TaqMan-MGB探针检测文森巴尔通体博格霍夫亚种实时荧光定量PCR方法的建立及应用

【目的】应用TaqMan-MGB探针技术,建立具有种水平特异性、高敏感性的荧光定量PCR方法,用于快速检测文森巴尔通体博格霍夫亚种。【方法】在序列特异性扩增区标记(Sequence characterized amplifiedregion,SCAR)技术基础上,依据文森巴尔通体博格霍夫亚种一段特有的基因序列设计探针和引物,分别优化扩增反应的退火温度、探针和引物的反应浓度;分析此方法的特异性、敏感性及重复性;绘制标准曲线,评估PCR反应的扩增效率和稳定性。【结果】本研究设计的TaqMan-MGB探针具有种水平特异性;最低检出限为每个PCR反应11个拷贝;组内和组间的变异系数CV值分别为0.12%-0.70%和0.14%-0.55%,在允许范围内;标准曲线线性关系良好(R2=1),扩增效率高(E=104.7%)。【结论】本研究建立的基于TaqMan-MGB探针技术的荧光定量PCR方法能够在种水平特异性、高灵敏度检出文森巴尔通体博格霍夫亚种,为这种巴尔通体所引起的一系列疾病的早期快速诊断、监测和流行病学调查等研究提供有效手段。     

三种常绿阔叶树光系统Ⅱ在低温胁迫下的光抑制及恢复

冬季低温胁迫对亚热带常绿阔叶树光合活性的主要影响之一,体现在光合机构的低温光抑制。为了阐明冬季低温胁迫下常绿阔叶树光系统Ⅱ的光抑制程度及光保护机制,该文研究了冬季自然低温胁迫(零下低温冻害和零上低温寒害)对红叶石楠、枇杷和猴樟三种亚热带常绿阔叶树光合机构光系统Ⅱ(PSⅡ)光抑制的影响以及春季气温回暖后的恢复情况。结果表明:冻害和寒害低温胁迫使猴樟的PSⅡ活性显著降低,PSⅡ受到较严重的光抑制,低温胁迫解除后PSⅡ活性未能完全恢复。红叶石楠PSⅡ活性下降程度和光抑制程度最轻,春季PSⅡ活性显著上升,光抑制显著下降。枇杷PSⅡ活性和光抑制程度介于猴樟和红叶石楠之间。低温胁迫下红叶石楠的非光化学猝灭(NPQ)接近常温水平; 枇杷的NPQ略有降低,春季恢复正常; 猴樟NPQ最低,春季低温解除后仍不能完全恢复。此外,三种常绿阔叶树在冬季低温胁迫和春季恢复时期的NPQ与PSⅡ的光抑制程度存在显著的负相关关系。综合以上结果分析表明,冬季低温对红叶石楠PSⅡ影响不大,对枇杷有一定影响但春季气温回暖后可以及时恢复,对猴樟PSⅡ有显著的光抑制且恢复过程较慢,同时NPQ对保护常绿阔叶树PSⅡ免受冬季低温光抑制有重要的贡献。     

烟酸转磷酸核糖激酶和丙酮酸羧化酶共表达对大肠杆菌BA002产丁二酸的影响

大肠杆菌BA002是敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸-甲酸裂解酶的编码基因 (pflB) 的工程菌。厌氧条件下NADH不能及时再生为NAD+,引起胞内辅酶NAD(H)的不平衡,最终导致厌氧条件下菌株不能利用葡萄糖生长代谢。pncB是烟酸转磷酸核糖激酶 (NAPRTase) 的编码基因,通过过量表达pncB基因能够提高NAD(H)总量与维持合适的NADH/NAD+,从而恢复了厌氧条件下重组菌E. coli BA014 (BA002/pTrc99a-pncB) 的生长和产丁二酸的性能。然而,BA014在厌氧发酵过程中有大量丙酮酸积累,为进一步提高菌株的丁二酸生产能力,减少副产物丙酮酸的生成,共表达NAPRTase和来自于乳酸乳球菌 NZ9000中丙酮酸羧化酶 (PYC) 的编码基因pyc,构建了重组菌E. coli BA016 (BA002/pTrc99a-pncB-pyc)。3 L发酵罐结果表明,BA016发酵112 h后,共消耗了35.00 g/L的葡萄糖。发酵结束时,菌体OD600为4.64,产生了25.09 g/L丁二酸。通过共表达pncB和pyc基因,使BA016的丙酮酸积累进一步降低,丁二酸产量进一步提高。     

