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Topics associated with the chemical sciences form a significant part of the curriculum in science at the primary school level in the U.K. In this methodology paper, we demonstrate how a wide range of research articles associated with the chemical sciences can be disseminated to an elementary school audience and how children can carry out investigations associated with cutting-edge research in the classroom. We discuss how the Primary Science Teaching Trust's (PSTT's) "I bet you did not know" (IBYDK) articles and their accompanying Teacher Guides benefit children, primary (elementary) school teachers, and other stakeholders including the researchers themselves. We define three types of research articles; ones describing how children can reproduce the research themselves without much adaptation, others where children can mirror the research using similar methods, and some where an analogy can be used to explain the research. We provide exemplars of each type and some preliminary feedback on articles written.

Climate change is of great concern to all age groups but in particular to children. "Simple" climate models have been in place for a long time and can be used effectively with post-16 students. For younger children, modifications are required, and we describe in this paper the development and use of two such models. The first (the Granny Model) is a pictorial version of the model that has been used extensively with primary and early secondary school aged children (14 and younger). The second is an online version of the simple climate model that can be used without recourse to the underpinning mathematics and science but allows children to experiment with changing variables and how these changes affect the average surface temperature of the Earth.

All learners have a contribution to make to the development of the Chemical Sciences, be that in novel ways to teach, and their perspectives and contexts, but also in research, both in chemical education and the wider Chemical Sciences. Through four case studies, this paper explores interactions with diverse groups and how this has altered perspectives on both teaching and research. The case studies include work with visually impaired adults, a project bringing together First Peoples in Australia with academics to explore old ways (traditional science) and new ways (modern approaches), primary (elementary) school perspectives on teaching science, and a project in South Africa to connect university and township communities. Not only do these case studies demonstrate the immense value these diverse groups bring to our understanding about how to learn, but they also bring new perspectives on how to view and solve chemical problems.

Postgraduate engagement in delivering outreach activities is more commonplace than it once was. However, the impact on postgraduate students (typically studying for a Ph.D. degree) of participating in the delivery of these outreach activities has rarely, if ever, been recorded. The Bristol ChemLabS Outreach program has been running for ca. 17 years, and in that time, many postgraduate students have been involved (approximately 500), with around 250 typically for up to 3 years. We sought to investigate the impact of outreach engagement on postgraduate alumni who were involved in the program for over 3 years (32) and how the experiences and training of the outreach program had impacted on their careers postgraduation. Thirty of the 32 postgraduates engaged and ~70% reported that their outreach experience had influenced their decision making on future careers. Many respondents reported that the skills and experiences gained through outreach participation had contributed to success in applying for and interviewing at their future employers. All respondents reported that outreach had helped them to develop key skills that were valued in the workplace, specifically, communication, teamwork, organizational skills, time planning, event planning, and event management. Rather than a pleasant distraction or an opportunity to supplement income, all participants noted that they felt there were many additional benefits and that this was time well spent. Outreach should not be viewed as a distraction to science research but rather an important enhancement to it provided that the program is well constructed and seeks to develop those delivering the outreach activities.

School counselling is a well-established means of supporting the mental health of children. Counsellors are most effective when they collaborate with parents, so it is important that parents have a good understanding of and access to school counselling services. Despite this, little is known about parents' perceptions of counselling in Hong Kong schools. We interviewed 27 parents in Hong Kong to investigate how they perceived the counselling services provided by their children's local and international schools, and analysed the data thematically. International school parents recognised the potential of school counselling as a means of support for their children and wanted to work more closely with counsellors to a greater extent than local school parents. Parents were confused about the roles of counsellors and experienced stigma and concerns about confidentiality which inhibited them from engaging with counselling services. Our findings suggest that school principals should work with counsellors to establish and communicate roles more clearly. Greater recognition of counsellors' professionalism, and clearer role differentiation between counsellors and other mental health and educational professionals may improve parental engagement with and support for school counselling.

Purpose of Review: Diabetes is the most common cause of kidney disease in individuals that receive a kidney transplant, and those without pre-existing diabetes are at greater risk of developing diabetes following kidney transplant. A class of diabetes treatment medications called glucagon-like peptide-1 receptor agonists (GLP-1RA) has seen recent widespread use for people with diabetes or obesity, with efficacy for improved glycemic control, weight loss, and reduced risk of cardiovascular events. Given these benefits, and indications for use that often co-occur in kidney transplant recipients, use of GLP-1RAs warrants consideration in this population. Therefore, we sought to review the current literature to better understand the mechanisms of action, clinical application, and person-centred considerations of GLP-1RAs in kidney transplant recipients. Sources of Information: Original articles were identified between December 2023 and July 2024 from electronic databases including the Ovid MEDLINE database, PubMed, and Google Scholar using terms “kidney transplant,” “GLP-1,” “glucagon-like peptide-1 receptor agonist,” and “diabetes.” Methods: A comprehensive review of the literature was conducted to explore the relationship between GLP-1RAs and kidney transplant recipients. We reviewed the current state of evidence across the research disciplines of basic or fundamental science, clinical and health services research, and person-centred equity science, and highlighted important knowledge gaps that offer opportunities for future research. Key Findings: Numerous clinical studies have demonstrated the benefit of GLP-1RAs in people with and without diabetic kidney disease, including decreased risk of cardiovascular events. However, there is a paucity of high-quality randomized controlled trials and observational studies analyzing use of GLP-1RAs in kidney transplant recipients. Evidence of benefit in this population is therefore limited to small studies or inferred from research conducted in nontransplant populations. Growing evidence from preclinical and clinical studies may elucidate renoprotective mechanisms of GLP-1RAs and remove barriers to application of these drugs in the transplant recipient population. Individuals who are female, non-white, have lower socioeconomic status, and live in rural communities are at greater risk of diabetes and have lower uptake of GLP-1RAs. There is a need for clinical trials across diverse kidney transplant populations to estimate the efficacy of GLP-1RAs on important health outcomes. Limitations: The search strategy for this narrative review may not have been sensitive to identify all relevant articles. Our search was limited to English language articles.

We studied seasonal and interannual variability in the diet of grey seals (Halichoerus grypus) using faecal samples collected from Sable Island, Nova Scotia between 1991 and 1998. More than 28,000 prey from at least 28 taxa were identified from 1,245 faecal samples collect mainly in spring, fall and winter. Sand lance (Ammodytes dubius) dominated the diet in all seasons and years, but the importance of this and other species varied over time. There was also evidence of seasonal and interannual variation in the size of prey consumed both within and among species. We compared diet composition with estimates of prey numbers and biomass from annual researchtrawl surveys conducted in March and July. Species-specific numerical corrections were applied to otolith counts to account for the complete digestion of otoliths, and fish catchability correction factors applied to trawl survey catches to account for trawl selectivity. Based on an odds ratio index of prey selectivity, grey seals positively selected sand lance in both seasons. Other species were either relatively avoided or eaten roughly in proportion to their estimated abundance.

