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The spread of drug-resistant bacteria into the community is an urgent threat. In most low-middle-income countries (LMICs) settings, community-acquired infection (CAI) is empirically treated with no data to support the choice of antibiotics, hence contributing to resistance development. Continuous antimicrobial resistance (AMR) data on community-acquired pathogens are needed to draft empirical treatment guidelines, especially for areas with limited culture and susceptibility testing. Despite the importance of addressing antibiotic-resistant pathogens in the community setting, protocols for the surveillance of AMR bacterial infections are lacking in most (LMICs). We present a protocol for surveillance of AMR in LMICs using urinary tract infection (UTI) as a proxy for CAI to enable users to quantify and establish the drivers of AMR bacteria causing UTI. The protocol intends to assist users in designing a sustainable surveillance program for AMR in the community involving children above two years of age and adults presenting to a primary health facility for healthcare. Implementation of the protocol requires initial preparation of the laboratories to be involved, surveillance areas, selection of priority bacteria and antimicrobials to be used, and the design of a coordinated sampling plan. Recruitment should occur continuously in selected health facilities for at least 12 months to observe seasonal trends of AMR. At least 10 mL of clean-catch mid-stream urine must be collected into 20 mL calibrated sterile screw-capped universal bottles lined with 0.2 mg boric acid and transported to the testing laboratory. Utilise the data system that generates standard reports for patient care to be shared internally and externally in the regions and the world through global platforms such as the Global Antimicrobial Resistance Surveillance System.

Abstract Background Antimicrobials are extensively used in cattle and poultry production in Tanzania. However, there is dearth of information on its quantitative use. A questionnaire-based cross-sectional study was conducted from August to September 2019 in randomly selected poultry and small-scale dairy farms, in three districts of Dar es Salaam City eastern, Tanzania, to assess the practice and quantify antimicrobial use. Descriptive and statistical analyses were performed at a confidence interval of 95%. The ratio of Used Daily Dose (UDD) and Defined Daily Dose (DDD) were used to determine whether the antimicrobial was overdosed or under dosed. Results A total of 51 poultry and 65 small-scale dairy farms were involved in the study. The route of antimicrobial administration was 98% orally via drinking water and 2% in feeds for poultry and for small-scale dairy farms, all through parenteral route. Seventeen types of antimicrobials comprising seven classes were recorded in poultry farms while nine belonging to six classes in the small dairy farms. Majority of the farms (poultry, 87.7% and small scale dairy, 84.3%) used antimicrobials for therapeutic purposes. About 41% of the poultry and one third (34%) of the dairy farmers’ were not compliant to the drug withdrawal periods. Beta-lactams, fluoroquinolones, sulphonamides, tetracyclines and macrolides were the commonly used antimicrobials on these farms. In the poultry farms both those with records and those which relied on recall, antimicrobials were overdosed whereas in the small dairy farms, sulfadimidine, oxytetracycline and neomycin were within the appropriate dosing range (0.8–1.2). The majority (58.6%) of farmers had adequate level of practices (favorable) regarding antimicrobial use in cattle and poultry production. This was associated with the age and level of education of the cattle and poultry farmers. Conclusion The study revealed a widespread misuse of antimicrobials of different types and classes in both poultry and small-scale dairy farming in Dar es Salaam, Tanzania. This result gives insight into the antimicrobial use practices and its quantification. The information obtained can guide and promote prudent use of antimicrobials among the farmers by developing mitigate strategies that reduce antimicrobial resistance risk potentials.

Accuracy in the diagnosis is a key step to identify HIV infected individuals for appropriate management. Insufficient supply of manufacture´s buffers in relation to the number of strips per kit has negative impact on patient’s results hence improper patient´s management. In resource limited settings some laboratory staff use different substitute buffers which has never been validated on their reliability. This study aimed at comparing the performance of 0.85% saline and SD-Bioline manufacture’s buffer in detection of HIV antibodies. A total of 220 whole blood specimens: 110 HIV positive specimens from patients attending care and treatment center (CTC) and 110 HIV negative specimens from blood donors were re-tested for HIV status using SD-Bioline HIV rapid test using manufacturer´s buffer and 0.85% saline separately. Data and laboratory results were recorded in Microsoft excel sheet followed by analysis using STATA version 13. For all tested samples, the level of agreement between 0.85% saline and manufacturer’s buffer was 98.64% (kappa=0.9727).The value of kappa indicates very good agreement between 0.85% saline and manufacturer’s buffer. In incidents where manufacturers´ buffer is not sufficient, 0.85% saline can give reliable results. Further studies to evaluate the suitable buffer for other rapid tests for HIV and other diseases are recommended especially in resource limited settings.

The unique asymmetry of the Gram-negative outer membrane, with glycerophospholipids (GPLs) in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet, works to resist external stressors and prevent the entry of toxic compounds. Thus, GPL and LPS synthesis must be tightly controlled to maintain the integrity of this essential structure. We sought to decipher why organisms like Escherichia coli possess two redundant pathways-PlsB and PlsX/Y-for synthesis of the GPL precursor lysophosphatidic acid (LPA). LPA is then converted by PlsC to the universal precursor for GPL synthesis, phosphatidic acid (PA). PlsB and PlsC are essential in E. coli , indicating they serve as the major pathway for PA synthesis. While loss of PlsX or PlsY individually has little consequence on the cell, the absence of both was lethal. To understand the synthetic lethality of this seemingly redundant PlsX/Y pathway, we performed a suppressor screen. Suppressor analysis indicated that ∆ plsXY requires increased levels of glycerol-3-phosphate (G3P), a GPL precursor. In agreement, ∆ plsXY required supplementation with G3P for survival. Furthermore, loss of PlsX dysregulated fatty acid synthesis, resulting in increased long-chain fatty acids. We show that although PlsX/Y together contribute to PA synthesis, they also contribute to the regulation of overall membrane biogenesis. Thus, synthetic lethality of ∆ plsXY is multifactorial, suggesting that PlsX/Y has been maintained as a redundant system to fine-tune the synthesis of major lipids and promote cell envelope homeostasis.IMPORTANCEGram-negative bacteria must maintain optimal ratios of glycerophospholipids and lipopolysaccharide within the cell envelope for viability. Maintenance of proper outer membrane asymmetry allows for resistance to toxins and antibiotics. Here, we describe additional roles of PlsX and PlsY in Escherichia coli beyond lysophosphatidic acid synthesis, a key precursor of all glycerophospholipids. These findings suggest that PlsX and PlsY also play a larger role in impacting homeostasis of lipid synthesis., Competing Interests: The authors declare no conflict of interest.

Rhabdoid tumor predisposition syndrome type-1 (RTPS1) is characterized by germline pathogenic variants in SMARCB1 and development of INI1-deficient rhabdoid tumors in early childhood. Due to its poor prognosis, the risk of subsequent tumor development and the impact of surveillance at later ages are poorly understood. We retrospectively reviewed individuals referred to the Cancer Genetics Program at The Hospital for Sick Children for SMARCB1 genetic testing and/or surveillance for RTPS1. In addition, to explore characteristics of late-onset tumors in RTPS1, a literature review was conducted. Of eighty-three individuals (55 probands and 28 family members), 12 probands and 4 family members were genetically confirmed with RTPS1. Four pediatric probands with RTPS1 underwent surveillance. An additional three individuals, including one patient with 22q11.2 distal deletion without history of tumor, one patient with negative genetic testing results but clinically diagnosed with RTPS1, and one sibling identified through cascade testing, underwent surveillance. Three patients with RTPS1 developed tumors between the ages of 9 and 17, including malignant rhabdoid tumors (N = 3), schwannomas (N = 4), and epithelioid malignant peripheral nerve sheath tumor (N = 1). Three of these lesions were asymptomatically detected by surveillance. A literature review revealed 17 individuals with RTPS1 who developed INI1-deficient tumors after age five. Individuals with RTPS1 remain at elevated risk for developing INI1-deficient tumors after the peak age of rhabdoid tumor in early childhood. Extension of surveillance beyond 5 years of age could lead to improved survival and reduced morbidity for these patients, and prospective evaluation of revised approaches will be important., Competing Interests: Competing interests The authors declare no competing interests. Ethics approval This study was approved by the Research Ethics Board (REB#1000053261) at the Hospital for Sick Children (SickKids; Toronto, Canada)., (© 2024. The Author(s), under exclusive licence to European Society of Human Genetics.)

