Functional characterisation of bacterial tripartite ATP-independent periplasmic (TRAP) transporters

Tripartite ATP-independent periplasmic (TRAP) transporters are extracytoplasmic solute receptor (ESR)-dependent secondary transporters that are widespread in bacteria, but not eukaryotes. TRAP transporters are composed of an ESR component and 2 unequally sized integral membrane components. The large membrane component has 12 predicted transmembrane helices (TMHs) and is thought to form the translocation channel. The small membrane component is composed of 4 TMHs and is of unknown, but essential function. TRAP transporters combine an ESR, which is normally associated with ATP-binding cassette (ABC) transporters, with the utilisation of electrochemical gradients across the membrane, using an undefined mechanism. Analysis of the data contained within the relational database, TRAPdb (www.trapdb.org), has revealed a number of new insights into TRAP transporter function, including the observation that marine-dwelling organisms have a propensity for high numbers of TRAP transporters - possibly indicating a role for Na+ in the transport cycle. Structural and functional analysis of TRAP transporters has been limited due to the recalcitrant nature of integral membrane proteins. The work presented here details the first overexpression and purification of the integral membrane proteins of a TRAP transporter. Using Escherichia coli and Lactococcus lactis expression systems, the integral membrane proteins from the E. coli TRAP transporter, YiaMNO, and the Haemophilus influenzae TRAP transporter, SiaPQM, have been expressed and purified. The entire sialic acid-specific TRAP transporter, SiaPQM, has been functionally reconstituted into liposomes. The energetic requirements of transport by SiaPQM have been elucidated revealing that a Na+ gradient in combination with an applied membrane potential are required to achieve maximal transport rates. Under normal conditions, SiaPQM is a unidirectional transporter, unlike most other secondary transporters, however, the presence of excess unliganded ESR is able to induce efflux of substrate from the proteoliposomes

Paediatric Injury from Powered Off-Road Vehicles

Background Powered off-road vehicles, such as quad-bikes and two-wheeled motorcycles, are popularly used recreationally by children, and are also used in a variety of farming and agricultural contexts. However, crashes can result in serious injuries and deaths, and as such are an important public health concern for Australian children. Compared to adults, children have different riding patterns, injury causes and risk factors relating to their level of development, as well as different patterns of injury outcomes and severity. Aims This work aims to investigate paediatric quad-bike and motorcycle riding patterns and behaviours, along with risk factors, injury severity, outcomes and potential avenues for injury prevention counter-measures. The work consists of five related studies. Methods and Results The first study was a survey of paediatric off-road vehicle riders, which was undertaken to characterise patterns of use, rider behaviours, experience and attitudes. Recreational motorcycle riding was most common, with children having high rates of previous riding experience and often participating in ‘structured’ riding within organised motorsports competitions and events. There was a small proportion of ‘unstructured’ riding in the context of agriculture or on private properties. Riders generally rode frequently and used helmets and other protective gear. The second study examined a group of children admitted to a paediatric hospital as a result of injury sustained from an off-road vehicle crash. In-depth crash investigation techniques examined injury mechanisms and related them to injury outcomes, along with rider, environmental and vehicular risk factors. A variety of recurring mechanisms emerged, particularly loss-of-control events leading to impacts with the ground or vehicle. A wide range of injuries were also observed, particularly to the extremities. A case-control component of the study identified a lower rider to vehicle weight ratio as a factor associated with higher crash risk. The third and fourth studies used large, linked population-level hospital admission and mortality datasets. The first of these examined injury epidemiology and outcomes for paediatric off-road vehicle crashes across New South Wales over a 17-year time period. The outcomes for different vehicle types were compared. The findings demonstrated the large burden of injury caused by ORV crashes in terms of hospital admissions and operative interventions. Furthermore, although on an individual level, quad-bike injuries were associated with higher injury severity, the far greater number of two-wheeled off-road motorcycle crash admissions suggest that motorcycles should be an injury prevention priority. The second linked data study examined the subset of children who have multiple hospital admissions following off-road vehicle crashes. Children who re-present to hospital repeatedly are a particularly vulnerable group, prone to more serious injury, and higher overall costs to the healthcare system. ‘Recidivist’ riders were compared to non-recidivists across various demographic, vehicle and injury outcome factors, highlighting the areas in which potential recidivists may be identified or targeted for injury prevention interventions. The fifth study was a systematic review of injury prevention countermeasures delivered through clinical environments such as hospitals and Emergency Departments, centred around the concept of the ‘Teachable Moment’: that children may be more receptive or amenable to behaviour change after sustaining an injury. The strengths and weaknesses of interventions that apply this model were identified, which may inform potential interventions pertaining to off-road crashes. The review found that multi-modal approaches combining face-to-face counselling or teaching, supplemented with other forms of written or visual communication, often paired with the provision of safety equipment and accurate monitoring as the most effective means of intervention. However, injury prevention programs are often limited by short-term and largely self-reported outcome measures, so research and programs applying these findings to off-road vehicle riders should be designed with robust design methods and appropriate measures of behaviour change, knowledge gain or injury reduction. Conclusion The results of the project overall demonstrate that off-road vehicle injuries are a large and important cause of injury, disability and death for Australian children. An overarching theme of this project is to demonstrate that children who participate in off-road riding are a unique and vulnerable population. Riding patterns are largely recreational, and the resultant injuries are common, spanning the spectrum of severity. Factors associated with worse injury outcomes and recidivism are explored, which, along with the review of injury prevention countermeasures delivered in clinical settings, may help re-prioritise and target injury prevention resources and research. Ultimately the goal of this project, as with any injury research, is to try to prevent or mitigate the severity off-road crashes. The studies investigate various aspects of paediatric off-road riding and provide important foundational knowledge for researchers and clinicians to work towards real-world applications and interventions

