AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

PbrARF4 contributes to calyx shedding of fruitlets in ‘Dangshan Suli’ pear by partly regulating the expression of abscission genes

Fruitlet calyx shedding in pear plants is apparently regulated via numerous pathways that involve both environmental triggers and phytohormones cues such as auxin. In this study, we found at 10 days after full bloom (DAFB) higher levels of indoleacetic acid (IAA) and tryptophan (Trp) in calyx persistence fruitlet (CPF) than calyx shedding fruitlet (CSF) ofDanshan Suli’ pear (Pyrus bretschneideri Rhed.). Consisting with this, the activity of indolealdehyde oxidase (IAAIdO), which promotes IAA synthesis, was remarkably increased, and that of peroxidase(POD), which degrades IAA, dropped markedly in CPF but not in CSF. Further, qRT-PCR results revealed that most of 31 PbrARFs (encoding auxin response factors) in Pyrus bretschneideri were highly expressed in CPF, whereas PbrARF4, PbrARF24 and PbrARF26 were significantly downregulated in CPF vis-a-vis CSF. Phylogenetic analysis revealed that 6 PbrARFs clustered in the group III, where PbrARF4 showed the closest affinity with AtARF1 that promotes organ abscission, indicating a putative role of PbrARF4 in mediating the process of calyx shedding in pear. In fact, the ectopic overexpression of PbrARF4 in Solanum lycopersicum resulted in an earlier-formed and deeper abscission layer (AL) in the transgenic plants, whose calyxes were more prone to wilt at the mature red stage (MR) compared with the control plants (wild-type). More importantly, expression levels of the abscission genes SILS and Sl Cel2 in transgenic plants overexpressing PbrARF4 were significantly upregulated in comparation with the WT, whereas those of Sl BI and Sl TAPG2 were considerably inhibited. Further, PbrJOINTLESS and PbrIDA,the two genes related to calyx shedding in pear, were up-regulated more in CSF than CPF. The findings contribute to a better understanding of PbrARFs involved in fruitlet calyx shedding of pear, which could prove beneficial to improving the quality of pear fruit.

Quantitative trait loci identification reveals zinc finger protein CONSTANS-LIKE 4 as the key candidate gene of stigma color in watermelon(Citrullus lanatus)

Stigma color is a critical agronomic trait in watermelon that plays an important role in pollination.However,there are few reports on the regulation of stigma color in watermelon.In this study,a genetic analysis of the F2 population derived from ZXG1553(P1,with orange stigma)and W1-17(P2,with yellow stigma)indicated that stigma color is a quantitative trait and the orange stigma is recessive compared with the yellow stigma.Bulk segregant analysis sequencing(BSA-seq)revealed a 3.75 Mb segment on chromosome 6 that is related to stigma color.Also,a major stable effective QTL Clqsc6.1(QTL stigma color)was detected in two years between cleaved amplified polymorphic sequencing(CAPS)markers Chr06_8338913 and Chr06_9344593 spanning a~1.01 Mb interval that harbors 51 annotated genes.Cla97C06G117020(annotated as zinc finger protein CONSTANS-LIKE 4)was identified as the best candidate gene for the stigma color trait through RNA-seq,quantitative real-time PCR(qRT-PCR),and gene structure alignment analysis among the natural watermelon panel.The expression level of Cla97C06G117020 in the orange stigma accession was lower than in the yellow stigma accessions with a significant difference.A nonsynonymous SNP site of the Cla97C06G117020 coding region that causes amino acid variation was related to the stigma color variation among nine watermelon accessions according to their re-sequencing data.Stigma color formation is often related to carotenoids,and we also found that the expression trend of ClCHYB(annotated asβ-carotene hydroxylase)in the carotenoid metabolic pathway was consistent with Cla97C06G117020,and it was expressed in low amounts in the orange stigma accession.These data indicated that Cla97C06G117020 and ClCHYB may interact to form the stigma color.This study provides a theoretical basis for gene fine mapping and mechanisms for the regulation of stigma color in watermelon.

