Sequence variability of the 5, UTR in isolates of hepatitis C virus in China

Background: Hepatitis C virus infection is a great is- sue in China; however, there is very little informa- tion on genotyping investigations based on sequence variability in the 5' untranslated (5'UTR) reported. The present study was to define the sequence varia- bility based on the sequence divergences of the 5' UTR of the virus. Methods: Sequences of 91 isolates from patients with chronic hepatitis C from Yunnan, southwest China, were sequenced and genotypes were defined accord- ing to the sequence divergences of the 5' UTR of the virus. Results: Eighty-six isolates were classified into 3 clades (previously termed groups or major types) by the methods proposed by Chan et al in 1992 and phy- logenetic analysis based on nucleotide sequence diver- gences within the 5' UTR. Fifty-six percent of the i- solates were classified into clade 3, 35% into clade 1, and 34.9% into clade 2. New genotypes 1f, 2h, 3h and 3i were defined. In addition, 3 novel sequences were discovered, respectively with an 18-nt sequence deletion (corresponding to nucleotide position -173 to -156), a 28-nt sequence insertion, and a 40-nt se- quence insertion, between -56 and -55. Of these i- solates, 56% possessed a 'G' at position -66 in place of the 'T' that is present in all previously re- ported sequences. Conclusions: These HCV variants, evolved or re- mained in this area, may be of great significance in diagnosis and treatment of hepatitis C patients.

Genomic structure analysis of SNC6, a progesterone-receptor associated protein gene, and cloning and characterization of its 5＇-flanking region

Objective: To analyze the genomic structure of SNC6, a progesterone-receptor associated protein gene and its regulatory elements in its 5'-flanking region. Methods: Genomic sequence from C, enllank database ( accession number: Z98048) covering the whole SNC6 gene was used to analyze the genonfic stnmture of SNC6 and design primers for PCR amplification of its 5'-flanking region. A 1894 bp fragrnem of the 5’-flanking region ( - 1814 to + 75) was cloned by PCR using genomic DNA from a healthy donor peripheral blood lymphocyte as template. This fragment, as well as 3 shorter derivative fragments ( 1423 bp, 632 bp and 416bp, which correspond to - 1344 to + 75, - 552 to + 75 and - 337 to + 75 respectively), were subeloned into pGL2 series luciferase reporter vectors. These constructs were introduced into colorectal cancer cell line SW620 for transient expression of reporter gene and luefferase activities were measured. Results: The genomic structure analysis showed there are 12 exons for SNC6 gene, which spans 32017 bp (nt71529 to nt39513 in Z98048 sequence). All tmnsfected SW620 cells with the above 5-flanking region-containing constructs showed lueiferase activities. The highest lueiferase activities were measured in transfected cells with vectors containing 1894 bp fragments, and the lowest luefferase activities were measured in tmnsfected cells with vectors containing 416 bp fragments, lmeiferase activities were higher in transfected cells with vectors containing 632 bp fragments than that in tmnsfected cells with vectors containing 1423 bp fragments. Conclusion: The basle tran-scription-promoting element (promoter) for SNC6 expression resides between 0 to - 337, and two transcription-enhancing dements (enhancer) resides between - 337 to - 552 and - 1344 to - 1814, whereas one transcription-inhibiting element (silencer) exists between -552 to - 1344.

Effect of Impregnation Sequence on Propane Dehydrogenation Performance of PtSnNa/ZSM-5 Catalyst

The effects of the sequence for impregnation of metal precursors on the performance of PtSnNa/ZSM-5 catalyst for propane dehydrogenation to propene were studied in this paper.Some methods such as XRD,TPDA,BET,H2-TPR,XPS,ICP,TEM and hydrogen chemisorption were used to characterize the catalysts.The structure of ZSM-5 zeolite was not destroyed by the introduction of metal components.Meanwhile the different impregnation sequence of metal precursors could affect the behavior of Sn4+species entering the ZSM-5 channel,and the interaction between platinum and tin species,as well as the degree for reduction of Pt and Sn components.As a result,the prepared catalysts exhibited different reaction activity and selectivity.Compared with the co-impregnation treated catalyst,the catalysts prepared by the sequential impregnation method showed better catalytic activity in propane dehydrogenation,especially the one prepared through impregnation with tin precursor at first.Finally,a model for the effect of impregnation sequence on the distribution of Pt and Sn species in PtSnNa/ZSM-5 catalyst was proposed.

