人PINK1-shRNA载体的构建及对神经母细胞瘤细胞线粒体的影响

摘要 目的：构建筛选人源靶向特异PINK1-shRNA敲减质粒,转染神经母细胞瘤SH-SY5Y细胞并验证该质粒转染后对PINK1基因的敲减效率,观察对细胞线粒体形态的影响。方法：构建两对人源PINK1-shRNA序列(编号分别为PINK1-shRNA-39和PINK1-shRNA-42),将这2对干扰序列连接在载体上形成重组载体,经测序验证后,将空载体和两对敲减质粒分别转染SH-SY5Y细胞,获得基因敲减的细胞模型,用CCK-8法检测细胞的存活率,用荧光定量PCR方法确定PINK1基因的敲减效率,用蛋白质免疫印迹法验证细胞内PINK1的表达水平是否发生改变,用激光共聚焦显微镜观察线粒体的形态是否发生了改变。结果：我们提取的质粒,经测序结果显示,质粒载体构建成功;转染细胞后,CCK-8 法检测细胞存活率发生了降低,与正常组比较,PINK1-shRNA-39和PINK1-shRNA-42敲减质粒组,细胞的存活率分别降低了13.7%（P＜0.05）和14.1%（P＜0.05）;荧光定量PCR结果显示,与正常组相比,PINK1-shRNA-39组和PINK1-shRNA-42组敲减质粒转染的细胞内PINK1基因的表达分别降低了24.1%（P＜0.01）和36.7%（P＜0.01）;蛋白质印迹法结果显示,与正常组相比,两对质粒分别转染细胞后,PINK1-shRNA-39和PINK1-shRNA-42敲减质粒转染的细胞内PINK1蛋白的表达水平降低,差异有统计学意义(P＜0.01),PINK1-shRNA-42敲减质粒转染的细胞内PINK1蛋白的表达水平有效降低更明显;与正常组比较,激光共聚焦显微镜观察到基因敲减两组细胞的线粒体部分发生断裂,碎片较多,基因敲减两组的线粒体的形态因子显著降低,差异有统计学意义（P＜ 0.01）。结论：成功构建了人源的PINK1基因敲减的质粒,并将敲减的质粒成功转染至SH-SY5Y 细胞中,细胞内PINK1基因的mRNA和蛋白的表达水平降低,且细胞线粒体的形态发生了改变。     

Complexities and pitfalls in analyzing and interpreting mitochondrial DNA content in human cancer

Mutations in the human mitochondrial genome have been observed in all types of human cancer, indicating that mutations might contribute to tumorigenesis, metastasis, recurrence, or drug response. This possibility is appealing because of the known shift from oxidative metabolism to glycolysis, known as the Warburg effect, that occurs in malignancy. Mitochondrial DNA (mtDNA) mutations could either be maternally inherited and predispose to cancer (germ line mutations) or occur sporadically in the mtDNA of specific tissues (tissue- or tumor-specific somatic mutations) and contribute to the tumor initiation and progression process. High-throughput sequencing technologies now enable comprehensive detection of mtDNA variation in tissues and bodily fluids, with the potential to be used as an early detection tool that may impact the treatment of cancer. Here, we discuss insights into the roles of mtDNA mutations in carcinogenesis, highlighting the complexities involved in the analysis and interpretation of mitochondrial genomic content, technical challenges in studying their contribution to pathogenesis, and the value of mtDNA mutations in developing early detection, diagnosis, prognosis, and therapeutic strategies for cancer.     

基于线粒体基因分析蓝孔雀与绿孔雀的遗传差异

种间的遗传差异是物种分类和确定保护管理单元的基础,本研究利用DNA条形码技术对未知样本进行鉴定,通过NCBI进行BLAST得到结果是:与绿孔雀的同源性为96%。近一步通过对蓝孔雀(Pavo cristatus)和绿孔雀(Pavo muticus)线粒体细胞色素C氧化酶Ⅰ(cytochrome coxidaseⅠ,COⅠ)基因及线粒体基因组的比较分析,结果表明两物种间的COⅠ基因在碱基组成、核苷酸多样性等各项指标上均具有明显差异。遗传距离分析结果表明蓝孔雀与绿孔雀种内遗传距离为分别0和0.012,种间遗传距离为0.045,表明种间仍具有明显的遗传差异。通过对两物种线粒体基因组各基因的比较分析,发现ND1基因变异位点所占比例相对较高,考虑作为绿孔雀和蓝孔雀种群遗传学研究的最优分子标记。本研究将为分析孔雀类群间的系统发育及制定绿孔雀的保护措施提供了更多科学依据。     

