Broad Ligand Specificity in the Small Multidrug Resistance Transporter EmrE

EmrE, an E. coli small multidrug resistance transporter, effluxes a diverse range of toxic polyaromatic cations, thus imparting resistance to drug compounds of this type. Transporters must interconvert between inward- and outward-facing structures during the transport cycle to alternate access of its binding site between the two sides of the membrane. As a secondary active antiporter, EmrE must couple drug binding to this conformational exchange and its energy source, the proton gradient across the inner membrane. The transport cycle involves the import of two protons to drive the export of one substrate molecule. As a multidrug resistance transporter, EmrE has particularly broad specificity and a large variation in affinities for these transported substrates, yet all substrates must trigger the same conformational change between inward- and outward-facing states in order for transport to occur. My research focuses on this coupling between substrate binding and conformational exchange and the functional outcome of this coupling, transport. As one of the smallest transporters, EmrE serves as an ideal system to study the minimal requirements for multidrug recognition and transport. EmrE transports polyaromatic cations that vary in geometry (i.e. planar vs. tetrahedral), charge (i.e. +1 vs. +2), and overall size. By combining a variety of biophysical techniques, I have investigated the thermodynamics and kinetics of broad substrate recognition and the conformational interconversion that it triggers. In particular, I have focused on two ligand series: i) a series of related tetrahedral ligands with an identical charge of +1, and ii) a series of planar ligands that vary in size and charge. EmrE binds these substrates with affinities that span several orders of magnitude and transports them with varying efficiency. We have directly monitored conformational exchange in substrate-bound EmrE using ZZ-exchange NMR spectroscopy and found that the rate of this key step in transport varies considerably with the identity of the bound ligand. These studies have interesting implications for the energetics of the transport cycle. Substrate binding alters both the ground and transition state energies of EmrE. A common theme has emerged in the literature suggesting that enzymes pre-sample their functional motions in the absence of substrate. However, to be a functional antiporter, EmrE cannot interconvert in the absence of substrate. Thus, the protein alone cannot determine the barrier to exchange between the inward- and outward-facing states, and substrate must play an important role

EmrE dimerization depends on membrane environment

AbstractThe small multi-drug resistant (SMR) transporter EmrE functions as a homodimer. Although the small size of EmrE would seem to make it an ideal model system, it can also make it challenging to work with. As a result, a great deal of controversy has surrounded even such basic questions as the oligomeric state. Here we show that the purified protein is a homodimer in isotropic bicelles with a monomer–dimer equilibrium constant (KMD2D) of 0.002–0.009mol% for both the substrate-free and substrate-bound states. Thus, the dimer is stabilized in bicelles relative to detergent micelles where the KMD2D is only 0.8–0.95mol% (Butler et al. 2004). In dilauroylphosphatidylcholine (DLPC) liposomes KMD2D is 0.0005–0.0008mol% based on Förster resonance energy transfer (FRET) measurements, slightly tighter than bicelles. These results emphasize the importance of the lipid membrane in influencing dimer affinity

Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells

Neural crest stem cells (NCSCs) persist in peripheral nerves throughout late gestation but their function is unknown. Current models of nerve development only consider the generation of Schwann cells from neural crest, but the presence of NCSCs raises the possibility of multilineage differentiation. We performed Cre-recombinase fate mapping to determine which nerve cells are neural crest derived. Endoneurial fibroblasts, in addition to myelinating and non-myelinating Schwann cells, were neural crest derived, whereas perineurial cells, pericytes and endothelial cells were not. This identified endoneurial fibroblasts as a novel neural crest derivative, and demonstrated that trunk neural crest does give rise to fibroblasts in vivo, consistent with previous studies of trunk NCSCs in culture. The multilineage differentiation of NCSCs into glial and non-glial derivatives in the developing nerve appears to be regulated by neuregulin, notch ligands, and bone morphogenic proteins, as these factors are expressed in the developing nerve, and cause nerve NCSCs to generate Schwann cells and fibroblasts, but not neurons, in culture. Nerve development is thus more complex than was previously thought, involving NCSC self-renewal, lineage commitment and multilineage differentiation

Randomized controlled trial of a primary care–based screening program to identify older women with prevalent osteoporotic vertebral fractures: Cohort for skeletal health in Bristol and Avon (COSHIBA)

Approximately 12% of postmenopausal women have osteoporotic vertebral fractures (VFs); these are associated with excess morbidity and mortality and a high risk of future osteoporotic fractures. Despite this, less than one-third come to clinical attention, partly due to lack of clear clinical triggers for referral for spinal radiographs. The aim of this study was to investigate whether a novel primary care–based screening tool could be used to identify postmenopausal women with osteoporotic VFs and increase appropriate management of osteoporosis. A randomized controlled trial was undertaken in 15 general practices within the Bristol area of the UK. A total of 3200 women aged 65 to 80 years were enrolled, with no exclusion criteria. A simple screening tool was carried out by a nurse in primary care to identify women at high risk of osteoporotic VFs. All identified high-risk women were offered a diagnostic thoracolumbar radiograph. Radiographs were reported using standard National Health Service (NHS) reporting, with results sent back to each participant's general practitioner (GP). Participants in the control arm did not receive the screening tool or radiographs. The main outcome measure was self-reported prescription of medication for osteoporosis at 6 months with a random 5% subsample verified against electronic GP records. Secondary outcome was self-reported incidence of new fractures. Results showed that allocation to screening increased prescription of osteoporosis medications by 124% (odds ratio [OR] for prescription 2.24 at 6 months; 95% confidence interval [CI], 1.16 to 4.33). Allocation to screening also reduced fracture incidence at 12-month follow-up (OR for new fracture 0.60; 95% CI, 0.35–1.03; p = 0.063), although this did not reach statistical significance. This study supports the use of a simple screening tool administered in primary care to increase appropriate prescription of medications for osteoporosis in postmenopausal women in the UK. © 2012 American Society for Bone and Mineral Researc

