AAV2-PDE6B restores retinal structure and function in the retinal degeneration 10 mouse model of retinitis pigmentosa by promoting phototransduction and inhibiting apoptosis

Retinitis pigmentosa is a group of inherited diseases that lead to retinal degeneration and photoreceptor cell death.However,there is no effective treatment for retinitis pigmentosa caused by PDE6B mutation.Adeno-associated virus(AAV)-mediated gene therapy is a promising strategy for treating retinitis pigmentosa.The aim of this study was to explore the molecular mechanisms by which AAV2-PDE6B rescues retinal function.To do this,we injected retinal degeneration 10(rd10)mice subretinally with AAV2-PDE6B and assessed the therapeutic effects on retinal function and structure using dark-and light-adapted electroretinogram,optical coherence tomography,and immunofluorescence.Data-independent acquisition-mass spectrometry-based proteomic analysis was conducted to investigate protein expression levels and pathway enrichment,and the results from this analysis were verified by real-time polymerase chain reaction and western blotting.AAV2-PDE6B injection significantly upregulated PDE6βexpression,preserved electroretinogram responses,and preserved outer nuclear layer thickness in rd10 mice.Differentially expressed proteins between wild-type and rd10 mice were closely related to visual perception,and treating rd10 mice with AAV2-PDE6B restored differentially expressed protein expression to levels similar to those seen in wild-type mice.Kyoto Encyclopedia of Genes and Genome analysis showed that the differentially expressed proteins whose expression was most significantly altered by AAV2-PDE6B injection were enriched in phototransduction pathways.Furthermore,the phototransductionrelated proteins Pde6α,Rom1,Rho,Aldh1a1,and Rbp1 exhibited opposite expression patterns in rd10 mice with or without AAV2-PDE6B treatment.Finally,Bax/Bcl-2,p-ERK/ERK,and p-c-Fos/c-Fos expression levels decreased in rd10 mice following AAV2-PDE6B treatment.Our data suggest that AAV2-PDE6B-mediated gene therapy promotes phototransduction and inhibits apoptosis by inhibiting the ERK signaling pathway and upregulating Bcl-2/Bax expression in retinitis pigmentosa.

Phylogenetic Relationships and Status Quo of Colonies for Gayal Based on Analysis of Cytochrome b Gene Partial Sequences

Thirty-three mutations and four different haplotypes were found when cytochrome b（Cytb） gene partial sequences of 12 gayals were analyzed. Together with sequences of Bos indicus, Bos taurus, Bos grunniens, and Bos gaurus with Bubalus bubalis as the out group, the partial sequences of Cytb gene of gayals were aligned and base composition and nucleotide variation of Cytb gene were analyzed. The phylogenetic trees were constructed by the NJ method and the MP method respectively, both supporting almost the same topology. Gayal is an independent species of Bos from Bos indicus, Bos taurus, and Bos gaurus. The results also indicate that a great proportion of gayal bloodline was invaded by other species, and the protection of gayal is facing a formidable situation.

Phylogenetic Relationships of Monal Pheasants Lophophorus Inferred from Sequences of Mitochondrial Cytochrome b Gene

The phylogeny of the monal pheasants (Lophophorus) and their relationships to some species of the genera Tragopan,Pucrasia and Ithaginis were studied by comparing mitochondrial cytochrome b (cyt b) nucleotide sequences.The molecular phylogenetic trees show that:①the genus Tragopan and the genus Pucrasia share a common ancestor which is the sister taxon of the ancestor of the genus Lophophorus;②the genus Lophophorus had evolved into two branches:One was the Sclaters Monal;the other included the Chinese Monal and the Himalayan Monal.Considering its molecular phylogeny,distribution patterns and morphological evidences,the genus Lophophorus might originate in the Hengduan mountains region of southwestern China.

抗虫基因CryIA(b)植物表达载体的构建

苏云金芽孢杆菌杀虫晶体蛋白基因具有对鳞翅目昆虫的毒杀作用,因此被广泛用于转基因研究,以提高植物的抗虫能力。故以含有CryIA(b)基因的PKUB质粒为模板,利用PCR技术克隆出抗虫基因〔CryIA(b)〕,将其构建到PGEMT-Easy上,获得重组质粒PGEMT-CryIA(b),并测定全部序列,将PGEMT-CryIA(b)用BamH I和Sm aI酶切后插入表达载体PB I121的CaMV35S启动子和NOS终止子之间,构建成准备用于丽江云杉等针叶树种遗传转化CryIA(b)基因的植物表达载体PB I-CryIA(b),采用液氮冻融法将其转化到根癌农杆菌LBA4404和C58中,为下一步的转化工作奠定了基础。

