Characterization of a thiopurine S-methyltransferase from Leptospira borgpetersenii and assessment of pre and posttesting in the middle school classroom

ABSTRACT Spirochaete is a diverse phylum of Gram-negative bacteria that is located under the order, Spirochaetales, which is divided into three families: Spirochaetacea, Brachyspiraceae, and Leptospiraeae. Research was focused on the latter, specifically, the genus, Leptospira. Leptospira consists of many saprophytic, intermediate pathogenic, and pathogenic species, with L. interrogans (transmitted via contaminated water) and L. borpetersenii (acquired via host to host transmission) causing the majority of leptospirosis cases. To gain insight into the pathogenesis of Leptospira, a putative thiopurine S-methyltransferase (LBJ0800) was exploited. Because this gene is not present in any of the sequenced spirochetes, Brachyspira, Borrelia, Treponema, except Leptospira, and even then, only in L. borgpetersenii, work was concentrated on this novel gene. The open reading frame predicted to encode the putative thiopurine methyl S-methyltransferase (645 bp) was PCR amplified and directionally cloned into pET101 with a polyhistidine tag at the C terminus to provide plasmid, LBJ0800/pET101. To confirm the presence of a functional recombinant thiopurine S-methyltransferase, the expression construct was introduced into E. coli BL21 Star (DE3) cells, which were then grown in LB broth for IPTG-initiated induction of the protein. Cells were harvested by centrifugation, resuspended in Lysis Buffer and purified by a Ni-NTA metal-affinity column. Western blot analysis confirmed the purified, recombinant protein with a molecular mass of approximately 28 kDa. In the second chapter, in vivo characterization of the putative thiopurine S-methyltransferase was analyzed. It was determined by ELISA experiments and immunohistochemistry studies that although LBJ0800 is an unique protein, it was not expressed during infection at detectable levels. In chapter 3, research efforts focused on characterizing the gene enzymatically. Demonstrating substrate specificity, LBJ0800 methylated 6-thioguanine, validating its putative function as a thiopurine S-methyltransferase

Longitudinal assessment of self-harm statements of youth in foster care: Rates, reporters, and related factors

Self-harm in youth is a risk factor related to mental health and future morbidity, yet, relatively little is known about the rates and course of self-harm in youth residing in foster care. This study examined self-harm talk in foster youth based on caregiver and child report for 135 children between the ages of 8- and 11-years old. Longitudinal data on course of self-harm talk from both youth and caregivers also are provided. Caregivers identified that 24% of youth participants had disclosed a desire to die or to hurt themselves. Youth self-report revealed that 21% of children indicated a desire for self-harm, and rates of self-harm from both reporters decreased over time. While overall rates were similar across reporters, findings show discrepancies between youth self-report and caregiver report within individuals. Also, caregivers for youth in residential facilities were more likely to report youth self-harm talk than caregivers from foster home settings

Untangling the relative contribution of maltreatment severity and frequency to type of behavioral outcome in foster youth

Within maltreatment research, type, frequency, and severity of abuse are often confounded and not always specifically documented. The result is that samples are often heterogeneous, and the role of components of maltreatment in predicting outcome is unclear. The purpose of the present study was to identify and test the potential unique role of type, frequency, and severity of maltreatment to elucidate each variable’s role in predicting outcome behavior. Data from 309 youth in foster care (ages 8–22) and their caregivers were collected using the Modified Maltreatment Classification System and the Behavioral Assessment System for Children, 2nd Edition (BASC2), to measure maltreatment exposure and behavioral outcome respectively. A measurement model of the BASC2 was completed to determine model fit within the sample data. A second confirmatory factor analysis (CFA) was completed to determine the unique contributions of frequency and severity of maltreatment across four types of abuse to externalizing, internalizing, and adaptive behavior. The result of the CFA determined good fit of the BASC2 to the sample data after a few modifications. The result of the second CFA indicated that the paths from severity to externalizing behavior and adaptive behavior (reverse loading) were significant. Paths from frequency of abuse were not predictive of behavioral outcome. Maltreatment is a complex construct and researchers are encouraged to avoid confounding components of abuse that may be differentially related to outcome behavior for youth. Untangling the multifaceted nature of abuse is important and may have implications for identifying specific outcomes for youth exposed to maltreatment

Using Multiple Informants to Assess Child Maltreatment: Condordance Between Case File and Youth Self-Report

This is an Accepted Manuscript of an article published by Taylor & Francis in Journal of Aggression, Maltreatment and Trauma in 2014, available online: http://www.tandfonline.com/10.1080/10926771.2014.933463.To understand the psychosocial implications of child maltreatment, methods used to document prevalence must be clear. Yet, rates of maltreatment found in child self-report are generally inconsistent with data found in case files from state social service agencies. Although self-reports and case file reports of abuse disagree on occurrence of specific events, it is unclear if reporters agree when overall categories of abuse are considered. This study investigated differences between case file and youth report of abuse by examining four types of abuse: physical, sexual, neglect, and psychological, in a within-subjects design using a sample of 97 youth in foster care aged 8 to 22. Case files were coded for the presence of any indication of each type of abuse. Self-report of abuse was also assessed for any indication of each type of abuse. Results indicated that, overall, youth reported more physical and psychological abuse, and younger youth reported more sexual abuse than documented in their file. Implications for research and service provision for maltreated youth are discussed

