Identification and Characterization of a Bacterial Periplasmic Solute Binding Protein That Binds l-Amino Acid Amides.

Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from Mesorhizobium sp. A09 (MeAmi_SBP) and show that MeAmi_SBP binds l-amino acid amides but not the corresponding l-amino acids. An X-ray crystal structure of MeAmi_SBP bound to l-serinamide highlights the residues that impart distinct specificity for l-amino acid amides and reveals a structural Ca2+ binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca2+ binding are conserved among the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake, and metabolism of l-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that MeAmi_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with l-amino acid amides and bacterial nitrogen utilization.

Improving feedback literacy in a primary care rotation.

Primary care education is a unique clinical experience for medical students. It is community-based and provides an opportunity for students to learn consultation skills with multiple sources of workplace-based feedback. Meaningful and demonstrable utilisation of this feedback by students remains an educational challenge. We showcase achievable changes to educational tasks in an established curriculum, which aim to improve student feedback literacy and create a feedback loop which improves on previous provision of unidirectional, terminal feedback. The changes have been well-received, with student and educator engagement being positive. Students have demonstrated critical reflection on feedback, and development in consultation and clinical reasoning skills.

Development and implementation of the lung volume reduction pulmonary rehabilitation tool to identify eligibility for lung volume reduction in people with chronic obstructive pulmonary disease during pulmonary rehabilitation.

BACKGROUND: Completion of pulmonary rehabilitation is recognised in chronic obstructive pulmonary disease (COPD) guidelines as a key opportunity to consider systematically whether a respiratory review to assess potential suitability for a lung volume reduction (LVR) procedure might be appropriate. We describe the development of a simple decision-support tool (the LVR-PR tool) to aid clinicians working in pulmonary rehabilitation, to operationalise this process. METHODS: We took an iterative mixed methods approach, which was partnership-based and involved an initial consensus survey, focus groups and an observational study cohort at multiple pulmonary rehabilitation centres. RESULTS: Diagnosis (97%), exercise capacity (84%), breathlessness (78%) and co-morbidities (76%) were acknowledged to be essential items for assessing basic LVR eligibility. Collating prior investigations and assessing patient understanding were considered useful but not essential. Clinician concerns included; streamlining the tool; access to clinical information and investigations; and care needed around introducing LVR therapies to patients in a PR setting. Access to clearer information about LVR procedures, the clinician's role in considering eligibility and how educational resources should be delivered were identified as important themes from patient group discussions. The LVR-PR tool was considered to be feasible and valid for implementation in a variety of PR services across the UK subject to the provision of appropriate health professional training. Clinicians working in specialist LVR centres across the UK who were not otherwise involved in the development process confirmed the tool's validity using the content validity index (CVI). INTERPRETATION: The LVR-PR tool appears to be an acceptable tool that can be feasibly implemented in PR services subject to good quality educational resources for both patients and healthcare professionals.

The extinct Sicilian wolf shows a complex history of isolation and admixture with ancient dogs.

The Sicilian wolf remained isolated in Sicily from the end of the Pleistocene until its extermination in the 1930s-1960s. Given its long-term isolation on the island and distinctive morphology, the genetic origin of the Sicilian wolf remains debated. We sequenced four nuclear genomes and five mitogenomes from the seven existing museum specimens to investigate the Sicilian wolf ancestry, relationships with extant and extinct wolves and dogs, and diversity. Our results show that the Sicilian wolf is most closely related to the Italian wolf but carries ancestry from a lineage related to European Eneolithic and Bronze Age dogs. The average nucleotide diversity of the Sicilian wolf was half of the Italian wolf, with 37-50% of its genome contained in runs of homozygosity. Overall, we show that, by the time it went extinct, the Sicilian wolf had high inbreeding and low-genetic diversity, consistent with a population in an insular environment.

Oxygen in interstitial lung diseases.

