Llanfrechfa Grange Hospital

The efficacy of cyproterone acetate in the treatment of sexual overactivity has been assessed in fifty male patients over a period of five years. Homosexual activity was reduced in its intensity but not in its direction. Masturbation was diminished in subnormal and chronic schizophrenic patients and there was a social improvement in elderly men guilty of sexual misbehaviour. Two sado-masochists showed greater clinical improvement when cyproterone acetate was added to psychotherapy than occurred with the latter therapy alone. Physical aggression exhibited by two subnormal patients which was resistant to conventional therapy, responded dramatically to cyproterone acetate administration. There were no significant side-effects.

UNLABELLED: Autistic spectrum disorder (ASD) is a developing area for dietetic referrals. There is little published data on current dietetic practice. Some children with ASD are referred for gluten/casein free diet. The theory is that abnormal metabolites in the urine may be a result of incomplete breakdown of gluten and casein in the gut. There are some published open studies that support the efficiency of such a diet [Knivsberg et al. (1995) Scand. J. Educ. Res.39: 223; Lucarelli et al. (1995) Panminerva Med.37: 137; Whiteley et al. (1999) Int. J. Res. Practice 3: 45] and also that there are many anecdotal reports that the diet helps some children. AIMS AND OBJECTIVES: This study aimed to audit the types of referral made to the dietetic service to identify key dietetic issues and to describe factors which may influence outcome/disease management. METHODS: Dietetic records were used to audit the referrals to the dietetic service over a 3-month period. Seven-day diet histories were assessed using computer food composition tables and topics of interest recorded against a draft protocol agreed within the profession. RESULTS: Requests for gluten-free and casein-free dietetic advice, and/or the management of food selectivity and dysfunctional feeding behaviour constituted the majority of referrals. In many cases, child's environment was rarely simple. CONCLUSIONS: Despite the limitations of this small study, the findings suggest that the management of these referrals is highly complex. A dietitian's input should ensure that the nutritional adequacy of the diet is maintained or restored.

AIMS AND OBJECTIVES: To review hospital passports currently in use for people with intellectual disabilities in the UK and to make recommendations for practice. BACKGROUND: Hospital passports have been introduced internationally to address communication barriers that may limit access to appropriate health care for people with intellectual disabilities. They are viewed as promoting patient safety and person-centred care but their format may vary, they are not always used appropriately, and hence, their effectiveness may be limited. DESIGN: Qualitative content analysis. METHODS: Sixty hospital passports in use in the UK were reviewed against a coding frame by two members of the research team. Areas of interest included key patient and primary care information, support network details, consent and capacity, support required in relation to activities of daily living, length of the document and completion details. Results were entered into Excel. RESULTS: Considerable variation was found between documents in terms of terminology, length and format. Most included information regarding communication and support needs although some omitted important information such as allergies, risk assessment and need for reasonable adjustments. CONCLUSIONS: Considerable variation exists between current hospital passports, which may limit their effectiveness: key information required may not be included and/or it may not be easy to locate. Greater standardisation of documents is required, but this process should include input from all key stakeholders. RELEVANCE TO CLINICAL PRACTICE: Internationally nurses provide care for people with intellectual disabilities and others with communication difficulties. Hospital passports are one way of enhancing safety and person-centred care, need to be accessed and used as a basis for care planning. However, variation in format may limit this effectiveness and nurses should work with others to develop a more standardised approach, which better meets the needs of all stakeholders.

This paper examines the link between a self‐report Anger Inventory (Benson & Ivins, 1992) and incidents of challenging behaviour by people with intellectual disabilities. Records of challenging behaviour were kept by significant others while the people with intellectual disabilities themselves were asked to complete the Anger Inventory on a number of different occasions. The inventory was used to evaluate and follow up the impact of an anger management group, and in some cases other associated interventions. Data are presented on five individuals for time periods of between two and three years. Visual inspection of the data suggests a degree of correspondence between the levels of challenging behaviour reported by staff and scores reported on the Anger Inventory by participants and this relationship is confirmed statistically. The implications of these results are discussed.