Cellular Senescence and SENEX Gene on the Peripheral CD4+CD25+ Treg Cells Enhancement in Elderly

Cellular senescence is a signal transduction process which maintained genomic stability and stopped mammalian cell growth. Furthermore, cellular senescence induces a protective response to a variety of DNA damage. However, this process is also associated with apoptosis, upregulated secretion of inflammatory cytokine, and promoted surrounding tissue damage. When cellular senescence accumulates to a certain extent, it triggers geriatric diseases, such as chronic inflammation, immune senescence-associated tumors and incontrollable infections. Cellular senescence gene SENEX, which was cloned in 2004, has been demonstrated to play a unique gatekeeper function in human endothelial cells when stress-induced pre-mature senescence and apoptosis occurr. The phenomenon that CD4+CD25+ Treg cells accumulated in the aged population has been well studied in recent years. Now Treg accumulation related to immune-pathology has attracted more interest. CD4+CD25+ Treg did not decline and age, but accumulated and suppressed immunoreaction. The enhanced Treg number and function may be associated with stress-induced premature senescence-mediated unique cellular senescence protection mechanisms, and SENEX may play a critical role in this process. In this article, we summarize the cellular senescence and SENEX gene in the accumulation and functional activity of CD4+CD25+ Treg in the elderly.

Genetic Polymorphism of TLR4 Gene and Correlation with Mastitis in Cattle

Toll-like receptor 4 （TLR4） recognizes pathogen ligands and mediates signaling to initiate innate and adaptive immune responses. In this experiment, a 316 bp and 382 bp fragments of TLR4 gene named T4CRBR1 and T4CRBR2, of Chinese Holstein, Sanhe cattle, and Chinese Simmental was amplified by polymerase chain reaction （PCR）, respectively. The genetic polymorphisms in the three populations were detected by Single-Strand Conformational Polymorphism （SSCP） in the first locus and by digesting the fragments with restriction endonuclease Alu I in the second one. Results showed that both alleles （A and B） of two loci were found in all the three populations and the value of polymorphism information content （PIC） indicated that these were a moderate polymorphism. Statistical results of X^2 test indicated that two polymorphism sites in the three populations fitted with Hardy-Weinberg equilibrium （P 〉 0.05）. After sequencing, A-G single nucleotide polymorphism （SNP） was identified at nucleotide 4,525 in intron 1 of TLR4 gene and C-T SNP was identified at nucleotide 1,397 in exon 3 of TLR4 gene. Meanwhile, the effect of polymorphism of TLR4 gene on somatic cell score （SCS） was analyzed, the results indicated that the cattle with allele A in T4CRBR1 showed lower somatic cell score than that of allele B （P 〈 0.05）. In short, the allele A might play an important role in mastiffs resistance in bovine.

水稻谷蛋白GluB-4基因启动子克隆及载体构建

GluB-4是水稻种子谷蛋白的编码基因,在种子成熟过程中,由GluB-4启动子调控,在胚乳中特异地表达。以水稻基因组DNA为模板,通过PCR扩增技术得到GluB-4启动子片段,序列分析结果表明:获得的启动子片段的大小为1 560 bp,与已报道的该启动子序列相比较,其核苷酸序列同源性为99.8%。该启动子区域含有TATA-box,CAAT-box,GCN4基序,Skn-1基序等胚乳特异表达启动子所必需的正调控元件,同时还含有高水平转录调控因子5UTR Py-rich stretch序列。利用该启动子构建了植物种子特异表达载体pCGB4P,为进一步的研究工作奠定了基础。

