Functional analysis of epilepsy-associated GABAA receptor mutations using Caenorhabditis elegans.

OBJECTIVE: GABAA receptor subunit mutations pose a significant risk for genetic generalized epilepsy; however, there are over 150 identified variants, many with unknown or unvalidated pathogenicity. We aimed to develop in vivo models for testing GABAA receptor variants using the model organism, Caenorhabditis elegans. METHODS: CRISPR-Cas9 gene editing was used to create a complete deletion of unc-49, a C. elegans GABAA receptor, and to create homozygous epilepsy-associated mutations in the endogenous unc-49 gene. The unc-49 deletion strain was rescued with transgenes for either the C. elegans unc-49B subunit or the α1, ß3, and γ2 subunits for the human GABAA receptor. All newly created strains were analyzed for phenotype and compared against existing unc-49 mutations. RESULTS: Nematodes with a full genetic deletion of the entire unc-49 locus were compared with existing unc-49 mutations in three separate phenotypic assays-coordinated locomotion, shrinker frequency and seizure-like convulsions. The full unc-49 deletion exhibited reduced locomotion and increased shrinker frequency and PTZ-induced convulsions, but were not found to be phenotypically stronger than existing unc-49 mutations. Rescue with the unc-49B subunit or creation of humanized worms for the GABAA receptor both showed partial phenotypic rescue for all three phenotypes investigated. Finally, two epilepsy-associated variants were analyzed and deemed to be loss of function, thus validating their pathogenicity. SIGNIFICANCE: These findings establish C. elegans as a genetic model to investigate GABAA receptor mutations and delineate a platform for validating associated variants in any epilepsy-associated gene. PLAIN LANGUAGE SUMMARY: Epilepsy is a complex human disease that can be caused by mutations in specific genes. Many possible mutations have been identified, but it is unknown for most of them whether they cause the disease. We tested the role of mutations in one specific gene using a small microscopic worm as an animal model. Our results establish this worm as a model for epilepsy and confirm that the two unknown mutations are likely to cause the disease.

Tissue distribution of cysteine string protein/DNAJC5 in C. elegans analysed by CRISPR/Cas9-mediated tagging of endogenous DNJ-14.

Cysteine string protein (CSP) is a member of the DnaJ/Hsp40 family of molecular chaperones. CSP is enriched in neurons, where it mainly localises to synaptic vesicles. Mutations in CSP-encoding genes in flies, worms, mice and humans result in neuronal dysfunction, neurodegeneration and reduced lifespan. Most attention has therefore focused on CSP's neuronal functions, although CSP is also expressed in non-neuronal cells. Here, we used genome editing to fluorescently tag the Caenorhabditis elegans CSP orthologue, dnj-14, to identify which tissues preferentially express CSP and hence may contribute to the observed mutant phenotypes. Replacement of dnj-14 with wrmScarlet caused a strong chemotaxis defect, as seen with other dnj-14 null mutants. In contrast, inserting the reporter in-frame to create a DNJ-14-wrmScarlet fusion protein had no effect on chemotaxis, indicating that C-terminal tagging does not impair DNJ-14 function. WrmScarlet fluorescence appeared most obvious in the intestine, head/pharynx, spermathecae and vulva/uterus in the reporter strains, suggesting that DNJ-14 is preferentially expressed in these tissues. Crossing the DNJ-14-wrmScarlet strain with GFP marker strains confirmed the intestinal and pharyngeal expression, but only a partial overlap with neuronal GFP was observed. DNJ-14-wrmScarlet fluorescence in the intestine was increased in response to starvation, which may be relevant to mammalian CSPα's role in microautophagy. DNJ-14's enrichment in worm reproductive tissues (spermathecae and vulva/uterus) parallels the testis-specific expression of CSPß and CSPγ isoforms in mammals. Furthermore, CSPα messenger RNA is highly expressed in the human proximal digestive tract, suggesting that CSP may have a conserved, but overlooked, function within the gastrointestinal system.

Proximity labelling reveals effects of disease-causing mutation on the DNAJC5/cysteine string protein α interactome.