Domiciliary oxygen is essential in the care of hypoxaemic interstitial lung disease (ILD) patients. Guidelines concur in advising prescription of long-term oxygen therapy (LTOT) for ILD patients with severe hypoxaemia at rest, in view of its beneficial impact on breathlessness/disability and extrapolating potential survival benefits seen in COPD patients. A less severe hypoxaemia threshold for initiation of LTOT is recommended for patients with pulmonary hypertension (PH)/right heart failure, requiring careful evaluation in all ILD patients. In light of evidence suggesting a link between nocturnal hypoxaemia, development of PH and poor survival, studies assessing the impact of nocturnal oxygen are urgently needed. Severe exertional hypoxaemia is frequent in ILD patients, with impact on exercise tolerance, quality of life and mortality. Ambulatory oxygen therapy (AOT) has been associated with improvement in breathlessness and quality of life in ILD patients with exertional hypoxaemia. However, given the paucity of evidence, not all current AOT guidelines are in agreement. Ongoing clinical trials will provide further useful data. Despite its beneficial effects, supplemental oxygen imposes burdens and challenges to patients. A key unmet area of need is the development of less cumbersome and more efficient oxygen delivery systems to reduce the negative impact of AOT on patients' lives.

Chemistry-led investigations into the mode of action of NAMPT activators, resulting in the discovery of non-pyridyl class NAMPT activators.

The cofactor nicotinamide adenine dinucleotide (NAD+) plays a key role in a wide range of physiological processes and maintaining or enhancing NAD+ levels is an established approach to enhancing healthy aging. Recently, several classes of nicotinamide phosphoribosyl transferase (NAMPT) activators have been shown to increase NAD+ levels in vitro and in vivo and to demonstrate beneficial effects in animal models. The best validated of these compounds are structurally related to known urea-type NAMPT inhibitors, however the basis for the switch from inhibitory activity to activation is not well understood. Here we report an evaluation of the structure activity relationships of NAMPT activators by designing, synthesising and testing compounds from other NAMPT ligand chemotypes and mimetics of putative phosphoribosylated adducts of known activators. The results of these studies led us to hypothesise that these activators act via a through-water interaction in the NAMPT active site, resulting in the design of the first known urea-class NAMPT activator that does not utilise a pyridine-like warhead, which shows similar or greater activity as a NAMPT activator in biochemical and cellular assays relative to known analogues.

Techniques for Oxygenation and Ventilation in Coronavirus Disease 2019.

This paper discusses mechanisms of hypoxemia and interventions to oxygenate critically ill patients with COVID-19 which range from nasal cannula to noninvasive and mechanical ventilation. Noninvasive ventilation includes continuous positive airway pressure ventilation (CPAP) and high-flow nasal cannula (HFNC) with or without proning. The evidence for each of these modalities is discussed and thereafter, when to transition to mechanical ventilation (MV). Various techniques of MV, again with and without proning, and rescue strategies which would include extra corporeal membrane oxygenation (ECMO) when it is available and permissive hypoxemia where it is not, are discussed.

An overview of dynamical methods for studying transitions between states in sheared plasma flows.

The self-organization of structures in a tokamak plasma as it undergoes an [Formula: see text]-mode transition shows properties similar to simpler shear flow configurations. We will describe recent dynamical studies of plasma shear flows, including the idea of tracking the edge of chaos that separates two bistable states, computing the nonlinear minimal seed that can lead to turbulence, finding the attractor solution on the edge and seeing how starting from this solution we can understand the stability of relative period orbits that permeate the turbulent basin of attraction. We present a modus operandi developed for these simple configurations that can be adapted to understand the [Formula: see text]-mode transition. This article is part of a discussion meeting issue 'H-mode transition and pedestal studies in fusion plasmas'.

The Successful Management of an Adult Patient With Unrepaired Tetralogy of Fallot for Caesarian Section: A Case Report From South Africa.