Small cell lung carcinoma with KIF5B-RET fusion partially responded to the 4^(th)-line therapy with anlotinib:A case report

BACKGROUND Small cell lung cancer(SCLC)exhibits a pronounced tendency for metastasis and relapse,and the acquisition of resistance to chemotherapy and radiotherapy,leading to complexity in treatment outcomes.It is crucial to tackle these challenges by advancing targeted therapeutic approaches in ongoing research endeavors.Variant RET fusions have been reported in several solid tumors,but are rarely reported in SCLC.CASE SUMMARY We present the first case of a KIF5B-RET fusion in a 65-year-old male patient with SCLC.To date,the patient has received the 4th line chemotherapy with anlotinib for one year and has shown a sustained favorable partial response.According to the results of next generation sequencing,this SCLC patient harbors the KIF5BRET fusion,suggesting that RET fusion could serve as a promising molecular target for SCLC treatment.Next-generation sequencing(NGS)plays a critical rolein comprehensively assessing the genotype and phenotype of cancer.CONCLUSION NGS can provide SCLC patients with personalized and targeted therapy options,thereby improving their likelihood of survival.

THE ABNORMAL EXPRESSION OF CLONED REPEATED SEQUENCE DNA, L5B-4, IN RAT HEPATOMA BERH-2

A repeated sequence DNA fragment, L5B-4, was cloned from the 5 kb BamHI DNA fragments of rat genomic DNA. The expressions of the L5B-4 DNA fragment are different in liver and hepatoma cells. The amounts of transcripts in hepatoma cells are lower in nucleus and higher in cytoplasm, especially in polysomal RNA, as compared with that in liver cells. The alteration shown in polysomal RNA of hepatoma cells seems to be specific. These results are discussed with respect to the possible function of this repeated DNA and its variation in hepatoma cells.

The Reaction Sequence and Dielectric Properties of BaAl₂Ti₅O₁₄Ceramics

To investigate the correct reaction sequence of BaO-Al2O3-5TiO2 system, powders calcined at different temperatures are analyzed by x-ray diffraction. The results show that the source phase BaCO3 decomposes below 800°C, TiO2 and Al2O3 start to consume at 900 and 1100°C, respectively. BaTi4O9 phase appears at 1000°C while BaAl2Ti5O14 phase starts to reveal at 1200°C. As the temperature increases, the density, dielectric constant and quality factor of the BaAl2Ti5O14 ceramic increase and keep unchanged at 1350°C. The dielectric properties of BaAl2Ti5O14 ceramic sintered at 1350°C for 3h are: εr=35.8, Q×f=5130GHz, τf=-6.8ppm/°C.

Molecular cloning and analysis of the partial sequence of Rhinopithecus roxellanae growth hormone gene

Growth hormone gene (GH) ofRhinopithecus roxellanae was amplified by PCR based on the sequences of the reported mammalian growth hormone gene for the first time. The amplified fragment was about 1.8 kb. It was cloned and its upper stream was sequenced. This sequencing region consists of a 5′flanking regulatory region, exon I and part of exon II, intron I of growth hormone gene. Comparing the corresponding sequences of growth hormone gene betweenRhinopithecus roxellanae and the porcine, we concluded that the homology reached 81% in the region, and there was high conservation in the 5′flanking sequence. The kinds of amino acids of exon I and exon II for about 90% were the same to those in pig. Many mutations occurred in the degenerate site of the triplet code. In the nucleotides of intron I, there were only 72% homologies with those in pig. It means that introns and 3′flanking sequence maybe play an important part in growth hormone gene regulation of the different animals.

