Perturbed cholesterol and vesicular trafficking associated with dengue blocking in Wolbachia-infected Aedes aegypti cells

Wolbachia are intracellular maternally inherited bacteria that can spread through insect populations and block virus transmission by mosquitoes, providing an important approach to dengue control. To better understand the mechanisms of virus inhibition, we here perform proteomic quantification of the effects of Wolbachia in Aedes aegypti mosquito cells and midgut. Perturbations are observed in vesicular trafficking, lipid metabolism and in the endoplasmic reticulum that could impact viral entry and replication. Wolbachia-infected cells display a differential cholesterol profile, including elevated levels of esterified cholesterol, that is consistent with perturbed intracellular cholesterol trafficking. Cyclodextrins have been shown to reverse lipid accumulation defects in cells with disrupted cholesterol homeostasis. Treatment of Wolbachia-infected Ae. aegypti cells with 2-hydroxypropyl-β-cyclodextrin restores dengue replication in Wolbachia-carrying cells, suggesting dengue is inhibited in Wolbachia-infected cells by localised cholesterol accumulation. These results demonstrate parallels between the cellular Wolbachia viral inhibition phenotype and lipid storage genetic disorders

Novel function for the p38-MK2 signaling pathway in circulating CD1c1 (BDCA-11) myeloid dendritic cells from healthy donors and advanced cancer patients; inhibition of p38 enhances IL-12 whilst suppressing IL-10

There is growing interest in myeloid (my) dendritic cells (DC) as an alternative to monocyte-derived DC (moDC) for immunotherapy. However, in contrast to moDC, little is known regarding the effect of malignancy on the function, abundance or use of intracellular signaling pathways in myDC. Understanding the molecular detail of circulating myDC is therefore important for future use in advanced cancer. Advanced cancer patients had similar numbers of circulating myDC to cancer-free patients and healthy individuals, and secreted similar levels of IL-1b, IL-6, IL-10, IL-12 and IL-23. However, myDC from some patients failed to secrete the Th1-cytokine IL-12. Surprisingly, inhibiting p38 (p38i) signaling (using BIRB0796 or SB203580) markedly increased IL-12 secretion by myDC. This is in complete contrast to what is established for moDC where inhibiting p38 ablates IL-12. Interestingly, this was specific to IL-12, since IL-10 was suppressed by p38i in both DC types. The opposing effect of p38i on IL-12 was evident at the transcriptional level and in both DC types was mediated through the p38-MK2 pathway but did not involve differential phosphorylation of the distal Rsk kinase. Importantly, where patient myDC did not secrete IL-12 (or after treatment with suppressive melanoma lysate), p38i restored IL-12 to normal levels. In contrast to p38, inhibiting the other MAPK pathways had similar consequences in both DC types. We show for the first time the differential use of a major intracellular signaling pathway by myDC. Importantly, there are sufficient circulating myDC in advanced cancer patients to consider development of adoptive immunotherapy

Widespread genetic heterogeneity of human ribosomal RNA genes

Polymorphism drives survival under stress and provides adaptability. Genetic polymorphism of ribosomal RNA (rRNA) genes derives from internal repeat variation of this multicopy gene, and from interindividual variation. A considerable amount of rRNA sequence heterogeneity has been proposed but has been challenging to estimate given the scarcity of accurate reference sequences. We identified four rDNA copies on chromosome 21 (GRCh38) with 99% similarity to recently introduced reference sequence KY962518.1. We customized a GATK bioinformatics pipeline using the four rDNA loci, spanning a total 145 kb, for variant calling and used high-coverage whole-genome sequencing (WGS) data from the 1000 Genomes Project to analyze variants in 2504 individuals from 26 populations. We identified a total of 3791 variant positions. The variants positioned nonrandomly on the rRNA gene. Invariant regions included the promoter, early 5 ' ETS, most of 18S, 5.8S, ITS1, and large areas of the intragenic spacer. A total of 470 variant positions were observed on 28S rRNA. The majority of the 28S rRNA variants were located on highly flexible human-expanded rRNA helical folds ES7L and ES27L, suggesting that these represent positions of diversity and are potentially under continuous evolution. Several variants were validated based on RNA-seq analyses. Population analyses showed remarkable ancestry-linked genetic variance and the presence of both high penetrance and frequent variants in the 5 ' ETS, ITS2, and 28S regions segregating according to the continental populations. These findings provide a genetic view of rRNA gene array heterogeneity and raise the need to functionally assess how the 28S rRNA variants affect ribosome functions.Peer reviewe

