Identification of sika deer and red deer using partial cytochrome b and 12s ribosomal RNA genes

A study was conducted on the identifications of the degraded samples of sika deer （Cervus nippon） and red deer （Cervus elaphus） by phylogenetic and nucleotide distance analysis of partial Cytb and 12s rRNA genes sequences. 402 bp Cytb genes were achieved by PCR-sequencing using DNA extracted from 8 case samples, and contrasted with 27 sequences of Cytb gene downloaded from GenBank database. The values of three nucleotide distance between three suspected samples and sika deer were identical （0.026±0.006）, which was smaller than the smallest nucleotide distance between eastern red deer and sika deer （0.036）. Furthermore, phylogenetic analysis of sika deer and red deer indicated that the evidences located within the same cluster as sika deer. The evidences were sika deer materials. As the same way, other three suspected samples were derived from red deer. The results were further confirmed by phylogenetic and nucleotide distance analysis of 387 bp 12s rRNA gene. The method was powerful and less time-consuming and helpful to reduce the related cases with wildlife.

Genes Expressing Differences in Upland Cotton Fiber Development Between 12 DPA and 16 DPA

Cotton is the major dominant natural fiber crop on the earth.Although some current cotton genetics and breeding programs had made great progresses in cotton lint yield increases and disease resistance(tolerance),fiber quality has little improvement.Global understanding genes expression

Construction of retroviral vector of p^(125FAK) specific ribozyme genes and its effects on BGC-823 cells

AIM： To construct the retroviral vector of p^125FAK specific ribozyme genes and to explore the feasibility of ribozyme in BGC-823 gene therapy in vitro. METHODS： A hammerhead ribozyme DNA targeting p^125FAK mRNA from nt 1010 to nt 1032 was synthesized and recombinated into the retroviral vector pLXSN forming pLRZXSN recon. Using the lipofectin-mediated DNA transfection technique, pLRZXSN was introduced into BGC-823 cells. The effects of ribozyme on the growth of BGC-823 cells and apoptosis were studied by cell colony assay, flow cytometry （FCM）, reverse transcriptasepolymerase chain reaction （RT-PCR）, detection of DNA fragmentation and electron microscopy. RESULTS： The number of BGC-823 cell colonies was inhibited by 56% after the cells were treated for 48 h. The cell proliferation was inhibited effectively by p^125FAK ribozyme and the inhibitory effect depended on the concentration and the time of incubation. The expression of p^125FAK mRNA and protein p^125 decreased sharply in BGC-823 cells treated with p^125FAK ribozyme. The characteristics of apoptosis, namely sub-G1 peak, DNA fragmentation and morphological changes, were revealed in BGC-823 cells treated with p^125FAK ribozyme. CONCLUSION： p^125FAK ribozyme decreases p^125FAK gene expression and induces apoptosis of human gastric cancer cells in vitro.

Identification of key genes and biological pathways in lung adenocarcinoma by integrated bioinformatics analysis

BACKGROUND The objectives of this study were to identify hub genes and biological pathways involved in lung adenocarcinoma(LUAD)via bioinformatics analysis,and investigate potential therapeutic targets.AIM To determine reliable prognostic biomarkers for early diagnosis and treatment of LUAD.METHODS To identify potential therapeutic targets for LUAD,two microarray datasets derived from the Gene Expression Omnibus(GEO)database were analyzed,GSE3116959 and GSE118370.Differentially expressed genes(DEGs)in LUAD and normal tissues were identified using the GEO2R tool.The Hiplot database was then used to generate a volcanic map of the DEGs.Weighted gene co-expression network analysis was conducted to cluster the genes in GSE116959 and GSE-118370 into different modules,and identify immune genes shared between them.A protein-protein interaction network was established using the Search Tool for the Retrieval of Interacting Genes database,then the CytoNCA and CytoHubba components of Cytoscape software were used to visualize the genes.Hub genes with high scores and co-expression were identified,and the Database for Annotation,Visualization and Integrated Discovery was used to perform enrichment analysis of these genes.The diagnostic and prognostic values of the hub genes were calculated using receiver operating characteristic curves and Kaplan-Meier survival analysis,and gene-set enrichment analysis was conducted.The University of Alabama at Birmingham Cancer data analysis portal was used to analyze relationships between the hub genes and normal specimens,as well as their expression during tumor progression.Lastly,validation of protein expression was conducted on the identified hub genes via the Human Protein Atlas database.RESULTS Three hub genes with high connectivity were identified;cellular retinoic acid binding protein 2(CRABP2),matrix metallopeptidase 12(MMP12),and DNA topoisomerase II alpha(TOP2A).High expression of these genes was associated with a poor LUAD prognosis,and the genes exhibited high diagnostic value.CONCLUSION Expression levels of CRABP2,MMP12,and TOP2A in LUAD were higher than those in normal lung tissue.This observation has diagnostic value,and is linked to poor LUAD prognosis.These genes may be biomarkers and therapeutic targets in LUAD,but further research is warranted to investigate their usefulness in these respects.