OBJECTIVE: The aim is to characterise early and late respiratory and bloodstream co-infection in patients admitted to intensive care units (ICUs) with SARS-CoV-2-related acute hypoxemic respiratory failure (AHRF) needing respiratory support in seven ICUs within Wales, during the first wave of the COVID-19 pandemic. We compare the rate of positivity of different secondary pathogens and their antimicrobial sensitivity in three different patient groups: patients admitted to ICU with COVID-19 pneumonia, Influenza A or B pneumonia, and patients without viral pneumonia. DESIGN: Multicentre, retrospective, observational cohort study with rapid microbiology data from Public Health Wales, sharing of clinical and demographic data from seven participating ICUs. SETTING: Seven Welsh ICUs participated between 10 March and 31 July 2020. Clinical and demographic data for COVID-19 disease were shared by each participating centres, and microbiology data were extracted from a data repository within Public Health Wales. Comparative data were taken from a cohort of patients without viral pneumonia admitted to ICU during the same period as the COVID-19 cohort (referred to as no viral pneumonia or 'no viral' group), and to a retrospective non-matched cohort of consecutive patients with Influenza A or B admitted to ICUs from 20 November 2017. The comparative data for Influenza pneumonia and no viral pneumonia were taken from one of the seven participating ICUs. PARTICIPANTS: A total of 299 consecutive patients admitted to ICUs with COVID-19 pneumonia were compared with 173 and 48 patients admitted with no viral pneumonia or Influenza A or B pneumonia, respectively. MAIN OUTCOME MEASURES: Primary outcome was to calculate comparative incidence of early and late co-infection in patients admitted to ICU with COVID-19, Influenza A or B pneumonia and no viral pneumonia. Secondary outcome was to calculate the individual group of early and late co-infection rate on a per-patient and per-sample basis, with their antimicrobial susceptibility and thirdly to ascertain any statistical correlation between clinical and demographic variables with rate of acquiring co-infection following ICU admission. RESULTS: A total of 299 adults (median age 57, M/F 2:1) were included in the COVID-19 ICU cohort. The incidence of respiratory and bloodstream co-infection was 40.5% and 15.1%, respectively. Staphylococcus aureus was the predominant bacterial pathogen within the first 48 h. Gram-negative organisms from Enterobacterales group were predominantly seen after 48 h in COVID-19 cohort. Comparative no viral pneumonia cohort had lower rates of respiratory tract infection and bloodstream infection. The influenza cohort had similar rates respiratory tract infection and bloodstream infection. Mortality in all three groups was similar, and no clinical or demographic variables were found to increase the rate of co-infection and ICU mortality. CONCLUSIONS: Higher incidence of bacterial co-infection was found in COVID-19 cohort as compared to the no viral pneumonia cohort admitted to ICUs for respiratory support.

A case of a chromatin positive, mentally deficient 51 year old male who is clinically an example of Klinefelter’s syndrome and has a sex chromosome constitution of XXYY is described. He has some of the features of the four cases previously described by Muldal <i>et al</i>. (1962), Carr <i>et al</i>. (1961), Ellis <i>et al</i>. (1961) and Vague <i>et al</i>. (1961), but also has abnormalities of the serum mucoproteins and the urine chondroitin sulphate levels and very prominent frontal sinuses. The possible aetiology of the chromosome, biochemical and x-ray abnormalities are discussed.

AIM: To define the characteristics of patients consulting with active dyspeptic symptoms in urban general practice, and to consider the implications of applying the British Society of Gastroenterology Dyspepsia management guidelines. DESIGN: Prospective observational study over a period of 12 months. SETTING: Two multipartner, two-centre general practices in the City of Leeds (UK) with a combined target population of 11 011 registered patients. SUBJECTS: A total of 340 patients consulting with active dyspeptic symptoms (52% male; mean age 53 years, range 16-89 years). RESULTS: Of the practice population, 3% consulted with dyspepsia (first-time consulter: 19%; previous consulter not yet investigated: 30%; previously investigated: 51%). Of 168 undiagnosed patients, 43% had upper abdominal pain (dysmotility-like symptoms in 42%), 35% had reflux symptoms, 22% had mixed symptoms, 12% had 'alarm' symptoms and 18% had a history of NSAID use. Patients < 45 years old with simple dyspepsia accounted for 32% of undiagnosed cases. A fifth of the workload was in dealing with undiagnosed dyspeptics over 45 years old. One per cent of the population would require endoscopy if all undiagnosed cases either > 45 years or with complicated dyspepsia were investigated. Of 172 previously investigated patients, 29% had negative tests, 25% had 'minor' findings, and 45% had evidence of acid-peptic disease. Patients with duodenal ulcer disease accounted for 12% of the total workload. CONCLUSIONS: A knowledge of the characteristics of patients consulting with dyspepsia in primary care should allow the adaptation of guidelines, to ensure advice is relevant to local case mix and compatible with local resources.

