Deep Mutational Scanning of SARS-CoV-2 Receptor Binding Domain Reveals Constraints on Folding and ACE2 Binding.

The receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein mediates viral attachment to ACE2 receptor and is a major determinant of host range and a dominant target of neutralizing antibodies. Here, we experimentally measure how all amino acid mutations to the RBD affect expression of folded protein and its affinity for ACE2. Most mutations are deleterious for RBD expression and ACE2 binding, and we identify constrained regions on the RBD's surface that may be desirable targets for vaccines and antibody-based therapeutics. But a substantial number of mutations are well tolerated or even enhance ACE2 binding, including at ACE2 interface residues that vary across SARS-related coronaviruses. However, we find no evidence that these ACE2-affinity-enhancing mutations have been selected in current SARS-CoV-2 pandemic isolates. We present an interactive visualization and open analysis pipeline to facilitate use of our dataset for vaccine design and functional annotation of mutations observed during viral surveillance.

Quadrivalent influenza nanoparticle vaccines induce broad protection.

Influenza vaccines that confer broad and durable protection against diverse viral strains would have a major effect on global health, as they would lessen the need for annual vaccine reformulation and immunization1. Here we show that computationally designed, two-component nanoparticle immunogens2 induce potently neutralizing and broadly protective antibody responses against a wide variety of influenza viruses. The nanoparticle immunogens contain 20 haemagglutinin glycoprotein trimers in an ordered array, and their assembly in vitro enables the precisely controlled co-display of multiple distinct haemagglutinin proteins in defined ratios. Nanoparticle immunogens that co-display the four haemagglutinins of licensed quadrivalent influenza vaccines elicited antibody responses in several animal models against vaccine-matched strains that were equivalent to or better than commercial quadrivalent influenza vaccines, and simultaneously induced broadly protective antibody responses to heterologous viruses by targeting the subdominant yet conserved haemagglutinin stem. The combination of potent receptor-blocking and cross-reactive stem-directed antibodies induced by the nanoparticle immunogens makes them attractive candidates for a supraseasonal influenza vaccine candidate with the potential to replace conventional seasonal vaccines3.

Improving the secretion of designed protein assemblies through negative design of cryptic transmembrane domains.

Computationally designed protein nanoparticles have recently emerged as a promising platform for the development of new vaccines and biologics. For many applications, secretion of designed nanoparticles from eukaryotic cells would be advantageous, but in practice, they often secrete poorly. Here we show that designed hydrophobic interfaces that drive nanoparticle assembly are often predicted to form cryptic transmembrane domains, suggesting that interaction with the membrane insertion machinery could limit efficient secretion. We develop a general computational protocol, the Degreaser, to design away cryptic transmembrane domains without sacrificing protein stability. The retroactive application of the Degreaser to previously designed nanoparticle components and nanoparticles considerably improves secretion, and modular integration of the Degreaser into design pipelines results in new nanoparticles that secrete as robustly as naturally occurring protein assemblies. Both the Degreaser protocol and the nanoparticles we describe may be broadly useful in biotechnological applications.

Energy Expenditure of Elite Male and Female Professional Tennis Players During Habitual Training.

Understanding the daily energy expenditure of athletes during training is important to support recovery, adaptation, and the maintenance of performance. The aim of the current research was to assess the total daily energy expenditure (TDEE) and the acute energy expenditure (EE) of tennis training sessions during habitual training of elite tennis players. Using a cohort study design, 27 (n = 10, male; age; 22.3 ± 3.2 years and n = 17, female; age: 23.8 ± 3.5 years) elite singles tennis players were assessed for TDEE and tennis training EE. Using Actiheart activity monitors during a 2- to 5-day training period, male players were analyzed for 26 days and 33 (1.3 ± 0.5 sessions/day) tennis training sessions, and female players for 43 days and 58 (1.2 ± 0.4 sessions/day) tennis training sessions. Male TDEE (4,708 ± 583 kcal/day) was significantly higher than female (3,639 ± 305 kcal/day). Male absolute and relative tennis training EEs (10.2 ± 2.3 kcal/min and 7.9 ± 1.4 kcal·hr-1·kg-1) were significantly higher than those of females (7.6 ± 1.0 kcal/min and 6.8 ± 0.9 kcal·hr-1·kg-1). The resting metabolic rate was assessed via indirect calorimetry. The physical activity level for both groups was 2.3 AU. The TDEE of male and female players during habitual training now highlights the continual cycle of high energy demands experienced by the elite tennis player. The broad ranges of TDEE and EE reported here suggest individual assessment and nutritional planning be prioritized, with a particular focus on carbohydrate requirements.