Key Clinical Message: Klippel-Trénaunay syndrome can present with atypical manifestations such as a bleeding vulvar hemangioma. This case report, the first documented in Uganda, highlights the need for awareness of such presentations and underscores the importance of continuous follow-up in female patients to manage potential complications throughout adolescence and pregnancy., Abstract: Klippel-Trénaunay syndrome (KTS) is a rare congenital disorder marked by bone and soft tissue hypertrophy, port-wine stains, and varicosities. Cases involving genital hemangiomas are rare. This report highlights a 5-year-old girl in Uganda with typical KTS features, including hypertrophy and port-wine stains, along with a bleeding vulvar hemangioma, emphasizing its uncommon presentation and potential complications. Treatment involved compression bandaging and timolol 0.2% solution. This case underscores the importance of awareness of atypical manifestations of hemangiomas with KTS and continuous follow-up for female patients, especially through adolescence and pregnancy, due to potential complications such as prepubertal per vaginal bleeding, menorrhagia, and post-partum bleeding., Competing Interests: The authors declare no competing interests., (© 2024 The Author(s). Clinical Case Reports published by John Wiley & Sons Ltd.)

Gram-negative bacteria produce a multilayered cell envelope in which their peptidoglycan is sandwiched between two membranes, an inner membrane made of glycerophospholipids and an asymmetric outer membrane with glycerophospholipids in the inner leaflet and lipopolysaccharide (LPS) in the outer leaflet. The Acinetobacter baumannii outer membrane contains lipooligosaccharide (LOS), a variant of LPS lacking O-antigen. LPS/LOS is typically essential, but A. baumannii can survive without LOS. Previously, we found that the peptidoglycan biogenesis protein NlpD becomes essential during LOS-deficiency. NlpD is typically redundant and is one of the cell's amidase activators for regulating peptidoglycan degradation, a process critical for cell division. We found that NlpD is essential under these conditions because a second putative amidase activator, termed WthA (cell w all turnover h ub protein A ), no longer functions in LOS-deficient cells. Mutants lacking WthA had severe cell division defects and were synthetically sick with loss of NlpD. Both Acinetobacter WthA and NlpD were found to activate an amidase activity of Oxa51, a chromosomally encoded β -lactamase. Further, WthA is homologous to Pseudomonas LbcA that impacts two other classes of peptidoglycan degradation enzymes, endopeptidases and lytic transglycosylases. WthA/LbcA homologs were identified across Proteobacteria, Bacteroidota, and Chlorobiota, suggesting they belong to a conserved family involved in regulation of peptidoglycan turnover. While Acinetobacter WthA may share functions of Pseudomonas LbcA, we found no evidence that LbcA is an amidase activator. Altogether, we have identified a missing player in Acinetobacter peptidoglycan biogenesis, a conserved hub protein that regulates multiple peptidoglycan turnover enzymes including cell division amidases., Significance Statement: Peptidoglycan is a rigid layer that provides structural support to bacterial cells. Peptidoglycan must be degraded to make room for new synthesis and for cells to divide, a process termed turnover. Turnover enzymes are tightly regulated to prevent their activities from lysing the cell. The critical pathogen Acinetobacter baumannii was missing known peptidoglycan amidases, a class of turnover enzymes, and the key activator that controls their activity during cell division. We have identified WthA as having a role in cell division most likely as an amidase activator. WthA homologs were widely distributed in bacteria and the closely related LbcA in Pseudomonas impacts two other types of turnover enzymes. We explore the possible functions of this new family of proteins that serves as a hub for impacting peptidoglycan turnover.

Gram-negative bacteria develop and exhibit resistance to antibiotics, owing to their highly asymmetric outer membrane maintained by a group of six proteins comprising the Mla (maintenance of lipid asymmetry) pathway. Here, we investigate the lipid binding preferences of one Mla protein, MlaC, which transports lipids through the periplasm. We used ultraviolet photodissociation (UVPD) to identify and characterize modifications of lipids endogenously bound to MlaC expressed in three different bacteria strains. UVPD was also used to localize lipid binding to MlaC residues 130-140, consistent with the crystal structure reported for lipid-bound MlaC. The impact of removing the bound lipid from MlaC on its structure was monitored based on collision cross section measurements, revealing that the protein unfolded prior to release of the lipid. The lipid selectivity of MlaC was evaluated based on titrimetric experiments, indicating that MlaC-bound lipids in various classes (sphingolipids, glycerophospholipids, and fatty acids) as long as they possessed no more than two acyl chains.

Background and Aim: Colorectal cancer (CRC) is the fourth leading cause of cancer death globally. CRC surveillance is a common indication for colonoscopy, representing a considerable burden for endoscopy services. Accurate identification of high-risk patients who would benefit from more intensive surveillance, as well as low-risk patients suitable for less frequent follow-up, could improve the effectiveness of surveillance protocols and resource use. Our aim was to identify and critically appraise published risk models for the occurrence of metachronous advanced colorectal neoplasia (ACN), defined here as CRC or advanced adenomas detected during surveillance colonoscopy., Methods: We searched PubMed and EMBASE for primary research studies reporting the development and/or validation of multivariable models that predict metachronous ACN risk. Screening of studies for inclusion, data extraction, and risk of bias assessment were conducted by two researchers independently., Results: We identified nine studies describing nine risk models. Six models were internally validated and two were externally validated. No model underwent both internal and external validation. Good model discrimination (concordance index > 0.7) was reported for two models during internal validation and for one model during external validation. Calibration was acceptable when assessed (n = 4). Methodological limitations and a high risk of bias were observed for all studies., Conclusions: Several published models predicting metachronous ACN risk showed some promise. However, adherence to methodological standards was limited, and only two models were externally validated. Head-to-head comparisons of existing models using populations independent from model development cohorts should be prioritized to identify models suitable for use in clinical practice., (© 2024 The Author(s). Journal of Gastroenterology and Hepatology published by Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.)

Objectives: The epidemiology of medication discrepancies during transitions from post-acute care (PAC) to home is poorly described. We sought to describe the frequency and types of medication discrepancies among hospitalized older adults transitioning from PAC to home., Design: A nested cohort analysis., Setting and Participants: Included participants enrolled in a patient-centered deprescribing trial, for patients (aged ≥50 years and taking at least 5 medications) transitioning from one of 22 PACs to home., Methods: We assessed demographic and medication measures at the initial hospitalization. The primary outcome measure was medication discrepancies, with the PAC discharge list serving as reference for comparison to the participant's self-reported medication list at 7 days following PAC discharge. Discrepancies were categorized as additions, omissions, and dose discrepancies and were organized by common medication classes and risk of harm (eg, 2015 Beers Criteria). Ordinal logistic regression assessed for patient risk factors for PAC discharge discrepancy count., Results: A total of 184 participants had 7-day PAC discharge medication data. Participants were predominately female (67%) and Caucasian (83%) with a median of 16 prehospital medications [interquartile range (IQR) 11, 20]. At the 7-day follow-up, 98% of participants had at least 1 medication discrepancy, with a median number of 7 medication discrepancies (IQR 4, 10) per person, 4 (IQR 2, 6) of which were potentially inappropriate medications as defined by the Beers Criteria. Higher medication discrepancies at index hospital admission and receipt of caregiver assistance with medications were 2 key predictors of medication discrepancies in the week after PAC discharge to home., Conclusions and Implications: Older patients transitioning home from a PAC facility are at high risk for medication discrepancies. This study underscores the need for interventions targeted at this overlooked transition period, especially as patients resume responsibility for managing their own medications after both a hospital and PAC stay., Competing Interests: Disclosure The authors have no relevant financial or personal conflicts of interest to report., (Published by Elsevier Inc.)

