Programmed Secretion Arrest and Receptor-Triggered Toxin Export during Antibacterial Contact-Dependent Growth Inhibition.

Contact-dependent growth inhibition (CDI) entails receptor-mediated delivery of CdiA-derived toxins into Gram-negative target bacteria. Using electron cryotomography, we show that each CdiA effector protein forms a filament extending ∼33 nm from the cell surface. Remarkably, the extracellular filament represents only the N-terminal half of the effector. A programmed secretion arrest sequesters the C-terminal half of CdiA, including the toxin domain, in the periplasm prior to target-cell recognition. Upon binding receptor, CdiA secretion resumes, and the periplasmic FHA-2 domain is transferred to the target-cell outer membrane. The C-terminal toxin region of CdiA then penetrates into the target-cell periplasm, where it is cleaved for subsequent translocation into the cytoplasm. Our findings suggest that the FHA-2 domain assembles into a transmembrane conduit for toxin transport into the periplasm of target bacteria. We propose that receptor-triggered secretion ensures that FHA-2 export is closely coordinated with integration into the target-cell outer membrane. VIDEO ABSTRACT.

Opioid analgesics for nociceptive cancer pain: A comprehensive review.

Pain is one of the most burdensome symptoms in people with cancer, and opioid analgesics are considered the mainstay of cancer pain management. For this review, the authors evaluated the efficacy and toxicities of opioid analgesics compared with placebo, other opioids, nonopioid analgesics, and nonpharmacologic treatments for background cancer pain (continuous and relatively constant pain present at rest), and breakthrough cancer pain (transient exacerbation of pain despite stable and adequately controlled background pain). They found a paucity of placebo-controlled trials for background cancer pain, although tapentadol or codeine may be more efficacious than placebo (moderate-certainty to low-certainty evidence). Nonsteroidal anti-inflammatory drugs including aspirin, piroxicam, diclofenac, ketorolac, and the antidepressant medicine imipramine, may be at least as efficacious as opioids for moderate-to-severe background cancer pain. For breakthrough cancer pain, oral transmucosal, buccal, sublingual, or intranasal fentanyl preparations were identified as more efficacious than placebo but were more commonly associated with toxicities, including constipation and nausea. Despite being recommended worldwide for the treatment of cancer pain, morphine was generally not superior to other opioids, nor did it have a more favorable toxicity profile. The interpretation of study results, however, was complicated by the heterogeneity in the study populations evaluated. Given the limited quality and quantity of research, there is a need to reappraise the clinical utility of opioids in people with cancer pain, particularly those who are not at the end of life, and to further explore the effects of opioids on immune system function and quality of life in these individuals.

The importance of m6A topology in chicken embryo mRNA: a precise mapping of m6A at the conserved chicken ß-actin zipcode.

N6-methyladenosine (m6A) in mRNA regulates almost every stage in the mRNA life cycle, and the development of methodologies for the high-throughput detection of methylated sites in mRNA using m6A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIPSeq) or m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) have revolutionized the m6A research field. Both of these methods are based on immunoprecipitation of fragmented mRNA. However, it is well documented that antibodies often have nonspecific activities, thus verification of identified m6A sites using an antibody-independent method would be highly desirable. We mapped and quantified the m6A site in the chicken ß-actin zipcode based on the data from chicken embryo MeRIPSeq results and our RNA-Epimodification Detection and Base-Recognition (RedBaron) antibody-independent assay. We also demonstrated that methylation of this site in the ß-actin zipcode enhances ZBP1 binding in vitro, while methylation of a nearby adenosine abolishes binding. This suggests that m6A may play a role in regulating localized translation of ß-actin mRNA, and the ability of m6A to enhance or inhibit a reader protein's RNA binding highlights the importance of m6A detection at nucleotide resolution.

Polymorphic Toxins and Their Immunity Proteins: Diversity, Evolution, and Mechanisms of Delivery.

All bacteria must compete for growth niches and other limited environmental resources. These existential battles are waged at several levels, but one common strategy entails the transfer of growth-inhibitory protein toxins between competing cells. These antibacterial effectors are invariably encoded with immunity proteins that protect cells from intoxication by neighboring siblings. Several effector classes have been described, each designed to breach the cell envelope of target bacteria. Although effector architectures and export pathways tend to be clade specific, phylogenetically distant species often deploy closely related toxin domains. Thus, diverse competition systems are linked through a common reservoir of toxin-immunity pairs that is shared via horizontal gene transfer. These toxin-immunity protein pairs are extraordinarily diverse in sequence, and this polymorphism underpins an important mechanism of self/nonself discrimination in bacteria. This review focuses on the structures, functions, and delivery mechanisms of polymorphic toxin effectors that mediate bacterial competition.

