domain in SpCrus6. These features indicated that SpCrus6 was a new member of crustin family. The SpCrus6 mRNA transcripts were up-regulated obviously after bacteria or virus challenge. These changes showed that SpCrus6 was involved in the antimicrobial and antiviral responses of Scylla paramamosain. Recombinant SpCrus6 (rSpCrus6) showed strong inhibitory abilities against Gram-positive bacteria (Bacillus megaterium, Staphylococcus aureus, and Bacillus subtilis). But the inhibitory abilities against four Gram-negative bacteria (Vibrio parahemolyticus, Vibrio alginolyticus, Vibrio harveyi and Escherichia coli) and two fungi (Pichia pastoris and Candida albicans) were not strong enough. Besides, rSpCrus6 could strongly bind to two Gram-positive bacteria (B. subtilis and B. megaterium) and three Gram-negative bacteria (V. alginolyticus, V. parahemolyticus, and V. harveyi). And the binding levels to S. aureus and two fungi (P. pastoris and C. albicans) were weak. The polysaccharides binding assays' results showed rSpCrus6 had superior binding activities to LPS, LTA, PGN and β-glucan. Through agglutinating assays, we found rSpCrus6 could agglutinate well three Gram-positive bacteria (S. aureus, B. subtilis and B. megaterium). And the agglutinating activities to Gram-negative bacteria and fungi were not found. In the aspect of antiviral functions, rSpCrus6 could bind specifically to the recombinant envelop protein 26 (rVP26) of white spot syndrome virus (WSSV) but not to recombinant envelop protein 28 (rVP28), whereas GST protein could not bind to rVP26 or rVP28. Besides, rSpCrus6 could suppress WSSV reproduction to some extent. Taken together, SpCrus6 was a multifunctional immunity effector in the innate immunity defending response of S. paramamosain.
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http://dx.doi.org/10.1016/j.fsi.2018.10.072DOI Listing
January 2019

High-throughput sequencing analysis of microRNAs in gills of red swamp crayfish, Procambarus clarkii infected with white spot syndrome virus.

Fish Shellfish Immunol 2018 Dec 7;83:18-25. Epub 2018 Sep 7.

School of Life Science and Technology, Inner Mongolia University of Science and Technology, Baotou, Inner Mongolia autonomous region, 014010, China. Electronic address:

MicroRNAs (miRNAs) are important posttranscriptional regulators. They play an important role in the antiviral innate immunity of invertebrates. In the present study, high-throughput small RNAs Illumina sequencing systems were carried out to identify differentially expressed miRNAs (DEMs) in the gills of Procambarus clarkii, which was challenged with white spot syndrome virus (WSSV). Our results identified 11,617 known and 6 novel miRNAs in normal group (NG) and WSSV-challenged group (WG) small RNA libraries. Additionally, 27 DEMs were shown to participate in the antiviral innate immunity of P. clarkii and were significantly upregulated or downregulated. In addition, the results of the KEGG pathway prediction of the DEMs target genes showed that putative target genes of these 27 DEMs were related mainly to the RNA transport pathway, tight junction pathway, mRNA surveillance pathway, regulation actin cytoskeleton pathway, focal adhesion pathway, and MAPK signaling pathway. These results provide important information for future studies about the antiviral innate immunity of crustaceans.
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http://dx.doi.org/10.1016/j.fsi.2018.09.007DOI Listing
December 2018

Reduced nucleic acid methylation impairs meiotic maturation and developmental potency of pig oocytes.

Theriogenology 2018 Nov 16;121:160-167. Epub 2018 Aug 16.

College of Animal Science and Technology, Northeast Agricultural University, Harbin, 150030, Heilongjiang, China. Electronic address:

