共查询到20条相似文献,搜索用时 0 毫秒
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David E. Gordon Lisa M. Bond Daniela A. Sahlender Andrew A. Peden 《Traffic (Copenhagen, Denmark)》2010,11(9):1191-1204
The role of SNAREs in mammalian constitutive secretion remains poorly defined. To address this, we have developed a novel flow cytometry‐based assay for measuring constitutive secretion and have performed a targeted SNARE and Sec1/Munc18 (SM) protein‐specific siRNA screen (38 SNAREs, 4 SNARE‐like proteins and 7 SM proteins). We have identified the endoplasmic reticulum (ER)/Golgi SNAREs syntaxin 5, syntaxin 17, syntaxin 18, GS27, SLT1, Sec20, Sec22b, Ykt6 and the SM protein Sly1, along with the post‐Golgi SNAREs SNAP‐29 and syntaxin 19, as being required for constitutive secretion. Depletion of SNAP‐29 or syntaxin 19 causes a decrease in the number of fusion events at the cell surface and in SNAP‐29‐depleted cells causes an increase in the number of docked vesicles at the plasma membrane as determined by total internal reflection fluorescence (TIRF) microscopy. Analysis of syntaxin 19‐interacting partners by mass spectrometry indicates that syntaxin 19 can form SNARE complexes with SNAP‐23, SNAP‐25, SNAP‐29, VAMP3 and VAMP8, supporting its role in Golgi to plasma membrane transport or fusion. Surprisingly, we have failed to detect any requirement for a post‐Golgi‐specific R‐SNARE in this process. 相似文献
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Priscille Brodin Yannick Poquet Florence Levillain Isabelle Peguillet Gerald Larrouy-Maumus Martine Gilleron Fanny Ewann Thierry Christophe Denis Fenistein Jichan Jang Mi-Seon Jang Sei-Jin Park Jean Rauzier Jean-Philippe Carralot Rachel Shrimpton Auguste Genovesio Jesus A. Gonzalo-Asensio Germain Puzo Carlos Martin Roland Brosch Graham R. Stewart Brigitte Gicquel Olivier Neyrolles 《PLoS pathogens》2010,6(9)
The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials. 相似文献
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A Simple Isothermal DNA Amplification Method to Screen Black Flies for Onchocerca volvulus Infection
Andy Alhassan Benjamin L. Makepeace Elwyn James LaCourse Mike Y. Osei-Atweneboana Clotilde K. S. Carlow 《PloS one》2014,9(10)
Onchocerciasis is a debilitating neglected tropical disease caused by infection with the filarial parasite Onchocerca volvulus. Adult worms live in subcutaneous tissues and produce large numbers of microfilariae that migrate to the skin and eyes. The disease is spread by black flies of the genus Simulium following ingestion of microfilariae that develop into infective stage larvae in the insect. Currently, transmission is monitored by capture and dissection of black flies and microscopic examination of parasites, or using the polymerase chain reaction to determine the presence of parasite DNA in pools of black flies. In this study we identified a new DNA biomarker, encoding O. volvulus glutathione S-transferase 1a (OvGST1a), to detect O. volvulus infection in vector black flies. We developed an OvGST1a-based loop-mediated isothermal amplification (LAMP) assay where amplification of specific target DNA is detectable using turbidity or by a hydroxy naphthol blue color change. The results indicated that the assay is sensitive and rapid, capable of detecting DNA equivalent to less than one microfilaria within 60 minutes. The test is highly specific for the human parasite, as no cross-reaction was detected using DNA from the closely related and sympatric cattle parasite Onchocerca ochengi. The test has the potential to be developed further as a field tool for use in the surveillance of transmission before and after implementation of mass drug administration programs for onchocerciasis. 相似文献
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目的:对产邻苯二酚菌株进行筛选。方法:采用前期筛得的产邻苯二酚菌为出发菌株,通过硫酸二乙酯诱变的方法使该菌株突变,同时建立96孔板培养和酶标仪检测方法对产邻苯二酚菌株进行高通量筛选。结果:硫酸二乙酯的终浓度为0.1%,诱变时间为8 min的条件下,突变菌致死率为84.5%,突变效果最好。筛选培养基中吸光值(495nm)和富集培养基中菌液浊度值(OD630)的加和值较大的突变菌株产邻苯二酚能力高。通过诱变和筛选得到的菌株,产邻苯二酚浓度可达0.87mg/ml,比出发菌株的提高了262.5%。经过形态学和生理生化反应,初步鉴定该菌株属于假单胞菌属(Pseudomonas sp.)。结论:硫酸二乙酯诱变和96孔板筛选的方法能以高通量方式快速筛选出产邻苯二酚菌株。 相似文献