Review on the impacts of indoor vector control on domiciliary pests: good intentions challenged by harsh realities.

Arthropod vectored diseases have been a major impediment to societal advancements globally. Strategies to mitigate transmission of these diseases include preventative care (e.g. vaccination), primary treatment and most notably, the suppression of vectors in both indoor and outdoor spaces. The outcomes of indoor vector control (IVC) strategies, such as long-lasting insecticide-treated nets (LLINs) and indoor residual sprays (IRSs), are heavily influenced by individual and community-level perceptions and acceptance. These perceptions, and therefore product acceptance, are largely influenced by the successful suppression of non-target nuisance pests such as bed bugs and cockroaches. Adoption and consistent use of LLINs and IRS is responsible for immense reductions in the prevalence and incidence of malaria. However, recent observations suggest that failed control of indoor pests, leading to product distrust and abandonment, may threaten vector control programme success and further derail already slowed progress towards malaria elimination. We review the evidence of the relationship between IVC and nuisance pests and discuss the dearth of research on this relationship. We make the case that the ancillary control of indoor nuisance and public health pests needs to be considered in the development and implementation of new technologies for malaria elimination.

Physician workload and practice management in pediatric gastroenterology.

OBJECTIVES: The pediatric gastroenterology workforce has grown in the last few decades. The North American Society of Pediatric Gastroenterology, Hepatology and Nutrition (NASPGHAN) formed a task force to understand current pediatric gastroenterology organizations' practice structures. METHODS: 19-item electronic survey was distributed to NASPGHAN members who were clinical or academic division directors. RESULTS: 30% responded to the survey, all directors of academic practices. The median number of clinical sessions per week was seven sessions, and the median individual work relative value unit (wRVU) target for practices was 4000-4500. Healthcare team ratios compared to provider clinical full-time equivalent were reported as the following: Nursing 0.80, medical assistant (MA) 0.29, dietitian 0.29, social worker 0.14, and psychologist 0.13. Regarding compensation, 68.0% were salaried with bonus based on billing or director decision, 28.0% were salaried with no incentive pay, and 4.0% were salaried with a portion at risk if the target was not met, and a bonus was given if the target was met. Most practices participated in a wellness activity with the most common strategies being didactic lectures about physician burnout (80%), annual burnout check-ins (68%), and/or after-hours social activities (60%). CONCLUSIONS: Pediatric gastroenterology practices vary regarding clinical sessions per week and annual wRVU targets with the median at seven sessions per week and an annual goal of 4000-4500 wRVUs, similar to reported national benchmark goals at the 50th percentile. Healthcare teams, including nursing, MAs, dietitians, social workers, and psychologists, had similar ratios of staff to providers for all sizes and types of practices. Most practices are engaging in wellness initiatives.

Paradoxical Activation of a Type VI Secretion System Phospholipase Effector by Its Cognate Immunity Protein.

Type VI secretion systems (T6SSs) deliver cytotoxic effector proteins into target bacteria and eukaryotic host cells. Antibacterial effectors are invariably encoded with cognate immunity proteins that protect the producing cell from self-intoxication. Here, we identify transposon insertions that disrupt the tli immunity gene of Enterobacter cloacae and induce autopermeabilization through unopposed activity of the Tle phospholipase effector. This hyperpermeability phenotype is T6SS dependent, indicating that the mutants are intoxicated by Tle delivered from neighboring sibling cells rather than by internally produced phospholipase. Unexpectedly, an in-frame deletion of tli does not induce hyperpermeability because Δtli null mutants fail to deploy active Tle. Instead, the most striking phenotypes are associated with disruption of the tli lipoprotein signal sequence, which prevents immunity protein localization to the periplasm. Immunoblotting reveals that most hyperpermeable mutants still produce Tli, presumably from alternative translation initiation codons downstream of the signal sequence. These observations suggest that cytosolic Tli is required for the activation and/or export of Tle. We show that Tle growth inhibition activity remains Tli dependent when phospholipase delivery into target bacteria is ensured through fusion to the VgrG ß-spike protein. Together, these findings indicate that Tli has distinct functions, depending on its subcellular localization. Periplasmic Tli acts as a canonical immunity factor to neutralize incoming effector proteins, while a cytosolic pool of Tli is required to activate the phospholipase domain of Tle prior to T6SS-dependent export. IMPORTANCE Gram-negative bacteria use type VI secretion systems deliver toxic effector proteins directly into neighboring competitors. Secreting cells also produce specific immunity proteins that neutralize effector activities to prevent autointoxication. Here, we show the Tli immunity protein of Enterobacter cloacae has two distinct functions, depending on its subcellular localization. Periplasmic Tli acts as a canonical immunity factor to block Tle lipase effector activity, while cytoplasmic Tli is required to activate the lipase prior to export. These results indicate Tle interacts transiently with its cognate immunity protein to promote effector protein folding and/or packaging into the secretion apparatus.