Evolution of a designed protein assembly encapsulating its own RNA genome.

The challenges of evolution in a complex biochemical environment, coupling genotype to phenotype and protecting the genetic material, are solved elegantly in biological systems by the encapsulation of nucleic acids. In the simplest examples, viruses use capsids to surround their genomes. Although these naturally occurring systems have been modified to change their tropism and to display proteins or peptides, billions of years of evolution have favoured efficiency at the expense of modularity, making viral capsids difficult to engineer. Synthetic systems composed of non-viral proteins could provide a 'blank slate' to evolve desired properties for drug delivery and other biomedical applications, while avoiding the safety risks and engineering challenges associated with viruses. Here we create synthetic nucleocapsids, which are computationally designed icosahedral protein assemblies with positively charged inner surfaces that can package their own full-length mRNA genomes. We explore the ability of these nucleocapsids to evolve virus-like properties by generating diversified populations using Escherichia coli as an expression host. Several generations of evolution resulted in markedly improved genome packaging (more than 133-fold), stability in blood (from less than 3.7% to 71% of packaged RNA protected after 6 hours of treatment), and in vivo circulation time (from less than 5 minutes to approximately 4.5 hours). The resulting synthetic nucleocapsids package one full-length RNA genome for every 11 icosahedral assemblies, similar to the best recombinant adeno-associated virus vectors. Our results show that there are simple evolutionary paths through which protein assemblies can acquire virus-like genome packaging and protection. Considerable effort has been directed at 'top-down' modification of viruses to be safe and effective for drug delivery and vaccine applications; the ability to design synthetic nanomaterials computationally and to optimize them through evolution now enables a complementary 'bottom-up' approach with considerable advantages in programmability and control.

Active site architecture reveals coordination sphere flexibility and specificity determinants in a group of closely related molybdoenzymes.

MtsZ is a molybdenum-containing methionine sulfoxide reductase that supports virulence in the human respiratory pathogen Haemophilus influenzae (Hi). HiMtsZ belongs to a group of structurally and spectroscopically uncharacterized S-/N-oxide reductases, all of which are found in bacterial pathogens. Here, we have solved the crystal structure of HiMtsZ, which reveals that the HiMtsZ substrate-binding site encompasses a previously unrecognized part that accommodates the methionine sulfoxide side chain via interaction with His182 and Arg166. Charge and amino acid composition of this side chain-binding region vary and, as indicated by electrochemical, kinetic, and docking studies, could explain the diverse substrate specificity seen in closely related enzymes of this type. The HiMtsZ Mo active site has an underlying structural flexibility, where dissociation of the central Ser187 ligand affected catalysis at low pH. Unexpectedly, the two main HiMtsZ electron paramagnetic resonance (EPR) species resembled not only a related dimethyl sulfoxide reductase but also a structurally unrelated nitrate reductase that possesses an Asp-Mo ligand. This suggests that contrary to current views, the geometry of the Mo center and its primary ligands, rather than the specific amino acid environment, is the main determinant of the EPR properties of mononuclear Mo enzymes. The flexibility in the electronic structure of the Mo centers is also apparent in two of three HiMtsZ EPR-active Mo(V) species being catalytically incompetent off-pathway forms that could not be fully oxidized.

Combining physics-based and Kriging models to improve the estimation of noise exposure.