The circ_0002538/miR-138-5p/plasmolipin axis regulates Schwann cell migration and myelination in diabetic peripheral neuropathy

Circular RNAs(circRNAs)play a vital role in diabetic peripheral neuropathy.However,their expression and function in Schwann cells in individuals with diabetic peripheral neuropathy remain poorly understood.Here,we performed protein profiling and circRNA sequencing of sural nerves in patients with diabetic peripheral neuropathy and controls.Protein profiling revealed 265 differentially expressed proteins in the diabetic peripheral neuropathy group.Gene Ontology indicated that differentially expressed proteins were mainly enriched in myelination and mitochondrial oxidative phosphorylation.A real-time polymerase chain reaction assay performed to validate the circRNA sequencing results yielded 11 differentially expressed circRNAs.circ_0002538 was markedly downregulated in patients with diabetic peripheral neuropathy.Further in vitro experiments showed that overexpression of circ_0002538 promoted the migration of Schwann cells by upregulating plasmolipin(PLLP)expression.Moreover,overexpression of circ_0002538 in the sciatic nerve in a streptozotocin-induced mouse model of diabetic peripheral neuropathy alleviated demyelination and improved sciatic nerve function.The results of a mechanistic experiment showed that circ_0002538 promotes PLLP expression by sponging miR-138-5p,while a lack of circ_0002538 led to a PLLP deficiency that further suppressed Schwann cell migration.These findings suggest that the circ_0002538/miR-138-5p/PLLP axis can promote the migration of Schwann cells in diabetic peripheral neuropathy patients,improving myelin sheath structure and nerve function.Thus,this axis is a potential target for therapeutic treatment of diabetic peripheral neuropathy.

鸡MDA5的序列分析与结构域预测

为了解鸡黑色素瘤分化相关基因-5(Melanoma differentiation-associated protein 5,MDA5)的序列和结构域,试验使用PCR方法扩增了鸡MDA5基因并构建真核表达质粒,实现了鸡MDA5蛋白的真核表达,同时利用MegAlign等软件对鸡MDA5氨基酸序列进行分析。结果显示:鸡MDA5全长3 006 bp,编码1 001个氨基酸;鸡MDA5与人、猕猴、野猪、小鼠、绿头鸭、灰雁、鸿雁、鲫鱼、草鱼的氨基酸相似性分别为62.4%、62.9%、62.7%、64.1%、86.8%、87.3%、87.4%、48.1%、46.3%;鸡MDA5与鸟类进化关系较近,与哺乳动物次之,与鱼类进化关系较远;鸡MDA5由N端两个半胱天冬酶激活招募结构域、DExD/H box RNA解旋酶结构域和C端结构域组成;鸡MDA5二级结构包含α螺旋(50.95%)、延伸链(11.19%)、β转角(3.9%)和无规则卷曲(33.97%),三级结构与人MDA5类似,在空间上呈现半闭合环状结构。研究成功鉴定了鸡MDA5基因,分析了其基因序列并预测其结构域,为深入研究鸡MDA5结构与功能及其在鸡抗病毒天然免疫应答中的作用奠定基础。

18种观赏植物F3′5′H基因生物信息学分析

采用生物信息学方法对18种观赏植物类黄酮-3′5′-羟化酶基因(flavonoid-3′5′-hydroxylase,F3′5′H)的mRNA和氨基酸序列的理化性质、跨膜结构域、保守结构域、亚细胞定位、二级结构、三级结构和同源性进行预测与分析。结果表明,绝大多数观赏植物的F3′5′H为亲水性稳定蛋白质,以α螺旋为主、无信号肽的跨膜蛋白质;大多数定位于内质网膜上;其三级结构模型为5ylw.1.A铁锈醇合成酶,为单链蛋白,属于细胞色素P450基因家族;同源保守氨基酸序列为“LPPGP”“AGTDTS”和“PFGAGRRICAG”。

Differential expression and significance of 5-hydroxymethylcytosine modification in hepatitis B virus carriers and patients with liver cirrhosis and liver cancer

