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This study examined the prevalence of food insecurity and associated factors among state university students. Participants (n = 943) completed an online questionnaire that combined the US Department of Agriculture 6-Item Short Form US Household Food Security Survey with demographic and food assistance use questions in fall, 2015. One-fourth of participants (25%) reported food insecurity within the past year. Students who received financial aid and identified as first generation were more likely to be food insecure. Food insecurity is a significant problem among university students. Additional research is needed to better understand and address food insecurity in higher education. [ABSTRACT FROM AUTHOR]

A indústria siderúrgica representa um dos setores de maior importância para a economia, visto que é o principal responsável pelo fornecimento de matéria-prima para diversos segmentos. Diferente de outros segmentos, as organizações que compõem o setor, sejam elas nacionais ou internacionais, vem passando por um cenário de reestruturação com a finalidade de garantir a liderança ou até a manutenção de suas posições no mercado. O objetivo desse estudo é apresentar as core competences ou competências essenciais de uma empresa pertencente ao setor siderúrgico. Este estudo deriva de uma pesquisa qualitativa com o objetivo de evidenciar as competências essenciais de uma empresa do setor siderúrgico. Após um levantamento bibliográfico, pesquisa de campo na empresa estudada e clientes, os resultados demonstraram que a organização analisada possui duas core competences analisadas pelos principais autores da área, sendo elas: competência de orientação para o cliente e competência de excelência operacional. Ainda, foram identificadas vantagens percebidas pelos clientes, que apontaram três principais artifícios pautados pela economia, qualidade e praticidade.

Este artigo elabora um balanço internacional da produção científica sobre a temática de desenvolvimento sustentável na área de administração, verifica as principais características da sua disseminação e permeação dentro do mundo acadêmico internacional, realizando um breve regresso histórico para compreender a evolução do tema. O estudo foi desenvolvido na perspectiva de uma pesquisa bibliométrica, modelo de pesquisa com caráter exploratório, aplicando métodos de observação, registro, classificação, análise e interpretação dos resultados, utilizando como base o banco de dados da Web of Science, abordando as pesquisas realizadas entre 1991 e 2012. Com o resultado, foi possível identificar os principais campos de estudo, as áreas temáticas, a distribuição temporal das publicações, os países de origem, as categorias da Web of Science, os institutos responsáveis, os principais autores e journals. Também foi utilizada a ferramenta TextStat para identificar os principais tópicos do estudo (Hot Topics).

Abstract Background To examine demographic and behavioral associations with self-reported health-related quality of life (HRQOL) among persons with HIV infection or AIDS. Methods Analysis of interviews with persons ≥ 18 years of age reported through routine disease surveillance with HIV infection or AIDS to nine state and local health departments from January 1995 through December 1996. Scales were constructed from validated measures of HRQOL, and mean scores were calculated (lower scores signified poorer HRQOL). Measures of HRQOL included Overall Health, Pain, Physical Functioning, Role Functioning, Social Functioning, Mental Health, Energy/Fatigue, and Cognitive Functioning. Differences in HRQOL were examined by various demographic and behavioral factors, including taking antiretroviral medication. Results HRQOL data were available for 3778 persons. Factors associated with lower HRQOL scores included older age, female sex, black or Hispanic race/ethnicity, injection drug use, lower education and income, no private health insurance, and lower CD4 count. In multivariate analysis, lower CD4 count was the factor most consistently associated with lower HRQOL. Taking antiretroviral medication was not associated with differences in HRQOL regardless of CD4 count. Conclusions Perception of HRQOL varied in a population with HIV infection or AIDS. On most HRQOL measures, lower CD4 count was associated with lower HRQOL. Measurement of HRQOL can assist in understanding the long-term effects of disease and treatment on persons with HIV.

In biological systems, the activities of macromolecular complexes must sometimes be turned off. Thus, a wide variety of protein inhibitors has evolved for this purpose. These inhibitors function through diverse mechanisms, including steric blocking of crucial interactions, enzymatic modification of key residues or substrates, and perturbation of post-translational modifications 1 . Anti-CRISPRs-proteins that block the activity of CRISPR-Cas systems-are one of the largest groups of inhibitors described, with more than 90 families that function through diverse mechanisms 2-4 . Here, we characterize the anti-CRISPR AcrIF25, and we show that it inhibits the type I-F CRISPR-Cas system by pulling apart the fully assembled effector complex. AcrIF25 binds to the predominant CRISPR RNA-binding components of this complex, comprising six Cas7 subunits, and strips them from the RNA. Structural and biochemical studies indicate that AcrIF25 removes one Cas7 subunit at a time, starting at one end of the complex. Notably, this feat is achieved with no apparent enzymatic activity. To our knowledge, AcrIF25 is the first example of a protein that disassembles a large and stable macromolecular complex in the absence of an external energy source. As such, AcrIF25 establishes a paradigm for macromolecular complex inhibitors that may be used for biotechnological applications., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The ongoing advancements in CRISPR-Cas technologies can significantly accelerate the preclinical development of both in vivo and ex vivo organ genome-editing therapeutics. One of the promising applications is to genetically modify donor organs prior to implantation. The implantation of optimized donor organs with long-lasting immunomodulatory capacity holds promise for reducing the need for lifelong potent whole-body immunosuppression in recipients. However, assessing genome-targeting interventions in a clinically relevant manner prior to clinical trials remains a major challenge owing to the limited modalities available. This study introduces a novel platform for testing genome editing in human lungs ex vivo , effectively simulating preimplantation genetic engineering of donor organs. We identified gene regulatory elements whose disruption via Cas nucleases led to the upregulation of the immunomodulatory gene interleukin 10 ( IL-10) . We combined this approach with adenoviral vector-mediated IL-10 delivery to create favorable kinetics for early (immediate postimplantation) graft immunomodulation. Using ex vivo organ machine perfusion and precision-cut tissue slice technology, we demonstrated the feasibility of evaluating CRISPR genome editing in human lungs. To overcome the assessment limitations in ex vivo perfused human organs, we conducted an in vivo rodent study and demonstrated both early gene induction and sustained editing of the lung. Collectively, our findings lay the groundwork for a first-in-human-organ study to overcome the current translational barriers of genome-targeting therapeutics.

Aim: This constructivist grounded theory study aimed to (1) explore patients' experiences of and roles in interprofessional collaborative practice for chronic conditions in primary care and (2) consider the relevance and alignment of an existing theoretical framework on patients' roles and based on the experiences of patient advocates., Background: High-quality management of chronic conditions requires an interprofessional collaborative practice model of care considering an individual's mental, physical, and social health situation. Patients' experiences of this model in the primary care setting are relatively unknown., Methods: A constructivist grounded theory approach was taken. Interview data were collected from primary care patients with chronic conditions across Australia in August 2020 - February 2022. Interviews were recorded, transcribed verbatim, and thematically analysed by (1) initial line-by-line coding, (2) focused coding, (3) memo writing, (4) categorisation, and (5) theme and sub-theme development. Themes and sub-themes were mapped against an existing theoretical framework to expand and confirm the results from a previous study with a similar research aim., Findings: Twenty adults with chronic conditions spanning physical disability, diabetes, heart disease, cancer, autoimmune, and mental health conditions participated. Two themes were developed: (1) Adapting to Change with two sub-themes describing how patients adapt to interprofessional team care and (2) Shifting across the spectrum of roles , with five sub-themes outlining the roles patients enact while receiving care. The findings suggest that patients' roles are highly variable and fluid in interprofessional collaborative practice, and further work is recommended to develop a resource to support greater patient engagement in interprofessional collaborative practice.