Worker exposure to occupational hazards is traditionally measured by equipping workers with wearable exposure monitors. An emerging alternative measurement first generates time-varying hazard maps from permanent monitors within the facility, then estimating worker exposure by integrating hazard levels traversed in map, following the tracked movement of workers. Complex environments may require many monitors to produce a hazard map with the necessary accuracy, but effective interpolation functions can reduce the required number of monitors needed. This work assesses the effectiveness of three models for accurately interpolating hazard levels among monitors: a traditional Kriging model, a physics-based model, and a hybrid model that combines the Kriging and physics-based models. The effectiveness of each interpolation function was tested with sound levels collected in four environmental settings. These detailed experimental data were used to generate over 10,000 simulation trials, where each trial configured the experimental data into a unique arrangement of simulated monitoring and sampling positions. For each simulation trial, the effectiveness of the three models was assessed with the root mean square error of the sound levels at the simulated sampling positions, using the simulated monitoring positions as input. The interpolated values between the monitored positions were analyzed separately from the extrapolated values beyond the monitored positions. The hybrid model consistently reported among the lowest errors in each trial. The Kriging model performed best for the densest networks (those with the most monitors). Even in these cases, the hybrid model performed within 10% of the Kriging model with less than a third of the monitors. The experiment demonstrates that the hybrid model is highly effective at estimating hazardous sound levels; future work may demonstrate similar advantages for gas and aerosol hazards.

Carbohydrate improves exercise capacity but does not affect subcellular lipid droplet morphology, AMPK and p53 signalling in human skeletal muscle.

KEY POINTS: Muscle glycogen and intramuscular triglycerides (IMTG, stored in lipid droplets) are important energy substrates during prolonged exercise. Exercise-induced changes in lipid droplet (LD) morphology (i.e. LD size and number) have not yet been studied under nutritional conditions typically adopted by elite endurance athletes, that is, after carbohydrate (CHO) loading and CHO feeding during exercise. We report for the first time that exercise reduces IMTG content in both central and peripheral regions of type I and IIa fibres, reflective of decreased LD number in both fibre types whereas reductions in LD size were exclusive to type I fibres. Additionally, CHO feeding does not alter subcellular IMTG utilisation, LD morphology or muscle glycogen utilisation in type I or IIa/II fibres. In the absence of alterations to muscle fuel selection, CHO feeding does not attenuate cell signalling pathways with regulatory roles in mitochondrial biogenesis. ABSTRACT: We examined the effects of carbohydrate (CHO) feeding on lipid droplet (LD) morphology, muscle glycogen utilisation and exercise-induced skeletal muscle cell signalling. After a 36 h CHO loading protocol and pre-exercise meal (12 and 2 g kg-1 , respectively), eight trained males ingested 0, 45 or 90 g CHO h-1 during 180 min cycling at lactate threshold followed by an exercise capacity test (150% lactate threshold). Muscle biopsies were obtained pre- and post-completion of submaximal exercise. Exercise decreased (P < 0.01) glycogen concentration to comparable levels (∼700 to 250 mmol kg-1 DW), though utilisation was greater in type I (∼40%) versus type II fibres (∼10%) (P < 0.01). LD content decreased in type I (∼50%) and type IIa fibres (∼30%) (P < 0.01), with greater utilisation in type I fibres (P < 0.01). CHO feeding did not affect glycogen or IMTG utilisation in type I or II fibres (all P > 0.05). Exercise decreased LD number within central and peripheral regions of both type I and IIa fibres, though reduced LD size was exclusive to type I fibres. Exercise induced (all P < 0.05) comparable AMPKThr172 (∼4-fold), p53Ser15 (∼2-fold) and CaMKIIThr268 phosphorylation (∼2-fold) with no effects of CHO feeding (all P > 0.05). CHO increased exercise capacity where 90 g h-1 (233 ± 133 s) > 45 g h-1 (156 ± 66 s; P = 0.06) > 0 g h-1 (108 ± 54 s; P = 0.03). In conditions of high pre-exercise CHO availability, we conclude CHO feeding does not influence exercise-induced changes in LD morphology, glycogen utilisation or cell signalling pathways with regulatory roles in mitochondrial biogenesis.

Pleural Effusions Following Liver Transplantation: A Single-Center Experience.