Oocyte meiosis is a complex process coordinated by multiple endocrinal and molecular circuits. Recently, N-methyladenosine (mA) epigenetic modification on RNA is revealed to be important for meiotic maturation. However, the molecular mechanism of how mA modification exerts its effect on oocyte maturation is largely unknown. Here, we showed that endogenous mA writers (Mettl3 and Wtap) and eraser (Fto) elevated their transcript levels during meiotic maturation of pig oocytes. From germinal vesicle (GV) to metaphase II (MII) stages, global mA level significantly increased, and existed mostly in ooplasm. Methyl donor (betaine, 16 mM) treatment of porcine cumulus-oocyte complexes (COCs) during in vitro maturation (IVM) significantly boosted nucleic acid mA level within oocytes, but unchanged meiotic process and oocyte subsequent development. By contrast, methylation inhibitor (cycloleucine, 20 mM) reduced nucleic acid mA level, and significantly decreased the germinal vesicle breakdown (GVBD) rate, the extrusion rate of the first polar body, and the cleavage and blastocyst rates of parthenotes. In addition, in cycloleucine-treated oocytes Wtap increased but Lin28 decreased their abundances significantly, along with the higher incidence of spindle defects and chromosome misalignment. Furthermore, pT161-CDK1 protein level in pig oocytes was confirmed to be decreased after cycloleucine treatment for 24 h. Taken together, chemical induced reduction of nucleic acid mA methylation during pig oocyte meiosis could impair meiotic maturation and subsequent development potency, possibly through down-regulating pluripotency marker Lin28 mRNA abundance and disturbing MPF-regulated chromosome/spindle organization.
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http://dx.doi.org/10.1016/j.theriogenology.2018.08.009DOI Listing
November 2018

Production of transgenic broilers by non-viral vectors via optimizing egg windowing and screening transgenic roosters.

Poult Sci 2019 Jan;98(1):430-439

Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture and Rural Affairs, Harbin 150030, Heilongjiang, China.

The generation of transgenic chickens is of both biomedical and agricultural significance, and recently chicken transgenesis technology has been greatly advanced. However, major issues still exist in the efficient production of transgenic chickens. This study was designed to optimize the production of enhanced green fluorescence protein (EGFP)-transgenic broilers, including egg windowing at the blunt end (air cell) of egg, and the direct transfection of circulating primordial germ cells by microinjection of the Tol2 plasmid-liposome complex into the early embryonic dorsal aorta. For egg windowing, we discovered that proper manipulation of the inner shell membrane at the blunt end could improve the rate of producing G0 transgenic roosters. From 27 G0 roosters, we successfully collected semen with EGFP-positive sperms from 16 and 19 roosters after direct fluorescence observation and fluorescence-activated cell sorting analyses (13 detected by both methods), respectively. After artificial insemination using the G0 rooster with the highest number of EGFP fluorescent sperm, one G1 EGFP transgenic broiler (1/81, 1.23%) was generated. Our results indicate that appropriate egg windowing and screening of potentially transgene-positive roosters can improve the production of germline-transmitted transgenic birds.
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http://dx.doi.org/10.3382/ps/pey321DOI Listing
January 2019

Rational approach to improve ansamitocin P-3 production by integrating pathway engineering and substrate feeding in Actinosynnema pretiosum.

Biotechnol Bioeng 2018 10 20;115(10):2456-2466. Epub 2018 Jul 20.

State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China.

Ansamitocin P-3 (AP-3) produced by Actinosynnema pretiosum is an important antitumor agent for cancer treatment, but its market supply suffers from a low production titer. The role of AP-3 unusual glycolate unit supply on its biosynthesis was investigated in this work by overexpressing the responsible gene cluster asm13-17 in A. pretiosum (WT). As a result, the accumulation of AP-3 and its intermediate glyceryl-S-ACP in the asm13-17-overexpressed strain (Oasm13-17) versus WT was enhanced by 1.94 and 1.49-fold, respectively. To provide a higher supply of another precursor 3-amino-5-hydroxybenzoic acid, asmUdpg was also overexpressed in Oasm13-17 (Oasm13-17:asmUdpg), and an improved AP-3 titer of 680.5 mg/L was achieved in shake flasks. To further enhance the AP-3 titer, a rational fed-batch strategy was developed in bioreactor fermentation of Oasm13-17:asmUdpg; and by pulse feeding 15 g/L fructose and 1.64 g/L isobutanol at 60, 96, and 120 hr, the AP-3 production level reached 757.7 mg/L, which is much higher than ever reported in bioreactors. This work demonstrated that a rational approach combining precursor pathway engineering with substrate feeding was very effective in enhancing the AP-3 titer, and this enabling methodology would be helpful to industrial production of this eye-catching drug.
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http://dx.doi.org/10.1002/bit.26775DOI Listing
October 2018

Heat shock protein 90α couples with the MAPK-signaling pathway to determine meiotic maturation of porcine oocytes.

J Anim Sci 2018 Jul;96(8):3358-3369

College of Animal Science and Technology, Northeast Agricultural University, Harbin, Heilongjiang, China.