Rurality of Medical Provider and Race of Patient as Risk Factors for Overdose in Opioid Use Disorder Populations

Title: Rurality of medical provider and race of patient as risk factors for overdose in opioid use disorder populations Background This study examines the outcomes of medication assisted treatment (MAT) for opioid use disorders (OUD) based on location of treatment and race of the individual seeking treatment. Opioid use in the United States has been disproportionately rising in the last decade and there is evidence of unequal treatment based on different social disparities, namely rurality and race. Discriminatory distribution of medication and treatment for individuals seeking OUD along the lines of race and rurality is an issue of grave importance in both medical and ethical fields. Methods Individuals seeking treatment for OUD were identified and the use of MAT and/or mental health treatment (MHT) was analyzed. Data analysis of which course of treatment (MAT or MHT) were protective factors against overdose were analyzed. MAT most commonly used to treat OUD, buprenorphine, methadone, and naltrexone, were analyzed for risk of overdose following treatment seeking for OUD. Using administrative Medicaid data, persons with a first episode OUD diagnosis (N=9538) that were admitted to an emergency medical treatment facility in 2016-2018 and had no previous claims for OUD were identified using ICD 10 diagnostic codes. The number that initiated medication within 30 days of diagnosis were identified, separated by type of prescription: naltrexone=564, methadone=117, buprenorphine=2722, and no MAT=6187. Demographic factors (age, race, zip code of housing, and gender) were obtained from patient records, and pharmacy claims following OUD diagnosis were used to determine MAT group (i.e., buprenorphine, naltrexone, methadone, or no MAT). Emergency department and hospital claims were used to identify overdoses in the year following OUD diagnosis, and Cox regression was used to analyze risk of overdose. Proposed Hypothesis We predict that being treated for OUD in rural areas or being of non-white race is a leading factor of an individual recieving care that leads to overdose following treatment for being diagnosed with OUD

Mechanism of transport modulation by an extracellular loop in an archaeal excitatory amino acid transporter (EAAT) homolog

Secondary transporters in the excitatory amino acid transporter family terminate glutamatergic synaptic transmission by catalyzing Na(+)-dependent removal of glutamate from the synaptic cleft. Recent structural studies of the aspartate-specific archaeal homolog, Glt(Ph), suggest that transport is achieved by a rigid body, piston-like movement of the transport domain, which houses the substrate-binding site, between the extracellular and cytoplasmic sides of the membrane. This transport domain is connected to an immobile scaffold by three loops, one of which, the 3-4 loop (3L4), undergoes substrate-sensitive conformational change. Proteolytic cleavage of the 3L4 was found to abolish transport activity indicating an essential function for this loop in the transport mechanism. Here, we demonstrate that despite the presence of fully cleaved 3L4, Glt(Ph) is still able to sample conformations relevant for transport. Optimized reconstitution conditions reveal that fully cleaved Glt(Ph) retains some transport activity. Analysis of the kinetics and temperature dependence of transport accompanied by direct measurements of substrate binding reveal that this decreased transport activity is not due to alteration of the substrate binding characteristics but is caused by the significantly reduced turnover rate. By measuring solute counterflow activity and cross-link formation rates, we demonstrate that cleaving 3L4 severely and specifically compromises one or more steps contributing to the movement of the substrate-loaded transport domain between the outward- and inward-facing conformational states, sparing the equivalent step(s) during the movement of the empty transport domain. These results reveal a hitherto unknown role for the 3L4 in modulating an essential step in the transport process

