Pathogenesis Mediated by Bacterial Membrane Vesicles.

The release of extracellular vesicles (EVs) is a process conserved across the three domains of life. Amongst prokaryotes, EVs produced by Gram-negative bacteria, termed outer membrane vesicles (OMVs), were identified more than 50 years ago and a wealth of literature exists regarding their biogenesis, composition and functions. OMVs have been implicated in benefiting numerous metabolic functions of their parent bacterium. Additionally, OMVs produced by pathogenic bacteria have been reported to contribute to pathology within the disease setting. By contrast, the release of EVs from Gram-positive bacteria, known as membrane vesicles (MVs), has only been widely accepted within the last decade. As such, there is a significant disproportion in knowledge regarding MVs compared to OMVs. Here we provide an overview of the literature regarding bacterial membrane vesicles (BMVs) produced by pathogenic and commensal bacteria. We highlight the mechanisms of BMV biogenesis and their roles in assisting bacterial survival, in addition to discussing their functions in promoting disease pathologies and their potential use as novel therapeutic strategies.

Safety first: Beliefs of older peers supplying alcohol to underage friends.

ISSUE ADDRESSED: Underage drinkers most commonly source alcohol from older peers. However, few studies have examined older peers' supply-related beliefs and motivations. METHODS: A sample of 270 risky drinkers aged 18-19 years were interviewed in Australia where the legal purchase age is 18. They were asked about their provision to underage friends, awareness of secondary supply legislation (intended to prohibit such supply) and 24 psycho-legal beliefs around supply. RESULTS: Half (49%) provided alcohol to a 16- to 17-year-old friend to drink at a party they were both attending at least twice a year. Three-quarters reported provision was okay so long as the recipient(s) were in a safe environment, and 46% reported "everyone gives alcohol to teenagers if they are in a safe environment." There was significantly higher agreement that "my friends would think I was mean if I did not give alcohol to a friend under the age of 18" (37%), compared to "my friends would think I was uncool if I did not give alcohol to a friend under the age of 18" (26%). Two thirds (69%) felt more responsible for an underage friend's safety if they provided the alcohol. A multivariate logistic regression revealed supply was more likely if the supplier: was aged 18 compared to 19 (95% CI OR: 1.57, 4.84), male (1.06, 3.27), of a higher SES quintile (1.08, 1.80) and believed alcohol supply to minors was morally acceptable (1.01, 1.33) and normal (1.04, 1.38). Knowledge of regulatory strategies (68%) designed to prevent supply to minors, and their perceived deterrent value did not significantly impact supply. CONCLUSIONS: Supply of alcohol to underage peers was perceived as morally and socially acceptable in a group of 18- to 19-year-old risky drinkers. SO WHAT?: Opportunities include harm reduction initiatives that prioritise caring responsibilities towards friends, as opposed to relying on external enforcement measures alone.

Alcohol supply as a favour for a friend: Scenarios of alcohol supply to younger friends and siblings.

ISSUES ADDRESSED: Adolescents under the legal purchase age primarily source their alcohol through social networks. This study assessed the provision context from the perspective of both underage recipients and their suppliers who were older peers and siblings. METHODS: Interviewer-administered surveys were conducted with 590 risky-drinking (50 g alcohol per session, at least monthly) adolescents. Participants of legal purchase age (18- to 19-year-olds; n = 269) reported their provision to 16- to 17-year-olds under eight scenarios. Those aged 14-17 (n = 321) reported receipt of alcohol under the same scenarios plus two parental supply contexts. RESULTS: Purchase-age participants reported supply: to an underage friend (67%), an acquaintance (44%) or a sibling (16%) to drink at the same party; to a friend (43%) or sibling (20%) to take to another party (20%) and to a stranger near a bottle shop (5%). Supply to a friend at the same party was more likely if money was exchanged (60% vs 40%; P < 0.001). Almost all (98%) 14- to 17-year-olds reported receiving alcohol from an adult (including 36% from a parent for consumption away from the parent), with a similar pattern of receipt scenarios as those reported by the 18- to 19-year-olds. CONCLUSIONS: Provision of alcohol was more frequent with a friend than a sibling or stranger, in close environmental proximity, and if money was exchanged. SO WHAT?: As supply may be sensitive to monetary considerations, the incidence of underage receipt may be affected by community-wide pricing measures. Traditional alcohol availability regulations should be supplemented by strategies relating to the social nature of supply and demand.