Over a period of 11 years, 33 sheep or goat holdings lost their maedi-visna or caprine arthritis-encephalitis accredited status (mean 2.8 per year [0.09 per cent] of the accredited holdings in Great Britain). Of these, 22 sheep flocks and two goat herds eradicated the infection and regained their accredited status. In addition, 10 sheep flocks and two goat herds managed to eradicate infection, having joined the scheme with infected animals. In flocks and herds with a high initial prevalence of infection, the adoption of an indirect ELISA, with greater sensitivity than the agar gel immunodiffusion test, improved success rates. A strategy was devised to interpret the ELISA results depending upon the prevalence of infection at the time. Eighteen of the 33 flocks/herds (54.5 per cent) that had introductions of infection also owned non-accredited stock.

BACKGROUND: several different models of out-of-hours primary care now exist in the UK. Important outcomes of care include users' satisfaction and enablement to manage their illness or condition, but the determinants of these outcomes in the unscheduled care domain are poorly understood. Aim. To identify predictors of user satisfaction and enablement across unscheduled care or GP out-of-hours service providers in Wales. The design of the study is a cross-sectional survey. The setting of the study is nine GP out-of-hours services, three Accident and Emergency units and an all Wales telephone advice service in Wales. METHODS: postal survey using the Out-of-hours Patient Questionnaire. Logistic regression was used to fit both satisfaction and enablement models, based on demographic variables, service provider and treatment received and perceptions or ratings of the care process. RESULTS: eight hundred and fifty-five of 3250 users responded (26% response rate, range across providers 14-41%, no evidence of non-response bias for age or gender). Treatment centre consultations were significantly associated with decreased patient satisfaction and decreased enablement compared with telephone advice. Delays in call answering or callback for triage and shorter consultations were significantly associated with lower satisfaction. Waiting more than a minute for initial call answering was associated with lower enablement. CONCLUSIONS: giving users more time to discuss their illness in consultations may enhance satisfaction and enablement but this may be resource intensive. More simple interventions to improve access by quicker response and triage, and keeping users informed of waiting times, could also serve to increase satisfaction and ultimately impact on their enablement.

Accessible summary • We ran a group for 6 people with a learning disability who worry a lot. The group last for 12 weeks and each person in the group brought someone with them. • People said they found the group helpful. People said they learnt ways to cope when they get worried. • This research will help staff to think about how they can help people who worry a lot. More research is needed to find out which bits of the group were most helpful. Summary An anxiety management group utilizing a cognitive behavioural intervention, of 12 weeks duration, for six people with mild to moderate learning disabilities is described. A number of techniques to assist in developing clients’ understanding of their anxiety, cognitive and behavioural coping strategies and maximizing generalizability of skills learnt were implemented. Outcome measures demonstrated that two participants showed a clinically significant reduction in anxiety levels. Qualitative feedback from both participants and carers suggested that all group members developed coping strategies. Measures also indicated that carers demonstrated a better understanding of the individual's difficulties and were able to support them more effectively. Adopting a cognitive behavioural approach proved valuable and recommendations for future interventions are made.