BACKGROUND The relationship between hepatitis B surface antigen(HBsAg)-positive carrier status and liver cancer has been extensively studied.However,the epigenetic changes that occur during progression from HBsAg-positive carrier status or cirrhosis to liver cancer are unknown.The epigenetic modification of DNA hydroxymethylation is critical in tumor development.Further,5-hydroxymethylcytosine(5hmC)is an important base for DNA demethylation and epigenetic regulation.It is also involved in the assembly of chromosomes and the regulation of gene expression.However,the mechanism of action of 5hmC in HBsAgpositive carriers or patients with cirrhosis who develop liver cancer has not been fully elucidated.AIM To investigate the possible epigenetic mechanism of HBsAg-positive carriers and hepatocellular carcinoma(HCC)progression from cirrhosis.METHODS Forty HBsAg-positive carriers,forty patients with liver cirrhosis,and forty patients with liver cancer admitted to the First People's Hospital of Yongkang between March 2020 and November 2021 were selected as participants.Free DNA was extracted using a cf-DNA kit.cfDNA was extracted by 5hmC DNA sequencing for principal component analysis,the expression profiles of the three groups of samples were detected,and the differentially expressed genes(DEGs)modified by hydroxymethylation were screened.Bioinformatic analysis was used to enrich DEGs,such as in biological pathways.RESULTS A total of 16455 hydroxymethylated genes were identified.Sequencing results showed that 32 genes had significant 5hmC modification differences between HBsAg carriers and liver cancer patients,of which 30 were upregulated and 2 downregulated in patients with HCC compared with HBsAg-positive carriers.Significant 5hmC modification differences between liver cirrhosis and liver cancer patients were identified in 20 genes,of which 17 were upregulated and 3 were downregulated in patients with HCC compared with those with cirrhosis.These genes may have potential loci that are undiscovered or unelucidated,which contribute to the development and progression of liver cancer.Analysis of gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes showed that the major signaling pathways involved in the differential genes were biliary secretion and insulin secretion.The analysis of protein interactions showed that the important genes in the protein-protein interaction network were phosphoenolpyruvate carboxykinase and solute carrier family 2.CONCLUSION The occurrence and development of liver cancer involves multiple genes and pathways,which may be potential targets for preventing hepatitis B carriers from developing liver cancer.

基于双向门控循环单元的5-甲基胞嘧啶位点预测

5-甲基胞嘧啶(5-methylcytosine,m5C)是一种重要的转录后修饰,大量证据表明,m5C在许多生物学过程中起着至关重要的作用.准确鉴定m5C位点有助于更好地了解其生物学功能.为此提出了一个名为pm5C-BGRU的模型,该模型通过拼接独热编码(One-hot encoding)和核苷酸化学性质(nucleotide chemical property,NCP)进而对RNA序列进行特征提取,并基于双向门控循环单元(Bidirectional Gated Recurrent Unit,BiGRU)来识别m5C位点.将该方法在人类、小鼠和拟南芥三个物种的m5C数据集上进行建模和测试,并对照已有的预测模型进行评估.结果表明,pm5C-BGRU在交叉验证和独立数据集测试中均取得优异效果,该模型有望成为鉴定m5C位点的有力工具.

耐盐转基因大豆事件AtARA6-A001外源插入片段侧翼序列及其应用

转基因大豆AtARA6-A001事件采用农杆菌介导大豆遗传转化法获得,其受体为沈农9号,具有耐盐特性,目前已经进入环境释放阶段。为明确该转基因大豆材料的分子特征及其转基因检测方法,推进该转基因事件生物安全评价工作。本研究以转基因大豆AtARA6-A001为研究对象,利用Southern杂交方法及基因组重测序技术鉴定了外源基因的拷贝数及插入位点的位置和方向。同时利用PCR扩增技术获得了外源T-DNA的左右侧翼序列,并基于左右旁侧序列,建立了转AtARA6基因耐盐大豆A001事件的特异性定性PCR检测方法。此方法能特异性检测转基因大豆植株AtARA6-A001根、茎、叶、花和种子样品,并且能够特异性识别转基因大豆事件的身份,这为后续转基因大豆及其产品的检测和监管提供技术支持。