Transformation of Two VP1 Genes of O-and Asia 1-Type Foot-and-Mouth Disease Virus into Maize

The expression of antigens in transgenic plants has increasingly been used as an alternative to the classical methodologies for the development of experimental vaccines.This paper reports here the development of a novel oral immunization system for foot-and-mouth disease （FMD） in transgenic maize with two serotypes of the structural protein VP1 of the foot-and-mouth disease virus （FMDV） viz.,O-and Asia 1-type,respectively.The transgenic plantlets were identified and investigated by polymerase chain reaction （PCR）,Southern blot,and real-time PCR.Moreover,it was found that the VP1 genes in transgenic plants could be transmitted stably to the next generation through PCR detection.To our knowledge,this is the first report in an attempt to induce a protective systemic antibody response in animals by feeding the transgenic plants in which two serotypes antigen protein of FMDV expressed together.Results of the experiment provide the possibility of using plant-based vaccines as feedstuff or feedstuff additives.

EFFECTS OF IFN-a COMBINED WITH IL-6 ON GROWTH AND EXPRESSION OF THE GENES RELATED TO CELL-GROWTH AND APOPTOSIS OF BONE MARROW CELLS FROM PATIENTS WITH CML

Objective:To investigate the effects of interferon-a (IFN-a) and IFN-a combined with interleukin-6 (IL-6) on growth and expression of bcr-abl, bcl-2 and c--myc genes in the mononuclear cells (MNCs) from bone marrow (BM) of patients with chronic myelogenous leukemia (CML). Methods: MNCs were collected from BM of the patients with CML in chronic phase by centrifugation in lymphocyte separation medium and cultured in liquid with IFN-a (200U/ml) or IFN-a (200U/ml) plus IL-6 (100 ng/ml). The growing cells were counted every day. The expression levels of b-actin, bcr-abl, bcl-2 and c-myc genes in the MNCs incubated for 24 h were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and relatively quantitative analysis of the amplified fragments by optical density scanning for the bands on gel. Results: The cell growth was markedly suppressed by IFN-a but the degree of cell-growth inhibition was slightly decreased by IL-6 on the basis of IFN-a effect. The expression of bcr-abl chimeric gene was intensely inhibited by IFN-a or IFN-a plus IL-6. The expression of bcl-2 gene was suppressed by either IFN-a or IFN-a plus IL-6, whereas that of c-myc gene was also inhibited by IFN-a but strongly elevated by IL-6 on the basis of IFN-a action. Conclusions: Both IFN-a and IFN-a plus IL-6 can inhibit the expression of anti-apoptosis gene such as bcr-abl and bcl-2 and regulate the expression of the cs.hn.cn gene related to cell proliferation and differentiation such as c-myc. Either IFN-a or IFN-a combined with IL-6 will serve as a trustful strategy of clinical treatment for CML.