Atmospheric electricity measurements, principally of the hourly potential gradient (PG), were made continuously at Eskdalemuir Observatory, Scotland (55.314° N, 3.206° W), between 1911 and 1981. Air ion properties were also determined. The sensing apparatus for PG measurement at Eskdalemuir initially used a Kelvin water dropper potential equaliser (1911–1936), followed by a radioactive probe from 1936 and, from 1965, a horizontal stretched wire sensor at 0.5 m, all attached to recording devices. Monthly mean PG data from these instruments are now available digitally. Originally, the data were classified into undisturbed and disturbed days, using the chart record (electrogram). This approach has deficiencies at Eskdalemuir due to mist, fog and calm conditions, which can influence the mean PG despite the day appearing undisturbed on the electrogram. Nevertheless, a correlation with Pacific Ocean temperature fluctuations is apparent in the Eskdalemuir PG data between 1911 and 1950. As at Lerwick, there was an abrupt decrease in the PG caused by nuclear weapon detonations in the late 1950s and early 1960s. The 1950s PG decrease began at Eskdalemuir before that at Lerwick, for which possible additional local factors are evaluated.

Rationale & Objective: Cilastatin is an inhibitor of drug metabolism in the proximal tubule that demonstrates nephroprotective effects in animals. It has been used in humans in combination with the antibiotic imipenem to block imipenem’s renal metabolism. This systematic review and meta-analysis evaluated the nephroprotective effects of cilastatin in humans. Study Design: Systematic review and meta-analysis of observational (comparative effectiveness) studies or randomized clinical trials (RCTs). Setting & Study Populations: People of any age at risk of acute kidney injury (AKI). Selection Criteria for Studies: We systematically searched MEDLINE, Embase, Web of Science, and the Cochrane Controlled Trials registry from database inception to November 2023 for observational studies or RCTs that compared kidney outcomes among groups treated with cilastatin, either alone or as combination imipenem-cilastatin, versus an inactive or active control group not treated with cilastatin. Data Extraction: Two reviewers independently evaluated studies for inclusion and risk of bias. Analytical Approach: Treatment effects were estimated using random-effects models, and heterogeneity was quantified using the I2 statistic. Results: We identified 10 studies (5 RCTs, n = 531 patients; 5 observational studies, n = 6,321 participants) that met the inclusion criteria, including 4 studies with comparisons to inactive controls and 6 studies with comparisons to alternate antibiotics. Based on pooled results from 7 studies, the risk of AKI was lower with imipenem-cilastatin (risk ratio [RR], 0.52; 95% confidence intervals [CI], 0.40-0.67; I2 = 26.5%), with consistent results observed in RCTs (3 RCTs, RR, 0.26; 95% CI, 0.09-0.77; I2 = 44.4%) and observational studies (4 studies, RR, 0.54; 95% CI, 0.41-0.72; I2 = 44.4%). Based on results from 6 studies, serum creatinine concentration was lower following treatment with imipenem-cilastatin than comparators (weighted mean difference in serum creatinine −0.14 mg/dL (95% CI, −0.21 to −0.07; I2 = 0%). The overall certainty of the evidence was low due to heterogeneity of the results, high risk of bias, and indirectness among the identified studies. Limitations: Clinical and statistical heterogeneity could not be fully explained due to a limited number of studies. Conclusions: Patients treated with imipenem-cilastatin developed AKI less frequently and had lower serum creatinine concentration following treatment than control groups or those who had received comparator antibiotics. Larger clinical trials with less risk of detection bias due to lack of allocation concealment and blinding are needed to establish the efficacy of cilastatin for AKI prevention. Plain-Language Summary: Cilastatin, used with the antibiotic imipenem, has shown kidney-protective effects in animals and preclinical studies of acute kidney injury (AKI). This systematic review and meta-analysis identified 10 studies (5 randomized controlled trials and 5 observational studies) of imipenem-cilastatin involving people at risk of AKI. Pooled estimates of treatment effects indicated that patients who received imipenem-cilastatin had a lower incidence of AKI and lower serum creatinine concentrations following treatment compared to comparator groups. Despite these promising findings, the overall certainty of the evidence was low due to heterogeneity among studies, high risk of bias, and indirectness of the data. Although cilastatin appears to be a promising medication for preventing AKI, larger, well-designed trials are needed to establish its effectiveness.

Summary: Background: The ubiquitin regulatory X (UBX) domain-containing proteins (UBXNs) are putative adaptors for ubiquitin ligases and valosin-containing protein; however, their in vivo physiological functions remain poorly characterised. We recently showed that UBXN3B is essential for activating innate immunity to DNA viruses and controlling DNA/RNA virus infection. Herein, we investigate its role in adaptive immunity. Methods: We evaluated the antibody responses to multiple viruses and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza in tamoxifen-inducible global and constitutive B cell-specific Ubxn3b knockout mice; quantified various immune populations, B lineage progenitors/precursors, B cell receptor (BCR) signalling and apoptosis by flow cytometry, immunoblotting and immunofluorescence microscopy. We also performed bone marrow transfer, single-cell and bulk RNA sequencing. Findings: Both global and B cell-specific Ubxn3b knockout mice present a marked reduction in small precursor B-II (>60%), immature (>70%) and mature B (>95%) cell numbers. Transfer of wildtype bone marrow to irradiated global Ubxn3b knockouts restores normal B lymphopoiesis, while reverse transplantation does not. The mature B population shrinks rapidly with apoptosis and higher pro and activated caspase-3 protein levels were observed following induction of Ubxn3b knockout. Mechanistically, Ubxn3b deficiency leads to impaired pre-BCR signalling and cell cycle arrest. Ubxn3b knockout mice are highly vulnerable to respiratory viruses, with increased viral loads and prolonged immunopathology in the lung, and reduced production of virus-specific IgM/IgG. Interpretation: UBXN3B is essential for B lymphopoiesis by maintaining constitutive pre-BCR signalling and cell survival in a cell-intrinsic manner. Funding: United States National Institutes of Health grants, R01AI132526 and R21AI155820.