The association of body temperature with antibiotic therapy and mortality in patients attending the emergency department with suspected infection

BACKGROUND AND IMPORTANCE: Previous studies found that septic patients with normothermia have higher mortality than patients with fever. We hypothesize that antibiotic therapy is less frequently initiated if infectious patients present with normothermia to the emergency department (ED). OBJECTIVES: To examine the association of body temperature with the initiation of antibiotic therapy in patients attending the ED with suspected and proven infection. Additionally, the association of temperature with 30-day mortality was assessed. DESIGN, SETTINGS AND PARTICIPANTS: We conducted a retrospective cohort study between 2012 and 2016 at a tertiary university hospital. Adult patients attending the ED with a blood culture taken (i.e. suspected infection) and a positive blood culture (i.e. proven bacteremia) were included. EXPOSURE: Tympanic temperature at arrival was categorized as hypothermia (38.0°C). OUTCOME MEASURES AND ANALYSIS: Primary outcome was the initiation of antibiotic therapy. A secondary outcome was 30-day mortality. Multivariable logistic regression was used to control for covariates. MAIN RESULTS: Of 5997 patients with a suspected infection, 45.8% had normothermia, 44.6% hyperthermia and 5.6% hypothermia. Patients with hyperthermia received more often antibiotic therapy (53.5%) compared to normothermic patients (27.6%, adjusted odds ratio [95% confidence interval], 2.59 [2.27–2.95]). Patients with hyperthermia had lower mortality (4.7%) than those with normothermia (7.4%, adjusted odds ratio [95% confidence interval], 0.50 [0.39–0.64]). Sensitivity analyses in patients with proven bacteremia (n = 934) showed similar results. CONCLUSION: Normothermia in patients presenting with infection was associated with receiving less antibiotic therapy in the ED compared to presentations with hyperthermia. Moreover, normothermia was associated with a higher mortality risk than hyperthermia

Identification of an E3 ligase that targets the catalytic subunit of RNA Polymerase I upon transcription stress

Publisher Copyright: © 2022 The AuthorsRNA Polymerase I (Pol I) synthesizes rRNA, which is the first and rate-limiting step in ribosome biogenesis. Factors governing the stability of the polymerase complex are not known. Previous studies characterizing Pol I inhibitor BMH-21 revealed a transcriptional stress-dependent pathway for degradation of the largest subunit of Pol I, RPA194. To identify the E3 ligase(s) involved, we conducted a cell-based RNAi screen for ubiquitin pathway genes. We establish Skp–Cullin–F-box protein complex F-box protein FBXL14 as an E3 ligase for RPA194. We show that FBXL14 binds to RPA194 and mediates RPA194 ubiquitination and degradation in cancer cells treated with BMH-21. Mutation analysis in yeast identified lysines 1150, 1153, and 1156 on Rpa190 relevant for the protein degradation. These results reveal the regulated turnover of Pol I, showing that the stability of the catalytic subunit is controlled by the F-box protein FBXL14 in response to transcription stress.Peer reviewe

Non-adherence to antimicrobial guidelines in patients with bloodstream infection visiting the emergency department