Tetralogy of Fallot (TOF) is a congenital heart defect characterized by four interlinked cardiac anomalies, leading to cyanosis and significant health risks if left untreated. We present the management and outcome of an adult patient with unrepaired TOF at 34 weeks of pregnancy at a central Johannesburg hospital. Uncorrected TOF in adulthood, especially in the late stages of pregnancy, is rare, presenting challenges for both the mother and baby. This case report outlines our successful management strategy, highlighting the importance of careful hemodynamic stability. The primary goal in managing this complex case was to prevent significant changes in systemic vascular resistance (SVR) and pulmonary vascular resistance (PVR). We diligently avoided triggers for worsening right-sided pressures and aimed to maintain haemodynamic stability and therefore optimise oxygen delivery and minimise blood loss and dehydration. Our approach focused on preventing a worsening right-to-left shunt, which exacerbates hypoxia and haemodynamic instability. Regarding the anaesthetic technique, we discuss the advantages and disadvantages of both general and regional anaesthesia. While there is no gold standard, the choice should be individualized based on the patient's condition and treating unit practice. We describe the use of a graded epidural anaesthesia technique, which proved to be a safe and effective method for managing a parturient with significant cardiac disease during caesarean section. This technique resulted in minimal hemodynamic changes and superior post-operative pain control and avoided potential side effects associated with general anaesthesia. Notably, the technique relied solely on a neuraxial anaesthetic technique, minimising the risk of neonatal cardiopulmonary depression. This case report serves as the first documented instance from South Africa of successfully anaesthetizing an adult patient with unrepaired TOF in late pregnancy for a caesarean section. The graded epidural technique emerged as a secure option for anaesthetic management in a challenging case, providing important insights into the care of patients with complex cardiac conditions during pregnancy.

Foundations for fairness in digital health apps.

Digital mental health applications promise scalable and cost-effective solutions to mitigate the gap between the demand and supply of mental healthcare services. However, very little attention is paid on differential impact and potential discrimination in digital mental health services with respect to different sensitive user groups (e.g., race, age, gender, ethnicity, socio-economic status) as the extant literature as well as the market lack the corresponding evidence. In this paper, we outline a 7-step model to assess algorithmic discrimination in digital mental health services, focusing on algorithmic bias assessment and differential impact. We conduct a pilot analysis with 610 users of the model applied on a digital wellbeing service called Foundations that incorporates a rich set of 150 proposed activities designed to increase wellbeing and reduce stress. We further apply the 7-step model on the evaluation of two algorithms that could extend the current service: monitoring step-up model, and a popularity-based activities recommender system. This study applies an algorithmic fairness analysis framework for digital mental health and explores differences in the outcome metrics for the interventions, monitoring model, and recommender engine for the users of different age, gender, type of work, country of residence, employment status and monthly income. Systematic Review Registration: The study with main hypotheses is registered at: https://osf.io/hvtf8.

Cat Scratch Disease: 9 Years of Experience at a Pediatric Center.

Background: A more complete understanding of the epidemiology, risk factors, and clinical features of cat scratch disease (CSD) in children could help guide patient care. Methods: We conducted a retrospective analysis of children presenting to a tertiary pediatric hospital system in Atlanta, Georgia between January 1, 2010 and December 31, 2018 who had serology, polymerase chain reaction, and/or cytopathological results consistent with a Bartonella henselae infection. We also retrospectively reviewed veterinary diagnostic results performed at the University of Georgia from 2018 to 2020 to ascertain the burden of bartonellosis in companion animals within the state. Results: We identified 304 children with CSD over 9 years with the largest proportion of diagnoses made during August (41 of 304, 13.5%) and September (47 of 304, 15.5%). The median age of child cases was 8.1 years (interquartile range [IQR], 5.4-12.1); 156 (51.3%) were female; 242 of 262 (92.4%) reported feline exposure; and 55 of 250 (22%) reported canine exposure of those with exposure histories documented in the medical record. Although lymphadenopathy was present on physical examination in the majority of cases (78.8%), atypical presentations lacking lymphadenopathy were also common (63 of 304, 20.7%). Among children with radiographic imaging, 20 of 55 (36.4%) had splenomegaly and 21 of 55 (38.1%) had splenic and/or hepatic microabscesses. Among veterinary data, Bartonella seroprevalence was 12 of 146 (8.2%), all among canines, with a geographic distribution that spanned the state of Georgia. Conclusions: Distinguishing clinical features of CSD included subacute regional lymphadenopathy in school-aged children in the late summer, almost all of whom had cat exposure. Atypical clinical manifestations of CSD were also commonly identified.

