Functional analyses reveal an important role for tyrosine residues in the staphylococcal multidrug efflux protein QacA

<p>Abstract</p> <p>Background</p> <p>The staphylococcal QacA multidrug efflux protein confers resistance to an exceptional number of structurally unrelated antimicrobial compounds. Aromatic amino acid residues have been shown to be highly important for the transport function of several multidrug transporters and are intimately involved in multidrug binding. This study investigated the structural and functional importance of the seven tyrosine residues in QacA by examining the phenotypic effect of incorporating conservative (aromatic) and non-conservative (non-aromatic) substitutions for these residues.</p> <p>Results</p> <p>Determination of the resistance profiles and analysis of drug transport assays revealed that non-conservative substitutions for most tyrosine residues influenced the QacA drug recognition spectrum. However, an aromatic residue at three tyrosine positions, 63, 410 and 429, was of importance for QacA-mediated transport and resistance to the majority of substrates tested.</p> <p>Conclusion</p> <p>A tyrosine or phenylalanine residue at amino acid positions corresponding to 63 of QacA in related drug efflux proteins is found to be highly conserved. Therefore, an aromatic side chain at this position is likely to partake in a function common to these drug transporters, such as proton translocation or essential intramolecular contacts, whereas aromatic residues at the non-conserved 410 and 429 positions are expected to mediate a QacA-specific function, possibly forming or stabilising part of the QacA drug binding region.</p

Homozygous in-frame deletion in CATSPERE in a man producing spermatozoa with loss of CatSper function and compromised fertilizing capacity

STUDY QUESTIONDoes a man (patient 1) with a previously described deficiency in principle cation channel of sperm (CatSper) function have a mutation in the CatSper-epsilon (CATSPERE) and/or CatSper-zeta (CATSPERZ) gene?SUMMARY ANSWERPatient 1 has a homozygous in-frame 6-bp deletion in exon 18 (c.2393_2398delCTATGG, rs761237686) of CATSPERE.WHAT IS KNOWN ALREADYCatSper is the principal calcium channel of mammalian spermatozoa. Spermatozoa from patient 1 had a specific loss of CatSper function and were unable to fertilize at IVF. Loss of CatSper function could not be attributed to genetic abnormalities in coding regions of seven CatSper subunits. Two additional subunits (CatSper-epsilon (CATPSERE) and CatSper-zeta (CATSPERZ)) were recently identified, and are now proposed to contribute to the formation of the mature channel complex.STUDY DESIGN, SIZE, DURATIONThis was a basic medical research study analysing genomic data from a single patient (patient 1) for defects in CATSPERE and CATSPERZ.PARTICIPANTS/MATERIALS, SETTING, METHODSThe original exome sequencing data for patient 1 were analysed for mutations in CATSPERE and CATSPERZ. Sanger sequencing was conducted to confirm the presence of a rare variant.MAIN RESULTS AND THE ROLE OF CHANCEPatient 1 is homozygous for an in-frame 6-bp deletion in exon 18 (c.2393_2398delCTATGG, rs761237686) of CATSPERE that is predicted to be highly deleterious.LIMITATIONS, REASONS FOR CAUTIONThe nature of the molecular deficit caused by the rs761237686 variant and whether it is exclusively responsible for the loss of CatSper function remain to be elucidated.WIDER IMPLICATIONS OF THE FINDINGSPopulation genetics are available for a significant number of predicted deleterious variants of CatSper subunits. The consequence of homozygous and compound heterozygous forms on sperm fertilization potential could be significant. Selective targeting of CatSper subunit expression maybe a feasible strategy for the development of novel contraceptives.STUDY FUNDING/COMPETING INTEREST(S)This study was funded by project grants from the MRC (MR/K013343/1 and MR/012492/1), Chief Scientist Office/NHS research Scotland. This work was also supported by NIH R01GM111802, Pew Biomedical Scholars Award 00028642 and Packer Wentz Endowment Will to P.V.L. C.L.R.B is the editor-in-chief of Molecular Human Reproduction, has received lecturing fees from Merck and Ferring, and is on the Scientific Advisory Panel for Ohana BioSciences. C.L.R.B was chair of the World Health Organization Expert Synthesis Group on Diagnosis of Male infertility (2012–2016)

Diversity and Function of Capsular Polysaccharide in Acinetobacter baumannii

The Gram-negative opportunistic bacterium Acinetobacter baumannii is a significant cause of hospital-borne infections worldwide. Alarmingly, the rapid development of antimicrobial resistance coupled with the remarkable ability of isolates to persist on surfaces for extended periods of time has led to infiltration of A. baumannii into our healthcare environments. A major virulence determinant of A. baumannii is the presence of a capsule that surrounds the bacterial surface. This capsule is comprised of tightly packed repeating polysaccharide units which forms a barrier around the bacterial cell wall, providing protection from environmental pressures including desiccation and disinfection regimes as well as host immune responses such as serum complement. Additionally, capsule has been shown to confer resistance to a range of clinically relevant antimicrobial compounds. Distressingly, treatment options for A. baumannii infections are becoming increasingly limited, and the urgency to develop effective infection control strategies and therapies to combat infections is apparent. An increased understanding of the contribution of capsule to the pathobiology of A. baumannii is required to determine its feasibility as a target for new strategies to combat drug resistant infections. Significant variation in capsular polysaccharide structures between A. baumannii isolates has been identified, with over 100 distinct capsule types, incorporating a vast variety of sugars. This review examines the studies undertaken to elucidate capsule diversity and advance our understanding of the role of capsule in A. baumannii pathogenesis

Speaking out about gender imbalance in invited speakers improves diversity.

