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991.
Conrad MD Gorman AW Schillinger JA Fiori PL Arroyo R Malla N Dubey ML Gonzalez J Blank S Secor WE Carlton JM 《PLoS neglected tropical diseases》2012,6(3):e1573
Background
Trichomonas vaginalis is the causative agent of human trichomoniasis, the most common non-viral sexually transmitted infection world-wide. Despite its prevalence, little is known about the genetic diversity and population structure of this haploid parasite due to the lack of appropriate tools. The development of a panel of microsatellite makers and SNPs from mining the parasite''s genome sequence has paved the way to a global analysis of the genetic structure of the pathogen and association with clinical phenotypes.Methodology/Principal Findings
Here we utilize a panel of T. vaginalis-specific genetic markers to genotype 235 isolates from Mexico, Chile, India, Australia, Papua New Guinea, Italy, Africa and the United States, including 19 clinical isolates recently collected from 270 women attending New York City sexually transmitted disease clinics. Using population genetic analysis, we show that T. vaginalis is a genetically diverse parasite with a unique population structure consisting of two types present in equal proportions world-wide. Parasites belonging to the two types (type 1 and type 2) differ significantly in the rate at which they harbor the T. vaginalis virus, a dsRNA virus implicated in parasite pathogenesis, and in their sensitivity to the widely-used drug, metronidazole. We also uncover evidence of genetic exchange, indicating a sexual life-cycle of the parasite despite an absence of morphologically-distinct sexual stages.Conclusions/Significance
Our study represents the first robust and comprehensive evaluation of global T. vaginalis genetic diversity and population structure. Our identification of a unique two-type structure, and the clinically relevant phenotypes associated with them, provides a new dimension for understanding T. vaginalis pathogenesis. In addition, our demonstration of the possibility of genetic exchange in the parasite has important implications for genetic research and control of the disease. 相似文献992.
Julia U. Sprenger Ruwan K. Perera Konrad R. G?tz Viacheslav O. Nikolaev 《Journal of visualized experiments : JoVE》2012,(66)
Förster resonance energy transfer (FRET) microscopy continues to gain increasing interest as a technique for real-time monitoring of biochemical and signaling events in live cells and tissues. Compared to classical biochemical methods, this novel technology is characterized by high temporal and spatial resolution. FRET experiments use various genetically-encoded biosensors which can be expressed and imaged over time in situ or in vivo1-2. Typical biosensors can either report protein-protein interactions by measuring FRET between a fluorophore-tagged pair of proteins or conformational changes in a single protein which harbors donor and acceptor fluorophores interconnected with a binding moiety for a molecule of interest3-4. Bimolecular biosensors for protein-protein interactions include, for example, constructs designed to monitor G-protein activation in cells5, while the unimolecular sensors measuring conformational changes are widely used to image second messengers such as calcium6, cAMP7-8, inositol phosphates9 and cGMP10-11. Here we describe how to build a customized epifluorescence FRET imaging system from single commercially available components and how to control the whole setup using the Micro-Manager freeware. This simple but powerful instrument is designed for routine or more sophisticated FRET measurements in live cells. Acquired images are processed using self-written plug-ins to visualize changes in FRET ratio in real-time during any experiments before being stored in a graphics format compatible with the build-in ImageJ freeware used for subsequent data analysis. This low-cost system is characterized by high flexibility and can be successfully used to monitor various biochemical events and signaling molecules by a plethora of available FRET biosensors in live cells and tissues. As an example, we demonstrate how to use this imaging system to perform real-time monitoring of cAMP in live 293A cells upon stimulation with a β-adrenergic receptor agonist and blocker. 相似文献
993.
