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91.
The study of protein function usually requires the use of a cloned version of the gene for protein expression and functional assays. This strategy is particularly important when the information available regarding function is limited. The functional characterization of the thousands of newly identified proteins revealed by genomics requires faster methods than traditional single‐gene experiments, creating the need for fast, flexible, and reliable cloning systems. These collections of ORF clones can be coupled with high‐throughput proteomics platforms, such as protein microarrays and cell‐based assays, to answer biological questions. In this tutorial, we provide the background for DNA cloning, discuss the major high‐throughput cloning systems (Gateway® Technology, Flexi® Vector Systems, and CreatorTM DNA Cloning System) and compare them side‐by‐side. We also report an example of high‐throughput cloning study and its application in functional proteomics. This tutorial is part of the International Proteomics Tutorial Programme (IPTP12). 相似文献
92.
93.
Seok-Hyung Kim Sarah A. Scott Michael J. Bennett Robert P. Carson Joshua Fessel H. Alex Brown Kevin C. Ess 《PLoS genetics》2013,9(6)
Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) is a severe mitochondrial disorder featuring multi-organ dysfunction. Mutations in either the ETFA, ETFB, and ETFDH genes can cause MADD but very little is known about disease specific mechanisms due to a paucity of animal models. We report a novel zebrafish mutant dark xavier (dxavu463) that has an inactivating mutation in the etfa gene. dxavu463 recapitulates numerous pathological and biochemical features seen in patients with MADD including brain, liver, and kidney disease. Similar to children with MADD, homozygote mutant dxavu463 zebrafish have a spectrum of phenotypes ranging from moderate to severe. Interestingly, excessive maternal feeding significantly exacerbated the phenotype. Homozygous mutant dxavu463 zebrafish have swollen and hyperplastic neural progenitor cells, hepatocytes and kidney tubule cells as well as elevations in triacylglycerol, cerebroside sulfate and cholesterol levels. Their mitochondria were also greatly enlarged, lacked normal cristae, and were dysfunctional. We also found increased signaling of the mechanistic target of rapamycin complex 1 (mTORC1) with enlarged cell size and proliferation. Treatment with rapamycin partially reversed these abnormalities. Our results indicate that etfa gene function is remarkably conserved in zebrafish as compared to humans with highly similar pathological, biochemical abnormalities to those reported in children with MADD. Altered mTORC1 signaling and maternal nutritional status may play critical roles in MADD disease progression and suggest novel treatment approaches that may ameliorate disease severity. 相似文献
94.
Alexandra M. Lopes Kenneth I. Aston Emma Thompson Filipa Carvalho Jo?o Gon?alves Ni Huang Rune Matthiesen Michiel J. Noordam Inés Quintela Avinash Ramu Catarina Seabra Amy B. Wilfert Juncheng Dai Jonathan M. Downie Susana Fernandes Xuejiang Guo Jiahao Sha António Amorim Alberto Barros Angel Carracedo Zhibin Hu Matthew E. Hurles Sergey Moskovtsev Carole Ober Darius A. Paduch Joshua D. Schiffman Peter N. Schlegel Mário Sousa Douglas T. Carrell Donald F. Conrad 《PLoS genetics》2013,9(3)
Gonadal failure, along with early pregnancy loss and perinatal death, may be an important filter that limits the propagation of harmful mutations in the human population. We hypothesized that men with spermatogenic impairment, a disease with unknown genetic architecture and a common cause of male infertility, are enriched for rare deleterious mutations compared to men with normal spermatogenesis. After assaying genomewide SNPs and CNVs in 323 Caucasian men with idiopathic spermatogenic impairment and more than 1,100 controls, we estimate that each rare autosomal deletion detected in our study multiplicatively changes a man''s risk of disease by 10% (OR 1.10 [1.04–1.16], p<2×10−3), rare X-linked CNVs by 29%, (OR 1.29 [1.11–1.50], p<1×10−3), and rare Y-linked duplications by 88% (OR 1.88 [1.13–3.13], p<0.03). By contrasting the properties of our case-specific CNVs with those of CNV callsets from cases of autism, schizophrenia, bipolar disorder, and intellectual disability, we propose that the CNV burden in spermatogenic impairment is distinct from the burden of large, dominant mutations described for neurodevelopmental disorders. We identified two patients with deletions of DMRT1, a gene on chromosome 9p24.3 orthologous to the putative sex determination locus of the avian ZW chromosome system. In an independent sample of Han Chinese men, we identified 3 more DMRT1 deletions in 979 cases of idiopathic azoospermia and none in 1,734 controls, and found none in an additional 4,519 controls from public databases. The combined results indicate that DMRT1 loss-of-function mutations are a risk factor and potential genetic cause of human spermatogenic failure (frequency of 0.38% in 1306 cases and 0% in 7,754 controls, p = 6.2×10−5). Our study identifies other recurrent CNVs as potential causes of idiopathic azoospermia and generates hypotheses for directing future studies on the genetic basis of male infertility and IVF outcomes. 相似文献
95.