Treating alcohol-related liver disease from a public health perspective.

Herein, we describe the evolving landscape of alcohol-related liver disease (ALD) including the current global burden of disease and cost to working-aged people in terms of death and disability, in addition to the larger spectrum of alcohol-related heath complications and its wider impact on society. We further review the most effective and cost-effective public health policies at both a population and individual level. Currently, abstinence is the only effective treatment for ALD, and yet because the majority of ALD remains undetected in the community abstinence is initiated too late to prevent premature death in the majority of cases. We therefore hope that this review will help inform clinicians of the "public health treatment options" for ALD to encourage engagement with policy makers and promote community-based hepatology as a speciality, expanding our patient cohort to allow early detection, and thereby a reduction in the enormous morbidity and mortality associated with this disease.

Generation of Nkx2-5/CreER transgenic mice for inducible Cre expression in developing hearts.

Nkx2-5 is a homeobox-containing transcriptional regulator that serves as one of the earliest markers of cardiac lineage commitment. To study the role of Nkx2-5-expressing progenitors at specific time points in cardiac development, we have generated a novel and inducible NKX2-5 mouse line by knocking in a CreER cassette into the Nkx2-5 genomic locus, while preserving the endogenous Nkx2-5 gene to avoid haploinsufficiency. We evaluated the specificity and efficiency of CreER activity after 4-OHT injection by crossing Nkx2-5CreER/+ mice with a Rosa26tdT/+ reporter strain. Our immunohistochemistry results confirmed Cre-induced tdTomato expression specifically in cells expressing Nkx2-5. These cells were mainly cardiomyocytes and were observed in the embryonic heart as early as day 9.5. Additionally, quantitative polymerase chain reaction on postnatal hearts showed enriched expression of Nkx2-5 in isolated tdTomato-expressing cells. No tdTomato expression was observed in Nkx2-5CreER/+ ;Rosa26tdT/+ mice in the absence of 4-OHT, confirming the inducible nature of CreER activity. The Nkx2-5/CreER mouse model described in this article will serve as an invaluable tool to trace myocardial lineage and to temporally induce genetic manipulation in a selective population of cardiac progenitors during embryonic development and in the adult heart.

Most American Association of Oral and Maxillofacial Surgeons Members Have Not Adopted the American Society of Anesthesiologists-Recommended Nil Per Os Guidelines.

PURPOSE: The purpose of this study was to determine if American Association of Oral and Maxillofacial Surgeons members have integrated the current American Society of Anesthesiologists (ASA) nil per os (NPO) guidelines into their preoperative instructions. MATERIALS AND METHODS: We designed and implemented a cross-sectional study and enrolled a random sample of private-practice American Association of Oral and Maxillofacial Surgeons members who practice in the United States. The predictor variables were year of graduation from residency, dual degree (MD and DDS or DMD) or single degree, and region. The primary outcome variable was adoption of the ASA NPO guidelines, defined as recommending fasting times of 2 hours for clear liquids and 6 hours for solid foods. To collect data, a systematic online search was implemented. Appropriate univariate and bivariate statistics were computed, and the level of significance was set at .05; in addition, 95% confidence intervals were calculated. RESULTS: The study sample was composed of 431 oral and maxillofacial surgeons (OMSs). Almost all of the study sample (99.1%) did not adopt the ASA guidelines. The fasting recommendations were different from 2 hours for clear liquids and 6 hours for solid foods. However, recommendations of 2 hours or greater for clear liquids were made by 99.8% of OMSs, and recommendations of 6 hours or greater for solid foods were made by 99.3%. Only 4.4% of OMSs made different recommendations for clear liquids and solid foods. No substantial association was found between whether OMSs adopted the most current ASA guidelines and the year they graduated from residency or the obtainment of dual degrees. CONCLUSIONS: OMSs in private practice are overwhelmingly recommending longer fasting times for clear liquids and solid foods on their Web sites when compared with the current ASA guidelines before ambulatory anesthesia. The ASA guidelines are based on meta-analysis; therefore, deviations in practice, although not incorrect, may call for discussion.

