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1.
SM Lank BA Golbach HM Creager RW Wiseman DB Keskin EL Reinherz V Brusic DH O'Connor 《BMC genomics》2012,13(1):378
ABSTRACT: BACKGROUND: High-resolution HLA genotyping is a critical diagnostic and research assay. Current methods rarely achieve unambiguous high-resolution typing without making population-specific frequency inferences due to a lack of locus coverage and difficulty in exon-phase matching. Achieving high-resolution typing is also becoming more challenging with traditional methods as the database of known HLA alleles increases. RESULTS: We designed a cDNA amplicon-based pyrosequencing method to capture 94% of the HLA class I open-reading-frame with only two amplicons per sample, and an analogous method for class II HLA genes, with a primary focus on sequencing the DRB loci. We present a novel Galaxy server-based analysis workflow for determining genotype. During assay validation, we performed two GS Junior sequencing runs to determine the accuracy of the HLA class I amplicons and DRB amplicon at different levels of multiplexing. When 116 amplicons were multiplexed, we unambiguously resolved 99%of class I alleles to four- or six-digit resolution, as well as 100% unambiguous DRB calls. The second experiment, with 271 multiplexed amplicons, missed some alleles, but generated high-resolution, concordant typing for 93% of class I alleles, and 96% for DRB1 alleles. In a third, preliminary experiment we attempted to sequence novel amplicons for other class II loci with mixed success. CONCLUSIONS: The presented assay is higher-throughput and higher-resolution than existing HLA genotyping methods, and suitable for allele discovery or large cohort sampling. The validated class I and DRB primers successfully generated unambiguously high-resolution genotypes, while further work is needed to validate additional class II genotyping amplicons. 相似文献
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BackgroundAddiction, or substance dependence, is nowadays considered a chronic relapsing condition. However, perceptions of addiction vary widely, also among healthcare professionals. Perceptions of addiction are thought to contribute to attitude and stigma towards patients with addiction. However, studies into perceptions of addiction among healthcare professionals are limited and instruments for reliable assessment of their perceptions are lacking. The Illness Perception Questionnaire (IPQ) is widely used to evaluate perceptions of illness. The aim of this study was to evaluate the psychometric properties of the IPQ: factor structure, internal consistency, and discriminant validity, when applied to evaluate healthcare professionals’ perceptions of addiction.MethodsParticipants were 1072 healthcare professionals in training and master students from the Netherlands and Indonesia, recruited from various addiction-training programs. The revised version of the IPQ was adapted to measure perceptions of addiction (IPQ-A). Maximum likelihood method was used to explore the best-fit IPQ factor structure. Internal consistency was evaluated for the final factors. The final factor structure was used to assess discriminant validity of the IPQ, by comparing illness perceptions of addiction between 1) medical students from the Netherlands and Indonesia, 2) medical students psychology students and educational science students from the Netherlands, and 3) participants with different training levels: medical students versus medical doctors.ResultsFactor analysis revealed an eight-factor structure for the perception subscale (demoralization, timeline chronic, consequences, personal control, treatment control, illness coherence, timeline cyclical emotional representations) and a four-factor structure for the attribution subscale (psychological attributions, risk factors, smoking/alcohol, overwork). Internal reliability was acceptable to good. The IPQ-A was able to detect differences in perceptions between healthcare professionals from different cultural and educational background and level of training.ConclusionsThe IPQ-A is a valid and reliable instrument to assess healthcare professionals’ perceptions of addiction. 相似文献
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Boots AM Hubers H Kouwijzer M den Hoed-van Zandbrink L Westrek-Esselink BM van Doorn C Stenger R Bos ES van Lierop MJ Verheijden GF Timmers CM van Staveren CJ 《Arthritis research & therapy》2007,9(4):R71