Cysteine string protein α (CSPα), also known as DNAJC5, is a member of the DnaJ/Hsp40 family of co-chaperones. The name derives from a cysteine-rich domain, palmitoylation of which enables localization to intracellular membranes, notably neuronal synaptic vesicles. Mutations in the DNAJC5 gene that encodes CSPα cause autosomal dominant, adult-onset neuronal ceroid lipofuscinosis (ANCL), a rare neurodegenerative disease. As null mutations in CSP-encoding genes in flies, worms and mice similarly result in neurodegeneration, CSP is evidently an evolutionarily conserved neuroprotective protein. However, the client proteins that CSP chaperones to prevent neurodegeneration remain unclear. Traditional methods for identifying protein-protein interactions such as yeast 2-hybrid and affinity purification approaches are poorly suited to CSP, due to its requirement for membrane anchoring and its tendency to aggregate after cell lysis. Therefore, we employed proximity labelling, which enables identification of interacting proteins in situ in living cells via biotinylation. Neuroendocrine PC12 cell lines stably expressing wild type or L115R ANCL mutant CSP constructs fused to miniTurbo were generated; then the biotinylated proteomes were analysed by liquid chromatographymass spectrometry (LCMS) and validated by western blotting. This confirmed several known CSP-interacting proteins, such as Hsc70 and SNAP-25, but also revealed novel binding proteins, including STXBP1/Munc18-1. Interestingly, some protein interactions (such as Hsc70) were unaffected by the L115R mutation, whereas others (including SNAP-25 and STXBP1/Munc18-1) were inhibited. These results define the CSP interactome in a neuronal model cell line and reveal interactions that are affected by ANCL mutation and hence may contribute to the neurodegeneration seen in patients.

A Caenorhabditis elegans model of autosomal dominant adult-onset neuronal ceroid lipofuscinosis identifies ethosuximide as a potential therapeutic.

Autosomal dominant adult-onset neuronal ceroid lipofuscinosis (ANCL) is a rare neurodegenerative disorder characterized by progressive dementia and premature death. Four ANCL-causing mutations have been identified, all mapping to the DNAJC5 gene that encodes cysteine string protein α (CSPα). Here, using Caenorhabditis elegans, we describe an animal model of ANCL in which disease-causing mutations are introduced into their endogenous chromosomal locus, thereby mirroring the human genetic disorder. This was achieved through CRISPR/Cas9-mediated gene editing of dnj-14, the C. elegans ortholog of DNAJC5. The resultant homozygous ANCL mutant worms exhibited reduced lifespans and severely impaired chemotaxis, similar to isogenic dnj-14 null mutants. Importantly, these phenotypes were also seen in balanced heterozygotes carrying one wild-type and one ANCL mutant dnj-14 allele, mimicking the heterozygosity of ANCL patients. We observed a more severe chemotaxis phenotype in heterozygous ANCL mutant worms compared with haploinsufficient worms lacking one copy of CSP, consistent with a dominant-negative mechanism of action. Additionally, we provide evidence of CSP haploinsufficiency in longevity, as heterozygous null mutants exhibited significantly shorter lifespan than wild-type controls. The chemotaxis phenotype of dnj-14 null mutants was fully rescued by transgenic human CSPα, confirming the translational relevance of the worm model. Finally, a focused compound screen revealed that the anti-epileptic drug ethosuximide could restore chemotaxis in dnj-14 ANCL mutants to wild-type levels. This suggests that ethosuximide may have therapeutic potential for ANCL and demonstrates the utility of this C. elegans model for future larger-scale drug screening.

Value of p53 sequencing in the prognostication of head and neck cancer: a systematic review and meta-analysis.