Omissions of qualified women scientists from major meeting programs continue to occur despite a surge in articles indicating persistent gender-discriminatory practices in hiring and promotion, and calls for gender balance in conference organizing committees

Reflecting on loss in Papua New Guinea

This article takes up the conundrum of conducting anthropological fieldwork with people who claim that they have 'lost their culture,' as is the case with Suau people in the Massim region of Papua New Guinea. But rather than claiming culture loss as a process of dispossession, Suau claim it as a consequence of their own attempts to engage with colonial interests. Suau appear to have responded to missionization and their close proximity to the colonial-era capital by jettisoning many of the practices characteristic of Massim societies, now identified as 'kastom.' The rejection of kastom in order to facilitate their relations with Europeans during colonialism, followed by the mourning for kastom after independence, both invite consideration of a kind of reflexivity that requires action based on the presumed perspective of another

Molecular-orbital-free algorithm for excited states in time-dependent perturbation theory

A non-linear conjugate gradient optimization scheme is used to obtain excitation energies within the Random Phase Approximation (RPA). The solutions to the RPA eigenvalue equation are located through a variational characterization using a modified Thouless functional, which is based upon an asymmetric Rayleigh quotient, in an orthogonalized atomic orbital representation. In this way, the computational bottleneck of calculating molecular orbitals is avoided. The variational space is reduced to the physically-relevant transitions by projections. The feasibility of an RPA implementation scaling linearly with system size, N, is investigated by monitoring convergence behavior with respect to the quality of initial guess and sensitivity to noise under thresholding, both for well- and ill-conditioned problems. The molecular- orbital-free algorithm is found to be robust and computationally efficient providing a first step toward a large-scale, reduced complexity calculation of time-dependent optical properties and linear response. The algorithm is extensible to other forms of time-dependent perturbation theory including, but not limited to, time-dependent Density Functional theory.Comment: 9 pages, 7 figure

Mechanisms underlying the exquisite sensitivity of Candida albicans to combinatorial cationic and oxidative stress that enhances the potent fungicidal activity of phagocytes

Copyright © 2014 Kaloriti et al.Peer reviewedPublisher PD

Total Body Irradiation–Based Myeloablative Haploidentical Stem Cell Transplantation Is a Safe and Effective Alternative to Unrelated Donor Transplantation in Patients Without Matched Sibling Donors

AbstractWe enrolled 30 patients on a prospective phase II trial utilizing a total body irradiation (TBI)–based myeloablative preparative regimen (fludarabine 30 mg/m2/day × 3 days and TBI 150 cGy twice per day on day -4 to -1 [total dose 1200 cGy]) followed by infusion of unmanipulated peripheral blood stem cells from a haploidentical family donor (haplo). Postgrafting immunosuppression consisted of cyclophosphamide 50 mg/kg/day on days 3 and 4, mycophenolate mofetil through day 35, and tacrolimus through day 180. Median patient age was 46.5 years (range, 24 to 60). Transplantation diagnosis included acute myelogenous leukemia (n = 16), acute lymphoblastic leukemia (n = 6), chronic myelogenous leukemia (n = 5), myelodysplastic syndrome (n = 1), and non-Hodgkin's lymphoma (n = 2). Using the Dana Farber/Center for International Blood and Marrow Transplant Research/Disease Risk Index (DRI), patients were classified as low (n = 4), intermediate (n = 12), high (n = 11), and very high (n = 3) risk. All patients engrafted with a median time to neutrophil and platelet recovery of 16 and 25 days, respectively. All evaluable patients achieved sustained complete donor T cell and myeloid chimerism by day +30. Acute graft-versus-host disease (GVHD) grades II to IV and III and IV was seen in 43% and 23%, respectively. The cumulative incidence of chronic GVHD was 56% (severe in 10%). After a median follow-up of 24 months, the estimated 2-year overall survival (OS), disease-free survival (DFS), nonrelapse mortality, and relapse rate were 78%, 73%, 3%, and 24%, respectively. Two-year DFS and relapse rate in patients with low/intermediate risk disease was 100% and 0%, respectively, compared with 39% and 53% for patients with high/very high risk disease. When compared with a contemporaneously treated cohort of patients at our institution receiving myeloablative HLA-matched unrelated donor (MUD) transplantation (acute myelogenous leukemia [n = 17], acute lymphoblastic leukemia [n = 15], chronic myelogenous leukemia [n = 7], myelodysplastic syndrome [n = 7], non-Hodgkin lymphoma [n = 1], chronic lymphoblastic leukemia [n = 1]), outcomes were statistically similar, with 2-yr OS and DFS being 78% and 73%, respectively after haplo transplantation versus 71% and 64%, respectively, after MUD transplantation. In patients with DRI low/intermediate risk disease, 2-yr DFS was superior after haplo compared with MUD transplantations (100% versus 74%, P = .032), whereas there was no difference in DFS in patients with high/very high risk disease (39% versus 37% for haplo and MUD respectively, P = .821). Grade II to IV acute GVHD was seen less often after haplo compared with MUD transplantation (43% versus 63%, P = .049), as was moderate-to-severe chronic GVHD (22% versus 58%, P = .003). Myeloablative haplo transplantation using this regimen is a valid option for patients with advanced hematologic malignancies who lack timely access to a conventional donor. Outcomes appear at least equivalent to those seen in contemporaneous patients who underwent transplantation from MUD