96.
Tibor Németh Adél Tóth Judit Szenzenstein Péter Horváth Joshua D. Nosanchuk Zsuzsanna Grózer Renáta Tóth Csaba Papp Zsuzsanna Hamari Csaba Vágv?lgyi Attila Gácser 《PloS one》2013,8(7)
The C. parapsilosis sensu lato group involves three closely related species, C. parapsilosis sensu stricto,
C
. orthopsilosis
and
C
. metapsilosis
. Although their overall clinical importance is dramatically increasing, there are few studies regarding the virulence properties of the species of the psilosis complex. In this study, we tested 63 C. parapsilosis sensu stricto, 12
C
. metapsilosis
and 18
C
. orthopsilosis
isolates for the ability to produce extracellular proteases, secrete lipases and form pseudohyphae. Significant differences were noted between species, with the
C
. metapsilosis
strains failing to secrete lipase or to produce pseudohyphae. Nine different clinical isolates each of C. parapsilosis sensu stricto,
C
. orthopsilosis
and
C
. metapsilosis
were co-cultured with immortalized murine or primary human macrophages. C. parapsilosis sensu stricto isolates showed a significantly higher resistance to killing by primary human macrophages compared to
C
. orthopsilosis
and
C
. metapsilosis
isolates. In contrast, the killing of isolates by J774.2 mouse macrophages did not differ significantly between species. However, C. parapsilosis sensu stricto isolates induced the most damage to murine and human macrophages, and
C
. metapsilosis
strains were the least toxic. Furthermore, strains that produced lipase or pseudohyphae were most resistant to macrophage-mediated killing and produced the most cellular damage. Finally, we used 9 isolates of each of the C. parapsilosis sensus lato species to examine their impact on the survival of
Galleria
mellonella
larvae. The mortality rate of
G
. mellonella
larvae infected with
C
. metapsilosis
isolates was significantly lower than those infected with C. parapsilosis sensu stricto or
C
. orthopsilosis
strains. Taken together, our findings demonstrate that
C
. metapsilosis
is indeed the least virulent member of the psilosis group, and also highlight the importance of pseudohyphae and secreted lipases during fungal-host interactions. 相似文献
97.
Maria M. James Oliver Laeyendecker Jin Sun Donald R. Hoover Caroline E. Mullis Matthew M. Cousins Thomas Coates Richard D. Moore Gabor D. Kelen Mary Glenn Fowler Johnstone J. Kumwenda Lynne M. Mofenson Newton I. Kumwenda Taha E. Taha Susan H. Eshleman 《PloS one》2013,8(2)
Introduction
The Post-exposure Prophylaxis in Infants (PEPI)-Malawi trial evaluated infant antiretroviral regimens for prevention of post-natal HIV transmission. A multi-assay algorithm (MAA) that includes the BED capture immunoassay, an avidity assay, CD4 cell count, and viral load was used to identify women who were vs. were not recently infected at the time of enrollment (MAA recent, N = 73; MAA non-recent, N = 2,488); a subset of the women in the MAA non-recent group known to have been HIV infected for at least 2 years before enrollment (known non-recent, N = 54). Antibody maturation and viral diversification were examined in these women.Methods
Samples collected at enrollment (N = 2,561) and 12–24 months later (N = 1,306) were available for serologic analysis using the BED and avidity assays. A subset of those samples was used for analysis of viral diversity, which was performed using a high resolution melting (HRM) diversity assay. Viral diversity analysis was performed using all available samples from women in the MAA recent group (61 enrollment samples, 38 follow-up samples) and the known non-recent group (43 enrollment samples, 22 follow-up samples). Diversity data from PEPI-Malawi were also compared to similar data from 169 adults in the United States (US) with known recent infection (N = 102) and known non-recent infection (N = 67).Results
In PEPI-Malawi, results from the BED and avidity assays increased over time in the MAA recent group, but did not change significantly in the MAA non-recent group. At enrollment, HIV diversity was lower in the MAA recent group than in the known non-recent group. HRM diversity assay results from women in PEPI-Malawi were similar to those from adults in the US with known duration of HIV infection.Conclusions
Antibody maturation and HIV diversification patterns in African women provide additional support for use of the MAA to identify populations with recent HIV infection. 相似文献98.