Loss of the Escherichia coli inner membrane protein YhcB results in pleomorphic cell morphology and clear growth defects. Prior work suggested that YhcB was directly involved in cell division or peptidoglycan assembly. We found that loss of YhcB is detrimental in genetic backgrounds in which lipopolysaccharide (LPS) or glycerophospholipid (GPL) synthesis is altered. The growth defect of Δ yhcB could be rescued through inactivation of the Mla pathway, a system responsible for the retrograde transport of GPLs that are mislocalized to the outer leaflet of the outer membrane. Interestingly, this rescue was dependent upon the outer membrane phospholipase PldA that cleaves GPLs at the bacterial surface. Since the freed fatty acids resulting from PldA activity serve as a signal to the cell to increase LPS synthesis, this result suggested that outer membrane lipids are imbalanced in Δ yhcB . Mutations that arose in Δ yhcB populations during two independent suppressor screens were in genes encoding subunits of the acetyl coenzyme A carboxylase complex, which initiates fatty acid biosynthesis (FAB). These mutations fully restored cell morphology and reduced GPL levels, which were increased compared to wild-type bacteria. Growth of Δ yhcB with the FAB-targeting antibiotic cerulenin also increased cellular fitness. Furthermore, genetic manipulation of FAB and lipid biosynthesis showed that decreasing FAB rescued Δ yhcB filamentation, whereas increasing LPS alone could not. Altogether, these results suggest that YhcB may play a pivotal role in regulating FAB and, in turn, impact cell envelope assembly and cell division.IMPORTANCESynthesis of the Gram-negative cell envelope is a dynamic and complex process that entails careful coordination of many biosynthetic pathways. The inner and outer membranes are composed of molecules that are energy intensive to synthesize, and, accordingly, these synthetic pathways are under tight regulation. The robust nature of the Gram-negative outer membrane renders it naturally impermeable to many antibiotics and therefore a target of interest for antimicrobial design. Our data indicate that when the inner membrane protein YhcB is absent in Escherichia coli , the pathway for generating fatty acid substrates needed for all membrane lipid synthesis is dysregulated which leads to increased membrane material. These findings suggest a potentially novel regulatory mechanism for controlling the rate of fatty acid biosynthesis., Competing Interests: The authors declare no conflict of interest.

Photoprotective properties of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) to reduce UV-induced DNA damage have been established in several studies. UV-induced DNA damage in skin such as single or double strand breaks is known to initiate several cellular mechanisms including activation of poly(ADP-ribose) (pADPr) polymerase-1 (PARP-1). DNA damage from UV also increases extracellular signal-related kinase (ERK) phosphorylation, which further increases PARP activity. PARP-1 functions by using cellular nicotinamide adenine dinucleotide (NAD+) to synthesise pADPr moieties and attach these to target proteins involved in DNA repair. Excessive PARP-1 activation following cellular stress such as UV irradiation may result in excessive levels of cellular pADPr. This can also have deleterious effects on cellular energy levels due to depletion of NAD+ to suboptimal levels. Since our previous work indicated that 1,25(OH) 2 D 3 reduced UV-induced DNA damage in part through increased repair via increased energy availability, the current study investigated the effect of 1,25(OH) 2 D 3 on UV-induced PARP-1 activity using a novel whole-cell enzyme- linked immunosorbent assay (ELISA) which quantified levels of the enzymatic product of PARP-1, pADPr. This whole cell assay used around 5000 cells per replicate measurement, which represents a 200-400-fold decrease in cell requirement compared to current commercial assays that measure in vitro pADPr levels. Using our assay, we observed that UV exposure significantly increased pADPr levels in human keratinocytes, while 1,25(OH) 2 D 3 significantly reduced levels of UV-induced pADPr in primary human keratinocytes to a similar extent as a known PARP-1 inhibitor, 3-aminobenzamide (3AB). Further, both 1,25(OH) 2 D 3 and 3AB as well as a peptide inhibitor of ERK-phosphorylation significantly reduced DNA damage in UV-exposed keratinocytes. The current findings support the proposal that reduction in pADPr levels may be critical for the function of 1,25(OH) 2 D 3 in skin to reduce UV-induced DNA damage.

In this review, we discuss some of the recent advances in our understanding of the physiology of the air pollution and exercise. The key areas covered include the effect of exercise intensity, the effects of pre-exposure to air pollution, acclimation to air pollution, and the utility of masks during exercise. Although higher intensity exercise leads to an increase in the inhaled dose of pollutants for a given distance traveled, the acute effects of (diesel exhaust) air pollution do not appear to be more pronounced. Second, exposure to air pollution outside of exercise bouts seems to have an effect on exercise response, although little research has examined this relationship. Third, humans appear to have an ability to acclimate to ground level ozone, but not other pollutants. And finally, masks may have beneficial effects on certain outcomes at low intensity exercise in pollution with significant levels of particles, but more study is required in realistic conditions., (© 2024 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

A fundamental feature of Gram-negative bacteria is their outer membrane that protects the cell against environmental stressors. This defense is predominantly due to its asymmetry, with glycerophospholipids located in the inner leaflet and lipopolysaccharide (LPS) or lipooligosaccharide (LOS) confined to the outer leaflet. LPS consists of a lipid A anchor, a core oligosaccharide, and a distal O-antigen while LOS lacks O-antigen. While LPS/LOS is typically essential for growth, this is not the case for Acinetobacter baumannii . Despite this unique property, the synthesis of the core oligosaccharide of A. baumannii LOS is not well-described. Here, we characterized the LOS chemotypes of A. baumannii strains with mutations in a predicted core oligosaccharide locus via tandem mass spectrometry. This allowed for an extensive identification of genes required for core assembly that can be exploited to generate precise structural LOS modifications in many A. baumannii strains. We further investigated two chemotypically identical yet phenotypically distinct mutants, ∆ 2903 and ∆ lpsB , that exposed a possible link between LOS and the peptidoglycan cell wall-two cell envelope components whose coordination has not yet been described in A. baumannii . Selective reconstruction of the core oligosaccharide via expression of 2903 and LpsB revealed that these proteins rely on each other for the unusual tandem transfer of two residues, KdoIII and N-acetylglucosaminuronic acid. The data presented not only allow for better usage of A. baumannii as a tool to study outer membrane integrity but also provide further evidence for a novel mechanism of core oligosaccharide assembly., Importance: Acinetobacter baumannii is a multidrug-resistant pathogen that produces lipooligosaccharide (LOS), a glycolipid that confers protective asymmetry to the bacterial outer membrane. The core oligosaccharide is a ubiquitous component of LOS that typically follows a well-established model of synthesis. In addition to providing an extensive analysis of the genes involved in the synthesis of the core region, we demonstrate that this organism has evidently diverged from the long-held archetype of core synthesis. Moreover, our data suggest that A. baumannii LOS assembly is important for cell division and likely intersects with the synthesis of the peptidoglycan cell wall, another essential component of the Gram-negative cell envelope. This connection between LOS and cell wall synthesis provides an intriguing foundation for a unique method of outer membrane biogenesis and cell envelope coordination., Competing Interests: The authors declare no conflict of interest.

Polyphosphates (polyP) are chains of inorganic phosphates that can reach over 1,000 residues in length. In Escherichia coli, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the molecular pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work, we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP (Δppk) during starvation to identify novel pathways regulated by PPK. In response to starvation, we found 92 proteins significantly differentially expressed between wild-type and Δppk mutant cells. Wild-type cells were enriched for proteins related to amino acid biosynthesis and transport, while Δppk mutants were enriched for proteins related to translation and ribosome biogenesis, suggesting that without PPK, cells remain inappropriately primed for growth even in the absence of the required building blocks. From our data set, we were particularly interested in Arn and EptA proteins, which were down-regulated in Δppk mutants compared to wild-type controls, because they play a role in lipid A modifications linked to polymyxin resistance. Using western blotting, we confirm differential expression of these and related proteins in K-12 strains and a uropathogenic isolate, and provide evidence that this mis-regulation in Δppk cells stems from a failure to induce the BasRS two-component system during starvation. We also show that Δppk mutants unable to up-regulate Arn and EptA expression lack the respective L-Ara4N and pEtN modifications on lipid A. In line with this observation, loss of ppk restores polymyxin sensitivity in resistant strains carrying a constitutively active basR allele. Overall, we show a new role for PPK in lipid A modification during starvation and provide a rationale for targeting PPK to sensitize bacteria towards polymyxin treatment. We further anticipate that our proteomics work will provide an important resource for researchers interested in the diverse pathways impacted by PPK., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Baijal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Lipopolysaccharide (LPS) is a fundamental tripartite glycolipid found on the surface of nearly all Gram-negative bacteria. It acts as a protective shield for the bacterial cell and is a potent agonist of the innate immune system. This primer serves to introduce the basic properties of LPS, its function in bacterial physiology and pathogenicity, and its use as a therapeutic target.