血清4型禽腺病毒的分离鉴定及致病性分析

旨在从送检的病鸡中分离鉴定出禽腺病毒(fowl adenovirus,FAdV),为临床诊断和疫病防控提供参考依据。本研究将阳性样品接种SPF鸡胚并传代,利用PCR检测、基因序列分析、动物回归试验等方法鉴定病毒。结果:经过鸡胚传代及病毒特异性检测获得了纯净的FAdV,命名为HB2306;分析Hexon基因序列可知,HB2306株属于血清4型分支,与国内外分离的FAdV-4毒株核苷酸序列同源性为98.2%~99.9%,HB2306株与国内外的4型高致病性毒株氨基酸序列相似性在97.9%~99.8%;受试动物临床病理变化显示,HB2306株以104.5EID50/只的剂量经胸部肌肉注射后,引起宿主心包积液,肝脏变黄肿胀、淤血出血,肾出血、肿胀等典型的病理变化,与流行的高致病性毒株所致临床症状相似,且死亡率为100%。综上,本研究成功分离并鉴定出一株FAdV-4高致病力毒株,丰富了毒种库,为该病毒感染的流行情况调查和综合防控奠定了基础。

香蕉枯萎病菌内源报告基因Foc4carS的鉴定及其应用

香蕉枯萎病是由尖孢镰刀菌古巴转化型(Fusarium oxysporum f. sp. cubense, Foc)引起的香蕉毁灭性土传病害,其中4号生理小种(Foc4)能感染几乎所有的香蕉品系,危害最严重。carS基因通过调控下游car结构基因参与调控镰刀菌类胡萝卜素的生物合成,本研究克隆鉴定了Foc4carS基因(FOIG_05085),Foc4carS蛋白具有典型的RING-finger蛋白结构域。利用分割标记法(Split-marker PCR)获得Foc4carS基因的融合片段,同时构建含有Foc4carS基因sgRNA591序列的pUC-fFuCas9-HTBNLS-hph-Foc4carS基因编辑载体,通过PEG介导的原生质体转化获得该基因的敲除突变体、回补突变体以及基因编辑敲除体,并对敲除和回补突变体的生物学特性和致病力进行分析。结果显示:ΔFoc4carS突变体的菌落直径、产孢量和致病力等生物学表型与野生菌株Foc4无显著差异,而ΔFoc4carS突变体菌落颜色呈深橙色,Foc4carS基因的缺失影响了次生代谢产物类胡萝卜素的生物合成;基因编辑的ΔFoc4carS(HDR)突变体不论是再生筛选板还是继代后的PDA平板,其菌落均出现典型的深橙色,表明Foc4carS可作为内源报告基因,在香蕉枯萎菌Foc4中进行基因质粒型CRISPR/Cas9编辑可行。

冠脉支架内再狭窄患者TLR4基因rs4986790、rs4986791位点多态性及其与临床关系的研究

目的 探讨冠脉支架内再狭窄(ISR)患者Toll样受体4(TLR4)外显子基因突变情况及其与临床的关系。方法 选取2019年1月至2022年12月在右江民族医学院附属医院治疗的ISR患者137例,检测体重指数(BMI)、血压、血脂、血糖、血尿酸、C反应蛋白(CRP)、β2微球蛋白(β2MG)、白细胞介素6(IL-6)和TLR4基因rs4986790、rs4986791位点碱基。并与131例同期行冠脉支架植入术支架内无再狭窄(NISR)患者比较。结果 ISR组患者BMI、收缩压、总胆固醇、甘油三酯、空腹血糖、血尿酸、CRP、β2MG和IL-6水平高于NISR组(P <0.05);ISR组TLR4基因rs4986790位点碱基突变率高于NISR组(P <0.05),TLR4基因rs4986791位点碱基突变率高于NISR组(P <0.05);TLR4基因突变表型组BMI、收缩压、总胆固醇、甘油三酯、空腹血糖、血尿酸、CRP、β2MG和IL-6水平高于TLR4野生表型组(P <0.05)。结论 冠脉支架内再狭窄患者TLR4基因外显子突变率高,TLR4基因外显子突变的患者其代谢和炎症因子异常情况有叠加作用。

Gene interference regulates aquaporin-4 expression in swollen tissue of rats with cerebral ischemic edema Correlation with variation in apparent diffusion coefficient

