National-scale antimicrobial resistance surveillance in wastewater: A comparative analysis of HT qPCR and metagenomic approaches.

Wastewater serves as an important reservoir of antimicrobial resistance (AMR), and its surveillance can provide insights into population-level trends in AMR to inform public health policy. This study compared two common high-throughput screening approaches, namely (i) high-throughput quantitative PCR (HT qPCR), targeting 73 antimicrobial resistance genes, and (ii) metagenomic sequencing. Weekly composite samples of wastewater influent were taken from 47 wastewater treatment plants (WWTPs) across Wales, as part of a national AMR surveillance programme, alongside 4 weeks of daily wastewater effluent samples from a large municipal hospital. Metagenomic analysis provided more comprehensive resistome coverage, detecting 545 genes compared to the targeted 73 genes by HT qPCR. It further provided contextual information critical to risk assessment (i.e. potential bacterial hosts). In contrast, HT qPCR exhibited higher sensitivity, quantifying all targeted genes including those of clinical relevance present at low abundance. When limited to the HT qPCR target genes, both methods were able to reflect the spatiotemporal dynamics of the complete metagenomic resistome, distinguishing that of the hospital and the WWTPs. Both approaches revealed correlations between resistome compositional shifts and environmental variables like ammonium wastewater concentration, though differed in their interpretation of some potential influencing factors. Overall, metagenomics provides more comprehensive resistome profiling, while qPCR permits sensitive quantification of genes significant to clinical resistance. We highlight the importance of selecting appropriate methodologies aligned to surveillance aims to guide the development of effective wastewater-based AMR monitoring programmes.

Molecular and Clinical Epidemiology of SARS-CoV-2 Infection among Vaccinated and Unvaccinated Individuals in a Large Healthcare Organization from New Jersey.

New Jersey was among the first states impacted by the COVID-19 pandemic, with one of the highest overall death rates in the nation. Nevertheless, relatively few reports have been published focusing specifically on New Jersey. Here we report on molecular, clinical, and epidemiologic observations, from the largest healthcare network in the state, in a cohort of vaccinated and unvaccinated individuals with laboratory-confirmed SARS-CoV-2 infection. We conducted molecular surveillance of SARS-CoV-2-positive nasopharyngeal swabs collected in nine hospitals from December 2020 through June 2022, using both whole genome sequencing (WGS) and a real-time RT-PCR screening assay targeting spike protein mutations found in variants of concern (VOCs) within our region. De-identified clinical data were obtained retrospectively, including demographics, COVID-19 vaccination status, ICU admission, ventilator support, mortality, and medical history. Statistical analyses were performed to identify associations between SARS-CoV-2 variants, vaccination status, clinical outcomes, and medical risk factors. A total of 5007 SARS-CoV-2-positive nasopharyngeal swabs were successfully screened and/or sequenced. Variant screening identified three predominant VOCs, including Alpha (n = 714), Delta (n = 1877), and Omicron (n = 1802). Omicron isolates were further sub-typed as BA.1 (n = 899), BA.2 (n = 853), or BA.4/BA.5 (n = 50); the remaining 614 isolates were classified as "Other". Approximately 31.5% (1577/5007) of the samples were associated with vaccine breakthrough infections, which increased in frequency following the emergence of Delta and Omicron. Severe clinical outcomes included ICU admission (336/5007 = 6.7%), ventilator support (236/5007 = 4.7%), and mortality (430/5007 = 8.6%), with increasing age being the most significant contributor to each (p < 0.001). Unvaccinated individuals accounted for 79.7% (268/336) of ICU admissions, 78.3% (185/236) of ventilator cases, and 74.4% (320/430) of deaths. Highly significant (p < 0.001) increases in mortality were observed in individuals with cardiovascular disease, hypertension, cancer, diabetes, and hyperlipidemia, but not with obesity, thyroid disease, or respiratory disease. Significant differences (p < 0.001) in clinical outcomes were also noted between SARS-CoV-2 variants, including Delta, Omicron BA.1, and Omicron BA.2. Vaccination was associated with significantly improved clinical outcomes in our study, despite an increase in breakthrough infections associated with waning immunity, greater antigenic variability, or both. Underlying comorbidities contributed significantly to mortality in both vaccinated and unvaccinated individuals, with increasing risk based on the total number of comorbidities. Real-time RT-PCR-based screening facilitated timely identification of predominant variants using a minimal number of spike protein mutations, with faster turnaround time and reduced cost compared to WGS. Continued evolution of SARS-CoV-2 variants will likely require ongoing surveillance for new VOCs, with real-time assessment of clinical impact.