Heat shock protein 90 (Hsp90) functions as a molecular chaperone in its interaction with clients to influence multiple cellular and physiological processes. However, our current understanding on Hsp90's relationship with mammalian oocyte maturation is still very limited. Here, we aimed to investigate Hsp90's effect on pig oocyte meiotic maturation. Endogenous Hsp90α was constantly expressed at both mRNA and protein levels in porcine maturing oocytes. Addition of 2 µM 17-allylamino-17-demethoxygeldanamycin (17-AAG), the Hsp90 inhibitor, to in vitro mature cumulus-oocyte complexes (COC) significantly decreased Hsp90α protein level (P < 0.05), delayed germinal vesicle breakdown (GVBD) (P < 0.05), and impeded the first polar body (PB1) extrusion (P < 0.01) of porcine oocytes. 2 µM 17-AAG treatment during in vitro maturation also decreased the subsequent development competence as indicated by the lower cleavage (P < 0.001) and higher fragmentation (P < 0.001) rates of parthenotes, whereas no effects on the percentage and average cell number of blastocysts were found. Immunodepletion of Hsp90α by antibody microinjection into porcine oocytes at germinal vesicle and metaphase II stages induced similar defects of meiotic maturation and parthenote development, to that resulted from 2 µM inhibitor 17-AAG. For oocytes treated by 2 µM 17-AAG, the cytoplasm and membrane actin levels were weakened (P < 0.01), and the spindle assembly was disturbed (P < 0.05), due to decreased p-ERK1/2 level (P < 0.05). However, the mitochondrial function and early apoptosis were not affected, as demonstrated by rhodamine 123 staining and Annexin V assays. Our findings indicate that Hsp90α can couple with mitogen-activated protein kinase to regulate cytoskeletal structure and orchestrate meiotic maturation of porcine oocytes.
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http://dx.doi.org/10.1093/jas/sky213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6095267PMC
July 2018

Embryonic transcriptome and proteome analyses on hepatic lipid metabolism in chickens divergently selected for abdominal fat content.

BMC Genomics 2018 May 23;19(1):384. Epub 2018 May 23.

Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture, Key Laboratory of Animal Genetics, Breeding and Reproduction, Education Department of Heilongjiang Province, College of Animal Science and Technology, Northeast Agricultural University, Harbin, 150030, People's Republic of China.

Background: In avian species, liver is the main site of de novo lipogenesis, and hepatic lipid metabolism relates closely to adipose fat deposition. Using our fat and lean chicken lines of striking differences in abdominal fat content, post-hatch lipid metabolism in both liver and adipose tissues has been studied extensively. However, whether molecular discrepancy for hepatic lipid metabolism exists in chicken embryos remains obscure.

Results: We performed transcriptome and proteome profiling on chicken livers at five embryonic stages (E7, E12, E14, E17 and E21) between the fat and lean chicken lines. At each stage, 521, 141, 882, 979 and 169 differentially expressed genes were found by the digital gene expression, respectively, which were significantly enriched in the metabolic, PPAR signaling and fatty acid metabolism pathways. Quantitative proteomics analysis found 20 differentially expressed proteins related to lipid metabolism, PPAR signaling, fat digestion and absorption, and oxidative phosphorylation pathways. Combined analysis showed that genes and proteins related to lipid transport (intestinal fatty acid-binding protein, nucleoside diphosphate kinase, and apolipoprotein A-I), lipid clearance (heat shock protein beta-1) and energy metabolism (NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 and succinate dehydrogenase flavoprotein subunit) were significantly differentially expressed between the two lines.

Conclusions: For hepatic lipid metabolism at embryonic stages, molecular differences related to lipid transport, lipid clearance and energy metabolism exist between the fat and lean chicken lines, which might contribute to the striking differences of abdominal fat deposition at post-hatch stages.
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http://dx.doi.org/10.1186/s12864-018-4776-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5966864PMC
May 2018

Ascorbic acid induces global epigenetic reprogramming to promote meiotic maturation and developmental competence of porcine oocytes.

Sci Rep 2018 04 17;8(1):6132. Epub 2018 Apr 17.

College of Animal Science and Technology, Northeast Agricultural University, Harbin, 150030, Heilongjiang, China.

