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Reminders, alerting patients to the need for vaccines that will be due in the future, and recall messages, informing patients about vaccines that are overdue, have been shown to improve immunization rates for children and adolescents in numerous systematic reviews. Therefore, reminder and recall interventions (R/R) are recommended by the Task Force on Community Preventive Services for increasing immunization rates on the basis of strong evidence. R/R messages can be delivered by mail (letter or postcard), via personal or auto-dialer phone calls, by text or e-mail or via patient-portals and can simply be alerts to action or can include educational material with the aim of motivating patients to seek vaccination. R/R has also been shown to be a relatively low-cost intervention with high cost-effectiveness compared with other recommended strategies. However, although R/R as a strategy is consistently effective and cost-effective overall, there is wide variation in the impact of R/R by 1) modality of how it is delivered, 2) the targeted vaccine, 3) the age group, and 4) whether the R/R is conducted centrally by a health system or Immunization Information System or by individual practices. This narrative review summarizes the literature about effectiveness of R/R within each of these categories. We also discuss limitations of R/R, with a focus on the potential impact of parental vaccine hesitancy in blunting its effectiveness and problems with data integrity, on which R/R relies. We also discuss challenges to sustaining R/R efforts, including potential methods of funding for R/R efforts.

Background Female reproductive anatomy and physiology, compounded by low health literacy, can result in limited patient understanding of gynecologic procedures, complicating the already challenging patient decision-making process. Objective Describe a process of developing an effective visual aid that accurately communicates the risks, benefits, and possible outcomes of hysterectomy surgical approaches. Patient Involvement None. Methods This qualitative study was performed in a private practice and an academic institution with a total of 8 female non-clinical employees in the English-speaking group and 9 non-employees in the Spanish-speaking group. A 5-minute video focusing on the hysterectomy procedure was developed and shown to focus group participants. A discussion led by a sociologist obtained feedback regarding understanding and perceptions of video content to validate the tool, which was analyzed to extract themes that could guide refinements. Results Focus group participants wanted more information on post-hysterectomy quality of life and desired a longer video that included more general information about hysterectomies. All participants felt the video would be a valuable aid if watched prior to undergoing a hysterectomy and would be an effective tool for improving patient communication. Discussion While there was strong support for the video-based patient education program, participants found some elements confusing (e.g., type and magnitude of risk, scope of aftercare) and expressed interest in more in-depth information. Our development process would have been enhanced by involving patients before the initial version of the video was created, holding more and more diverse focus groups and ensuring that translation was both accurate and culturally appropriate. Practical Value This study offers lessons learned in the process of developing a video-based visual aid for improving women's knowledge of hysterectomy and may inform other efforts to help patients understand complex medical pathology and procedures.

Purpose Female adolescents seeking emergency department (ED) care are at high risk of unintended pregnancy, primarily because of contraceptive nonuse; yet, few ED patients follow up for reproductive care when referred. The objective of this cohort study was to determine the feasibility, acceptability, adoption, fidelity, and potential efficacy of a personalized and interactive ED-based pregnancy prevention mobile health intervention (Emergency Room Interventions to improve the Care of Adolescents [Dr. Erica]). Methods We conducted a prospective cohort study with sexually active female ED patients aged 14–19 years who were not using highly effective contraceptives. Dr. Erica consists of a 10-week, automated, two-way texting intervention based on an evidence-based sexual health curriculum, the Social Cognitive Theory, and motivational interviewing techniques. At 12 weeks, we conducted follow-up via online survey and phone call to measure feasibility, acceptability, adoption, fidelity, and preliminary efficacy data (contraception initiation). Results We screened 209 female ED patients to enroll 42. The average age was 17.5 years (standard deviation ± 1.4); the majority were Hispanic (n = 37, 88%) and had a primary provider (n = 40, 95%). One participant opted out (1/42, 2%), and a total of 35 participants (83%) completed follow-up. Although interactivity diminished with time, 83% of participants (35/42) replied to one or more text. Ninety-four percent of participants (29/31) liked the messages, and 83% (25/30) would recommend the program. Hormonal contraceptives were initiated by 46% of participants (16/35). Conclusions Dr. Erica was feasible and acceptable among female adolescent ED patients and demonstrated high fidelity and adoption. The intervention also showed potential to increase highly effective contraceptive use among high-risk females.

OBJECTIVE Type II odontoid fractures may be managed operatively or nonoperatively. If managed with bracing, bony union may never occur despite stability. This phenomenon is termed fibrous union. The authors aimed to determine associations with stable fibrous union and compare the morbidity of patients managed operatively and nonoperatively. METHODS The authors performed a retrospective review of their spine trauma database for adults with type II odontoid fractures between 2015 and 2019. Two-sample t-tests and Fisher’s exact tests identified associations with follow-up stability and were used to compare operative and nonoperative outcomes. Sensitivity, specificity, and predictive values were calculated to validate initial stable upright cervical radiographs related to follow-up stability. RESULTS Among 88 patients, 10% received upfront surgical fixation, and 90% were managed nonoperatively, of whom 22% had fracture instability on follow-up. Associations with instability after nonoperative management include myelopathy (OR 0.04, 95% CI 0.0–0.92), cerebrovascular disease (OR 0.23, 95% CI 0.06–1.0), and dens displacement ≥ 2 mm (OR 0.29, 95% CI 0.07–1.0). Advanced age was not associated with follow-up instability. Initial stability on upright radiographs was associated with stability on follow-up (OR 4.29, 95% CI 1.0–18) with excellent sensitivity and positive predictive value (sensitivity 89%, specificity 35%, positive predictive value 83%, and negative predictive value 46%). The overall complication rate and respiratory failure requiring ventilation on individual complication analysis were more common in operatively managed patients (33% vs 3%, respectively; p = 0.007), even though they were generally younger and healthier than those managed nonoperatively. Operative or nonoperative management conferred no difference in length of hospital or ICU stay, discharge disposition, or mortality. CONCLUSIONS The authors delineate the validity of upright cervical radiographs on presentation in association with follow-up stability in type II odontoid fractures. In their experience, factors associated with instability included cervical myelopathy, cerebrovascular disease, and fracture displacement but not increased age. Operatively managed patients had higher complication rates than those managed without surgery. Fibrous union, which can occur with nonoperative management, provided adequate stability.

The characterization of cancer genomes has provided insight into somatically altered genes across tumors, transformed our understanding of cancer biology, and enabled tailoring of therapeutic strategies. However, the function of most cancer alleles remains mysterious, and many cancer features transcend their genomes. Consequently, tumor genomic characterization does not influence therapy for most patients. Approaches to understand the function and circuitry of cancer genes provide complementary approaches to elucidate both oncogene and non-oncogene dependencies. Emerging work indicates that the diversity of therapeutic targets engendered by non-oncogene dependencies is much larger than the list of recurrently mutated genes. Here we describe a framework for this expanded list of cancer targets, providing novel opportunities for clinical translation.

The research field of ferroptosis has seen exponential growth over the past few years, since the term was coined in 2012. This unique modality of cell death, driven by iron-dependent phospholipid peroxidation, is regulated by multiple cellular metabolic pathways, including redox homeostasis, iron handling, mitochondrial activity and metabolism of amino acids, lipids and sugars, in addition to various signalling pathways relevant to disease. Numerous organ injuries and degenerative pathologies are driven by ferroptosis. Intriguingly, therapy-resistant cancer cells, particularly those in the mesenchymal state and prone to metastasis, are exquisitely vulnerable to ferroptosis. As such, pharmacological modulation of ferroptosis, via both its induction and its inhibition, holds great potential for the treatment of drug-resistant cancers, ischaemic organ injuries and other degenerative diseases linked to extensive lipid peroxidation. In this Review, we provide a critical analysis of the current molecular mechanisms and regulatory networks of ferroptosis, the potential physiological functions of ferroptosis in tumour suppression and immune surveillance, and its pathological roles, together with a potential for therapeutic targeting. Importantly, as in all rapidly evolving research areas, challenges exist due to misconceptions and inappropriate experimental methods. This Review also aims to address these issues and to provide practical guidelines for enhancing reproducibility and reliability in studies of ferroptosis. Finally, we discuss important concepts and pressing questions that should be the focus of future ferroptosis research.