Background: Non-drug interventions are recommended for chronic condition prevention and management yet are underused in clinical practice. Understanding barriers and enablers to using non-drug interventions may help implement non-drug interventions in primary care. We aimed to conduct an overview of reviews to identify and summarise common barriers and enablers for using non-drug interventions for common chronic conditions in primary care., Methods: We included qualitative and quantitative reviews that used systematic process or methods to examine barriers and enablers to using non-drug interventions for chronic condition prevention and management in primary care settings. We searched 5 electronic databases (PubMed, Cochrane Database of Systematic Reviews, EMBASE, PsycInfo and CINAHL) from inception to September 2022. Two authors independently screened reviews. One author extracted and deductively coded data to Consolidated Framework of Implementation Research (CFIR) (and where relevant, Theoretical Domains Framework [TDF]). A second author validated 10% of extracted data and coding. Data was synthesised thematically using CFIR and TDF. One author assessed the methodological quality of included reviews using a modified AMSTAR 2 tool, with 10% validated by a second author. We assessed overlap between primary studies in included reviews., Results: From 5324 records, we included 25 reviews, with data predominately from patients. Overall, 130 subthemes (71 barrier and 59 enabler) were identified across 4 CFIR domains (Innovation, Outer Setting, Inner Setting, and Individuals), and all TDF domains. Common barrier and enabler subthemes were identified for CFIR constructs of Innovation Adaptability, Innovation Cost, Innovation Relative Advantage, Local Attitudes, External Pressure, Local Conditions, Relational Connections, Available Resources, and Access to Knowledge and Information. For TDF domains, important barrier and enabler subthemes were identified for Knowledge, Skills, Environmental Context and Resources, Beliefs about Consequences, Reinforcement, and Emotion., Conclusions: We synthesised reviews to provide new insight into common barriers and enablers for using non-drug interventions to prevent and manage chronic conditions in primary care. The factors identified can inform the development of generalisable implementation interventions to enhance uptake of multiple non-drug interventions simultaneously., Trial Registration: This study was registered in PROSPERO (CRD42022357583)., (© 2024. The Author(s).)

Bacteria have evolved diverse antiviral defence mechanisms to protect themselves against phage infection. Phages integrated into bacterial chromosomes, known as prophages, also encode defences that protect the bacterial hosts in which they reside. Here, we identify a type of anti-phage defence that interferes with the virion assembly pathway of invading phages. The protein that mediates this defence, which we call Tab (for 'Tail assembly blocker'), is constitutively expressed from a Pseudomonas aeruginosa prophage. Tab allows the invading phage replication cycle to proceed, but blocks assembly of the phage tail, thus preventing formation of infectious virions. While the infected cell dies through the activity of the replicating phage lysis proteins, there is no release of infectious phage progeny, and the bacterial community is thereby protected from a phage epidemic. Prophages expressing Tab are not inhibited during their own lytic cycle because they express a counter-defence protein that interferes with Tab function. Thus, our work reveals an anti-phage defence that operates by blocking virion assembly, thereby both preventing formation of phage progeny and allowing destruction of the infected cell due to expression of phage lysis genes., (© 2024. The Author(s).)

Background: Chronic conditions can lead to physical, cognitive and social decline; thus, increasing an individual's dependence on family who assist with activities of daily living. Interprofessional collaborative practice (IPCP), involving two or more health professionals working with the patient and their family, is one model of care for the high-quality management of individuals with chronic conditions in primary care. Nevertheless, family carers have reported a disconnect between themselves and healthcare providers in previous research. This study aimed to explore the experiences and perspectives of family carers for individuals with chronic conditions, regarding their involvement in IPCP., Methods: Aspects of constructivist grounded theory methodology were used. Family carers of individuals with chronic conditions were invited to participate in a one-on-one, semistructured interview about their experiences with IPCP in the care of their loved one. Interview transcripts were analysed using Charmaz's four-step iterative process: (1) line-by-line coding, (2) focused coding, (3) categorisation of codes and (4) potential theme and subtheme development with memo writing to support each phase of analysis. The research team collaborated on reflexivity exercises, the conceptualisation of categories and the development of themes., Results: Constructivist data analysis of interviews (average 40 min) with 10 family carers resulted in two themes. (1) Stepping in for my loved one represents the notion that carers take on external roles on behalf of their loved ones (subthemes: working with interprofessional teams, supporting independence and learning as I go). (2) Taking on the carer role, represents the internal factors that influence the external roles described in theme 1 (subthemes: feeling obligated to be involved and changing relationship dynamics)., Conclusion: This study outlines the external actions and internal influences on family carer involvement in an interprofessional team. The required knowledge and support to care for their loved ones is currently learned in an ad hoc manner, and carers' resources should be better promoted by health professionals. Additionally, the relationship dynamics between a carer and their loved one change as the carer becomes more involved in IPCP and influences how and the extent health professionals involve family carers., Patient or Public Contribution: Carers were the study population involved in this qualitative study. Patient advocates who have chronic conditions, and are informal family carers, were involved in the creation and design of this study, including a review of the research question, participant information sheet and the interview guide., (© 2023 The Authors. Health Expectations published by John Wiley & Sons Ltd.)