Phylogenomics of the world's otters.

Comparative whole-genome analyses hold great power to illuminate commonalities and differences in the evolution of related species that share similar ecologies. The mustelid subfamily Lutrinae includes 13 currently recognized extant species of otters,1-5 a semiaquatic group whose evolutionary history is incompletely understood. We assembled a dataset comprising 24 genomes from all living otter species, 14 of which were newly sequenced. We used this dataset to infer phylogenetic relationships and divergence times, to characterize patterns of genome-wide genealogical discordance, and to investigate demographic history and current genomic diversity. We found that genera Lutra, Aonyx, Amblonyx, and Lutrogale form a coherent clade that should be synonymized under Lutra, simplifying the taxonomic structure of the subfamily. The poorly known tropical African Aonyx congicus and the more widespread Aonyx capensis were found to be reciprocally monophyletic (having diverged 440,000 years ago), supporting the validity of the former as a distinct species. We observed variable changes in effective population sizes over time among otters within and among continents, although several species showed similar trends of expansions and declines during the last 100,000 years. This has led to different levels of genomic diversity assessed by overall heterozygosity, genome-wide SNV density, and run of homozygosity burden. Interestingly, there were cases in which diversity metrics were consistent with the current threat status (mostly based on census size), highlighting the potential of genomic data for conservation assessment. Overall, our results shed light on otter evolutionary history and provide a framework for further in-depth comparative genomic studies targeting this group.

Probing the genomic limits of de-extinction in the Christmas Island rat.

Three principal methods are under discussion as possible pathways to "true" de-extinction; i.e., back-breeding, cloning, and genetic engineering.1,2 Of these, while the latter approach is most likely to apply to the largest number of extinct species, its potential is constrained by the degree to which the extinct species genome can be reconstructed. We explore this question using the extinct Christmas Island rat (Rattus macleari) as a model, an endemic rat species that was driven extinct between 1898 and 1908.3-5 We first re-sequenced its genome to an average of >60× coverage, then mapped it to the reference genomes of different Rattus species. We then explored how evolutionary divergence from the extant reference genome affected the fraction of the Christmas Island rat genome that could be recovered. Our analyses show that even when the extremely high-quality Norway brown rat (R. norvegicus) is used as a reference, nearly 5% of the genome sequence is unrecoverable, with 1,661 genes recovered at lower than 90% completeness, and 26 completely absent. Furthermore, we find the distribution of regions affected is not random, but for example, if 90% completeness is used as the cutoff, genes related to immune response and olfaction are excessively affected. Ultimately, our approach demonstrates the importance of applying similar analyses to candidates for de-extinction through genome editing in order to provide critical baseline information about how representative the edited form would be of the extinct species.

The effect of renewable energy incorporation on power grid stability and resilience.

Contemporary proliferation of renewable power generation is causing an overhaul in the topology, composition, and dynamics of electrical grids. These low-output, intermittent generators are widely distributed throughout the grid, including at the household level. It is critical for the function of modern power infrastructure to understand how this increasingly distributed layout affects network stability and resilience. This paper uses dynamical models, household power consumption, and photovoltaic generation data to show how these characteristics vary with the level of distribution. It is shown that resilience exhibits daily oscillations as the grid's effective structure and the power demand fluctuate. This can lead to a substantial decrease in grid resilience, explained by periods of highly clustered generator output. Moreover, the addition of batteries, while enabling consumer self-sufficiency, fails to ameliorate these problems. The methodology identifies a grid's susceptibility to disruption resulting from its network structure and modes of operation.

Discovery and structure-activity relationships of a novel oxazolidinone class of bacterial type II topoisomerase inhibitors.