Anti-cancer activity of biosynthesized silver nanoparticles using Avicennia marina against A549 lung cancer cells through ROS/mitochondrial damages

The biosynthesized Ag NPs was synthesized by using marine mangrove plant extract Avicennia marina. The synthesized Ag NPs was confirmed by various physiochemical characterization including UV-spectrometer and XRD analysis. In addition, the shape and of the synthesized Ag NPs was morphologically identified by SEM initially and TEM finally. After confirmation, the anti-cancer property of synthesized Ag NPs was confirmed at 50 µg/mL concentration against A549 lung cancer cells by MTT assay. Further, the ability to stimulate the ROS generation and mitochondrial membrane at the IC₅₀ concentration of Ag NPs was confirmed by fluorescence microscopy using DCFH-DA and rhodamine 123 dyes respectively. Finally, the result was concluded that the synthesized Ag NPs has improved anti-cancer activity against A549 cells at lowest concentration.     

PGAM5: A crucial role in mitochondrial dynamics and programmed cell death

In response to mitochondrial damage, mitochondria activate mitochondrial dynamics to maintain normal functions, and an imbalance in mitochondrial dynamics triggers multiple programmed cell death processes. Recent studies have shown that phosphoglycerate mutase 5 (PGAM5) is associated with mitochondrial damage. PGAM5 activates mitochondrial biogenesis and mitophagy to promote a cellular compensatory response when mitochondria are mildly damaged, whereas severe damage to mitochondria leads to PGAM5 inducing excessive mitochondria fission, disruption to mitochondrial movement, and amplification of apoptosis, necroptosis and mitophagic death signals, which eventually evoke cell death. PGAM5 functions mainly through protein-protein interactions and specific Ser/Thr/His protein phosphatase activity. PGAM5 is also regulated by mitochondrial proteases. Detection of PGAM5 and its interacting protein partners should enable a more accurate evaluation of mitochondrial damage and a more precise method for the diagnosis and treatment of diseases.     

基于线粒体D-loop区和Cyt b基因分析广西禾花鲤三个群体遗传结构

研究基于线粒体D-loop区和Cyt b基因部分序列, 分析了广西全州、融水、环江三地禾花鲤(Cyprinus carpio)的遗传结构。在3个群体中共鉴定到19种D-loop与Cyt b序列的组合单倍型, 总的单倍型多样性(H_d)和核苷酸多样性(π)分别为0.916±0.010和0.008±0.004, 禾花鲤线粒体DNA具有单倍型多样性高和核苷酸多样性低的特点。环江群体单倍型多样性最低, 但是核苷酸多样性却最高, 反映了环江群体存在最明显的谱系混杂。分子方差分析(AMOVA)显示遗传变异主要来源于群体内部(71.54%), 禾花鲤三个群体间有显著的遗传分化(F_st=0.285; P<0.01)。系统发育分析表明, 多数样本的线粒体单倍型属于华南鲤(C. carpio rubrefusus)类型, 同时三地禾花鲤中均检测到一定频率的西鲤(C. carpio carpio)或远东鲤(C. carpio haematopterus)类型的线粒体单倍型, 提示禾花鲤可能受到鲤养殖品种的杂交渐渗。研究初步揭示了广西禾花鲤的种质资源现状, 为禾花鲤这一地方特色“稻鱼共作”品种的选育和苗种管理提供了必要的依据。     

妊娠期母体甲状腺功能减退对胎鼠骨骼肌胰岛素抵抗和线粒体功能的影响

摘要 目的：研究妊娠期母体甲状腺功能减退对胎鼠骨骼肌胰岛素抵抗和线粒体功能的影响。方法：构建妊娠期母体甲状腺功能减退小鼠模型,制备胎鼠骨骼肌线粒体,同时选取健康正常胎鼠做本次实验的对照组。采用酶联免疫吸附法和放射免疫分析法测定两组小鼠甲状腺功能;通过全自动生化分析仪检测两组胎鼠胰岛素抵抗结果;利用Clark氧电极测定密闭反应体系评价两组胎鼠线粒体功能结果,并分析母体甲状腺功能减退与胎鼠骨骼肌胰岛素抵抗和线粒体功能的相关性。结果：两组小鼠甲状腺功能结果、两组胎鼠骨骼肌胰岛素抵抗和线粒体结果对比分析之间均有显著差异（P<0.05）。甲减组小鼠血清中促甲状腺激素（Thyroid Stimulating Hormone,TSH）、丙氨酸转氨酶（alanine aminotransferase,ALT）和门冬氨酸氨基转移酶（aspartate aminotransferase,AST）均较对照组低,胎鼠的空腹血糖（fasting blood-glucose,FBG）、动态3（state3,ST3）、动态4（state4,ST4）呼吸速率和呼吸控制比（respiratory control,RCR）也均较对照组低;而甲减组小鼠游离甲状腺素（free thyroxine,FT4）却较对照组高,胎鼠的胰岛素（insulin,INS）和胰岛素抵抗结果（homeostasis model assessment of insulin resistance,HOMA-IR）也较对照组高（P<0.05）。且母体甲状腺功能减退指标中FT4和RCR、ALT和FBG以及RCR之间有负相关关系,母体甲状腺功能减退的其他指标则与胎鼠骨骼肌胰岛素抵抗和线粒体功能的其他相关指标之间呈正相关关系（P<0.05）。结论：妊娠期母体甲状腺功能减退会降低胎鼠骨骼肌胰岛素抵抗和线粒体功能,影响胎鼠的正常发育。     