Screening key target genes for coronavirus disease 2019 based on bioinformatics and gene network

Objective:To use bioinformatics and gene networks to screen key target genes of coronavirus disease 2019,which provides references for clinical research and development of drugs for coronavirus disease 2019.Methods:Target genes related to coronavirus disease 2019 were screened in the GeneCards and National Center for Biotechnology Information databases,and the obtained gene data were imported into the Database for Annotation,Visualization and Integrated Discovery(Version 6.8)database to collect the related information about pathways and genes.The genes enriched in the first 20 pathways and the genes whose occurrence frequency≥5 were imported into the String database respectively to construct protein-protein interaction network diagram and compare the two network diagrams.Results:TNF,IL-6,IL-2,IL-8,CXCL8,IL1B,CCL2,IFNG,STAT1,MAPK1,MAPK3,MAPK8,TP53 and RELA are ranked top in the two network diagrams,and the frequency of occurrence in the first 20 pathways was≥5.Conclusion:The incidence of coronavirus disease 2019 is associated with multiple signaling pathways,including influenza A,pathways in cancer,toll-like receptor signaling pathway,hypoxia-inducible factor-1 signaling pathway,et al.TNF,IL-6,IL-2,IL-8,CXCL8,IL1B,CCL2,IFNG,STAT1,MAPK1,MAPK3,MAPK8,TP53 and RELA are closely related to coronavirus disease 2019,which needs to be further studied.By analyzing the pathways of the genes related to coronavirus disease 2019 and the interactive network diagrams between the genes,it is helpful to understand the pathogenesis of the disease and provide a reference for clinical research and development of effective drugs for coronavirus disease 2019.

Improve meat production and virus resistance by simultaneously editing multiple genes in livestock using Cas12i^(Max)

The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated gene(Cas) system is continually optimized to achieve the most efficient gene editing effect. The Cas12i^(Max), a Cas12i variant, exhibits powerful DNA editing activity and enriches the gene editing toolbox. However, the application of Cas12i^(Max)in large domestic animals has not yet been reported. To verify the efficiency and feasibility of multiple gene editing in large animals, we generated porcine fibroblasts with simultaneous knockouts of IGF2, ANPEP, CD163,and MSTN via Cas12i^(Max)in one step. Phenotypically stable pigs were created through somatic cell nuclear transfer technology. They exhibited improved growth performance and muscle quality. Furthermore, we simultaneously edited three genes in bovine fibroblasts. A knockout of MSTN and PRNP was created and the amino acid Q-G in CD18 was precisely substituted. Meanwhile, no off-target phenomenon was observed by sum-type analysis or off-target detection. These results verified the effectiveness of Cas12i^(Max)for gene editing in livestock animals and demonstrated the potential application of Cas12i^(Max)in the field of animal trait improvement for agricultural production.

The involvement of interleukin-22 in the expression of pancreatic beta cell regenerative Reg genes

Background:In Type 1 diabetes,the insulin-producingβ-cells within the pancreatic islets of Langerhans are destroyed.We showed previously that immunotherapy with Bacillus Calmette-Guerin(BCG)or complete Freund’s adjuvant(CFA)of non-obese diabetic(NOD)mice can prevent disease process and pancreaticβ-cell loss.This was associated with increased islet Regenerating(Reg)genes expression,and elevated IL-22-producing Th17 T-cells in the pancreas.Results:We hypothesized that IL-22 was responsible for the increased Reg gene expression in the pancreas.We therefore quantified the Reg1,Reg2,and Reg3δ(INGAP)mRNA expression in isolated pre-diabetic NOD islets treated with IL-22.We measured IL-22,and IL-22 receptor(R)-αmRNA expression in the pancreas and spleen of pre-diabetic and diabetic NOD mice.Our results showed:1)Reg1 and Reg2 mRNA abundance to be significantly increased in IL-22-treated islets in vitro;2)IL-22 mRNA expression in the pre-diabetic mouse pancreas increased with time following CFA treatment;3)a reduced expression of IL-22Rαfollowing CFA treatment;4)a down-regulation in Reg1 and Reg2 mRNA expression in the pancreas of pre-diabetic mice injected with an IL-22 neutralizing antibody;and 5)an increased isletβ-cell DNA synthesis in vitro in the presence of IL-22.Conclusions:We conclude that IL-22 may contribute to the regeneration ofβ-cells by up-regulating Regenerating Reg1 and Reg2 genes in the islets.