Physician Burnout in Pediatric Gastroenterology.

OBJECTIVES: Physicians are prone to burnout which can negatively affect the quality of patient care and lead to medical errors. Burnout can also affect physicians by impacting their personal relationships, their sense of career fulfillment, and job satisfaction. The North American Society of Pediatric Gastroenterology, Hepatology and Nutrition (NASPGHAN) formed a taskforce to investigate burnout among pediatric gastroenterologists. METHODS: A 35-item electronic survey was developed to collect demographic and practice information and characterize the well-being of pediatric gastroenterologists. Burnout was assessed employing 2 single-item measures adapted from the Maslach Burnout Inventory. The survey was distributed to NASPGHAN members 3 times from February 2020 to March 2020. Descriptive statistics, Chi-square, and Fisher exact tests were used. RESULTS: One thousand seven hundred ninety-one e-mails were successfully sent and 408 participants (22.7%) returned surveys. A total of 28.8% reported high risk for emotional exhaustion, 17.5% reported high risk for depersonalization, and 33% reported overall burnout. Participants 44 years of age or younger reported significantly more burnout than those 45 years and older ( P = 0.018). Contributors to high burnout identified included increased patient load/demands, insufficient nursing support, electronic health record (EHR) use, insufficient administrative staff, excessive on-call coverage, and more complex patients. Forty-four percent reported not having enough time for their personal life including family. A total of 16.2% of participants reported that they would not choose to be a pediatric gastroenterologist again. CONCLUSIONS: Pediatric gastroenterologists are at risk for emotional exhaustion, depersonalization, and overall burnout. Strategies to prevent physician burnout should be implemented as soon as feasibly possible to improve individual mental health and patient care.

The ß-encapsulation cage of rearrangement hotspot (Rhs) effectors is required for type VI secretion.

Bacteria deploy rearrangement hotspot (Rhs) proteins as toxic effectors against both prokaryotic and eukaryotic target cells. Rhs proteins are characterized by YD-peptide repeats, which fold into a large ß-cage structure that encapsulates the C-terminal toxin domain. Here, we show that Rhs effectors are essential for type VI secretion system (T6SS) activity in Enterobacter cloacae (ECL). ECL rhs- mutants do not kill Escherichia coli target bacteria and are defective for T6SS-dependent export of hemolysin-coregulated protein (Hcp). The RhsA and RhsB effectors of ECL both contain Pro-Ala-Ala-Arg (PAAR) repeat domains, which bind the ß-spike of trimeric valine-glycine repeat protein G (VgrG) and are important for T6SS activity in other bacteria. Truncated RhsA that retains the PAAR domain is capable of forming higher-order, thermostable complexes with VgrG, yet these assemblies fail to restore secretion activity to ∆rhsA ∆rhsB mutants. Full T6SS-1 activity requires Rhs that contains N-terminal transmembrane helices, the PAAR domain, and an intact ß-cage. Although ∆rhsA ∆rhsB mutants do not kill target bacteria, time-lapse microscopy reveals that they assemble and fire T6SS contractile sheaths at ∼6% of the frequency of rhs+ cells. Therefore, Rhs proteins are not strictly required for T6SS assembly, although they greatly increase secretion efficiency. We propose that PAAR and the ß-cage provide distinct structures that promote secretion. PAAR is clearly sufficient to stabilize trimeric VgrG, but efficient assembly of T6SS-1 also depends on an intact ß-cage. Together, these domains enforce a quality control checkpoint to ensure that VgrG is loaded with toxic cargo before assembling the secretion apparatus.

Impact of local rivers on coastal acidification.