P53-inducible Gene 3(PIG-3)在弥漫性大B细胞淋巴瘤中的表达及意义

本研究旨在探究P53-inducible gene 3(PIG-3)在弥漫性大B细胞淋巴瘤(DLBCL)中的表达情况及其与淋巴瘤发病机制的相关性。应用免疫印迹(Western blot)和RT-PCR等方法,检测弥漫性大B细胞淋巴瘤患者和健康成年人PIG-3蛋白的表达情况,并判断其与淋巴瘤发病机制的相关性。结果表明,Western blot检测弥漫性大B细胞淋巴瘤细胞中PIG-3蛋白表达明显低于对照组,化疗后6个月PIG-3蛋白表达较化疗前升高。RT-PCR结果显示,扩增产物大小为1285 bp,与理论值吻合。结论:PIG-3表达下调可能与弥漫性大B细胞淋巴瘤发生密切相关,故PIG-3有可能作为弥漫性大B细胞淋巴瘤治疗及预后检测的一个重要指标。

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.

Identification of the Rice Vacuolar ATPase B Subunit Gene and Its Expression Pattern Analysis Under Phosphorus Deficiency

A vacuolar ATPase (V-ATPase.) B subunit gene has been cloned and characterized front a phosphorus starvation induced rice root subtractive cDNA library by suppression subtractive hybridization (SSH) method and RT-PCR amplification. This gene encodes a polypeptide of 487 amino acid residues, containing a conservative ATP binding site and with a molecular weight of 54.06 kD and an isoelectric point of 4.99, southern analysis of the. genomic DNA indicates that V-ATPase B subunit is encoded by a single gene in rice genome. The amino acid homologies of V-ATPase B subunits among different organisms range from 76% to 97% and reveals that the evolution of V-ATPase B subunit is accompanied with the biological evolution. Expression pattern analysis indicated that the maximal expression of V-ATPase B subunit gene occurred at an early stage (6 - 12 h) after phosphorus starvation in roots, and lately stage (24 - 48 It) in leaves. Under phosphorus deficiency, the up-regulated expression of V-ATPase gene was presumed to strengthen the proton transport and provide the required energy to maintain an electrochemical gradient across the tonoplast to facilitate Phosphorus transport.

Construction of Recombinant Adenovirus Vector Containing CEVB2L Gene

[Objective] Sheep contagious ecthyma virus B2L gene recombinant adenovirus was built by adenovirus vector system.[Method] Genome DNA extracted from sheep contagious ecthyma virus strain JLSY04 as a template,Gene fragments obtained from B2L by PCR amplification;B2L gene cloning was cloned into PDNR-CMD vector,screening positive clones and plasmid CTC572-6 was obtained;CTC572-6 plasmid for homologous was recombined with the adenoviral vector.Screening positive clones and bacilli PCR,digestion and sequencing and so on were identified.[Result] After identified by enzyme digestion and gene sequencing,recombinant adenovirus vector CTC572Ade-30 of carrying sheep contagious ecthyma virus B2L gene was constructed successfully.[Conclusion] Which laid the foundation for sheep contagious ecthyma genetically engineered vaccine.

DGGE Analysis on Mitochondrial Cyt b Gene of Eriocheir sinensis and Eriocheir hepuensis

[Objective] The aim was to investigate the possibility to analyze the genetic diversity of Eriocheir sinensis and Eriocheir hepuensis by using the technique denaturing gradient gel electrophoresis（DGGE）.[Method] Mitochondrial cyt b gene fragment was amplified from 180 individuals of five populations of E.sinensis and a population of E.hepuensis and then analyzed by using DGGE.[Result] All PCR products showed two kinds of electrophoretic mobility on DGGE.The PCR products of all individuals from E.hepuensis showed the same mobility with that of the individuals from 46.7% of Jiangdu population,23.3% of Yizheng population and 20.0% of Wenzhou population of E.sinensis,while the rest of the individuals from the three populations of E.sinensis mentioned above as well as all the individuals of Nanjing and Panjin populations showed the same mobility,which was higher compared with that of E.hepuensis.The results indicated that there was the same genetic marker in E.sinensis populations as that of E.hepuensis population,which was consistent with previous studies.[Conclusion] DGGE technique could be used to analyze the genetic diversity of Chinese mitten crab.