Vitamin B_(12)-auxotrophy in dinoflagellates caused by incomplete or absent cobalamin-independent methionine synthase genes(metE)

Dinoflagellates are responsible for most marine harmful algal blooms (HABs) and play vital roles in many ocean processes.More than 90% of dinoflagellates are vitamin B_(12) auxotrophs and that B_(12) availability can control dinoflagellate HABs,yet the genetic basis of B_(12) auxotrophy in dinoflagellates in the framework of the ecology of dinoflagellates and particularly HABs,which was the objective of this work.Here,we investigated the presence,phylogeny,and transcription of two methionine synthase genes(B_(12)-dependent metH and B_(12)-independent metE)via searching and assembling transcripts and genes from transcriptomic and genomic databases,cloning 38 cDNA isoforms of the two genes from 14 strains of dinoflagellates,measuring the expression at different scenarios of B_(12),and comprehensive phylogenetic analyses of more than 100 organisms.We found that 1)metH was present in all 58 dinoflagellates accessible and metE was present in 40 of 58 species,2)all metE genes lacked N-terminal domains,3)metE of dinoflagellates were phylogenetically distinct from other known metE genes,and 4)expression of metH in dinoflagellates was responsive to exogenous B_(12) levels while expression of metE was not responding as that of genuine metE genes.We conclude that most,hypothetically all,dinoflagellates have either non-functional metE genes lacking N-terminal domain for most species,or do not possess metE for other species,which provides the genetic basis for the widespread nature of B_(12) auxotrophy in dinoflagellates.The work elucidated a fundamental aspect of the nutritional ecology of dinoflagellates.

RDH12-associated retinal degeneration caused by a homozygous pathogenic variant of 146C>T and literature review

AIM:To describe the clinical,electrophysiological,and genetic features of an unusual case with an RDH12 homozygous pathogenic variant and reviewed the characteristics of the patients reported with the same variant.METHODS:The patient underwent a complete ophthalmologic examination including best-corrected visual acuity,anterior segment and dilated fundus,visual field,spectral-domain optical coherence tomography(OCT)and electroretinogram(ERG).The retinal disease panel genes were sequenced through chip capture high-throughput sequencing and Sanger sequencing was used to confirm the result.Then we reviewed the characteristics of the patients reported with the same variant.RESULTS:A 30-year male presented with severe early retinal degeneration who complained night blindness,decreased visual acuity,vitreous floaters and amaurosis fugax.The best corrected vision was 0.04 OD and 0.12 OS,respectively.The fundus photo and OCT showed bilateral macular atrophy but larger areas of macular atrophy in the left eye.Autofluorescence shows bilateral symmetrical hypo-autofluorescence.ERG revealed that the amplitudes of a-and b-wave were severely decreased.Multifocal ERG showed decreased amplitudes in the local macular area.A homozygous missense variant c.146C>T(chr14:68191267)was found.The clinical characteristics of a total of 13 patients reported with the same pathologic variant varied.CONCLUSION:An unusual patient with a homozygous pathogenic variant in the c.146C>T of RDH12 which causes late-onset and asymmetric retinal degeneration are reported.The clinical manifestations of the patient with multimodal retinal imaging and functional examinations have enriched our understanding of this disease.

Analysis of Mutations of Two Gitelman Syndrome Family SLC12A3 Genes and Proposed Treatments Using Chinese Medicine

