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11.
Vincenzo Forgetta Gary Leveque Joana Dias Deborah Grove Robert Lyons Jr. Suzanne Genik Chris Wright Sushmita Singh Nichole Peterson Michael Zianni Jan Kieleczawa Robert Steen Anoja Perera Doug Bintzler Scottie Adams Will Hintz Volker Jacobi Louis Bernier Roger Levesque Ken Dewar 《Journal of biomolecular techniques》2013,24(1):39-49
As part of the DNA Sequencing Research Group of the Association of Biomolecular Resource Facilities, we have tested the reproducibility of the Roche/454 GS-FLX Titanium System at five core facilities. Experience with the Roche/454 system ranged from <10 to >340 sequencing runs performed. All participating sites were supplied with an aliquot of a common DNA preparation and were requested to conduct sequencing at a common loading condition. The evaluation of sequencing yield and accuracy metrics was assessed at a single site. The study was conducted using a laboratory strain of the Dutch elm disease fungus Ophiostoma novo-ulmi strain H327, an ascomycete, vegetatively haploid fungus with an estimated genome size of 30–50 Mb. We show that the Titanium System is reproducible, with some variation detected in loading conditions, sequencing yield, and homopolymer length accuracy. We demonstrate that reads shorter than the theoretical minimum length are of lower overall quality and not simply truncated reads. The O. novo-ulmi H327 genome assembly is 31.8 Mb and is comprised of eight chromosome-length linear scaffolds, a circular mitochondrial conti of 66.4 kb, and a putative 4.2-kb linear plasmid. We estimate that the nuclear genome encodes 8613 protein coding genes, and the mitochondrion encodes 15 genes and 26 tRNAs. 相似文献
12.
13.
A previous study on the evolutionary patterns of Tarentola mauritanica demonstrated that low levels of mitochondrial diversity observed in the European populations relative to nuclear markers were consistent with a selective sweep hypothesis. In order to unravel the mitochondrial evolutionary history in this European population and two other lineages of T. mauritanica (Iberian and North African clades), variation within 22 nearly complete mitogenomes was analyzed. Surprisingly, each clade seems to have a distinct evolutionary history; with both the European and Iberian clades presenting a decrease of polymorphism, which in the former is consistent with departure from neutrality of the mtDNA (positive or background selection), but in the latter seems to be the result of a bottleneck after a population expansion. The pattern exhibited by the North African clade seems to be a consequence of adaptation to certain mtDNA variants by positive selection. 相似文献
14.
Rolando Perdomo-Morales Vivian Montero-Alejo Gerardo Corzo Vladimir Besada Yamile Vega-Hurtado Yamile González-González Erick Perera Marlene Porto-Verdecia 《The Journal of biological chemistry》2013,288(44):31867-31879
The melanization reaction promoted by the prophenoloxidase-activating system is an essential defense response in invertebrates subjected to regulatory mechanisms that are still not fully understood. We report here the finding and characterization of a novel trypsin inhibitor, named panulirin, isolated from the hemocytes of the spiny lobster Panulirus argus with regulatory functions on the melanization cascade. Panulirin is a cationic peptide (pI 9.5) composed of 48 amino acid residues (5.3 kDa), with six cysteine residues forming disulfide bridges. Its primary sequence was determined by combining Edman degradation/N-terminal sequencing and electrospray ionization-MS/MS spectrometry. The low amino acid sequence similarity with known proteins indicates that it represents a new family of peptidase inhibitors. Panulirin is a competitive and reversible tight-binding inhibitor of trypsin (Ki = 8.6 nm) with a notable specificity because it does not inhibit serine peptidases such as subtilisin, elastase, chymotrypsin, thrombin, and plasmin. The removal of panulirin from the lobster hemocyte lysate leads to an increase in phenoloxidase response to LPS. Likewise, the addition of increasing concentrations of panulirin to a lobster hemocyte lysate, previously depleted of trypsin-inhibitory activity, decreased the phenoloxidase response to LPS in a concentration-dependent fashion. These results indicate that panulirin is implicated in the regulation of the melanization cascade in P. argus by inhibiting peptidase(s) in the pathway toward the activation of the prophenoloxidase enzyme. 相似文献
15.