Web-Based Virtual Microscopy of Digitized Blood Slides for Malaria Diagnosis: An Effective Tool for Skills Assessment in Different Countries and Environments.

BACKGROUND: Morphological examination of blood films remains the reference standard for malaria diagnosis. Supporting the skills required to make an accurate morphological diagnosis is therefore essential. However, providing support across different countries and environments is a substantial challenge. OBJECTIVE: This paper reports a scheme supplying digital slides of malaria-infected blood within an Internet-based virtual microscope environment to users with different access to training and computing facilities. The feasibility of the approach was established, allowing users to test, record, and compare their own performance with that of other users. METHODS: From Giemsa stained thick and thin blood films, 56 large high-resolution digital slides were prepared, using high-quality image capture and 63x oil-immersion objective lens. The individual images were combined using the photomerge function of Adobe Photoshop and then adjusted to ensure resolution and reproduction of essential diagnostic features. Web delivery employed the Digital Slidebox platform allowing digital microscope viewing facilities and image annotation with data gathering from participants. RESULTS: Engagement was high with images viewed by 38 participants in five countries in a range of environments and a mean completion rate of 42/56 cases. The rate of parasite detection was 78% and accuracy of species identification was 53%, which was comparable with results of similar studies using glass slides. Data collection allowed users to compare performance with other users over time or for each individual case. CONCLUSIONS: Overall, these results demonstrate that users worldwide can effectively engage with the system in a range of environments, with the potential to enhance personal performance through education, external quality assessment, and personal professional development, especially in regions where educational resources are difficult to access.

Pre-Drinking Behavior of Young Heavy Drinkers.

BACKGROUND: Pre-drinking has been linked to subsequent heavy drinking and the engagement in multiple risky behaviors. OBJECTIVES: The present study examined a group of adolescents who recently had a "big night out" to determine whether there were differences in their pre-drinking behavior based on age, gender, geographic location, and social setting. METHODS: Participants (n = 351, aged 16-19) representing the heaviest 20-25% of drinkers in their age group were recruited using nonrandom sampling from metropolitan (Melbourne, Sydney, Perth) or regional (Bunbury) locations across Australia and administered a survey by a trained interviewer. RESULTS: Almost half the sample pre-drank (n = 149), most commonly at a friend's house. Those aged 18-19 were more likely to pre-drink, and did so at higher quantities compared to their younger counterparts. Males and females reported similar pre-drinking duration, quantity and amount spent on alcohol. Compared to those in cities, regional participants consumed greater quantities over longer periods of time. Two-thirds of participants consumed alcohol in excess of national guidelines during their pre-drinking session. These participants were more likely to nominate price as a motivation to pre-drink and were less likely to report that someone else provided them alcohol. CONCLUSIONS: This study sheds light on the pre-drinking habits of a population of young risky drinkers, and highlights the need for policy makers to address this form of drinking to reduce alcohol-related harm among young people.

The Wild West: Associations between mining and violence in Western Australia.

OBJECTIVE: To investigate the association between mining activity and police-reported assault offences across Western Australia. DESIGN: A cross-sectional multivariable negative binomial regression analysis at the local government area level. SETTING: Local government areas in Western Australia. PARTICIPANTS: Victims of reported assault offences occurring in 2008-2009. MAIN OUTCOME MEASURES: Eight reported assault measures by gender of victim and type of assault. The analysis controlled for a range of potentially confounding variables, including numbers of licensed outlets and alcohol sales. RESULTS: Compared with females in other areas, females in mining regions had a 64% increased risk of assault, a 59% increased risk of non-domestic assault and a 136% increased risk of sexual assault. Risk of domestic assault was 64% higher for males in mining regions. CONCLUSIONS: Regions where mining is a major employer of people usually or temporarily residing in the area (i.e. usual residents or temporary fly-in fly-out residents) are associated with higher risk of assaults among females and domestic assaults among males, and these associations appear to be independent of licensed outlet numbers and alcohol sales. Mining communities appear to present a special case for the management and reduction of violence; public health and safety intervention needs to identify and address risk factors independent of alcohol use.

Monomeric C-reactive protein and Notch-3 co-operatively increase angiogenesis through PI3K signalling pathway.