Conversations about open science have reached the mainstream, yet many open science practices such as data sharing remain uncommon. Our efforts towards openness therefore need to increase in scale and aim for a more ambitious target. We need an ecosystem not only where research outputs are openly shared but also in which transparency permeates the research process from the start and lends itself to more rigorous and collaborative research. To support this vision, this Essay provides an overview of a selection of open science initiatives from the past 2 decades, focusing on methods transparency, scholarly communication, team science, and research culture, and speculates about what the future of open science could look like. It then draws on these examples to provide recommendations for how funders, institutions, journals, regulators, and other stakeholders can create an environment that is ripe for improvement., Competing Interests: AB is an Academic Editor for PLOS Biology. AB is a founder of SciCrunch Inc, a company that works with publishers to improve the representation of research resources in scientific literature. She is also a member of the board, and serves as the CEO. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. OBA is a member of the eLife Global South Advisory Committee and co-founder of the Brazilian Reproducibility Network. N.D. is an external consultant and animal welfare officer at Medizinisches Kompetenzzentrum c/o HCx Consulting, Brandenburg, Germany. All other authors declare no conflict of interest., (Copyright: © 2023 Thibault et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Inflammatory injury in the donor lung remains a persistent challenge in lung transplantation that limits donor organ usage and post-transplant outcomes. Inducing immunomodulatory capacity in donor organs could address this unsolved clinical problem. We sought to apply clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) technologies to the donor lung to fine-tune immunomodulatory gene expression, exploring for the first time the therapeutic use of CRISPR-mediated transcriptional activation in the whole donor lung., Methods: We explored the feasibility of CRISPR-mediated transcriptional upregulation of interleukin 10 (IL-10), a key immunomodulatory cytokine, in vitro and in vivo. We first evaluated the potency, titratability, and multiplexibility of the gene activation in rat and human cell lines. Next, in vivo CRISPR-mediated IL-10 activation was characterized in rat lungs. Finally, the IL-10-activated donor lungs were transplanted into recipient rats to assess the feasibility in a transplant setting., Results: The targeted transcriptional activation induced robust and titrable IL-10 upregulation in vitro. The combination of guide RNAs also facilitated multiplex gene modulation, that is, simultaneous activation of IL-10 and IL1 receptor antagonist. In vivo profiling demonstrated that adenoviral delivery of Cas9-based activators to the lung was feasible with the use of immunosuppression, which is routinely applied to organ transplant recipients. The transcriptionally modulated donor lungs retained IL-10 upregulation in isogeneic and allogeneic recipients., Conclusions: Our findings highlight the potential of CRISPR epigenome editing to improve lung transplant outcomes by creating a more favorable immunomodulatory environment in the donor organ, a paradigm that may be extendable to other organ transplants., Competing Interests: Disclosure Statement M.C. and S.K. are co-founders of Perfusix Canada and Scientific Advisors to Lung Bioengineering. S.K. is CMO of Traferox Technologies. University Health Network has a patent filed and pending for technologies related to donor organ modification, on which K.M., S.J., M.C., M.L., and S.K. are inventors. A.R.D. is on the Scientific Advisory Board of Acrigen. B.P.K is an inventor on patents and patent applications filed by Mass General Brigham that describe genome engineering technologies. B.P.K. is a consultant for EcoR1 capital and is a scientific advisory board member of Acrigen Biosciences, Life Edit Therapeutics, and Prime Medicine. This work was supported by the University Health Network Foundation, University of Toronto’s Medicine by Design (MbDPEFR1–2021-01), New Frontiers in Research Funding (NFRFT-2020–00787), and the Canadian Institutes of Health Research (CIHR PJT-173392). We thank Spatio-temporal Targeting and Amplification of Radiation Response (STTARR) at UHN and Advanced Optical Microscopy Facility (AOMF) at UHN for histology and imaging service. K.M. was supported by UHN Transplant Competition 2020, University of Toronto’s Institute of Medical Science Open Fellowship 2020–2023, and Canadian Society of Transplantation (CST) Research Training Award 2022. A.R.D. is supported by a CIHR Foundation grant (FDN-15427) and a Tier 1 Canada Research Chair. B.P.K. acknowledges support from the National Institutes of Health (NIH) P01 HL142494., (Copyright © 2023 International Society for the Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Most Pseudomonas aeruginosa strains produce bacteriocins derived from contractile or noncontractile phage tails known as R- and F-type pyocins, respectively. These bacteriocins possess strain-specific bactericidal activity against P. aeruginosa and likely increase evolutionary fitness through intraspecies competition. R-type pyocins have been studied extensively and show promise as alternatives to antibiotics. Although they have similar therapeutic potential, experimental studies on F-type pyocins are limited. Here, we provide a bioinformatic and experimental investigation of F-type pyocins. We introduce a systematic naming scheme for genes found in R- and F-type pyocin operons and identify 15 genes invariably found in strains producing F-type pyocins. Five proteins encoded at the 3' end of the F-type pyocin cluster are divergent in sequence and likely determine bactericidal specificity. We use sequence similarities among these proteins to define eleven distinct F-type pyocin groups, five of which had not been previously described. The five genes encoding the variable proteins associate in two modules that have clearly reassorted independently during the evolution of these operons. These proteins are considerably more diverse than the specificity-determining tail fibers of R-type pyocins, suggesting that F-type pyocins may have emerged earlier. Experimental studies on six F-type pyocin groups show that each displays a distinct spectrum of bactericidal activity. This activity is strongly influenced by the lipopolysaccharide O-antigen type, but other factors also play a role. F-type pyocins appear to kill as efficiently as R-type pyocins. These studies set the stage for the development of F-type pyocins as antibacterial therapeutics. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen that causes antibiotic-resistant infections with high mortality rates, particularly in immunocompromised individuals and cystic fibrosis patients. Due to the increasing frequency of multidrug-resistant P. aeruginosa infections, there is great need for the development of alternative therapeutics. In this study, we investigate one such potential therapeutic: F-type pyocins, which are bacteriocins naturally produced by P. aeruginosa that resemble noncontractile phage tails. We show that they are potent killers of P. aeruginosa and identify their probable bactericidal specificity determinants, which opens up the possibility of engineering them to precisely target strains of pathogenic bacteria. The resemblance of F-type pyocins to well-characterized phage tails will greatly facilitate their development into effective antibacterials., Competing Interests: The authors declare no conflict of interest.

Bacteria in the genus Streptomyces are found ubiquitously in nature and are known for the number and diversity of specialized metabolites they produce, as well as their complex developmental lifecycle. Studies of the viruses that prey on Streptomyces, known as phages, have aided the development of tools for genetic manipulation of these bacteria, as well as contributing to a deeper understanding of Streptomyces and their behaviours in the environment. Here, we present the genomic and biological characterization of twelve Streptomyces phages. Genome analyses reveal that these phages are closely related genetically, while experimental approaches show that they have broad overlapping host ranges, infect early in the Streptomyces lifecycle, and induce secondary metabolite production and sporulation in some Streptomyces species. This work expands the group of characterized Streptomyces phages and improves our understanding of Streptomyces phage-host dynamics., (© 2023. The Author(s).)

Anti-CRISPR proteins inhibit CRISPR-Cas immune systems through diverse mechanisms. Previously, the anti-CRISPR protein AcrIIC5 Smu was shown to potently inhibit a type II-C Cas9 from Neisseria meningitidis (Nme1Cas9). In this work, we explore the mechanism of activity of the AcrIIC5 homologue from Neisseria chenwenguii (AcrIIC5 Nch ) and show that it prevents Cas9 binding to target DNA. We show that AcrIIC5 Nch targets the PAM-interacting domain (PID) of Nme1Cas9 for inhibition, agreeing with previous findings for AcrIIC5 Smu , and newly establish that strong binding of the anti-CRISPR requires guide RNA be pre-loaded on Cas9. We determined the crystal structure of AcrIIC5 Nch using X-ray crystallography and identified amino acid residues that are critical for its function. Using a protein docking algorithm we show that AcrIIC5 Nch likely occupies the Cas9 DNA binding pocket, thereby inhibiting target DNA binding through a mechanism similar to that previously described for AcrIIA2 and AcrIIA4., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [K.L.M. and A.R.D. are co-inventors on patents pertaining to anti-CRISPRs. A.R.D. is on the Scientific Advisory Board of Acrigen Biosciences.], (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Aims: Foodservices are a strong contributor to global environmental impact. Systemic change is required to lead the transformation towards environmentally sustainable foodservices. However, guidance to support foodservices to be more environmentally sustainable is lacking. The aim was to explore food-related environmentally sustainable strategies and their transferability to foodservices in a range of settings, to inform a framework for future application and research., Methods: A constructivist grounded theory study design was used. Semi-structured interviews with foodservice sustainability consultants, who support foodservice organisations to improve environmental sustainability, were conducted. Interviews were recorded, transcribed, and coded line-by-line. Ten consultants were purposively sampled for diversity in location, organisation type, funding model, and services provided. Codes were collapsed into categories, to inform the development of themes and a framework for the implementation of strategies., Results: Four sub-themes were created under an overarching theme of 'Transforming the Foodservice System': embedding leadership, shifting perspective, constructing collaborative networks, and fostering momentum. A range of implementation strategies were captured within the sub-themes., Conclusion: These themes informed the development of a practical application framework for implementing sustainable strategies in foodservices that is useful for practice and future research in the area., (© 2023 The Authors. Nutrition & Dietetics published by John Wiley & Sons Australia, Ltd on behalf of Dietitians Australia.)