We sought to identify an altered peptide ligand (APL) based on the endogenously expressed synovial auto-epitope of human cartilage glycoprotein-39 (HC gp-39) for modulation of cognate, HLA-DR4-restricted T cells. For this purpose we employed a panel of well-characterized T cell hybridomas generated from HC gp-39-immunized HLA-DR4 transgenic mice. The hybridomas all respond to the HC gp-39(263-275) epitope when bound to HLA-DR4(B1*0401) but differ in their fine specificities. First, the major histocompatibility complex (MHC) and T-cell receptor (TCR) contact residues were identified by analysis of single site substituted analogue peptides for HLA-DR4 binding and cognate T cell recognition using both T hybridomas and polyclonal T cells from peptide-immunized HLA-DR4 transgenic mice. Analysis of single site substituted APL by cognate T cells led to identification of Phe265 as the dominant MHC anchor. The amino acids Ala268, Ser269, Glu271 and Thr272 constituted the major TCR contact residues, as substitution at these positions did not affect HLA-DR4(B1*0401) binding but abrogated T cell responses. A structural model for visualisation of TCR recognition was derived. Second, a set of non-classical APLs, modified at the MHC key anchor position but with unaltered TCR contacts, was developed. When these APLs were analysed, a partial TCR agonist was identified and found to modulate the HC gp-39(263-275)-specific, pro-inflammatory response in HLA-DR4 transgenic mice. We identified a non-classical APL by modification of the p1 MHC anchor in a synovial auto-epitope. This APL may qualify for rheumatoid arthritis immunotherapy. 相似文献
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Havelaar AH van Rosse F Bucura C Toetenel MA Haagsma JA Kurowicka D Heesterbeek JH Speybroeck N Langelaar MF van der Giessen JW Cooke RM Braks MA 《PloS one》2010,5(11):e13965
Background
To support the development of early warning and surveillance systems of emerging zoonoses, we present a general method to prioritize pathogens using a quantitative, stochastic multi-criteria model, parameterized for the Netherlands.Methodology/Principal Findings
A risk score was based on seven criteria, reflecting assessments of the epidemiology and impact of these pathogens on society. Criteria were weighed, based on the preferences of a panel of judges with a background in infectious disease control.Conclusions/Significance
Pathogens with the highest risk for the Netherlands included pathogens in the livestock reservoir with a high actual human disease burden (e.g. Campylobacter spp., Toxoplasma gondii, Coxiella burnetii) or a low current but higher historic burden (e.g. Mycobacterium bovis), rare zoonotic pathogens in domestic animals with severe disease manifestations in humans (e.g. BSE prion, Capnocytophaga canimorsus) as well as arthropod-borne and wildlife associated pathogens which may pose a severe risk in future (e.g. Japanese encephalitis virus and West-Nile virus). These agents are key targets for development of early warning and surveillance. 相似文献6.
Shen J Liu X Yu WM Liu J Nibbelink MG Guo C Finkel T Qu CK 《Molecular and cellular biology》2011,31(24):4902-4916
Mitochondria are highly dynamic organelles that play multiple roles in cells. How mitochondria cooperatively modulate embryonic stem (ES) cell function during development is not fully understood. Global disruption of Ptpmt1, a mitochondrial Pten-like phosphatidylinositol phosphate (PIP) phosphatase, resulted in developmental arrest and postimplantation lethality. Ptpmt1(-/-) blastocysts failed to outgrow, and inner-cell-mass cells failed to thrive. Depletion of Ptpmt1 in conditional knockout ES cells decreased proliferation without affecting energy homeostasis or cell survival. Differentiation of Ptpmt1-depleted ES cells was essentially blocked. This was accompanied by upregulation of cyclin-dependent kinase inhibitors and a significant cell cycle delay. Reintroduction of wild-type but not of catalytically deficient Ptpmt1 C132S or truncated Ptpmt1 lacking the mitochondrial localization signal restored the differentiation capabilities of Ptpmt1 knockout ES cells. Intriguingly, Ptpmt1 is specifically important for stem cells, as ablation of Ptpmt1 in differentiated embryonic fibroblasts did not disturb cellular function. Further analyses demonstrated that oxygen consumption of Ptpmt1-depleted cells was decreased, while glycolysis was concomitantly enhanced. In addition, mitochondrial fusion/dynamics were compromised in Ptpmt1 knockout cells due to accumulation of PIPs. These studies, while establishing a crucial role for Ptpmt1 phosphatase in embryogenesis, reveal a mitochondrial metabolic stress-activated checkpoint in the control of ES cell differentiation. 相似文献
