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Effects of lysophosphatidic acid on human periodontal ligament stem cells from teeth extracted from dental patients

The Journal of Biomedical Research 2019年第2期33卷 122-130页

作者： Byung Cheol Kim Jae-In Song Kyoung-Ha So Sang-Hwan Hyun Laboratory of Veterinary Embryology and Biotechnology Veterinary Medical Center and College of Veterinary MedicineChungbuk National University Institute of Stem Cell & Regenerative Medicine Chungbuk National University

Despite their potential applications in future regenerative medicine, periodontal ligament stem cells(PDLSCs) are difficult to obtain in large amounts from patients. Therefore, maintaining sternness while expanding the cell numbers for medical use is the key to transitioning PDLSCs from the bench to the clinic. Lysophosphatidic acid(LPA), which is present in the human body and saliva, is a signaling molecule derived from phospholipids. In this study, we examined the effects of LPA on sternness maintenance in human PDLSCs. Several spindle-shaped and fibroblast-like periodontal ligament stem-like cell lines were established from PDLSC isolation. Among these cell lines, the most morphologically appropriate cell line was characterized. The expression levels of OCT4, NANOG(a stem cell marker), and CD90(a mesenchymal stem cell marker) were high. However, CD73(a negative marker of mesenchymal stem cells) expression was not observed. Notably, immunofluorescence analysis identified the expression of STRO-1, CD146(a mesenchymal stem cell marker), and sex determining region Y-box 2 at the protein level. In addition, lipid droplets were stained by Oil red O after the induction of adipogenesis for 21 days, and mineralized nodules were stained by Alizarin Red S after the induction of osteogenesis for 14 days. Alkaline phosphate staining also demonstrated the occurrence of osteogenesis. In summary, we established a human PDLSC line, which could be applied as a cell source for tissue regeneration in dental patients. However, further studies are needed to determine the detailed effects of LPA on PDLSCs.

关键词： periodontal ligament stem cell lysophosphatidic acid stemness primary cell culture

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Effect of zeaxanthin on porcine embryonic development during in vitro maturation

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The Journal of Biomedical Research 2017年第2期31卷 154-161页

作者： Seo-jin Park Kyoung-Ha So Sang-Hwan Hyun Laboratory of Veterinary Embryology and Biotechnology Veterinary Medical Center and College of Veterinary Medicine Chungbuk National University Seowon-gu Cheonglu 28644 Republic of Korea Institute of Stem Cell & Regenerative Medicine Chungbuk National University Seowon-gu Cheongiu 28644 Republic of Korea

Zeaxanthin is a common carotenoid, which is a powerful antioxidant that protects against damage caused by reactive oxygen species. The aim of the present study was to investigate the effects of zeaxanthin supplementation on in vitro maturation of porcine embryo development. We investigated nuclear maturation, intracellular glutathione （GSH）, and reactive oxygen species （ROS） levels during in vitro maturation, and subsequent embryonic development following parthenogenetic activation and in vitro fertilization OVF）. The oocytes were maturated and used at the metaphase II stage. After 42 hours of in vitro maturation, the zeaxanthin-treated group （0.5 μmol/L） showed significant increases in nuclear maturation （89.6%） than the control group （83.4%）（P 〈 0.05）. The intracellular GSH levels increased significantly （P 〈 0.05） as zeaxanthin concentrations increased; ROS generation levels decreased with increased zeaxanthin concentrations, but there were no significant differences. There were no significant differences in subsequent embryonic development, cleavage rate, blastocyst stage rate, and total blastocyst cell numbers following parthenogenetic activation and IVF when in vitro maturation media was supplemented with zeaxanthin. These results suggest that treatment with zeaxanthin during in vitro maturation improved the nuclear maturation of porcine oocytes by increasing the intracellular GSH level, thereby slightly decreasing the intracellular ROS level.

关键词： in vitro maturation porcine embryonic development zeaxanthin antioxidant

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Continued evolution of the Eurasian avian-like H1N1 swine influenza viruses in China

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Science China(Life Sciences) 2023年第2期66卷 269-282页

作者： Fei Meng Yan Chen Zuchen Song Qiu Zhong Yijie Zhang Chuanling Qiao Cheng Yan Huihui Kong Liling Liu Chengjun Li Huanliang Yang Hualan Chen State Key laboratory of veterinary biotechnology Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069China