3.0T MRI T2Mapping序列对0~5岁正常儿童髋关节软骨评估的探索性分析

目的利用3.0T MRI T2Mapping序列对0~5岁正常儿童髋关节软骨行磁共振扫描并量化分析,探究不同阶段髋关节软骨T2值变化规律,初步建立0~5岁正常儿童髋关节软骨T2值的参考范围。方法收集0~5岁发育正常儿童196名,其中男92名,女104名。根据年龄分成5组,分别对所有儿童行髋关节常规序列、T2-mapping序列扫描,在髋臼软骨及与其对应的股骨头软骨划分上、前、后三个区域,共6个亚区,测量其T2值。观察分析组间及组内不同亚区T2值的差异并计算其正常值范围,分析相关因素。结果0~5岁正常儿童髋关节软骨,不同性别各亚区间T2值比较差异均无统计学意义(均P>0.05)。左、右两侧对应亚区T2值比较差异均无统计学意义(均P>0.05)。不同髋关节亚区各年龄组T2值比较,年龄组间T2值差异有统计学意义(均P<0.05)。结论3.0T MRI T2 mapping序列能定量分析儿童髋关节软骨T2值,0~5岁正常儿童髋关节软骨各亚区软骨T2值与年龄相关,与性别和左右无关。

投掷动作发展序列与4~5岁幼儿上手投掷 能力的关联性研究

该文采用录像解析、数理统计等方法,旨在探究投掷动作发展序列与投掷能力的关联性,以便日后的幼儿体育工作者和研究者运用投掷动作发展序列客观评价幼儿的投掷能力。研究结果:(1)整体序列与投掷距离关联性P=0.005<0.05,y=2.930x+0.411与投掷精准度关联性P=0.301>0.05;(2)部分序列与投掷距离关联性P=0.000<0.001,y=1.378x+0.288与投掷精准度关联性P=0.222>0.05;(3)整体序列与部分序列关联性P=0.000<0.001,y=5.536x+1.354。研究结论:(1)4~5岁幼儿上手投掷动作处于低、中两阶段的人数最多;(2)上手投掷动作序列与投掷距离呈正相关,与投掷精准度无显著相关性;(3)整体序列与部分序列的发展呈正相关。

Analysis and validation of genome-specific DNA variations in 5′flanking conserved sequences of wheat low-molecular-weight glutenin subunit genes

The thirty-three 5′ flanking conserved sequences of the known low-molecular-weight subunit (LMW-GS) genes have been divided into eight clusters, which was in agreement with the classification based on the deduced N-terminal protein sequences. The DNA polymorphism between the eight clusters was obtained by sequence alignment, and a total of 34 polymorphic positions were observed in the approximately 200 bp regions, among which 18 polymorphic positions were candidate SNPs. Seven cluster-specific primer sets were designed for seven out of eight clusters containing cluster-specific bases, with which the genomic DNA of the ditelosomic lines of group 1 chromosomes of a wheat variety ‘Chinese Spring’ was employed to carry out chromosome assignment. The subse-quent cloning and DNA sequencing of PCR fragments validated the sequences specificity of the 5′ flanking conserved sequences between LMW-GS gene groups in different genomes. These results suggested that the coding and 5′ flanking regions of LMW-GS genes are likely to have evolved in a concerted fashion. The seven primer sets developed in this study could be used to isolate the com-plete ORFs of seven groups of LMW-GS genes, respectively, and therefore possess great value for further research in the contributions of a single LMW-GS gene to wheat quality in the complex genetic background and the efficient selections of quality-related components in breeding programs.

鳜鱼(Siniperca chuatsi)β-肌动蛋白基因cDNA全序列与5′侧翼区的克隆与分析

采用RT-PCR及RACE法,分离、克隆鳜鱼β-肌动蛋白基因cDNA全序列,再用基因组步行法(Genome Walker)克隆鳜鱼β-肌动蛋白基因5′调控区。序列分析结果表明,鳜鱼β-肌动蛋白基因全长1897bp,其中5′-UTR长94bp,3′-UTR长675bp,编码区长1128bp,编码375个氨基酸。将所得序列与其它动物类群的β-肌动蛋白基因序列进行比较分析显示,鱼类、两栖类、鸟类、哺乳类等不同类群脊椎动物β-肌动蛋白氨基酸序列同源性均在96%以上,说明该基因在生物进化过程中高度保守。通过鳜鱼与其它脊椎动物β-肌动蛋白基因的核苷酸序列构建的进化树显示,脊椎动物β-肌动蛋白聚类成3个分支,鱼类β-肌动蛋白基因形成一个独立的分化群,说明鱼类β-肌动蛋白基因起源于一个共同祖先。克隆得到的鳜鱼β-肌动蛋白基因5'侧翼序列长1399bp,对其进行序列分析,在其起始密码字ATG上游200bp范围内发现含有CAATbox、CC(A/T)6GG(CArGbox)、TATA box对转录调控起重要作用的顺式元件,同时在侧翼区也发现含有GC box、MYOD、YY1、SP1、GATA等多个潜在调控元件。鳜鱼β-肌动蛋白基因5′侧翼序列的克隆成功,为今后转基因鳜鱼的研究工作奠定了基础。