Diverse bacterial species produce extracellular contractile injection systems (eCISs). Although closely related to contractile phage tails, eCISs can inject toxic proteins into eukaryotic cells. Thus, these systems are commonly viewed as cytotoxic defense mechanisms that are not central to other aspects of bacterial biology. Here, we provide evidence that eCISs appear to participate in the complex developmental process of the bacterium Streptomyces coelicolor. In particular, we show that S. coelicolor produces eCIS particles during its normal growth cycle, and that strains lacking functional eCIS particles exhibit pronounced alterations in their developmental program. Furthermore, eCIS-deficient mutants display reduced levels of cell death and altered morphology during growth in liquid media. Our results suggest that the main role of eCISs in S. coelicolor is to modulate the developmental switch that leads to aerial hyphae formation and sporulation, rather than to attack other species., (© 2023. The Author(s).)

Recent studies show that antiviral systems are remarkably conserved from bacteria to mammals, demonstrating that unique insights into these systems can be gained by studying microbial organisms. Unlike in bacteria, however, where phage infection can be lethal, no cytotoxic viral consequence is known in the budding yeast Saccharomyces cerevisiae even though it is chronically infected with a double-stranded RNA mycovirus called L-A. This remains the case despite the previous identification of conserved antiviral systems that limit L-A replication. Here, we show that these systems collaborate to prevent rampant L-A replication, which causes lethality in cells grown at high temperature. Exploiting this discovery, we use an overexpression screen to identify antiviral functions for the yeast homologs of polyA-binding protein (PABPC1) and the La-domain containing protein Larp1, which are both involved in viral innate immunity in humans. Using a complementary loss of function approach, we identify new antiviral functions for the conserved RNA exonucleases REX2 and MYG1 ; the SAGA and PAF1 chromatin regulatory complexes; and HSF1 , the master transcriptional regulator of the proteostatic stress response. Through investigation of these antiviral systems, we show that L-A pathogenesis is associated with an activated proteostatic stress response and the accumulation of cytotoxic protein aggregates. These findings identify proteotoxic stress as an underlying cause of L-A pathogenesis and further advance yeast as a powerful model system for the discovery and characterization of conserved antiviral systems.

Interprofessional collaborative practice has been shown to be an appropriate model of care for chronic disease management in primary care. However, how patients play a role in this model is relatively unknown. The aim of this constructivist grounded theory focus group study was to explore the perceptions of patient advocates regarding the role of patients in interprofessional collaborative practice for chronic conditions in primary care. Primary data were collected from patient advocates, from public and private Australian organisations and who represent patients with chronic disease in primary care, through focus groups in July-August 2020. Videoconference focus groups were recorded, transcribed verbatim and inductively, thematically analysed using the five-step approach by Charmaz: (1) initial line-by-line coding, (2) focused coding, (3) memo writing, (4) categorisation and (5) theme and sub-theme development. Three focus groups comprising 17 patient advocates with diverse cultural and professional backgrounds participated. Two themes and five sub-themes relating to interprofessional collaborative practice teams were constructed from the data. In theme 1, patients 'shifted across the spectrum of roles' from 'relinquishing control to the team', 'joining the team' to 'disengaging from the team'. The second theme was the need for 'juggling roles' by 'integrating patient role with life roles', and 'learning about the patient role'. The diversity and variability of patient roles as described by patient advocates highlight the challenges of working with people with chronic conditions. The diverse patient roles described by advocates are an important finding that may better inform communication between patients and health professionals when managing chronic conditions. From the health professional perspective, identification of the role of a patient may be challenging. Therefore, future research should explore the development of a tool to assist both patients and health professionals to identify patient roles as they move across the spectrum, with the support of policy makers. This tool should aim to identify and promote patient engagement in interprofessional collaborative practice in primary care settings., (© 2022 The Authors. Health and Social Care in the Community published by John Wiley & Sons Ltd.)

Aim: The aim of this scoping review was to provide an overview of the development of practice nurses, and the learnings that could be applied to improve the profile of dietetics practice in primary care., Methods: A scoping review synthesising peer-reviewed and other literature relevant to the development of Australian practice nurses was conducted. Structured searches using keywords 'general practice', 'nurse' and 'Australia' were conducted in PubMed and Google Scholar in June 2021. Key government websites, Department of Health and Services Australia, were searched to identify grey literature. One reviewer screened the titles and abstracts against inclusion criteria; two reviewers conducted full-text screening independently. Data on the evolution of practice nursing were extracted based on its interest and transferability to the dietetics workforce., Results: A total of 102 results (82 peer-reviewed and 20 other literature) were included in the review. Key drivers for practice nurse role development in Australia were government funding, practice nurse practice standards, cost-benefit analyses of practice nurses, career and education opportunities, general practitioner and patient perspectives of practice nurses and, individual, community and local needs., Conclusions: The practice nurse role has grown and strengthened and there are three key learnings that could be translated to strengthen the dietetics workforce in primary care. (1) Use and expansion of government funding, (2) furthering post-tertiary education and career opportunities, including dietetic primary care practice standards and (3) targeting underserviced areas such as those that are rural and remote and building positive relationships with other stakeholders including practice nurses, general practitioners, patients and the broader primary care team., (© 2022 The Authors. Nutrition & Dietetics published by John Wiley & Sons Australia, Ltd on behalf of Dietitians Australia.)

