Construction of competitive endogenous RNA network mediated by lung ischemia-reperfusion core genes / 器官移植

Objective To analyze the core genes of lung ischemia-reperfusion injury and construct a competitive endogenous RNA (ceRNA) network. Methods Original data of GSE145989 were downloaded from the Gene Expression Omnibus (GEO) database as the training set, and the GSE172222 and GSE9634 datasets were used as the validation sets, and the differentially-expressed genes (DEG) were identified. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed. Protein-protein interaction (PPI) network was constructed, and the core genes were screened, and the diagnostic values of these core genes and the immune infiltration levels of immune cells were evaluated. The ceRNA network was constructed and validated. The targeted drugs based on ceRNA network were assessed. Results A total of 179 DEG were identified, including 61 down-regulated and 118 up-regulated genes. GO analysis showed that DEGs were associated with multiple biological processes, such as cell migration, differentiation and regulation, etc. They were correlated with cell components, such as vesicle membrane, serosa and membrane raft, etc. They were also associated with multiple molecular functions, such as chemokine receptor, G protein-coupled receptor, immune receptor activity and antigen binding, etc. KEGG pathway enrichment analysis revealed that DEG were involved in tumor necrosis factor (TNF), Wnt, interleukin (IL)-17 and nuclear factor (NF)-κB signaling pathways, etc. PPI network suggested that CD8A, IL2RG, STAT1, CD3G and SYK were the core genes of lung ischemia-reperfusion injury. The ceRNA network prompted that miR-146a-3p, miR-28-5p and miR-593-3p were related to the expression level of CD3G. The miR-149-3p, miR-342-5p, miR-873-5p and miR-491-5p were correlated with the expression level of IL-2RG. The miR-194-3p, miR-512-3p, miR-377-3p and miR-590-3p were associated with the expression level of SYK. The miR-590-3p and miR-875-3p were related to the expression level of CD8A. The miR-143-5p, miR-1231, miR-590-3p and miR-875-3p were associated with the expression level of STAT1. There were 13 targeted drugs for CD3G, 4 targeted drugs for IL-2RG, 28 targeted drugs for SYK and 3 targeted drugs for lncRNA MUC2. No targeted drugs were identified for CD8A, STAT1 and other ceRNA network genes. Conclusions CD8A, IL2RG, STAT1, CD3G and SYK are the core genes of lung ischemia-reperfusion injury. The research and analysis of these core genes probably contribute to the diagnosis of lung ischemia-reperfusion injury and providing novel research ideas and therapeutic targets.

Mining Novel Anti-Nocardia Drug Targets Based on Pan-genomics and Subtractive Proteomics / 中山大学学报(医学科学版)

ObjectiveNocardia is an apathogen that causes opportunistic infections in humans and has a global distribution. In recent years， resistance of Nocardia to commonly used drugs have been observed， highlighting the urgent need for the identification of new drug targets and the development of novel antimicrobial agents against Nocardia. MethodsThirty-one complete genome sequences of Nocardia strains were retrieved from the GenBank database. Pan-genomic analysis was performed using BPGA， and drug target candidates were screened using subtractive proteomics. Homology modeling was employed to predict the 3D structures of target proteins， and potential drugs targeting these proteins were predicted using DrugBank. Molecular docking techniques were utilized to validate the binding activity between the drugs and target proteins. ResultsThe pan-genomic analysis of the 31 Nocardia strains revealed 1 421 core proteins. Fifteen candidate drug target proteins were identified through subtractive proteomics analysis. Among them， the physicochemical properties of the OG1493 protein （such as amino acid count， molecular weight， isoelectric point， grand average of hydropathicity， fat index，and instability index Ⅱ） were found to be most suitable for a drug target protein. Using the DrugBank database， seven compounds， namely Adenosine-5'-Rp-Alpha-Thio-Triphosphate， alpha，beta-Methyleneadenosine 5'-triphosphate， Phosphoaminophosphonic Acid-Adenylate Ester ，Radicicol，2-Hydroxyestradiol， p-Coumaric acid， and Ethylmercurithiosalicylic acid were identified as potential compounds capable of exerting anti-Nocardia effects by targeting this protein. Molecular docking results indicated a strong binding affinity between the target protein and these compounds. The experimental result showed that that Radicicol could be a potential antibacterial drug targeting this particular protein. ConclusionPan-genomic analysis and subtractive proteomics are valuable approaches for mining novel anti-Nocardia drug targets.