Phylogenetic relationships of some Sylviidae species based on complete mtDNA cyt b and partial COI sequence data

Members of the passerine family Sylviidae are distributed widely around the Old World and the phylogenetic relationships of many species still remain controversial.In this study,we investigated the phylogeny and relationships among 37 species of 11 genera by analyzing DNA sequences obtained from the complete mitochondrial cytochrome b (cyt b) and partial cytochrome oxidase I (COI) genes.The data were analyzed by maximum-likelihood analysis and Bayesian inference.The results demonstrate that the current genus Phylloscopus is non-monophyletic,while Seicercus is synonymized with Phylloscopus or is a group within the genus Phylloscopus.We may conclude that within Phylloscopus there are close relations between P.magnirostris and P.borealis;among P.proregulus,P.yunnanensis,P.pulcher and P.maculipennis,as well as among P.occisinensis,P.schwarzi,P.armandii,P.collybita and P.fuscatus.Monophyly of the genus Cettia could not be corroborated;it is closely related to the genera Abroscopus and Tesia.However monophyly of Acrocephalus is supported.Furthermore,the close relationships between Sylvia and Zosterops and among Prinia,Orthotomus and Cisticola are also supported.

B亚型新等位基因803delC的分子生物学研究

目的分析研究1例新的B亚型等位基因血清学特点和分子生物学机制。方法应用血清学方法检测患者ABO血型。应用序列特异性引物-聚合酶链反应(PCR-SSP)检测ABO血型基因。应用Sanger基因序列分析检测ABO基因1~7外显子编码区域,确定基因突变位点。结果血清学鉴定患者正定型为O型,反定型为B型。PCR-SSP基因分型结果为A/O型,存在A基因,与血清学结果不符。进一步Sanger双链测序结果显示该标本在ABO^(*)B.01/ABO^(*)O.01.01的基础上,第7外显子803位置缺失C碱基。该突变最终导致多肽链上发生p.Ala268Gly和p.Phe269Ser的氨基酸替换,并且从269位置开始产生新的开放阅读框,新的开放阅读框第20号氨基酸为终止密码子,导致B基因表达终止。进一步ABO基因克隆测序证明该突变点位于ABO*B.01基因上,该突变已提交NCBI数据库,收录编号为OR343908。结论在中国人群中发现1种新的导致B变异型的ABO等位基因,基因检测方法可辅助鉴定血清学正、反定型不符的疑难血型。

Phylogenetic Relationships of 11 Bumblebee Species (Hymenoptera:Apidae) Based on Mitochondrial Cytochrome b Gene Sequences

Phylogenetic relationships of 11 bumblebee species,including 5 subgenera:Bombus (5 species),Thoracobombus (3 species),Mendacibombus (1 species),Fervidobombus (1 species) and Pyrobombus (1 species),were analyzed based on the 357?bp mitochondrial cytochrome b gene sequences.There are 65 singleton polymorphic sites and 71 parsimony informative polymorphic sites in this DNA segment,and 45 polymorphic sites within the total 119 translated amino acids segment.Both NJ tree and MP tree show that Mendacibombus (B.avinovielllus) is basal to others,followed by Fervidobombus (B.pensylvanicus);Pyrobombus (B.impatiens) and Bombus are sister subgenera;the subgenus of Bombus is monophyletic,in which B.ignitus diverged first.

基于线粒体Cyt b基因序列的4个黄尾鲴养殖群体遗传多样性分析

黄尾鲴(Xenocypris davidi)是浙江省自然水域增殖放流的主要鱼类,为了解人工繁育对黄尾鲴遗传多样性的影响,利用线粒体DNA细胞色素b基因(Cyt b)对4个养殖群体的遗传多样性进行研究,旨在为黄尾鲴增殖放流策略制定和实施提供基础数据。结果显示,在编码Cyt b的1 140 bp序列中,检测到157个变异位点,界定了21种单倍型,其中长兴、双浦、八里店和醴陵群体的单倍型数目分别为10个、11个、7个和2个,单倍型多样性介于0.226~0.794,核苷酸多样性介于0.006 14~0.023 86。除醴陵群体遗传多样性较低外,其余3个养殖群体的遗传多样性具有高单倍型数和高核苷酸多样性的特点。4个黄尾鲴养殖群体间的遗传距离为0.018 74~0.092 74,遗传分化指数为0.808 63(P<0.01),其中长兴和八里店群体的分化程度较低,双浦和醴陵群体的分化程度较高,且遗传变异主要发生在群体间。本研究结果可从分子水平为黄尾鲴的资源保护和人工增殖放流提供参考依据。