Abstract Background and Aims High tissue sodium accumulation and intermuscular adipose tissue (IMAT) are associated with aging, type 2 diabetes, and chronic kidney disease. In this study, we aim to investigate whether high lower‐extremity tissue sodium accumulation relates to IMAT quantity and whether systemic inflammatory mediators and adipocytokines contribute to such association. Methods Tissue sodium content and IMAT accumulation (percentage of IMAT area to muscle area) were measured in 83 healthy individuals using sodium imaging (23Na‐MRI) and proton (1H‐MRI) imaging of the calf. Insulin sensitivity was assessed by glucose disposal rate (GDR) measured with the hyperinsulinemic‐euglycemic clamp. Results Median (interquartile range) muscle and skin sodium contents were 16.6 (14.9, 19.0) and 12.6 (10.9, 16.7) mmol/L, respectively. Median IMAT was 3.69 (2.80, 5.37) %. In models adjusted for age, sex, BMI, GDR, adiponectin, and high‐sensitivity C‐reactive protein, increasing tissue sodium content was significantly associated with higher IMAT quantity (p = 0.018 and 0.032 for muscle and skin tissue sodium, respectively). In subgroup analysis stratified by sex, skin sodium was significantly associated with IMAT only among men. In interaction analysis, the association between skin sodium and IMAT was greater with increasing levels of high‐sensitivity C‐reactive protein and interleukin‐6 (p for interaction = 0.022 and 0.006, respectively). Conclusions Leg muscle and skin sodium are associated with IMAT quantity among healthy individuals. The relationship between skin sodium and IMAT may be mediated by systemic inflammation.

Abstract The Retinoic acid-Inducible Gene I (RIG-I) like receptors (RLRs) are the major viral RNA sensors essential for the initiation of antiviral immune responses. RLRs are subjected to stringent transcriptional and posttranslational regulations, of which ubiquitination is one of the most important. However, the role of ubiquitination in RLR transcription is unknown. Here, we screen 375 definite ubiquitin ligase knockout cell lines and identify Ubiquitin Protein Ligase E3 Component N-Recognin 5 (UBR5) as a positive regulator of RLR transcription. UBR5 deficiency reduces antiviral immune responses to RNA viruses, while increases viral replication in primary cells and mice. Ubr5 knockout mice are more susceptible to lethal RNA virus infection than wild type littermates. Mechanistically, UBR5 mediates the Lysine 63-linked ubiquitination of Tripartite Motif Protein 28 (TRIM28), an epigenetic repressor of RLRs. This modification prevents intramolecular SUMOylation of TRIM28, thus disengages the TRIM28-imposed brake on RLR transcription. In sum, UBR5 enables rapid upregulation of RLR expression to boost antiviral immune responses by ubiquitinating and de-SUMOylating TRIM28.

School counselling has the potential to deliver significant support for the wellbeing of children. However, much of the research on school counsellors has been conducted in developed Western countries, with very limited research into factors influencing the effectiveness of counsellors in lower middle-income countries or in Asia. The aim of this qualitative study was to investigate the perceptions of Filipino counsellors about their roles, and factors that supported or impeded their effectiveness. Seventeen school counsellors in the Philippines were interviewed, and the data were analysed thematically. Our findings suggest that Filipino school counsellors often carry out dual roles, experience a lack of role clarity, and are systemically disempowered in their schools. Relationships with school principals have a significant influence on counsellors' roles and positioning in schools, and therefore on their effectiveness. The ability of principals to foster a school ethos supportive of counselling is essential in enabling counsellors to leverage the multifunctional nature of their work, become embedded and centrally positioned in the school community, and enhance their effectiveness. Doing so can enable counselling to be more culturally accessible to young people.

Abstract Chimeric antigen receptor (CAR) T cells have transformed the treatment landscape for hematological malignancies. However, CAR T cells are less efficient against solid tumors, largely due to poor infiltration resulting from the immunosuppressive nature of the tumor microenvironment (TME). Here, we assessed the efficacy of Lewis Y antigen (LeY)-specific CAR T cells in patient-derived xenograft (PDX) models of prostate cancer. In vitro, LeY CAR T cells directly killed organoids derived from androgen receptor (AR)-positive or AR-null PDXs. In vivo, although LeY CAR T cells alone did not reduce tumor growth, a single prior dose of carboplatin reduced tumor burden. Carboplatin had a pro-inflammatory effect on the TME that facilitated early and durable CAR T cell infiltration, including an altered cancer-associated fibroblast phenotype, enhanced extracellular matrix degradation and re-oriented M1 macrophage differentiation. In a PDX less sensitive to carboplatin, CAR T cell infiltration was dampened; however, a reduction in tumor burden was still observed with increased T cell activation. These findings indicate that carboplatin improves the efficacy of CAR T cell treatment, with the extent of the response dependent on changes induced within the TME.

During 4 decades, from 1950 to 1990, atmospheric electricity measurements as well as other environmental measurements were made by Reinhold Reiter at several sites close to Garmisch-Partenkirchen. The quantities determined included the atmospheric potential gradient, the vertical current, and the ion concentrations. Observations made at the Mount Wank site (47∘30′ N, 11∘09′ E; 1780 m) from 1 August 1972 to 31 December 1983 are available in digital form.

Background: The kidney failure risk equation (KFRE) can be used to predict progression to end-stage kidney disease in a clinical setting. Objective: Evaluate implementation of a formalized risk-based approach in nephrologists’ outpatient clinics and multidisciplinary chronic kidney disease (CKD) clinics to determine candidacy for multidisciplinary care, and the impact of CKD care selection on clinical outcomes. Design: Population-based descriptive cohort study. Setting: Alberta Kidney Care South. Patients: Adults attending or considered for a multidisciplinary CKD clinic between April 1, 2017, and March 31, 2019. Measurements: Exposure —The course of CKD care assigned by the nephrologist: management at multidisciplinary CKD clinic; management by a nephrologist or primary care physician. Primary Outcome —CKD progression, defined as commencement of kidney replacement therapy (KRT). Secondary Outcomes —Death, emergency department visits, and hospitalizations. Methods: We linked operational data from the clinics (available until March 31, 2019) with administrative health and laboratory data (available until March 31, 2020). Comparisons among patient groups, courses of care, and clinical settings with negative binomial regression count models and calculated unadjusted and fully adjusted incidence rate ratios. For the all-cause death outcome, we used Cox survival models to calculate unadjusted and fully adjusted hazard ratios. Results: Of the 1748 patients for whom a KFRE was completed, 1347 (77%) remained in or were admitted to a multidisciplinary CKD clinic, 310 (18%) were managed by a nephrologist only, and 91 (5%) were referred back for management by their primary care physician. There was a much higher kidney failure risk among patients who remained at or were admitted to a multidisciplinary CKD clinic (median 2-year risk of 34.7% compared with 3.6% and 0.8% who remained with a nephrologist or primary care physician, respectively). None of the people managed by their primary care physician alone commenced KRT, while only 2 (0.6%) managed by a nephrologist without multidisciplinary CKD care commenced KRT. The rates of emergency department visits, hospitalizations, and death were lower in those assigned to management outside the multidisciplinary CKD clinics when compared with those managed in the multidisciplinary care setting. Limitations: The follow-up period may not have been long enough to determine outcomes, and potentially limited generalizability given variability of care in multidisciplinary clinics. Conclusions: Our findings indicate that a portion of patients can be directed to less resource-intensive care without a higher risk of adverse events. Trial registration: Not applicable.