L-ascorbic acid (Vitamin C) can enhance the meiotic maturation and developmental competence of porcine oocytes, but the underlying molecular mechanism remains obscure. Here we show the role of ascorbic acid in regulating epigenetic status of both nucleic acids and chromatin to promote oocyte maturation and development in pigs. Supplementation of 250 μM L-ascorbic acid 2-phosphate sesquimagnesium salt hydrate (AA2P) during in vitro maturation significantly enhanced the nuclear maturation (as indicated by higher rate of first polar body extrusion and increased Bmp15 mRNA level), reduced level of reactive oxygen species, and promoted developmental potency (higher cleavage and blastocyst rates of parthenotes, and decreased Bax and Caspase3 mRNA levels in blastocysts) of pig oocytes. AA2P treatment caused methylation erasure in mature oocytes on nucleic acids (5-methylcytosine (5 mC) and N -methyladenosine (mA)) and histones (Histone H3 trimethylations at lysines 27, H3K27me3), but establishment of histone H3 trimethylations at lysines 4 (H3K4me3) and 36 (H3K36me3). During the global methylation reprogramming process, levels of TET2 (mRNA and protein) and Dnmt3b (mRNA) were significantly elevated, but simultaneously DNMT3A (mRNA and protein), and also Hif-1α, Hif-2α, Tet3, Mettl14, Kdm5b and Eed (mRNA) were significantly inhibited. Our findings support that ascorbic acid can reprogram the methylation status of not only DNA and histone, but also RNA, to improve pig oocyte maturation and developmental competence.
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http://dx.doi.org/10.1038/s41598-018-24395-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5904140PMC
April 2018

Single-cell transcriptome sequencing reveals that cell division cycle 5-like protein is essential for porcine oocyte maturation.

J Biol Chem 2018 02 8;293(5):1767-1780. Epub 2017 Dec 8.

From the Key Laboratory of Animal Cellular and Genetic Engineering of Heilongjiang Province, College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China

The brilliant cresyl blue (BCB) test is used in both basic biological research and assisted reproduction to identify oocytes likely to be developmentally competent. However, the underlying molecular mechanism targeted by the BCB test is still unclear. To explore this question, we first confirmed that BCB-positive porcine oocytes had higher rates of meiotic maturation, better rates of cleavage and development into blastocysts, and lower death rates. Subsequent single-cell transcriptome sequencing on porcine germinal vesicle (GV)-stage oocytes identified 155 genes that were significantly differentially expressed between BCB-negative and BCB-positive oocytes. These included genes such as , , , , , , and , which are enriched in functionally important signaling pathways including cell cycle regulation, oocyte meiosis, spliceosome formation, and nucleotide excision repair. In BCB-positive GV oocytes that additionally had a lower frequency of DNA double-strand breaks, the CDC5L protein was significantly more abundant. /CDC5L inhibition by short interference (si)RNA or antibody microinjection significantly impaired porcine oocyte meiotic maturation and subsequent parthenote development. Taken together, our single-oocyte sequencing data point to a potential new role for CDC5L in porcine oocyte meiosis and early embryo development, and supports further analysis of this protein in the context of the BCB test.
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http://dx.doi.org/10.1074/jbc.M117.809608DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798306PMC
February 2018

Identification and characterization of intestine microRNAs and targets in red swamp crayfish, Procambarus clarkii infected with white spot syndrome virus.

PLoS One 2017 9;12(11):e0187760. Epub 2017 Nov 9.

East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai, China.

MicroRNAs (miRNAs) are small non-coding endogenous RNA molecules that play important roles in the innate immunity system of invertebrates, especially in the aspect of antivirus. In the present study, high-throughput small RNA Illumina sequencing systems were used to identify differentially expressed miRNAs (DEMs) from the intestines of Procambarus clarkii that were infected with white spot syndrome virus (WSSV). As a result, 39 known and 12 novel miRNAs were identified in both NG and WG small RNA libraries. Seven DEMs were determined to be involved in the antiviral innate immunity in the intestines of P. clarkii. The results of the target gene predictions of the DEMs showed that the putative target genes of these 7 DEMs are related to tight junctions, vascular smooth muscle contraction regulation of the actin cytoskeleton, focal adhesion, RNA transport, mRNA surveillance, viral carcinogenesis, and Salmonella infection. These results provide theoretical insights for future studies on the antiviral immunity of crustaceans.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187760PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5679607PMC
November 2017
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