Objective： To determine the gene location of two Gitelman syndrome （GS） family SLC12A3 genes and explore treatments using Chinese medicine （CM） prescriptions. Methods： In order to locate the two GS mutations, samples were collected from 11 people from two different pedigrees for direct genetic sequencing and comparison of the 26 exons of SLC12A3. Furthermore, the change of serum potassium was monitored throughout the therapy and those two probands undertook a sequential superposition of Western medicine （including potassium, Panangin and potassium-sparing diuretics） with CM prescription based on Buyang Huanwu Decoction （补阳还五汤） and Sijunzi Decoction （四君子汤）. The treatment included three stages, oral potassium chloride for the first 2 weeks （stage 1）, potassium-sparing diuretic and Panangin with potassium chloride for the next 2 weeks （stage 2）, CM along with the medicine in stage 2 for the final 2 weeks （stage 3）. Results： The three mutations occurring in proband 1 from pedigree 1 were Thr60Met, 965-1_976de113ins12 （small indels mutation） and Ala122Ala （homozygous silent mutation）. Likewise, three mutations, Asn359Lys, Thr382Met and Arg913GIn, appeared in the proband 2 from pedigree Ⅱ. The serum potassium levels increasing from baseline to sequential stages were 1.63 mmol/L （baseline）, 2.5 mmol/L （stage 1）, 3.1 mmol/L （stage 2） and 3.9 mmol/L （stage 3） in the proband 1, and 2.8 mmol/L （baseline）, 3.1 mmol/L （stage 1）, 3.5 mmol/L （stage 2） and 4.3 mmol/L （stage 3） in the proband 2, respectively. The symptoms （numbness of limbs, weakness, palpitations, etc.） of both probands were all alleviated. Conclusions： The mutations of both GS pedigrees can be defined as compound heterozygous mutations, most of which are known as missense mutations. Applying CM could be an appropriate choice for future intervention of GS.

MiR-3653 blocks autophagy to inhibit epithelial-mesenchymal transition in breast cancer cells by targeting the autophagy-regulatory genes ATG12 and AMBRA1

Background:Metastasis is the main cause of tumor-associated death and mainly responsible for treatment failure of breast cancer.Autophagy accelerates tumor metastasis.In our work,we aimed to investigate the possibility of microRNAs(miRNAs)which participate in the regulation of autophagy to inhibit tumor metastasis.Methods:MiRNA array and comprehensive analysis were performed to identify miRNAs which participated in the regulation of autophagy to inhibit tumor metastasis.The expression levels of miR-3653 in breast cancer tissues and cells were detected by quantitative real-time polymerase chain reaction.In vivo and in vitro assays were conducted to determine the function of miR-3653.The target genes of miR-3653 were detected by a dual luciferase reporter activity assay and Western blot.The relationship between miR-3653 and epithelial-mesenchymal transition(EMT)was assessed by Western blot.Student’s t-test was used to analyze the difference between any two groups,and the difference among multiple groups was analyzed with one-way analysis of variance and a Bonferroni post hoc test.Results:miR-3653 was downregulated in breast cancer cells with high metastatic ability,and high expression of miR-3653 blocked autophagic flux in breast cancer cells.Clinically,low expression of miR-3653 in breast cancer tissues(0.054±0.013 vs.0.131±0.028,t=2.475,P=0.014)was positively correlated with lymph node metastasis(0.015±0.004 vs.0.078±0.020,t=2.319,P=0.023)and poor prognosis(P<0.001).miR-3653 ameliorated the malignant phenotypes of breast cancer cells,including proliferation,migration(MDA-MB-231:0.353±0.013 vs.1.000±0.038,t=16.290,P<0.001;MDA-MB-468:0.200±0.014 vs.1.000±0.043,t=17.530,P<0.001),invasion(MDA-MB-231:0.723±0.056 vs.1.000±0.035,t=4.223,P=0.013;MDA-MB-468:0.222±0.016 vs.1.000±0.019,t=31.050,P<0.001),and colony formation(MDA-MB-231:0.472±0.022 vs.1.000±0.022,t=16.620,P<0.001;MDA-MB-468:0.650±0.040 vs.1.000±0.098,t=3.297,P=0.030).The autophagy-associated genes autophagy-related gene 12(ATG12)and activating molecule in beclin 1-regulated autophagy protein 1(AMBRA1)are target genes of miR-3653.Further studies showed that miR-3653 inhibited EMT by targeting ATG12 and AMBRA1.Conclusions:Our findings suggested that miR-3653 inhibits the autophagy process by targeting ATG12 and AMBRA1,thereby inhibiting EMT,and provided a new idea and target for the metastasis of breast cancer.

