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Ma C.,University of Leeds | Ma C.,Institute of Molecular and Experimental Medicine | Hao Z.,Nankai University | Hao Z.,University of Leeds | And 15 more authors.
PLoS ONE | Year: 2015

Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs. However, their investigation is hampered by difficulties in their production and purification on a scale suitable for structural studies. In particular, the nature and location of affinity tags introduced for the purification of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purification. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of divalent metal cation transporters and from the concentrative nucleoside transporter family. The proteins are expressed in E. coli in a correctly folded, functional state and can be purified in amounts suitable for structural investigations. © 2015 Ma et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Source

Gobat N.,University of Cardiff | Kinnersley P.,University of Cardiff | Gregory J.W.,Institute of Molecular and Experimental Medicine | Robling M.,University of Cardiff
Patient Education and Counseling | Year: 2015

Objective: To establish consensus on the core domains of agenda setting in consultations. Methods: We reviewed the healthcare literature and, using a modified Delphi technique to embrace both patient and clinician perspectives, conducted an iterative online survey, with 30 experts in health communication. Participants described agenda setting and rated the importance of proposed domains. Consensus was determined where the group median was ≥5 on a 7-point Likert-like response scale, and the interquartile range fell to within one point on this scale. Results: Relevant publications were identified in three overlapping bodies of healthcare literature. Survey respondents considered that agenda setting involved a process whereby patients and clinicians establish a joint focus for both their conversation and their working relationship. Consensus was obtained on six core domains: identifying patient talk topics, identifying clinician talk topics, agreement of shared priorities, establishing conversational focus, collaboration and engagement. New terminology - agenda mapping and agenda navigation - is proposed. Conclusion: We identified core agenda setting domains that embraced patient and clinician perspectives. Practice implications: An integrated conceptualization of agenda setting may now be used by researchers and educators in both clinician and patient focused interventions. © 2015 Elsevier Ireland Ltd. Source

Rees D.A.,Institute of Molecular and Experimental Medicine | Udiawar M.,Institute of Molecular and Experimental Medicine | Udiawar M.,University of Cardiff | Berlot R.,Kings College London | And 4 more authors.
Journal of Clinical Endocrinology and Metabolism | Year: 2016

Context: Polycystic ovary syndrome (PCOS) is a disorder characterized by insulin resistance and hyperandrogenism, which leads to an increased risk of type 2 diabetes in later life. Androgens and insulin signaling affect brain function but little is known about brain structure and function in younger adults with PCOS. Objective: To establish whether young women with PCOS display altered white matter microstructure and cognitive function. Patients, interventions, and main outcome measures: Eighteen individuals with PCOS (age, 31 = 6 y; body mass index [BMI] 30=6 kg/m2) and 18 control subjects (age, 31=7 y; BMI, 29=6 kg/m2), matched for age, IQ, and BMI, underwent anthropometric and metabolic evaluation, diffusion tensor MRI, a technique especially sensitive to brain white matter structure, and cognitive assessment. Cognitive scores and white matter diffusion metrics were compared between groups. White matter microstructure was evaluated across the whole white matter skeleton using tract-based spatial statistics. Associations with metabolic indices were also evaluated. Results: PCOS was associated with a widespread reduction in axial diffusivity (diffusion along the main axis of white matter fibers) and increased tissue volume fraction (the proportion of volume filled by white or grey matter rather than cerebrospinal fluid) in the corpus callosum. Cognitive performance was reduced compared with controls (first principal component, t = 2.9, P = .007), reflecting subtle decrements across a broad range of cognitive tests, despite similar education and premorbid intelligence. In PCOS, there was a reversal of the relationship seen in controls between brain microstructure and both androgens and insulin resistance. Conclusions: White matter microstructure is altered, and cognitive performance is compromised, in young adults with PCOS. These alterations in brain structure and function are independent of age, education and BMI. If reversible, these changes represent a potential target for treatment. Source