The bacterial peptidoglycan (PG) cell wall is remodeled during growth and division, releasing fragments called muropeptides. Muropeptides can be internalized and reused in a process called PG recycling. Escherichia coli is highly devoted to recycling muropeptides and is known to have at least two transporters, AmpG and OppBCDF, that import them into the cytoplasm. While studying mutants lacking AmpG, we unintentionally isolated mutations that led to the altered expression of a third transporter, CadB. CadB is normally upregulated under acidic pH conditions and is an antiporter for lysine and cadaverine. Here, we explored if CadB was altering PG recycling to assist in the absence of AmpG. Surprisingly, CadB overexpression was able to restore PG recycling when both AmpG and OppBCDF were absent. CadB was found to import freed PG peptides, a subpopulation of muropeptides, through a promiscuous activity. Altogether, our data support that CadB is a third transporter capable of contributing to PG recycling. IMPORTANCE Bacteria produce a rigid mesh cell wall. During growth, the cell wall is remodeled, which releases cell wall fragments. If released into the extracellular environment, cell wall fragments can trigger inflammation by the immune system of a host. Gastrointestinal bacteria, like Escherichia coli , have dedicated pathways to recycle almost all cell wall fragments they produce. E. coli contains two known recycling transporters, AmpG and Opp, that we previously showed are optimized for growth in different environments. Here, we identify that a third transporter, CadB, can also contribute to cell wall recycling. This work expands our understanding of cell wall recycling and highlights the dedication of organisms like E. coli to ensure high recycling in multiple growth environments., Competing Interests: The authors declare no conflict of interest.

Purpose: The impact of COVID-19 is challenging for many health and social care workers. The impact has been more felt by all ethnic groups, but during the course of its tenure, it has become more apparent that the black community has been affected more than others. They have been reported to suffer more fatalities from the pandemic compared to their white counterparts. Blacks are reported to make a significant percentage of health care workers. They are sometimes undervalued, lowly paid, with many on insecure contracts and experiencing professional inequality. This study sought to explore the challenges experienced by Black Sub-Saharan African (BSSA) front-line workers in health care during COVID-19 pandemic., Methodology: The study utilised an explorative qualitative approach (EQA). Forty research participants were recruited for the study. Semi-structured interviews were used to collect data through online platforms which included Zoom, WhatsApp and Teams. A thematic approach was used to analyse data., Results: Following data analysis, the research found that the research participants experienced undermining of expertise, lack of appreciation and unfair allocation of tasks and were overlooked for promotion and perceived as carriers of COVID-19., Conclusion: This group was over-represented in agency and self-employed roles. There is need for a strong government commitment to prevent discrimination through enacting a comprehensive legislation to support tackling the problem. Race equality training awareness needs to be rolled out into healthcare organisations and empower managers to deal with equality issues at work., (© 2024. Crown.)

Regenerative agriculture is an alternative approach to farming that has been gaining traction and interest among farmers due to its potential to reduce input costs, improve soil health, and increase the resilience of farming systems. This paper undertakes a practice-based analysis of farmers, applying a lens of regenerative agriculture. Surveys were developed as a part of a broader project using an established methodological framework. Topics were developed and adapted with input from local stakeholders before being mailed out to three farming regions across Australia (the Western Australian Wheatbelt, the Eyre Peninsula in South Australia and Central West New South Wales). The research clustered farmers into two groups: those who are using best-practices that fall inside the scope of regenerative agriculture, and those who are not. The similarities and differences in farmer attributes, as well as self-reported knowledge levels and information sources used by each group are explored. Results indicate that a belief in anthropogenic climate change may be one of the primary divides between the two groups, and therefore a possible driver of best-practice implementation. The findings provide insight into perceptions of regenerative agriculture for Australian farmers, and may assist with knowledge dissemination amongst those managing our environment., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Dr Hanabeth Luke reports financial support was provided by Cooperative Research Centre for High Performance Soils (Soil CRC). Mathew Alexanderson reports financial support was provided by Cooperative Research Centre for High Performance Soils (Soil CRC)., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Although thrombocytopenia in neonatal intensive care patients is rarely due to inherited disorders, the number of genetic variants implicated in platelet defects has grown dramatically with increasing genome-wide sequencing. Here we describe a case of severe, oligogenic neonatal thrombocytopenia and reinterpret a reportedly benign mutation that is likely pathogenic. Despite this patient's synonymous mutation ( GFI1B 576 C>T, Phe192=) being annotated as benign, GFI1B is a well-known regulator of megakaryopoiesis, this variant alters splicing and megakaryocyte maturation, and our analysis of existing genome-wide associated studies demonstrates that it likely causes gray platelet syndrome. This variant has not been reported in a case of life-threatening thrombocytopenia. We propose that the severity of this patient's phenotype is due to synergistic epistasis between the intrinsic platelet defect caused by this mutation and her concomitant inherited PMM2 congenital glycosylation disorder neither of which have been associated with such a severe phenotype. This case highlights the importance of whole-exome/genome sequencing for critically ill patients, reexamining variant interpretation when clinically indicated, and the need to study diverse genetic variation in hematopoiesis.

Bacteria produce a structural layer of peptidoglycan (PG) that enforces cell shape, resists turgor pressure, and protects the cell. As bacteria grow and divide, the existing layer of PG is remodeled and PG fragments are released. Enterics such as Escherichia coli go to great lengths to internalize and reutilize PG fragments. E. coli is estimated to break down one-third of its cell wall, yet only loses ~0 to 5% of meso-diaminopimelic acid, a PG-specific amino acid, per generation. Two transporters were identified early on to possibly be the primary permease that facilitates PG fragment recycling, i) AmpG and ii) the Opp ATP binding cassette transporter in conjunction with a PG-specific periplasmic binding protein, MppA. The contribution of each transporter to PG recycling has been debated. Here, we have found that AmpG and MppA/Opp are differentially regulated by carbon source and growth phase. In addition, MppA/Opp is uniquely capable of high-affinity scavenging of muropeptides from growth media, demonstrating that AmpG and MppA/Opp allow for different strategies of recycling PG fragments. Altogether, this work clarifies environmental contexts under which E. coli utilizes distinct permeases for PG recycling and explores how scavenging by MppA/Opp could be beneficial in mixed communities.

The modification of lipopolysaccharide (LPS) in Escherichia coli and Salmonella spp. is primarily controlled by the two-component system PmrAB. LPS modification allows bacteria to avoid killing by positively charged antibiotics like polymyxin B (PMB). We previously demonstrated that in uropathogenic E. coli (UPEC), the sensor histidine kinase PmrB also activates a non-cognate transcription factor, QseB, and this activation somehow augments PMB tolerance in UPEC. Here, we demonstrate-for the first time-that in the absence of the canonical LPS transcriptional regulator, PmrA, QseB can direct some modifications on the LPS. In agreement with this observation, transcriptional profiling analyses demonstrate regulatory overlaps between PmrA and QseB in terms of regulating LPS modification genes. However, both PmrA and QseB must be present for UPEC to mount robust tolerance to PMB. Transcriptional and metabolomic analyses also reveal that QseB transcriptionally regulates the metabolism of glutamate and 2-oxoglutarate, which are consumed and produced during the modification of lipid A. We show that deletion of qseB alters glutamate levels in the bacterial cells. The qseB deletion mutant, which is susceptible to positively charged antibiotics, is rescued by exogenous addition of 2-oxoglutarate. These findings uncover a previously unknown mechanism of metabolic control of antibiotic tolerance that may be contributing to antibiotic treatment failure in the clinic. IMPORTANCE Although antibiotic prescriptions are guided by well-established susceptibility testing methods, antibiotic treatments oftentimes fail. The presented work is significant because it uncovers a mechanism by which bacteria transiently avoid killing by antibiotics. This mechanism involves two closely related transcription factors, PmrA and QseB, which are conserved across Enterobacterales . We demonstrate that PmrA and QseB share regulatory targets in lipid A modification pathway and prove that QseB can orchestrate modifications of lipid A in Escherichia coli in the absence of PmrA. Finally, we show that QseB controls glutamate metabolism during the antibiotic response. These results suggest that rewiring of QseB-mediated metabolic genes could lead to stable antibiotic resistance in subpopulations within the host, thereby contributing to antibiotic treatment failure., Competing Interests: The authors declare no conflict of interest.