Tailed bacteriophages are abundant and extremely diverse. Understanding this diversity is a challenge, and here we examine a small slice of that diversity in some detail. We contrast and compare the small genome, virulent, non-contractile tailed phages that infect the bacterial order Enterobacteriales. These phages, with genomes in the 35-60 kbp range, have very similar virions that are often difficult to distinguish by negative stain electron microscopy. There are currently 651 genome sequences of such phages in the public database. We show that these can be robustly parsed into fifteen well-defined clusters that have very different nucleotide sequences. We examine the similarities and differences among these clusters, as well as genetic exchange among clusters and the relationships between host species and phage clusters., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Emerging evidence supports the health benefits of ginger for a range of conditions and symptoms; however, there is a lack of synthesis of literature to determine which health indications are supported by quality evidence., Objectives: In this umbrella review of systematic reviews we aimed to determine the therapeutic effects and safety of any type of ginger from the Zingiber family administered in oral form compared with any comparator or baseline measures on any health and well-being outcome in humans., Methods: Five databases were searched from inception to April 2021. Review selection and quality were assessed in duplicate using the Assessment of Multiple Systematic Reviews-2 (AMSTAR-2) checklist and the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) method, with results presented in narrative form., Results: Twenty-four systematic reviews were included with 3% overlap of primary studies. The strongest evidence was found for the antiemetic effects of ginger in pregnant women (effect size: large; GRADE: high), analgesic effects for osteoarthritis (effect size: small; GRADE: high), and glycemic control (effect size: none to very large; GRADE: very low to moderate). Ginger also had a statistically significant positive effect on blood pressure, weight management, dysmenorrhea, postoperative nausea, and chemotherapy-induced vomiting (effect size: moderate to large; GRADE: low to moderate) as well as blood lipid profile (effect size: small; GRADE: very low) and anti-inflammatory and antioxidant biomarkers (effect size: unclear; GRADE: very low to moderate). There was substantial heterogeneity and poor reporting of interventions; however, dosage of 0.5-3 g/d in capsule form administered for up to 3 mo was consistently reported as effective., Conclusions: Dietary consumption of ginger appears safe and may exert beneficial effects on human health and well-being, with greatest confidence in antiemetic effects in pregnant women, analgesic effects in osteoarthritis, and glycemic control. Future randomized controlled and dose-dependent trials with adequate sample sizes and standardized ginger products are warranted to better inform and standardize routine clinical prescription., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Objectives: This study examined older adults' experiences of participating in the Ballistic Exercise of the Lower Limb (BELL) trial, involving 12-weeks of group-based hardstyle kettlebell training., Methods: In the BELL trial, 28 insufficiently active older adults (15 women, 13 men, 59-79 years) completed six weeks of face-to-face group training, and six weeks of home-based training. In-depth semi-structured interviews were audio recorded, transcribed, and inductively coded, with themes constructed from patterns of shared meaning., Results: Four higher-order themes were developed that reflect older adults' experiences participating in a group-exercise program of hardstyle kettlebell training. These included: (1) "It's one of the best things we've done"-enjoying the physical and psychosocial benefits, (2) "It's improved it tremendously!"-change in a long-term health condition, (3) "It put me on a better course"-overcoming challenges, and (4) "I wasn't just a number"-feeling part of a group/community., Discussion: Findings highlight the perceived physical and psychological benefits of older adults participating in hardstyle group kettlebell training, and the value attributed to being part of an age-matched community of like-minded people engaged in group exercise. Implications for program design and delivery, and future research, are discussed., (© 2022. The Author(s).)

Phages, plasmids, and other mobile genetic elements express inhibitors of CRISPR-Cas immune systems, known as anti-CRISPR proteins, to protect themselves from targeted destruction. These anti-CRISPR proteins have been shown to function through very diverse mechanisms. In this work we investigate the activity of an anti-CRISPR isolated from a prophage in Haemophilus parainfluenzae that blocks CRISPR-Cas9 DNA cleavage activity. We determine the three-dimensional crystal structure of AcrIIC4 Hpa and show that it binds to the Cas9 Recognition Domain. This binding does not prevent the Cas9-anti-CRISPR complex from interacting with target DNA but does inhibit DNA cleavage. AcrIIC4 Hpa likely acts by blocking the conformational changes that allow the HNH and RuvC endonuclease domains to contact the DNA sites to be nicked., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Improving the patient experience is one of the quadruple aims of healthcare. Therefore, understanding patient experiences and perceptions of healthcare interactions is paramount to quality improvement. This integrative review aimed to explore how patients with chronic conditions experience Interprofessional Collaborative Practice in primary care., Methods: An integrative review was conducted to comprehensively synthesize primary studies that used qualitative, quantitative, and mixed methods. Databases searched were Medline, Embase, CINAHL and Web of Science on June 1st, 2021. Eligible studies were empirical full-text studies in primary care that reported experiences or perceptions of Interprofessional Collaborative Practice by adult patients with a chronic condition, in any language published in any year. Quality appraisal was conducted on included studies using the Mixed Method Appraisal Tool. Data on patients' experiences and perceptions of Interprofessional Collaborative Practice in primary care were extracted, and findings were thematically analyzed through a meta-synthesis., Results: Forty-eight (n = 48) studies met the inclusion criteria with a total of n = 3803 participants. Study quality of individual studies was limited by study design, incomplete reporting, and the potential for positive publication bias. Three themes and their sub-themes were developed inductively: (1) Interacting with Healthcare Teams, subthemes: widening the network, connecting with professionals, looking beyond the condition, and overcoming chronic condition collectively; (2) Valuing Convenient Healthcare, subthemes: sharing space and time, care planning creates structure, coordinating care, valuing the general practitioner role, and affording healthcare; (3) Engaging Self-care, subthemes: engaging passively is circumstantial, and, engaging actively and leading care., Conclusions: Patients overwhelmingly had positive experiences of Interprofessional Collaborative Practice, signaling it is appropriate for chronic condition management in primary care. The patient role in managing their chronic condition was closely linked to their experience. Future studies should investigate how the patient role impacts the experience of patients, carers, and health professionals in this context., Systematic Review Registration: PROSPERO: CRD42020156536., (© 2022. The Author(s).)

Tape measure (TM) proteins are essential for the formation of long-tailed phages. TM protein assembly into tails requires the action of tail assembly chaperones (TACs). TACs (e.g. gpG and gpT of E. coli phage lambda) are usually produced in a short (TAC-N) and long form (TAC-NC) with the latter comprised of TAC-N with an additional C-terminal domain (TAC-C). TAC-NC is generally synthesized through a ribosomal frameshifting mechanism. TAC encoding genes have never been identified in the intensively studied Escherichia coli phage T4, or any related phages. Here, we have bioinformatically identified putative TAC encoding genes in diverse T4-like phage genomes. The frameshifting mechanism for producing TAC-NC appears to be conserved in several T4-like phage groups. However, the group including phage T4 itself likely employs a different strategy whereby TAC-N and TAC-NC are encoded by separate genes (26 and 51 in phage T4)., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Phages and other mobile genetic elements express anti-CRISPR proteins (Acrs) to protect their genomes from destruction by CRISPR-Cas systems. Acrs usually block the ability of CRISPR-Cas systems to bind or cleave their nucleic acid substrates. Here, we investigate an unusual Acr, AcrIF9, that induces a gain-of-function to a type I-F CRISPR-Cas (Csy) complex, causing it to bind strongly to DNA that lacks both a PAM sequence and sequence complementarity. We show that specific and non-specific dsDNA compete for the same site on the Csy:AcrIF9 complex with rapid exchange, but specific ssDNA appears to still bind through complementarity to the CRISPR RNA. Induction of non-specific DNA-binding is a shared property of diverse AcrIF9 homologues. Substitution of a conserved positively charged surface on AcrIF9 abrogated non-specific dsDNA-binding of the Csy:AcrIF9 complex, but specific dsDNA binding was maintained. AcrIF9 mutants with impaired non-specific dsDNA binding activity in vitro displayed a reduced ability to inhibit CRISPR-Cas activity in vivo. We conclude that misdirecting the CRISPR-Cas complex to bind non-specific DNA is a key component of the inhibitory mechanism of AcrIF9. This inhibitory mechanism is distinct from a previously characterized anti-CRISPR, AcrIF1, that sterically blocks DNA-binding, even though AcrIF1and AcrIF9 bind to the same site on the Csy complex., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Stress is a potent environmental factor that can confer potent and enduring effects on brain structure and function. Exposure to stress during early life (ELS) has been linked to a wide range of consequences later in life. In particular, ELS exerts lasting effects on neurogenesis in the adult hippocampus, suggesting that ELS is a significant regulator of adult neural stem cell numbers and function. Here, we investigated the effect of ELS on cell proliferation and the numbers of neural stem/precursor cells in another neurogenic region: the hypothalamus of adult mice. We show that ELS has long-term suppressive effects on cell proliferation in the hypothalamic parenchyma and reduces the numbers of putative hypothalamic neural stem/precursor cells at 4 months of age. Specifically, ELS reduced the number of PCNA + cells present in hypothalamic areas surrounding the 3rd ventricle with a specific reduction in the proliferation of Sox2+/Nestin-GFP + putative stem cells present in the median eminence at the base of the 3rd ventricle. Furthermore, ELS reduced the total numbers of β-tanycytes lining the ventral 3rd ventricle, without affecting α-tanycyte numbers in more dorsal areas. These results are the first to indicate that ELS significantly reduces proliferation and β-tanycyte numbers in the adult hypothalamus, and may have (patho)physiological consequences for metabolic regulation or other hypothalamic functions in which β-tanycytes are involved.