Animal influenza viruses continue to pose a threat to human public health. The Eurasian avian-like H1N1(EA H1N1) viruses are widespread in pigs throughout Europe and China and have caused human infections in several countries, indicating their pandemic potential. To carefully monitor the evolution of the EA H1N1 viruses in nature, we collected nasal swabs from 103,110pigs in 22 provinces in China between October 2013 and December 2019, and isolated 855 EA H1N1 viruses. Genomic analysis of 319 representative viruses revealed that these EA H1N1 viruses formed eight different genotypes through reassortment with viruses of other lineages circulating in humans and pigs, and two of these genotypes(G4 and G5) were widely distributed in pigs.Animal studies indicated that some strains have become highly pathogenic in mice and highly transmissible in ferrets via respiratory droplets. Moreover, two-thirds of the EA H1N1 viruses reacted poorly with ferret serum antibodies induced by the currently used H1N1 human influenza vaccine, suggesting that existing immunity may not prevent the transmission of the EA H1N1 viruses in humans. Our study reveals the evolution and pandemic potential of EA H1N1 viruses and provides important insights for future pandemic preparedness.

关键词： H1N1 influenza immunity

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H7N9 virus infection triggers lethal cytokine storm by activating gasdermin E-mediated pyroptosis of lung alveolar epithelial cells

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National Science Review 2022年第1期9卷 47-55页

作者： Xiaopeng Wan Jiqing Li Yupeng Wang Xiaofei Yu Xijun He Jianzhong Shi Guohua Deng Xianying Zeng Guobin Tian Yanbing Li Yongping Jiang Yuntao Guan Chengjun Li Feng Shao Hualan Chen State Key laboratory of veterinary biotechnology Harbin Veterinary Research Institute Chinese Academy of Agricultural Sciences National Institute of Biological Sciences

The H7 N9 influenza virus emerged in China in 2013,causing more than 1560 human infections,39% of which were fatal.A ’cytokine storm’ in the lungs of H7 N9 patients has been linked to a poor prognosis and death;however,the underlying mechanism that triggers the cytokine storm is unknown.Here,we found that efficient replication of the H7 N9 virus in mouse lungs activates gasdermin E(GSDME)-mediated pyroptosis in alveolar epithelial cells,and that the released cytosolic contents then trigger a cytokine storm.Knockout of Gsdme switched the manner of death of A549 and human primary alveolar epithelial cells from pyroptosis to apoptosis upon H7 N9 virus infection,and Gsdme knockout mice survived H7 N9 virus lethal infection.Our findings reveal that GSDME activation is a key and unique mechanism for the pulmonary cytokine storm and lethal outcome of H7 N9 virus infection and thus opens a new door for the development of antivirals against the H7 N9 virus.

关键词： H7N9 virus cytokine storm gasdermin E pyroptosis

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TRIM35 mediates protection against influenza infection by activating TRAF3 and degrading viral PB2

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Protein & Cell 2020年第12期11卷 894-914页

作者： Nan Sun Li Jiang Miaomiao Ye Yihan Wang Guangwen Wang Xiaopeng Wan Yuhui Zhao Xia Wen Libin Liang Shujie Ma Liling Liu Zhigao Bu Hualan Chen Chengjun Li State Key laboratory of veterinary biotechnology Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069China

Tripartite motif(TRIM)family proteins are important effectors of innate immunity against viral infections.Here we identified TRIM35 as a regulator of TRAF3 activation.Deficiency in or inhibition of TRIM35 suppressed the production of type I interferon(IFN)in response to viral infection.777m35-deficient mice were more susceptible to influenza A virus(IAV)infection than were wild-type mice.TRIM35 promoted the RIG-Imediated signaling by catalyzing Lys63-linked polyubiquitination of TRAF3 and the subsequent formation of a signaling complex with VISA and TBK1.IAV PB2 polymerase countered the innate antiviral immune response by impeding the Lys63-linked polyubiquitination and activation of TRAF3.TRIM35 mediated Lys48-linked polyubiquitination and proteasomal degradation of IAV PB2,thereby antagonizing its suppression of TRAF3 activation.Our in vitro and in vivo findings thus reveal novel roles of TRIM35,through catalyzing Lys63-or Lys48-linked polyubiquitination,in RIG-I antiviral immunity and mechanism of defense against IAV infection.