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Milou E. W. M. Silkens Onyebuchi A. Arah Albert J. J. A. Scherpbier Maas Jan Heineman Kiki M. J. M. H. Lombarts 《PloS one》2016,11(1)
Background
A department’s learning climate is known to contribute to the quality of postgraduate medical education and, as such, to the quality of patient care provided by residents. However, it is unclear how the learning climate is perceived over time.Objectives
This study investigated whether the learning climate perceptions of residents changed over time.Methods
The context for this study was residency training in the Netherlands. Between January 2012 and December 2014, residents from 223 training programs in 39 hospitals filled out the web-based Dutch Residency Educational Climate Test (D-RECT) to evaluate their clinical department’s learning climate. Residents had to fill out 35 validated questions using a five point Likert-scale. We analyzed data using generalized linear mixed (growth) models.Results
Overall, 3982 D-RECT evaluations were available to investigate our aim. The overall mean D-RECT score was 3.9 (SD = 0.3). The growth model showed an increase in D-RECT scores over time (b = 0.03; 95% CI: 0.01–0.06; p < 0.05).Conclusions
The observed increase in D-RECT scores implied that residents perceived an improvement in the learning climate over time. Future research could focus on factors that facilitate or hinder learning climate improvement, and investigate the roles that hospital governing committees play in safeguarding and improving the learning climate. 相似文献8.
R. Milou Schuurmans J. Merijn Schuurmans Martijn Bekker Jacco C. Kromkamp Hans C.P. Matthijs Klaas J. Hellingwerf 《Plant physiology》2014,165(1):463-475
A method is presented for rapid extraction of the total plastoquinone (PQ) pool from Synechocystis sp. strain PCC 6803 cells that preserves the in vivo plastoquinol (PQH2) to -PQ ratio. Cells were rapidly transferred into ice-cold organic solvent for instantaneous extraction of the cellular PQ plus PQH2 content. After high-performance liquid chromatography fractionation of the organic phase extract, the PQH2 content was quantitatively determined via its fluorescence emission at 330 nm. The in-cell PQH2-PQ ratio then followed from comparison of the PQH2 signal in samples as collected and in an identical sample after complete reduction with sodium borohydride. Prior to PQH2 extraction, cells from steady-state chemostat cultures were exposed to a wide range of physiological conditions, including high/low availability of inorganic carbon, and various actinic illumination conditions. Well-characterized electron-transfer inhibitors were used to generate a reduced or an oxidized PQ pool for reference. The in vivo redox state of the PQ pool was correlated with the results of pulse-amplitude modulation-based chlorophyll a fluorescence emission measurements, oxygen exchange rates, and 77 K fluorescence emission spectra. Our results show that the redox state of the PQ pool of Synechocystis sp. strain PCC 6803 is subject to strict homeostatic control (i.e. regulated between narrow limits), in contrast to the more dynamic chlorophyll a fluorescence signal.The photosynthetic apparatus of oxygenic phototrophs consists of two types of photosynthetic reaction centers: PSII and PSI. Both photosystems are connected in series, with electrons flowing from PSII toward PSI through an intermediate electron transfer chain, which comprises the so-called plastoquinone (PQ) pool, plastocyanin and/or cytochrome c553, and the cytochrome b6f complex. The redox potential of the PQ pool is clamped by the relative rates of electron release into and uptake from this pool. Within the PSII complex, electrons are extracted from water at the lumenal side of the thylakoid membrane and transferred to the primary accepting quinone (QA) at the stromal side. The electron is subsequently transferred to a PQ molecule in the secondary accepting quinone (QB) of PSII. The intermediate QB semiquinone, which is formed accordingly, is stable in the QB site for several seconds (Diner et al., 1991; Mitchell, 1993) and subsequently can be reduced to plastoquinol (PQH2). The midpoint potential of QA reduction is approximately −100 mV (Krieger-Liszkay and Rutherford, 