Determining virulence towards race‐specific resistance genes is a prerequisite to understanding the response of pathogen populations to resistant cultivars, and therefore to assess the durability of these resistance genes and the performance of resistance management strategies. In Phytophthora infestans , virulence testing began shortly after the introduction of R‐genes from Solanum demissum into S. tuberosum cultivars. However, the characteristics of R‐gene expression, the sensitivity of the phenotype to environmental and physiological parameters, and the diversity of experimental protocols make the comparison of data from different studies problematic. This prompted European teams working on P. infestans diversity to: (i) design a joint protocol, using detached leaflets from greenhouse‐grown plants of a shared set of differential cultivars inoculated with standardized suspensions of inoculum, and (ii) assess the performance of this protocol in a blind ring test involving 12 laboratories and 10 European isolates of the pathogen. A high level of consensus in the determination of virulence/avirulence to R1, R3, R4, R7, R8, R10 and R11 was achieved among the collaborators, showing that the protocol could be robustly applied across a range of laboratories. However, virulence to R2, R5 or R9 was detected more frequently in some laboratories, essentially from northern Europe; these genes are known to be highly sensitive to host and environmental conditions. The consensus determination was often markedly different from the original virulence phenotype of the isolates, suggesting virulence instability in stored P. infestans isolates. This indicates that creating reliable core collections of pathogen isolates with known virulences could be difficult.

The increasing rates of youth unemployment have given rise to concern in academic and political circles as well as amongst the general public. So much so that successive governments have introduced employment schemes specifically for this age group. Although a variety of opinions have been expressed concerning the value of such schemes and much thought and planning has gone into the design of successive generations of schemes, there appears to be little hard evidence concerning the effects they have on the well-being of young people. In the course of a study of the wider aspects of unemployment amongst school leavers, the authors collected evidence which they believe bears directly on this question. In a prospective study, which began by assessing fifth formers before they left school, it was found that in general those who went on to YOP schemes occupied an intermediate position between those who obtained work and those who remained unemployed, on a variety of measures of psychological well-being. However, it was found that those on Schemes were less likely than those in either of the other two groups to believe that they had a measure of personal control over their lives. The findings are discussed in relation to the design of employment schemes and the longer term implications.

AIMS: To conduct a meta-analysis of randomized controlled trials (RCTs) to assess the effect of sodium-glucose cotransporter-2 (SGLT2) inhibitors on inflammatory biomarkers. METHODS: Medline, Embase and the Cochrane Library were searched for RCTs investigating the effect of SGLT2 inhibitors on inflammatory biomarkers, adipokine profiles and insulin sensitivity. RESULTS: Thirty-eight RCTs were included (14 967 participants, 63.3% male, mean age 62 ± 8.6 years) with a median (interquartile range) follow-up of 16 (12-24) weeks. Meta-analysis showed that SGLT2 inhibitors significantly improved adiponectin, interleukin-6, tumour necrosis factor receptor-1 (vs. placebo alone: standardized mean difference [SMD] 0.34 [95% confidence interval {CI} 0.23, 0.45], mean difference [MD] -0.85 pg/mL [95% CI -1.32, -0.38], SMD -0.13 [95% CI -0.20, -0.06], respectively), leptin and homeostatic model assessment of insulin resistance index (vs. CONTROL: SMD -0.20 [95% CI -0.33, -0.07], MD -0.83 [95% CI -1.32, -0.33], respectively). There were no significant changes in C-reactive protein (CRP), tumour necrosis factor-α, plasminogen activator inhibitor-1, fibroblast growth factor-21 or monocyte chemoattractant protein-1. CONCLUSIONS: Our analysis shows that SGLT2 inhibitors likely improve adipokine biomarkers and insulin sensitivity, but there is little evidence that SGLT2 inhibitors improve other inflammatory biomarkers including CRP.

The use of continuous-flow isotope-ratio mass spectrometry (CF-IRMS) as a tool in soil analysis has been assessed as part of a larger study using a number of geological techniques applied in a forensic context. Carbon and nitrogen isotopic ratios, delta13C and delta15N, have been analysed to investigate situations which have arisen from crime casework. Three questions have been addressed: the role of spatial variation found over the short-scale (less than 20 m), temporal variation over a period of almost 2 years, and the variation found between source soils and soil transferred to footwear soles during a simple one-stage transfer process. Results are presented for the three experiments. The use of carbon and nitrogen isotopes has been shown to be useful in discriminating between soil types and sample locations, even when sampling occurs at a different time (as might be the case with a crime scene). In cases of primary transfer (from a source soil by a one-stage transfer to another surface, in this case, shoes and boots), the combination of carbon and nitrogen isotope ratios is a valuable tool in discriminating between sites and in showing the relationship of the transferred samples to the relevant source soils. Used in combination with other analytical techniques, isotopic analysis may prove to be a useful tool in a forensic context.