Anti-CRISPRs are protein inhibitors of CRISPR-Cas systems. They are produced by phages and other mobile genetic elements to evade CRISPR-Cas-mediated destruction. Anti-CRISPRs are remarkably diverse in sequence, structure, and functional mechanism; thus, structural and mechanistic investigations of anti-CRISPRs continue to yield exciting new insights. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of AcrIE2, an anti-CRISPR that inhibits the type I-E CRISPR-Cas system of Pseudomonas aeruginosa. Guided by the structure, we used site-directed mutagenesis to identify key residues that are required for AcrIE2 function. Using affinity purification experiments, we found that AcrIE2 binds the type I-E CRISPR-Cas complex (Cascade). In vivo transcriptional assays, in which Cascade was targeted to promoter regions, demonstrated that Cascade still binds to DNA in the presence of AcrIE2. This is the first instance of a type I anti-CRISPR that binds to a CRISPR-Cas complex but does not prevent DNA-binding. Another unusual property of AcrIE2 is that the effect of Cascade:AcrIE2 complex binding to promoter regions varied depending on the position of the binding site. Most surprisingly, Cascade:AcrIE2 binding led to transcriptional activation in some cases rather than repression, which did not occur when Cascade alone bound to the same sites. We conclude that AcrIE2 operates through a distinct mechanism compared to other type I anti-CRISPRs. While AcrIE2 does not prevent Cascade from binding DNA, it likely blocks subsequent recruitment of the Cas3 nuclease to Cascade thereby preventing DNA cleavage., Competing Interests: Conflict of Interest Statement A.R.D., K.L.M., and A.P. are co-inventors on patents pertaining to anti-CRISPRs. A.R.D. is on the Scientific Advisory Board of Acrigen Biosciences., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The arms race between bacteria and phages has led to the evolution of diverse anti-phage defenses, several of which are controlled by quorum-sensing pathways. In this work, we characterize a quorum-sensing anti-activator protein, Aqs1, found in Pseudomonas phage DMS3. We show that Aqs1 inhibits LasR, the master regulator of quorum sensing, and present the crystal structure of the Aqs1-LasR complex. The 69-residue Aqs1 protein also inhibits PilB, the type IV pilus assembly ATPase protein, which blocks superinfection by phages that require the pilus for infection. This study highlights the remarkable ability of small phage proteins to bind multiple host proteins and disrupt key biological pathways. As quorum sensing influences various anti-phage defenses, Aqs1 provides a mechanism by which infecting phages might simultaneously dampen multiple defenses. Because quorum-sensing systems are broadly distributed across bacteria, this mechanism of phage counter-defense may play an important role in phage-host evolutionary dynamics., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

To initiate their life cycle, phages must specifically bind to the surface of their bacterial hosts. Long-tailed phages often interact with the cell surface using fibers, which are elongated intertwined trimeric structures. The folding and assembly of these complex structures generally requires the activity of an intra- or intermolecular chaperone protein. Tail fiber assembly (Tfa) proteins are a very large family of proteins that serve as chaperones for fiber folding in a wide variety of phages that infect diverse species. A recent structural study showed that the Tfa protein from Escherichia coli phage Mu (Tfa Mu ) mediates fiber folding and stays bound to the distal tip of the fiber, becoming a component of the mature phage particle. This finding revealed the potential for Tfa Mu to also play a role in cell surface binding. To address this issue, we have here shown that Tfa Mu binds to lipopolysaccharide (LPS), the cell surface receptor of phage Mu, with a similar strength as to the fiber itself. Furthermore, we have found that Tfa Mu and the Tfa protein from E. coli phage P2 bind LPS with distinct specificities that mirror the host specificity of these two phages. By comparing the sequences of these two proteins, which are 93% identical, we identified a single residue that is responsible for their distinct LPS-binding behaviors. Although we have not yet found conditions under which Tfa proteins influence host range, the potential for such a role is now evident, as we have demonstrated their ability to bind LPS in a strain-specific manner. IMPORTANCE With the growing interest in using phages to combat antibiotic-resistant infections or manipulate the human microbiome, establishing approaches for the modification of phage host range has become an important research topic. Tfa proteins are a large family of proteins known previously to function as chaperones for the folding of phage fibers, which are crucial determinants of host range for long-tailed phages. Here, we reveal that some Tfa proteins are bi-functional, with the additional activity of binding to LPS, the surface binding receptor for many phages. This discovery opens up new potential avenues for altering phage host range through engineering of the surface binding specificity of Tfa proteins., (Copyright © 2021 American Society for Microbiology.)

Whilst it is universally accepted that the energy support of the brain is glucose, the form in which the glucose is taken up by neurones is the topic of intense debate. In the last few decades, the concept of lactate shuttling between glial elements and neural elements has emerged in which the glial cells glycolytically metabolise glucose/glycogen to lactate, which is shuttled to the neural elements via the extracellular fluid. The process occurs during periods of compromised glucose availability where glycogen stored in astrocytes provides lactate to the neurones, and is an integral part of the formation of learning and memory where the energy intensive process of learning requires neuronal lactate uptake provided by astrocytes. More recently sleep, myelination and motor end plate integrity have been shown to involve lactate shuttling. The sequential aspect of lactate production in the astrocyte followed by transport to the neurones is vulnerable to interruption and it is reported that such disparate pathological conditions as Alzheimer's disease, amyotrophic lateral sclerosis, depression and schizophrenia show disrupted lactate signalling between glial cells and neurones.