1998; Allakhverdiev et al., 2011), whereas the corresponding midpoint potential of the QB semiquinone is close to zero (Nicholls and Ferguson, 2013). PQH2 equilibrates with the PQ pool in the thylakoid membranes, which has a size that is approximately 1 order of magnitude larger than the number of PSII reaction centers (Melis and Brown, 1980; Aoki and Katoh, 1983).PQ is a lipophilic, membrane-bound electron carrier, with a midpoint potential of +80 mV (Okayama, 1976), that can accept two electrons and two protons to form PQH2 (Rich and Bendall, 1980). PQH2 can donate both electrons to the cytochrome b6f complex, one to low-potential cytochrome b6, by which reduced high-potential cytochrome b6 is formed, and one to the cytochrome f moiety on the lumenal side of the thylakoid membrane, where the two protons are released. High-potential cytochrome b6 then donates an electron back to PQ on the stromal side of the membrane, rendering a semiquinone in the PQ-binding pocket on the cytoplasmic face of the b6f complex ready as an acceptor of another electron from PSII, and reduced cytochrome f feeds an electron to a water-soluble electron carrier (i.e. either plastocyanin or cytochrome c553) for subsequent transfer to the reaction center of PSI or to cytochrome c oxidase, respectively (Rich et al., 1991; Geerts et al., 1994; Schubert et al., 1995; Paumann et al., 2004; Mulkidjanian, 2010).Electron transfer through the cytochrome b6f complex proceeds according to the Q-cycle mechanism (Rich et al., 1991). As a result, maximally two protons from the stroma are released into the lumen per electron transferred. This electrochemical proton gradient can be used for the synthesis of ATP by the ATP synthase complex (Walker, 1998). In PSI, another transthylakoid membrane charge separation process is energized by light. Electron transfer within the PSI complex involves iron-sulfur clusters and quinones and leads to the reduction of ferredoxin, the reduced form of which serves as the electron donor for NADPH by the ferredoxin:NADP+ oxidoreductase enzyme (van Thor et al., 1999). The ATP and NADPH generated this way are used for CO2 fixation in a mutual stoichiometry that is close to the stoichiometry at which these two energy-rich compounds are formed at the thylakoid membrane. Normally, this ratio is ATP:NADPH = 3:2 (Behrenfeld et al., 2008).Photosynthetic and respiratory electron transport in cyanobacteria share a single PQ pool (Aoki and Katoh, 1983; Aoki et al., 1983; Matthijs et al., 1984; Scherer, 1990). Respiratory electron transfer provides cells the ability to form ATP in the dark, but this ability is not limited to those conditions. Transfer of electrons into the PQ pool is the result of the joint activity of PSII, respiratory dehydrogenases [in particular those specific for NAD(P)H and succinate], and cyclic electron transport around PSI (Mi et al., 1995; Cooley et al., 2000; Howitt et al., 2001;Yeremenko et al., 2005), whereas oxidation of PQH2 is catalyzed by the PQH2 oxidase, the cytochrome b6f complex, the respiratory cytochrome c oxidase (Nicholls et al., 1992; Pils and Schmetterer, 2001; Berry et al., 2002), and possibly plasma terminal oxidase (Peltier et al., 2010). Multiples of these partial reactions can proceed simultaneously, including respiratory electron transfer during illumination (Schubert et al., 1995), which includes oxygen uptake through a Mehler-like reaction (Helman et al., 2005; Allahverdiyeva et al., 2013).Because of its central location between the two photosystems, the redox state of the PQ pool has been identified as an important parameter that can signal photosynthetic imbalances (Mullineaux and Allen, 1990; Allen, 1995; Ma et al., 2010; Allen et al., 2011). Yet, an accurate estimation of the in vivo redox state of this pool has not been reported in cyanobacteria so far. Instead, the redox state of the PQ pool is widely assumed to be reflected in, or related to, the intensity of the chlorophyll a fluorescence emissions (Prasil et al., 1996; Yang et al., 2001; Gotoh et al., 2010; Houyoux et al., 2011). Imbalance in electron transport through the two photosystems may lead to a loss of excitation energy and, hence, to a loss of chlorophyll a fluorescence emission (Schreiber et al., 1986). Therefore, patterns of chlorophyll a fluorescence (pulse-amplitude modulated [PAM] fluorimetry; Baker, 2008) have widely been adopted for the analysis of (un)balanced photosynthetic electron transfer and, by inference, for indirect recording