Coastal ecosystems are highly dynamic areas for carbon cycling and are likely to be negatively impacted by increasing ocean acidification. This research focused on dissolved inorganic carbon (DIC) and total alkalinity (TA) in the Mississippi Sound to understand the influence of local rivers on coastal acidification. This area receives large fluxes of freshwater from local rivers, in addition to episodic inputs from the Mississippi River through a human-built diversion, the Bonnet Carré Spillway. Sites in the Sound were sampled monthly from August 2018 to November 2019 and weekly from June to August 2019 in response to an extended spillway opening. Prior to the 2019 spillway opening, the contribution of the local, lower alkalinity rivers to the Sound may have left the study area more susceptible to coastal acidification during winter months, with aragonite saturation states (Ωar) < 2. After the spillway opened, despite a large increase in TA throughout the Sound, aragonite saturation states remained low, likely due to hypoxia and increased CO2 concentrations in subsurface waters. Increased Mississippi River input could represent a new normal in the Sound's hydrography during spring and summer months. The spillway has been utilized more frequently over the last two decades due to increasing precipitation in the Mississippi River watershed, which is primarily associated with climate change. Future increases in freshwater discharge and the associated declines in salinity, dissolved oxygen, and Ωar in the Sound will likely be detrimental to oyster stocks and the resilience of similar ecosystems to coastal acidification.

Mapping the interaction between eukaryotic initiation factor 4A (eIF4A) and the inhibitor hippuristanol using carbene footprinting and mass spectrometry.

Protein-ligand interactions are central to protein activity and cell functionality. Improved knowledge of these relationships greatly benefits our understanding of key biological processes and aids in rational drug design towards the treatment of clinically relevant diseases. Carbene footprinting is a recently developed mass spectrometry-based chemical labelling technique that provides valuable information relating to protein-ligand interactions, such as the mapping of binding sites and associated conformational change. Here, we show the application of carbene footprinting to the interaction between eIF4A helicase and a natural product inhibitor, hippuristanol, found in the coral Isis hippuris. Upon addition of hippuristanol we identified reduced carbene labelling (masking) in regions of eIF4A previously implicated in ligand binding. Additionally, we detected hippuristanol-associated increased carbene labelling (unmasking) around the flexible hinge region of eIF4A, indicating ligand-induced conformational change. This work represents further development of the carbene footprinting technique and demonstrates its potential in characterising medicinally relevant protein-ligand interactions.

Target highlights in CASP14: Analysis of models by structure providers.

The biological and functional significance of selected Critical Assessment of Techniques for Protein Structure Prediction 14 (CASP14) targets are described by the authors of the structures. The authors highlight the most relevant features of the target proteins and discuss how well these features were reproduced in the respective submitted predictions. The overall ability to predict three-dimensional structures of proteins has improved remarkably in CASP14, and many difficult targets were modeled with impressive accuracy. For the first time in the history of CASP, the experimentalists not only highlighted that computational models can accurately reproduce the most critical structural features observed in their targets, but also envisaged that models could serve as a guidance for further studies of biologically-relevant properties of proteins.

Diazophosphonates: Effective Surrogates for Diazoalkanes in Pyrazole Synthesis.

Diazophosphonates, readily prepared from α-ketophosphonates by oxidation of the corresponding hydrazones in batch or in flow, are useful partners in 1,3-dipolar cycloaddition reactions to alkynes to give N-H pyrazoles, including the first intramolecular examples of such a process. The phosphoryl group imbues a number of desirable properties into the diazo 1,3-dipole. The electron-withdrawing nature of the phosphoryl stabilizes the diazo compound making it easier to handle, whilst the ability of the phosphoryl group to migrate readily in a [1,5]-sigmatropic rearrangement enables its transfer from C to N to aromatize the initial cycloadduct, and hence its facile removal from the final pyrazole product. Overall, the diazophosphonate acts as a surrogate for the much less stable diazoalkane in cycloadditions, with the phosphoryl group playing a vital, but traceless, role. The cycloaddition proceeds more readily with alkynes bearing electron-withdrawing groups, and is regiospecific with asymmetrical alkynes. The potential of diazophosphonates for use in bioorthogonal cycloadditions is demonstrated by their facile addition to strained alkynes.

Early Antibiotic Administration Is Associated with a Reduced Infection Risk When Combined with Primary Wound Closure in Patients with Open Tibia Fractures.