Abstract Background People with kidney failure often require surgery and experience worse postoperative outcomes compared to the general population, but existing risk prediction tools have excluded those with kidney failure during development or exhibit poor performance. Our objective was to derive, internally validate, and estimate the clinical utility of risk prediction models for people with kidney failure undergoing non-cardiac surgery. Design, setting, participants, and measures This study involved derivation and internal validation of prognostic risk prediction models using a retrospective, population-based cohort. We identified adults from Alberta, Canada with pre-existing kidney failure (estimated glomerular filtration rate [eGFR]

Background: Despite efforts to provide evidence-based care for people living with kidney disease, health care provider goals and priorities are often misaligned with those of individuals with lived experience of disease. Coupled with competing interests of time, resources, and an abundance of suitable guideline topics, identifying and prioritizing areas of focus for the Canadian nephrology community with a patient-oriented perspective is necessary and important. Similar priority-setting exercises have been undertaken to establish research priorities for kidney disease and to standardize outcomes for kidney disease research and clinical care; however, research priorities are distinct from priorities for guideline development. Inclusion of people living with health conditions in the selection and prioritization of guideline topics is suggested by patient engagement frameworks, though the process to operationalizing this is variable. We propose that the Canadian Society of Nephrology Clinical Practice Guideline Committee (CSN CPGC) takes the opportunity at this juncture to incorporate evidence-based prioritization exercises with involvement of people living with kidney disease and their caregivers to inform future guideline activities. In this protocol, we describe our planned research methods to address this. Objective: To establish consensus-based guideline topic priorities for the CSN CPGC using a modified Delphi survey with involvement of multidisciplinary stakeholders, including people living with kidney disease and their caregivers. Study design: Protocol for a Modified Delphi Survey. Setting: Pilot-tested surveys will be distributed via email and conducted using the online platform SurveyMonkey, in both French and English. Participants: We will establish a group of multidisciplinary clinical and research stakeholders (both within and outside CSN membership) from Canada, in addition to people living with kidney disease and/or their caregivers. Methods: A comprehensive literature search will be conducted to generate an initial list of guideline topics, which will be organized into three main categories: (1) International nephrology-focused guidelines that may require Canadian commentary, (2) Non-nephrology specific guidelines from Canada that may require CSN commentary, and (3) Novel topics for guideline development. Participants will engage in a multi-round Modified Delphi Survey to prioritize a set of “important guideline topics.” Measures: Consensus will be reached for an item based on both median score on the Likert-type scale (≥ 7) and the percentage agreement (≥ 75%); the Delphi process will be complete when consensus is reached on each item. Guideline topics will then be given a priority score calculated from the total Likert ratings across participants, adjusted for the number of participants. Limitations: Potential limitations include participant response rates and compliance to survey completion. Conclusions: We propose to incorporate evidence-based prioritization exercises with the engagement of people living with kidney disease and their caregivers to establish consensus-based guideline topics and inform future guidelines activities of the CSN CPGC.

Abstract Extracellular vesicles (EVs) recently emerged as important players in the pathophysiology of parasitic infections. While the protist parasite Giardia duodenalis can produce EVs, their role in giardiasis remains obscure. Giardia can disrupt gut microbiota biofilms and transform commensal bacteria into invasive pathobionts at sites devoid of colonizing trophozoites via unknown mechanisms. We hypothesized that Giardia EVs could modify gut bacterial behaviour via a novel mode of trans‐kingdom communication. Our findings indicate that Giardia EVs exert bacteriostatic effects on Escherichia coli HB101 and Enterobacter cloacae TW1, increasing their swimming motility. Giardia EVs also decreased the biofilm‐forming ability of E. coli HB101 but not by E. cloacae TW1, supporting the hypothesis that these effects are, at least in part, bacteria‐selective. E. coli HB101 and E. cloacae TW1 exhibited increased adhesion/invasion onto small intestine epithelial cells when exposed to Giardia EVs. EVs labelled with PKH67 revealed colocalization with E. coli HB101 and E. cloacae TW1 bacterial cells. Small RNA sequencing revealed a high abundance of ribosomal RNA (rRNA)‐ and transfer RNA (tRNA)‐derived small RNAs, short‐interfering RNAs (siRNAs) and micro‐RNAs (miRNAs) within Giardia EVs. Proteomic analysis of EVs uncovered the presence of RNA chaperones and heat shock proteins that can facilitate the thermal stability of EVs and its sRNA cargo, as well as protein‐modifying enzymes. In vitro, RNase heat‐treatment assays showed that total RNAs in EVs, but not proteins, are responsible for modulating bacterial swimming motility and biofilm formation. G. duodenalis small RNAs of EVs, but not proteins, were responsible for the increased bacterial adhesion to intestinal epithelial cells induced upon exposure to Giardia EVs. Together, the findings indicate that Giardia EVs contain a heat‐stable, RNase‐sensitive cargo that can trigger the development of pathobiont characteristics in Enterobacteria, depicting a novel trans‐kingdom cross‐talk in the gut.