of the redox state of the PQ pool. However, the multitude of electron transfer pathways in the thylakoid membranes of cyanobacteria (see above) makes it much more complex to explain PAM signals in these organisms than in chloroplasts (Campbell et al., 1998). Additional regulatory mechanisms of nonphotochemical quenching, via the xanthophyll cycle in chloroplasts (Demmig-Adams et al., 2012) and the orange carotenoid protein (Kirilovsky and Kerfeld, 2012) in cyanobacteria, and energy redistribution via state transitions (Allen, 1995; Van Thor et al., 1998) complicate such comparisons even further.Several years ago, an HPLC-based technique was developed for the detection of the redox state of PQH2 in isolated thylakoids (Kruk and Karpinski, 2006), but these results have neither been related to physiological conditions nor to the results of chlorophyll a fluorescence measurements. In this report, we describe an adaptation of this method with elements of a method for estimation of the redox state of the ubiquinone pool in Escherichia coli (Bekker et al., 2007). This modified method allows for reliable measurements of the redox state of the PQ pool of Synechocystis sp. strain PCC 6803 under physiologically relevant conditions. The method uses rapid cell lysis in an organic solvent to arrest all physiological processes, followed by extraction and identification of PQH2 by HPLC separation with fluorescence detection. Next, we manipulated the redox state of the PQ pool with various redox-active agents, with inhibitors of photosynthetic electron flow, and by illumination with light specific for either PSII or PSI. The measured redox state of the PQ pool was then related to the chlorophyll a fluorescence signal and 77 K fluorescence emission spectra of cell samples taken in parallel and to oxygen-exchange rates measured separately. These experiments reveal that, despite highly fluctuating conditions of photosynthetic and respiratory electron flow, a remarkably stable redox state of the PQ pool is maintained. This homeostatically regulated redox state correlates poorly in many of the conditions tested with the more dynamic signal of chlorophyll a fluorescence emission, as measured with PAM fluorimetry. The latter signal only reflects the redox state of QA and not that of the PQ pool. 相似文献
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Susana RostroGarcía Jan F. Kamler Christin Minge Anthony Caragiulo Rachel Crouthers Milou Groenenberg Thomas N. E. Gray Visattha In Chanratana Pin Prum Sovanna Marc Kry David W. Macdonald 《Ecology and evolution》2021,11(9):4205
Dry deciduous dipterocarp forests (DDF) cover about 15%–20% of Southeast Asia and are the most threatened forest type in the region. The jungle cat (Felis chaus) is a DDF specialist that occurs only in small isolated populations in Southeast Asia. Despite being one of the rarest felids in the region, almost nothing is known about its ecology. We investigated the ecology of jungle cats and their resource partitioning with the more common leopard cats (Prionailurus bengalensis) in a DDF‐dominated landscape in Srepok Wildlife Sanctuary, Cambodia. We used camera‐trap data collected from 2009 to 2019 and DNA‐confirmed scats to determine the temporal, dietary and spatial overlap between jungle cats and leopard cats. The diet of jungle cats was relatively diverse and consisted of murids (56% biomass consumed), sciurids (15%), hares (Lepus peguensis; 12%), birds (8%), and reptiles (8%), whereas leopard cats had a narrower niche breadth and a diet dominated by smaller prey, primarily murids (73%). Nonetheless, dietary overlap was high because both felid species consumed predominantly small rodents. Both species were primarily nocturnal and had high temporal overlap. Two‐species occupancy modelling suggested jungle cats were restricted to DDF and had low occupancy, whereas leopard cats had higher occupancy and were habitat generalists. Our study confirmed that jungle cats are DDF specialists that likely persist in low numbers due to the harsh conditions of the dry season in this habitat, including annual fires and substantial decreases in small vertebrate prey. The lower occupancy and more diverse diet of jungle cats, together with the broader habitat use of leopard cats, likely facilitated the coexistence of these species. The low occupancy of jungle cats in DDF suggests that protection of large areas of DDF will be required for the long‐term conservation of this rare felid in Southeast Asia. 相似文献