Exploring Academic Performance of Medical Students in an Integrated Hybrid Curriculum by Gender.

Gender gaps in academic performance have been reported at a variety of educational levels including several national standardized exams for medical education, with men scoring higher than women. These gaps potentially impact medical school acceptance and residency matching and may be influenced by curricular design. Performance data for our 4-year integrated hybrid curriculum, which features a large proportion of active learning, revealed a gender gap with men performing better early in the curriculum and on the first national standardized exam. This gap in performance almost entirely disappeared for years 2-4 of the curriculum and the second national standardized exam.

Peer-Assisted Learning in Undergraduate Medical Education for Resilience and Well-being.

Peers are a powerful resource in the learning process. Peer-assisted learning (PAL) is a high value activity for the peer and the coach. We report here a PAL activity focused on resilience and well-being for first-year undergraduate medical students. The model and lessons learned are described.

Curating a Case Catalog: Development and Implementation of a Process for Revising Small Group Teaching Cases for Pre-clerkship Medical Education.

Small group, case-based learning (CBL) is an integral component of many pre-clerkship undergraduate medical education (UME) curricula. We report here an institutional process for curating a catalog of CBL cases utilized in a pre-clerkship curriculum, providing a practical guide for faculty. We describe the structured revision process conducted by a team of foundational and clinical science faculty, which incorporates student and faculty feedback. Revisions take into account core attributes of a case catalog, producing a collection of cases that are more relevant and instructional, realistic, challenging, consistent, current, diverse and inclusive, patient-centered, and mission-centered. Measurable outcomes after implementation of this process include increased focus on primary care as well as humanization and diversification of the case patients.

Using focus groups to plan culturally acceptable primary cervical cancer screening in Grenada, West Indies.

Objective: To document Grenadian women's knowledge about cervical cancer and human papillomavirus (HPV) infection, as well as their attitudes towards primary cervical cancer screening methods. Methods: In this qualitative study, we used focus groups in Grenada to gather information concerning women's knowledge about, attitudes towards and perceptions of screening for cervical cancer and general knowledge about HPV. Ten focus groups comprising 73 participants representing 5 of the 6 parishes in Grenada were conducted with women aged 19-59. Participants were asked about pelvic exams, Pap smears, HPV, reasons for seeking or avoiding cervical cancer screening and how different modalities of testing might affect their decision-making. Responses were then coded and organized into common themes. Results: While many respondents had heard of HPV, far fewer knew about its causative role in cervical cancer, how to prevent HPV infection or testing for the high-risk HPV types that cause almost all cases of cervical cancer. Many participants were aware that cervical cancer screening was beneficial, but numerous barriers to obtaining that screening were noted, including concerns about privacy and stigma, potential discomfort, and the cost and inconvenience involved. Conclusions: Our findings have implications for future cervical cancer screening efforts in Grenada. Central to these efforts should be a focus on educating Grenadians about the role of HPV infection in cervical cancer and the importance of early detection through screening. In addition, addressing issues of stigma and privacy are key to eliminating cervical cancer in Grenada.