Genomic evolution characteristics of pathogenicity islands of enteropatho-genic Escherichia coli Deng strain / 中国感染控制杂志

Objective To analyze the genomic evolution characteristics of pathogenicity islands (PAIs)in Deng strain of enteropathogenic Escherichia coli (E.coli,EPEC Deng).Methods EPEC Deng was isolated from infant stool specimen,serotypes were identified and antimicrobial susceptibility testing was performed;whole-genome se-quencing was performed by Illumina 2000 system,the locations of prophages(PPs)in the chromosome were detected using PHAST software,collinearity analysis was performed by MUMmer software,phylogenetic trees of homolo-gous gene were constructed in order to understand the evolutional rule of homology gene.PAIs prediction was per-formed using PAI finder software,the homologous evolutionary rule of PAIs core region(LEE)and core genes were clarified,genetic polymorphism was analyzed.Results The serotype of EPEC Deng strain was O119:H6,the strain was resistant to ciprofloxacin,levofloxacin,and ampicillin,but sensitive to other antimicrobial agents.The complete circular chromosome contained 5 025 482 bp with a GC content of 50.52 %,and the plasmid contained 207 564 bp with a GC content of 49.50%.A total of 17 PPs in the chromosomal genome were discovered,phyloge-netic trees analysis suggested that EPEC Deng strain was highly homologous with O26:H11 and O111 :H strains;PAIs and core genes were highly homologous with RDEC-1 and O26:H413/89-1 strains;genetic diversity analysis showed that the intimin (eae)and its receptor tir had high polymorphism,with the pi (π)value>0.10,the genes in type III secretion system was relatively stable.Conclusion The study clarified the genomic evolution characteris-tics of EPEC Deng genome and it’s PAIs,and is helpful for understanding genetic characteristics of native EPEC.

Inhibition of HBV-DNA replication and expression by siRNA based on magnetic nanoparticles transfering in HepG2 2.2.15 cells / 中南大学学报(医学版)

Objective To investigate the inhibitory effect of downregulation of hepatitis B virus (HBV) core gene (HBcAg) expression by RNA interference and magnetic nanoparticles on both HBV DNA replication and expression in vitro. Methods HepG2 2.2.15 cells were transfected with U6 promoter plasmids coding for small interfering RNA (siRNA) targeting HBV core gene using magnetic nanoparticles. RT-PCR and Western blot were used to assess the mRNA and protein expression HBV core antigen. Real-time PCR was used to evaluate the suppression efficiency of HBV-DNA replication and expression; and radioimmunoassay was used for HBV surface antigen (HBsAg), core antigen (HBcAg), and e antigen (HBeAg) detection. Results We successfully constructed nanoparticles with siRNA plasmid targeting HBV core antigen; HBcAg mRNA and HBV core antigen protein levels were significantly reduced in the transfected cells. HBV-DNA downregulation was estimated at 4-5 logs and the HBsAg and HBeAg levels were also reduced compared with the controls. Conclusion Downregulation of HBV core gene using RNAi technology and magnetic nanoparticles can potentially be used as a therapeutic strategy for Hepatitis B.

Detection of Core Gene Deletion and Its Clinical Significance in Chronic Hepatitis B Virus infection / 대한진단검사의학회지

BACKGROUND: In the course of chronic hepatitis B virus (HBV) infections, a point mutation or core gene deletion has been attributed to play a role in persistence of HBV infection. This study was undertaken to determine the prevalence of core gene deletion in chronic HBV infections, molecular characteristics, and its clinical significance. METHODS: Among 276 patients with positive results in HBV PCR for precore and core region, patients with smaller bands in addition to the band of expected wild type size, suggestive of deletion, were analyzed by direct sequencing, and hospital records were reviewed on 217 patients. RESULTS: The prevalence of core gene deletion among patients with positivity in HBV PCR was 12.7% (35/276) and they always existed together with wild type. Seventeen patients were further studied by direct sequencing and 16 patients had similar positions of deletion in the center of core gene (nt 2000-2200). Of 35 patients with core gene deletion, 26 (74.3%) were HBeAg-positive and 7 (20.0%) were anti-HBe-positive. There were no significant differences in AST, ALT and HBV DNA quantitation between the wild type and the deletion groups. CONCLUSIONS: HBV core gene deletion is frequently found in chronic HBV infections and has some common features in deleted position, serologic markers and clinical state. However, the mechanism and time at which deletion mutants appear and disappear, and their clinical significances are not fully understood. By longitudinal studies for chronic hepatitis B patients, we should be able to demonstrate the immunologic significance of the core gene deletion mutation.

Effect of hepatitis B virus X gene and hepatitis C virus C gene coexpression protein on telomerase activity of HepG2 cells / 第三军医大学学报

Objective To establish an experimental model of HCV C-HBV X coexpression protein and explore its effect on tolemerase activity. Methods The HBV X gene was recovered by enzyme digestion, cloned into PBK-CMV and PBK-HCV C, and the recombinant plasmids PBK-X and PBK-X-C were obstained. The plasmids PBK-CMV, PBK-X, PBK-HCV C and PBK-X-C were transfected into HepG2 cells with liposome. After selected with G418, positive colonies were obtained. The reverse transcription PCR and Western blotting were used to detect HBV X and HCV core protein expression and PCR-ELISA for tolemerase activity. Results The recombinant plasmid PBK-X-C could express HBV X and HCV core protein efficiently. The telomerase activity of the cells coexpressed HCV C-HBV X protein was higher than that of cells expessed HBV X, HCV C and vector only. Conclusion HBV X-HCV C coexpression protein can increase the telomerase activity of HepG2 cells, which suggests that HBV and HCV can cooperate with carcinogenesis.