Abstract A survey examined the differences between managers and direct carers working in community homes for people with intellectual disabilities in terms of the nature and causes of stress. Fifty‐seven managers and 49 direct carers provided data relating to perceived levels of pressure and support from a variety of sources. The questionnaire administered consisted of the Powell questionnaire of sources of stress and support ratings, a stress measure, and information on the background characteristics of participants. Analysis of the data revealed that managers of group homes were reporting higher levels of anxiety and pressure than direct carers. This was associated with managers reporting higher ratings for sources of stress. The implications of these findings are discussed.

This study evaluates the implementation of Safewards on an assessment and treatment unit (ATU) for people with an intellectual disability. There are no previous studies evaluating this model in this context and previous research has focused largely on acute mental health services. The 'Patient-Staff Conflict Shift Report' was used at baseline for 1 month and 1 year later, after all the interventions had been implemented, to evaluate the impact of Safewards. Significant reductions were found in conflict and containment measures used within the service after the implementation of Safewards. Staff who led on the interventions were also asked to give feedback on their experiences, the challenges they faced and how they would like to move forward. Safewards was generally seen as a positive approach by the team. Limitations of this study are highlighted and suggestions for future research are made.

BACKGROUND: Several models of GP out-of-hours provision exist in the UK but there is little detail about their effectiveness to meet users' needs and expectations. AIM: To explore users' needs, expectations, and experiences of out-of-hours care, and to identify proposals for service redesign. SETTING: Service providers in urban (GP cooperative), mixed (hospital based), rural (private) locations in Wales. PARTICIPANTS: Sixty recent service users or carers (20 in each location). METHOD: Semi-structured telephone interviews; thematic analysis. RESULTS: Users' concerns were generally consistent across the three different services. Efficiency was a major concern, with repetitive triage procedures and long time delays at various stages in the process being problematic. Access to a doctor when required was also important to users, who perceived an obstructive gatekeeping function of preliminary contacts. Expectations moderated the relationship between user concerns and satisfaction. Where expectations of outcome were unfulfilled, participants reported greater likelihood of reconsulting with the same or alternative services for the same illness episode. Accurate expectations concerning contacts with the next administrative, nursing, or medical staff professional were managed by appropriate information provision. CONCLUSION: Users require more streamlined and flexible triage systems. Their expectations need to be understood and incorporated into how services advise and provide services for users, and actively managed to meet the aims of both enhancing satisfaction and enabling users to cope with their condition. Better information and education about services are needed if users are to derive the greatest benefit and satisfaction. This may influence choices about using the most appropriate forms of care.

Background In the UK, Early Intensive Behavioural Intervention [EIBI] programmes typically are conducted within the homes of children with autism. Despite evidence for their effectiveness in producing appreciable developmental gains in children with autism, a concern expressed about EIBI programmes is that stressful effects from the high levels of demand they place on family resources could undermine their effectiveness [The Effectiveness of Early Interventions for Children with Autistic Spectrum Disorders (ASD). A report for the DfES South East Regional Special Educational Needs Partnership. SERSEN Website, 2004]. This study investigates the positive impacts and the stressors experienced by families running EIBI programmes. Method Sixteen parents from nine different families participated in semi‐structured qualitative interviews on their experiences of running a home‐based EIBI programme. Data were analysed using the Grounded Theory process. Results Positive and negative impacts of the programmes were reported. Analysis indicated that sources of support obtained through the programmes’ benefits offset sources of stress through the programmes’ demands. Conclusions The interaction between programme demands and benefits and the resources available to each family strongly influences the impact of running a home‐based EIBI programme.