Using focus groups to plan culturally acceptable primary cervical cancer screening in Grenada, West Indies

[ABSTRACT]. Objective. To document Grenadian women’s knowledge about cervical cancer and human papillomavirus (HPV) infection, as well as their attitudes towards primary cervical cancer screening methods. Methods. In this qualitative study, we used focus groups in Grenada to gather information concerning women’s knowledge about, attitudes towards and perceptions of screening for cervical cancer and general knowledge about HPV. Ten focus groups comprising 73 participants representing 5 of the 6 parishes in Grenada were conducted with women aged 19–59. Participants were asked about pelvic exams, Pap smears, HPV, reasons for seeking or avoiding cervical cancer screening and how different modalities of testing might affect their decision-making. Responses were then coded and organized into common themes. Results. While many respondents had heard of HPV, far fewer knew about its causative role in cervical can- cer, how to prevent HPV infection or testing for the high-risk HPV types that cause almost all cases of cervical cancer. Many participants were aware that cervical cancer screening was beneficial, but numerous barriers to obtaining that screening were noted, including concerns about privacy and stigma, potential discomfort, and the cost and inconvenience involved. Conclusions. Our findings have implications for future cervical cancer screening efforts in Grenada. Central to these efforts should be a focus on educating Grenadians about the role of HPV infection in cervical cancer and the importance of early detection through screening. In addition, addressing issues of stigma and privacy are key to eliminating cervical cancer in Grenada.

[RESUMEN]. Objetivo. Documentar los conocimientos de las mujeres granadinas sobre el cáncer cervicouterino y la infección por el virus de los papilomas humanos (VPH), así como sus actitudes hacia los métodos primarios de detección del cáncer cervicouterino. Métodos. En este estudio cualitativo, se han empleado grupos focales en Granada para recopilar información sobre los conocimientos, las actitudes y las percepciones de las mujeres sobre la detección del cáncer cervicouterino y nociones generales sobre el VPH. Participaron 73 mujeres de 19 a 59 años de edad, representantes de 5 de las 6 parroquias de Granada. Se formaron diez grupos focales, a los que se les preguntó sobre los exámenes pélvicos, las pruebas de Papanicolaou, el VPH, las razones para buscar o evitar la detección del cáncer cervicouterino y cómo las diferentes modalidades de examen podrían afectar sus decisiones. Luego se codificaron las respuestas y se organizaron en temas comunes. Resultados. Si bien muchas participantes habían oído hablar del VPH, un número considerablemente menor conocía su relación causal con el cáncer cervicouterino, cómo prevenir la infección por VPH o los exámenes de detección de los tipos del VPH de alto riesgo que causan casi todos los casos de cáncer cervicouterino. Muchas participantes sabían que los exámenes de detección del cáncer de cuello uterino eran convenientes, pero mencionaron numerosos obstáculos para obtenerlos, como las preocupaciones sobre la privacidad y la estigmatización, posibles molestias, así como el costo y los inconvenientes relacionados. Conclusiones. Nuestros hallazgos tienen implicaciones para la futura labor de detección del cáncer cervicouterino en Granada. En esta labor debería ser esencial adoptar un enfoque dirigido a educar a las granadinas sobre la relación de la infección por VPH con el cáncer cervicouterino y la importancia de la detección temprana mediante exámenes. Además, combatir los problemas de estigmatización y privacidad es clave para eliminar el cáncer cervicouterino en Granada.

[RESUMO]. Objetivo. Documentar o conhecimento das mulheres de Granada sobre o câncer do colo do útero e a infecção por papilomavírus humano (HPV), bem como suas atitudes em relação aos métodos de rastreamento de câncer do colo do útero primário. Métodos. Neste estudo qualitativo, usamos grupos focais em Granada para coletar informações sobre conhecimentos, atitudes e percepções das mulheres sobre o rastreamento de câncer do colo do útero e conhecimentos gerais sobre HPV. Foram conduzidos dez grupos focais, incluindo 73 participantes e representando 5 das 6 paróquias de Granada, com mulheres de 19 a 59 anos de idade. As participantes responderam perguntas sobre exames ginecológicos, Papanicolau, HPV, razões para procurar ou evitar o rastreamento de câncer do colo do útero e como diferentes modalidades de testes podem afetar sua tomada de decisão. As respostas foram codificadas e organizadas por temas comuns. Resultados. Muitas participantes já tinham ouvido falar do HPV, mas um número muito menor conhecia sua relação causal com o câncer do colo do útero, formas de prevenir a infecção por HPV ou os testes para os tipos de HPV de alto risco, que causam quase todos os casos de câncer do colo do útero. Muitas participantes sabiam que o rastreamento de câncer do colo do útero era benéfico, mas várias barreiras para o rastreamento foram indicadas, incluindo preocupações relacionadas à privacidade e ao estigma, o potencial desconforto e o custo e inconveniência envolvidos. Conclusões. Nossos achados têm implicações para as futuras iniciativas de rastreamento de câncer do colo do útero em Granada. Essas iniciativas devem se focar em educar a população de Granada sobre o papel da infecção por HPV no câncer do colo do útero e a importância da detecção precoce por meio do rastreamento. Além disso, é fundamental abordar questões de estigma e privacidade para eliminar o câncer do colo do útero em Granada.