C-reactive protein (CRP) is the most acute-phase reactant serum protein of inflammation and a strong predictor of cardiovascular disease. Its expression is associated with atherosclerotic plaque instability and the formation of immature micro-vessels. We have previously shown that CRP upregulates endothelial-derived Notch-3, a key receptor involved in vascular development, remodelling and maturation. In this study, we investigated the links between the bioactive monomeric CRP (mCRP) and Notch-3 signalling in angiogenesis. We used in vitro (cell counting, wound-healing and tubulogenesis assays) and in vivo (chorioallantoic membrane) angiogenic assays and Western blotting to study the angiogenic signalling pathways induced by mCRP and Notch-3 activator chimera protein (Notch-3/Fc). Our results showed an additive effect on angiogenesis of mCRP stimulatory effect combined with Notch-3/Fc promoting bovine aortic endothelial cell (BAEC) proliferation, migration, tube formation in Matrigel(TM) with up-regulation of phospho-Akt expression. The pharmacological blockade of PI3K/Akt survival pathway by LY294002 fully inhibited in vitro and in vivo angiogenesis induced by mCRP/Notch-3/Fc combination while blocking Notch signalling by gamma-secretase inhibitor (DAPT) partially inhibited mCRP/Notch-3/Fc-induced angiogenesis. Using a BAEC vascular smooth muscle cell co-culture sprouting angiogenesis assay and transmission electron microscopy, we showed that activation of both mCRP and Notch-3 signalling induced the formation of thicker sprouts which were shown later by Western blotting to be associated with an up-regulation of N-cadherin expression and a down-regulation of VE-cadherin expression. Thus, mCRP combined with Notch-3 activator promote angiogenesis through the PI3K/Akt pathway and their therapeutic combination has potential to promote and stabilize vessel formation whilst reducing the risk of haemorrhage from unstable plaques.

'Something's brewing': The changing trends in alcohol coverage in Australian newspapers 2000-2011.

AIMS: The portrayal of alcohol in the news media, including newspapers, plays an important role in influencing societal norms and setting public agendas. We present the first large-scale examination of news coverage of alcohol-related issues in Australian newspapers. METHOD: Content analysis was performed on a sample of alcohol-related newspaper articles (n = 4217) published across Australia from 2000 to 2011. Articles were coded for type, theme, prominence, topic slant, opinion slant and sources/spokesperson. RESULTS: Across the period, the most common themes were promotion (21%), drink-driving (16%) and restrictions/policy (16%). Themes of restrictions/policy and responsible beverage services became more common over time. Promotion and business-related articles significantly declined over time. Overall, the topic slant of the majority of news related articles disapproved of alcohol use. Disapproval increased over time while approval of alcohol use decreased. While the slant of opinion pieces was predominantly approving of alcohol, this decreased over time. Presence of an alcohol industry representative in articles declined over time. CONCLUSION: The presentation of alcohol use in Australian newspapers became more disapproving over time, which may suggest that harmful alcohol use has become less acceptable among the broader Australian community.

Association between Bar Closing Time, Alcohol Use Disorders and Blood Alcohol Concentration: A Cross-Sectional Observational Study of Nightlife-Goers in Perth, Australia.

Introduction and aims: Associations between bar trading hours, a government lever for controlling alcohol availability, nightlife-goer intoxication levels and their likelihood of alcohol use disorder (AUD) have not been explored. We investigated whether: (i) participant AUD was associated with blood alcohol concentration (BAC); and, (ii) any association between AUD and BAC was moderated by participant preferred bar (i.e., venue spent most time at) closing time. Design and methods: A cross-sectional observational study using a sample of nightlife-goers who went out drinking in Perth, Western Australia, on weekends in 2015-16. Participants who reported alcohol use that night and spent most time in a bar (n = 667) completed street intercept surveys including AUDIT-C (n = 459) and provided a breath sample to estimate BAC (n = 651). We used gender-specific multinomial logistic regression models to explore associations between participant AUDIT-C score (1−4, lower risk; 5−7, hazardous; 8−12, active AUD), preferred bar type (standard vs. late closing time based on absence or presence of an extended trading permit) and BAC (male: 0−0.049, 0.05−0.099, ≥0.1 g/100 mL; female: 0−0.049, 0.05−0.079, ≥0.08 g/100 mL). Results: Males with active AUD (RR = 3.31; 95% CI 1.30−8.42; p = 0.01) and females with hazardous/active AUD (RR = 9.75; 95% CI 2.78−34.21; p < 0.001) were both more likely to have high-range BAC than their counterparts typically drinking at lower risk. We also found preferred bar type moderated the association between AUDIT-C score and BAC for some males but no females. Males with active AUD and high-range BAC were less likely to prefer late closing bars than males usually drinking at lower risk and high-range BAC (RR = 0.12; 95% CI 0.02−0.96; p = 0.046). Discussion and conclusions: Our study provides evidence of positive associations between AUD and acute intoxication among nightlife-goers and on the moderating effect of bar closing times among males.