关键词： influenza A virus PB2 TRIM35 TRAF3 ubiquitination antiviral immunity

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Next-generation sequencing and single-cell RT-PCR reveal a distinct variable gene usage of porcine antibody repertoire following PEDV vaccination

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Science China(Life Sciences) 2020年第8期63卷 1240-1250页

作者： Ren Li Fang Fu Li Feng PingHuang Liu State Key laboratory of veterinary biotechnology Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150001China

Porcine epidemic diarrhea virus(PEDV)is the most common diarrhea-causing pathogen in newborn piglets.The clarifications of the overall antibody repertoire and antigen-specific antibody repertoire are essential to provide important insights into the B-cell response and reshape new vaccines.Here,we applied next-generation sequencing(NGS)technology to investigate immunoglobulin(Ig)variable(V)gene segment usage of swine B-cells from peripheral blood lymphocytes(PBL)and mesenteric lymph node(MLN)cells following PEDV vaccination.We identified the transcripts of all functional Ig V-genes in antibody repertoire.IgHV1 S2,IgKV1-11,and IgLV3-4 were the most prevalent gene segments for heavy,kappa,and lambda chains,respectively,in PBL and MLN.Unlike previous studies,IgKV1,instead of IgKV2,and IgLV3,instead of IgLV8,were the prevalent Ig V-gene families for kappa and lambda light chains,respectively.We further examined the antibody repertoire of PEDV spike-specific B cells by single-cell RT-PCR.In contrast to the overall antibody repertoire,Ig V-gene segments of PEDV spike-specific B cells preferentially adopted IgHV1-4 and IgHV1-14 for heavy chain,IgKV1-11 for kappa chain,and IgLV3-3 for lambda chain.These results represent a comprehensive analysis to characterize the Ig V-gene segment usage in the overall and PEDV spike-specific antibody repertoire in PBL and MLN.

关键词： porcine epidemic diarrhea virus(PEDV) next-generation sequencing(NGS) single-cell RT-PCR antibody repertoire

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Generation and application of two monoclonal antibodies targeting conserved linear epitopes in the NP protein of influenza A virus

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Journal of Integrative Agriculture 2022年第7期21卷 2095-2105页

作者： ZHAO Yu-hui WEN Xia LI Qi-bing JIANG Li WANG Guang-wen LIANG Li-bin WANG Xiu-rong CHEN Hua-lan LI Cheng-jun State Key laboratory of veterinary biotechnology Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069P.R.China

Monoclonal antibodies(mAbs) are widely used in virus research and disease diagnosis. The nucleoprotein(NP) of influenza A virus(IAV) plays important roles in multiple stages of the virus life cycle. Therefore, generating conserved mAbs against NP and characterizing their properties will provide useful tools for IAV research. In this study, two mAbs against the NP protein, 10 E9 and 3 F3, were generated with recombinant truncated NP proteins(NP-1 and NP-2) as immunogens. The heavy-chain subclass of both 10 E9 and 3 F3 was determined to be IgG2α, and the light-chain type was κ. Truncation and site-specific mutation analyses showed that the epitopes of mAbs 10 E9 and 3 F3 were located in the N terminal 84–89 amino acids and the C terminal 320–324 amino acids of the NP protein, respectively. We found that mAbs 10 E9 and 3 F3 reacted well with the NP protein of H1–H15 subtypes of IAV. Both 10 E9 and 3 F3 can be used in immunoprecipitation assay, and 10 E9 was also successfully applied in confocal microscopy. Furthermore, we found that the 10 E9-recognized _(84) SAGKDP_(89) epitope and 3 F3-recognized 320 ENPAH324 epitope were highly conserved in NP among all avian and human IAVs. Thus, the two mAbs we developed could be used as powerful tools in the development of diagnostic methods of IAV, and also surely promote the basic research in understanding the replication mechanisms of IAV.

关键词： influenza A virus nucleoprotein monoclonal antibody application

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Generation and application of replication-competent Venus-expressing H5N1,H7N9,and H9N2 influenza A viruses

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Science Bulletin 2018年第3期63卷 176-186页

作者： guangwen wang jie zhang fandi kong qibing li jinliang wang shujie ma yuhui zhao libin liang junping li nan sun lizheng guan yuan zhou chenchen zhou shanyu huang zhigao bu li jiang hualan chen chengjun li State Key laboratory of veterinary biotechnology Harbin Veterinary Research Institute Chinese Academy of Agricultural Sciences Harbin 150069 China

The generation and application of replication-competent influenza A virus(IAV) expressing a reporter gene represent a valuable tool to elucidate the mechanism of viral pathogenesis and establish new countermeasures to combat the threat of influenza. Here, replication-competent IAVs with a neuraminidase(NA) segment harboring a fluorescent reporter protein, Venus, were generated in the background of H5N1, H7N9, and H9N2 influenza viruses, the three subtypes of viruses with imminent pandemic potential. All three reporter viruses maintained virion morphology, replicated with similar or slightly reduced titers relative to their parental viruses, and stably expressed the fluorescent signal for at least two passages in embryonated chicken eggs. As a proof of concept, we demonstrated that these reporter viruses,used in combination with a high-content imaging system, can serve as a convenient and rapid tool for the screening of antivirals and host factors involved in the virus life cycle. Moreover, the reporter viruses demonstrated similar growth properties and tissue tropism as their parental viruses in mice, among which the H7N9 NA-Venus virus could potentially be used in ex vivo studies to better understand H7N9 pathogenesis or to develop novel therapeutics.