Using focus groups to plan culturally acceptable primary cervical cancer screening in Grenada, West Indies / Uso de grupos focales para planificar exámenes primarios de detección del cáncer cervicouterino culturalmente aceptables en Granada, Caribe / Uso de grupos focais para planejar estratégias culturalmente aceitáveis para rastreamento de câncer do colo do útero primário em Granada, Índias Ocidentais

ABSTRACT Objective. To document Grenadian women's knowledge about cervical cancer and human papillomavirus (HPV) infection, as well as their attitudes towards primary cervical cancer screening methods. Methods. In this qualitative study, we used focus groups in Grenada to gather information concerning women's knowledge about, attitudes towards and perceptions of screening for cervical cancer and general knowledge about HPV. Ten focus groups comprising 73 participants representing 5 of the 6 parishes in Grenada were conducted with women aged 19-59. Participants were asked about pelvic exams, Pap smears, HPV, reasons for seeking or avoiding cervical cancer screening and how different modalities of testing might affect their decision-making. Responses were then coded and organized into common themes. Results. While many respondents had heard of HPV, far fewer knew about its causative role in cervical cancer, how to prevent HPV infection or testing for the high-risk HPV types that cause almost all cases of cervical cancer. Many participants were aware that cervical cancer screening was beneficial, but numerous barriers to obtaining that screening were noted, including concerns about privacy and stigma, potential discomfort, and the cost and inconvenience involved. Conclusions. Our findings have implications for future cervical cancer screening efforts in Grenada. Central to these efforts should be a focus on educating Grenadians about the role of HPV infection in cervical cancer and the importance of early detection through screening. In addition, addressing issues of stigma and privacy are key to eliminating cervical cancer in Grenada.

RESUMEN Objetivo. Documentar los conocimientos de las mujeres granadinas sobre el cáncer cervicouterino y la infección por el virus de los papilomas humanos (VPH), así como sus actitudes hacia los métodos primarios de detección del cáncer cervicouterino. Métodos. En este estudio cualitativo, se han empleado grupos focales en Granada para recopilar información sobre los conocimientos, las actitudes y las percepciones de las mujeres sobre la detección del cáncer cervicouterino y nociones generales sobre el VPH. Participaron 73 mujeres de 19 a 59 años de edad, representantes de 5 de las 6 parroquias de Granada. Se formaron diez grupos focales, a los que se les preguntó sobre los exámenes pélvicos, las pruebas de Papanicolaou, el VPH, las razones para buscar o evitar la detección del cáncer cervicouterino y cómo las diferentes modalidades de examen podrían afectar sus decisiones. Luego se codificaron las respuestas y se organizaron en temas comunes. Resultados. Si bien muchas participantes habían oído hablar del VPH, un número considerablemente menor conocía su relación causal con el cáncer cervicouterino, cómo prevenir la infección por VPH o los exámenes de detección de los tipos del VPH de alto riesgo que causan casi todos los casos de cáncer cervicouterino. Muchas participantes sabían que los exámenes de detección del cáncer de cuello uterino eran convenientes, pero mencionaron numerosos obstáculos para obtenerlos, como las preocupaciones sobre la privacidad y la estigmatización, posibles molestias, así como el costo y los inconvenientes relacionados. Conclusiones. Nuestros hallazgos tienen implicaciones para la futura labor de detección del cáncer cervicouterino en Granada. En esta labor debería ser esencial adoptar un enfoque dirigido a educar a las granadinas sobre la relación de la infección por VPH con el cáncer cervicouterino y la importancia de la detección temprana mediante exámenes. Además, combatir los problemas de estigmatización y privacidad es clave para eliminar el cáncer cervicouterino en Granada.