Multi trace element profiling in pathogenic and non-pathogenic fungi

Acknowledgements SW and EM were funded by an MRC NIRG to AB (G0900211/90671). AP was funded by a British Mycological Society Summer Studentship. AB was funded by a Royal Society URF (UF080611) and a Senior Wellcome Research Fellowship (206412/A/17/Z), which also funded TB. DW was funded by a Senior Wellcome Research Fellowship (214317/A/18/Z). The work was carried out in the MRC Centre for Medical Mycology (MR/N006364/2). This article is part of the Fungal Adaptation to Hostile Challenges special issue for the third International Symposium on Fungal Stress (ISFUS), which is supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo grant 2018/20571-6 and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior grant 88881.289327/2018-01.Peer reviewedproofPublisher PD

Prospectus, February 13, 2020

THE SCIENCE OF LOVE; Love is in the air; Perimeter Road to host second annual music festival this May; Letter to the editor; Jacarra Lee receives Outstanding Black Student Award; Study abroad deadlines quickly approaching; Black History Month figure; Arthur Ashe; Support soars for Illini Basketballhttps://spark.parkland.edu/prospectus_2020/1003/thumbnail.jp

Wicked Urban Challenges in Western Sydney: Researchers Respond

The purpose of this publication is to provide critical insights and perspectives around how to tackle four of Western Sydney’s wicked urban challenges, and ensure our region is prepared for the future, namely: job/housing imbalances and inadequate infrastructure investment; declining housing affordability; cultural infrastructure disparities; extreme urban heat. Our aim is that this publication continues the debate generated in the online forum, ‘Wicked urban challenges in Western Sydney: researchers respond’, held in October 2021. The event was sponsored by Western Sydney University (WSU). The university is a modern, forward-thinking, research-led university, located at the heart of the Western Sydney region. Boasting 12 campuses (many in CBD locations) and more than 170,000 alumni, 48,000 students and 3,000 staff, the university has 14 Schools with an array of well-designed programs and courses carefully structured to meet the demands of future industry. The event was organised through the University’s Urban Living Futures and Society Research Theme and formed part of the University’s 2021 Research Week, called ‘Bold Research Futures’. This theme had real resonance with what was discussed that day. Over 160 people attended this highly interactive forum, right across the built environment profession and other key professions. The invitation, however, had gone wider, to many people living and working in Western Sydney and beyond. The event brought together our researchers, government, industry, and our local community to challenge conventional policy thinking and offer new ways to solve these four wicked urban challenges in Western Sydney (as outlined above). The remainder of this report provides a summary of four of WSU’s leading urban researchers’ presentations, as delivered on the day. Each of the academics draw from the strategic programs of work being carried out by multi-disciplinary teams across our university. Each brings fresh perspectives and insights to our understanding of the challenges that Western Sydney faces and offers bold policy solutions and initiatives

Functional gene analysis suggests different acetogen populations in the Bovine Rumen and Tammar Wallaby Forestomach

Reductive acetogenesis via the acetyl coenzyme A (acetyl-CoA) pathway is an alternative hydrogen sink to methanogenesis in the rumen. Functional gene-based analysis is the ideal approach for investigating organisms capable of this metabolism (acetogens). However, existing tools targeting the formyltetrahydrofolate synthetase gene (fhs) are compromised by lack of specificity due to the involvement of formyltetrahydrofolate synthetase (FTHFS) in other pathways. Acetyl-CoA synthase (ACS) is unique to the acetyl-CoA pathway and, in the present study, acetyl-CoA synthase genes (acsB) were recovered from a range of acetogens to facilitate the design of acsB-specific PCR primers. fhs and acsB libraries were used to examine acetogen diversity in the bovine rumen and forestomach of the tammar wallaby (Macropus eugenii), a native Australian marsupial demonstrating foregut fermentation analogous to rumen fermentation but resulting in lower methane emissions. Novel, deduced amino acid sequences of acsB and fhs affiliated with the Lachnospiraceae in both ecosystems and the Ruminococcaeae/Blautia group in the rumen. FTHFS sequences that probably originated from nonacetogens were identified by low "homoacetogen similarity" scores based on analysis of FTHFS residues, and comprised a large proportion of FTHFS sequences from the tammar wallaby forestomach. A diversity of FTHFS and ACS sequences in both ecosystems clustered between the Lachnospiraceae and Clostridiaceae acetogens but without close sequences from cultured isolates. These sequences probably originated from novel acetogens. The community structures of the acsB and fhs libraries from the rumen and the tammar wallaby forestomach were different (LIBSHUFF, P < 0.001), and these differences may have significance for overall hydrogenotrophy in both ecosystems

Prospectus, March 5, 2020

THE RETIRED; Parkland\u27s LEED scorecard explained; Career closet coming to Parkland; Student actors and designers showcased in Machinal ; Student Leadership Academy proving successful; Sustainable style; sayonara winter, salutations spring; Get ready for some March Madness; Evodie Tshipamba Receives Outstanding Black Student Award; The women of Machinalhttps://spark.parkland.edu/prospectus_2020/1004/thumbnail.jp