关键词：流行性感冒病毒 H9N2 维纳斯复制 H5N1 tial 种子类型联合使用

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Development of a recombinant pB602L-based indirect ELISA assay for detecting antibodies against African swine fever virus in pigs

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Journal of Integrative Agriculture 2022年第3期21卷 819-825页

作者： WANG Peng-fei WANG Ming SHI Zhi-bin SUN Zhen-zhao WEI Li-li LIU Zai-si WANG Shi-da HE Xi-jun WANG Jing-fei State Key laboratory of veterinary biotechnology Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin 150069P.R.China

African swine fever(ASF),caused by the African swine fever virus(ASFV),is a devastating disease of domestic and wild pigs.There is no effective vaccine,and the control of the disease relies mainly on surveillance and early detection of infected pigs.Previously,serological assays,such as ELISA,have been developed mainly based on recombinant structural viral proteins of ASFV,including p72,p54,and p30.However,the antibodies against these proteins do not provide efficient protection against ASFV infection in pigs.Therefore,new serological assays that can be applied for clinical diagnosis and evaluating serological immune response in vaccinated pigs are still required.In this study,we expressed and purified a recombinant p B602 L protein.The purified p B602 L protein was then used as an antigen to develop an indirect ELISA assay.This assay has no cross-reaction with the anti-sera against the 15 most common pig pathogens in China,such as classical swine fever virus,pseudorabies virus,and porcine parvovirus.This assay and a commercial ELISA kit were then used to detect 60 field pig serum samples,including an unknown number of antiASFV sera.The coincidence of the two assays was 95%.Furthermore,the p B602 L-based ELISA was employed to test the antibody responses to the seven-gene-deleted ASFV strain HLJ/18-7 GD in pigs.The results showed that the antibody levels in all vaccinated pigs,starting from the 10 th day post-inoculation,have increased continuously during the observation period of 45 days.Our results indicate that this p B602 L-based indirect ELISA assay can be employed potentially in the field of ASFV diagnosis.

关键词： African swine fever virus pB602L recombinant protein indirect ELISA assay pig

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Influenza A virus use of BinCARD1 to facilitate the binding of viral NP to importinα7 is counteracted by TBK1-p62 axis-mediated autophagy

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Cellular & Molecular Immunology 2022年第10期19卷 1168-1184页

作者： Xuyuan Wang Li Jiang Guangwen Wang Wenjun Shi Yuzhen Hu Bo Wang Xianying Zeng Guobin Tian Guohua Deng Jianzhong Shi Liling Liu Chengjun Li Hualan Chen College of veterinary Medicine Gansu Agricultural UniversityLanzhou730070China Guangdong laboratory for Lingnan Modern Agriculture Guangzhou510642China State Key laboratory of veterinary biotechnology Harbin Veterinary Research InstituteChinese Academy of Agricultural SciencesHarbin150069China

As a major component of the viral ribonucleoprotein(vRNP)complex in influenza A virus(IAV),nucleoprotein(NP)interacts with isoforms of importinαfamily members,leading to the import of itself and vRNP complex into the nucleus,a process pivotal in the replication cycle of IAV.In this study,we found that BinCARD1,an isoform of Bcl10-interacting protein with CARD(BinCARD),was leveraged by IAV for efficient viral replication.BinCARD1 promoted the nuclear import of the vRNP complex and newly synthesized NP and thus enhanced vRNP complex activity.Moreover,we found that BinCARD1 interacted with NP to promote NP binding to importinα7,an adaptor in the host nuclear import pathway.However,we also found that BinCARD1 promoted RIG-I-mediated innate immune signaling by mediating Lys63-linked polyubiquitination of TRAF3,and that TBK1 appeared to degrade BinCARD1.We showed that BinCARD1 was polyubiquitinated at residue K103 through a Lys63 linkage,which was recognized by the TBK1-p62 axis for autophagic degradation.Overall,our data demonstrate that IAV leverages BinCARD1 as an important host factor that promotes viral replication,and two mechanisms in the host defense system are triggered—innate immune signaling and autophagic degradation—to mitigate the promoting effect of BinCARD1 on the life cycle of IAV.

关键词： Influenza A virus BinCARD1 TRAF3 TBK1 p62

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