Relationship Between Core Gene Mutations of Hepatitis B Virus and Response to Alpha Interferon Therapy in Chronic Hepatitis B / 대한간학회지

BACKGROUND/AIMS: Treatment of chronic hepatitis B with interferon results in a sustained loss of hepatitis B virus DNA and hepatitis B e antigen (HBeAg) and remission of liver disease only in a proportion of cases. Recently, mutations of hepatitis B virus (HBV) core gene have been reported as being related to the failure of interferon treatment in chronic hepatitis B. This study investigated whether core gene mutations of HBV are related to non-response to interferon therapy and whether the recurrence of HBeAg and HBV DNA in initial responders to interferon therapy is associated with the emergence of HBV core gene mutants. METHODS: The precore/core gene sequence was determined by polymerase chain reaction (PCR) and direct sequencing of PCR product in serum samples obtained before interferon treatment from 10 responders and 10 non-responders to interferon therapy. In addition, precore/core gene sequence was determined in serum samples obtained before interferon treatment and after recurrence from 10 patients who showed recurrence of HBeAg and HBV DNA after initial response to interferon therapy. RESULTS: In samples from 10 responders, there were 7 missense mutations and 71 silent mutations. However, there were 43 missense mutations and 109 silent mutations in samples from 10 non-responders. In samples obtained before interferon treatment from the 10 patients who showed recurrence after initial response, 8 missense mutations and 74 silents mutations were found. The nucleotide sequences from the samples obtained after the recurrence showed 6 silent nucleotide substitutions compared with the sequences from the samples obtained before interferon treatment. CONCLUSIONS: Mutations in the core protein of HBV occur more frequently in non-responders than responders to interferon therapy of chronic hepatitis B and may be a factor responsible for the failure of interferon treatment. The recurrence of HBeAg and HBV-DNA in initial responders to interferon therapy is not associated with the emergence of the HBV core gene mutants.

CONSTRUCTION,IDENTIFICATION AND EXPRESSION OF FRAMEWORK PLASMID pAd.HCV-CE1 OF ADENOVIRUS EXPRESSION VECTOR OF HCV-CE1 / 解放军医学杂志

The HCV core gene and E1 gene derived from the plasmid pBRTM/HCV1 3011 by using polymerase chain reaction(PCR) was inserted into the backward position of cytomegalovirus(CMV) immediate early promotor element of framework plasmid (pAd.CMV Link 1) of adenovirus expression vector, then the recombinant plasmid pAd.HCV CE1 was obtained. The inserted DNA of pAd.HCV CE1 was confirmed to be HCV core and E1 gene by endonuclease, PCR and sequencing. HCV core gene was expressed transiently with lipofectamine 2000 coated in human hepatoblastoma 7721 cells which was confirmed by immunofluorescence. The results indicate that the recombinant framework plasmid of adenovirus expression vector pAd.HCV CE1 can express HCV core and E1 gene. This should be useful to pack adenovirus expression vector which can express HCV core and E1 gene.

THE STUDY ON QUASISPECIES OF HEPATITIS B VIRUS : PRE-CORE/CORE GENE AS AN EXAMPLE / 解放军医学杂志

To investigate the HBV quasispecies groups in the patients with chronic HBV infection. A set of specific primers was synthesized according to HBV DNA sequence of the Chinese strain, and the whole pre core/core region was amplified by PCR method from the serum of 4 patients with chronic HBV infection.Then the PCR products were subcloned into pGEM Teasy vectors. Clones were selected to be sequenced,and then sequence comparison was made to find the difference. Each sequence of selected clones was found to be different. The homology of the nucleic acid sequence from the same patient was above 98 0%. There were many mutation types in this region, including point substitution, core region internal deletion, as well as frame shift reading. The results indicated that there were HBV quasispecies groups in chronically infected patients. The mutation in this region may influence the prognosis of HBV infection.

Cloning and Sequencing of Core Gene cDNA of Chinese Hepatitis C Vims / 第二军医大学学报

One hepatitis C virus(HCV) cDNA fragment, 534bp in length and designated as Q534, was obtained by means of PCR amplification with self-designed oligonucleotide primers. Q534 was doned into Hinc II site of pUC18 and the- recombinant plasmid pQ534 was then selected from the bacterial transformants. The sequence data indicated the Q534 was a cDNA fragment of HCV core gene, and located in HCV genome from positions 320 to 853 in correspondence with Chiron's prototype sequence. The homologies between Q534 and the prototype at the levels of nucleotides and amino adds were 90.9% and 97.6%, respectively. The homologies of Q534 with Japanese HCV-J and HCV-BK strains were 96.8% and 97.0% at the nucleotide level, and 98.2% and 98.8% at the amino add level. Compared with the corresponding sequences of other HCV isolates, this Chinese HCV isolate we obtained in the present study belongs to HCV group II .