白洋淀富营养化湖泊湿地厌氧氨氧化菌的分布及对氮循环的影响

随着生物反应器和海洋生态系统中厌氧氨氧化反应的发现,自然生态系统的氮循环过程被重新认识,但是目前厌氧氨氧化过程是否也存在于富营养化湖泊湿地并发挥着重要作用,还未见报道。结合15N同位素示踪与分子生物学技术研究了白洋淀湖泊湿地沉积物中厌氧氨氧化菌的分布、菌群结构特性、生物多样性及其活性。结果表明,在藻类影响导致的高氨氮沉积物中,厌氧氨氧化菌具有广泛存在性。通过构建16S rRNA克隆文库发现,沉积物中厌氧氨氧化菌的生物多样性相对较低,在2%差异度的条件下,30个克隆序列只分为5个操作分类单元(OTUs),代表序列与Genebank数据库中已探明的厌氧氨氧化菌Candidatus Brocadia fulgida和Candidatus Brocadia anammoxidans同源性最高,分别可达97%和96%。同位素示踪结果表明,白洋淀湖泊湿地沉积物中厌氧氨氧化活性为0.19—7.78 nmol N g-1h-1,空间差异较大,产生的氮气占此沉积物总氮气生成量的0.64%—20.65%,体现了湿地的异质性。通过得出的厌氧氨氧化反应速率推算每年由厌氧氨氧化反应损失的氮量为1.8—78gN m-2a-1,对白洋淀氮循环起到非常重要的作用。富营养化湖泊湿地沉积物中厌氧氨氧化反应的发现为研究厌氧氨氧化对氮循环的重要影响提供了新证据。     

Over-expression of fibroblast activation protein alpha increases tumor growth in xenografts of ovarian cancer cells

Fibroblast activation protein alpha （FAPα） is a 95-kDa serine protease of post-prolyl peptidase family on cell surface. FAPoL is widely expressed in tumor microenviron- ment. The wide spread association of FAPα expression with cancer suggests that it has important functions in the disease. However, the nature of FAPα＇s roles in cancer cell activity is not well-determined. It has been showed that FAPα silencing in SKOV3 cells induces ovarian tumors but significantly reduces tumor growth in a xenograft mouse model. To further determine the role of FAPoL in epithelial ovarian cancer cells, SKOV3-FAPα and HO8910-FAPα cell lines, which over-expressed FAPα stably, were con- structed and then their biological behaviors were investi- gated. It was found that FAPoL promoted ovarian cancer cell proliferation, drug resistance, invasiveness, and migra- tion in vitro. Immunochemistry assay showed that FAPα significantly facilitated tumor growth in xenograft tumor tissues. These results suggested that FAPα might directly promote tumor growth and invasiveness in ovarian cancer cells.     

多方法组合调查新疆阿勒泰地区6种常见鼠传致病菌流行状况

【背景】鼠传疾病是对人类危害较大的一种人兽共患病,全球化使得鼠传疾病流行区域不断扩大,出现了多种新发鼠传疾病的发生及旧传染病的复燃。【目的】调查新疆阿勒泰地区常见的鼠传致病菌在啮齿动物中的流行状况,为当地自然疫源性疾病防控提供科学依据。【方法】采用夹夜法捕获啮齿动物,无菌收集其脾脏和肾脏组织,提取基因组DNA。应用TaqMan探针法的荧光定量聚合酶链式反应(quantitative polymerase chain reaction, qPCR)检测巴尔通体(Bartonella spp.)、问号钩端螺旋体(Leptospirainterrogans)、恙虫病东方体(Orientiatsutsugamushi)、莫氏立克次体(Rickettsia mooseri)、嗜吞噬细胞无形体(Anaplasma phagocytophilum)和土拉弗朗西斯菌(Francisella tularensis)6种常见的鼠传致病菌。采用16SrRNA基因的通用引物进行常规PCR扩增后,应用Illumina测序和Nanopore测序进一步检测致病菌,同时对脾脏组织进行巴尔通体体外分离培养。比较qPCR...     