INTRODUCTION: This was a single-center retrospective study to evaluate incidence, prognosis, and risk factors in patients with postoperative pleural effusions, a common pulmonary complication following liver transplantation. METHODS: A retrospective review was performed on 374 liver transplantation cases through a database within the timeframe of January 1, 2009 through December 31, 2015. Demographics, pulmonary and cardiac function testing, laboratory studies, intraoperative transfusion/infusion volumes, postoperative management, and outcomes were analyzed. RESULTS: In the immediate postoperative period, 189 (50.5%) developed pleural effusions following liver transplantation of which 145 (76.7%) resolved within 3 months. Those who developed pleural effusions demonstrated a lower fibrinogen (149.6 ± 66.3 mg/dL vs 178.4 ± 87.3 mg/dL; P = .009), total protein (5.8 ± 1.0 mg/dL vs 6.1 ± 1.2 mg/dL; P = .04), and hemoglobin (9.8 ± 1.8 mg/dL vs 10.3 ± 1.9 mg/dL; P = .004). There was not a statistically significant difference in 1-year all-cause mortality and in-hospital mortality between liver transplant recipients with and without pleural effusions. Liver transplant recipients who developed pleural effusions had a longer hospital length of stay (16.4 ± 10.9 days vs 14.0 ± 16.5 days; P = .1), but the differences were not statistically significant. However, there was a significant difference in tracheostomy rates (11.6% vs 5.4%; P = .03) in recipients who developed pleural effusions compared to recipients who did not. CONCLUSIONS: In summary, pleural effusions are common after liver transplantation and are associated with increased morbidity. Pre- and intraoperative risk factors can offer both predictive and prognostic value for post-transplantation pleural effusions. Further prospective studies will be needed to further evaluate the relevance of these findings to limit instances of postoperative pleural effusions.

Analysis of a wooden specimen's mechanical properties through acoustic measurements in the very near field.

This paper presents the method for prediction of some basic mechanical parameters of a wood specimen by using simple instrumentation. The standard acoustic method for assessment of modulus of elasticity using the impulse excitation is extended by using a recording in the very near field. In this way, visualization of vibration patterns of the analyzed sample was accomplished for all modes below 5 kHz. This enables detection of signals with a good signal-to-noise ratio over a wide frequency range which is then used for loss factor assessment.

New Vaccine Design and Delivery Technologies.

Technological advances in immunology, protein design, and genetic delivery have unlocked new possibilities for vaccine concepts and delivery technologies that were previously inaccessible. These next-generation vaccine design efforts are particularly promising in their potential to provide solutions to challenging targets for which conventional approaches have proven ineffective-for example, a universal influenza vaccine. In this perspective, we discuss emerging approaches to vaccine design and engineering based on recent insights into immunology, structural biology, computational biology, and immunoengineering. We anticipate that these cutting-edge, interdisciplinary approaches will lead to breakthrough vaccine concepts for ever-evolving and (re)emerging influenza viruses, with important ramifications for global public health.

No barrier to emergence of bathyal king crabs on the Antarctic shelf.

Cold-water conditions have excluded durophagous (skeleton-breaking) predators from the Antarctic seafloor for millions of years. Rapidly warming seas off the western Antarctic Peninsula could now facilitate their return to the continental shelf, with profound consequences for the endemic fauna. Among the likely first arrivals are king crabs (Lithodidae), which were discovered recently on the adjacent continental slope. During the austral summer of 2010 ‒ 2011, we used underwater imagery to survey a slope-dwelling population of the lithodid Paralomis birsteini off Marguerite Bay, western Antarctic Peninsula for environmental or trophic impediments to shoreward expansion. The population density averaged ∼ 4.5 individuals × 1,000 m(-2) within a depth range of 1,100 ‒ 1,500 m (overall observed depth range 841-2,266 m). Images of juveniles, discarded molts, and precopulatory behavior, as well as gravid females in a trapping study, suggested a reproductively viable population on the slope. At the time of the survey, there was no thermal barrier to prevent the lithodids from expanding upward and emerging on the outer shelf (400- to 550-m depth); however, near-surface temperatures remained too cold for them to survive in inner-shelf and coastal environments (<200 m). Ambient salinity, composition of the substrate, and the depth distribution of potential predators likewise indicated no barriers to expansion of lithodids onto the outer shelf. Primary food resources for lithodids--echinoderms and mollusks--were abundant on the upper slope (550-800 m) and outer shelf. As sea temperatures continue to rise, lithodids will likely play an increasingly important role in the trophic structure of subtidal communities closer to shore.