Phylogenetic Relationships among 12 Species of Tetrigidae (Orthoptera:Tetrigoidea) Based on Partial Sequences of 12S and 16S Ribosomal RNA

Mitochondrial 12S and 16S ribosomal RNA genes sequences were sequenced using dye-labeled terminator on an ABI 377 automated sequencer in 11 individuals and 1 species' sequences were gained from GenBank,representing 6 genera of family Tetrigidae.The collated sequences were aligned using Clustal X version 1.81 and then,the sequence variability and heredity distances based on Kimura 2-parameter model were calculated using Mega 2.1.In obtained sequences (736 bp),the average A+T content is 73.9%,ranging from 71.2% to 77.5%;the overall G+C content is 26.1%,ranging from 22.5% to 28.8%.Based on alignment of the combined sequences,185 parsimony-informative sites were revealed in 755 available base pairs.Phylogenetic trees were reconstructed using NJ,MP and ML methods with Cylindraustralia kochii as outgroup.The results indicated that the monophyletic nature of Tetrix is questioned and suggest that T.tubercarina may be given tribal rank.Our results also show that Coptltettix huanjiangensis and C.gongshanensis are the same species,i.e.Coptltettix gongshanensis Zheng,and C.huanjiangensis is the synonyms of C.gongshanensis.

Cloning of Cotton Delta-12 Oleate Desaturase Gene FAD2-1 and Construction of Its ihpRNA and amiRNA Interference Vectors

Delta-12 oleate desaturase gene （FAD2-1） which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of cottonseed oil. By employing RT-PCR method, full length cDNA of cotton delta-12 oleate desat- urase gene GhFAD2-1 containing an open reading frame of 1 158 bp was cloned for constructing RNAi vector. A 515 bp long specific fragment of this gene was se- lected for constructing ihpRNA vector under the control of a seed-specific promoter NAPIN, named pFGC1008-NAPIN-FAD2-1; meanwhile miRNA gene-silencing vector pCAMBIA1302-amiRNA-FAD2-1 targeting GhFAD2-1 was also constructed.

Gene-viral vectors: a promising way to target tumor cells and express anticancer genes simultaneously

OBJECTIVE: To develop a new kind of vector system called gene-viral vector, which combines the advantages of gene and virus therapies. METHODS: Using recombinant technology, an anti-tumor gene was inserted into the genome of replicative virus specific for tumor cells. The cell killing effect, reporter gene expression of the green fluorescence protein, anti-tumor gene expression of mouse interleukin-12 (mIL-12) and replication of virus were observed by the methods of cell pathology, fluorescence microscopy, ELISA and electron microscopy, respectively. RESULTS: A new kind of gene-viral vector system of adenovirus, in which the E1b-55 kD gene was deleted but the E1a gene was preserved, was constructed. The vector system, like the replicative virus ONYX-015, replicated and proliferated in tumor cells but not in normal ones. Our vector had an advantage over ONYX-015 in that it carried different kinds of anti-tumor genes to enhance its therapeutic effect. The reporter gene expression of the green fluorescence protein in tumor cells was much better than the adenovirus vector employed in conventional gene the rapy, and the expression in our vector system was as low as or even less than that in the conventional adenovirus gene therapy system. Similar results were observed in experiments with this vector system carrying the anti-tumor gene mIL-12. Replication and proliferation of the virus carrying the mIL-12 gene in tumor cells were confirmed by electron microscopy. CONCLUSIONS: Gene-viral vectors are new vectors with an anti-tumor gene inserted into the genome of replicative virus specific for tumor cells. Because of the specific replication and proliferation of the virus in tumor cells, expression of the anti-tumor gene is increased hundreds to thousands of times. This approach takes full advantages of gene therapy and virus therapy to enhance the effect on the tumor. It overcomes the disadvantages of conventional gene therapy, such as low transfer rate, low gene expression, lack of target tropism, and low anti-tumor activity. We believe that this is a promising means for future tumor treatment.