UHRF1 is regulated by miR-124-3p and promotes cell proliferation in intrahepatic cholangiocarcinoma

Ubiquitin-like with PHD and ring finger domains 1 (UHRF1) is abnormally overexpressed in multiple cancers and closely correlated with tumor-promoting effects, such as high proliferation. However, how UHRF1 functions in intrahepatic cholangiocarcinoma (ICC) has not yet been determined. Herein, we found that UHRF1 is overexpressed in ICC tissues. Downregulated UHRF1 attenuated the transition of the G1/S cell cycle and then suppressed cell proliferation in vitro and tumor growth in vivo. Moreover, upstream regulators of the UHRF1 expression were predicted, and we found that direct binding of miR-124-3p inhibited the UHRF1 expression. Elevated miR-124-3p suppressed proliferation and led to the arrest of the cell cycle. Furthermore, the expression of UHRF1 was positively correlated with PCNA. Clinically, we showed that elevated UHRF1 was associated with poor prognosis, and served as an independent prognostic factor in ICC patients. Together, these findings demonstrate that UHRF1, regulated by miR-124-3p, acts as a tumor promoter by promoting cell proliferation in ICC.     

Cloning and characterization of the <Emphasis Type="Italic">Tne</Emphasis>DI restriction–modification system of <Emphasis Type="Italic">Thermotoga neapolitana</Emphasis>

A putative Type II restriction–modification system of Thermotoga neapolitana, TneDI, was cloned into Escherichia coli XL1-Blue MRF′ and characterized. Gene CTN_0339 specifies the endonuclease R.TneDI, while CTN_0340 encodes the cognate DNA methyltransferase M.TneDI. Both enzymes were purified simply by heating the cell lysates of E. coli followed by centrifugation. The enzymes were active over a broad range of temperatures, from 42°C to at least 77°C, with the highest activities observed at 77°C. R.TneDI cleaved at the center of the recognition sequence (CG↓CG) and generated blunt-end cuts. Overexpression of R.TneDI in BL21(DE3) was confirmed by both SDS-PAGE and Western blotting.     

Phenolic Compounds and Antioxidant Activity of Different Organs of Potentilla fruticosa L. from Two Main Production Areas of China

This report compared the phenolic compounds and antioxidant activity of the leaves, flowers, and stems of Potentilla fruticosa L. collected from two main production areas of P. R. China (Taibai Mountains and the Qinghai Huzhu Northern Mountains). The results indicated that there were significant differences in the phenol contents and antioxidant activities among the different organs and between the two productions. High‐performance liquid‐chromatography analysis indicated that hyperoside, (+)‐catechin, ellagic acid, and rutin were the primary compounds in leaves and flowers; for stems, the content of six phenolic compounds, from two productions, were the lowest. The 1,1‐diphenyl‐2‐picryl hydrazyl (DPPH), 2,2‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) di‐ammonium salt (ABTS), ferric reducing power (FRAP), lipid peroxidation assays, and microbial test system (MTS) were used to evaluate the antioxidant activity. The results demonstrated that the leaves from two productions exhibited powerful antioxidant activity than other organs, which did not significantly differ from that of the positive control (rutin), followed by the flowers and stems. The correlation between the content of phytochemicals and the antioxidant activities of different organs showed that the total phenol, tannin, hyperoside, and (+)‐catechin contents may influence the antioxidant activity, and these compounds can be used as markers for the quality control of P. fruticosa.     

Congenital semilunar valvulogenesis defect in mice deficient in phospholipase C epsilon

Phospholipase Cepsilon is a novel class of phosphoinositide-specific phospholipase C, identified as a downstream effector of Ras and Rap small GTPases. We report here the first genetic analysis of its physiological function with mice whose phospholipase Cepsilon is catalytically inactivated by gene targeting. The hearts of mice homozygous for the targeted allele develop congenital malformations of both the aortic and pulmonary valves, which cause a moderate to severe degree of regurgitation with mild stenosis and result in ventricular dilation. The malformation involves marked thickening of the valve leaflets, which seems to be caused by a defect in valve remodeling at the late stages of semilunar valvulogenesis. This phenotype has a remarkable resemblance to that of mice carrying an attenuated epidermal growth factor receptor or deficient in heparin-binding epidermal growth factor-like growth factor. Smad1/5/8, which is implicated in proliferation of the valve cells downstream of bone morphogenetic protein, shows aberrant activation at the margin of the developing semilunar valve tissues in embryos deficient in phospholipase Cepsilon. These results suggest a crucial role of phospholipase Cepsilon downstream of the epidermal growth factor receptor in controlling semilunar valvulogenesis through inhibition of bone morphogenetic protein signaling.