Islet Neogenesis Associated Protein (INGAP) induces the differentiation of an adult human pancreatic ductal cell line into insulin-expressing cells through stepwise activation of key transcription factors for embryonic beta cell development.

Regeneration of ß-cells in diabetic patients is an important goal of diabetes research. Islet Neogenesis Associated Protein (INGAP) was discovered in the partially duct-obstructed hamster pancreas. Its bioactive fragment, pentadecapeptide 104-118 (INGAP-P), has been shown to reverse diabetes in animal models and to improve glucose homeostasis in patients with diabetes in clinical trials. Further development of INGAP as a therapy for diabetes requires identification of target cells in the pancreas and characterization of the mechanisms of action. We hypothesized that adult human pancreatic ductal cells retain morphogenetic plasticity and can be induced by INGAP to undergo endocrine differentiation. To test this hypothesis, we treated the normal human pancreatic ductal cell line (HPDE) with either INGAP-P or full-length recombinant protein (rINGAP) for short-term periods. Our data show that this single drug treatment induces both proliferation and transdifferentiation of HPDE cells, the latter being characterized by the rapid sequential activation of endocrine developmental transcription factors Pdx-1, Ngn3, NeuroD, IA-1, and MafA and subsequently the expression of insulin at both the mRNA and the protein levels. After 7 days, C-peptide was detected in the supernatant of INGAP-treated cells, reflecting their ability to secrete insulin. The magnitude of differentiation was enhanced by embedding the cells in Matrigel, which led to islet-like cluster formation. The islet-like clusters cells stained positive for nuclear Pdx-1 and Glut 2 proteins, and were expressing Insulin mRNA. These new data suggest that human adult pancreatic ductal cells retain morphogenetic plasticity and demonstrate that a short exposure to INGAP triggers their differentiation into insulin-expressing cells in vitro. In the context of the urgent search for a regenerative and/or cellular therapy for diabetes, these results make INGAP a promising therapeutic candidate.

Assessing the prediction of arterial CO2 from end tidal CO2 in adult blunt trauma patients.

BACKGROUND: The control of PaCO2 in ventilated patients is known to be of particular importance in the management and prognosis of trauma patients. Although EtCO2 is often used as a continuous, non-invasive, surrogate marker for PaCO2 in ventilated trauma patients in the emergency department (ED), previous studies suggest a poor correlation in this cohort. However, previous data has predominantly been collected retrospectively, raising the possibility that the elapsed time between PaCO2 sampling and EtCO2 recording may contribute to the poor correlation. As such this study aimed to analyse the correlation of PaCO2 to EtCO2 in the ventilated blunt trauma patient presenting to the ED through contemporaneous sampling. METHODS: This study was conducted as a prospective observational study analysing the near simultaneous recording of EtCO2 and Arterial Blood Gas sampling of ventilated adult trauma patients in the ED of a Level 1 trauma centre over a 12-month period. Data was analysed using linear regression and subgroup analysis by Injury Severity Score (ISS) and Abbreviated Injury Score (AIS) of the Chest. RESULTS: Linear regression of EtCO2 vs PaCO2 demonstrated a moderate correlation with r = 0.54 (p < 0.01, n = 51, 95 % CI 0.31-0.71). Subgroup analysis by ISS, revealed a stronger correlation in those with minor ISS (0-11) (r = 0.76, p < 0.01, n = 13, 95 % CI 0.36-0.92) compared to those more severely injured patients (ISS > 15) (r = 0.44, P < 0.01, n = 38, 95 % CI 0.14-0.67). Analysis by AIS Chest demonstrated similar correlation between patients without chest injuries (AIS 0) (r = 0.55, n = 29, p < 0.01, 95 % CI 0.23-0.76) and those with an AIS >1 (r = 0.51, n = 22, p = 0.02, 95 % CI 0.11-0.77). In patients with traumatic head injuries who had an EtCO2 between 30 and 39 mmHg, only 57 % had a measured PaCO2 within 5 mmHg. CONCLUSIONS: As patients transition from minor to seriously injured, a decreasing strength of PaCO2 to EtCO2 correlation is observed, decreasing the reliability of EtCO2 as a surrogate marker of PaCO2 in this patient group. This inconsistency cannot be accounted for by the presence of chest injuries and worryingly is frequently seen in those with traumatic brain injuries.