Aim Investigate changes in alcohol use and related harm using the first multisite, controlled, longitudinal study of Managed Alcohol Programs (MAPs). MAPs provide regular doses of alcohol, accommodation, social supports and healthcare to unstably housed people with alcohol dependence. Methods A multisite, quasi-experimental, longitudinal study was conducted in day centres, shelters and residential programs for unstably housed people. There were 59 MAP participants from six Canadian cities and 116 local controls. Self-reported alcohol consumption and harms were assessed at 0-2, 6 and 12 months. Liver function test results were accessed for MAP participants. Results Both groups had similar reductions in mean drinks per day (MAP: -8.11; controls: -8.54 controls, P < 0.001) and days drinking per month (MAP: -2.51 days, P < 0.05; control: -4.81 days, P = 0.0001) over 6–-12 months. Both reduced non-beverage alcohol consumption. MAP participants reported significantly fewer harms at both 0-2 and 6 months than controls. MAP participants had similar total consumption to controls, but spread out over more days (25.41 versus 19.64 days per month, P = 0.001). After leaving a MAP, participants’ liver status deteriorated, with increases in both aspartate transaminase and bilirubin levels. MAP sites with effective policies on outside drinking drank less and had fewer harms. Conclusion MAP participants drank less hazardously than controls, especially with effective management of non-MAP drinking. Reductions in alcohol use and harms occurred for both groups, although MAP participants reported fewer harms at 0-6 months. Departing an MAP was associated with deterioration in liver status. Although providing stable housing, MAPs did not worsen health or increase alcohol use.

The employment of directed acyclic graphs to advance the tracking, control and appraisal of crystallographic phasing strategies is discussed., Crystallographic phasing strategies increasingly require the exploration and ranking of many hypotheses about the number, types and positions of atoms, molecules and/or molecular fragments in the unit cell, each with only a small chance of being correct. Accelerating this move has been improvements in phasing methods, which are now able to extract phase information from the placement of very small fragments of structure, from weak experimental phasing signal or from combinations of molecular replacement and experimental phasing information. Describing phasing in terms of a directed acyclic graph allows graph-management software to track and manage the path to structure solution. The crystallographic software supporting the graph data structure must be strictly modular so that nodes in the graph are efficiently generated by the encapsulated functionality. To this end, the development of new software, Phasertng, which uses directed acyclic graphs natively for input/output, has been initiated. In Phasertng, the codebase of Phaser has been rebuilt, with an emphasis on modularity, on scripting, on speed and on continuing algorithm development. As a first application of phasertng, its advantages are demonstrated in the context of phasertng.xtricorder, a tool to analyse and triage merged data in preparation for molecular replacement or experimental phasing. The description of the phasing strategy with directed acyclic graphs is a generalization that extends beyond the functionality of Phasertng, as it can incorporate results from bioinformatics and other crystallographic tools, and will facilitate multifaceted search strategies, dynamic ranking of alternative search pathways and the exploitation of machine learning to further improve phasing strategies.

INTRODUCTION Policy changes may contribute to increased alcohol-related risks to populations. These include privatisation of alcohol retailing, which influences density of alcohol outlets, location of outlets, hours of sale and prevention of alcohol sales to minors or intoxicated customers. Meta-analyses, reviews and original research indicate enhanced access to alcohol is associated with elevated risk of and actual harm. We assess the 10 Canadian provinces on two alcohol policy domains-type of alcohol control system and physical availability of alcohol-in order to track changes over time, and document shifting changes in alcohol policy. METHODS Our information was based on government documents and websites, archival statistics and key informant interviews. Policy domains were selected and weighted for their degree of effectiveness and population reach based on systematic reviews and epidemiological evidence. Government representatives were asked to validate all the information for their jurisdiction. RESULTS The province-specific reports based on the 2012 results showed that 9 of 10 provinces had mixed retail systems-a combination of government-run and privately owned alcohol outlets. Recommendations in each provincial report were to not increase privatisation. However, by 2017 the percentage of off-premise private outlets had increased in four of these nine provinces, with new private outlet systems introduced in several. DISCUSSION AND CONCLUSIONS Decision-making protocols are oriented to commercial interests and perceived consumer convenience. If public health and safety considerations are not meaningfully included in decision-making protocols on alcohol policy, then it will be challenging to curtail or reduce harms.

Autistic people, by definition, differ in social behavior from non-autistic individuals. One characteristic common to many autistic people is a special interest in a particular topic—something spoken about with such frequency and intensity that it may be stigmatized by non-autistic peers. We investigated college students’ interest in interacting with peers described as behaving in ways characteristic of autism (or not), and additionally described as having a special interest (or not). As expected, autistic characters were more stigmatized, but autistic characters with a special interest were not more stigmatized than those without. Only among non-autistic characters was having a special interest associated with greater stigmatization. Findings give further insight into factors influencing the stigmatization of autistic college students.

The representation of non-standard and regional accent and dialect in literary fiction has been framed mainly sociolinguistically and treated as an index of authenticity, within an account of characterisation. The reader’s attitude to such speakers in literary fiction is manipulated narratorially and authorially. Since readerly effects, impressions and evaluations are the key issues involved, it seems plausible that a cognitive poetic approach to the reading of dialect in literature would also be productive. In the current deictic theory, the dimension of social deixis captures a broad range of stylistic features including register and dialectal representations. Cognitive deictic theory draws on an explicitly spatial metaphor in which characters are positioned in conceptual space. However, insufficient attention has been paid to the effect of readerly positioning and dispositioning. This article revisits social deixis and its points of transition and textural variation from a theoretical perspective. It develops a new angle on the representation and significance of accented and dialectal forms in literary fiction, with some illustrative examples drawn from 19th and 20th century British novels.

Excessive alcohol use is a leading cause of preventable death in the United States (1) and costs associated with it, such as those from losses in workplace productivity, health care expenditures, and criminal justice, were $249 billion in 2010 (2). CDC used the Alcohol-Related Disease Impact (ARDI) application* to estimate national and state average annual alcohol-attributable deaths and years of potential life lost (YPLL) during 2011-2015, including deaths from one's own excessive drinking (e.g., liver disease) and from others' drinking (e.g., passengers killed in alcohol-related motor vehicle crashes). This study found an average of 93,296 alcohol-attributable deaths (255 deaths per day) and 2.7 million YPLL (29 years of life lost per death, on average) in the United States each year. Of all alcohol-attributable deaths, 51,078 (54.7%) were caused by chronic conditions, and 52,361 (56.0%) involved adults aged 35-64 years. Age-adjusted alcohol-attributable deaths per 100,000 population ranged from 20.3 in New Jersey and New York to 52.3 in New Mexico. YPLL per 100,000 population ranged from 613.8 in New York to 1,651.7 in New Mexico. Implementation of effective strategies for preventing excessive drinking, including those recommended by the Community Preventive Services Task Force (e.g., increasing alcohol taxes and regulating the number and concentration of alcohol outlets), could reduce alcohol-attributable deaths and YPLL.†.

The deferred antagonism technique has been utilized for several decades for detecting antibiosis activity. Most protocols require the elimination of antibiotic-producing cells by exposing them to chloroform vapour, UV radiation or filter sterilizing the filtrate steps that require additional time and expense to complete. We provide a modified approach to current soft agar overlay practices, which involves addition of antibiotics to the soft agar overlay to inhibit growth of the producer but not the indicator strain. This technique can be used to reproducibly and efficiently screen for antibiotic production with ease. We demonstrate the effectiveness of this technique with three bacterial systems: inhibition of the bacterial spot of tomato pathogen, Xanthomonas euvesicatoria, by its pathogenic competitor Xanthomonas perforans; and inhibition of the fire blight pathogen, Erwinia amylovora, by Pantoea vagans C9-1 or Pseudomonas fluorescens A506. Significance and Impact of the Study Deferred antagonism assays are used commonly to observe antibiotic production by micro-organisms. Killing or removing the producer cells prior to introduction of the indicator strain is a standard practice but requires additional time and special handling procedures. We evaluated a modification of the assay, where the overlay medium is amended with an antibiotic to which the indicator strain is resistant and the producer strain is sensitive. This modification obviates extra steps to kill the producer strain prior to overlaying with the indicator strain and provides a rapid, consistent and cost-effective method to detect antibiosis.