BACKGROUND: Early administration of antibiotics and wound coverage have been shown to decrease the deep infection risk in all patients with Type 3 open tibia fractures. However, it is unknown whether early antibiotic administration decreases infection risk in patients with Types 1, 2, and 3A open tibia fractures treated with primary wound closure. QUESTIONS/PURPOSES: (1) Does decreased time to administration of the first dose of antibiotics decrease the deep infection risk in all open tibia fractures with primary wound closure? (2) What patient demographic factors are associated with an increased deep infection risk in Types 1, 2, and 3A open tibia fractures with primary wound closure? METHODS: We identified 361 open tibia fractures over a 5-year period at a Level I regional trauma center that receives direct admissions and transfers from other hospitals which produces large variation in the timing of antibiotic administration. Patients were excluded if they were younger than 18 years, had associated plafond or plateau fractures, associated with compartment syndrome, had a delay of more than 24 hours from injury to the operating room, underwent repeat débridement procedures, had incomplete data, and were treated with negative-pressure dressings or other adjunct wound management strategies that would preclude primary closure. Primary closure was at the descretion of the treating surgeon. We included patients with a minimum follow-up of 6 weeks with assessment at 6 months and 12 months. One hundred forty-three patients with were included in the analysis. Our primary endpoint was deep infection as defined by the CDC criteria. We obtained chronological data, including the time to the first dose of antibiotics and time to surgical débridement from ambulance run sheets, transferring hospital records, and the electronic medical record to answer our first question. We considered demographics, American Society of Anesthesiologists classification, mechanism of injury, smoking status, presence of diabetes, and Injury Severity Score in our analysis of other factors. These were compared using one-way ANOVA, chi-square, or Fisher's exact tests. Binary regression was used to to ascertain whether any factors were associated with postoperative infection. Receiver operator characteristic curves were used to identify threshold values. RESULTS: Increased time to first administration of antibiotics was associated with an increased infection risk in patients who were treated with primary wound closure; the greatest inflection point on that analysis occurred at 150 minutes, when the increased infection risk was greatest (20% [8 of 41] versus 4% [3 of 86]; odds ratio 5.6 [95% CI 1.4 to 22.2]; p = 0.01). After controlling for potential confounding variables like age, diabetes and smoking status, none of the variables we evaluated were associated with an increased risk of deep infection in Type 1, 2, and 3A open tibia fractures in patients treated with primary wound closure. CONCLUSION: Our findings suggest that in open tibia fractures, which receive timely antibiotic administration, primary wound closure is associated with a decreased infection risk. We recognize that more definitive studies need to be performed to confirm these findings and confirm feasibility of early antibiotic administration, especially in the pre-hospital context. LEVEL OF EVIDENCE: Level III, therapeutic study.

The Impact of Macrocycle Conformation on the Taxadiene-Forming Carbocation Cascade: Insight Gained from Sobralene, a Recently Discovered Verticillene Isomer.

DFT calculations on the carbocation intermediates that connect the biosynthetic pathways leading to the sand fly pheromone sobralene and taxadiene have been made. Establishment of the conformation of the macrocyclic carbocation intermediate required to produce the (Z)-C8,C9 alkene bond in sobralene has identified new conformations of the verticillyl carbocation intermediates on the taxadiene biosynthetic pathway. These "sobralene-like" carbocation conformations provide an exothermic pathway to taxadiene and are validated by comparison to closely related structures (X-ray and NMR).

Beyond the black stump: rapid reviews of health research issues affecting regional, rural and remote Australia.