The DmsABC S-oxide reductase is an essential component of a novel, hypochlorite-inducible system of extracellular stress defense in Haemophilus influenzae.

Defenses against oxidative damage to cell components are essential for survival of bacterial pathogens during infection, and here we have uncovered that the DmsABC S-/N-oxide reductase is essential for virulence and in-host survival of the human-adapted pathogen, Haemophilus influenzae. In several different infection models, H. influenzae ΔdmsA strains showed reduced immunogenicity as well as lower levels of survival in contact with host cells. Expression of DmsABC was induced in the presence of hypochlorite and paraquat, closely linking this enzyme to defense against host-produced antimicrobials. In addition to methionine sulfoxide, DmsABC converted nicotinamide- and pyrimidine-N-oxide, precursors of NAD and pyrimidine for which H. influenzae is an auxotroph, at physiologically relevant concentrations, suggesting that these compounds could be natural substrates for DmsABC. Our data show that DmsABC forms part of a novel, periplasmic system for defense against host-induced S- and N-oxide stress that also comprises the functionally related MtsZ S-oxide reductase and the MsrAB peptide methionine sulfoxide reductase. All three enzymes are induced following exposure of the bacteria to hypochlorite. MsrAB is required for physical resistance to HOCl and protein repair. In contrast, DmsABC was required for intracellular colonization of host cells and, together with MtsZ, contributed to resistance to N-Chlorotaurine. Our work expands and redefines the physiological role of DmsABC and highlights the importance of different types of S-oxide reductases for bacterial virulence.

Macromolecular Cargo Encapsulation via In Vitro Assembly of Two-Component Protein Nanoparticles.

Self-assembling protein nanoparticles are a promising class of materials for targeted drug delivery. Here, the use of a computationally designed, two-component, icosahedral protein nanoparticle is reported to encapsulate multiple macromolecular cargoes via simple and controlled self-assembly in vitro. Single-stranded RNA molecules between 200 and 2500 nucleotides in length are encapsulated and protected from enzymatic degradation for up to a month with length-dependent decay rates. Immunogenicity studies of nanoparticles packaging synthetic polymers carrying a small-molecule TLR7/8 agonist show that co-delivery of antigen and adjuvant results in a more than 20-fold increase in humoral immune responses while minimizing systemic cytokine secretion associated with free adjuvant. Coupled with the precise control over nanoparticle structure offered by computational design, robust and versatile encapsulation via in vitro assembly opens the door to a new generation of cargo-loaded protein nanoparticles that can combine the therapeutic effects of multiple drug classes.

Hfe Permease and Haemophilus influenzae Manganese Homeostasis.

Haemophilus influenzae is a commensal of the human upper respiratory tract that can infect diverse host niches due, at least in part, to its ability to withstand both endogenous and host-mediated oxidative stresses. Here, we show that hfeA, a gene previously linked to iron import, is essential for H. influenzae manganese recruitment via the HfeBCD transporter. Structural analyses show that metal binding in HfeA uses a unique mechanism that involves substantial rotation of the C-terminal lobe of the protein. Disruption of hfeA reduced H. influenzae manganese acquisition and was associated with decreased growth under aerobic conditions, impaired manganese-superoxide dismutase activity, reduced survival in macrophages, and changes in biofilm production in the presence of superoxide. Collectively, this work shows that HfeA contributes to H. influenzae manganese acquisition and virulence attributes. High conservation of the hfeABCD permease in Haemophilus species suggests that it may serve similar roles in other pathogenic Pasteurellaceae.