Varus-valgus constrained (VVC) inserts are used in primary total knee arthroplasty (TKA) when stability cannot be achieved with a traditional insert. Concern has been raised regarding premature loosening and failure of these primary TKAs due to the increased load transfer through the prosthesis. This study seeks to assess the survival, clinical outcomes and radiographic assessment of VVC total knee inserts used in a single primary TKA system without diaphyseal stem extensions.A consecutive cohort of 74 primary TKAs with VVC inserts was identified from an institutional database. A two-to-one matched group of 136 posterior-stabilized (PS) primary TKAs was generated from the same database. Survival analysis was assessed for all-cause revision surgery. Patient outcome measures were the Oxford Knee Score (OKS) and patient reported satisfaction. Radiographs were assessed in accordance with the Knee Society radiographic scoring system for radiolucency.Survival rates at three and five years were 98.1% and 95.1% for the VVC liner group and 98.1% and 98.1% for the PS liner group, respectively. OKS improved from pre-op to post-op for both groups (p .001). OKS was reduced in the VVC liner group compared to the PS liner group at latest available follow-up (p = .012). However, clinical satisfaction rates did not differ between the two groups. Small, non-progressive radiolucent lines existed on several radiographs; however, no components in either group were deemed radiographically loose.The survivorship, clinical, and radiographic outcomes of VVC knees were similar to the PS comparison group at short- to mid-term follow-up.

Social wasps of the genus Vespula have spread to nearly all landmasses worldwide and have become significant pests in their introduced ranges, affecting economies and biodiversity. Comprehensive genome assemblies and annotations for these species are required to develop the next generation of control strategies and monitor existing chemical control. We sequenced and annotated the genomes of the common wasp (Vespula vulgaris), German wasp (Vespula germanica), and the western yellowjacket (Vespula pensyl- vanica). Our chromosome-level Vespula assemblies each contain 176–179 Mb of total sequence assembled into 25 scaffolds, with 10–200 unanchored scaffolds, and 16,566–18,948 genes. We annotated gene sets relevant to the applied management of invasive wasp populations, including genes associated with spermatogenesis and development, pesticide resistance, olfactory receptors, immunity and venom. These genomes provide evidence for active DNA methylation in Vespidae and tandem duplications of venom genes. Our genomic resources will contribute to the development of next-generation control strategies, and monitoring potential resistance to chemical control.

Cell death is an essential feature of development in multicellular organisms, a critical driver of degenerative diseases, and can be harnessed for treating some cancers. Understanding the mechanisms governing cell death is critical for addressing its role in disease. Similarly, metabolism is essential for normal energy and biomolecule production, and goes awry in many diseases. Metabolism and cell death are tightly linked in the phenomenon of ferroptosis, a form of regulated cell death driven by peroxidation of phospholipids. Glutathione peroxidase 4 (GPX4) uses glutathione to protect cells from ferroptosis by eliminating phospholipid peroxides. Recent data have revealed glutathione/GPX4-independent axes for suppressing ferroptosis, and insight into the regulation of iron and mitochondria in ferroptosis. Ferroptosis has recently been implicated in multiple diseases, and functions as a tumor suppression mechanism. Ferroptosis induction is a promising approach in treating a number of conditions, including neoplastic diseases. Here, we summarize these recent advances.

MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, MlklD139V, that alters the two-helix ‘brace’ that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of MlklD139V homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO)., Necroptosis is a regulated form of inflammatory cell death driven by activated MLKL. Here, the authors identify a mutation in the brace region that confers constitutive activation, leading to lethal inflammation in homozygous mutant mice and providing insight into human mutations in this region.

Objective: Media coverage of alcohol-related policy measures can influence public debate and is often more aligned with interests of the alcohol industry than public health. The purpose of this study was to examine the framing of news coverage of alcohol warning label (AWL) initiatives that included a cancer message on alcohol containers in two different countries. Policy contexts and industry perspectives were also evaluated. Method: We identified and systematically reviewed news articles published between 2017–2019 covering an AWL academic study in Yukon, Canada, and labeling provisions in a Public Health (Alcohol) Bill in Ireland. Both included a cancer message. News stories were coded for media type and topic slant; inclusion of alcohol industry perspectives was examined using content analysis. Results: Overall, 68.4% of media articles covering the Yukon Study (n = 38) and 18.9% covering the Ireland Bill (n = 37) were supportive of AWLs with a cancer message. The majority of articles in both sites presented alcohol industry perspectives (Yukon, 65.8%; Ireland, 86.5%), and industry arguments opposing AWLs were similar across both contexts. In articles with statements from industry representatives, the label message was frequently disputed by distorting or denying the evidence that alcohol causes cancer (n = 33/43). Conclusions: News coverage of AWLs with a cancer message was more supportive in Canada than Ireland, where alcohol industry perspectives were consistently foregrounded. Industry arguments opposing the cancer label bore similarities across contexts, often distorting or denying the evidence. Increasing awareness of industry messaging strategies may generate more critical coverage of industry lobbying activities and increase public support for alcohol policies. (J. Stud. Alcohol Drugs, 81, 273–283, 2020)

OBJECTIVES The purpose of this study was to determine correlation and temporal association between automated pupillary measurements and intracranial pressure in pediatric patients with brain injury or encephalopathy requiring intracranial pressure monitoring. We hypothesized that abnormal pupillary measurements would precede increases in intracranial pressure. DESIGN A prospective cohort study was performed. Automated pupillometry measurements were obtained at the same frequency as the patients' neurologic assessments with concurrent measurement of intracranial pressure, for up to 72 hours. Pupillary measurements and the Neurologic Pupil index, an algorithmic score that combines measures of pupillary reactivity, were assessed for correlation with concurrent and future intracranial pressure measurements. SETTING Single-center pediatric quaternary ICU, from July 2017 to October 2018. PATIENTS Pediatric patients 18 years or younger with a diagnosis of acute brain injury or encephalopathy requiring an intracranial pressure monitor. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS Twenty-eight patients were analyzed with a total of 1,171 intracranial pressure measurements. When intracranial pressure was elevated, the Neurologic Pupil index, percent change in pupillary size, constriction velocity, and dilation velocity were significantly lower than when intracranial pressure was within normal range (p < 0.001 for all). There were mild to moderate negative correlations between concurrent intracranial pressure and pupillary measurements. However, there was an inconsistent pattern of abnormal pupillary measurements preceding increases in intracranial pressure; some patients had a negative association, while others had a positive relationship or no relationship between Neurologic Pupil index and intracranial pressure. CONCLUSIONS Our data indicate automated assessments of pupillary reactivity inversely correlate with intracranial pressure, demonstrating that pupillary reactivity decreases as intracranial pressure increases. However, a temporal association in which abnormal pupillary measurements precede increases in intracranial pressure was not consistently observed. This work contributes to limited data available regarding automated pupillometry in neurocritically ill patients, and the even more restricted subset available in pediatrics.

Objective:Although the World Health Organization (WHO) declared alcohol a Class 1 carcinogen 30 years ago, few governments have communicated this fact to the public. We illustrate how alcohol indus...

The highly productive northern Bering and Chukchi marine shelf ecosystem has long been dominated by strong seasonality in sea-ice and water temperatures. Extremely warm conditions from 2017 into 2019—including loss of ice cover across portions of the region in all three winters—were a marked change even from other recent warm years. Biological indicators suggest that this change of state could alter ecosystem structure and function. Here, we report observations of key physical drivers, biological responses and consequences for humans, including subsistence hunting, commercial fishing and industrial shipping. We consider whether observed state changes are indicative of future norms, whether an ecosystem transformation is already underway and, if so, whether shifts are synchronously functional and system wide or reveal a slower cascade of changes from the physical environment through the food web to human society. Understanding of this observed process of ecosystem reorganization may shed light on transformations occurring elsewhere. Exceptionally warm years in 2017–2019 have caused changes in the physical and biological characteristics of the Pacific Arctic Ocean. What these changes mean for the ecosystem and societal consequences will depend on if they are evidence of a transformation or anomalies in the system.