The zebrafish is an important animal system for modeling human diseases. This includes kidney dysfunction as the embryonic kidney (pronephros) shares considerable molecular and morphological homology with the human nephron. A key clinical indicator of kidney disease is proteinuria, but a high-throughput readout of proteinuria in the zebrafish is currently lacking. To remedy this, we used the Tol2 transposon system to generate a transgenic zebrafish line that uses the fabp10a liver-specific promoter to over-express a nanoluciferase molecule fused with the D3 domain of Receptor-Associated Protein (a type of molecular chaperone) which we term NL-D3. Using a luminometer, we quantified proteinuria in NL-D3 zebrafish larvae by measuring the intensity of luminescence in the embryo medium. In the healthy state, NL-D3 is not excreted, but when embryos were treated with chemicals that affected either proximal tubular reabsorption (cisplatin, gentamicin) or glomerular filtration (angiotensin II, Hanks Balanced Salt Solution, Bovine Serum Albumin), NL-D3 is detected in fish medium. Similarly, depletion of several gene products associated with kidney disease (nphs1, nphs2, lrp2a, ocrl, col4a3, and col4a4) also induced NL-D3 proteinuria. Treating col4a4 depleted zebrafish larvae (a model of Alport syndrome) with captopril reduced proteinuria in this system. Thus, our findings validate the use of the NL-D3 transgenic zebrafish as a robust and quantifiable proteinuria reporter. Hence, given the feasibility of high-throughput assays in zebrafish, this novel reporter will permit screening for drugs that ameliorate proteinuria, thereby prioritizing candidates for further translational studies. The zebrafish is an important animal system for modeling human diseases. This includes kidney dysfunction as the embryonic kidney (pronephros) shares considerable molecular and morphological homology with the human nephron. A key clinical indicator of kidney disease is proteinuria, but a high-throughput readout of proteinuria in the zebrafish is currently lacking. To remedy this, we used the Tol2 transposon system to generate a transgenic zebrafish line that uses the fabp10a liver-specific promoter to over-express a nanoluciferase molecule fused with the D3 domain of Receptor-Associated Protein (a type of molecular chaperone) which we term NL-D3. Using a luminometer, we quantified proteinuria in NL-D3 zebrafish larvae by measuring the intensity of luminescence in the embryo medium. In the healthy state, NL-D3 is not excreted, but when embryos were treated with chemicals that affected either proximal tubular reabsorption (cisplatin, gentamicin) or glomerular filtration (angiotensin II, Hanks Balanced Salt Solution, Bovine Serum Albumin), NL-D3 is detected in fish medium. Similarly, depletion of several gene products associated with kidney disease (nphs1, nphs2, lrp2a, ocrl, col4a3, and col4a4) also induced NL-D3 proteinuria. Treating col4a4 depleted zebrafish larvae (a model of Alport syndrome) with captopril reduced proteinuria in this system. Thus, our findings validate the use of the NL-D3 transgenic zebrafish as a robust and quantifiable proteinuria reporter. Hence, given the feasibility of high-throughput assays in zebrafish, this novel reporter will permit screening for drugs that ameliorate proteinuria, thereby prioritizing candidates for further translational studies. Translational StatementThe zebrafish has become a useful tool for modeling kidney disease. However, proteinuria, an important clinical indicator of kidney dysfunction, is not easily detected in the zebrafish system. Here, we describe a novel transgenic zebrafish line that measures proteinuria associated with both glomerular and tubular dysfunction. This new resource will impact future treatments for patients by permitting rapid and economical preclinical screening of drugs to treat kidney disease associated with glomerular or tubular dysfunction. The zebrafish has become a useful tool for modeling kidney disease. However, proteinuria, an important clinical indicator of kidney dysfunction, is not easily detected in the zebrafish system. Here, we describe a novel transgenic zebrafish line that measures proteinuria associated with both glomerular and tubular dysfunction. This new resource will impact future treatments for patients by permitting rapid and economical preclinical screening of drugs to treat kidney disease associated with glomerular or tubular dysfunction. Proteinuria is a key clinical indicator of kidney disease.1National Kidney FoundationK/DOQI clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification.Am J Kidney Dis. 2002; 39: S1-266PubMed Google Scholar Using urine dipsticks, the presence of protein in the urine can be determined in seconds. Further evaluation of the molecular size of proteins in the urine indicates the site of functional deficit along the nephron; the presence of large proteins in the urine suggests dysfunction in the glomerular filter, whereas the presence of low-molecular-weight proteins indicates proximal tubule dysfunction.2Butt L. Unnersjö-Jess D. Höhne M. et al.A molecular mechanism explaining albuminuria in kidney disease.Nat Metab. 