To investigate the effects of mRNA interference on aquaporin-4 expression in swollen tissue of rats with ischemic cerebral edema, and diagnose the significance of diffusion-weighted MRI, we injected 5 pL shRNA- aquaporin-4 （control group） or siRNA- aquaporin-4 solution （1：800） （RNA interference group） into the rat right basal ganglia immediately before occlusion of the middle cerebral artery. At 0.25 hours after occlusion of the middle cerebral artery, diffusion-weighted MRI displayed a high signal; within 2 hours, the relative apparent diffusion coefficient decreased markedly, aquaporin-4 expression increased rapidly, and intracellular edema was obviously aggravated; at 4 and 6 hours, the relative apparent diffusion coefficient slowly returned to control levels, aquaporin-4 expression slightly increased, and angioedema was observed. In the RNA interference group, during 0.25- 6 hours after injection of siRNA- aquaporin-4 solution, the relative apparent diffusion coefficient slightly fluctuated and aquaporin-4 expression was upregulated; during 0.5 4 hours, the relative apparent diffusion coefficient was significantly higher, while aquaporin-4 expression was significantly lower when compared with the control group, and intracellular edema was markedly reduced; at 0.25 and 6 hours, the relative apparent diffusion coefficient and aquaporin-4 expression were similar when compared with the control group; obvious angioedema remained at 6 hours. Pearson＇s correlation test results showed that aquaporin-4 expression was negatively correlated with the apparent diffusion coefficient （r = -0.806, P 〈 0.01）. These findings suggest that upregulated aquaporin-4 expression is likely to be the main molecular mechanism of intracellular edema and may be the molecular basis for decreased relative apparent diffusion coefficient. Aquaporin-4 gene interference can effectively inhibit the upregulation of aquaporin-4 expression during the stage of intracelfular edema with time-effectiveness. Moreover, diffusion-weighted MRI can accurately detect intracellular edema.

Polymorphism of p16INK4a gene and rare mutation of p15INK4b gene exon2 in primary hepatocarcinoma

INTRODUCTION Hepatocellular carcinoma(HCC)is the mostcommon cause of death from cancer in China.Themechanisms of hepatocarcinogenesis are not yetknown clearly,p16INK4a gene,the multiple tumorsuppressor gene 1(MTS1),encodes P16 protein,which acts as an inhibitor by binding directly toCDK4 and CDK6 and preventing its association

Association of Graves’ disease and Graves’ ophthalmopathy with the polymorphisms in promoter and exon 1 of cytotoxic T lymphocyte associated antigen-4 gene

Objective: To investigate the association of Graves’ disease and Graves’ ophthalmopathy with the C/T transition polymorphism at position –318 of promoter and the A/G transition polymorphism at position 49 of exon 1 within cytotoxic T lymphocyte associated antigen-4 (CTLA-4) gene. Methods: Thirty-three patients with ophthalmopathy of Graves’ disease, fifty-six Graves’ patients without ophthalmopathy and sixty normal subjects as control were involved in the present case-control study. The polymorphisms were evaluated by polymerase chain reaction fragment length polymorphism (PCR-RFLP). Com-parisons were made of gene frequencies and allele frequencies between the groups. Results: The gene frequencies of CT and allele frequencies of T were much higher in Graves’ patients with ophthalmopathy than that in the group without ophthalmopathy (P=0.020, P=0.019). The gene frequencies of GG and allele frequencies of G in patients with Graves’ disease were significantly increased as compared with control group (P=0.008, P=0.007). The data suggest that smokers with Graves’ disease seemed to be more predisposed to ophthalmopathy than non-smokers (P=0.018). Conclusion: Our results suggest that an allele of T at position –318 of promoter is associated with genetic susceptibility to Graves’ ophthalmopathy while an allele of G at position 49 of exon 1 is associated with genetic susceptibility to Graves’ disease instead. Smoking is believed to be a major risk factor for ophthalmo-pathy.