This review aimed to examine the relationship between TP53 mutational status, as determined by genomic sequencing, and survival in squamous cell carcinoma of the head and neck. The databases Medline, Embase, Web of Science (core collection), Scopus and Cochrane Library were searched from inception to April 2021 for studies assessing P53 status and survival. Qualitative analysis was carried out using the REMARK criteria. A meta-analyses was performed and statistical analysis was carried out to test the stability and reliability of results. Twenty-five studies met the inclusion criteria, of which fifteen provided enough data for quantitative evaluation. TP53 mutation was associated with worse overall survival (HR 1.75 [95% CI 1.45-2.10], p < 0.001), disease-specific survival (HR 4.23 [95% CI 1.19-15.06], p = 0.03), and disease-free survival (HR 1.80 [95% CI 1.28-2.53], p < 0.001). Qualitative assessment identified room for improvement and the pooled analysis of all anatomical subsites leads to heterogeneity that may erode the validity of the observed overall effect and its subsequent extrapolation and application to individual patients. Our systematic review and meta-analysis supports the utility of TP53 mutational as a prognostic factor for survival in head and neck squamous cell carcinoma. A well designed prospective, multi-centre trial is needed to definitively answer this question.

The Current State and Validity of Digital Assessment Tools for Psychiatry: Systematic Review.

BACKGROUND: Given the role digital technologies are likely to play in the future of mental health care, there is a need for a comprehensive appraisal of the current state and validity (ie, screening or diagnostic accuracy) of digital mental health assessments. OBJECTIVE: The aim of this review is to explore the current state and validity of question-and-answer-based digital tools for diagnosing and screening psychiatric conditions in adults. METHODS: This systematic review was based on the Population, Intervention, Comparison, and Outcome framework and was carried out in accordance with the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. MEDLINE, Embase, Cochrane Library, ASSIA, Web of Science Core Collection, CINAHL, and PsycINFO were systematically searched for articles published between 2005 and 2021. A descriptive evaluation of the study characteristics and digital solutions and a quantitative appraisal of the screening or diagnostic accuracy of the included tools were conducted. Risk of bias and applicability were assessed using the revised tool for the Quality Assessment of Diagnostic Accuracy Studies 2. RESULTS: A total of 28 studies met the inclusion criteria, with the most frequently evaluated conditions encompassing generalized anxiety disorder, major depressive disorder, and any depressive disorder. Most of the studies used digitized versions of existing pen-and-paper questionnaires, with findings revealing poor to excellent screening or diagnostic accuracy (sensitivity=0.32-1.00, specificity=0.37-1.00, area under the receiver operating characteristic curve=0.57-0.98) and a high risk of bias for most of the included studies. CONCLUSIONS: The field of digital mental health tools is in its early stages, and high-quality evidence is lacking. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR2-10.2196/25382.

Are we asking the right questions? Working with the LGBTQ+ community to prioritise healthcare research themes.

BACKGROUND: Conversations about research priorities with members of the public are rarely designed specifically to include people who identify as Lesbian, Gay, Bisexual, Transgender and Queer (LGBTQ+) and are not researchers. METHODS: Generally, to address this gap, and specifically, to inform future research for CLS, we carried out a rapid review of published research priority sets covering LGBTQ+ topics, and an online workshop to prioritise identified themes. RESULTS: Rapid review: results. The rapid review identified 18 LGBTQ+ research priority sets. Some focussed on specific populations such as women or men, younger or older people or people living within families. Five addressed transgender and gender non- conforming populations. All of the research priority sets originated from English-speaking, high and middle-income countries (UK, US, Canada, and Australia), and date from 2016 onwards. Prioritization approaches were wide-ranging from personal commentary to expert workshops and surveys. Participants involved in setting priorities mostly included research academics, health practitioners and advocacy organisations, two studies involved LGBTQ+ public in their process. Research priorities identified in this review were then grouped into themes which were prioritised during the workshop. Workshop: results. For the workshop, participants were recruited using local (Cambridge, UK) LGBTQ+ networks and a national advert to a public involvement in research matching website to take part in an online discussion workshop. Those that took part were offered payment for their time in preparing for the workshop and taking part. Participants personal priorities and experiences contributed to a consensus development process and a final ranked list of seven research themes and participants' experiences of healthcare, mental health advocacy, care homes, caring responsibilities, schools and family units added additional context. CONCLUSIONS: From the workshop the three research themes prioritised were: healthcare services delivery, prevention, and particular challenges / intersectionality of multiple challenges for people identifying as LGBTQ+. Research themes interconnected in many ways and this was demonstrated by the comments from workshop participants. This paper offers insights into why these priorities were important from participants' perspectives and detail about how to run an inclusive and respectful public involvement research exercise. On a practical level these themes will directly inform future research direction for CLS.