RESUMO Objetivo. Documentar o conhecimento das mulheres de Granada sobre o câncer do colo do útero e a infecção por papilomavírus humano (HPV), bem como suas atitudes em relação aos métodos de rastreamento de câncer do colo do útero primário. Métodos. Neste estudo qualitativo, usamos grupos focais em Granada para coletar informações sobre conhecimentos, atitudes e percepções das mulheres sobre o rastreamento de câncer do colo do útero e conhecimentos gerais sobre HPV. Foram conduzidos dez grupos focais, incluindo 73 participantes e representando 5 das 6 paróquias de Granada, com mulheres de 19 a 59 anos de idade. As participantes responderam perguntas sobre exames ginecológicos, Papanicolau, HPV, razões para procurar ou evitar o rastreamento de câncer do colo do útero e como diferentes modalidades de testes podem afetar sua tomada de decisão. As respostas foram codificadas e organizadas por temas comuns. Resultados. Muitas participantes já tinham ouvido falar do HPV, mas um número muito menor conhecia sua relação causal com o câncer do colo do útero, formas de prevenir a infecção por HPV ou os testes para os tipos de HPV de alto risco, que causam quase todos os casos de câncer do colo do útero. Muitas participantes sabiam que o rastreamento de câncer do colo do útero era benéfico, mas várias barreiras para o rastreamento foram indicadas, incluindo preocupações relacionadas à privacidade e ao estigma, o potencial desconforto e o custo e inconveniência envolvidos. Conclusões. Nossos achados têm implicações para as futuras iniciativas de rastreamento de câncer do colo do útero em Granada. Essas iniciativas devem se focar em educar a população de Granada sobre o papel da infecção por HPV no câncer do colo do útero e a importância da detecção precoce por meio do rastreamento. Além disso, é fundamental abordar questões de estigma e privacidade para eliminar o câncer do colo do útero em Granada.

The genetic architecture underlying prey-dependent performance in a microbial predator.

Natural selection should favour generalist predators that outperform specialists across all prey types. Two genetic solutions could explain why intraspecific variation in predatory performance is, nonetheless, widespread: mutations beneficial on one prey type are costly on another (antagonistic pleiotropy), or mutational effects are prey-specific, which weakens selection, allowing variation to persist (relaxed selection). To understand the relative importance of these alternatives, we characterised natural variation in predatory performance in the microbial predator Dictyostelium discoideum. We found widespread nontransitive differences among strains in predatory success across different bacterial prey, which can facilitate stain coexistence in multi-prey environments. To understand the genetic basis, we developed methods for high throughput experimental evolution on different prey (REMI-seq). Most mutations (~77%) had prey-specific effects, with very few (~4%) showing antagonistic pleiotropy. This highlights the potential for prey-specific effects to dilute selection, which would inhibit the purging of variation and prevent the emergence of an optimal generalist predator.

Refining our understanding of metal bioavailability in sediments using information from porewater: Application of a multimetal biotic ligand model as an extension of the equilibrium partitioning sediment benchmarks.