Effect of deleted pancreatic cancer locus 4 gene transfection on biological behaviors of human colorectal carcinoma cells

AIM: To investigate the effect of deleted pancreatic cancer locus 4 (DPC4) gene transfection on biological behaviors of human colorectal carcinoma cells and the role of DPC4 gene in colorectal carcinogenesis. METHODS: PcDNA3.1-DPC4 plasmid was re-constructed by gene-recombination technology. SW620 cells, a human colorectal carcinoma cell line, were transfected with PcDNA3.1-DPC4 plasmid using lipofectamine transfecting technique. Transfected cells were selected with G418. Expression of Smad4 protein was detected in cells transfected with DPC4 gene by immunohistochemistry and Western blot. Biological characterristics of transfected cells were evaluated by population-doubling time and cloning efficiency. Alterations of percentage of S phage cells (S%) and apoptosis rate were determined by flow-cytometry. RESULTS: PcDNA3.1-DPC4 plasmid was constructed successfully. SW620 cells transfected with PcDNA3.1-DPC4 plasmid (DPC4+-SW620 cells) showed a strong intracellular expression of Smad4 protein, and the positive signal was localized in cytoplasm and nuclei, mainly in cytoplasm, where the expressions of Smad4 protein in SW620 cells transfected with PcDNA3.1 plasmid (PcDNA3.1-SW620 cells) and non-transfected SW620 cells (SW620 cells) were weaker than those in DPC4+-SW620 cells. The population-doubling time in DPC4+-SW620 cells (116 h) was significantly longer than that in SW620 cells (31 h) and PcDNA3.1-Sw620 cells (29 h) (P<0.01). The cloning efficiencies of DPC4+-SW620 cells (12%) were markedly lower than those of SW620 cells (69%) and PcDNA3.1-Sw620 cells (67%) (P<0.01). Compared with SW620 cells and PcDNA3.1-Sw620 cells, the Go-G1% of DPC4+-SW620 cells was obviously higher and the S% was markedly lower (P<0.05). Apoptosis rate of DPC4+-SW620 cells was significantly higher than that of SW620 cells and PcDNA3.1-SW620 cells. CONCLUSION: PcDNA3.1-DPC4 plasmid can be successfully re-constructed and stably transfected into human SW620cells, thereby the cells can steadily express Smad4. DPC4 protein may regulate proliferation of colorectal carcinoma cells by inhibiting cell growth and inducing cell apoptosis.

Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination

The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtil＆ was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone a-factor （MFals）, and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction （PCR）-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae （SC-βG） was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A （PrA） activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-glucanase expression level with the maximum of 69.3 U/（h·ml） after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-1,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.

Bioinformatics analysis of microarray data to explore the key genes involved in HSF4 mutation-induced cataract

AIM： To reveal the mechanisms of heat-shock transcription factor 4 （HSF4） mutation-induced cataract.METHODS： GSE22362, including 3 HSF4-null lens and 3 wild-type lens, was obtained from Gene Expression Omnibus database. After data preprocessing, the differentially expressed genes （DEGs） were identified using the limma package. Based on Database for Annotation, Visualization and Integrated Discovery （DAVID） tool, functional and pathway enrichment analyses were performed for the DEGs. Followed by protein-protein interaction （PPI） network was constructed using STRING database and Cytoscape software. Furthermore, the validated microRNA （miRNA）-DEG pairs were obtained from miRWalk2.0 database, and then miRNA-DEG regulatory network was visualized by Cytoscape software. RESULTS： A total of 176 DEGs were identified in HSF4-null lens compared with wild-type lens. In the PPI network, FBJ osteosarcoma oncogene （FOS）, early growth response 1 （EGR1） and heme oxygenase （decycling） 1 （HMOX1） had higher degrees and could interact with each other. Besides, mmu-miR-15a-5p and mmu-miR-26a-5p were among the top 10 miRNAs in the miRNA-DEG regulatory network. Additionally, mmu-miR-26a-5p could target EGR1 in the regulatory network. CONCLUSION： FOS, EGR1, HMOX1, mmu-miR-26a-5p and mmu-miR-15a-5p might function in the pathogenesis of HSF4 mutation-induced cataract.

