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.

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.

Progress in gene transfer by germ cells in mammals

Use of germ cells as vectors for transgenesis in mammals has been well developed and offers exciting prospects for experimental and applied biology, agricultural and medical sciences. Such approach is referred to as either male germ cell mediated gene transfer （MGCMGT） or female germ cell mediated gene transfer （FGCMGT） technique. Sperm-mediated gene transfer （SMGT）, including its alternative method, testis-mediated gene transfer （TMGT）, becomes an established and reliable method for transgenesis. They have been extensively used for producing transgenic animals. The newly developed approach of FGCMGT, ovary-mediated gene transfer （OMGT） is also a novel and useful tool for efficient transgenesis. This review highlights an overview of the recent progress in germ cell mediated gene transfer techniques, methods developed and mechanisms of nucleic acid uptake by germ cells.

COOH-terminal deletion of HBx gene is a frequent event in HBV-associated hepatocellular carcinoma

AIM:To investigate the hepatitis B virus (HBV) x gene (HBx) state in the tissues of HBV-related hepatocellular carcinoma (HCC) in Chinese patients and whether there were particular HBx mutations. METHODS: HBx gene was amplified and direct sequencing was used in genomic DNA samples from 20 HCC and corresponding non-cancerous liver tissues from HBsAg-positive patients. HBV DNA integration and HBx deleted mutation were validated in 45 HCC patients at different stages by Southern blot analysis and polymerase chain reaction methods. RESULTS: The frequencies of HBx point mutations were significantly lower in HCC than their corresponding non- cancerous liver tissues (11/19 vs 18/19, P = 0.019). In contrast, deletions in HBx gene were significantly higher in HCC than their non-cancerous liver tissues (16/19 vs 4/19, P < 0.001). The deletion of HBx COOH-terminal was detected in 14 HCC tissues. A specific integration of HBx at 17p13 locus was also found in 8 of 16 HCC, and all of them also exhibited full-length HBx deletions. Integrated or integrated coexistence with replicated pattern was obtained in 45.5% (20/45) - 56.8% (25/45) tumors and 40.9% (18/45) - 52.3% (23/45) non-tumor tissues. CONCLUSION: HBx deletion, especially the COOH- terminal deletion of HBx is a frequent event in HBV-associated HCC tissues in China. HBV integration had also taken place in partial HCC tissues. This supporting the hypothesis that deletion and probably integrated forms of the HBx gene may be implicated in liver carcinogenesis.

Determining Nodulation Regulatory (Rj) Genes of Myanmar Soybean Cultivars and Their Symbiotic Effectiveness with <i>Bradyrhizobium japonicum</i>USDA110

Soybean (Glycine max L.) plays an essential role in human nutrition as a protein source, and in plant nutrition as a N source. The rate of N fixation varies depending on the cultivars and compatibility between the inoculated Rhizobium strain and the host cultivar. Characterizing the nodulation regulatory (Rj) genes is necessary to determine the compatibility of cultivars and Rhizobium strains. Rj genes were previously identified based on inoculation tests and PCR analyses. The six cultivars Yezin-3, Yezin-7, Yezin-11, Shan Seine (Local), Madaya (Local), and Hinthada (Local) were identified as harboring the Rj4 gene. Two cultivars, Yezin-6 and Yezin-8, were classified as non-Rj-gene harboring. Two other cultivars, Yezin-9 and Yezin-10, were identified as Rj3- and Rj2Rj3-gene harboring, respectively. Ours is the first report on Rj3- and Rj2Rj3-gene harboring cultivars in Myanmar. We evaluated Myanmar soybean cultivars for symbiotic effectiveness, relying on the standard strain Bradyrhizobium japonicum USDA110. In our first experiment, the soybean cultivar Yezin-11 (Rj4) showed the highest N fixing potential. Based on their potential for fixing N and nodulation, the top six soybean cultivars were Yezin-11 (Rj4), Yezin-9 (Rj3), Yezin-6 (non-Rj), Yezin-8 (non-Rj), Yezin-3 (Rj4) and Yezin-10 (Rj2Rj3). These cultivars were selected for a second experiment, which revealed that the N fixation, nodulation, and plant growth of Yezin-11 (Rj4) *Corresponding author. A. Z. Htwe et al. 2800 were superior to the other cultivars. We conclude that Yezin-11 (Rj4) is the most efficient cultivar for nodulation and N fixation when inoculated with B. japonicum USDA110.

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.

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.

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.

基于线粒体Cyt b基因的中国南方唇(Hermibarbus labeo)和间(Hermibarbus medius)种群分化及亲缘地理研究

为了解中国南方唇(Hermibarbus labeo)和间(Hermibarbus medius)的种群分化、亲缘地理格局及物种有效性,作者对唇8个水系及间9个水系共148尾样本的Cyt b基因全序列进行了测定。在所有序列中,共有128个变异位点,共检测出了41个单倍型,单倍型多样性为0.954,核苷酸多样性为0.02153。基于Cyt b基因全序列构建的NJ树显示,中国南方的唇与间合聚为两大支,其中韩江和九龙江的全部唇样本组成了Ⅰ支,而其他水系的唇和间样本则组成了Ⅱ支。两支系间的遗传距离为5.1%,而唇和间之间的遗传距离为3.2%。现有证据不支持间与唇达到种一级的分化。单倍型网络图显示,韩江、九龙江种群和其他水系的种群分化较大;海南岛三大水系种群和漠阳江种群的单倍型分支与代表珠江水系单倍型的分支之间的亲缘关系较近,与其他地理区间种群的亲缘关系则相对较远;西江可能为珠江水系、漠阳江水系和海南岛三大水系间种群的扩散中心,其中一支往东向北江和东江扩散;另一支往南向海南岛扩散,海南岛三大水系种群尔后在冰期海退时向北扩散至漠阳江水系及珠江水系。AMOVA分析表明,唇和间地理区之间变异约占54.50%,地理区内种群间变异约占18.64%,种群内的变异占26.86%,这说明,唇和间种群Cyt b的遗传分化主要是来自地理区之间。错配分析及中性检验显示,全部种群、唇种群、间种群在历史上均没有发生过明显的扩张,两个mt DNA支系亦未发生过扩张,而海南岛的昌化江种群曾发生过种群扩张。

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.

牛皮蝇Hypoderm in B基因的克隆及原核表达载体构建

参照牛皮蝇HypoderminB(HB)基因的核苷酸序列,设计一对引物,以牛皮蝇总RNA为模板利用RT-PCR扩增牛皮蝇HB基因,并对扩增基因进行克隆测序。序列分析表明,克隆的牛皮蝇HA基因与GenBank中已经登录的核苷酸和氨基酸序列的同源性分别为97.2%和99.3%。同时,将该基因与原核表达载体PGEX-4T-1连接,构建并获得阳性重组表达载体。

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。