More than 155,000 New Yorkers were trained in Mental Health First Aid (MHFA) between 2016 and 2020. Free citywide trainings were made available to all New Yorkers and were disseminated through city agencies and community-based settings. RAND Corporation researchers conducted a mixed-methods study that included a web-based survey of past trainees and a series of focus groups with leaders of community-based organizations and city agency staff to assess the impact of the MHFA trainings and needs for future training. In this article, the authors describe the evaluation activities that took place; the methods behind them; and the results at the individual, agency, and community levels. They also offer recommendations for ways to improve future mental health education efforts. Respondents applied MHFA skills extensively and broadly across their social networks. Nine in ten respondents had contact with an individual with a mental health problem in the past six months. Among those who had contact, 84 percent indicated using their MHFA skills to help a friend or family member, and nearly half reported applying skills with a co-worker, neighbor, or acquaintance. Because MHFA was offered through city agency workplaces and community-based settings, tens of thousands of New Yorkers were given tools to come to the aid of individuals in their personal and professional lives. MHFA may be a promising approach to building supportive social networks, organizations, and communities that are primed to recognize and assist those experiencing mental health challenges., (Copyright © 2023 RAND Corporation.)

Plant cell walls are composed of a heterogeneous mixture of polysaccharides that require several different enzymes to degrade. These enzymes are important for a variety of biotechnological processes, from biofuel production to food processing. Several classical mannanolytic enzyme functions of glycoside hydrolases (GH), such as β-mannanase, β-mannosidase and α-galactosidase activities, are helpful for efficient mannan hydrolysis. In this light, we bring three enzymes into the model of mannan degradation that have received little or no attention. By linking their three-dimensional structures and substrate specificities, we have predicted the interactions and cooperativity of these novel enzymes with classical mannanolytic enzymes for efficient mannan hydrolysis. The novel exo-β-1,4-mannobiohydrolases are indispensable for the production of mannobiose from the terminal ends of mannans, this product being the preferred product for short-chain mannooligosaccharides (MOS)-specific β-mannosidases. Second, the side-chain cleaving enzymes, acetyl mannan esterases (AcME), remove acetyl decorations on mannan that would have hindered backbone cleaving enzymes, while the backbone cleaving enzymes liberate MOS, which are preferred substrates of the debranching and sidechain cleaving enzymes. The nonhydrolytic expansins and swollenins disrupt the crystalline regions of the biomass, improving their accessibility for AcME and GH activities. Finally, lytic polysaccharide monooxygenases have also been implicated in promoting the degradation of lignocellulosic biomass or mannan degradation by classical mannanolytic enzymes, possibly by disrupting adsorbed mannan residues. Modelling effective enzymatic mannan degradation has implications for improving the saccharification of biomass for the synthesis of value-added and upcycling of lignocellulosic wastes., (© 2023. The Author(s).)

Purpose: Advanced genomic and genetic testing technologies are quickly diffusing into clinical practice, but standardized approaches to assessing their clinical utility are limited. Previous work developed and generated preliminary evidence of validity for a novel outcome measure, the Clinician-reported Genetic testing Utility InDEx (C-GUIDE). C-GUIDE is a 17-item measure that captures the utility of genetic testing from the providers' perspective. Preliminary evidence of its inter-rater reliability was obtained through a clinical vignette study. The purpose of this study was to further assess its inter-rater reliability using actual clinical cases., Methods: One genetic counselor and one medical geneticist independently completed C-GUIDE Version 1.1 after genetic test results were disclosed to a shared set of 42 patients. Raters also completed a case description questionnaire, including information about the patient's age, indication for testing, and type of test performed. Inter-rater reliability was assessed by comparing the raters' C-GUIDE scores using ANOVA to generate intra-class correlation coefficients (ICCs), absolute agreement, and mixed repeated measures ANOVA., Findings: Of the 42 patients studied, the most common indications for testing were hearing loss (n = 18) and craniosynostosis (n = 11), and the most common tests ordered were gene panels (n = 20) and microarrays (n = 10). Test results were diagnostic or partially diagnostic for 11 patients, potentially diagnostic for 14 patients, or nondiagnostic for 17 patients. The overall ICC was 0.95 (95% CI, 0.89-0.97) and absolute agreement was acceptable (>70%) for 15 individual items. Inter-rater agreement was excellent (ICC > 0.90) for 8 items, good (ICC = 0.75-0.89) for 3 items, moderate (ICC = 0.50-0.74) for 4 items and poor (ICC < 0.50) for 2 items. Absolute agreement was unacceptable (<70%), and rater agreement was fair (ICC = 0.40-0.59) for 2 items. For the global rating, the ICC was 0.62 (95% CI, 0.39-0.77), and the absolute agreement was 61.9%., Implications: Rater instructions for item completion have been modified to improve consistency of item interpretation. Although further assessments of reliability are warranted after modifications, these findings provide additional tentative evidence of C-GUIDE's inter-rater reliability and suggest that it may be useful as a strategy for measuring the value of genetic testing, as perceived by genetics providers., Competing Interests: Declaration of Competing Interest Dr Hayeems is supported by the Canada Research Chair in Genomics and Health Policy (Tier 2); and is a member of the Ontario Genetics Advisory Committee and the Provincial Genetics Advisory Committee. Dr Meyn received an honorarium for review of the National Institutes of Health intramural program; holds a patent for PhenoTips software; is a member of the Directors Committee of the National Organization for Rare Disease Centers of Excellence (USA); and has 1% stock ownership in Gene42. Dr Ungar is supported by the Canada Research Chair in Economic Evaluation and Technology Assessment in Child Health; and is the Chair of the Ontario Genetics Advisory Committee. The authors have indicated that they have no other conflicts of interest regarding the content of this article. The Canadian Institutes of Health Research did not have any role in the study design, collection, analysis and interpretation of data, or in the writing and decision to submit the manuscript., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Ultraviolet radiation (UVR) induces immunosuppression and DNA damage, both of which contribute to the rising global incidence of skin cancer including melanoma. Nucleotide excision repair, which is activated upon UVR-induced DNA damage, is linked to expression of interleukin-12 (IL-12) which serves to limit immunosuppression and augment the DNA repair process. Herein, we report an immunomodulating peptide, designated IK14800, that not only elicits secretion of IL-12, interleukin-2 (IL-2) and interferon-gamma (IFN-γ) but also reduces DNA damage in the skin following exposure to UVR. Combined with re-invigoration of exhausted CD4+ T cells, inhibition of UVR-induced MMP-1 release and suppression of B16F10 melanoma metastases, IK14800 offers an opportunity to gain further insight into mechanisms underlying the development and progression of skin cancers., (© 2023. The Author(s).)