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在中国农业微生物菌种中心(ACCC)菌株的定期转接保藏过程中,发现解淀粉芽孢杆菌ACCC 19742在同一培养基上出现两种不同的菌落形态,将这两个不同形态的菌株编号为19742-1和19742-2。通过形态学、生理生化及基因组分析相结合鉴定不同菌落形态ACCC 19742,并进一步确定该菌株的分类地位。首先将菌株进行分离与纯化,其次将纯化后的菌株进行16S rRNA及gyrB基因扩增及序列分析,通过MEGA 7.0软件构建系统发育树;API 20NE、BIOLOG及脂肪酸等分析菌株的生理生化特性;全基因组分析菌株的ANI和DDH值。两株菌在API 20NE中,仅葡萄糖酸盐同化反应存在差异;脂肪酸检测中主要组成相同,仅是百分含量方面略有差别;两株菌的16S rRNA基因相似性为100%,gyrB基因的相似性为99.4%;全基因组测序表明,两株菌的ANI值为99.95%,DDH值为99.62%。综合遗传学特征和表型特征,证实两者为来源于同一菌株不同的形态变异型,而并非污染所致。同时,19742-1和19742-2与Bacillus velezensis NRRL_B 41580~T的ANI及DDH值最高,分别为97%和77%,且16S rRNA和gyrB系统进化分析也表明,该菌株在分类地位上属于贝莱斯芽孢杆菌(Bacillus velezensis),而非解淀粉芽孢杆菌。这为菌株的保藏提供了一定的参考价值。 相似文献
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Harsh B. Pathak Yan Zhou Geetika Sethi Jeff Hirst Russell J. Schilder Erica A. Golemis Andrew K. Godwin 《PloS one》2015,10(12)
Molecular targeted therapies have been the focus of recent clinical trials for the treatment of patients with recurrent epithelial ovarian cancer (EOC). The majority have not fared well as monotherapies for improving survival of these patients. Poor bioavailability, lack of predictive biomarkers, and the presence of multiple survival pathways can all diminish the success of a targeted agent. Dasatinib is a tyrosine kinase inhibitor of the Src-family kinases (SFK) and in preclinical studies shown to have substantial activity in EOC. However, when evaluated in a phase 2 clinical trial for patients with recurrent or persistent EOC, it was found to have minimal activity. We hypothesized that synthetic lethality screens performed using a cogently designed siRNA library would identify second-site molecular targets that could synergize with SFK inhibition and improve dasatinib efficacy. Using a systematic approach, we performed primary siRNA screening using a library focused on 638 genes corresponding to a network centered on EGFR, HER2, and the SFK-scaffolding proteins BCAR1, NEDD9, and EFS to screen EOC cells in combination with dasatinib. We followed up with validation studies including deconvolution screening, quantitative PCR to confirm effective gene silencing, correlation of gene expression with dasatinib sensitivity, and assessment of the clinical relevance of hits using TCGA ovarian cancer data. A refined list of five candidates (CSNK2A1, DAG1, GRB2, PRKCE, and VAV1) was identified as showing the greatest potential for improving sensitivity to dasatinib in EOC. Of these, CSNK2A1, which codes for the catalytic alpha subunit of protein kinase CK2, was selected for additional evaluation. Synergistic activity of the clinically relevant inhibitor of CK2, CX-4945, with dasatinib in reducing cell proliferation and increasing apoptosis was observed across multiple EOC cell lines. This overall approach to improving drug efficacy can be applied to other targeted agents that have similarly shown poor clinical activity. 相似文献
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Although the genetic linkage map of Triticum tauschil, the D-genome progenitor of wheat its available, its use for linkage analysis of hexaploid wheat chromosome regions is hampered by the lack of polymorphism in wheat. Here we describe a strategy to identity probes that detect a high degree of polymorphism in wheat. The strategy involves the use of DNA probes that detect null alleles. About 16% of the Pstl genomic clones from Triticum tauschil detect null alleles in the species. The probes that detect null alleles reveal high degree of polymorphism among hexaploid wheat cultivars. The probes selected following this strategy are expected to detect null alleles throughout the tribe Triticeae, therefore, reveal high degree of polymorphism. 相似文献
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鸡冠花耐热性评价方法研究 总被引:3,自引:0,他引:3
以探讨快速鉴定鸡冠花耐热特性的方法和指标为目的,选用6个鸡冠花品种,以30℃为起始温度,每4 h升高5℃,最高处理温度为50℃,分别对种子和10叶龄幼苗进行热胁迫处理,划分了热萎蔫指数,观测了种子发芽率、成苗率、相对电导率、丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性。结果表明,高温下鸡冠花种子的发芽率和成苗率显著降低,鸡冠花幼苗的顶芽是最易受热害部位,热害萎蔫指数4是幼苗恢复极限,在高温胁迫下,幼苗的相对电导率、MDA显著增加,SOD活性为先升高后降低,且45℃高温处理时,相对电导率和MDA增加最快,SOD的活性最高。6个品种的耐热顺序为:世纪绿叶和洋娃娃最强,新象和城堡其次,和服和世纪铜叶最弱。种子发芽率、成苗率、相对电导率、丙二醛含量、超氧化物歧化酶活性均可作为鸡冠花耐热性评价指标。 相似文献
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Transformation of Plants with Multiple Cassettes Generates Simple Transgene Integration Patterns and High Expression Levels 总被引:1,自引:0,他引:1
Agrawal Pawan K. Kohli Ajay Twyman Richard M. Christou Paul 《Molecular breeding : new strategies in plant improvement》2005,16(3):247-260
We transformed rice (Oryza sativa L.) simultaneously with five minimal cassettes, each containing a promoter, coding region and polyadenylation site but no
vector backbone. We found that multi-transgene cotransformation was achieved with high efficiency using multiple cassettes,