Association of polymorphisms of interleukin-18 gene promoter region with chronic hepatitis B in Chinese Han population

AIM: To investigate the polymorphisms of interleukin-18 (IL-18) gene promoters, and to disclose whether such polymorphisms are associated with susceptibility to chronic hepatitis B in Chinese Han population. METHODS: Using polymerase chain reaction with sequence specific primers (PCR-SSP) method, the single nucleotide polymorphisms (SNPs) of the promoter region of IL-18 gene at position -607 and -137 were detected in 231 patients with chronic hepatitis B and 300 normal controls. RESULTS: Allele C at position -607 in the promoter of IL-18 gene was detected in 48.7% of normal controls and 51.9% of patients, while allele A at position -607 was detected in 51.3% of normal controls and 48.1% of patients. The frequencies of -607CC, -607 CA and -607AA genotypes in normal controls were 22.0%, 53.3% and 24.7% respectively and in chronic hepatitis B patients were 26.8%, 50.2% and 23.0% respectively. Allele G at position -137 in the promoter of IL-18 gene was detected in 82.3% of normal controls and 88.5% of chronic hepatitis B patients, while allele C at position -137 was detected in 17.7% of normal controls and 11.5% of patients. The frequencies of -137GG, GC and CC genotype were 67.3%, 30.0% and 2.7% in normal controls respectively, while in chronic hepatitis B patients were 78.8%, 19.5% and 1.7% respectively. The frequency of-137GG genotype in chronic hepatitis B groups was significantly higher than that in normal controls (x2=8.55, P=0.003 <0.05), whereas the frequencies of -607C/-137C and -607A/-137C haplotypes in chronic hepatitis B groups were significantly lower than that in normal controls. The association between genotypes of IL-18 promoter region polymorphisms and HBV copies showed that the frequency of -607AA genotype in high HBV-DNA copies groups was lower than that in low HBV-DNA copies groups (x2=6.03, P=0.014 <0.05). CONCLUSION: The polymorphisms of the promoter region of IL-18 gene at position -607 and -137 are closely associated with susceptibility to chronic hepatitis B. The people with allele C at position -137 in the promoter of IL-18 gene may be protected against HBV infection; moreover AA genotype at position -607 may be closely linked to inhibit HBV-DNA replication. These findings give some new clues to the study of pathogenesis of chronic hepatitis B.

Interleukin-1β gene polymorphism associated with hepatocellular carcinoma in hepatitis B virus infection

AIM： To examine the effect of interleukin-l-beta （IL-113） promoter region C-511T and IL-1 receptor antagonist （IL-1RN） polymorphism among the patients with chronic hepatitis B virus （HBV） infection （HCC and non-HCC）. METHODS： Genomic DNA from 136 Thai patients with chronic HBV infection （HCC =46 and non-HCC= 90） and 152 healthy individuals was genotyped for IL-113 gene polymorphism （-511） using polymerase chain reaction with sequence specific primers （PCR-SSP）. The variable number of tandem repeats （VNTR） of IL-1RN gene was assessed by a PCR-based assay. The association between these genes and status of the disease was evaluated by X^2 test. RESULTS： IL-1B-511 genotype c/c was found to be significantly different in patients with HCC when compared with healthy individuals （P = 0.036, OR = 2.29, 95%CI = 1.05-4.97） and patients without HCC （P=0.036, OR= 2.52, 95%CI=1.05-6.04）. Analysis of allele frequencies of IL-1B-511 showed that IL-1B-511 C allele was also significantly increased in patients with HCC, compared to that in healthy control （P=0.033, OR= 1.72, 95%CI=1.04-2.84）. However, no significant association in IL-1RN gene was found between the two groups. CONCLUSION： IL-1B-511C allele, which may be associated with high IL-1B production in the liver, is a genetic marker for the development of HCC in chronic hepatitis B patients in Thai population.