Clinical significance of NOD2/CARD15 and Toll-like receptor 4 gene single nucleotide polymorphisms in inflammatory bowel disease

AIM： To evaluate the role of genetic factors in the pathogenesis of Crohn＇s disease （CD） and ulcerative colitis （UC）, we investigated the single nucleotide polymorphisms （SNPs） of NOD2/CARD15 （R702W, Gg08R and L1007finsC）, and Toll-like receptor 4 （TLR4） genes （D299G and T399I） in a selected inflammatory bowel disease （IBD） population coming from Southern Italy. METHODS： Allele and genotype frequencies of NOD2/ CARD15 （R702W, Gg08R and L1007finsC） and TLR4 （D299G and T399I） SNPs were examined in 133 CD patients, in 45 UC patients, and in 103 healthy controls. A genotype-phenotype correlation was performed. RESULTS： NOD2/CARD15 R702W mutation was significantly more frequent in CD （9.8%） than in controls （2.4%, P = 0.001） and in UC （2.3%, P = 0.03）. No significant difference was found between UC patients and control group （P 〉 0.05）. In CD and UC patients, no significant association with G908R variant was found. L1007finsC SNP showed an association with CD （9.8%） compared with controls （2.9%, P = 0.002） and UC patients （2.3%, P = 0.01）. Moreover, in CD patients, G908R and L1007finsC mutations were significantly associated with different phenotypes compared to CD wild-type patients. No association of IBD with the TLR4 SNPs was found in either cohort （allele frequencies： D299G-controls 3.9%, CD 3.7%, UC 3.4%, P 〉 0.05; T399I-controls 2.9%, CD 3.0%, UC 3.4%, P 〉 0.05）. CONCLUSION： These findings confirm that, in our IBD patients selected from Southern Italy, the NOD2/ CARD15, but not TLR4 SNPs, are associated with increased risk of CD.

Cloning and Expression of C4B Gene in Siji Goose

[Objective] This study aimed to clone C4B gene in Siji goose and detect its expression level in different tissues. [Method] cDNA sequence of C4B gene was cloned with RACE-PCR method. Amino acid sequences in multiple species were aligned in GenBank, and a phylogenetic tree was constructed for homology analysis. [Result] C4B gene in Siji goose shared relatively high homology with chicken and quail; Siji goose C4B gene was expressed highly in liver and lung of adult geese and expressed lowly in epididymis, seminiferous duct, brain, kidney, testis, heart, oviduct and smal intestine. [Conclusion] In the present study, mRNA expression lev-el of C4B gene in different tissues and organs of Siji goose was determined by flu-orescence quantitative PCR, which provided basis for rapid diagnosis of specific an-imal diseases.

Cytotoxic T-lymphocyte antigen 4 gene polymorphisms and susceptibility to chronic hepatitis B

AIM： To assess the three polymorphJsm regions within cytotoxic T-lymphocyte antigen 4 （CTLA-4） gene, a C/T base exchange in the promoter region-318 （CTLA-4 -318C/T）, an A/G substitution in the exon 1 position 49 （CTLA-4 49A/G）, a T/C substitution in 1172 （CTLA-4 -1172T/C） in patients with chronic hepatitis B. METHODS： Fifty-one patients with chronic hepatitis B virus infection and 150 healthy subjects were recruited sequentially as they presented to the hepatic clinic. Classification of chronic hepatitis B virus （HBV）-infected patients was as asymptomatic carrier state （26 patients） and chronic hepatitis B （25 patients）. Genomic DNA was isolated from anti-coagulated peripheral blood Bully coat using Miller＇s salting-out method. The presence of the CTLA-4 gene polymorphisms was determined using polymerase chain reaction amplification refractory mutation system （ARMS）. RESULTS： We observed a significant association between -318 genotypes frequency （T＋C-, T＋C＋, T-C＋） and susceptibility to chronic hepatitis B （P=0.012, OR=0.49, 95%CI： 0.206-1.162）. However, we did not observe a significant association for ＋49 genotype frequency （T＋C＋, T＋C- T-C＋） and -1172 genotype frequency （C＋T＋, T＋C- C＋T-） and state of disease. CONCLUSION： Our results suggest that CTLA-4 gene polymorphisms may partially be involved in the susceptibility to chronic hepatitis B.