Bacterial CRISPR-Cas systems employ RNA-guided nucleases to destroy phage (viral) DNA. Phages, in turn, have evolved diverse "anti-CRISPR" proteins (Acrs) to counteract acquired immunity. In Listeria monocytogenes, prophages encode two to three distinct anti-Cas9 proteins, with acrIIA1 always present. However, the significance of AcrIIA1's pervasiveness and its mechanism are unknown. Here, we report that AcrIIA1 binds with high affinity to Cas9 via the catalytic HNH domain. During lysogeny in Listeria, AcrIIA1 triggers Cas9 degradation. During lytic infection, however, AcrIIA1 fails to block Cas9 due to its multi-step inactivation mechanism. Thus, phages encode an additional Acr that rapidly binds and inactivates Cas9. AcrIIA1 also uniquely inhibits a highly diverged Cas9 found in Listeria (similar to SauCas9) and Type II-C Cas9s, likely due to Cas9 HNH domain conservation. In summary, Listeria phages inactivate Cas9 in lytic growth using variable, narrow-spectrum inhibitors, while the broad-spectrum AcrIIA1 stimulates Cas9 degradation for protection of the lysogenic genome., Competing Interests: Declaration of Interests J.B.-D. is a scientific advisory board member of SNIPR Biome and Excision Biotherapeutics, a scientific advisory board member and co-founder of Acrigen Biosciences, and an inventor on patents relating to anti-CRISPR proteins. B.P.K. is an inventor on various patents and patent applications that describe gene editing and epigenetic editing technologies, a consultant for Avectas Inc., and an advisor to Acrigen Biosciences. A.R.D. is a scientific advisory board member for Acrigen Biosciences and an inventor on patents relating to anti-CRISPR proteins., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Clustered regularly interspaced short palindromic repeats (CRISPR) together with their accompanying cas (CRISPR-associated) genes are found frequently in bacteria and archaea, serving to defend against invading foreign DNA, such as viral genomes. CRISPR-Cas systems provide a uniquely powerful defense because they can adapt to newly encountered genomes. The adaptive ability of these systems has been exploited, leading to their development as highly effective tools for genome editing. The widespread use of CRISPR-Cas systems has driven a need for methods to control their activity. This review focuses on anti-CRISPRs (Acrs), proteins produced by viruses and other mobile genetic elements that can potently inhibit CRISPR-Cas systems. Discovered in 2013, there are now 54 distinct families of these proteins described, and the functional mechanisms of more than a dozen have been characterized in molecular detail. The investigation of Acrs is leading to a variety of practical applications and is providing exciting new insight into the biology of CRISPR-Cas systems.

Bacteria have evolved sophisticated adaptive immune systems, called CRISPR-Cas, that provide sequence-specific protection against phage infection. In turn, phages have evolved a broad spectrum of anti-CRISPRs that suppress these immune systems. Here we report structures of anti-CRISPR protein IF9 (AcrIF9) in complex with the type I-F CRISPR RNA-guided surveillance complex (Csy). In addition to sterically blocking the hybridization of complementary dsDNA to the CRISPR RNA, our results show that AcrIF9 binding also promotes non-sequence-specific engagement with dsDNA, potentially sequestering the complex from target DNA. These findings highlight the versatility of anti-CRISPR mechanisms utilized by phages to suppress CRISPR-mediated immune systems.

CRISPR-Cas9 systems provide powerful tools for genome editing. However, optimal employment of this technology will require control of Cas9 activity so that the timing, tissue specificity, and accuracy of editing may be precisely modulated. Anti-CRISPR proteins, which are small, naturally occurring inhibitors of CRISPR-Cas systems, are well suited for this purpose. A number of anti-CRISPR proteins have been shown to potently inhibit subgroups of CRISPR-Cas9 systems, but their maximal inhibitory activity is generally restricted to specific Cas9 homologs. Since Cas9 homologs vary in important properties, differing Cas9s may be optimal for particular genome-editing applications. To facilitate the practical exploitation of multiple Cas9 homologs, here we identify one anti-CRISPR, called AcrIIA5, that potently inhibits nine diverse type II-A and type II-C Cas9 homologs, including those currently used for genome editing. We show that the activity of AcrIIA5 results in partial in vivo cleavage of a single-guide RNA (sgRNA), suggesting that its mechanism involves RNA interaction., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Phage tail fibres are elongated protein assemblies capable of specific recognition of bacterial surfaces during the first step of viral infection 1-4 . The folding of these complex trimeric structures often requires a phage-encoded tail fibre assembly (Tfa) protein 5-7 . Despite the wide occurrence of Tfa proteins, their functional mechanism has not been elucidated. Here, we investigate the tail fibre and Tfa of Escherichia coli phage Mu. We demonstrate that Tfa forms a stable complex with the tail fibre, and present a 2.1 Å resolution X-ray crystal structure of this complex. We find that Tfa proteins are comprised of two domains: a non-conserved N-terminal domain that binds to the C-terminal region of the fibre and a conserved C-terminal domain that probably mediates fibre oligomerization and assembly. Tfa forms rapidly exchanging multimers on its own, but not a stable trimer, implying that Tfa does not specify the trimeric state of the fibre. We propose that the key conserved role of Tfa is to ensure that fibre assembly and multimerization initiates at the C terminus, ensuring that the intertwined and repetitive structural elements of fibres come together in the correct sequence. The universal importance of correctly aligning the C termini of phage fibres is highlighted by our work.

Phages express anti-CRISPR (Acr) proteins to inhibit CRISPR-Cas systems that would otherwise destroy their genomes. Most acr genes are located adjacent to anti-CRISPR-associated (aca) genes, which encode proteins with a helix-turn-helix DNA-binding motif. The conservation of aca genes has served as a signpost for the identification of acr genes, but the function of the proteins encoded by these genes has not been investigated. Here we reveal that an acr-associated promoter drives high levels of acr transcription immediately after phage DNA injection and that Aca proteins subsequently repress this transcription. Without Aca activity, this strong transcription is lethal to a phage. Our results demonstrate how sufficient levels of Acr proteins accumulate early in the infection process to inhibit existing CRISPR-Cas complexes in the host cell. They also imply that the conserved role of Aca proteins is to mitigate the deleterious effects of strong constitutive transcription from acr promoters., (Copyright © 2019 Elsevier Inc. All rights reserved.)

CRISPR-Cas adaptive immune systems function to protect bacteria from invasion by foreign genetic elements. The CRISPR-Cas9 system has been widely adopted as a powerful genome-editing tool, and phage-encoded inhibitors, known as anti-CRISPRs, offer a means of regulating its activity. Here, we report the crystal structures of anti-CRISPR protein AcrIIC2 Nme alone and in complex with Nme1Cas9. We demonstrate that AcrIIC2 Nme inhibits Cas9 through interactions with the positively charged bridge helix, thereby preventing sgRNA loading. In vivo phage plaque assays and in vitro DNA cleavage assays show that AcrIIC2 Nme mediates its activity through a large electronegative surface. This work shows that anti-CRISPR activity can be mediated through the inhibition of Cas9 complex assembly.