Background: People with kidney failure have high risk of postoperative morbidity and mortality. Although the revised cardiac risk index (RCRI) is used to estimate the risk of major postoperative events, it has not been validated in this population. We aimed to externally validate the RCRI and determine whether updating the model improved predictions for people with kidney failure. Methods: We derived a retrospective, population-based cohort of adults with kidney failure (maintenance dialysis or sustained estimated glomerular filtration rate < 15 mL/min per 1.73 m2) who had surgery in Alberta, Canada between 2005 and 2019. We categorized participants based on RCRI variables and assigned risk estimates of death or major cardiac events, and then estimated predictive performance. We re-estimated the coefficients for each RCRI variable and internally validated the updated model. Net benefit was estimated with decision curve analysis. Results: After 38,541 surgeries, 1204 events (3.1%) occurred. The estimated C-statistic for the original RCRI was 0.64 (95% confidence interval: 0.62, 0.65). Examination of calibration revealed significant risk overestimation. In the re-estimated RCRI model, discrimination was marginally different (C-statistic 0.67 [95% confidence interval: 0.66, 0.69]), though calibration was improved. No net benefit was observed when the data were examined with decision curve analysis, whereas the original RCRI was associated with harm. Conclusions: The RCRI performed poorly in a Canadian kidney failure cohort and significantly overestimated risk, suggesting that RCRI use in similar kidney failure populations should be limited. A re-estimated kidney failure-specific RCRI may be promising but needs external validation. Novel perioperative models for this population are urgently needed. Résumé: Contexte: Les personnes atteintes d’insuffisance rénale présentent un risque élevé de mortalité et de morbidité postopératoires. L’indice de risque cardiaque révisé (IRCR) est utilisé pour estimer le risque d’événements postopératoires majeurs, mais il n’a pas été validé au sein de cette po-pulation. Nous avons cherché à réaliser une validation externe de l’IRCR et à déterminer si une modification du modèle pourrait permettre une meilleure valeur prédictive pour les patients atteints d’insuffisance rénale. Méthodologie: Nous avons étudié rétrospectivement une cohorte populationnelle d’adultes atteints d’insuffisance rénale (sous dialyse d’entretien ou avec un débit de filtration glomérulaire estimé < 15 ml/min/1,73 m2, de façon soutenue) ayant subi une intervention chirurgicale en Alberta (Canada) entre 2005 et 2019. Les participants ont été classifiés selon les variables de l’IRCR, et une estimation du risque de décès ou d’événement cardiovasculaire majeur leur a été attribuée; la performance prédictive a ensuite été évaluée. Nous avons réestimé les coefficients pour chacune des variables de l’IRCR et nous avons validé de manière interne le modèle modifié. Le bénéfice net a été estimé avec une analyse de la courbe décisionnelle. Résultats: Après 38 541 interventions chirurgicales, des événements cardiovasculaires sont survenus dans 1 204 cas (3,1 %). La statistique C estimée obtenue avec l’IRCR initial était de 0,64 (intervalle de confiance [IC] à 95 %, de 0,62 à 0,65). Un examen de la calibration de l’indice a révélé une surestimation significative du risque. Avec le modèle d’IRCR modifié, la discrimination présentait une légère différence (statistique C de 0,67 [IC à 95 %, de 0,66 à 0,69]), bien que la calibration ait été améliorée. Pour l’indice modifié, aucun bénéfice net n’a été observé lors de l’examen des données par une analyse décisionnelle, alors qu’un préjudice était associé à l’IRCR initial. Conclusions: L’IRCR s’est révélé peu concluant dans une cohorte populationnelle de patients canadiens atteints d’insuffisance rénale et il a significativement surestimé les risques pour ces patients, ce qui suggère que l’utilisation de l’IRCR dans des populations similaires atteintes d’insuffisance rénale devrait être limitée. Un IRCR réestimé, propre à la population des patients atteints d’insuffisance rénale, pourrait être prometteur, mais requiert une validation externe. De nouveaux modèles périopératoires sont indispensables pour cette population.

Rationale & Objective: To evaluate follow-up care of critically ill patients with acute kidney injury (AKI). Study Design: Retrospective cohort study. Setting & Participants: Patients admitted to the intensive care unit (ICU) with AKI in Alberta, Canada from 2005 to 2018, who survived to discharge without kidney replacement therapy or estimated glomerular filtration rate

ABSTRACT Plant-associated microbial assemblages are known to shift at time scales aligned with plant phenology, as influenced by the changes in plant-derived nutrient concentrations and abiotic conditions observed over a growing season. But these same factors can change dramatically in a sub-24-hour period, and it is poorly understood how such diel cycling may influence plant-associated microbiomes. Plants respond to the change from day to night via mechanisms collectively referred to as the internal “clock,” and clock phenotypes are associated with shifts in rhizosphere exudates and other changes that we hypothesize could affect rhizosphere microbes. The mustard Boechera stricta has wild populations that contain multiple clock phenotypes of either a 21- or a 24-hour cycle. We grew plants of both phenotypes (two genotypes per phenotype) in incubators that simulated natural diel cycling or that maintained constant light and temperature. Under both cycling and constant conditions, the extracted DNA concentration and the composition of rhizosphere microbial assemblages differed between time points, with daytime DNA concentrations often triple what were observed at night and microbial community composition differing by, for instance, up to 17%. While we found that plants of different genotypes were associated with variation in rhizosphere assemblages, we did not see an effect on soil conditioned by a particular host plant circadian phenotype on subsequent generations of plants. Our results suggest that rhizosphere microbiomes are dynamic at sub-24-hour periods, and those dynamics are shaped by diel cycling in host plant phenotype. IMPORTANCE We find that the rhizosphere microbiome shifts in composition and extractable DNA concentration in sub-24-hour periods as influenced by the plant host’s internal clock. These results suggest that host plant clock phenotypes could be an important determinant of variation in rhizosphere microbiomes.

Atmospheric electricity measurements were made at Lerwick Observatory, Shetland, between 1925 and 1984. These principally provide a long series of hourly potential gradient (PG) measurements at an unpolluted site but also include air–Earth current density measurements during the late 1970s and early 1980s. An especially notable aspect was investigating the dramatic atmospheric electrical changes caused by nuclear weapon detonations in the late 1950s and early 1960s, which has parallels with the discovery of the Antarctic ozone hole. The methodology employed at Lerwick to provide the PG measurements is described. There is renewed international interest in such measurements, not least because the Lerwick PG data have been shown to be linked to Pacific Ocean temperature anomalies. The past measurements described have characterised the Lerwick site exceptionally well in atmospheric electrical terms, which also indicate its suitability for future, similar measurements.

Background: Catheter malfunction in hemodialysis (HD) is increasingly managed with recombinant tissue plasminogen activator (rt-PA, alteplase), though evidence of improved catheter function is lacking. Objective: To evaluate the effect of a standardized rt-PA administration protocol on rt-PA usage, catheter function, and adverse events. Design: Observational quality improvement study. Setting: Single, urban, community HD unit in Calgary, Alberta. Patients: Patients treated with maintenance in-center HD through central venous catheter. Outcomes: Incidence of rt-PA usage, catheter interventions, hospitalizations, and measures of dialysis efficacy. Methods: The rt-PA protocol was designed following a consultative and iterative design period with dialysis shareholders, which included focusing on standard objective criteria before use and targeting use to the problematic lumen. Protocol implementation occurred over a 6-month period in 2021. Patient and dialysis data were collected through our regional dialysis electronic health record. Results: Implementation of the rt-PA protocol resulted in decreased rt-PA use (standardized per 100 dialysis sessions) compared to the preprotocol period (incidence rate ratio [IRR] of 0.57, 95% confidence interval [CI]: [0.34, 0.94]). Line procedures were also less frequent (IRR = 0.42, 95% CI: [0.18, 0.89]). Hospitalization rates and measures of dialysis efficacy were similar in both periods. Limitations: Small sample size with single dialysis center and short duration of follow-up. Conclusions: Implementation of a multidisciplinary designed rt-PA administration protocol decreased incident rt-PA usage