MDM2 and MDMX, negative regulators of the tumor suppressor p53, can work separately and as a heteromeric complex to restrain p53's functions. MDM2 also has pro-oncogenic roles in cells, tissues, and animals that are independent of p53. There is less information available about p53-independent roles of MDMX or the MDM2–MDMX complex. We found that MDM2 and MDMX facilitate ferroptosis in cells with or without p53. Using small molecules, RNA interference reagents, and mutant forms of MDMX, we found that MDM2 and MDMX, likely working in part as a complex, normally facilitate ferroptotic death. We observed that MDM2 and MDMX alter the lipid profile of cells to favor ferroptosis. Inhibition of MDM2 or MDMX leads to increased levels of FSP1 protein and a consequent increase in the levels of coenzyme Q10, an endogenous lipophilic antioxidant. This suggests that MDM2 and MDMX normally prevent cells from mounting an adequate defense against lipid peroxidation and thereby promote ferroptosis. Moreover, we found that PPARα activity is essential for MDM2 and MDMX to promote ferroptosis, suggesting that the MDM2–MDMX complex regulates lipids through altering PPARα activity. These findings reveal the complexity of cellular responses to MDM2 and MDMX and suggest that MDM2–MDMX inhibition might be useful for preventing degenerative diseases involving ferroptosis. Furthermore, they suggest that MDM2/MDMX amplification may predict sensitivity of some cancers to ferroptosis inducers.

Energy stress depletes ATP and induces cell death. Here, we identify an unexpected inhibitory role of energy stress on ferroptosis, a form of regulated cell death induced by iron-dependent lipid peroxidation. We found that ferroptotic cell death and lipid peroxidation can be inhibited by treatments that induce or mimic energy stress. Inactivation of AMP-activated protein kinase (AMPK), a sensor of cellular energy status, largely abolishes the protective effects of energy stress on ferroptosis in vitro and on ferroptosis-associated renal ischemia/reperfusion injury in vivo. Cancer cells with high basal AMPK activation are resistant to ferroptosis, and AMPK inactivation sensitizes these cells to ferroptosis. Functional and lipidomic analyses further link AMPK regulation of ferroptosis to AMPK-mediated phosphorylation of acetyl-CoA carboxylase (ACC) and polyunsaturated fatty acid biosynthesis. Together, our study demonstrates that energy stress inhibits ferroptosis partly through AMPK, and reveals an unexpected coupling between ferroptosis and AMPK-mediated energy stress signaling.

There is a compelling need for new therapeutic strategies for glioblastoma multiforme (GBM). Preclinical target and therapeutic discovery for GBMs is primarily conducted using cell lines grown in serum-containing media, such as U-87 MG, which do not reflect the gene expression profiles of tumors found in GBM patients. To address this lack of representative models, we sought to develop a panel of patient-derived GBM models and characterize their genomic features, using RNA sequencing (RNA-seq) and growth characteristics, both when grown as neurospheres in culture, and grown orthotopically as xenografts in mice. When we compared these with commonly used GBM cell lines in the Cancer Cell Line Encyclopedia (CCLE), we found these patient-derived models to have greater diversity in gene expression and to better correspond to GBMs directly sequenced from patient tumor samples. We also evaluated the potential of these models for targeted therapy, by using the genomic characterization to identify small molecules that inhibit the growth of distinct subsets of GBMs, paving the way for precision medicines for GBM.

OBJECTIVE: Policy makers require evidence-based estimates of the economic costs of substance use-attributable lost productivity to set strategies aimed at reducing substance use-related harms. Building on a study by Rehm et al. (2006), we provide estimates of workplace costs using updated methods and data sources. METHODS: We estimated substance use-attributable productivity losses due to premature mortality, long-term disability, and presenteeism/absenteeism in Canada between 2007 and 2014. Lost productivity was estimated using a hybrid prevalence and incidence approach. Substance use prevalence data were drawn from three national self-report surveys. Premature mortality data were from the Canadian Vital Statistics Death Database, and long-term disability and workplace interference data were from the Canadian Community Health Survey. RESULTS: In 2014, the total cost of lost productivity due to substance use was $15.7 billion, or approximately $440 per Canadian, an increase of 8% from 2007. Substances responsible for the greatest economic costs were alcohol (38% of per capita costs), tobacco (37%), opioids (12%), other central nervous system (CNS) depressants (4%), other CNS stimulants (3%), cannabis (2%), cocaine (2%), and finally other psychoactive substances (2%). CONCLUSION: In 2014, alcohol and tobacco represent three quarters of substance use-related lost productivity costs in Canada, followed by opioids. These costs provide a valuable baseline that can be used to assess the impact of future substance use policy, practice, and other interventions, especially important given Canada’s opioid crisis and recent cannabis legalization. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.17269/s41997-019-00271-8) contains supplementary material, which is available to authorized users.

Background The Enquiring About Tolerance (EAT) study examined whether the early introduction of 6 allergenic foods from 3 months of age in exclusively breastfed infants prevented the development of food allergy. The intervention was effective in the per-protocol analysis for allergy to 1 or more foods and for egg and peanut individually, but only 42% of early introduction group (EIG) children met the per-protocol criteria. Objective We sought to identify which factors were responsible for nonadherence in the EAT study. Methods Factors influencing adherence within the key early introduction period in the EIG (up to 6 months of age) were divided into enrollment and postenrollment factors, and their association with nonadherence was explored. Results In an adjusted analysis, at enrollment, increased maternal age, nonwhite ethnicity, and lower maternal quality of life were independently and significantly associated with overall nonadherence in the EIG. Enrollment eczema and enrollment serum allergen-specific IgE sensitization to 1 or more foods (≥0.1 kU/L) were not related to overall nonadherence. After enrollment, 2 factors were significantly related to EIG overall nonadherence: parent-reported IgE-type symptoms with infant allergenic food consumption by 6 months of age and reported feeding difficulties by 4 months of age. Conclusion If early introduction of allergenic foods were to be considered a strategy to prevent food allergy, families of nonwhite ethnicity, those with older mothers, and those with infants with reported feeding difficulties or early-onset eczema would benefit from support to promote early and sustained consumption., Graphical abstract

Background The twin problems of severe alcohol dependence and homelessness are associated with precarious living and multiple acute, social and chronic harms. While much attention has been focused on harm reduction services for illicit drug use, there has been less attention to harm reduction for this group. Managed alcohol programs (MAPs) are harm reduction interventions that aim to reduce the harms of severe alcohol use, poverty and homelessness. MAPs typically provide accommodation, health and social supports alongside regularly administered sources of beverage alcohol to stabilize drinking patterns and replace use of non-beverage alcohol (NBA). Methods We examined impacts of MAPs in reducing harms and risks associated with substance use and homelessness. Using case study methodology, data were collected from five MAPs in five Canadian cities with each program constituting a case. In total, 53 program participants, 4 past participants and 50 program staff were interviewed. We used situational analysis to produce a series of “messy”, “ordered” and “social arenas” maps that provide insight into the social worlds of participants and the impact of MAPs. Results Prior to entering a MAP, participants were often in a revolving world of cycling through multiple arenas (health, justice, housing and shelters) where abstinence from alcohol is often required in order to receive assistance. Residents described living in a street-based survival world characterized by criminalization, unmet health needs, stigma and unsafe spaces for drinking and a world punctuated by multiple losses and disconnections. MAPs disrupt these patterns by providing a harm reduction world in which obtaining accommodation and supports are not contingent on sobriety. MAPs represent a new arena that focuses on reducing harms through provision of safer spaces and supply of alcohol, with opportunities for reconnection with family and friends and for Indigenous participants, Indigenous traditions and cultures. Thus, MAPs are safer spaces but also potentially spaces for healing. Conclusions In a landscape of limited alcohol harm reduction options, MAPs create a new arena for people experiencing severe alcohol dependence and homelessness. While MAPs reduce precarity for participants, programs themselves remain precarious due to ongoing challenges related to lack of understanding of alcohol harm reduction and insecure program funding.