Background: Hospitalisation for an eating disorder is rare, however treatment in the acute medical setting can be a life-saving admission. While the multidisciplinary team delivers overall patient care, medical decisions are the responsibility of the treating physicians. Treatment decisions directly impact on patient care and outcomes. This study aimed to explore the considerations that influence the medical decisions of physicians when treating patients with eating disorders in the acute setting., Method: Semi-structured interviews were conducted with ten medical physicians who had previously treated eating disorders on a general medical unit in two Australian tertiary hospitals. An interview schedule, based on the literature and four relevant domains from the Consolidated Framework for Implementation Research, was developed. Interviews were audio recorded, transcribed verbatim and analyzed thematically. Coding and interim themes and sub-themes were developed by two dietitian researchers; these were further refined through researcher discussion and triangulation with two additional dietitian researchers., Results: Ten doctors were interviewed (3 consultants (1 adult general medical and 2 paediatricians: 13-16 years medical experience), 2 registrars (4-7 years experience), 1 resident (1 year experience), and 4 interns (< 1 year experience). Doctors described memorable patient cases, related to hospital stays over several weeks. Interviews ranged in length from 58 min to 91 min. Four themes ( with five sub-themes ) were developed: 1) navigating uncertainty ( focusing on processes and goals and seeking information ), 2) being "the good doctor" ( doing the right thing), 3) seeing the big picture ( depending on key players and considering short and long-term ), and 4) involving family and patient., Conclusions: Non-specialist physicians described challenges in the treatment of eating disorders in the inpatient setting. They take a holistic approach that considers both short and longer-term goals, relying on specialist colleagues, the wider multidisciplinary team and sometimes family members to guide treatment decisions during admissions on general medical wards. Additional support, education and training centered on the key themes may increase physicians' confidence and ability to make effective treatment decisions for this patient group. The results are relevant to all health professionals working in this field to better understand the priorities of medical physicians and to support them to achieve positive outcomes in the inpatient treatment of patients with eating disorders., Competing Interests: Three of the authors, SB, LD and DPR currently work or have previously worked in acute hospital settings with eating disorder patients. DPR is an Accredited Practising Dietitian (Australia) and a Registered Dietitian (UK), SB and LD are Accredited Practising Dietitians (Australia), and ARD was a student dietitian in a Master of Nutrition and Dietetic Practice program during the study.Approval was gained from (Bond University Human) Research Ethics Committee, Ref: 015660. All participants were provided with a Participant Information Sheet and Consent Form and signed prior to commencement of interviews.Not applicable.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

In their natural settings, CRISPR-Cas systems play crucial roles in bacterial and archaeal adaptive immunity to protect against phages and other mobile genetic elements, and they are also widely used as genome engineering technologies. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017). We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature (K. L. Maxwell, Mol Cell 68:8-14, 2017, https://doi.org/10.1016/j.molcel.2017.09.002; A. Pawluk, A. R. Davidson, and K. L. Maxwell, Nat Rev Microbiol 16:12-17, 2018, https://doi.org/10.1038/nrmicro.2017.120; E. J. Sontheimer and A. R. Davidson, Curr Opin Microbiol 37:120-127, 2017, https://doi.org/10.1016/j.mib.2017.06.003). We have identified new anti-CRISPRs using the same bioinformatic approach that successfully identified previous Acr proteins (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017) against Neisseria meningitidis Cas9 (NmeCas9). In this work, we report two novel anti-CRISPR families in strains of Haemophilus parainfluenzae and Simonsiella muelleri , both of which harbor type II-C CRISPR-Cas systems (A. Mir, A. Edraki, J. Lee, and E. J. Sontheimer, ACS Chem Biol 13:357-365, 2018, https://doi.org/10.1021/acschembio.7b00855). We characterize the type II-C Cas9 orthologs from H. parainfluenzae and S. muelleri , show that the newly identified Acrs are able to inhibit these systems, and define important features of their inhibitory mechanisms. The S. muelleri Acr is the most potent NmeCas9 inhibitor identified to date. Although inhibition of NmeCas9 by anti-CRISPRs from H. parainfluenzae and S. muelleri reveals cross-species inhibitory activity, more distantly related type II-C Cas9s are not inhibited by these proteins. The specificities of anti-CRISPRs and divergent Cas9s appear to reflect coevolution of their strategies to combat or evade each other. Finally, we validate these new anti-CRISPR proteins as potent off-switches for Cas9 genome engineering applications. IMPORTANCE As one of their countermeasures against CRISPR-Cas immunity, bacteriophages have evolved natural inhibitors known as anti-CRISPR (Acr) proteins. Despite the existence of such examples for type II CRISPR-Cas systems, we currently know relatively little about the breadth of Cas9 inhibitors, and most of their direct Cas9 targets are uncharacterized. In this work we identify two new type II-C anti-CRISPRs and their cognate Cas9 orthologs, validate their functionality in vitro and in bacteria, define their inhibitory spectrum against a panel of Cas9 orthologs, demonstrate that they act before Cas9 DNA binding, and document their utility as off-switches for Cas9-based tools in mammalian applications. The discovery of diverse anti-CRISPRs, the mechanistic analysis of their cognate Cas9s, and the definition of Acr inhibitory mechanisms afford deeper insight into the interplay between Cas9 orthologs and their inhibitors and provide greater scope for exploiting Acrs for CRISPR-based genome engineering., (Copyright © 2018 Lee et al.)

The viruses that infect bacteria, known as phages, play a critical role in controlling bacterial populations in many diverse environments, including the human body. This control stems not only from phages killing bacteria but also from the formation of lysogens. In this state, the phage replication cycle is suppressed, and the phage genome is maintained in the bacterial cell in a form known as a prophage. Prophages often carry genes that benefit the host bacterial cell, since increasing the survival of the host cell by extension also increases the fitness of the prophage. These highly diverse and beneficial phage genes, which are not required for the life cycle of the phage itself, have been referred to as "morons," as their presence adds "more on" the phage genome in which they are found. While individual phage morons have been shown to contribute to bacterial virulence by a number of different mechanisms, there have been no systematic investigations of their activities. Using a library of phages that infect two different clinical isolates of P. aeruginosa , PAO1 and PA14, we compared the phenotypes imparted by the expression of individual phage morons. We identified morons that inhibit twitching and swimming motilities and observed an inhibition of the production of virulence factors such as rhamnolipids and elastase. This study demonstrates the scope of phage-mediated phenotypic changes and provides a framework for future studies of phage morons. IMPORTANCE Environmental and clinical isolates of the bacterium Pseudomonas aeruginosa frequently contain viruses known as prophages. These prophages can alter the virulence of their bacterial hosts through the expression of nonessential genes known as "morons." In this study, we identified morons in a group of Pseudomonas aeruginosa phages and characterized the effects of their expression on bacterial behaviors. We found that many morons confer selective advantages for the bacterial host, some of which correlate with increased bacterial virulence. This work highlights the symbiotic relationship between bacteria and prophages and illustrates how phage morons can help bacteria adapt to different selective pressures and contribute to human diseases., (Copyright © 2018 American Society for Microbiology.)

Since phages present a major challenge to survival in most environments, bacteria express a battery of anti-phage defences including CRISPR-Cas, restriction-modification and abortive infection systems 1-4 . Such strategies are effective, but the phage genome-which encodes potentially inhibitory gene products-is still allowed to enter the cell. The safest way to preclude phage infection is to block initial phage adsorption to the cell. Here, we describe a cell-surface modification that blocks infection by certain phages. Strains of the opportunistic pathogen Pseudomonas aeruginosa express one of five different type IV pilins (T4P) 5 , two of which are glycosylated with O-antigen units 6 or polymers of D-arabinofuranose 7-9 . We propose that predation by bacteriophages that use T4P as receptors selects for strains that mask potential phage binding sites using glycosylation. Here, we show that both modifications protect P. aeruginosa from certain pilus-specific phages. Alterations to pilin sequence can also block phage infection, but glycosylation is considered less likely to create disadvantageous phenotypes. Through construction of chimeric phages, we show that specific phage tail proteins allow for infection of strains with glycosylated pili. These studies provide insight into first-line bacterial defences against predation and ways in which phages circumvent them, and provide a rationale for the prevalence of pilus glycosylation in nature.