Lopes P.H.,Butantan Institute | Bertani R.,Butantan Institute | Goncalves-de-Andrade R.M.,Butantan Institute | Nagahama R.H.,Butantan Institute | And 2 more authors.
PLoS Neglected Tropical Diseases | Year: 2013

Background:The spider family Sicariidae includes two genera, Sicarius and Loxosceles. Bites by Sicarius are uncommon in humans and, in Brazil, a single report is known of a 17-year old man bitten by a Sicarius species that developed a necrotic lesion similar to that caused by Loxosceles. Envenomation by Loxosceles spiders can result in dermonecrosis and severe ulceration. Sicarius and Loxosceles spider venoms share a common characteristic, i.e., the presence of Sphingomyelinases D (SMase D). We have previously shown that Loxosceles SMase D is the enzyme responsible for the main pathological effects of the venom. Recently, it was demonstrated that Sicarius species from Africa, like Loxosceles spiders from the Americas, present high venom SMase D activity. However, despite the presence of SMase D like proteins in venoms of several New World Sicarius species, they had reduced or no detectable SMase D activity. In order to contribute to a better understanding about the toxicity of New World Sicarius venoms, the aim of this study was to characterize the toxic properties of male and female venoms from the Brazilian Sicarius ornatus spider and compare these with venoms from Loxosceles species of medical importance in Brazil.Methodology/Principal Findings:SDS-PAGE analysis showed variations in the composition of Loxosceles spp. and Sicarius ornatus venoms. Differences in the electrophoretic profiles of male and female venoms were also observed, indicating a possible intraspecific variation in the composition of the venom of Sicarius spider. The major component in all tested venoms had a Mr of 32-35 kDa, which was recognized by antiserum raised against Loxosceles SMases D. Moreover, male and female Sicarius ornatus spiders' venoms were able to hydrolyze sphingomyelin, thus showing an enzymatic activity similar to that determined for Loxosceles venoms. Sicarius ornatus venoms, as well as Loxosceles venoms, were able to render erythrocytes susceptible to lysis by autologous serum and to induce a significant loss of human keratinocyte cell viability; the female Sicarius ornatus venom was more efficient than male.Conclusion:We show here, for the first time, that the Brazilian Sicarius ornatus spider contains active Sphingomyelinase D and is able to cause haemolysis and keratinocyte cell death similar to the South American Loxosceles species, harmful effects that are associated with the presence of active SMases D. These results may suggest that envenomation by this Sicarius spider has the potential to cause similar pathological events as that caused by Loxosceles envenomation. Our results also suggest that, in addition to the interspecific differences, intraspecific variations in the venoms composition may play a role in the toxic potential of the New World Sicarius venoms species. © 2013 Lopes et al. Source

Chakraborty P.K.,Newborn Screening Ontario | Schmitz-Abe K.,Boston Childrens Hospital | Kennedy E.K.,University of Ottawa | Mamady H.,Newborn Screening Ontario | And 31 more authors.
Blood | Year: 2014

Mutationsingenes encoding proteins that are in volved in mitochondrial heme synthesis, iron-sulfur cluster biogenesis, and mitochondrial protein synthesis have previously been implicated in the pathogenesis of the congenital sideroblastic anemias (CSAs). We recently described a syndromic form of CSA associated with B-cell immunodeficiency, periodic fevers, and developmental delay (SIFD). Here we demonstrate that SIFD is caused by biallelic mutations in TRNT1, the gene encoding the CCA-adding enzyme essential for maturationofboth nuclear and mitochondrial transfer RNAs. Using budding yeast lacking the TRNT1 homolog, CCA1, we confirm that the patient-associated TRNT1 mutations resultin partial lossoffunction of TRNT1 and lead to metabolic defectsinboth the mitochondria and cytosol, which can account for the phenotypic pleiotropy. Copyright © 2011 by The American Society of Hematology; all rights reserved. Source

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