Purpose Personalized and interactive text messaging interventions may increase participant engagement; yet, how to design messages that retain adolescent attention and positively affect sexual health behaviors remains unclear. The purpose of this study was to identify the characteristics of sexual health text messages perceived as engaging by sexually active adolescent females. Methods We conducted semistructured, open-ended interviews with sexually active females aged 14–19 in one urban emergency department. Participants received automated sexual health information sent via an interactive, two-way texting format. The 343 messages viewed by participants were based on key stakeholder input, relevant theoretical models, and existing evidence-based guidelines. Interviews elicited feedback. Enrollment continued until saturation of themes. Interviews were recorded, transcribed, and coded based on thematic analysis using NVivo 10. Results Participants (n = 31) were predominantly Hispanic (28; 90%), insured (29; 94%), and recently sexually active (24; 77%). Themes were as follows: (1) Tone: messages should be direct, factual, entertaining, and respect adolescent autonomy; messages should not be intrusive, presumptive, or preachy. (2) Emotion evoked: participants preferred messages that provoked thought, validated feelings, and empowered. Messages from a reliable source felt comforting, making participants feel cared for and special. (3) Interactivity: participants favored messages that offered choices, such as a mini-conversation. (4) Personalization: messages should look similar to adolescent digital preferences but be individually tailored with relatable characters. Conclusions This study informs the tone, structure, and style of sexual health text messages directed toward adolescent females in the emergency department. Future work should consider these characteristics when designing digital interventions to engage adolescent females.

Interleukin 6 (IL6) is a cytokine that regulates a number of important immune and inflammatory pathways. We used the ability of IL6 to inhibit the clonal proliferation of the mouse M1 myeloid leukemia cell line in agar to positively screen a cDNA expression library for proteins that inhibited IL6 activity. We found three clones completely resistant to IL6 that contained the cDNA for the Membrane-Associated RING-CH E3 ubiquitin ligase MARCH2. MARCH2 is a member of a family of membrane-bound E3 ubiquitin ligases that target cell surface receptors for degradation. MARCH2 overexpressing M1 clones retained responsiveness to the related cytokines leukemia inhibitory factor and oncostatin M and we showed that its inhibitory effect was a result of selective down-regulation of the IL6 receptor alpha chain and not the shared receptor subunit, gp130 or other signalling molecules. This activity of MARCH2 was also shared with related proteins MARCH4, MARCH9 and an isoform of MARCH3. The transmembrane domains and C-terminal domains, as well as a functional RING domain, of MARCH proteins were all required for substrate recognition and down-regulation. Genetic deletion of individual MARCH proteins in mice had no or little effect on IL6Rα levels but combined deletions of MARCH2,3 and 4 displayed elevated steady-state levels of IL6Rα in selected haemopoietic cell subsets including CD8+ and CD4+ T cells. These studies extend the potential immunosuppressive roles of MARCH proteins to include down-regulation of IL6 inflammatory responses.

Healthcare-acquired infections (HAIs) continue to persist in hospitals, despite the use of increasingly strict infection-control precautions. Opportunistic airborne transmission of potentially pathogenic bioaerosols may be one possible reason for this persistence. Therefore, this study aimed to systematically review the concentrations and compositions of indoor bioaerosols in different areas within hospitals and the effects of different ventilation systems. Electronic databases (Medline and Web of Science) were searched to identify articles of interest. The search was restricted to articles published from 2000 to 2017 in English. Aggregate data was used to examine the differences in mean colony forming units per cubic metre (cfu/m3) between different hospital areas and ventilation types. A total of 36 journal articles met the eligibility criteria. The mean total bioaerosol concentrations in the different areas of the hospitals were highest in the inpatient facilities (77 cfu/m3, 95% confidence interval (CI): 55-108) compared with the restricted (13cfu/m3, 95% CI: 10-15) and public areas (14 cfu/m3, 95% CI: 10-19). Hospital areas with natural ventilation had the highest total bioaerosol concentrations (201 cfu/m3, 95% CI: 135-300) compared with areas using conventional mechanical ventilation systems (20 cfu/m3, 95% CI: 16-24). Hospital areas using sophisticated mechanical ventilation systems (such as increased air changes per hour, directional flow and filtration systems) had the lowest total bioaerosol concentrations (9 cfu/m3, 95% CI: 7-13). Operating sophisticated mechanical ventilation systems in hospitals contributes to improved indoor air quality within hospitals, which assists in reducing the risk of airborne transmission of HAIs.

Ferroptosis, a cell death process driven by cellular metabolism and iron-dependent lipid peroxidation, has been implicated in diseases such as ischaemic organ damage and cancer1,2. The enzyme glutathione peroxidase 4 (GPX4) is a central regulator of ferroptosis, and protects cells by neutralizing lipid peroxides, which are by-products of cellular metabolism. The direct inhibition of GPX4, or indirect inhibition by depletion of its substrate glutathione or the building blocks of glutathione (such as cysteine), can trigger ferroptosis3. Ferroptosis contributes to the antitumour function of several tumour suppressors such as p53, BAP1 and fumarase4–7. Counterintuitively, mesenchymal cancer cells—which are prone to metastasis, and often resistant to various treatments—are highly susceptible to ferroptosis8,9. Here we show that ferroptosis can be regulated non-cell-autonomously by cadherin-mediated intercellular interactions. In epithelial cells, such interactions mediated by E-cadherin suppress ferroptosis by activating the intracellular NF2 (also known as merlin) and Hippo signalling pathway. Antagonizing this signalling axis allows the proto-oncogenic transcriptional co-activator YAP to promote ferroptosis by upregulating several ferroptosis modulators, including ACSL4 and TFRC. This finding provides mechanistic insights into the observations that cancer cells with mesenchymal or metastatic property are highly sensitive to ferroptosis8. Notably, a similar mechanism also modulates ferroptosis in some non-epithelial cells. Finally, genetic inactivation of the tumour suppressor NF2, a frequent tumorigenic event in mesothelioma10,11, rendered cancer cells more sensitive to ferroptosis in an orthotopic mouse model of malignant mesothelioma. Our results demonstrate the role of intercellular interactions and intracellular NF2–YAP signalling in dictating ferroptotic death, and also suggest that malignant mutations in NF2–YAP signalling could predict the responsiveness of cancer cells to future ferroptosis-inducing therapies. Ferroptosis in cancer cells can be regulated by cadherin-mediated intercellular contacts, NF2–Hippo signalling, and activity of the YAP transcription co-activator.

The human papillomavirus virus (HPV) vaccine is effective at preventing various cancers, but coverage falls short of targets that are needed for community protection. Here, we use the RE-AIM implementation framework (Reach, Effectiveness, Adoption, Implementation, Maintenance) to understand how text, email, and electronic health record (EHR) reminders and social media campaigns can be used as part of policy and practice interventions to increase HPV vaccination. These technology-based interventions could be used together and mainstreamed into clinical and system-based practice to have the greatest impact. Of the interventions explored, text-based, email-based, and EHR reminders have the most evidence behind them to support their effectiveness. While there are several studies of promotion of the HPV vaccine on social media, more studies are needed to demonstrate their effects and better methods are needed to be able to attribute results to these interventions.