鲢鱼可溶性谷胱甘肽S-转移酶(sGST)基因cDNA全序列与5′调控区的克隆与分析

淡水鱼类可溶性谷胱甘肽S-转移酶(sGST)在微囊藻毒素去毒代谢过程中具有独特的关键作用,因而也称为微囊藻毒素去毒酶.从淡水食毒藻鱼类鲢鱼(Hypophthalmichthysmolitrix)肝脏通过简并引物克隆微囊藻毒素去毒酶基因cDNA核心序列,应用5′RACE和3′RACE技术分别扩增该序列的5′末端和3′末端序列,最后通过序列拼接获得鲢鱼肝脏微囊藻毒素去毒酶基因cDNA全序列.序列分析结果表明,鲢鱼肝脏微囊藻毒素去毒酶基因cDNA全长920bp,其中5′-UTR长74bp,3′-UTR长174bp,编码区长672bp,编码223个氨基酸.应用基因组步行法,在鲢鱼克隆得到淡水鱼类微囊藻毒素去毒酶基因5′侧翼区878bp序列.与哺乳动物及海水鱼sGST基因不同,鲢鱼微囊藻毒素去毒酶基因的5′侧翼区,发现存在多个脂多糖反应元件(LPSRE),表明来源于毒藻的脂多糖可能对鲢鱼微囊藻毒素去毒酶基因表达有潜在调控作用.

山羊FSHR基因5'端侧翼序列的克隆与分析

为找寻山羊FSH受体基因的SNP突变位点,进行FSH受体基因的多态性分析和探索该受体基因在山羊多胎中的可能作用,利用比较基因组学的方法,根据绵羊FSH受体基因序列设计引物,以湖南湘东黑山羊基因组为模板,PCR扩增了FSH受体基因5' 端侧翼调控区,通过克隆进行了序列测定及分析,并与牛、绵羊该区段序列进行了比较.结果表明,PCR扩增获得的片段为844 bp,与中国西门塔尔牛该段序列相比,同源性为94.08%,在-364 bp处的左翼发生了多碱基插入,湘东黑山羊该序列有5个碱基的插入,另在-625^-619 bp处的7个碱基缺失;与澳洲绵羊的FSHR基因比较,同源性为93.60%,在-367 bp处的左翼,湘东黑山羊该段序列有2个碱基的插入,在-630,-145,-97 bp 处各有1个碱基缺失.

鸡8个功能基因5′-侧翼区与内含子的SNP多样性比较

本研究旨在探讨鸡功能基因5′-侧翼区的单核苷酸多态性(SNP)水平。作者以白来航鸡、隐性白洛克鸡、杏花鸡、丝羽乌骨鸡、固始鸡、红色原鸡为材料,扩增了GH、DDBC1、RIKEN、RASGRP3、IGF1、THRSP、VIP及PRL共8个基因的5′-侧翼区DNA序列。扩增序列全长为8,399bp,SNP的总数为161个,平均每52bp出现一个SNP。8个功能基因5′-侧翼区核苷酸多样性的θ均值和π均值分别为0.00620±0.00110和0.00559±0.00100。比较检验表明,5′-侧翼区的SNP多样性显著低于内含子区域。5′-侧翼区是基因表达的一个重要调控区域,在分子进化和系统发生过程中承受着比内含子区域更大的选择压,其较低的SNP多样性是适应性较好的表现。Tajima检验与Fu和Li检验表明,与鸡繁殖性状显著关联的VIP基因和PRL基因很可能是人工选择或自然选择的目的基因。