Correction: Gilmore et al. Association between Nightlife Goers' Likelihood of an Alcohol Use Disorder and Their Preferred Bar's Closing Time: A Cross-Sectional Observational Study in Perth, Australia. Int. J. Environ. Res. Public Health 2021, 18, 13040.

In the original publication [...].

Disclosures of harming others during their most recent drinking session: Findings from a large national study of heavy-drinking adolescents.

INTRODUCTION: The extant Alcohol's Harms to Others (AHTO) literature is largely comprised of reports from victims. We investigated AHTO from perpetrators' perspectives, including how harms were associated with individual characteristics, and alcohol quantities consumed during the perpetration incident. METHODS: Participants (N = 2932) were 14-19 years old, recruited primarily through social media and screened as risky drinkers. They completed face-to-face (n = 594) or self-administered (n = 2338) surveys. They self-reported whether during their last risky drinking session (LRDS) they had perpetrated any verbal abuse, physical abuse or property damage. A multinomial logistic regression examined whether nine factors were associated with perpetrating zero, one or 2+ categories of AHTO. RESULTS: Eleven percent (n = 323) reported perpetrating at least one form of AHTO (7.5% verbal, 1.9% physical and 4.6% property). Perpetration of AHTO at LRDS was uniquely associated with: younger age, male gender, experiences of childhood physical punishment, greater perpetration incident-specific drinking, concurrent illicit drug use, and less frequent use of safety strategies while drinking in the past 12 months. Controlling for the other variables, an increase of six Australian standard drinks (60 g of alcohol) increased the odds of perpetration by 15% [95% confidence interval (CI) adjusted odds ratio (AOR) 1.08, 1.23], and an increase of 15 Australian standard drinks increased the odds by 42% (95% CI AOR 1.20, 1.69). DISCUSSION AND CONCLUSIONS: Individual characteristics, larger quantities of alcohol consumed, and a disinclination to practice harm reduction amplified risk of AHTO perpetration. This has implications for health promotion and risk prevention/reduction strategies.

Bacteroides fragilis outer membrane vesicles preferentially activate innate immune receptors compared to their parent bacteria.

The release of bacterial membrane vesicles (BMVs) has become recognized as a key mechanism used by both pathogenic and commensal bacteria to activate innate immune responses in the host and mediate immunity. Outer membrane vesicles (OMVs) produced by Gram-negative bacteria can harbor various immunogenic cargo that includes proteins, nucleic acids and peptidoglycan, and the composition of OMVs strongly influences their ability to activate host innate immune receptors. Although various Gram-negative pathogens can produce OMVs that are enriched in immunogenic cargo compared to their parent bacteria, the ability of OMVs produced by commensal organisms to be enriched with immunostimulatory contents is only recently becoming known. In this study, we investigated the cargo associated with OMVs produced by the intestinal commensal Bacteroides fragilis and determined their ability to activate host innate immune receptors. Analysis of B. fragilis OMVs revealed that they packaged various biological cargo including proteins, DNA, RNA, lipopolysaccharides (LPS) and peptidoglycan, and that this cargo could be enriched in OMVs compared to their parent bacteria. We visualized the entry of B. fragilis OMVs into intestinal epithelial cells, in addition to the ability of B. fragilis OMVs to transport bacterial RNA and peptidoglycan cargo into Caco-2 epithelial cells. Using HEK-Blue reporter cell lines, we identified that B. fragilis OMVs could activate host Toll-like receptors (TLR)-2, TLR4, TLR7 and nucleotide-binding oligomerization domain-containing protein 1 (NOD1), whereas B. fragilis bacteria could only induce the activation of TLR2. Overall, our data demonstrates that B. fragilis OMVs activate a broader range of host innate immune receptors compared to their parent bacteria due to their enrichment of biological cargo and their ability to transport this cargo directly into host epithelial cells. These findings indicate that the secretion of OMVs by B. fragilis may facilitate immune crosstalk with host epithelial cells at the gastrointestinal surface and suggests that OMVs produced by commensal bacteria may preferentially activate host innate immune receptors at the mucosal gastrointestinal tract.