Longer times in computed tomography for trauma patients result in a decrease in body temperature.

OBJECTIVE: To determine if lower ambient temperatures in computed tomography (CT) rooms contribute to accidental hypothermia (≤35°C) in trauma patients. METHODS: Prospective, observational study of trauma patients undergoing a CT scan at the Royal Adelaide Hospital. RESULTS: Among a cohort of 81 trauma patients, 54 met level 1 call-out criteria. Mean time in CT was 11 min. Longer time spent in CT contributes to greater decrease in body temperature (∆T/t = -0.1483°C/min, P = 0.0026). CONCLUSION: Increasing time spent in CT leads to a decrease in body temperature in trauma patients. Clinicians should actively reduce time spent in the CT room and take active warming measures.

Increased distance or time from a major trauma centre in South Australia is not associated with worse outcomes after moderate to severe traumatic brain injury.

OBJECTIVE: Considerations in traumatic brain injury (TBI) management include time to critical interventions and neurosurgical care, which can be influenced by the geographical location of injury. In Australia, these distances can be vast with varying degrees of first-responder experience. The present study aimed to evaluate the association that distance and/or time to a major trauma centre (MTC) had on patient outcomes with moderate to severe TBI. METHODS: A retrospective cohort study was conducted using data from the Royal Adelaide Hospital's (RAH) Trauma Registry over a 3-year period (1 January 2018 to 31 December 2020). All patients with a moderate to severe TBI (Glasgow Coma Scale [GCS] ≤13 and abbreviated injury score head of ≥2) were included. The association of distance and time to the RAH and patient outcomes were compared by calculating the odds ratio utilising a logistic regression model. RESULTS: A total of 378 patients were identified; of these, 226 met inclusion criteria and comprised our study cohort. Most patients were male (79%), injured in a major city (55%), with median age of 38 years old and median injury severity score (ISS) of 25. After controlling for age, ISS, ED GCS on arrival and pre-MTC intubation, increasing distance or time from injury site to the RAH was not shown to be associated with mortality or discharge destination in any of the models investigated. CONCLUSION: Our analysis revealed that increasing distance or time from injury site to a MTC for patients with moderate to severe TBI was not significantly associated with adverse patient outcomes.

An Observational Case Series Measuring the Energy Expenditure of Elite Tennis Players During Competition and Training by Using Doubly Labeled Water.

PURPOSE: An understanding of an athlete's total daily energy expenditure (TEE) is necessary to inform nutritional strategies, particularly where daily training and competitive demands are highly variable. This observational case series assessed the TEE of elite tennis players during high-level competition. METHODS: Senior female singles participants (FS: n = 3; 21 [1] y; ranked  Women's Tennis Association [WTA] top 125-375), an FS junior (n = 1; 16 y; ranked WTA top 350), and a men's doubles player (n = 1; 26 y; ranked Association of Tennis Professionals [ATP] top 5) were assessed for TEE (using the doubly labeled water method) during a 9- to 14-day period, which included training, Wimbledon Championships, WTA/ATP International Tournaments, Junior/Senior International Tennis Federation, and Wimbledon Junior Championships. One female (FS3) did not exercise from day 4 following injury. RESULTS: TEE for men's doubles was 4586 kcal·d-1 (67 kcal·kg-1 fat-free mass [FFM]; daily activity 98 [74] min). Noninjured adult female participants' TEEs were 3396 and 3948 kcal·d-1 (66 and 81 kcal·kg-1 FFM; daily activity durations were 139 [84] min and 150 [66] min, respectively), while TEE for the injured athlete was 2583 kcal·d-1 (45.7 kcal·kg-1; daily nonexercise activity duration was <45 min). The junior player TEE was 3988 kcal·d-1 (78.2 kcal·kg-1 FFM; daily activity of 131 [66] min). CONCLUSION: This observational case series positions tennis as a highly energetically demanding sport with variability evident between individuals (ie, TEE between 60 and 90 kcal·kg-1 FFM). Accordingly, nutritional strategies that promote sufficient energy availability should be emphasized with individual variability suitably assessed prior to prescription.