The hormonal form of vitamin D 3 , 1,25(OH) 2 D 3 , reduces UV-induced DNA damage. UV exposure initiates pre-vitamin D 3 production in the skin, and continued UV exposure photoisomerizes pre-vitamin D 3 to produce "over-irradiation products" such as lumisterol 3 (L 3 ). Cytochrome P450 side-chain cleavage enzyme (CYP11A1) in skin catalyzes the conversion of L 3 to produce three main derivatives: 24-hydroxy-L 3 [24(OH)L 3 ], 22-hydroxy-L 3 [22(OH)L 3 ], and 20,22-dihydroxy-L 3 [20,22(OH)L 3 ]. The current study investigated the photoprotective properties of the major over-irradiation metabolite, 24(OH)L 3 , in human primary keratinocytes and human skin explants. The results indicated that treatment immediately after UV with either 24(OH)L 3 or 1,25(OH) 2 D 3 reduced UV-induced cyclobutane pyrimidine dimers and oxidative DNA damage, with similar concentration response curves in keratinocytes, although in skin explants, 1,25(OH) 2 D 3 was more potent. The reductions in DNA damage by both compounds were, at least in part, the result of increased DNA repair through increased energy availability via increased glycolysis, as well as increased DNA damage recognition proteins in the nucleotide excision repair pathway. Reductions in UV-induced DNA photolesions by either compound occurred in the presence of lower reactive oxygen species. The results indicated that under in vitro and ex vivo conditions, 24(OH)L 3 provided photoprotection against UV damage similar to that of 1,25(OH) 2 D 3 .

Purpose: Mucosal melanoma is a rare, aggressive form of melanoma with extremely high recurrence rates despite definitive surgical resection with curative intent. Currently there is no consensus on adjuvant therapy. Data on checkpoint inhibitors for adjuvant therapy are lacking., Patients and Methods: We performed a single-arm, multicenter clinical trial using "flip dose" ipilimumab (1 mg/kg q3w × 4 cycles), and nivolumab (3 mg/kg q3w × 4 cycles), then nivolumab 480 mg q4w × 11 cycles to complete a year of adjuvant therapy. Participants must have had R0/R1 resection ≤90 days before registration, no prior systemic therapy (adjuvant radiotherapy allowed), ECOG 0/1, and no uncontrolled autoimmune disease or other invasive cancer. Patients were recruited through the Midwest Melanoma Partnership/Hoosier Oncology Network., Results: From September 2017 to August 2021, 35 patients were enrolled. Of these, 29 (83%) had R0 resections, and 7 (20%) received adjuvant radiotherapy. Median age was 67 years, 21 (60.0%) female. Recurrence-free survival (RFS) rates at 1 and 2 years were 50% [95% confidence interval (CI), 31%-66%] and 37% (95% CI, 19%-55%), respectively. Overall survival rates at 1 and 2 years were 87% (95% CI, 68%-95%) and 68% (95% CI, 46%-83%), respectively. Median RFS was 10.3 months (95% CI, 5.7-25.8). Most common grade 3 toxicities were diarrhea (14%), hypertension (14%), and hyponatremia (11%), with no grade 4/5 toxicities., Conclusions: Flip-dose ipilimumab and nivolumab after resection of mucosal melanoma is associated with outcomes improved over that of surgical resection alone. Long-term follow-up, subgroup analyses and correlative studies are ongoing., (©2023 American Association for Cancer Research.)

This work demonstrated the potential of CNC as a substitute for PEG as an additive in ultrafiltration membrane fabrication. Two sets of modified membranes were fabricated using the phase inversion technique, with polyethersulfone (PES) as the base polymer and 1-N-methyl-2 pyrrolidone (NMP) as the solvent. The first set was fabricated with 0.075 wt% CNC, while the second set was fabricated with 2 wt% PEG. All membranes were characterized using SEM, EDX, FTIR, and contact angle measurements. The SEM images were analyzed for surface characteristics using WSxM 5.0 Develop 9.1 software. The membranes were tested, characterized, and compared for their performance in treating both synthetic and real restaurant wastewater. Both membranes exhibited improved hydrophilicity, morphology, pore structure, and roughness. Both membranes also exhibited similar water flux for real and synthetic polluted water. However, the membrane prepared with CNC gave higher turbidity removal and COD removal when raw restaurant water was treated. The membrane compared well with the UF membrane containing 2 wt% PEG in terms of morphology and performance when synthetic turbid water and raw restaurant water were treated., Competing Interests: The authors declare no conflicts of interest.

Venezuelan equine encephalitis virus (VEEV) is a disease typically confined to South and Central America, whereby human disease is characterised by a transient systemic infection and occasionally severe encephalitis, which is associated with lethality. Using an established mouse model of VEEV infection, the encephalitic aspects of the disease were analysed to identify biomarkers associated with inflammation. Sequential sampling of lethally challenged mice (infected subcutaneously) confirmed a rapid onset systemic infection with subsequent spread to the brain within 24 h of the challenge. Changes in inflammatory biomarkers (TNF-α, CCL-2, and CCL-5) and CD45 + cell counts were found to correlate strongly to pathology (R>0.9) and present previously unproven biomarkers for disease severity in the model, more so than viral titre. The greatest level of pathology was observed within the olfactory bulb and midbrain/thalamus. The virus was distributed throughout the brain/encephalon, often in areas not associated with pathology. The principal component analysis identified five principal factors across two independent experiments, with the first two describing almost half of the data: (1) confirmation of a systemic Th1-biased inflammatory response to VEEV infection, and (2) a clear correlation between specific inflammation of the brain and clinical signs of disease. Targeting strongly associated biomarkers of deleterious inflammation may ameliorate or even eliminate the encephalitic syndrome of this disease.

Gram-negative bacteria have a unique cell surface that can be modified to maintain bacterial fitness in diverse environments. A well-defined example is the modification of the lipid A component of lipopolysaccharide (LPS), which promotes resistance to polymyxin antibiotics and antimicrobial peptides. In many organisms, such modifications include the addition of the amine-containing constituents 4-amino-4-deoxy-l-arabinose (l-Ara4N) and phosphoethanolamine (pEtN). Addition of pEtN is catalyzed by EptA, which uses phosphatidylethanolamine (PE) as its substrate donor, resulting in production of diacylglycerol (DAG). DAG is then quickly recycled into glycerophospholipid (GPL) synthesis by the DAG kinase A (DgkA) to produce phosphatidic acid, the major GPL precursor. Previously, we hypothesized that loss of DgkA recycling would be detrimental to the cell when LPS is heavily modified. Instead, we found that DAG accumulation inhibits EptA activity, preventing further degradation of PE, the predominant GPL of the cell. However, DAG inhibition of pEtN addition results in complete loss of polymyxin resistance. Here, we selected for suppressors to find a mechanism of resistance independent of DAG recycling or pEtN modification. Disrupting the gene encoding the adenylate cyclase, cyaA , fully restored antibiotic resistance without restoring DAG recycling or pEtN modification. Supporting this, disruptions of genes that reduce CyaA-derived cAMP formation (e.g., ptsI ) or disruption of the cAMP receptor protein, Crp, also restored resistance. We found that loss of the cAMP-CRP regulatory complex was necessary for suppression and that resistance arises from a substantial increase in l-Ara4N-modified LPS, bypassing the need for pEtN modification. IMPORTANCE Gram-negative bacteria can alter the structure of their LPS to promote resistance to cationic antimicrobial peptides, including polymyxin antibiotics. Polymyxins are considered last-resort antibiotics for treatment against multidrug-resistant Gram-negative organisms. Here, we explore how changes in general metabolism and carbon catabolite repression pathways can alter LPS structure and influence polymyxin resistance., Competing Interests: The authors declare no conflict of interest.