乳腺癌患者病理特征与Bcl-2、CXCL13、PAX8表达情况的关系分析

目的分析乳腺癌患者病理特征与B细胞淋巴瘤/白血病-2基因(Bcl-2)、趋化因子配体13(CXCL13)、配对盒基因8抗体(PAX8)表达情况的关系。方法收集2021年1月至2023年1月该院收治的160例乳腺癌患者临床资料。采用免疫组化法对其癌组织与癌旁组织Bcl-2、CXCL13、PAX8表达情况进行检测,并分析3项指标与患者病理特征的关系。结果与癌旁组织比较,癌组织Bcl-2、CXCL13、PAX8阳性率更高,差异有统计学意义(P<0.05)。与雌激素受体(ER)阴性、肿瘤最大径≥3 cm、孕激素受体(PR)阴性患者比较,ER阳性、肿瘤最大径<3 cm、PR阳性患者中Bcl-2高表达占比更高,差异有统计学意义(P<0.05);与无淋巴结转移、Ⅰ~Ⅱ期患者比较,淋巴结转移、Ⅲ~Ⅳ期患者中CXCL13高表达占比更高,差异有统计学意义(P<0.05);与Ⅰ~Ⅱ期、高/中分化、无淋巴结转移患者比较,Ⅲ~Ⅳ期、低分化、有淋巴结转移患者中PAX8高表达占比更高,差异有统计学意义(P<0.05)。ER、PR表达情况与Bcl-2表达情况呈正相关(P<0.05),肿瘤最大径与Bcl-2表达情况呈负相关(P<0.05);临床分期、淋巴结转移情况与CXCL13、PAX8表达情况呈正相关(P<0.05);分化程度与PAX8表达情况呈负相关(P<0.05)。结论乳腺癌患者Bcl-2、CXCL13、PAX8表达情况对疾病的发生和发展具有明显影响,有望成为评估乳腺癌患者病情严重程度的标志物。

ABO血型基因第7外显子695 T>C突变导致Bw11亚型的研究

目的:在中国彝族人群中发现罕见的ABO*BW.11/ABO*O.01.02血型基因亚型,探讨ABO*BW.11/ABO*O.01.02血型血清学特点、分子机制及遗传背景。方法:先证者为孕产妇,25岁,入院常规检测显示其ABO正反定型不符,在常规ABO血型血清学无法准确鉴定的情况下,对ABO基因第1-7外显子采用Sanger法进行测序分析,并利用野生型B糖基转移酶氨基酸系列同源建模,预测该位点突变对B糖基转移酶结构和功能的影响。结果:先证者及家系成员血型血清学结果与常见B亚型均不相符;ABO基因测序结果显示,先证者及家系成员4代共7人均存在ABO血型基因第7外显子存在c.695T>C错义突变,导致B糖基转移酶第232位亮氨酸被脯氨酸替换。同源建模显示,突变影响了蛋白质的肽键和氢键,导致其结构和功能发生改变,B糖基转移酶的活性降低,导致B抗原表达减弱。结论:ABO等位基因出现第7外显子c.695T>C.P.leu 232 Pro点突变是形成ABO*BW.11/ABO*O.01.02亚型的分子基础,此突变在此家系多成员中稳定遗传。

Expression of IGF-Ⅱ,p53,p21 and HBxAg in precancerous events of hepatocarcinogenesis induced by AFBI and/or HBV in tree shrews

INTRODUCTIONIn order to study the relationship between oncogeneexpression and HCC generation,we observed theprecancerous hepatic GGT loci,IGF-Ⅱ,p53 andp21 expression during hepatocarcinogenesis of treeshrew induced by hepatitis B virus (HBV) and/oraflatoxin B1 (AFB1).

Expression of hepatitis B virus genes in early embryonic cells originated from hamster ova and human spermatozoa transfected with the complete viral genome

Aim： To detect the expression of hepatitis B virus （HBV） genes （HB S and C genes） in early embryonic cells after introducing motile human sperm carrying HBV DNA into zona-free hamster oocytes via the in vitro fertilization （IVF） technique. Methods： Human sperm-mediated HBV genes were delivered into zona-free hamster oocytes by the IVF method. Polymerase chain reaction （PCR） was used to detect HB S and pre-Core/Core （pre-C/C） coding genes both in one- and two-cell embryos. Reverse transcription-PCR （RT-PCR） analysis was used to study the expression of the two genes. Fluorescence in situ hybridization （FISH） analysis using the full-length HBV DNA as the hybridization probe was performed to confirm the integration of viral DNA in the host embryonic genome. Results： Both HB S and pre-C/C coding genes are present and transcribed in one- and two-cell embryos originated from hamster ova IVF with human spermatozoa carrying HBV DNA sequences. Conclusion： Sperm-mediated HBV genes are able to replicate and express themselves in early embryonic cells. These results provide direct evidence that HBV DNA could transmit vertically to the next generation via the male germ line.