In a 2-alternative forced-choice (2AFC) discrimination task, observers choose which of two stimuli has the higher value. The psychometric function for this task gives the probability of a correct response for a given stimulus difference, . This paper proves four theorems about the psychometric function. Assuming the observer applies a transducer and adds noise, Theorem 1 derives a convenient general expression for the psychometric function. Discrimination data are often fitted with a Weibull function. Theorem 2 proves that the Weibull “slope” parameter, , can be approximated by , where is the of the Weibull function that fits best to the cumulative noise distribution, and depends on the transducer. We derive general expressions for and , from which we derive expressions for specific cases. One case that follows naturally from our general analysis is Pelli''s finding that, when , . We also consider two limiting cases. Theorem 3 proves that, as sensitivity improves, 2AFC performance will usually approach that for a linear transducer, whatever the actual transducer; we show that this does not apply at signal levels where the transducer gradient is zero, which explains why it does not apply to contrast detection. Theorem 4 proves that, when the exponent of a power-function transducer approaches zero, 2AFC performance approaches that of a logarithmic transducer. We show that the power-function exponents of 0.4–0.5 fitted to suprathreshold contrast discrimination data are close enough to zero for the fitted psychometric function to be practically indistinguishable from that of a log transducer. Finally, Weibull reflects the shape of the noise distribution, and we used our results to assess the recent claim that internal noise has higher kurtosis than a Gaussian. Our analysis of for contrast discrimination suggests that, if internal noise is stimulus-independent, it has lower kurtosis than a Gaussian. 相似文献
99.
James Grogan Andrew Plumptre Joshua Mabonga Simon Nampindo Mustapha Nsubuga Andrew Balmford 《African Journal of Ecology》2020,58(1):2-13
Elephant populations are in decline across the African continent, but recent aerial surveys show that populations in Uganda are increasing. However, threats such as poaching and habitat disturbance remain. Having a comprehensive knowledge of the ranging behaviour of Ugandan elephants is crucial to understanding where critical habitat for the species occurs. We investigated various aspects of ranging behaviour of 45 radio-collared elephants (Loxodonta africana) in three areas—Queen Elizabeth Protected Area (QEPA), Murchison Falls (MFPA) Protected Area and Kidepo Valley (KVCA) Conservation Area. We also set Ugandan analyses in a continental context by comparison with home ranges reported in published literature. Elephants within KVCA had larger core ranges than elephants in QEPA or MFPA. Wet season ranges in KVCA were much larger than dry season ranges. The most important core areas in all three national parks were centred around water resources. Home range size was negatively correlated with net primary productivity (NPP) at Ugandan (N = 39 individuals) and continental (N = 17 sites) scales. This study indicates that, at a local scale, factors such as water source location are important in shaping elephant ranging behaviour. At larger scales, factors such as NPP are good predictors of elephant home range size. 相似文献
100.
Joshua P. Moatt Eevi Savola Jennifer C. Regan Daniel H. Nussey Craig A. Walling 《BioEssays : news and reviews in molecular, cellular and developmental biology》2020,42(8):1900241
Dietary restriction (DR) is the most consistent environmental manipulation to extend lifespan. Originally thought to be caused by a reduction in caloric intake, recent evidence suggests that macronutrient intake underpins the effect of DR. The prevailing evolutionary explanations for the DR response are conceptualized under the caloric restriction paradigm, necessitating reconsideration of how or whether these evolutionary explanations fit this macronutrient perspective. In the authors’ opinion, none of the current evolutionary explanations of DR adequately explain the intricacies of observed results; instead a context-dependent combination of these theories is suggested which is likely to reflect reality. In reviewing the field, it is proposed that the ability to track the destination of different macronutrients within the body will be key to establishing the relative roles of the competing theories. Understanding the evolution of the DR response and its ecological relevance is critical to understanding variation in DR responses and their relevance outside laboratory environments. 相似文献