The biennial plant Gentianella bohemica is a subendemic of the Bohemian Massif, where it occurs in seminatural grasslands. It has become rare in recent decades as a result of profound changes in land use. Using amplified fragment length polymorphisms (AFLP) fingerprint data, we investigated the genetic structure within and among populations of G. bohemica in Bavaria, the Czech Republic, and the Austrian border region. The aim of our study was (1) to analyze the genetic structure among populations and to discuss these findings in the context of present and historical patterns of connectivity and isolation of populations, (2) to analyze genetic structure among consecutive generations (cohorts of two consecutive years), and (3) to investigate relationships between intrapopulational diversity and effective population size (N(e)) as well as plant traits. (1) The German populations were strongly isolated from each other (pairwise F(ST)= 0.29-0.60) and from all other populations (F(ST)= 0.24-0.49). We found a pattern of near panmixis among the latter (F(ST)= 0.15-0.35) with geographical distance explaining only 8% of the genetic variance. These results were congruent with a principal coordinate analysis (PCoA) and analysis using STRUCTURE to identify genetically coherent groups. These findings are in line with the strong physical barrier and historical constraints, resulting in separation of the German populations from the others. (2) We found pronounced genetic differences between consecutive cohorts of the German populations (pairwise F(ST)= 0.23 and 0.31), which can be explained by local population history (land use, disturbance). (3) Genetic diversity within populations (Shannon index, H(Sh)) was significantly correlated with N(e) (R(S)= 0.733) and reflected a loss of diversity due to several demographic bottlenecks. Overall, we found that the genetic structure in G. bohemica is strongly influenced by historical periods of high connectivity and isolation as well as by marked demographic fluctuations in declining populations. 相似文献
994.
Görgens A Beckmann J Ludwig AK Möllmann M Dürig J Horn PA Rajendran L Giebel B 《The international journal of biochemistry & cell biology》2012,44(7):1121-1132
Freshly isolated human hematopoietic stem and progenitor cells (HSPCs) are small and round cells which upon cultivation adopt a polarized morphology and redistribute certain cell surface antigens. To functionally dissect this polarization process, we addressed impacts of protein synthesis, HSPC trafficking, cytoskeleton organization or lipid raft integrity on the establishment and maintenance of the cell polarity of human HSPCs. Effects on the morphology, sub-cellular distribution of lipid raft-associated molecular polarization markers (Flotillin-1, Flotillin-2, ICAM-3) and in vitro migration capabilities of treated cells were studied. We could distinguish two levels of cellular polarization, a molecular and a morphological level. Our data suggest that protein synthesis, lipid raft integrity and enzymatic activities of PI3K and aPKC are required to organize the molecular cell polarity. The morphological cell polarization process, however, also depends on actin polymerization and rho-GTPase activities. In summary, our data qualify HSPC polarization processes as new pharmaceutical target to interfere with migratory and with homing capabilities of HSPCs. 相似文献
995.
996.
Cystic echinococcosis is a chronic, complex, and neglected disease. Novel therapeutical tools are needed to optimize human treatment. A number of compounds have been investigated, either using in vitro cultured parasites and/or applying in vivo rodent models. Although some of these compounds showed promising activities in vitro, and to some extent also in the rodent models, they have not been translated into clinical applications. Membrane enzyme activities in culture supernatants of treated protoscoleces with calcium modulator drugs and anthelmintic drugs were measured and provided an indication of compound efficacy. This work describes for the first time the detection of alkaline phosphatase, gamma-glutamyl-transpeptidase and acetylcholinesterase activities in supernatants of in vitro treated Echinococcus granulosus protoscoleces. Marked differences on the enzymatic activities in supernatants from drug treated cultures were detected. We demonstrated that those genes that show the highest degree of conservation when compared to orthologs, are constitutively and highly expressed in protoscoleces and metacestodes. Due to high sensibility and the lack of activity in supernatants of intact protoscoleces, gamma-glutamyl-transpeptidase is proposed as the ideal viability marker during in vitro pharmacological studies against E. granulosus protoscoleces. 相似文献
997.
The Finapres finger cuff recording system provides continuous calculations of beat-to-beat variations in cardiac output (CO), total peripheral resistance, heart rate (HR), and blood pressure (BP). This system is unique in that it allows experimental subjects to immediately, continuously, and noninvasively visualize changes in CO at rest and during exercise. This study provides evidence that using the Finapres system improves undergraduate student engagement, understanding, and learning of how the cardiovascular system responds to exercise. Second-year science students undertaking a physiology practical class in 2009 (n = 243) and 2010 (n = 263) used the Finapres system to record CO, BP, and HR during graded exercise on a cycle ergometer. Student experiences with the Finapres was evaluated with a survey (a 5-point scale from strongly disagree to strongly agree). This indicated that students appreciated the immediacy of the recordings (88% of students agreed or strongly agreed, average for 2009 and 2010), gained an understanding of how to record physiological data (84%), enjoyed the practical (81%), and would recommend the Finapres to other students (81%). To determine if the practical enhanced student learning of cardiovascular physiology, identical tests were given to the students at the beginning (pretest) and end (posttest) of the class. There was a significant improvement from the pretest to the posttest (4% in 2009 and 20% in 2010). In summary, the ability of the Finapres to continuously display CO, BP, and HR during experimental protocols provides students with immediate feedback and improves their understanding of cardiovascular physiology. 相似文献
998.