C. H. W. M. R. Chandrasekara Bhagya W. S. Wijesundera Sulochana N. Perera Hemamali 《Indian journal of human genetics》2013,19(1):78-83
CONTEXT:
Amplification of Guanine-Cytosine (GC) -rich sequences becomes important in screening and diagnosis of certain genetic diseases such as diseases arising due to expansion of GC-rich trinucleotide repeat regions. However, GC-rich sequences in the genome are refractory to standard polymerase chain reaction (PCR) amplification and require a special reaction conditions and/or modified PCR cycle parameters.AIM:
Optimize a cost effective PCR assay to amplify the GC-rich DNA templates.SETTINGS AND DESIGN:
Fragile X mental retardation gene (FMR 1) is an ideal candidate for PCR optimization as its GC content is more than 80%. Primers designed to amplify the GC rich 5’ untranslated region of the FMR 1 gene, was selected for the optimization of amplification using DNA extracted from buccal mucosal cells.MATERIALS AND METHODS:
A simple and rapid protocol was used to extract DNA from buccal cells. PCR optimization was carried out using three methods, (a) substituting a substrate analog 7-deaza-dGTP to dGTP (b) in the presence of a single PCR additive and (c) using a combination of PCR additives. All PCR amplifications were carried out using a low-cost thermostable polymerase.RESULTS:
Optimum PCR conditions were achieved when a combination of 1M betaine and 5% dimethyl sulfoxide (DMSO) was used.CONCLUSIONS:
It was possible to amplify the GC rich region of FMR 1 gene with reproducibility in the presence of betaine and DMSO as additives without the use of commercially available kits for DNA extraction and the expensive thermostable polymerases. 相似文献16.
Jeremy Howick Claire Friedemann Maria Tsakok Robert Watson Teresa Tsakok Jennifer Thomas Rafael Perera Susannah Fleming Carl Heneghan 《PloS one》2013,8(5)
BackgroundPlacebos are widely used in clinical practice in spite of ethical restrictions. Whether such use is justified depends in part on the relative benefit of placebos compared to ‘active’ treatments. A direct test for differences between placebo and ‘active’ treatment effects has not been conducted.ObjectivesWe aimed to test for differences between treatment and placebo effects within similar trial populations.ResultsIn trials with continuous outcomes (n = 115) we found no difference between treatment and placebo effects (MD = −0.29, 95% CI −0.62 to 0.05, P = 0.10). In trials with binary outcomes (n = 37) treatments were significantly more effective than placebos (RRR = 0.72, 95%CI = 0.61 to 0.86, P = 0.0003). Treatment and placebo effects were not different in 22 out of 28 predefined subgroup analyses. Of the six subgroups with differences treatments were more effective than placebos in five. However when all criteria for reducing bias were ruled out (continuous outcomes) placebos were more effective than treatments (MD = 1.59, 95% CI = 0.40 to 2.77, P = 0.009).
Conclusions and Implications
Placebos and treatments often have similar effect sizes. Placebos with comparatively powerful effects can benefit patients either alone or as part of a therapeutic regime, and trials involving such placebos must be adequately blinded. 相似文献17.
Mathew Seymour Omaththage P. Perera Howard W. Fescemyer Ryan E. Jackson Shelby J. Fleischer Craig A. Abel 《Ecology and evolution》2016,6(10):3198-3207
Seasonal climatic shifts create peripheral habitats that alternate between habitable and uninhabitable for migratory species. Such dynamic peripheral habitats are potential sites where migratory species could evolve high genetic diversity resulting from convergence of immigrants from multiple regionally distant areas. Migrant populations of Helicoverpa zea (Boddie) captured during two different seasons were assessed for genetic structure using microsatellite markers and for host plant type using stable carbon isotope analysis. Individuals (N = 568) were genotyped and divided into 13 putative populations based on collection site and time. Fixation indices (F‐statistics), analysis of molecular variance (AMOVA), and discriminant analysis of principal components (DAPC) were used to examine within and among population genetic variation. Mean number of alleles per locus was 10.25 (± 3.2 SD), and allelic richness ranged from 2.38 to 5.13 (± 3.2 SD). The observed and expected heterozygosity ranged from 0.07 to 0.48 and 0.08 to 0.62, respectively. Low FST (0.01 to 0.02) and high FIS (0.08 to 0.33) values suggest captured migrants originated from breeding populations with different allele frequencies. We postulate that high genetic diversity within migrant populations and low genetic differentiation among migrant populations of H. zea are the result of asymmetrical immigration due to the high dispersal and reproductive behavior of H. zea, which may hinder the adaptation and establishment of H. zea to peripheral habitat. These findings highlight the importance of assessing peripheral population structure in relation to ecological and evolutionary dynamics of this and other highly reproductive and dispersive species. 相似文献
18.