Abstract Background Peer support complements traditional models of chronic kidney disease (CKD) care through sharing of peer experiences, pragmatic advice, and resources to enhance chronic kidney disease self-management and decision-making. As peer support is variably offered and integrated into multi-disciplinary CKD care, we aimed to characterize healthcare providers’ experiences and views on peer support provision for people with non-dialysis-dependent CKD within Canada. Methods In this concurrent mixed methods study, we used a self-administered online survey to collect information from multi-disciplinary CKD clinic providers (e.g., nurses, nephrologists, allied health professionals) on peer support awareness, program characteristics and processes, perceived value, and barriers and facilitators to offering peer support in CKD clinics. Results were analyzed descriptively. We undertook semi-structured interviews with a sample of survey respondents to elaborate on perspectives about peer support in CKD care, which we analyzed using inductive, content analysis. Results We surveyed 113 providers from 49 clinics. Two thirds (66%) were aware of formal peer support programs, of whom 19% offered in-house peer support through their clinic. Peer support awareness differed by role and region, and most referrals were made by social workers. Likert scale responses suggested a high perceived need of peer support for people with CKD. Top cited barriers to offering peer support included lack of peer support access and workload demands, while facilitators included systematic clinic processes for peer support integration and alignment with external programs. Across 18 interviews, we identified themes related to peer support awareness, logistics, and accessibility and highlighted a need for integrated support pathways. Conclusions Our findings suggest variability in awareness and availability of peer support among Canadian multi-disciplinary CKD clinics. An understanding of the factors influencing peer support delivery will inform strategies to optimize its uptake for people with advanced CKD.

In atmospheric science, measurements above the surface have long been obtained by carrying instrument packages, radiosondes, aloft using balloons. Whilst occasionally used for research, most radiosondes – around 1000 are released daily – only generate data for routine weather forecasting. If meteorological radiosondes are modified to carry additional sensors, of either mass-produced commercial heritage or designed for a specific scientific application, a wide range of new measurements becomes possible. A programme to develop add-on devices for standard radiosondes, which retains the core meteorological use, is described here. Combining diverse sensors on a single radiosonde helps interpretation of findings and yields economy of equipment, consumables and effort. A self-configuring system has been developed to allow different sensors to be easily combined, enhancing existing weather balloons and providing an emergency monitoring capability for airborne hazards. This research programme was originally pursued to investigate electrical properties of extensive layer clouds and has expanded to include a wide range of balloon-carried sensors for solar radiation, cloud, turbulence, volcanic ash, radioactivity and space weather. For the cloud charge application, multiple soundings in both hemispheres have established that charging at the boundaries of extensive layer clouds is widespread and likely to be a global phenomenon. This paper summarises the Christiaan Huygens medal lecture given at the 2021 European Geosciences Union meeting.

Yang et al report that intrinsic cardiac adrenergic cell-derived norepinephrine promotes myocardial dysfunction in response to LPS. They demonstrate norepinephrine produced by these cells activated β1-adrenergic receptors on cardiomyocytes increasing the expression of tumor necrosis factor-a in a Ca2+/calmodulin-dependent protein kinase II dependent manner.

Persons with HIV (PWH) on long-term antiretroviral therapy (ART) have a higher incidence and prevalence of cardiometabolic diseases attributed, in part, to persistent inflammation despite viral suppression. In addition to traditional risk factors, immune responses to co-infections such as cytomegalovirus (CMV) may play an unappreciated role in cardiometabolic comorbidities and offer new potential therapeutic targets in a subgroup of individuals. We assessed the relationship of CX3CR1+, GPR56+, and CD57+/- T cells (termed CGC+) with comorbid conditions in a cohort of 134 PWH co-infected with CMV on long-term ART. We found that PWH with cardiometabolic diseases (non-alcoholic fatty liver disease, calcified coronary arteries, or diabetes) had higher circulating CGC+CD4+ T cells compared to metabolically healthy PWH. The traditional risk factor most correlated with CGC+CD4+ T cell frequency was fasting blood glucose, as well as starch/sucrose metabolites. While unstimulated CGC+CD4+ T cells, like other memory T cells, depend on oxidative phosphorylation for energy, they exhibited higher expression of carnitine palmitoyl transferase 1A compared to other CD4+ T cell subsets, suggesting a potentially greater capacity for fatty acid β-oxidation. Lastly, we show that CMV-specific T cells against multiple viral epitopes are predominantly CGC+. Together, this study suggests that among PWH, CGC+ CD4+ T cells are frequently CMV-specific and are associated with diabetes, coronary arterial calcium, and non-alcoholic fatty liver disease. Future studies should assess whether anti-CMV therapies could reduce cardiometabolic disease risk in some individuals.

IntroductionImproving outcomes for oral squamous cell carcinoma (OSCC) patients has been hindered by a lack of effective predictive animal models. Spontaneously occurring canine OSCC could help fill this gap. The objective of this study was to characterize the immune landscape of canine OSCC to advance understanding of how dogs could serve as a surrogate for human OSCC.Methods/ResultsCanine OSCC contains a heterogenous tumor immune microenvironment. CD3+ T cells were the predominant tumor infiltrating immune cell population; however, there was a wide range CD3+ T cell density across samples. The most common CD3+ T cell micro-anatomical distribution was defined as “pre-existing immunity”, but the remaining 20% of tumors were characterized as “immunologically ignorant” or “excluded infiltrates” patterns. When compared to normal oral mucosa, the tumor gene expression pattern suggests that canine OSCC microenvironment is highly inflamed and characterized by the presence of an anti-tumor immune response dominated by cytotoxic\effector T cells and NK cells (CD8a, GZMA, OX40, and HLA-A); however, overexpression of genes associated with effector T cell exhaustion and microenvironmental immunosuppression was also identified (PD-1, LAG3, CXCL2). Correlations between CD3+ T cell density and immune gene expression revealed key genes associated with cytotoxic anti-tumor T cell responses (GZMA, GZMB, PRF1), co-stimulation of T cells (CD27, CD28, ICOS), and other immune processes, including Type I IFN response (TNF, TNFSF10), and T cell exhaustion (CTLA4, PD-1). CD3+ T cell density in canine OSCC was significantly correlated with a cytolytic activity score (mean PRF1 and GZMA expression), suggestive of active effector CD8 T cell function. CD204+ macrophages were the second most abundant tumor infiltrating immune cell, and when comparing to normal oral mucosa, two differently expressed genes linked to tumor associated macrophages and myeloid derived suppressor cells (MDSC) were identified: CXCL2, CD70. Overexpression of CXCL2 was also identified in canine OSCC “T cell-high” tumors compared to “T cell-low” tumors.DiscussionThis study identified actionable immunotherapy targets which could inform future comparative oncology trials in canine OSCC: CTLA-4, PD-1, CXCL2. These data provide a good first step towards utilizing spontaneous canine OSCC as a comparative model for human OSCC radiation and immuno-oncology research.