The equilibrium partitioning sediment benchmarks (ESBs) derived by the US Environmental Protection Agency (USEPA) in 2005 provide a mechanistic framework for understanding metal bioavailability in sediments by considering equilibrium partitioning (EqP) theory, which predicts that metal bioavailability in sediments is determined largely by partitioning to sediment particles. Factors that favor the partitioning of metals to sediment particles, such as the presence of acid volatile sulfide (AVS) and sediment organic matter, reduce metal bioavailability to benthic organisms. Because ESBs link metal bioavailability to partitioning to particles, they also predict that measuring metals in porewater can lead to a more accurate assessment of bioavailability and toxicity to benthic organisms. At the time of their development, sediment ESBs based on the analysis of porewater metal concentrations were limited to comparison with hardness-dependent metals criteria for the calculation of interstitial water benchmark units (IWBUs). However, the multimetal biotic ligand model (mBLM) provides a more comprehensive assessment of porewater metal concentrations, because it considers factors in addition to hardness, such as pH and dissolved organic carbon, and allows for interactions between metals. To evaluate the utility of the various sediment and porewater ESBs, four Hyalella azteca bioassay studies were identified that included sediment and porewater measurements of metals and porewater bioavailability parameters. Evaluations of excess simultaneously extracted metals, IWBUs, and mBLM toxic units (TUs) were compared among the bioassay studies. For porewater, IWBUs and mBLM TUs were calculated using porewater metal concentrations from samples collected using centrifugation and peepers. The percentage of correct predictions of toxicity was calculated for each benchmark comparison. The mBLM-based assessment using peeper data provided the most accurate predictions for the greatest number of samples among the evaluation methods considered. This evaluation demonstrates the value of porewater-based evaluations in conjunction with sediment chemistry in understanding toxicity observed in bioassay studies. Integr Environ Assess Manag 2022;18:1335-1347. © 2021 SETAC.

Collection and use of porewater data from sediment bioassay studies for understanding exposure to bioavailable metals.

The US Environmental Protection Agency Procedures for the Derivation of Equilibrium Partitioning Sediment Benchmarks (ESBs) for the Protection of Benthic Organisms: Metal Mixtures (Cadmium, Copper, Lead, Nickel, Silver and Zinc) equilibrium partitioning approach causally link metal concentrations and toxicological effects; they apply to sediment and porewater (i.e., interstitial water). The evaluation of bioavailable metal concentrations in porewater, using tools such as the biotic ligand model, provides an advancement that complements sediment-based evaluations. However, porewater characterization is less commonly performed in sediment bioassays than sediment chemistry characterization due to the difficulty and expense of porewater collection as well as concerns about interpretation of porewater data. This study discusses the advantages and disadvantages of different porewater extraction methods for analysis of metals and bioavailability parameters during laboratory sediment bioassays, with a focus on peepers and centrifugation. The purpose is to provide recommendations to generate bioassay porewater data of sufficient quality for use in risk-based decision-making, such as for regulated cleanup actions. Comparisons of paired data from previous bioassay studies indicate that metal porewater concentrations collected via centrifugation tend to be higher than those collected via peepers. However, centrifugation disrupts the redox status of the sediment; also, metal concentrations can vary markedly based on centrifugation conditions. Data to compare the concentrations of peeper- and centrifugation-collected bioavailability parameters (e.g., major ions, pH) are much more limited, but indicate smaller differences than those observed for metal concentrations. While peepers can be sampled without altering the redox status of the porewater, the small volume of porewater peepers collected is enough for metal concentration analysis, but insufficient for analysis of all metal bioavailability parameters. Given the benefits of metal collection via peepers, it is optimal to use centrifugation and peepers in tandem for bioassay porewater collection to improve bioavailability predictions. Environ Assess Manag 2022;18:1321-1334. © 2021 SETAC.

Mutant resources for functional genomics in Dictyostelium discoideum using REMI-seq technology.

BACKGROUND: Genomes can be sequenced with relative ease, but ascribing gene function remains a major challenge. Genetically tractable model systems are crucial to meet this challenge. One powerful model is the social amoeba Dictyostelium discoideum, a eukaryotic microbe widely used to study diverse questions in the cell, developmental and evolutionary biology. RESULTS: We describe REMI-seq, an adaptation of Tn-seq, which allows high throughput, en masse, and quantitative identification of the genomic site of insertion of a drug resistance marker after restriction enzyme-mediated integration. We use REMI-seq to develop tools which greatly enhance the efficiency with which the sequence, transcriptome or proteome variation can be linked to phenotype in D. discoideum. These comprise (1) a near genome-wide resource of individual mutants and (2) a defined pool of 'barcoded' mutants to allow large-scale parallel phenotypic analyses. These resources are freely available and easily accessible through the REMI-seq website that also provides comprehensive guidance and pipelines for data analysis. We demonstrate that integrating these resources allows novel regulators of cell migration, phagocytosis and macropinocytosis to be rapidly identified. CONCLUSIONS: We present methods and resources, generated using REMI-seq, for high throughput gene function analysis in a key model system.