Background: Physical activity and sedentary behavior are modifiable risk factors for non-communicable disease and healthy ageing, however the majority of older adults remain insufficiently active. Digital behavior change interventions (DBCI) have the potential to reach many older adults to promote physical activity and reduce sedentary time. This study aims to assess the efficacy of DBCI interventions in older adults (≥50 years) on physical activity and sedentary behavior. Methods: A systematic review of major databases from inception to 03/2018 was undertaken. Randomized controlled trials (RCT) or pre-post interventions assessing effects of DBCI on physical activity and/or sedentary behavior in older adults (≥50 years) were included. Random effects meta-analyses were carried out. Results: Twenty-two studies were included, including 1757 older adults (mean age = 67 years, %male = 41), 68% showed moderate-high risk of bias. Meta-analyses suggested that DBCI increased total physical activity among RCT studies (n = 8) (SMD = 0.28; 95%CI 0.01, 0.56; p = 0.04) and pre-post studies (n = 6) (SMD = 0.25; 95%CI 0.09, 0.41; p = 0.002), increased moderate-to-vigorous physical activity (SMD = 0.47; 95%CI 0.32, 0.62, p < 0.001; MD = 52 min/week) and reduced sedentary time (SMD = −0.45; 95%CI −0.69, −0.19; p < 0.001; MD = 58 min/day). Reductions in systolic blood pressure (−11 bpm; p = 0.04) and improvements in physical functioning (p = 0.03) were also observed. Conclusions: DBCI may increase physical activity and physical functioning, and reduce sedentary time and systolic blood pressure in older adults, however more high-quality studies are required.

KRAS is the most commonly mutated oncogene in human cancer, with particularly high mutation frequencies in pancreatic cancers, colorectal cancers, and lung cancers [Ostrem, J. M., and Shokat, K. M. (2016) Nat. Rev. Drug Discovery 15, 771–785]. The high prevalence of KRAS mutations and its essential role in many cancers make it a potentially attractive drug target; however, it has been difficult to create small molecule inhibitors of mutant K-Ras proteins. Here, we identified a putative small molecule binding site on K-RasG12D using computational analyses of the protein structure and then used a combination of computational and biochemical approaches to discover small molecules that may bind to this pocket, which we have termed the P110 site, due to its adjacency to proline 110. We confirmed that one compound, named K-Ras allosteric ligand KAL-21404358, bound to K-RasG12D, as measured by microscale thermophoresis, a thermal shift assay, and nuclear magnetic resonance spectroscopy. KAL-21404358 did not bind...

Alcohol policy in North America is dominated by moderation and abstinence-based modalities that focus on controlling population-level alcohol consumption and modifying individual consumption patterns to prevent and reduce alcohol-related harms. However, conventional alcohol policies and interventions do not adequately address harms associated with high-risk drinking among individuals experiencing severe alcohol use disorder (AUD) and structural vulnerability such as poverty and homelessness. In this commentary we address this gap in alcohol harm reduction, and highlight the lack of, and distinct need for, alcohol-specific harm reduction for people experiencing structural vulnerability and severe AUD. These individuals, doubly impacted by structural oppression and severe AUD, engage in various high-risk drinking practices that contribute to a unique set of harms that conventional abstinence-based treatments and interventions fail to adequately attend to. Managed alcohol programs (MAPs) have been established to address these multiple intersecting harms, and though gaining momentum across Canada, have had a hard time finding their place within the harm reduction movement. We illustrate how MAPs play a crucial role in the harm reduction movement in their ability to not only address high-risk drinking practices among structurally marginalized individuals, but to respond to harms associated with broader structural inequities such as poverty and homelessness.

The Mekong River is a vital fisheries resource supporting millions of people in mainland Southeast Asia. However, numerous threats have the potential to negatively impact fish populations in this region including overfishing, pollution, climate change and increased urban, agriculture and upstream hydropower development. Although a few studies have examined the population genetic structure of fishes within the upper Mekong River, no known studies have explored that of fishes within the Mekong Delta (MD). Here, we examine the population structure of an important food fish within the MD, Polynemus melanochir, using a panel of 1735 single nucleotide polymorphisms (SNPs) generated by restriction site-associated DNA (RAD) sequencing across eight locations on the Tien (Mekong) and Hau (Bassac) Rivers in Vietnam. Pairwise FST values, principal component analysis and Structure analysis all indicate high levels of gene flow among the sites sampled across the MD. In contrast to the lack of genetic structure, high levels of relatedness were found, including 26 putatively related pairs, as well as an effective population size (Ne) of less than 500 across the MD. While panmixia indicates that fragmentation of this population is not presently an important threat, a low Ne estimate suggests this species may not be resilient to long-term environmental changes in the MD. The reliance on P. melanochir as a food resource may be contingent on management and mitigation of low effective population sizes.

BACKGROUND AND OBJECTIVE: Aerosol transmission of Pseudomonas aeruginosa has been suggested as a possible mode of respiratory infection spread in patients with cystic fibrosis (CF); however, whether this occurs in other suppurative lung diseases is unknown. Therefore, we aimed to determine if (i) patients with bronchiectasis (unrelated to CF) or chronic obstructive pulmonary disease (COPD) can aerosolize P. aeruginosa during coughing and (ii) if genetically indistinguishable (shared) P. aeruginosa strains are present in these disease cohorts.METHODS: People with bronchiectasis or COPD and P. aeruginosa respiratory infection were recruited for two studies. Aerosol study: Participants (n = 20) underwent cough testing using validated cough rigs to determine the survival of P. aeruginosa aerosols in the air over distance and duration. Genotyping study: P. aeruginosa sputum isolates (n = 95) were genotyped using the iPLEX20SNP platform, with a subset subjected to the enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) assay to ascertain their genetic relatedness.RESULTS: Aerosol study: Overall, 7 of 20 (35%) participants released P. aeruginosa cough aerosols during at least one of the cough aerosol tests. These cough aerosols remained viable for 4 m from the source and for 15 min after coughing. The mean total aerosol count of P. aeruginosa at 2 m was two colony-forming units. Typing study: No shared P. aeruginosa strains were identified.CONCLUSION: Low viable count of P. aeruginosa cough aerosols and a lack of shared P. aeruginosa strains observed suggest that aerosol transmission of P. aeruginosa is an unlikely mode of respiratory infection spread in patients with bronchiectasis and COPD.

Ferroptosis is a nonapoptotic, iron-dependent form of cell death that can be activated in cancer cells by natural stimuli and synthetic agents. Three essential hallmarks define ferroptosis, namely: the loss of lipid peroxide repair capacity by the phospholipid hydroperoxidase GPX4, the availability of redox-active iron, and oxidation of polyunsaturated fatty acid (PUFA)-containing phospholipids. Several processes including RAS/MAPK signaling, amino acid and iron metabolism, ferritinophagy, epithelial-to-mesenchymal transition, cell adhesion, and mevalonate and phospholipid biosynthesis can modulate susceptibility to ferroptosis. Ferroptosis sensitivity is also governed by p53 and KEAP1/NRF2 activity, linking ferroptosis to the function of key tumor suppressor pathways. Together these findings highlight the role of ferroptosis as an emerging concept in cancer biology and an attractive target for precision cancer medicine discovery.

The health and welfare of farmed fish are highly dependent on environmental conditions. Under suboptimal conditions, the negative impact on welfare can cause changes in fish behaviour. Acoustic tags can provide high resolution and high frequency data to monitor fish positioning within the cage, which can be used to infer swimming behaviour. In this study, implanted acoustic tags were used to monitor the three-dimensional positioning of Atlantic salmon (Salmo salar) at a commercial farm in Nova Scotia, Canada. The one-month study period allowed the characterisation of background behaviour and changes in behaviour in relation to different environmental conditions, namely, water characteristics in terms of dissolved oxygen and temperature caused by the fall overturn, storm conditions, and feeding activity. The three-dimensional position of 15 fish was recorded using high temporal resolution (3 s). Fish movement was characterised by calculating four fish variables: distance from the centre of the cage [m], depth [m], velocity [ms−1], and turning angle [°]. The population swam in a counterclockwise swimming direction around 4 ± 2 m depth at an average speed of 0.61 ± 0.38 ms−1. After the fall overturn, the population moved significantly towards cage centre while decreasing velocity, and non-significant differences in depth and turning angle were observed. During feeding periods, a significant increase in depth and velocity, as well as a reduction in turning angle were observed. The storm event did not cause any significant change in the four fish variables. While some of the behavioural changes were difficult to assess with respect to causation, the high resolution, high frequency data provide unique detailed positioning information to further our understanding of the swimming behaviour of farmed fish.