Expression and significance of HBV genes and their antigens in human primary intrahepatic cholangiocarcinoma

AIM To explore the etiology and pathogenesis of human primary intrahepatic cholangiocarcinoma, the expression of HBV genes and HBV-antigens was detected in the cancerous tissue and its surrounding hepatic tissues.METHODS HBV-antigens were detected by immunohistochemical technique and HBV genes were examined with in situ hybridization.RESULTS In 20 cases of cholangiocarcinoma, the positive detection rate of HBxAg, pre-S1, pre-S2, HBsAg and HBcAg was 75%, 40%, 40%, 10% and 0%, respectively, and in the surrounding hepatic tissues of 19 cases the positive rates were 84.2%, 47.9%, 47.9%, 31.6% and 31.6%. Among 40 cases of cholangiocarcinoma, the positive rate of HBV-DNA, x gene, pre-s gene, s gene and s gene fell on 77.5%, 70.0%, 47.5%, 40% and 42.5%, respectively, and of the surrounding hepatic tissues in 33 cases, 87.9%, 84.8%, 63.6%, 69.7% and 66.7%.CONCLUSION The development of human primary intrahepatic cholangiocarcinoma bears a close relationship with chronic persistent HBV infection. Particularly, the x gene of HBV and its protein (HBxAg) might play an important role in pathogenesis of hepatic carcinoma.A large number of studies indicate a close relationship between human primary hepatocellular carcinoma and hepatitis B virus (HBV) infection, which is considered generally as an important factor in the development of hepatic carcinoma[1,2]. In human primary hepatic carcinoma, hepatocellular carcinoma is more frequently encountered, while intrahepatic cholangiocarcinoma (ChC), including hepatocholangiocarcinoma (HChC), is relatively less, being 8%-10%[3]. For a long time, the etiology and pathogenesis of intrahepatic cholangiocarcinoma have been unclear. A few reports considered it to be related to infestation with clonorchiasis sinensis[4,5], but never involved with HBV infection. We used immunohistochemical technique and in situ hybridization methods to detect HBV genes and their -related antigens in the tissues of intrahepatic cholangiocarcinoma and its surrounding hepatic tissues for the purpose of exploring the etiology and pathogenesis of intrahepatic cholangiocarcinoma.

The cloning of 3'-truncated preS/S gene from HBV genomic DNA and its expression in transgenic mice

INTRODUCTIONHepatitis B virus (HBV) is regarded as one of themain etiologic factors involved in the developmentof human hepatocellular carcinoma (HCC).The open reading frame (orf)of X gene of HBVencoded a transactivating factor is the evidence thatstrongly supported the notion that the X gene ofHBV DNA integrated in HCC genomic DNA couldcontribute to the carcinogenesis of liver cells byactivation of some related cellular genes

XbaⅠpolymorphisms of apolipoprotein B gene:Another risk factor of gallstone formation after radical gastrectomy

AIM:To prospectively investigate the association between the XbaⅠpolymorphisms of apolipoprotein B (APOB)gene and gallstone formation following gastrectomy.METHODS:The study was conducted between January 2005 and December 2006.A total of 186 gastric cancer patients who had undergone radical gastrectomy were grouped according to XbaⅠpolymorphisms of APOB gene(X+X-group,n=24 and X-X-group,n =162)and compared.The XbaⅠpolymorphisms of APOB gene were detected by polymerase chain reaction-restriction fragment length polymorphism(PCRRFLP).RESULTS:The incidence of gallstone was significantly higher in the X + X-group than in the X-X-group[54.2% vs 9.3%,RR=5.85(2.23-15.32),P<0.001].The serum levels of total cholesterol(TC)and low-density lipoprotein(LDL)were higher in the X + X-than in the X-X-group(4.02±1.12 vs 3.48±0.88,P=0.004 before surgery and 3.88±1.09 vs 3.40±0.86,P=0.008 after surgery).LDL was 2.21±0.96 vs 1.89±0.84(P =0.042)before surgery and 2.09±0.95 vs 1.72±0.85 (P=0.029)after surgery in the two groups.No relationship was found between XbaⅠpolymorphisms and gallbladder motility.CONCLUSION:In Chinese patients after radical gastrectomy,X + allele of APOB gene is another risk factor for the development of gallstone besides the gallbladder motility disorder after surgery.