Amino acid deletions at positions 893 and 894 of cytotoxinassociated gene A protein affect Helicobacter pylori gastric epithelial cell interactions

BACKGROUND Helicobacter pylori(H.pylori)persistently colonizes the human gastric mucosa in more than 50%of the global population,leading to various gastroduodenal diseases ranging from chronic gastritis to gastric carcinoma.Cytotoxin-associated gene A(CagA)protein,an important oncoprotein,has highly polymorphic Glu-Pro-Ile-Tyr-Ala segments at the carboxyl terminus,which play crucial roles in pathogenesis.Our previous study revealed a significant association between amino acid deletions at positions 893 and 894 and gastric cancer.AIM To investigate the impact of amino acid deletions at positions 893 and 894 on CagA function.METHODS We selected a representative HZT strain from a gastric cancer patient with amino acid deletions at positions 893 and 894.The cagA gene was amplified and mutated into cagA-NT and cagA-NE(sequence characteristics of strains from nongastric cancer patients),cloned and inserted into pAdtrack-CMV,and then transfected into AGS cells.The expression of cagA and its mutants was examined using realtime polymerase chain reaction and Western blotting,cell elongation via cell counting,F-actin cytoskeleton visualization using fluorescence staining,and interleukin-8(IL-8)secretion via enzyme-linked immunosorbent assay.RESULTS The results revealed that pAdtrack/cagA induced a more pronounced hummingbird phenotype than pAdtrack/cagA-NT and pAdtrack/cagA-NE(40.88±3.10 vs 32.50±3.17,P<0.001 and 40.88±3.10 vs 32.17±3.00,P<0.001)at 12 hours after transfection.At 24 hours,pAdtrack/cagA-NE induced significantly fewer hummingbird phenotypes than pAdtrack/cagA and pAdtrack/cagA-NT(46.02±2.12 vs 53.90±2.10,P<0.001 and 46.02±2.12 vs 51.15±3.74,P<0.001).The total amount of F-actin caused by pAdtrack/cagA was significantly lower than that caused by pAdtrack/cagA-NT and pAdtrack/cagA-NE(27.54±17.37 vs 41.51±11.90,P<0.001 and 27.54±17.37 vs 41.39±14.22,P<0.001)at 12 hours after transfection.Additionally,pAdtrack/cagA induced higher IL-8 secretion than pAdtrack/cagA-NT and pAdtrack/cagA-NE at different times after transfection.CONCLUSION Amino acid deletions at positions 893 and 894 enhance CagA pathogenicity,which is crucial for revealing the pathogenic mechanism of CagA and identifying biomarkers of highly pathogenic H.pylori.

Polymorphisms in interleukin-10 gene according to mutations of NOD2/CARD15 gene and relation to phenotype in Spanish patients with Crohn's disease

AIM： To examine the contribution of interleukin-10 （IL-10） gene polymorphisms to Crohn＇s disease （CD） phenotype, and the possible genetic epistasis between IL-10 gene polymorphisms and CARD15/NOD2 gene mutations. METHODS： A cohort of 205 Spanish unrelated patients with Crohn＇s disease recruited from a single center was studied. All patients were rigorously phenotyped and followed-up for at least 3 years （mean time, 12.5 years）. The clinical phenotype was established prior to genotyping. RESULTS： The correlation of genotype-Vienna classification groups showed that the Ueocolonic location was significantly associated with the -1082G allele in the NOD2/CARD15 mutation-positive patients （RR = 1.52, 95%CI, 1.21 to 1.91,P= 0.008）. The multivariate analysis demonstrated that the IL-10 G14 microsatellite allele in the NOD2/CARD15 mutation positive patients was associated with two risk factors, history of appendectomy （RR = 2.15, 95%CI = 1.1-4.30, P= 0.001） and smoking habit at diagnosis （RR= 1.29, 95%CI= 1.04-4.3, P= 0.04）. CONCLUSION： In Spanish population from Madrid, in CD patients carrying at least one NOD2/CARD15 mutation, the -1082G allele is assodated with ileocolonic disease and the IL-IOG14 microsatellite allele is associated with previous history of appendectomy and smoking habit at diagnosis. These data provide further molecular evidence for a genetic basis of the clinical heterogeneity of CD.