_Objective:_ Non-adherence to antimicrobial guidelines in patients with bloodstream infection can result in undertreatment, overtreatment, or equivalent treatment, and could lead to suboptimal care. Our aim was to examine the association between non-adherence and appropriate coverage as well as to assess the impact of non-adherence on 30-day mortality. _Methods:_ We conducted a retrospective cohort study between 2012 and 2017 at a tertiary university hospital. Adult patients attending the emergency department with a bloodstream infection were included. Adherence was defined as guideline-recommended antibiotic therapy. Non-adherence was either undertreatment (too narrow-spectrum), overtreatment (too broad-spectrum), or equivalent treatment. Outcomes were appropriate coverage (i.e. antibiotic therapy that matches in vitro susceptibility of the isolated bacteria) and 30-day mortality. _Results:_ We included 909 patients of whom 395 (43.5%) were treated adherently, 355 (39.1%) were undertreated, 87 (9.6%) were overtreated, and 72 (7.9%) received an equivalent treatment. Overtreated patients were more severely ill, whilst undertreated patients had more favorable patient characteristics. Overtreatment did not result in higher appropriate coverage, whereas undertreatment was associated with lower coverage (OR[95%CI]: 0.18 [0.12; 0.26]). Overtreatment and undertreatment were not associated with 30-day mortality. _Conclusions:_ Guideline adherence likely depends on disease severity, because overtreatment was more often observed in patients with high disease severity and undertreatment in less severely ill patients. Undertreatment was associated lower appropriate coverage but not with higher mortality. However, this can be the result of residual confounding. Overtreatment did not result in higher appropriate antibiotic coverage nor a survival benefit. Therefore, overtreatment seems not justifiable

Learning in and across communities of practice: health professions education students’ learning from boundary crossing

Learning to adapt to new contexts is crucial in health professions education (HPE). Boundaries between and within contexts challenge continuity in students’ learning processes. Little is known about how HPE students can make these “boundary experiences” productive for learning. We investigated how and what nursing students learn from boundary experiences while they are simultaneously growing into a community of practice (CoP). Using a boundary-crossing lens, experiences of discontinuity were identified in pre-placement and post-placement interviews and diary fragments with 14 nursing students during their placement in an academic hospital. We found that students experience discontinuity as a result of different approaches to nursing care and to learning, both between (academic and clinical) settings and within a setting. When students feel safe enough, they can convert boundary experiences into meaningful learning situations, such as critical discussions with staff. Successfully overcoming boundary experiences improves students’ understanding of healthcare and professional development and helps them to develop a personal approach to learning. Students critically address boundary experiences when they are motivated to learn and when they perceive a violation of ethical standards but not when they are concerned that it will affect their assessment. Objects designed to bridge theory and practice can generate additional barriers. This study adds to the HPE literature by demonstrating the learning potential of boundaries and to the broader literature by showing how responses to boundary experiences are intertwined with the process of growing into a CoP. The findings can be used to design future boundary objects

Predicting 30-day mortality using point-of-care testing; an external validation and derivation study

Background Early risk stratification for guiding treatment priority in the emergency department (ED) is becoming increasingly important. Existing prediction models typically use demographics, vital signs and laboratory parameters. Laboratory-based models require blood testing, which may cause substantial delay. However, these delays can be prevented by the use of point-of-care testing (POCT), where results are readily available. We aimed to externally validate a laboratory-based model for mortality and subsequently assessed whether a POCT model yields comparable performance. Methods All adult patients visiting the ED of a university hospital between January 1st, 2012 and December 31st, 2016 were retrospectively reviewed for inclusion. Primary outcome was defined as 30-day mortality after ED presentation. We externally validated one existing prediction model including age, glucose, urea, sodium, haemoglobin, platelet count and white blood cell count. We assessed the predictive performance by discrimination, expressed as Area under the Curve (AUC). We compared the existing model to an equivalent model using predictors that are available with POCT (i.e. glucose, urea, sodium and haemoglobin). Additionally, we internally validated these models with bootstrapping. Results We included 34,437 patients of whom 1,942 (5.6%) died within 30 days. The AUC of the laboratory-based model was 0.794. We refitted this model to our ED population and found an AUC of 0.812, which decreased only slightly to 0.790 with only POCT parameters. Conclusions Our POCT-model performs similar to existing laboratory-based models in identifying patients at high risk for mortality, with results available within minutes. Although the model needs further validation and evaluation, it shows the potential of POCT for early risk stratification in the ED