The hyper-variable V4 and V9 regions of the small subunit (SSU) rDNA have been targeted for assessing environmental diversity of microbial eukaryotes using next generation sequencing technologies. Here, we explore how the genetic distances among these short fragments compare with the distances obtained from near full-length SSU-rDNA sequences by comparing all pairwise estimates, as well as within and among species of ciliates. Results show that pairwise distances from V4 more closely match the near full-length SSU-rDNA and are more comparable with previous studies based on much longer SSU-rDNA fragments, then pairwise distances from V9. Thus, studies that use the V4 will estimate similar values of phylotype richness and community structure as would have been estimated using the full-length SSU-rDNA. 相似文献
999.
Mulji A Haslam C Brown F Randle R Karamshi B Smith J Eagle R Munoz-Muriedas J Taylor J Sheikh A Bridges A Gill K Jepras R Smee P Barker M Woodrow M Liddle J Thomas P Jones E Gordon L Tanner R Leveridge M Hutchinson S Martin M Brown M Kruidenier L Katso R 《Journal of biomolecular screening》2012,17(1):108-120
The biological complexity associated with the regulation of histone demethylases makes it desirable to configure a cellular mechanistic assay format that simultaneously encompasses as many of the relevant cellular processes as possible. In this report, the authors describe the configuration of a JMJD3 high-content cellular mechanistic imaging assay that uses single-cell multiparameter measurements to accurately assess cellular viability and the enzyme-dependent demethylation of the H3K27(Me)3 mark by exogenously expressed JMJD3. This approach couples robust statistical analyses with the spatial resolving power of cellular imaging. This enables segregation of expressing and nonexpressing cells into discrete subpopulations and consequently pharmacological quantification of compounds of interest in the expressing population at varying JMJD3 expression levels. Moreover, the authors demonstrate the utility of this hit identification strategy through the successful prosecution of a medium-throughput focused campaign of an 87 500-compound file, which has enabled the identification of JMJD3 cellular-active chemotypes. This study represents the first report of a demethylase high-content imaging assay with the ability to capture a repertoire of pharmacological tools, which are likely both to inform our mechanistic understanding of how JMJD3 is modulated and, more important, to contribute to the identification of novel therapeutic modalities for this demethylase enzyme. 相似文献
1000.
Finkel J Guptill V Khaibullina A Spornick N Vasconcelos O Liewehr DJ Steinberg SM Quezado ZM 《Nitric oxide》2012,26(2):81-88
Nitric oxide synthases (NOSs) have been shown to modulate thermal hyperalgesia and mechanical hypersensitivity in inflammatory and neuropathic pain. However, little is known about the effect of NOSs on baseline function of sensory nerve fibers. Using genetic deficiency and pharmacologic inhibition of NOSs, we examined the impact of the three isoforms NOS1, NOS2, and NOS3 on baseline nocifensive behavior by measuring current vocalization threshold in response to electrical stimulation at 5, 250, 2000 Hz that preferentially stimulate C, Aδ, and Aβ fibers. In response to 5, 250 and 2000 Hz, NOS1-deficient animals had significantly higher current vocalization thresholds compared with wild-type. Genetic deficiency of NOS2 was associated with higher current vocalization thresholds in response to 5 Hz (C-fiber) stimulation. In contrast, NOS3-deficient animals had an overall weak trend toward lower current vocalization thresholds at 5 Hz and significantly lower current vocalization threshold compared with wild-type animals at 250 and 2000 Hz. Therefore, NOSs distinctively affect baseline mouse current vocalization threshold and appear to play a role on nocifensive response to electrical stimulation of sensory nerve fibers. 相似文献