Association of gene and protein expression and genetic polymorphism of CC chemokine ligand 4 in colorectal cancer

BACKGROUND Leukocytes,such as T cells and macrophages,play an important role in tumorigenesis.CC chemokine ligand(CCL)4,which is produced by lymphocytes and macrophages,has been found to be expressed in the mucosa of the gastrointestinal tract and is a potent chemoattractant for various leukocytes.AIM To examine CCL4 expression and its genetic polymorphism rs10491121 in patients with colorectal cancer(CRC)and evaluate their prognostic significance.METHODS Luminex technology was used to determine CCL4 Levels in CRC tissue(n=98),compared with paired normal tissue,and in plasma from patients with CRC(n=103),compared with healthy controls(n=97).Included patients had undergone surgical resection for primary colorectal adenocarcinomas between 1996 and 2019 at the Department of Surgery,Ryhov County Hospital,Jönköping,Sweden.Reverse transcription quantitative PCR was used to investigate the CCL4 gene expression in CRC tissue(n=101).Paired normal tissue and TaqMan single nucleotide polymorphism assays were used for the CCL4 rs10491121 polymorphism in 610 CRC patients and 409 healthy controls.RESULTS The CCL4 protein and messenger RNA expression levels were higher in CRC tissue than in normal paired tissue(90%,P<0.001 and 45%,P<0.05,respectively).CRC tissue from patients with localized disease had 2.8-fold higher protein expression levels than that from patients with disseminated disease.Low CCL4 protein expression levels in CRC tissue were associated with a 30%lower cancer-specific survival rate in patients(P<0.01).The level of plasma CCL4 was 11%higher in CRC patients than in healthy controls(P<0.05)and was positively correlated(r=0.56,P<0.01)with the CCL4 protein level in CRC tissue.The analysis of CCL4 gene polymorphism rs10491121 showed a difference(P<0.05)between localized disease and disseminated disease in the right colon,with a dominance of allele A in localized disease.Moreover,the rate of the A allele was higher among CRC patients with mucinous cancer than among those with nonmucinous cancer.CONCLUSION The present study indicates that the CRC tissue levels of CCL4 and CCL4 gene polymorphism rs10491121,particularly in the right colon,are associated with clinical outcome in CRC patients.

EYA4 gene functions as a prognostic marker and inhibits the growth of intrahepatic cholangiocarcinoma

Background:The molecular prognostic markers and carcinogenesis of intrahepatic cholangiocarcinoma(ICC) have not been well documented.The purpose of this study was to investigate the prognostic value of the eyes absent homolog 4(EYA4) gene in ICC and its biological effects on ICC growth in vitro and in vivo.Methods:One hundred twelve patients with ICC who underwent hepatectomy were enrolled in the study.EYA4 mRNA and EYA4 protein levels in ICC and adjacent non-tumoral tissues were evaluated using real-time quantitative polymerase chain reaction and immunohistochemical staining,respectively.EYA4 protein levels in ICC cells were determined using western blot analysis.The associations between EYA4 expression and clinicopathologic features of ICC were analyzed.To identify independent prognostic factors,univariate and multivariate analyses were performed.The biological effects of EYA4 on ICC cells were evaluated by establishing stable EYA4-overexpressing transfectants in vitro,and EYA4's effects on tumor growth were evaluated by intra-tumoral injection of EVA4-expressing plasmids in a NOD/SCID murine model of xenograft tumors.Results:ICC tissues had significantly lower EYA4 mRNA and protein levels compared with adjacent non-tumoral tissues(both P < 0.001).Univariate and multivariate analyses showed that EYA4 protein level,tumor number,adjacent organ invasion,lymph node metastasis,and tumor differentiation were independent prognostic factors for diseasefree survival and overall survival(all P < 0.05).In vitro,EYA4 overexpression inhibited tumor cell growth,foci formation,and cell invasiveness.In vivo,intra-tumoral injection of EYA4-expressing plasmids significantly inhibited ICC growth in the murine xenograft model compared with the control group(P < 0.05).Conclusion:EYA4 gene functioned as a molecular prognostic marker in ICC,and its overexpression inhibited tumor growth in vitro and in vivo.