Isolation and characterization of human embryonic stem cell-derived heart field-specific cardiomyocytes unravels new insights into their transcriptional and electrophysiological profiles.

AIMS: We prospectively isolate and characterize first and second heart field- and nodal-like cardiomyocytes using a double reporter line from human embryonic stem cells. Our double reporter line utilizes two important transcription factors in cardiac development, TBX5 and NKX2-5. TBX5 expression marks first heart field progenitors and cardiomyocytes while NKX2-5 is expressed in nearly all myocytes of the developing heart (excluding nodal cells). We address the shortcomings of prior work in the generation of heart field-specific cardiomyocytes from induced pluripotent stem cells and provide a comprehensive early developmental transcriptomic as well as electrophysiological analyses of these three populations. METHODS AND RESULTS: Transcriptional, immunocytochemical, and functional studies support the cellular identities of isolated populations based on the expression pattern of NKX2-5 and TBX5. Importantly, bulk and single-cell RNA sequencing analyses provide evidence of unique molecular signatures of isolated first and second heart field cardiomyocytes, as well as nodal-like cells. Extensive electrophysiological analyses reveal dominant atrial action potential phenotypes in first and second heart fields in alignment with our findings in single-cell RNA sequencing. Lastly, we identify two novel surface markers, POPDC2 and CORIN, that enable purification of cardiomyocytes and first heart field cardiomyocytes, respectively. CONCLUSIONS: We describe a high-yield approach for isolation and characterization of human embryonic stem cell-derived heart field-specific and nodal-like cardiomyocytes. Obtaining enriched populations of these different cardiomyocyte subtypes increases the resolution of gene expression profiling during early cardiogenesis, arrhythmia modelling, and drug screening. This paves the way for the development of effective stem cell therapy to treat diseases that affect specific regions of the heart- or chamber-specific congenital heart defects.

Association between Nightlife Goers' Likelihood of an Alcohol Use Disorder and Their Preferred Bar's Closing Time: A Cross-Sectional Observational Study in Perth, Australia.

INTRODUCTION AND AIMS: Associations between longer-term alcohol-related conditions and licensed outlet trading hours are not well understood. We investigated the association between nightlife-goers' likelihood of an alcohol use disorder (AUD) and their preference for bars with special permits to remain open 'late' (i.e., spent more time there compared to any other venue) until 2 a.m. or 3 a.m. (Friday; Saturday) or midnight (Sunday) compared to bars with 'standard' closing times of midnight (Friday; Saturday) or 10 p.m. (Sunday). DESIGN AND METHODS: A cross-sectional observational study was conducted in four major nightlife areas of Perth, Australia, in 2015-2016. We conducted weekend street intercept surveys outside bars between 8 p.m. and 3 a.m. and screened participants who reported alcohol use prior to the survey and spent more time in a bar than any other venue type (n = 667) regarding their past year drinking pattern using AUDIT-C (n = 459). We used gender-specific logistic regression models to estimate associations between AUDIT-C categories (1-4, low risk; 5-7, hazardous; 8-12, active AUD) and preference for bars with different closing times (late vs. standard). RESULTS: A large proportion of participants were hazardous drinkers or had active AUD (83% males; 65% females), and over half preferred a late to a standard closing bar. We found evidence of a positive association between preference for late closing bars and hazardous drinking females (OR = 3.48; 95% CI 1.47-8.23; p = 0.01), but not for females with active AUD, male hazardous drinkers, nor males with active AUD. DISCUSSION AND CONCLUSIONS: Our study adds new evidence on associations between likelihood of AUD among nightlife-goers and trading hours. With increasing international relaxation of trading hours, evidence that late closing bars may be preferred by hazardous drinking females will be of concern to policymakers wanting to curb alcohol-related harms in the community.