Aquaculture, or the farmed production of fish and shellfish, has grown rapidly, from supplying just 7% of fish for human consumption in 1974 to more than half in 2016. This rapid expansion has led to the growth of Precision Aquaculture concept that aims to exploit data-driven management of fish production, thereby improving the farmer's ability to monitor, control, and document biological processes in farms. Fundamental to this paradigm is monitoring of environmental and animal processes within a cage, and processing those data toward farm insight using models and analytics. This paper presents an analysis of environmental and fish behaviour datasets collected at three salmon farms in Norway, Scotland, and Canada. Information on fish behaviour were collected using hydroacoustic sensors that sampled the vertical distribution of fish in a cage at high spatial and temporal resolution, while a network of environmental sensors characterised local site conditions. We present an analysis of the hydroacoustic datasets using AutoML (or automatic machine learning) tools that enables developers with limited data science expertise to train high-quality models specific to the data at hand. We demonstrate how AutoML pipelines can be readily applied to aquaculture datasets to interrogate the data and quantify the primary features that explains data variance. Results demonstrate that variables such as temperature, wind conditions, and hour-of-day were important drivers of fish motion at all sites. Further, there were distinct differences in factors that influenced in-cage variations driven by local variables such as water depth and ambient environmental conditions (particularly dissolved oxygen). The framework offers a transferable approach to interrogate fish behaviour within farm systems, and quantify differences between sites.

Background: To ensure safe consumption of gluten-free products, there is a need to understand all sources of unintentional contamination with gluten in the food chain. In this study, ryegrass (Lolium perenne), a common weed infesting cereal crop, is analysed as a potential source of gluten-like peptide contamination.Materials and Methods: Ten ryegrass cultivars were analysed using shotgun proteomics for the presence of proteins from the prolamin superfamily. A relative quantitative assay was developed to detect ryegrass gluten-like peptides in comparison with those found in 10 common wheat cultivars.Results: A total of 19 protein accessions were found across 10 cultivars of ryegrass for the protein families of PF00234-Tryp_alpha_amyl, PF13016-Gliadin, and PF03157-Glutenin_HMW. Protein and peptide homology searches revealed that gliadin-like peptides were similar to avenin and gamma-gliadin peptides. A total of 20 peptides, characteristic of prolamin superfamily proteins, were selected for liquid chromatography mass spectrometry (LC-MS) with multiple reaction monitoring (MRM). Only two of the monitored peptides were detected with high abundance in wheat, and all others were detected in ryegrass. Glutenin and alpha-amylase/trypsin inhibitor peptides were reported for the first time in ryegrass and were noted to be conserved across the Poaceae family.Conclusion: A suite of gluten-like peptides were identified using proteomics that showed consistent abundance across ryegrass cultivars but were not detected in wheat cultivars. These peptides will be useful for differentiating wheat gluten contamination from ryegrass gluten contamination.

Background While there is robust evidence for strategies to reduce harms of illicit drug use, less attention has been paid to alcohol harm reduction for people experiencing severe alcohol use disorder (AUD), homelessness, and street-based illicit drinking. Managed Alcohol Programs (MAPs) provide safer and regulated sources of alcohol and other supports within a harm reduction framework. To reduce the impacts of heavy long-term alcohol use among MAP participants, cannabis substitution has been identified as a potential therapeutic tool. Methods To determine the feasibility of cannabis substitution, we conducted a pre-implementation mixed-methods study utilizing structured surveys and open-ended interviews. Data were collected from MAP organizational leaders (n = 7), program participants (n = 19), staff and managers (n = 17) across 6 MAPs in Canada. We used the Consolidated Framework for Implementation Research (CFIR) to inform and organize our analysis. Results Five themes describing feasibility of CSP implementation in MAPs were identified. The first theme describes the characteristics of potential CSP participants. Among MAP participants, 63% (n = 12) were already substituting cannabis for alcohol, most often on a weekly basis (n = 8, 42.1%), for alcohol cravings (n = 15, 78.9%,) and withdrawal (n = 10, 52.6%). Most MAP participants expressed willingness to participate in a CSP (n = 16, 84.2%). The second theme describes the characteristics of a feasible and preferred CSP model according to participants and staff. Participants preferred staff administration of dry, smoked cannabis, followed by edibles and capsules with replacement of some doses of alcohol through a partial substitution model. Themes three and four highlight organizational and contextual factors related to feasibility of implementing CSPs. MAP participants requested peer, social, and counselling supports. Staff requested education resources and enhanced clinical staffing. Critically, program staff and leaders identified that sustainable funding and inexpensive, legal, and reliable sourcing of cannabis are needed to support CSP implementation. Conclusion Cannabis substitution was considered feasible by all three groups and in some MAPs residents are already using cannabis. Partial substitution of cannabis for doses of alcohol was preferred. All three groups identified a need for additional supports for implementation including peer support, staff education, and counselling. Sourcing and funding cannabis were identified as primary challenges to successful CSP implementation in MAPs.

Organisms exposed to endocrine disruptors in early life can show altered phenotype later in adulthood. Although the mechanisms underlying these long-term effects remain poorly understood, an increasing body of evidence points towards the potential role of epigenetic processes. In the present study, we exposed hatchlings of an isogenic lineage of the self-fertilizing fish mangrove rivulus for 28 days to 4 and 120 ng/L of 17-α-ethinylestradiol. After a recovery period of 140 days, reduced representation bisulphite sequencing (RRBS) was performed on the liver in order to assess the hepatic genome-wide methylation landscape. Across all treatment comparisons, a total of 146 differentially methylated fragments (DMFs) were reported, mostly for the group exposed to 4 ng/L, suggesting a non-monotonic effect of EE2 exposure. Gene ontology analysis revealed networks involved in lipid metabolism, cellular processes, connective tissue function, molecular transport and inflammation. The highest effect was reported for nipped-B-like protein B (NIPBL) promoter region after exposure to 4 ng/L EE2 (+ 21.9%), suggesting that NIPBL could be an important regulator for long-term effects of EE2. Our results also suggest a significant role of DNA methylation in intergenic regions and potentially in transposable elements. These results support the ability of early exposure to endocrine disruptors of inducing epigenetic alterations during adulthood, providing plausible mechanistic explanations for long-term phenotypic alteration. Additionally, this work demonstrates the usefulness of isogenic lineages of the self-fertilizing mangrove rivulus to better understand the biological significance of long-term alterations of DNA methylation by diminishing the confounding factor of genetic variability.

We describe a mammalian cell-based assay to identify coronavirus 3CL protease (3CLpro) inhibitors. This assay is based on rescuing protease-mediated cytotoxicity and does not require live virus. By enabling the facile testing of compounds across a range of 15 distantly related coronavirus 3CLpro enzymes, we identified compounds with broad 3CLpro-inhibitory activity. We also adapted the assay for use in compound screening and in doing so uncovered additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CLpro inhibitors. We observed strong concordance between data emerging from this assay and those obtained from live-virus testing. The reported approach democratizes the testing of 3CLpro inhibitors by developing a simplified method for identifying coronavirus 3CLpro inhibitors that can be used by the majority of laboratories, rather than the few with extensive biosafety infrastructure. We identified two lead compounds, GC376 and compound 4, with broad activity against all 3CL proteases tested, including 3CLpro enzymes from understudied zoonotic coronaviruses. IMPORTANCE Multiple coronavirus pandemics have occurred over the last 2 decades. This has highlighted a need to be proactive in the development of therapeutics that can be readily deployed in the case of future coronavirus pandemics. We developed and validated a simplified cell-based assay for the identification of chemical inhibitors of 3CL proteases encoded by a wide range of coronaviruses. This assay is reporter free, does not require specialized biocontainment, and is optimized for performance in high-throughput screening. By testing reported 3CL protease inhibitors against a large collection of 3CL proteases with variable sequence similarity, we identified compounds with broad activity against 3CL proteases and uncovered structural insights into features that contribute to their broad activity. Furthermore, we demonstrated that this assay is suitable for identifying chemical inhibitors of proteases from families other than 3CL proteases.