Adjunctive rifampicin for Staphylococcus aureus bacteraemia (ARREST): a multicentre, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Staphylococcus aureus bacteraemia is a common cause of severe community-acquired and hospital-acquired infection worldwide. We tested the hypothesis that adjunctive rifampicin would reduce bacteriologically confirmed treatment failure or disease recurrence, or death, by enhancing early S aureus killing, sterilising infected foci and blood faster, and reducing risks of dissemination and metastatic infection. METHODS: In this multicentre, randomised, double-blind, placebo-controlled trial, adults (≥18 years) with S aureus bacteraemia who had received ≤96 h of active antibiotic therapy were recruited from 29 UK hospitals. Patients were randomly assigned (1:1) via a computer-generated sequential randomisation list to receive 2 weeks of adjunctive rifampicin (600 mg or 900 mg per day according to weight, oral or intravenous) versus identical placebo, together with standard antibiotic therapy. Randomisation was stratified by centre. Patients, investigators, and those caring for the patients were masked to group allocation. The primary outcome was time to bacteriologically confirmed treatment failure or disease recurrence, or death (all-cause), from randomisation to 12 weeks, adjudicated by an independent review committee masked to the treatment. Analysis was intention to treat. This trial was registered, number ISRCTN37666216, and is closed to new participants. FINDINGS: Between Dec 10, 2012, and Oct 25, 2016, 758 eligible participants were randomly assigned: 370 to rifampicin and 388 to placebo. 485 (64%) participants had community-acquired S aureus infections, and 132 (17%) had nosocomial S aureus infections. 47 (6%) had meticillin-resistant infections. 301 (40%) participants had an initial deep infection focus. Standard antibiotics were given for 29 (IQR 18-45) days; 619 (82%) participants received flucloxacillin. By week 12, 62 (17%) of participants who received rifampicin versus 71 (18%) who received placebo experienced treatment failure or disease recurrence, or died (absolute risk difference -1·4%, 95% CI -7·0 to 4·3; hazard ratio 0·96, 0·68-1·35, p=0·81). From randomisation to 12 weeks, no evidence of differences in serious (p=0·17) or grade 3-4 (p=0·36) adverse events were observed; however, 63 (17%) participants in the rifampicin group versus 39 (10%) in the placebo group had antibiotic or trial drug-modifying adverse events (p=0·004), and 24 (6%) versus six (2%) had drug interactions (p=0·0005). INTERPRETATION: Adjunctive rifampicin provided no overall benefit over standard antibiotic therapy in adults with S aureus bacteraemia. FUNDING: UK National Institute for Health Research Health Technology Assessment

Minimal information for studies of extracellular vesicles 2018 (MISEV2018):a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles (“MISEV”) guidelines for the field in 2014. We now update these “MISEV2014” guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points

Peri-operative red blood cell transfusion in neonates and infants: NEonate and Children audiT of Anaesthesia pRactice IN Europe: A prospective European multicentre observational study

BACKGROUND: Little is known about current clinical practice concerning peri-operative red blood cell transfusion in neonates and small infants. Guidelines suggest transfusions based on haemoglobin thresholds ranging from 8.5 to 12 g dl-1, distinguishing between children from birth to day 7 (week 1), from day 8 to day 14 (week 2) or from day 15 (≥week 3) onwards. OBJECTIVE: To observe peri-operative red blood cell transfusion practice according to guidelines in relation to patient outcome. DESIGN: A multicentre observational study. SETTING: The NEonate-Children sTudy of Anaesthesia pRactice IN Europe (NECTARINE) trial recruited patients up to 60 weeks' postmenstrual age undergoing anaesthesia for surgical or diagnostic procedures from 165 centres in 31 European countries between March 2016 and January 2017. PATIENTS: The data included 5609 patients undergoing 6542 procedures. Inclusion criteria was a peri-operative red blood cell transfusion. MAIN OUTCOME MEASURES: The primary endpoint was the haemoglobin level triggering a transfusion for neonates in week 1, week 2 and week 3. Secondary endpoints were transfusion volumes, 'delta haemoglobin' (preprocedure - transfusion-triggering) and 30-day and 90-day morbidity and mortality. RESULTS: Peri-operative red blood cell transfusions were recorded during 447 procedures (6.9%). The median haemoglobin levels triggering a transfusion were 9.6 [IQR 8.7 to 10.9] g dl-1 for neonates in week 1, 9.6 [7.7 to 10.4] g dl-1 in week 2 and 8.0 [7.3 to 9.0] g dl-1 in week 3. The median transfusion volume was 17.1 [11.1 to 26.4] ml kg-1 with a median delta haemoglobin of 1.8 [0.0 to 3.6] g dl-1. Thirty-day morbidity was 47.8% with an overall mortality of 11.3%. CONCLUSIONS: Results indicate lower transfusion-triggering haemoglobin thresholds in clinical practice than suggested by current guidelines. The high morbidity and mortality of this NECTARINE sub-cohort calls for investigative action and evidence-based guidelines addressing peri-operative red blood cell transfusions strategies. TRIAL REGISTRATION: ClinicalTrials.gov, identifier: NCT02350348

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

The Science Performance of JWST as Characterized in Commissioning

This paper characterizes the actual science performance of the James Webb Space Telescope (JWST), as determined from the six month commissioning period. We summarize the performance of the spacecraft, telescope, science instruments, and ground system, with an emphasis on differences from pre-launch expectations. Commissioning has made clear that JWST is fully capable of achieving the discoveries for which it was built. Moreover, almost across the board, the science performance of JWST is better than expected; in most cases, JWST will go deeper faster than expected. The telescope and instrument suite have demonstrated the sensitivity, stability, image quality, and spectral range that are necessary to transform our understanding of the cosmos through observations spanning from near-earth asteroids to the most distant galaxies.Comment: 5th version as accepted to PASP; 31 pages, 18 figures; https://iopscience.iop.org/article/10.1088/1538-3873/acb29