CHAPTER 1: RETAIL INITIATIVES TO IMPROVE THE HEALTHINESS OF FOOD ENVIRONMENTS IN RURAL, REGIONAL AND REMOTE COMMUNITIES: Objective: To synthesise the evidence for effectiveness of initiatives aimed at improving food retail environments and consumer dietary behaviour in rural, regional and remote populations in Australia and comparable countries, and to discuss the implications for future food environment initiatives for rural, regional and remote areas of Australia. STUDY DESIGN: Rapid review of articles published between January 2000 and May 2020. DATA SOURCES: We searched MEDLINE (EBSCOhost), Health and Society Database (Informit) and Rural and Remote Health Database (Informit), and included studies undertaken in rural food environment settings in Australia and other countries. DATA SYNTHESIS: Twenty-one articles met the inclusion criteria, including five conducted in Australia. Four of the Australian studies were conducted in very remote populations and in grocery stores, and one was conducted in regional Australia. All of the overseas studies were conducted in rural North America. All of them revealed a positive influence on food environment or consumer behaviour, and all were conducted in disadvantaged, rural communities. Positive outcomes were consistently revealed by studies of initiatives that focused on promotion and awareness of healthy foods and included co-design to generate community ownership and branding. CONCLUSION: Initiatives aimed at improving rural food retail environments were effective and, when implemented in different rural settings, may encourage improvements in population diets. The paucity of studies over the past 20 years in Australia shows a need for more research into effective food retail environment initiatives, modelled on examples from overseas, with studies needed across all levels of remoteness in Australia. Several retail initiatives that were undertaken in rural North America could be replicated in rural Australia and could underpin future research. CHAPTER 2: WHICH INTERVENTIONS BEST SUPPORT THE HEALTH AND WELLBEING NEEDS OF RURAL POPULATIONS EXPERIENCING NATURAL DISASTERS?: Objective: To explore and evaluate health and social care interventions delivered to rural and remote communities experiencing natural disasters in Australia and other high income countries. STUDY DESIGN: We used systematic rapid review methods. First we identified a test set of citations and generated a frequency table of Medical Subject Headings (MeSH) to index articles. Then we used combinations of MeSH terms and keywords to search the MEDLINE (Ovid) database, and screened the titles and abstracts of the retrieved references. DATA SOURCES: We identified 1438 articles via database searches, and a further 62 articles via hand searching of key journals and reference lists. We also found four relevant grey literature resources. After removing duplicates and undertaking two stages of screening, we included 28 studies in a synthesis of qualitative evidence. DATA SYNTHESIS: Four of us read and assessed the full text articles. We then conducted a thematic analysis using the three phases of the natural disaster response cycle. CONCLUSION: There is a lack of robust evaluation of programs and interventions supporting the health and wellbeing of people in rural communities affected by natural disasters. To address the cumulative and long term impacts, evidence suggests that continuous support of people's health and wellbeing is needed. By using a lens of rural adversity, the complexity of the lived experience of natural disasters by rural residents can be better understood and can inform development of new models of community-based and integrated care services. CHAPTER 3: THE IMPACT OF BUSHFIRE ON THE WELLBEING OF CHILDREN LIVING IN RURAL AND REMOTE AUSTRALIA: Objective: To investigate the impact of bushfire events on the wellbeing of children living in rural and remote Australia. STUDY DESIGN: Literature review completed using rapid realist review methods, and taking into consideration the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) statement for systematic reviews. DATA SOURCES: We sourced data from six databases: EBSCOhost (Education), EBSCOhost (Health), EBSCOhost (Psychology), Informit, MEDLINE and PsycINFO. We developed search terms to identify articles that could address the research question based on the inclusion criteria of peer reviewed full text journal articles published in English between 1983 and 2020. We initially identified 60 studies and, following closer review, extracted data from eight studies that met the inclusion criteria. DATA SYNTHESIS: Children exposed to bushfires may be at increased risk of poorer wellbeing outcomes. Findings suggest that the impact of bushfire exposure may not be apparent in the short term but may become more pronounced later in life. Children particularly at risk are those from more vulnerable backgrounds who may have compounding factors that limit their ability to overcome bushfire trauma. CONCLUSION: We identified the short, medium and long term impacts of bushfire exposure on the wellbeing of children in Australia. We did not identify any evidence-based interventions for supporting outcomes for this population. Given the likely increase in bushfire events in Australia, research into effective interventions should be a priority. CHAPTER 4: THE ROLE OF NATIONAL POLICIES TO ADDRESS RURAL ALLIED HEALTH, NURSING AND DENTISTRY WORKFORCE MALDISTRIBUTION: Objective: Maldistribution of the health workforce between rural, remote and metropolitan communities contributes to longstanding health inequalities. Many developed countries have implemented policies to encourage health care professionals to work in rural and remote communities. This scoping review is