Wolf 1130: A Nearby Triple System Containing a Cool, Ultramassive White Dwarf

Following the discovery of the T8 subdwarf WISEJ200520.38+542433.9 (Wolf 1130C), with common proper motion to a binary (Wolf 1130AB) consisting of an M subdwarf and a white dwarf, we set out to learn more about the old binary in the system. We find that the A and B components of Wolf 1130 are tidally locked, which is revealed by the coherence of more than a year of V band photometry phase folded to the derived orbital period of 0.4967 days. Forty new high-resolution, near-infrared spectra obtained with the Immersion Grating Infrared Spectrometer (IGRINS) provide radial velocities and a projected rotational velocity (v sin i) of 14.7 +/- 0.7 km/s for the M subdwarf. In tandem with a Gaia parallax-derived radius and verified tidal-locking, we calculate an inclination of i=29 +/- 2 degrees. From the single-lined orbital solution and the inclination we derive an absolute mass for the unseen primary (1.24+0.19-0.15 Msun). Its non-detection between 0.2 and 2.5 microns implies that it is an old (>3.7 Gyr) and cool (Teff<7000K) ONe white dwarf. This is the first ultramassive white dwarf within 25pc. The evolution of Wolf 1130AB into a cataclysmic variable is inevitable, making it a potential Type Ia supernova progenitor. The formation of a triple system with a primary mass >100 times the tertiary mass and the survival of the system through the common-envelope phase, where ~80% of the system mass was lost, is remarkable. Our analysis of Wolf 1130 allows us to infer its formation and evolutionary history, which has unique implications for understanding low-mass star and brown dwarf formation around intermediate mass stars.Comment: 37 pages, 9 Figures, 5 Table

Flipping the switch: dynamic modulation of membrane transporter activity in bacteria

The controlled entry and expulsion of small molecules across the bacterial cytoplasmic membrane is essential for efficient cell growth and cellular homeostasis. While much is known about the transcriptional regulation of genes encoding transporters, less is understood about how transporter activity is modulated once the protein is functional in the membrane, a potentially more rapid and dynamic level of control. In this review, we bring together literature from the bacterial transport community exemplifying the extensive and diverse mechanisms that have evolved to rapidly modulate transporter function, predominantly by switching activity off. This includes small molecule feedback, inhibition by interaction with small peptides, regulation through binding larger signal transduction proteins and, finally, the emerging area of controlled proteolysis. Many of these examples have been discovered in the context of metal transport, which has to finely balance active accumulation of elements that are essential for growth but can also quickly become toxic if intracellular homeostasis is not tightly controlled. Consistent with this, these transporters appear to be regulated at multiple levels. Finally, we find common regulatory themes, most often through the fusion of additional regulatory domains to transporters, which suggest the potential for even more widespread regulation of transporter activity in biology

Short-Term Cardiac and Noncardiac Mortality Following Liver Transplantation

Objectives. To determine the importance of acute cardiac events as a cause of mortality compared to non-cardiac events in the four month period following liver transplantation (LT) using current preoperative cardiac screening strategies. Patients and Methods. We retrospectively reviewed timing, type, and outcome of adverse cardiac events, and all cause mortality in the 4 month postoperative period in 393 consecutive LT patients from October 1999 to February 2008. Results. Of 30 total deaths (7.6% overall mortality rate), 27 (90%) were due to surgical or medical complications and 3 (10%) were primary cardiac deaths (0.8% cardiac mortality rate). Acute cardiac events occurred in 26 patients (6.6%), including 13 arrhythmias (50%), 7 new onset heart failures (27%), and 6 myocardial infarctions (23%). Twelve of 13 intraoperative events were arrhythmias (92%) including two of three cardiac deaths. Conclusions. Using current preoperative screening recommendations, deaths from primary cardiac events within four months of LT are very uncommon (0.8%), especially compared with deaths related to medical and surgical complications (6.9%)

Frontal alpha asymmetry in response to stressor moderates the relation between parenting hassles and child externalizing problems

Inequitable urban environments are associated with toxic stress and altered neural social stress processing that threatens the development of self-regulation. Some children in these environments struggle with early onset externalizing problems that are associated with a variety of negative long-term outcomes. While previous research has linked parenting daily hassles to child externalizing problems, the role of frontal alpha asymmetry (FAA) as a potential modifier of this relationship has scarcely been explored. The present study examined mother-child dyads, most of whom were living in low socioeconomic status households in an urban environment and self-identified as members of racial minority groups. Analyses focused on frustration task electroencephalography (EEG) data from 67 children (mean age = 59.0 months, SD = 2.6). Mothers reported the frequency of their daily parenting hassles and their child’s externalizing problems. Frustration task FAA moderated the relationship between parenting daily hassles and child externalizing problems, but resting FAA did not. More specifically, children with left frontal asymmetry had more externalizing problems as their mothers perceived more hassles in their parenting role, but parenting hassles and externalizing problems were not associated among children with right frontal asymmetry. These findings lend support to the motivational direction hypothesis and capability model of FAA. More generally, this study reveals how individual differences in lateralization of cortical activity in response to a stressor may confer differential susceptibility to child behavioral problems with approach motivation (i.e., left frontal asymmetry) predicting externalizing problems under conditions of parental stress