with all transgenic plants we generated containing at least two transgenes and 16% containing all five. About 75% of the plants
had simple transgene integration patterns with a predominance of single-copy insertions. The expression levels for all transgenes,
and the overall coexpression frequencies, were much higher than previously reported in whole plasmid transformants. Four of
five lines analyzed for transgene expression stability in subsequent generations showed stable and high expression levels
over generations. A simple model is proposed, which accounts for differences in the molecular make-up and the expression profile
of transgenic plants generated using whole plasmid or minimal cassettes. We conclude that gene transfer using minimal cassettes
is an efficient and rapid method for the production of transgenic plants containing and stably expressing several different
transgenes. Our results facilitate effective manipulation of multi-gene pathways in plants in a single transformation step. 相似文献
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Recent advances in genotyping methodologies have allowed genome-wide association studies (GWAS) to accurately identify genetic variants that associate with common or pathological complex traits. Although most GWAS have focused on associations with single genetic variants, joint identification of multiple genetic variants, and how they interact, is essential for understanding the genetic architecture of complex phenotypic traits. Here, we propose an efficient stepwise method based on the Cochran-Mantel-Haenszel test (for stratified categorical data) to identify causal joint multiple genetic variants in GWAS. This method combines the CMH statistic with a stepwise procedure to detect multiple genetic variants associated with specific categorical traits, using a series of associated I × J contingency tables and a null hypothesis of no phenotype association. Through a new stratification scheme based on the sum of minor allele count criteria, we make the method more feasible for GWAS data having sample sizes of several thousands. We also examine the properties of the proposed stepwise method via simulation studies, and show that the stepwise CMH test performs better than other existing methods (e.g., logistic regression and detection of associations by Markov blanket) for identifying multiple genetic variants. Finally, we apply the proposed approach to two genomic sequencing datasets to detect linked genetic variants associated with bipolar disorder and obesity, respectively. 相似文献
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Geetika Sethi Harsh B. Pathak Hong Zhang Yan Zhou Margret B. Einarson Vinod Vathipadiekal Sumedha Gunewardena Michael J. Birrer Andrew K. Godwin 《PloS one》2012,7(10)
Targeted therapies have been used to combat many tumor types; however, few have effectively improved the overall survival in women with epithelial ovarian cancer, begging for a better understanding of this deadly disease and identification of essential drivers of tumorigenesis that can be targeted effectively. Therefore, we used a loss-of-function screening approach to help identify molecular vulnerabilities that may represent key points of therapeutic intervention. We employed an unbiased high-throughput lethality screen using a 24,088 siRNA library targeting over 6,000 druggable genes and studied their effects on growth and/or survival of epithelial ovarian cancer (EOC) cell lines. The top 300 “hits” affecting the viability of A1847 cells were rescreened across additional EOC cell lines and non-tumorigenic, human immortalized ovarian epithelial cell lines. Fifty-three gene candidates were found to exhibit effects in all tumorigenic cell lines tested. Extensive validation of these hits refined the list to four high quality candidates (HSPA5, NDC80, NUF2, and PTN). Mechanistic studies show that silencing of three genes leads to increased apoptosis, while HSPA5 silencing appears to alter cell growth through G1 cell cycle arrest. Furthermore, two independent gene expression studies show that NDC80, NUF2 and PTN were significantly aberrantly overexpressed in serous adenocarcinomas. Overall, our functional genomics results integrated with the genomics data provide an important unbiased avenue towards the identification of prospective therapeutic targets for drug discovery, which is an urgent and unmet clinical need for ovarian cancer. 相似文献