an international synthesis of those policies, examining their effectiveness at recruiting and retaining nursing, dental and allied health professionals in rural communities. STUDY DESIGN: Using scoping review methods, we included primary research - published between 1 September 2009 and 30 June 2020 - that reported an evaluation of existing policy initiatives to address workforce maldistribution in high income countries with a land mass greater than 100 000 km2 . DATA SOURCES: We searched MEDLINE, Ovid Embase, Ovid Emcare, Informit, Scopus, and Web of Science. We screened 5169 articles for inclusion by title and abstract, of which we included 297 for full text screening. We then extracted data on 51 studies that had been conducted in Australia, the United States, Canada, United Kingdom and Norway. DATA SYNTHESIS: We grouped the studies based on World Health Organization recommendations on recruitment and retention of health care workers: education strategies (n = 27), regulatory change (n = 11), financial incentives (n = 6), personal and professional support (n = 4), and approaches with multiple components (n = 3). CONCLUSION: Considerable work has occurred to address workforce maldistribution at a local level, underpinned by good practice guidelines, but rarely at scale or with explicit links to coherent overarching policy. To achieve policy aspirations, multiple synergistic evidence-based initiatives are needed, and implementation must be accompanied by well designed longitudinal evaluations that assess the effectiveness of policy objectives. CHAPTER 5: AVAILABILITY AND CHARACTERISTICS OF PUBLICLY AVAILABLE HEALTH WORKFORCE DATA SOURCES IN AUSTRALIA: Objective: Many data sources are used in Australia to inform health workforce planning, but their characteristics in terms of relevance, accessibility and accuracy are uncertain. We aimed to identify and appraise publicly available data sources used to describe the Australian health workforce. STUDY DESIGN: We conducted a scoping review in which we searched bibliographic databases, websites and grey literature. Two reviewers independently undertook title and abstract screening and full text screening using Covidence software. We then assessed the relevance, accessibility and accuracy of data sources using a customised appraisal tool. DATA SOURCES: We searched for potential workforce data sources in nine databases (MEDLINE, Embase, Ovid Emcare, Scopus, Web of Science, Informit, the JBI Evidence-based Practice Database, PsycINFO and the Cochrane Library) and the grey literature, and examined several pre-defined websites. DATA SYNTHESIS: During the screening process we identified 6955 abstracts and examined 48 websites, from which we identified 12 publicly available data sources - eight primary and four secondary data sources. The primary data sources were generally of modest quality, with low scores in terms of reference period, accessibility and missing data. No single primary data source scored well across all domains of the appraisal tool. CONCLUSION: We identified several limitations of data sources used to describe the Australian health workforce. Establishment of a high quality, longitudinal, linked database that can inform all aspects of health workforce development is urgently needed, particularly for rural health workforce and services planning. CHAPTER 6: RAPID REALIST REVIEW OF OPIOID TAPERING IN THE CONTEXT OF LONG TERM OPIOID USE FOR NON-CANCER PAIN IN RURAL AREAS: Objective: To describe interventions, barriers and enablers associated with opioid tapering for patients with chronic non-cancer pain in rural primary care settings. STUDY DESIGN: Rapid realist review registered on the international register of systematic reviews (PROSPERO) and conducted in accordance with RAMESES standards. DATA SOURCES: English language, peer-reviewed articles reporting qualitative, quantitative and mixed method studies, published between January 2016 and July 2020, and accessed via MEDLINE, Embase, CINAHL Complete, PsycINFO, Informit or the Cochrane Library during June and July 2020. Grey literature relating to prescribing,deprescribing or tapering of opioids in chronic non-cancer pain, published between January 2016 and July 2020, was identified by searching national and international government, health service and peek organisation websites using Google Scholar. DATA SYNTHESIS: Our analysis of reported approaches to tapering conducted across rural and non-rural contexts showed that tapering opioids is complex and challenging, and identified several barriers and enablers. Successful outcomes in rural areas appear likely through therapeutic relationships, coordination and support, by using modalities and models of care that are appropriate in rural settings and by paying attention to harm minimisation. CONCLUSION: Rural primary care providers do not have access to resources available in metropolitan centres for dealing with patients who have chronic non-cancer pain and are taking opioid medications. They often operate alone or in small group practices, without peer support and access to multidisciplinary and specialist teams. Opioid tapering approaches described in the literature include regulation, multimodal and multidisciplinary approaches, primary care provider support, guidelines, and patient-centred strategies. There is little research to inform tapering in rural contexts. Our review provides a synthesis of the current evidence in the form of a conceptual model. This preliminary model could inform the development of a model of care for use in implementation research, which could test a variety of mechanisms for supporting decision making, reducing primary care providers' concerns about potential harms arising from opioid tapering, and improving patient outcomes.