Genome sequencing (GS) is a powerful test for the diagnosis of rare genetic disorders. Although GS can enumerate most non-coding variation, determining which non-coding variants are disease-causing is challenging. RNA sequencing (RNA-seq) has emerged as an important tool to help address this issue, but its diagnostic utility remains understudied, and the added value of a trio design is unknown. We performed GS plus RNA-seq from blood using an automated clinical-grade high-throughput platform on 97 individuals from 39 families where the proband was a child with unexplained medical complexity. RNA-seq was an effective adjunct test when paired with GS. It enabled clarification of putative splice variants in three families, but it did not reveal variants not already identified by GS analysis. Trio RNA-seq decreased the number of candidates requiring manual review when filtering for de novo dominant disease-causing variants, allowing for the exclusion of 16% of gene-expression outliers and 27% of allele-specific-expression outliers. However, clear diagnostic benefit from the trio design was not observed. Blood-based RNA-seq can facilitate genome analysis in children with suspected undiagnosed genetic disease. In contrast to DNA sequencing, the clinical advantages of a trio RNA-seq design may be more limited., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Chagas disease is a significant public health risk in rural and semi-rural areas of Venezuela. Triatomine infection by the aetiological agent Trypanosoma cruzi is also observed in the Metropolitan District of Caracas (MDC), where foodborne T. cruzi outbreaks occasionally occur but active vector-to-human transmission (infection during triatomine bloodmeal) is considered absent. Citizen science-based domiciliary triatomine collection carried out between 2007 and 2013 in the MDC has advanced understanding of urban T. cruzi prevalence patterns and represents an important public awareness-building tool. The present study reports on the extension of this triatomine collection program from 2014 to 2019 and uses mitochondrial metabarcoding to assess feeding behavior in a subset of specimens. The combined, thirteen-year dataset (n = 4872) shows a high rate of T. cruzi infection (75.2%) and a predominance of Panstrongylus geniculatus (99.01%) among triatomines collected in domiciliary areas by MDC inhabitants. Collection also involved nymphal stages of P. geniculatus in 18 of 32 MDC parishes. Other collected species included Triatoma nigromaculata, Triatoma maculata, Rhodnius prolixus, and Panstrongylus rufotuberculatus. Liquid intestinal content indicative of bloodmeal was observed in 53.4% of analyzed specimens. Dissection pools representing 108 such visually blooded P. geniculatus specimens predominantly tested positive for human cytochrome b DNA (22 of 24 pools). Additional bloodmeal sources detected via metabarcoding analysis included key sylvatic T. cruzi reservoirs (opossum and armadillo), rodents, and various other synanthropic and domesticated animals. Results suggest a porous sylvatic-domiciliary transmission interface and ongoing adaptation of P. geniculatus to the urban ecotope. Although P. geniculatus defecation traits greatly limit the possibility of active T. cruzi transmission for any individual biting event, the cumulation of this low risk across a vast metropolitan population warrants further investigation. Efforts to prevent triatomine contact with human food sources also clearly require greater attention to protect Venezuela's capital from Chagas disease., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Segovia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The calcium-sensing receptor (CaSR) is an important regulator of epidermal function. We previously reported that knockdown of the CaSR or treatment with its negative allosteric modulator, NPS-2143, significantly reduced UV-induced DNA damage, a key factor in skin cancer development. We subsequently wanted to test whether topical NPS-2143 could also reduce UV-DNA damage, immune suppression, or skin tumour development in mice. In this study, topical application of NPS-2143 (228 or 2280 pmol/cm 2 ) to Skh:hr1 female mice reduced UV-induced cyclobutane pyrimidine dimers (CPD) ( p < 0.05) and oxidative DNA damage (8-OHdG) ( p < 0.05) to a similar extent as the known photoprotective agent 1,25(OH) 2 vitamin D3 (calcitriol, 1,25D). Topical NPS-2143 failed to rescue UV-induced immunosuppression in a contact hypersensitivity study. In a chronic UV photocarcinogenesis protocol, topical NPS-2143 reduced squamous cell carcinomas for only up to 24 weeks ( p < 0.02) but had no other effect on skin tumour development. In human keratinocytes, 1,25D, which protected mice from UV-induced skin tumours, significantly reduced UV-upregulated p-CREB expression ( p < 0.01), a potential early anti-tumour marker, while NPS-2143 had no effect. This result, together with the failure to reduce UV-induced immunosuppression, may explain why the reduction in UV-DNA damage in mice with NPS-2143 was not sufficient to inhibit skin tumour formation.

Background: Children with medical complexity (CMC) are a priority pediatric population, with high resource use and associated costs. Genome-wide sequencing is increasingly organized for CMC early in life as a diagnostic test. Polypharmacy becomes common as CMC age. Clinically relevant pharmacogenetic (PGx) information can be extracted from existing genome sequencing (GS) data via GS-PGx profiling. The role of GS-PGx profiling in the CMC population is unclear., Methods: Prescribed medications were extracted from care plans of 802 eligible CMC enrolled in a structured Complex Care Program over a 10-year period. Drug-gene associations were annotated using curated Clinical Pharmacogenetics Implementation Consortium data. GS-PGx profiling was then performed for a subset of 50 CMC., Results: Overall, 546 CMC (68%) were prescribed at least one medication with an established PGx association. In the GS-PGx subgroup, 24 (48%) carried variants in pharmacogenes with drug-gene guidelines for one or more of their current medications. All had findings of potential relevance to some medications, including 32 (64%) with variants in CYP2C19 that could affect their metabolism of proton-pump inhibitors., Conclusion: GS-PGx profiling at the time of diagnostics-focused genetic testing could be an efficient way to incorporate precision prescribing practices into the lifelong care of CMC., Impact: Polypharmacy and genetic test utilization are both common in children with medical complexity. The role of repurposing genome sequencing data for pharmacogenetic profiling in children with medical complexity was previously unclear. We identified a high rate of medication use with clinically relevant drug-gene associations in this priority pediatric population and demonstrated that relevant pharmacogenetic information can be extracted from their existing genome sequencing data. Pharmacogenetic profiling at the time of diagnostics-focused genetic testing could be an efficient way to incorporate precision prescribing practices into the lifelong care of children with medical complexity., (© 2022. The Author(s).)

Importance: Deprescribing is a promising approach to addressing the burden of polypharmacy. Few studies have initiated comprehensive deprescribing in the hospital setting among older patients requiring ongoing care in a postacute care (PAC) facility., Objective: To evaluate the efficacy of a patient-centered deprescribing intervention among hospitalized older adults transitioning or being discharged to a PAC facility., Design, Setting, and Participants: This randomized clinical trial of the Shed-MEDS (Best Possible Medication History, Evaluate, Deprescribing Recommendations, and Synthesis) deprescribing intervention was conducted between March 2016 and October 2020. Patients who were admitted to an academic medical center and discharged to 1 of 22 PAC facilities affiliated with the medical center were recruited. Patients who were 50 years or older and had 5 or more prehospital medications were enrolled and randomized 1:1 to the intervention group or control group. Patients who were non-English speaking, were unhoused, were long-stay residents of nursing homes, or had less than 6 months of life expectancy were excluded. An intention-to-treat approach was used., Interventions: The intervention group received the Shed-MEDS intervention, which consisted of a pharmacist- or nurse practitioner-led comprehensive medication review, patient or surrogate-approved deprescribing recommendations, and deprescribing actions that were initiated in the hospital and continued throughout the PAC facility stay. The control group received usual care at the hospital and PAC facility., Main Outcomes and Measures: The primary outcome was the total medication count at hospital discharge and PAC facility discharge, with follow-up assessments during the 90-day period after PAC facility discharge. Secondary outcomes included the total number of potentially inappropriate medications at each time point, the Drug Burden Index, and adverse events., Results: A total of 372 participants (mean [SD] age, 76.2 [10.7] years; 229 females [62%]) were randomized to the intervention or control groups. Of these participants, 284 were included in the intention-to-treat analysis (142 in the intervention group and 142 in the control group). Overall, there was a statistically significant treatment effect, with patients in the intervention group taking a mean of 14% fewer medications at PAC facility discharge (mean ratio, 0.86; 95% CI, 0.80-0.93; P < .001) and 15% fewer medications at the 90-day follow-up (mean ratio, 0.85; 95% CI, 0.78-0.92; P < .001) compared with the control group. The intervention additionally reduced patient exposure to potentially inappropriate medications and Drug Burden Index. Adverse drug event rates were similar between the intervention and control groups (hazard ratio, 0.83; 95% CI, 0.52-1.30)., Conclusions and Relevance: Results of this trial showed that the Shed-MEDS patient-centered deprescribing intervention was safe and effective in reducing the total medication burden at PAC facility discharge and 90 days after discharge. Future studies are needed to examine the effect of this intervention on patient-reported and long-term clinical outcomes., Trial Registration: ClinicalTrials.gov Identifier: NCT02979353.