James Weger-Lucarelli Claudia Rückert Nunya Chotiwan Chilinh Nguyen Selene M. Garcia Luna Joseph R. Fauver Brian D. Foy Rushika Perera William C. Black Rebekah C. Kading Gregory D. Ebel 《PLoS neglected tropical diseases》2016,10(10)
In 2015, Zika virus (ZIKV; Flaviviridae; Flavivirus) emerged in the Americas, causing millions of infections in dozens of countries. The rapid spread of the virus and the association with disease outcomes such as Guillain-Barré syndrome and microcephaly make understanding transmission dynamics essential. Currently, there are no reports of vector competence (VC) of American mosquitoes for ZIKV isolates from the Americas. Further, it is not clear whether ZIKV strains from other genetic lineages can be transmitted by American Aedes aegypti populations, and whether the scope of the current epidemic is in part facilitated by viral factors such as enhanced replicative fitness or increased vector competence. Therefore, we characterized replication of three ZIKV strains, one from each of the three phylogenetic clades in several cell lines and assessed their abilities to be transmitted by Ae. aegypti mosquitoes. Additionally, laboratory colonies of different Culex spp. were infected with an American outbreak strain of ZIKV to assess VC. Replication rates were variable and depended on virus strain, cell line and MOI. African strains used in this study outcompeted the American strain in vitro in both mammalian and mosquito cell culture. West and East African strains of ZIKV tested here were more efficiently transmitted by Ae. aegypti from Mexico than was the currently circulating American strain of the Asian lineage. Long-established laboratory colonies of Culex mosquitoes were not efficient ZIKV vectors. These data demonstrate the capacity for additional ZIKV strains to infect and replicate in American Aedes mosquitoes and suggest that neither enhanced virus replicative fitness nor virus adaptation to local vector mosquitoes seems likely to explain the extent and intensity of ZIKV transmission in the Americas. 相似文献
19.
Natalia V. Voge Rushika Perera Sebabrata Mahapatra Lionel Gresh Angel Balmaseda María A. Loro?o-Pino Amber S. Hopf-Jannasch John T. Belisle Eva Harris Carol D. Blair Barry J. Beaty 《PLoS neglected tropical diseases》2016,10(2)
Background
Epidemic dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are overwhelming public health capacity for diagnosis and clinical care of dengue patients throughout the tropical and subtropical world. The ability to predict severe dengue disease outcomes (DHF/DSS) using acute phase clinical specimens would be of enormous value to physicians and health care workers for appropriate triaging of patients for clinical management. Advances in the field of metabolomics and analytic software provide new opportunities to identify host small molecule biomarkers (SMBs) in acute phase clinical specimens that differentiate dengue disease outcomes.Methodology/Principal Findings
Exploratory metabolomic studies were conducted to characterize the serum metabolome of patients who experienced different dengue disease outcomes. Serum samples from dengue patients from Nicaragua and Mexico were retrospectively obtained, and hydrophilic interaction liquid chromatography (HILIC)-mass spectrometry (MS) identified small molecule metabolites that were associated with and statistically differentiated DHF/DSS, DF, and non-dengue (ND) diagnosis groups. In the Nicaraguan samples, 191 metabolites differentiated DF from ND outcomes and 83 differentiated DHF/DSS and DF outcomes. In the Mexican samples, 306 metabolites differentiated DF from ND and 37 differentiated DHF/DSS and DF outcomes. The structural identities of 13 metabolites were confirmed using tandem mass spectrometry (MS/MS). Metabolomic analysis of serum samples from patients diagnosed as DF who progressed to DHF/DSS identified 65 metabolites that predicted dengue disease outcomes. Differential perturbation of the serum metabolome was demonstrated following infection with different DENV serotypes and following primary and secondary DENV infections.Conclusions/Significance
These results provide proof-of-concept that a metabolomics approach can be used to identify metabolites or SMBs in serum specimens that are associated with distinct DENV infections and disease outcomes. The differentiating metabolites also provide insights into metabolic pathways and pathogenic and immunologic mechanisms associated with dengue disease severity. 相似文献20.
Sachiko?OhdeEmail author Emily?McFadden Gautam?A.?Deshpande Hiroshi?Yokomichi Osamu?Takahashi Tsuguya?Fukui Rafael?Perera Zentaro?Yamagata 《BMC endocrine disorders》2016,16(1):65