Activation of contact-dependent antibacterial tRNase toxins by translation elongation factors.

Contact-dependent growth inhibition (CDI) is a mechanism by which bacteria exchange toxins via direct cell-to-cell contact. CDI systems are distributed widely among Gram-negative pathogens and are thought to mediate interstrain competition. Here, we describe tsf mutations that alter the coiled-coil domain of elongation factor Ts (EF-Ts) and confer resistance to the CdiA-CTEC869 tRNase toxin from enterohemorrhagic Escherichia coli EC869. Although EF-Ts is required for toxicity in vivo, our results indicate that it is dispensable for tRNase activity in vitro. We find that CdiA-CTEC869 binds to elongation factor Tu (EF-Tu) with high affinity and this interaction is critical for nuclease activity. Moreover, in vitro tRNase activity is GTP-dependent, suggesting that CdiA-CTEC869 only cleaves tRNA in the context of translationally active GTP·EF-Tu·tRNA ternary complexes. We propose that EF-Ts promotes the formation of GTP·EF-Tu·tRNA ternary complexes, thereby accelerating substrate turnover for rapid depletion of target-cell tRNA.

Antibiotic cement spacer retention for chronic shoulder infection after minimum 2-year follow-up.

HYPOTHESIS: The treatment of periprosthetic shoulder infections and proximal humerus osteomyelitis is challenging. The outcomes of antibiotic cement spacer retention are poorly defined in the literature. The purpose of this study was to review long-term functional and patient-reported outcomes data of patients with retained antibiotic cement spacers. We predict reasonable functional outcomes and minimal pain. METHODS: We identified 22 patients of the senior author who have been treated with definitive antibiotic spacer placement. All patients were originally offered a 2-stage revision and declined. Twelve patients had a minimum follow-up of 2 years and were included in our cohort. Mean age was 70.7 (range 59-81), 8/12 patients were female, and the average body mass index was 27.8 (range = 17-45). Functional outcome assessments included the Standardized Shoulder Assessment Form, the Quick Disabilities of the Arm, Shoulder, and Hand Score (QuickDASH), and visual analog scale (VAS) along with clinical range of motion examination. RESULTS: The patients were followed up for a mean of 5.6 years. Eight patients had spacer placement for chronic shoulder arthroplasty infections, whereas 4 patients had spacer placement for chronic osteomyelitis of the proximal humerus. No patients were currently being treated with suppressive antibiotics. One patient had negative cultures at the time of antibiotic spacer placement. The most common organisms were Cutibacterium acnes (6), Staphylococcus epidermidis (6), and methicillin-resistant Staphylococcus aureus (4), with 4 patients growing more than 1 species. The average ASES score was 54 (range = 27-73), QuickDASH was 45 (range = 14-89), and VAS score 2.8 (range = 0-8). Average active range of motion was 68° of forward elevation and 35° of external rotation. CONCLUSIONS: Retention of antibiotic cement spacer is a viable option in the treatment algorithm for chronic shoulder infections. Long-term antibiotic cement spacer may be considered for those patients who are unwilling or unable to undergo a 2-stage revision. Patients can expect a reasonable amount of function and little to no pain with an antibiotic cement spacer.

Identification of a target cell permissive factor required for contact-dependent growth inhibition (CDI).

Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiB/CdiA family of two-partner secretion proteins. CdiA effector proteins are exported onto the surface of CDI(+) inhibitor cells, where they interact with susceptible bacteria and deliver effectors/toxins derived from their C-terminal regions (CdiA-CT). CDI(+) cells also produce an immunity protein that binds the CdiA-CT and blocks its activity to prevent autoinhibition. Here, we show that the CdiA-CT from uropathogenic Escherichia coli strain 536 (UPEC536) is a latent tRNase that requires activation by the biosynthetic enzyme CysK (O-acetylserine sulfhydrylase A). UPEC536 CdiA-CT exhibits no nuclease activity in vitro, but cleaves within transfer RNA (tRNA) anti-codon loops when purified CysK is added. CysK and CdiA-CT form a stable complex, and their binding interaction appears to mimic that of the CysK/CysE cysteine synthase complex. CdiA-CT activation is also required for growth inhibition. Synthesis of CdiA-CT in E. coli cysK(+) cells arrests cell growth, whereas the growth of ΔcysK mutants is unaffected by the toxin. Moreover, E. coli ΔcysK cells are completely resistant to inhibitor cells expressing UPEC536 CdiA, indicating that CysK is required to activate the tRNase during CDI. Thus, CysK acts as a permissive factor for CDI, providing a potential mechanism to modulate growth inhibition in target cells.

Target highlights in CASP13: Experimental target structures through the eyes of their authors.

The functional and biological significance of selected CASP13 targets are described by the authors of the structures. The structural biologists discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP13 experiment.