Predicting mortality in patients with suspected sepsis at the Emergency Department; A retrospective cohort study comparing qSOFA, SIRS and National Early Warning Score

Objective In hospitalized patients, the risk of sepsis-related mortality can be assessed using the quick Sepsis-related Organ Failure Assessment (qSOFA). Currently, different tools that predict deterioration such as the National Early Warning Score (NEWS) have been introduced in clinical practice in Emergency Departments (ED) worldwide. It remains ambiguous which screening tool for mortality at the ED is best. The objective of this study was to evaluate the predictive performance for mortality of two sepsis-based scores (i.e. qSOFA and Systemic Inflammatory Response Syndrome (SIRS)-criteria) compared to the more general NEWS score, in patients with suspected infection directly at presentation to the ED. Methods We performed a retrospective cohort study. Patients who presented to the ED between June 2012 and May 2016 with suspected sepsis in a large tertiary care center were included. Suspected sepsis was defined as initiation of intravenous antibiotics and/or collection of any culture in the ED. Outcome was defined as 10-day and 30-day mortality after ED presentation. Predictive performance was expressed as discrimination (AUC) and calibration using Hosmer-Lemeshow goodness-of-fit test. Subsequently, sensitivity, and specificity were calculated. Results In total 8,204 patients were included of whom 286 (3.5%) died within ten days and 490 (6.0%) within 30 days after presentation. NEWS had the best performance, followed by qSOFA and SIRS (10-day AUC: 0.837, 0.744, 0.646, 30-day AUC: 0.779, 0.697, 0.631). qSOFA (�2) lacked a high sensitivity versus SIRS (�2) and NEWS (�7) (28.5%, 77.2%, 68.0%), whilst entailing highest specificity versus NEWS and SIRS (93.7%, 66.5%, 37.6%). Conclusions NEWS is more accurate in predicting 10- and 30-day mortality than qSOFA and SIRS in patients presenting to the ED with suspected sepsis

The volume-outcome relationship in severely injured patients: A systematic review and meta-analysis

BACKGROUND The volume-outcome relationship in severely injured patients remains under debate and this has consequences for the designation of trauma centers. OBJECTIVES The aim of this study was to evaluate the relationship between hospital or surgeon volume and health outcomes in severely injured patients. METHODS Six electronic databases were searched from 1980 up to January 30, 2018, to identify studies that describe the relationship between hospital or surgeon volume and health outcomes in severely injured patients (preferably Injury Severity Score above 15). Selection of relevant studies, data extraction, and critical appraisal of the methodological quality were performed by two independent reviewers. Pooled adjusted and unadjusted estimates of the effect of volume on in-hospital mortality, only in study populations with Injury Severity Score greater than 15, were calculated with a random-effects meta-analysis. A mixed effects linear regression model was used to assess hospital volume as continuous parameter. RESULTS Eighteen observational cohort studies were included. The majority (13 [72%] of 18) reported an association between higher hospital or surgeon volume and lower mortality rate. Overall, the quality of the included studies was reasonable, with insufficient adjustment as one of the most common limitations. Eight studies were included in the meta-analysis with a total of 222,418 patients. High hospital volume (>240 admitted severely injured patients per year) was associated with a lower risk of mortality (adjusted odds ratio, 0.85; 95% confidence interval, 0.76-0.94). Four studies were included in the regression model, providing a beta of-0.17 per 10 patients (95% CI,-0.27 to-0.07). There was no clear association between surgeon volume and mortality rates based on three available studies. CONCLUSION Our systematic overview of the literature reveals a modest association between high-volume centers and lower mortality in severely injured patients, suggesting that designation of high-volume centers might improve outcomes among severely injured patients