A review of the genus Rusa in the indo-malayan archipelago and conservation efforts

Genus Rusa, belonging to the deer family Cervidae is native to the Indo-Malaya Archipelago (IMA). However, detailed information on the Rusa genus in the IMA is limited. This review provides comprehensive information on the Rusa genus in the IMA including, threats and conservation efforts. There are four species of deer in Rusa genus, which is Sambar deer (Rusa unicolor), Javan deer (Rusa timorensis), Visayan spotted deer (Rusa alfredi) and Philippine deer (Rusa marianna). Despite their wide distribution in the South Asian and Southeast Asian regions, they are under serious threats. Some conservation efforts that are being done to protect and conserve them among others are; (1) facilities protection, (2) habitat enrichment programme, (3) Ex-situ conservation, (4) legislations, and (5) captive breeding. Conservation through genetics is also an important step in conserving these species. Recommendations for conservation of the genus are also discussed; 1. maintenance of ecosystem. 2. more effective monitoring system on the existing protected area. 3. ex-situ conservation, and 4. habitat monitoring.     

Small and overlooked: Phylogeny of the genus Trigonodactylus (Squamata: Gekkonidae), with the first record of Trigonodactylus arabicus from Jordan

Geckos of the genus Trigonodactylus are widely distributed in the sand deserts of the Arabian Peninsula. Three species of this genus are currently recognized, with a fourth one, Stenodactylus pulcher, which placement within Trigonodactylus has been tentatively suggested, but not yet confirmed. We present a phylogenetic analysis of the genus Trigonodactylus with new specimens collected in central Saudi Arabia and southern Jordan. New genetic data has been generated from three mitochondrial markers to investigate the phylogenetic relationships of all species of the genus and to assess the putative generic assignment of S. pulcher. Our results confirm that S. pulcher indeed belongs within Trigonodactylus, branching as a sister lineage to all other species of the genus. The new samples cluster within Trigonodactylus arabicus, thus confirming the genetic homogeneity of the species across its large and seemingly inhospitable range. The new specimen collected in southern Jordan represents the first record for the country and a considerable range extension to the northwest from all previously reported localities. Our findings and discovery of a new species for Jordan highlight the need of more field surveys to be carried out in the underexplored parts of Jordan and northern Saudi Arabia, as these places still hold a potential for new discoveries and are crucial for understating the biogeography of the Arabian herpetofauna.     

Plant derived coumestrol phytochemical targets human skin carcinoma cells by inducing mitochondrial-mediated apoptosis,cell cycle arrest,inhibition of cell migration and invasion and modulation of m-TOR/PI3K/AKT signalling pathway

The current study was undertaken to investigate anticancer activity of coumestrol phytoestrogen against human skin cancer. MTT assay was performed for cell viability assessment and clonogenic assay for cell colony formation assessment. Apoptosis was analysed by Annexin V/FITC staining, AO/EB staining and western blotting assays. Effects on the m-TOR/PI3K/AKT signalling pathway were investigated by western blotting. Results indicated that coumestrol induced significant toxicity in human skin cancer cells in contrast to mouse skin cancer cells. The proliferation rate in normal skin cells remained almost intact. Annexin V-FITC and AO/EB staining assays indicated coumestrol induced cytotoxicity in skin cancer cells is mediated through apoptosis stimulation. The apoptosis in skin cancer cells was mediated through caspase-activation. Cell migration and invasion was inhibited by coumestrol in human skin cancer cells via inhibition of MMP-2 and MMP-9 expressions. Moreover, m-TOR/PI3K/AKT signalling pathway in SKEM-5 cells was blocked by coumestrol.