There is an increasingly urgent and unmet medical need for novel antibiotic drugs that tackle infections caused by multidrug-resistant (MDR) pathogens. Novel bacterial type II topoisomerase inhibitors (NBTIs) are of high interest due to limited cross-resistance with fluoroquinolones, however analogues with Gram-negative activity often suffer from hERG channel inhibition. A novel series of bicyclic-oxazolidinone inhibitors of bacterial type II topoisomerase were identified which display potent broad-spectrum anti-bacterial activity, including against MDR strains, along with an encouraging in vitro safety profile. In vivo proof of concept was achieved in a A. baumannii mouse thigh infection model.

CMC determination using isothermal titration calorimetry for five industrially significant non-ionic surfactants.

Surfactants are used in a vast array of products including pharmaceuticals, cosmetics and household formulations. From an industrial perspective, non-ionic surfactants are ideal for inclusion within such products as they are non-toxic, simple to formulate and economic to use. This study considers five non-ionic surfactants (Tween 20, Tween 80, Crodasol, Croduret and Etocas 35) to determine the critical micellar concentration (CMC) for each using isothermal titration calorimetry, thus avoiding issues regarding poor accuracy found with other techniques. Furthermore, this methodology has not previously been applied to this group of surfactants. For the most commonly used non-ionics (Tween 20 and Tween 80) a further study was undertaken to consider the influence of surfactant purity on the CMC determined, using standard grade (Tween 20 and 80), high purity (Tween 20 HP and Tween 80 HP) and Super Refined (SR PS20 and SR PS80). Results permitted calculation of the CMC for the surfactants whereupon the values were determined to range from 1.0 mM for Tween 20 HP to 2.9 mM for Tween 80 HP. Such information regarding the CMC event is useful from a formulation perspective as it can ensure that the most optimum concentration of surfactant is included within a formulation to maximize its efficacy.

An Evaluation of the Effect of Activation Methods on the Release of Growth Factors from Platelet-Rich Plasma.

BACKGROUND: Activation of platelets in platelet-rich plasma may improve growth factor release, thus enhancing regenerative properties. The authors investigated whether different methods of platelet-rich plasma activation affected growth factor release kinetics over time. METHODS: Platelet-rich plasma from 20 healthy volunteers was processed by six different methods: (1) control (nonactivated); (2) activation with calcium chloride; (3) activation with calcium chloride and ethanol; (4) activation with calcium chloride and ethanol at 4°C; (5) activation with calcium chloride and ethanol with vitamin C; (6) activation with calcium chloride and ethanol with vitamin C at 4°C. Concentration of secreted vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and insulin-like growth factor over 24 hours was measured by immunoassay. RESULTS: Calcium chloride-activated platelet-rich plasma produced significantly more insulin-like growth factor at 1 hour compared to cold and vitamin C platelet-rich plasma, and calcium chloride plus ethanol produced significantly more at 24 hours compared to vitamin C platelet-rich plasma. The addition of vitamin C reduced release of PDGF over time. Activation with calcium chloride and ethanol with or without cold temperature produced a gradual PDGF release as opposed to calcium chloride alone, which caused higher PDGF within 4 hours. There were no significant differences between groups for VEGF, although calcium chloride and cooled platelet-rich plasma approached significance for producing more than vitamin C platelet-rich plasma. CONCLUSIONS: Activation of platelet-rich plasma does not significantly improve growth factor secretion, which is made worse by the addition of vitamin C, a platelet inhibitor. Ethanol does not negatively impact growth factor production and may offer a more gradual release. CLINICAL RELEVANCE STATEMENT: These findings will help guide platelet-rich plasma preparation methods where therapeutic growth factors are used. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Histological analysis of fat grafting with platelet-rich plasma for diabetic foot ulcers-A randomised controlled trial.