Conversations about research priorities with members of the public who identify as Lesbian, Gay, Bisexual, Transgender and Queer (LGBTQ+) and are not researchers are not common.We reviewed published research priorities covering LGBTQ+ topics and held an online workshop. The review identified 18 LGBTQ+ research published priority sets. Some focussed on specific populations such as women or men, younger or older people or people living within families. Five were on transgender and gender non- conforming populations. Priorities were achieved by different methods such as workshops and surveys. People involved in setting priorities mostly included researchers, health practitioners and advocacy organisations, two studies involved LGBTQ+ public in their process. Research priorities identified in the review were grouped into themes which were prioritised during the workshop.For the online workshop, participants were recruited using local (Cambridge, UK) LGBTQ+ networks and a national advert and offered payment for their time. Participants personal priorities and experiences contributed to agreeing a final list of seven research themes in priority order. Participants' experiences of healthcare, mental health advocacy, care homes, caring responsibilities, schools and family units were helpful.From the workshop the three top research themes were: healthcare services delivery, prevention, and particular and multiple challenges of people identifying as lesbian, gay, bisexual, transgender or queer. Research themes interconnected in many ways as shown by the comments from workshop participants. This paper describes why these priorities were important from participants' perspective and offers information about how to run an inclusive and respectful public involvement research exercise.

Creating conference posters: Structure, form and content.

This article aims to provide an overview of the form, structure and content of conference posters for researchers who intend to submit an academic poster to a conference. It focuses in particular on the design and layout of academic conference posters, making some suggestions for possible poster layouts. It also provides information about factors influencing conference selection. Finally, it summarises some top tips to be considered when creating a conference poster such as font selection and use of images.

Systematic reviews: Structure, form and content.

This article aims to provide an overview of the structure, form and content of systematic reviews. It focuses in particular on the literature searching component, and covers systematic database searching techniques, searching for grey literature and the importance of librarian involvement in the search. It also covers systematic review reporting standards such as PRISMA-P and PRISMA, critical appraisal and tools and resources to support the review and ensure it is conducted efficiently and effectively. Finally, it summarizes the requirements when screening search results for inclusion in the review, and the statistical synthesis of included studies' findings.

Writing for publication: Structure, form, content, and journal selection.

This article provides an overview of writing for publication in peer-reviewed journals. While the main focus is on writing a research article, it also provides guidance on factors influencing journal selection, including journal scope, intended audience for the findings, open access requirements, and journal citation metrics. Finally, it covers the standard content of a scientific journal article, providing general advice and guidance regarding the information researchers would typically include in their published papers.

The Current State and Diagnostic Accuracy of Digital Mental Health Assessment Tools for Psychiatric Disorders: Protocol for a Systematic Review and Meta-analysis.

BACKGROUND: Despite the rapidly growing number of digital assessment tools for screening and diagnosing mental health disorders, little is known about their diagnostic accuracy. OBJECTIVE: The purpose of this systematic review and meta-analysis is to establish the diagnostic accuracy of question- and answer-based digital assessment tools for diagnosing a range of highly prevalent psychiatric conditions in the adult population. METHODS: The Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) will be used. The focus of the systematic review is guided by the population, intervention, comparator, and outcome framework (PICO). We will conduct a comprehensive systematic literature search of MEDLINE, PsychINFO, Embase, Web of Science Core Collection, Cochrane Library, Applied Social Sciences Index and Abstracts (ASSIA), and Cumulative Index to Nursing and Allied Health Literature (CINAHL) for appropriate articles published from January 1, 2005. Two authors will independently screen the titles and abstracts of identified references and select studies according to the eligibility criteria. Any inconsistencies will be discussed and resolved. The two authors will then extract data into a standardized form. Risk of bias will be assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) tool, and a descriptive analysis and meta-analysis will summarize the diagnostic accuracy of the identified digital assessment tools. RESULTS: The systematic review and meta-analysis commenced in November 2020, with findings expected by May 2021. CONCLUSIONS: This systematic review and meta-analysis will summarize the diagnostic accuracy of question- and answer-based digital assessment tools. It will identify implications for clinical practice, areas for improvement, and directions for future research. TRIAL REGISTRATION: PROSPERO International Prospective Register of Systematic Reviews CRD42020214724; https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42020214724. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/25382.