Background Persons with HIV have a higher prevalence of coronary artery disease compared with their HIV‐negative counterparts. Earlier identification of subclinical atherosclerosis may provide a greater opportunity for cardiovascular disease risk reduction. We investigated coronary cross‐sectional area (CorCSA) by noncontrasted computed tomography imaging as a noninvasive measure of arterial remodeling among virally suppressed persons with HIV. Methods and Results We assessed 105 persons with HIV with a spectrum of cardiometabolic health. All participants underwent computed tomography imaging to assess the mean corCSA of the proximal left anterior descending artery and 28 participants underwent additional coronary computed tomography angiography. Partial Spearman rank correlations adjusted for cardiovascular disease risk factors were used to assess relationships of corCSA with anthropometric measurements, HIV‐related factors, and plasma cytokines. Mean corCSA measured by noncontrast computed tomography and coronary computed tomography angiography were strongly correlated (ρ=0.91, P

Abstract Background People with kidney failure have a high incidence of major surgery, though the risk of perioperative outcomes at a population-level is unknown. Our objective was to estimate the proportion of people with kidney failure that experience acute myocardial infarction (AMI) or death within 30 days of major non-cardiac surgery, based on surgery type. Methods In this retrospective population-based cohort study, we used administrative health data to identify adults from Alberta, Canada with major surgery between April 12,005 and February 282,017 that had preoperative estimated glomerular filtration rates (eGFRs)

Cardiovascular disease is the major cause of morbidity and mortality in breast cancer survivors. Chemotherapy contributes to this risk. We aimed to define the mechanisms of long-term vascular dysfunction caused by neoadjuvant chemotherapy (NACT) and identify novel therapeutic targets. We studied arteries from postmenopausal women who had undergone breast cancer treatment using docetaxel, doxorubicin, and cyclophosphamide (NACT) and from women with no history of such treatment matched for key clinical parameters. We explored mechanisms in WT and Nox4–/– mice and in human microvascular endothelial cells. Endothelium-dependent, NO-mediated vasodilatation was severely impaired in patients after NACT, while endothelium-independent responses remained normal. This was mimicked by a 24-hour exposure of arteries to NACT agents ex vivo. When applied individually, only docetaxel impaired endothelial function in human vessels. Mechanistic studies showed that NACT increased inhibitory eNOS phosphorylation of threonine 495 in a Rho-associated protein kinase–dependent (ROCK-dependent) manner and augmented vascular superoxide and hydrogen peroxide production and NADPH oxidase activity. Docetaxel increased expression of the NADPH oxidase NOX4 in endothelial and smooth muscle cells and NOX2 in the endothelium. A NOX4 increase in human arteries may be mediated epigenetically by diminished DNA methylation of the NOX4 promoter. Docetaxel induced endothelial dysfunction and hypertension in mice, and these were prevented in Nox4–/– mice and by pharmacological inhibition of Nox4 or Rock. Commonly used chemotherapeutic agents and, in particular, docetaxel alter vascular function by promoting the inhibitory phosphorylation of eNOS and enhancing ROS production by NADPH oxidases.

To advance our understanding of the stratosphere, high-quality observational datasets of the stratosphere are needed. It is commonplace that reanalysis datasets are used to conduct stratospheric studies. However, the accuracy of these reanalyses at these heights is hard to infer due to a lack of in situ measurements. Satellite measurements provide one source of temperature information. As some satellite information is already assimilated into reanalyses, the direct comparison of satellite temperatures to the reanalysis is not truly independent. Stratospheric lidars use Rayleigh scattering to measure density in the middle and upper atmosphere, allowing temperature profiles to be derived for altitudes from 30 km (where Mie scattering due to stratospheric aerosols becomes negligible) to 80–90 km (where the signal-to-noise ratio begins to drop rapidly). The Network for the Detection of Atmospheric Composition Change (NDACC) contains several lidars at different latitudes that have measured atmospheric temperatures since the 1970s, resulting in a long-running upper-stratospheric temperature dataset. These temperature datasets are useful for validating reanalysis datasets in the stratosphere, as they are not assimilated into reanalyses. Here, stratospheric temperature data from lidars in the Northern Hemisphere between 1990–2017 were compared with the European Centre for Medium-Range Weather Forecasts ERA-Interim and ERA5 reanalyses. To give confidence to any bias found, temperature data from NASA's EOS Microwave Limb Sounder were also compared to ERA-Interim and ERA5 at points over the lidar sites. In ERA-Interim a cold bias of −3 to −4 K between 10 and 1 hPa was found when compared to both measurement systems. Comparisons with ERA5 found a small bias of magnitude 1 K which varies between cold and warm bias with height between 10 and 1 hPa, indicating a good thermal representation of the middle atmosphere up to 1 hPa. A further comparison was undertaken looking at the temperature bias by year to see the effects of the assimilation of the Advanced Microwave Sounding Unit-A (AMSU-A) satellite data and the Constellation Observing System for Meteorology, Ionosphere, and Climate GPS Radio Occultation (COSMIC GPSRO) data on stratospheric temperatures within the aforementioned ERA analyses. It was found that ERA5 was sensitive to the introduction of COSMIC GPSRO in 2007 with the reduction of the cold bias above 1 hPa. In addition to this, the introduction of AMSU-A data caused variations in the temperature bias between 1–10 hPa between 1997–2008.

Abstract Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) sense viral RNA and activate antiviral immune responses. Herein we investigate their functions in human epithelial cells, the primary and initial target of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A deficiency in MDA5, RIG-I or mitochondrial antiviral signaling protein (MAVS) enhanced viral replication. The expression of the type I/III interferon (IFN) during infection was impaired in MDA5 −/− and MAVS −/−, but not in RIG-I −/−, when compared to wild type (WT) cells. The mRNA level of full-length angiotensin-converting enzyme 2 (ACE2), the cellular entry receptor for SARS-CoV-2, was ~ 2.5-fold higher in RIG-I −/− than WT cells. These data demonstrate MDA5 as the predominant SARS-CoV-2 sensor, IFN-independent induction of ACE2 and anti-SARS-CoV-2 role of RIG-I in epithelial cells.