Diabetic foot ulcers are often unresponsive to conventional therapy and are a leading cause of amputation. Animal studies have shown stem cells and growth factors can accelerate wound healing. Adipose-derived stem cells are found in fat grafts and mixing them with platelet-rich plasma (PRP) may improve graft survival. This study aimed to establish the histological changes when diabetic foot ulcers are treated with fat grafts and PRP. A three-armed RCT was undertaken of 18 diabetic foot ulcer patients: fat grafting; fat grafting with PRP; and routine podiatry care. Biopsies were obtained at week 0, 1, and 4, and underwent quantitative histology/immunohistochemistry (H&E, CD31, and Ki67). Treatment with fat and PRP increased mean microvessel density at 1 week to 1645 (SD 96) microvessels/mm2 (+32%-45% to other arms, P = .035). PRP appeared to increase vascularity surrounding fat grafts, and histology suggested PRP may enhance fat graft survival. There was no clinical difference between arms. This study demonstrates PRP with fat grafts increased neovascularisation and graft survival in diabetic foot ulcers. The histology was not, however, correlated with wound healing time. Future studies should consider using apoptosis markers and fluorescent labelling to ascertain if enhanced fat graft survival is due to proliferation or reduced apoptosis. Trial registration NCT03085550.

Improved COVID-19 outcomes in a large non-invasive respiratory support cohort despite emergence of the alpha variant.

INTRODUCTION: Respiratory high-dependency units (rHDUs) are used to manage respiratory failure in COVID-19 outside of the intensive care unit (ICU). The alpha variant of COVID-19 has been linked to increased rates of mortality and admission to ICU; however, its impact on a rHDU population is not known. We aimed to compare rHDU outcomes between the two main UK waves of COVID-19 infection and evaluate the impact of the alpha variant on second wave outcomes. METHODS: We conducted a single-centre, retrospective analysis of all patients with a diagnosis of COVID-19 admitted to the rHDU of our teaching hospital for respiratory support during the first and second main UK waves. RESULTS: In total, 348 patients were admitted to rHDU. In the second wave, mortality (26.7% s vs 50.7% first wave, χ2=14.7, df=1, p=0.0001) and intubation rates in those eligible (24.3% s vs 58.8% first wave, χ2=17.3, df=2, p=0.0002) were improved compared with the first wave. In the second wave, the alpha variant had no effect on mortality (OR 1.18, 95% CI 0.60 to 2.32, p=0.64). Continuous positive airway pressure (CPAP) (89.5%) and awake proning (85.6%) were used in most patients in the second wave. DISCUSSION: Our single-centre experience shows that rHDU mortality and intubation rates have improved over time in spite of the emergence of the alpha variant. Our data support the use of CPAP and awake proning, although improvements in outcome are likely to be multifactorial.

Epidemiology of Clostridioides difficile in South Africa.

BACKGROUND: Clostridioides difficile (CD) is the most common healthcare-associated enteric infection. There is currently limited epidemiological evidence on CD incidence in South Africa. AIM: To estimate the burden of CD infection (CDI) in the South African public sector between 1 July 2016 and 30 June 2017. METHODS: A retrospective cohort study utilizing secondary data was conducted to describe the epidemiology of CD in South Africa. We assessed the patient-level association between variables of interest, CD, and CD recurrence, by undertaking both univariate and multivariable analysis. Adjusted incidence rate ratios (aIRR) were calculated utilizing multivariable Poisson regression. The incidence of CD, CD recurrence and CD testing was estimated by Poisson regression for various levels of care and provinces. RESULTS: A total of 14 023 samples were tested for CD during the study period. After applying exclusion criteria, we were left with a sample of 10 053 of which 1 860 (18.50%) tested CD positive. A positive and significant association between CDI and level of care is found, with patients treated in specialized tuberculosis (TB) hospitals having a five-fold increased adjusted incidence risk ratio (aIRR) for CDI (aIRR 4.96 CI95% 4.08-6.04,) compared to those managed in primary care. Patients receiving care at a secondary, tertiary, or central hospital had 35%, 66% and 41% increased adjusted incidence of CDI compared to those managed in primary care, respectively. National incidence of CDI is estimated at 53.89 cases per 100 000 hospitalizations (CI95% 51.58-56.29), the incidence of recurrence at 21.39 (CI95% 15.06-29.48) cases per 1 000 cases and a recurrence rate of 2.14% (CI95% 1.51-2.94). CONCLUSION: Compared to European countries, we found a comparable incidence of CD. However, our estimates are lower than those for the United States. Compared to high-income countries, this study found a comparatively lower CD recurrence.