Pain After Cochlear Implantation Without Signs of Inflammation: A Systematic Review.

OBJECTIVES: This review summarizes current evidence on causes and management strategies for delayed pain post-cochlear implantation (CI) surgery, without clinical evidence of inflammation or infection. METHODS: The systematic review was undertaken in line with Preferred Reporting Items for Systematic review and Meta-Analysis Protocols 2015 guidelines. A literature search was undertaken, with inclusion of patients who underwent CI and presented with delayed pain (>3 months post-operatively) around their device site without an identifiable cause. Analysis was undertaken using MATLAB (MathWorks, Natick, MA) and the R-software package (www.r-project.org). RESULTS: 4 articles (48 patients), all retrospective case series, met inclusion criteria. The mean onset of pain post-CI was 60 months and mean follow-up was 15.8 months, there was no difference in the prevalence of pain between device brands (p=0.13). The majority (90%) did not have any hearing deterioration, and investigations did not reveal a cause for the pain in any of the patients. In terms of management, medical therapies, including oral therapy (analgesia, non-steroidal anti-inflammatories, antibiotics) and local treatments (topical, injections) resolved pain in 41% and 63%, respectively. Surgical intervention (explantation, magnet replacement, tympanic neurectomy), where undertaken, resolved pain in 100%. A minority had an identifiable infective microorganism cultured from intra-operative soft tissue or biofilm samples. CONCLUSIONS: Evidence for the causes and management of delayed pain post-CI without clinical evidence of inflammation is scarce. A stepwise approach is deemed best, with decisions being made on an individual basis, evaluating each patient's specific circumstances and priorities. Further evaluation of explanted devices would allow for better understanding of the causes and treatment of this group of patients.

Learning how to teach unfamiliar subjects: developing training on writing for publication and presentation of research for health libraries.

Academic librarians with teaching responsibility have traditionally delivered training in discovering and organising information. However, in recent years, there has been an increased emphasis on supporting researchers through all stages of the research lifecycle. While librarians are ideally placed to provide training in writing for publication and presentation of research, very few in the United Kingdom appear to be doing so. However, there are clear benefits to teaching these subjects. Based on feedback from faculty on user needs, the University of Cambridge Medical Library's training programme was expanded to include training and support in the publication and presentation of research outputs. This article recounts the process by which the new courses were developed, and the techniques used by the library's teaching staff to gain understanding of conventions and requirements of forms of written communication with which they were unfamiliar. It also evaluates the impact of the new courses, discusses next steps and provides advice for other librarians wishing to develop similar courses. D.I.

A systematic review on prevention and management of wound infections from cochlear implantation.

OBJECTIVE OF REVIEW: Surgical site infections are a recognised complication of cochlear implant (CI) surgery with significant morbidity. Our aim was to search for the optimum prevention and management strategy to deal with this issue. TYPE OF REVIEW: Systematic review. SEARCH STRATEGY: A systematic literature search was undertaken from the databases of Embase, CINAHL, MEDLINE® , Web of Science, Scopus and Cochrane Library according to the predefined inclusion and exclusion criteria. EVALUATION METHOD: All relevant titles, abstracts and full-text articles were reviewed by two authors who resolved any differences by discussion and consultation with senior authors. RESULTS: Fourteen articles were included in our review. The overall quality of evidence was low with the vast majority of the studies being retrospective case series and expert opinions. No randomised controlled trials were noted. We found consistent reports that intraoperative prophylactic antibiotics should be given to all patients undergoing CI and that the vast majority of CI wound infections had grown Staphylococcal spp. or Pseudomonas spp. CONCLUSION: Our review has not identified any reliable or reproducible strategies to prevent and deal with wound infections after CI. We strongly encourage further research within this field and would suggest that a consensus of opinions from a multidisciplinary panel of experts may be a pragmatic way forward as an effective guide.