Menstrual experience of adolescents in the USA: protocol for a scoping review.

INTRODUCTION: In recent years, there has been a growing desire to address issues related to menstruation, particularly for adolescent girls. In low-income and middle-income countries, prior literature review of the adolescent menstrual experience suggests the need for further research into the impact and efficacy of interventions with this population. There is evidence to suggest the need for initiatives and research in higher-income countries like the USA. To date, the body of research on adolescent menstrual experience in the USA remains uncharacterised. Therefore, we propose a scoping review of the literature on this subject to better inform on areas for future primary study. METHODS AND ANALYSES: Using the framework proposed by Arksey and O'Malley and expounded on by Levac et al and the Joanna Briggs Institute, we will search electronic databases (MEDLINE, CINAHL, PsycINFO, Web of Science, ProQuest Public Health Database, Social Science Citation Index, Social Services Abstracts and SocINDEX) and grey literature for relevant studies in consultation with experienced librarians. The abstracts and full-text from each reference will be screened by two independent reviewers for inclusion. Bibliographic data, study characteristics and themes will be extracted from studies selected for inclusion using a rubric created by the research team. Findings will be summarised and a list of subject areas for future primary research will be generated in consultation with stakeholders. The review will be conducted using the Preferred Reporting Items from Systematic Reviews and Meta-Analyses extension for Scoping Reviews guidelines. ETHICS AND DISSEMINATION: Formal ethics training for this study is not required, as the research team will review publicly available studies. Stakeholders working in adolescent and menstrual health were consulted in designing this review. We will share key findings with stakeholders and in scholarly journals at the conclusion of the review.

A new mechanism for cannabidiol in regulating the one-carbon cycle and methionine levels in Dictyostelium and in mammalian epilepsy models.

BACKGROUND AND PURPOSE: Epidiolex™, a form of highly purified cannabidiol (CBD) derived from Cannabis plants, has demonstrated seizure control activity in patients with Dravet syndrome, without a fully elucidated mechanism of action. We have employed an unbiased approach to investigate this mechanism at a cellular level. EXPERIMENTAL APPROACH: We use a tractable biomedical model organism, Dictyostelium, to identify a protein controlling the effect of CBD and characterize this mechanism. We then translate these results to a Dravet syndrome mouse model and an acute in vitro seizure model. KEY RESULTS: CBD activity is partially dependent upon the mitochondrial glycine cleavage system component, GcvH1 in Dictyostelium, orthologous to the human glycine cleavage system component H protein, which is functionally linked to folate one-carbon metabolism (FOCM). Analysis of FOCM components identified a mechanism for CBD in directly inhibiting methionine synthesis. Analysis of brain tissue from a Dravet syndrome mouse model also showed drastically altered levels of one-carbon components including methionine, and an in vitro rat seizure model showed an elevated level of methionine that is attenuated following CBD treatment. CONCLUSIONS AND IMPLICATIONS: Our results suggest a novel mechanism for CBD in the regulating methionine levels and identify altered one-carbon metabolism in Dravet syndrome and seizure activity.

Impacting Growth Mindsets in Student Employment.

This chapter discusses the importance of a growth mindset for employability and how leadership educators and supervisors can assist students' development in this area. The ability to have a growth mindset ensures that students see an opportunity in setbacks and their own growth and development.

Ubiquitin-dependent chloroplast-associated protein degradation in plants.

Chloroplasts contain thousands of nucleus-encoded proteins that are imported from the cytosol by translocases in the chloroplast envelope membranes. Proteolytic regulation of the translocases is critically important, but little is known about the underlying mechanisms. We applied forward genetics and proteomics in Arabidopsis to identify factors required for chloroplast outer envelope membrane (OEM) protein degradation. We identified SP2, an Omp85-type ß-barrel channel of the OEM, and CDC48, a cytosolic AAA+ (ATPase associated with diverse cellular activities) chaperone. Both proteins acted in the same pathway as the ubiquitin E3 ligase SP1, which regulates OEM translocase components. SP2 and CDC48 cooperated to bring about retrotranslocation of ubiquitinated substrates from the OEM (fulfilling conductance and motor functions, respectively), enabling degradation of the substrates by the 26S proteasome in the cytosol. Such chloroplast-associated protein degradation (CHLORAD) is vital for organellar functions and plant development.