Abstract The greatest contributors to cancer mortality are metastasis and the consequences of its treatment. Here, we present a novel treatment of metastatic breast cancer that combines photothermal therapy with targeted single-walled carbon nanotubes (SWCNTs) and immunostimulation with a checkpoint inhibitor. We find that the selective near-infrared photothermal ablation of primary orthotopic EMT6 breast tumors in syngeneic BALB/cJ mice using an annexin A5 (ANXA5) functionalized SWCNT bioconjugate synergistically enhances an anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4)-dependent abscopal response, resulting in an increased survival (55%) at 100 days after tumor inoculation. In comparison, there was no survival at 100 days for either photothermal therapy by itself or immunostimulation by itself. Prior to photothermal therapy, the SWCNT-ANXA5 bioconjugate was administered systemically at a relatively low dose of 1.2 mg/kg, where it then accumulated in tumor vasculature via ANXA5-dependent binding. During photothermal therapy, the average maximum temperature in the tumor reached 54 °C (duration 175 s). The mechanism of prolonged survival resulting from combinatorial photothermal ablation and immune stimulation was evaluated by flow cytometric quantification of splenic antitumoral immune effector cells and serum cytokine quantification.

Abstract Modern metabolomic approaches that generate more comprehensive phytochemical profiles than were previously available are providing new opportunities for understanding plant‐animal interactions. Specifically, we can characterize the phytochemical landscape by asking how a larger number of individual compounds affect herbivores and how compounds covary among plants. Here we use the recent colonization of alfalfa (Medicago sativa) by the Melissa blue butterfly (Lycaeides melissa) to investigate the effects of indivdiual compounds and suites of covarying phytochemicals on caterpillar performance. We find that survival, development time, and adult weight are all associated with variation in nutrition and toxicity, including biomolecules associated with plant cell function as well as putative anti‐herbivore action. The plant‐insect interface is complex, with clusters of covarying compounds in many cases encompassing divergent effects on different aspects of caterpillar performance. Individual compounds with the strongest associations are largely specialized metabolites, including alkaloids, phenolic glycosides, and saponins. The saponins are represented in our data by more than 25 individual compounds with beneficial and detrimental effects on L. melissa caterpillars, which highlights the value of metabolomic data as opposed to approaches that rely on total concentrations within broad defensive classes.

ABSTRACT The composition of microbial communities found in association with plants is influenced by host phenotype and genotype. However, the ways in which specific genetic architectures of host plants shape microbiomes are unknown. Genome duplication events are common in the evolutionary history of plants and influence many important plant traits, and thus, they may affect associated microbial communities. Using experimentally induced whole-genome duplication (WGD), we tested the effect of WGD on rhizosphere bacterial communities in Arabidopsis thaliana. We performed 16S rRNA amplicon sequencing to characterize differences between microbiomes associated with specific host genetic backgrounds (Columbia versus Landsberg) and ploidy levels (diploid versus tetraploid). We modeled relative abundances of bacterial taxa using a hierarchical Bayesian approach. We found that host genetic background and ploidy level affected rhizosphere community composition. We then tested to what extent microbiomes derived from a specific genetic background or ploidy level affected plant performance by inoculating sterile seedlings with microbial communities harvested from a prior generation. We found a negative effect of the tetraploid Columbia microbiome on growth of all four plant genetic backgrounds. These findings suggest an interplay between host genetic background and ploidy level and bacterial community assembly with potential ramifications for host fitness. Given the prevalence of ploidy-level variation in both wild and managed plant populations, the effects on microbiomes of this aspect of host genetic architecture could be a widespread driver of differences in plant microbiomes. IMPORTANCE Plants influence the composition of their associated microbial communities, yet the underlying host-associated genetic determinants are typically unknown. Genome duplication events are common in the evolutionary history of plants and affect many plant traits. Using Arabidopsis thaliana, we characterized how whole-genome duplication affected the composition of rhizosphere bacterial communities and how bacterial communities associated with two host plant genetic backgrounds and ploidy levels affected subsequent plant growth. We observed an interaction between ploidy level and genetic background that affected both bacterial community composition and function. This research reveals how genome duplication, a widespread genetic feature of both wild and crop plant species, influences bacterial assemblages and affects plant growth.

Computation methods that predict the binding of peptides to MHC-I are important tools for screening and identifying immunogenic antigens and have the potential to accelerate vaccine and drug development. However, most available tools are sequence-based and optimized only for peptides containing the twenty canonical amino acids. This omits a large number of peptides containing non-canonical amino acids (NCAA), or residues that undergo varied post-translational modifications such as glycosylation or phosphorylation. These modifications fundamentally alter peptide immunogenicity. Similarly, existing structure-based methods are biased towards canonical peptide backbone structures, which may or may not be preserved when NCAAs are present. Rosetta FlexPepDock ab-initio is a structure-based computational protocol able to evaluate peptide-receptor interaction where no prior information of the peptide backbone is known. We benchmarked FlexPepDock ab-initio for docking canonical peptides to MHC-I, and illustrate for the first time the method's ability to accurately model MHC-I bound epitopes containing NCAAs. FlexPepDock ab-initio protocol was able to recapitulate near-native structures (≤1.5Å) in the top lowest-energy models for 20 out of 25 cases in our initial benchmark. Using known experimental binding affinities of twenty peptides derived from an influenza-derived peptide, we showed that FlexPepDock protocol is able to predict relative binding affinity as Rosetta energies correlate well with experimental values (r = 0.59, p = 0.006). ROC analysis revealed 80% true positive and a 40% false positive rate, with a prediction power of 93%. Finally, we demonstrate the protocol's ability to accurately recapitulate HLA-A*02:01 bound phosphopeptide backbone structures and relative binding affinity changes, the theoretical structure of the lymphocytic choriomeningitis derived glycosylated peptide GP392 bound to MHC-I H-2Db, and isolevuglandin-adducted peptides. The ability to use non-canonical amino acids in the Rosetta FlexPepDock protocol may provide useful insight into critical amino acid positions where the post-translational modification modulates immunologic responses.

ABSTRACT New approaches to characterizing microbiomes via high-throughput sequencing provide impressive gains in efficiency and cost reduction compared to approaches that were standard just a few years ago. However, the speed of method development has been such that staying abreast of the latest technological advances is challenging. Moreover, shifting laboratory protocols to include new methods can be expensive and time consuming. To facilitate adoption of new techniques, we provide a guide and review of recent advances that are relevant for single-locus sequence-based study of microbiomes—from extraction to library preparation—including a primer regarding the use of liquid-handling automation in small-scale academic settings. Additionally, we describe several amendments to published techniques to improve throughput, track contamination, and reduce cost. Notably, we suggest adding synthetic DNA molecules to each sample during nucleic acid extraction, thus providing a method of documenting incidences of cross-contamination. We also describe a dual-indexing scheme for Illumina sequencers that allows multiplexing of many thousands of samples with minimal PhiX input. Collectively, the techniques that we describe demonstrate that laboratory technology need not impose strict limitations on the scale of molecular microbial ecology studies. IMPORTANCE New methods to characterize microbiomes reduce technology-imposed limitations to study design, but many new approaches have not been widely adopted. Here, we present techniques to increase throughput and reduce contamination alongside a thorough review of current best practices.