INTRODUCTION Evidence-based alcohol policies have the potential to reduce a wide range of related harms. Yet, barriers to adoption and implementation within governments often exist. Engaging relevant stakeholders may be an effective way to identify and address potential challenges thereby increasing reach and uptake of policy evaluation research and strengthening jurisdictional responses to alcohol harms. METHODS As part of the 2019 Canadian Alcohol Policy Evaluation project, we conducted interviews with government stakeholders across alcohol-related sectors prior to a second round of researcher-led policy assessments in Canada's 13 provinces and territories. Stakeholders were asked for feedback on the design and impact of an earlier policy assessment in 2013 and for recommendations to improve the design and dissemination strategy for the next iteration. Content analysis was used to identify ways of improving stakeholder engagement. RESULTS We interviewed 25 stakeholders across 12 of Canada's 13 jurisdictions, including representatives from government health ministries and from alcohol regulation, distribution and finance departments. In providing feedback on our stakeholder engagement strategy, participants highlighted the importance of maintaining ongoing contact; presenting results in accessible online formats; providing advance notice of results; and offering jurisdiction-specific webinars. DISCUSSION AND CONCLUSIONS This study offers important insight into the engagement preferences of government stakeholders involved in the health, regulation, distribution and financial aspects of alcohol control policy. Findings suggest that seeking input from stakeholders as part of conducting evaluation research is warranted; increasing the relevance, reach and uptake of results. Specific stakeholder engagement strategies are outlined.

Background: Real-world data on non-vitamin K oral anticoagulants (NOACs) are essential in determining whether evidence from randomised controlled clinical trials translate into meaningful clinical benefits for patients in everyday practice. RIVER (RIVaroxaban Evaluation in Real life setting) is an ongoing international, prospective registry of patients with newly diagnosed non-valvular atrial fibrillation (NVAF) and at least one investigator-determined risk factor for stroke who received rivaroxaban as an initial treatment for the prevention of thromboembolic stroke. The aim of this paper is to describe the design of the RIVER registry and baseline characteristics of patients with newly diagnosed NVAF who received rivaroxaban as an initial treatment. Methods and results: Between January 2014 and June 2017, RIVER investigators recruited 5072 patients at 309 centres in 17 countries. The aim was to enroll consecutive patients at sites where rivaroxaban was already routinely prescribed for stroke prevention. Each patient is being followed up prospectively for a minimum of 2-years. The registry will capture data on the rate and nature of all thromboembolic events (stroke / systemic embolism), bleeding complications, all-cause mortality and other major cardiovascular events as they occur. Data quality is assured through a combination of remote electronic monitoring and onsite monitoring (including source data verification in 10% of cases). Patients were mostly enrolled by cardiologists (n = 3776, 74.6%), by internal medicine specialists 14.2% (n = 718) and by primary care/general practice physicians 8.2% (n = 417). The mean (SD) age of the population was 69.5 (11.0) years, 44.3% were women. Mean (SD) CHADS2 score was 1.9 (1.2) and CHA2DS2-VASc scores was 3.2 (1.6). Almost all patients (98.5%) were prescribed with once daily dose of rivaroxaban, most commonly 20 mg (76.5%) and 15 mg (20.0%) as their initial treatment; 17.9% of patients received concomitant antiplatelet therapy. Most patients enrolled in RIVER met the recommended threshold for AC therapy (86.6% for 2012 ESC Guidelines, and 79.8% of patients according to 2016 ESC Guidelines). Conclusions: The RIVER prospective registry will expand our knowledge of how rivaroxaban is prescribed in everyday practice and whether evidence from clinical trials can be translated to the broader cross-section of patients in the real world. Funding: This work is supported by an unrestricted research grant from Bayer AG (Berlin, Germany) to the Thrombosis Research Institute (London, UK), which sponsors the RIVER registry. The funding source had no involvement in the data collection, data analysis or data interpretation.

Deregulation of the cyclin-dependent kinases 4 and 6 (CDK4/6) is highly prevalent in cancer yet inhibitors against these kinases currently show use in restricted tumour contexts. The extent to which cancers depend on CDK4/6 and what may undermine such dependency is poorly understood. Here we report that signalling engaging the MET proto-oncogene receptor tyrosine kinase/focal adhesion kinase (FAK) axis leads to CDK4/6-independent CDK2-activation, involving as a critical mechanistic events loss of the CDK inhibitor p21CIP1 and gain of its regulator, the ubiquitin ligase subunit SKP2. Combined inhibition of MET/FAK and CDK4/6 eliminates proliferation capacity of cancer cells in culture, and enhances tumour growth inhibition in vivo. Activation of the MET/FAK axis is known to arise through cancer extrinsic and intrinsic cues. Our work predicts that such cues support cell division independent of the activity of the cell cycle-regulating CDK4/6 kinases and identifies MET/FAK as a tractable route to broaden the utility of CDK4/6 inhibitor-based therapies in the clinic.

The World Health Organization (WHO) is undertaking important work to develop a new action plan on alcohol for 2022–30. However, the WHO's published proposal for the action plan envisages that the alcohol industry be a co‐regulator with government of consumer information, including health warnings, on alcoholic beverage labels.

The COVID-19 pandemic is presenting significant challenges for health and social care systems globally. The implementation of unprecedented public health measures, alongside the augmentation of the treatment capacity for those severely affected by COVID-19, are compromising and limiting the delivery of essential care to people with severe substance use problems and, in some cases, widening extreme social inequities such as poverty and homelessness. This global pandemic is severely challenging current working practices. However, these challenges can provide a unique opportunity for a flexible and innovative learning approach, bringing certain interventions into the spotlight. Harm reduction responses are well-established evidenced approaches in the management of opioid dependence but not so well-known or implemented in relation to alcohol use disorders. In this position paper, we explore the potential for expanding harm reduction approaches during the COVID-19 crisis and beyond as part of substance use treatment services. We will examine alcohol use and related vulnerabilities during COVID-19, the impact of COVID-19 on substance use services, and the potential philosophical shift in orientation to harm reduction and outline a range of alcohol harm reduction approaches. We discuss relevant aspects of the Structured Preparation for Alcohol Detoxification (SPADe) treatment model, and Managed Alcohol Programs (MAPs), as part of a continuum of harm reduction and abstinence orientated treatment for alcohol use disorders. In conclusion, while COVID-19 has dramatically reduced and limited services, the pandemic has propelled the importance of alcohol harm reduction and created new opportunities for implementation of harm reduction philosophy and approaches, including programs that incorporate the provision of alcohol as medicine as part of the substance use treatment continuum.

Epidermal club cells (ECCs), along with mucus cells, are present in the skin of many fishes, particularly in the well-studied Ostariophysan family Cyprinidae. Most ECC-associated literature has focused on the potential role of ECCs as a component of chemical alarm cues released passively when a predator damages the skin of its prey, alerting nearby prey to the presence of an active predator. Because this warning system is maintained by receiver-side selection (senders are eaten), there is want of a mechanism to confer fitness benefits to the individual that invests in ECCs to explain their evolutionary origin and maintenance in this speciose group of fishes. In an attempt to understand the fitness benefits that accrue from investment in ECCs, we reviewed the phylogenetic distribution of ECCs and their histochemical properties. ECCs are found in various forms in all teleost superorders and in the chondrostei inferring either early or multiple independent origins over evolutionary time. We noted that ECCs respond to several environmental stressors/immunomodulators including parasites and pathogens, are suppressed by immunomodulators such as testosterone and cortisol, and their density covaries with food ration, demonstrating a dynamic metabolic cost to maintaining these cells. ECC density varies widely among and within fish populations, suggesting that ECCs may be a convenient tool with which to assay ecoimmunological tradeoffs between immune stress and foraging activity, reproductive state, and predator–prey interactions. Here, we review the case for ECC immune function, immune functions in fishes generally, and encourage future work describing the precise role of ECCs in the immune system and life history evolution in fishes.