Draft Genome Sequence of a Novel Rhabdovirus Isolated from Deinocerites Mosquitoes.

We present a draft genome of a novel rhabdovirus, called Grenada mosquito rhabdovirus 1 (GMRV1), with homology to Wuhan mosquito virus 9 (WMV9) (NCBI reference sequence NC_031303), isolated from Deinocerites mosquitoes. The genome has a length of 14,420 nucleotides and encodes five open reading frames.

Curcumin and derivatives function through protein phosphatase 2A and presenilin orthologues in Dictyostelium discoideum.

Natural compounds often have complex molecular structures and unknown molecular targets. These characteristics make them difficult to analyse using a classical pharmacological approach. Curcumin, the main curcuminoid of turmeric, is a complex molecule possessing wide-ranging biological activities, cellular mechanisms and roles in potential therapeutic treatment, including Alzheimer's disease and cancer. Here, we investigate the physiological effects and molecular targets of curcumin in Dictyostelium discoideum We show that curcumin exerts acute effects on cell behaviour, reduces cell growth and slows multicellular development. We employed a range of structurally related compounds to show the distinct role of different structural groups in curcumin's effects on cell behaviour, growth and development, highlighting active moieties in cell function, and showing that these cellular effects are unrelated to the well-known antioxidant activity of curcumin. Molecular mechanisms underlying the effect of curcumin and one synthetic analogue (EF24) were then investigated to identify a curcumin-resistant mutant lacking the protein phosphatase 2A regulatory subunit (PsrA) and an EF24-resistant mutant lacking the presenilin 1 orthologue (PsenB). Using in silico docking analysis, we then showed that curcumin might function through direct binding to a key regulatory region of PsrA. These findings reveal novel cellular and molecular mechanisms for the function of curcumin and related compounds.

Polyglycine Acts as a Rejection Signal for Protein Transport at the Chloroplast Envelope.

PolyGly is present in many proteins in various organisms. One example is found in a transmembrane ß-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins, the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.

In-frame amber stop codon replacement mutagenesis for the directed evolution of proteins containing non-canonical amino acids: identification of residues open to bio-orthogonal modification.

Expanded genetic code approaches are a powerful means to add new and useful chemistry to proteins at defined residues positions. One such use is the introduction of non-biological reactive chemical handles for site-specific biocompatible orthogonal conjugation of proteins. Due to our currently limited information on the impact of non-canonical amino acids (nAAs) on the protein structure-function relationship, rational protein engineering is a "hit and miss" approach to selecting suitable sites. Furthermore, dogma suggests surface exposed native residues should be the primary focus for introducing new conjugation chemistry. Here we describe a directed evolution approach to introduce and select for in-frame codon replacement to facilitate engineering proteins with nAAs. To demonstrate the approach, the commonly reprogrammed amber stop codon (TAG) was randomly introduced in-frame in two different proteins: the bionanotechnologically important cyt b(562) and therapeutic protein KGF. The target protein is linked at the gene level to sfGFP via a TEV protease site. In absence of a nAA, an in-frame TAG will terminate translation resulting in a non-fluorescent cell phenotype. In the presence of a nAA, TAG will encode for nAA incorporation so instilling a green fluorescence phenotype on E. coli. The presence of endogenously expressed TEV proteases separates in vivo target protein from its fusion to sfGFP if expressed as a soluble fusion product. Using this approach, we incorporated an azide reactive handle and identified residue positions amenable to conjugation with a fluorescence dye via strain-promoted azide-alkyne cycloaddition (SPAAC). Interestingly, best positions for efficient conjugation via SPAAC were residues whose native side chain were buried through analysis of their determined 3D structures and thus may not have been chosen through rational protein engineering. Molecular modeling suggests these buried native residues could become partially exposed on substitution to the azide containing nAA.