Efficient regeneration requires multiple cell types acting in coordination. To better understand the intercellular networks involved and how they change when regeneration fails, we profile the transcriptome of hematopoietic, stromal, myogenic, and endothelial cells over 14 days following acute muscle damage. We generate a time-resolved computational model of interactions and identify VEGFA-driven endothelial engagement as a key differentiating feature in models of successful and failed regeneration. In addition, the analysis highlights that the majority of secreted signals, including VEGFA, are simultaneously produced by multiple cell types. To test whether the cellular source of a factor determines its function, we delete VEGFA from two cell types residing in close proximity: stromal and myogenic progenitors. By comparing responses to different types of damage, we find that myogenic and stromal VEGFA have distinct functions in regeneration. This suggests that spatial compartmentalization of signaling plays a key role in intercellular communication networks., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Air pollution is among the leading environmental threats to health around the world today, particularly in the context of sports and exercise. With the effects of air pollution, pollution episodes (eg, wildfire conflagrations) and climate change becoming increasingly apparent to the general population, so have their impacts on sport and exercise. As such, there has been growing interest in the sporting community (ie, athletes, coaches, and sports science and medicine team members) in practical personal-level actions to reduce the exposure to and risk of air pollution. Limited evidence suggests the following strategies may be employed: minimising all exposures by time and distance, monitoring air pollution conditions for locations of interest, limiting outdoor exercise, using acclimation protocols, wearing N95 face masks and using antioxidant supplementation. The overarching purpose of this position statement by the Canadian Academy of Sport and Exercise Medicine and the Canadian Society for Exercise Physiology is to detail the current state of evidence and provide recommendations on implementing these personal strategies in preventing and mitigating the adverse health and performance effects of air pollution exposure during exercise while recognising the limited evidence base., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Purpose: Diagnosis of Mismatch Repair Deficiency (MMRD) is crucial for tumor management and early detection in patients with the cancer predisposition syndrome constitutional mismatch repair deficiency (CMMRD). Current diagnostic tools are cumbersome and inconsistent both in childhood cancers and in determining germline MMRD., Patients and Methods: We developed and analyzed a functional Low-pass Genomic Instability Characterization (LOGIC) assay to detect MMRD. The diagnostic performance of LOGIC was compared with that of current established assays including tumor mutational burden, immunohistochemistry, and the microsatellite instability panel. LOGIC was then applied to various normal tissues of patients with CMMRD with comprehensive clinical data including age of cancer presentation., Results: Overall, LOGIC was 100% sensitive and specific in detecting MMRD in childhood cancers (N = 376). It was more sensitive than the microsatellite instability panel (14%, P = 4.3 × 10 -12 ), immunohistochemistry (86%, P = 4.6 × 10 -3 ), or tumor mutational burden (80%, P = 9.1 × 10 -4 ). LOGIC was able to distinguish CMMRD from other cancer predisposition syndromes using blood and saliva DNA ( P < .0001, n = 277). In normal cells, MMRDness scores differed between tissues (GI > blood > brain), increased over time in the same individual, and revealed genotype-phenotype associations within the mismatch repair genes. Importantly, increased MMRDness score was associated with younger age of first cancer presentation in individuals with CMMRD ( P = 2.2 × 10 -5 )., Conclusion: LOGIC was a robust tool for the diagnosis of MMRD in multiple cancer types and in normal tissues. LOGIC may inform therapeutic cancer decisions, provide rapid diagnosis of germline MMRD, and support tailored surveillance for individuals with CMMRD.

Verheij syndrome (VRJS) is a rare craniofacial spliceosomopathy presenting with craniofacial dysmorphism, multiple congenital anomalies and variable neurodevelopmental delay. It is caused by single nucleotide variants (SNVs) in PUF60 or interstitial deletions of the 8q24.3 region. PUF60 encodes a splicing factor which forms part of the spliceosome. To date, 36 patients with a sole diagnosis of VRJS due to disease-causing PUF60 SNVs have been reported in peer-reviewed publications. Although the depth of their phenotyping has varied greatly, they exhibit marked phenotypic heterogeneity. We report 10 additional unrelated patients, including the first described patients of Khmer, Indian, and Vietnamese ethnicities, and the eldest patient to date, with 10 heterozygous PUF60 variants identified through exome sequencing, 8 previously unreported. All patients underwent deep phenotyping identifying variable dysmorphism, growth delay, neurodevelopmental delay, and multiple congenital anomalies, including several unique features. The eldest patient is the only reported individual with a germline variant and neither neurodevelopmental delay nor intellectual disability. In combining these detailed phenotypic data with that of previously reported patients (n = 46), we further refine the known frequencies of features associated with VRJS. These include neurodevelopmental delay/intellectual disability (98%), axial skeletal anomalies (74%), appendicular skeletal anomalies (73%), oral anomalies (68%), short stature (66%), cardiac anomalies (63%), brain malformations (48%), hearing loss (46%), microcephaly (41%), colobomata (38%), and other ocular anomalies (65%). This case series, incorporating three patients from previously unreported ethnic backgrounds, further delineates the broad pleiotropy and mutational spectrum of PUF60 pathogenic variants., (© 2022 Wiley Periodicals LLC.)

Genome sequencing (GS) outperforms other rare disease diagnostics, but standardized approaches to assessing its clinical utility are limited. This study assessed the validity of the Clinician-reported Genetic testing Utility InDEx (C-GUIDE), a novel tool for assessing the utility of genetic testing from a clinician's perspective, for GS. C-GUIDE ratings were completed for patients who received GS results. For each patient, total C-GUIDE and single item global scores were calculated. Construct validity was assessed using linear regression to determine the association between C-GUIDE total and global item scores and measure the effects of potential explanatory variables. Ratings were completed for 67 pediatric and 36 adult patients. GS indications were neurological for 70.9% and results were diagnostic for 28.2%. When the C-GUIDE assessed primary (PV), secondary (SV), and pharmacogenomic (PGx) variants, on average, a one unit increase in the global item score was associated with an increase of 7.3 in the C-GUIDE score (p < 0.05). Diagnostic results were associated with an increase in C-GUIDE score of 5.0 compared to non-diagnostic results (p < 0.05) and an increase of one SV was associated with an increase of 2.5 (p < 0.05). For children, decreased age of one year was associated with an increase in C-GUIDE score of 0.3 (p < 0.05). Findings provide evidence that C-GUIDE measures the construct of clinical utility in pediatric and adult rare disease populations and is sensitive to changes in utility related to variant type. Quantifying the clinical utility of GS using C-GUIDE can inform efforts to optimize its use in patient care., (© 2022. The Author(s).)

Post-traumatic stress disorder (PTSD) is a debilitating mental health condition for which current treatments have long-term efficacy in 50% of patients. There is a clear need for better understanding of the mechanisms underlying PTSD and the development of new treatment approaches. Analog trauma procedures in animals, such as the stress-enhanced fear learning (SEFL) procedure, can be used to produce behavioral and neurobiological changes that have validity in modeling PTSD. However, by necessity, the modeling of PTSD in animals requires them to potentially experience pain and suffering. Consistent with the '3Rs' (reduction, refinement and replacement) of animal research, this study aimed to determine whether the SEFL procedure can be refined to reduce potential animal pain and suffering while retaining the same behavioral and neurobiological changes. Here we showed that PTSD-relevant changes could be produced in both behavior and the brain of rats that were group- rather than single-housed and that received lower-magnitude electric shocks in the 'trauma analog' session. We also varied the number of shock exposures in the trauma analog session, finding SEFL-susceptible and SEFL-resilient populations at all levels of shock exposure, but with greater levels of shock increasing the proportion of rats showing the SEFL-susceptible phenotype. These data demonstrate that the SEFL procedure can be used as an animal analog of PTSD with reduced potential pain and suffering to the animals and that variations in the procedure could be used to generate specific proportions of SEFL-susceptible and SEFL-resilient animals in future studies., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)