2020; 2: 461-474Crossref PubMed Scopus (59) Google Scholar,3Lawrence M.G. Altenburg M.K. Sanford R. et al.Permeation of macromolecules into the renal glomerular basement membrane and capture by the tubules.Proc Natl Acad Sci U S A. 2017; 114: 2958-2963Crossref PubMed Scopus (78) Google Scholar The zebrafish is a popular model organism for the study of kidney development and disease,4Outtandy P. Russell C. Kleta R. Bockenhauer D. Zebrafish as a model for kidney function and disease.Pediatr Nephrol. 2019; 34: 751-762Crossref PubMed Scopus (50) Google Scholar, 5Poureetezadi S.J. Wingert R.A. Little fish, big catch: zebrafish as a model for kidney disease.Kidney Int. 2016; 89: 1204-1210Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 6Morales E.E. Wingert R.A. Zebrafish as a model of kidney disease.Results Probl Cell Differ. 2017; 60: 55-75Crossref PubMed Scopus (43) Google Scholar but a quantitative reporter of proteinuria is currently lacking. Embryonic zebrafish develop a functioning pronephric kidney that has molecular and structural analogy to a human nephron.7Drummond I.A. Majumdar A. Hentschel H. et al.Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function.Development. 1998; 125: 4655-4667Crossref PubMed Google Scholar The pronephros consists of a single midline fused glomerulus attached to bilateral tubules that are segmented along their anterior-posterior axis into distinct proximal and distal domains.7Drummond I.A. Majumdar A. Hentschel H. et al.Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function.Development. 1998; 125: 4655-4667Crossref PubMed Google Scholar,8Wingert R.A. Selleck R. Yu J. et al.The cdx genes and retinoic acid control the positioning and segmentation of the zebrafish pronephros.PLoS Genet. 2007; 3: 1922-1938Crossref PubMed Scopus (247) Google Scholar Until recently, the best method of determining kidney function in zebrafish embryos was the use of fluorescent dextrans of varying molecular weights that distinguish between glomerular and proximal tubule dysfunction.9Christou-Savina S. Beales P.L. Osborn D.P.S. Evaluation of zebrafish kidney function using a fluorescent clearance assay.J Vis Exp. 2015; 96e52540Google Scholar However, fluorescent dextran is not specific to the cellular machinery of protein endocytosis; it is passively internalized in the fluid bulk. As such, these methods are useful, but they are not quantitative, do not specifically identify megalin-mediated endocytosis defects in the proximal tubule, and cannot be used in a high-throughput manner. Recently, a zebrafish transgenic reporter (fabp10a:½vdbp-mCherry) has enabled the embryo medium to be collected and assayed for low-molecular-weight proteins.10Chen Z. Luciani A. Mateos J.M. et al.Transgenic zebrafish modeling low-molecular-weight proteinuria and lysosomal storage diseases.Kidney Int. 2020; 97: 1150-1163Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar This powerful system permits the quantification of proteinuria as a readout of dysfunction of the lrp2/megalin endocytosis pathway in the proximal tubule. It reports proximal tubular dysfunction after downstream processing with immunofluorescence imaging or enzyme-linked immunosorbent assays to quantify protein uptake in the proximal tubules and proteinuria. This genetic tool highlights the potential of transgenics to develop the reporters of proteinuria in the zebrafish. Here, we describe a novel nanoluciferase (NL)-based reporter of proteinuria in zebrafish. Excreted NL protein in the embryo medium is detected by luminescence after simple addition of its substrate luciferin. With this new system, perturbations in both proximal tubular and glomerular function can be assayed, and its simple and easy application makes it amenable to use in a high-throughput manner. For detailed methods see Supplementary Methods. Zebrafish were maintained and staged according to established protocols11Kimmel C.B. Ballard W.W. Kimmel S.R. et al.Stages of embryonic development of the zebrafish.Dev Dyn. 1995; 203: 253-310Crossref PubMed Scopus (8997) Google Scholar and in accordance with the project licenses of Martin Lowe (70/9091) and Rachel Lennon (P1AE9A736) under the current guidelines of the UK Animals Act 1986. Embryos were injected at the stage indicated in the text and were treated with drugs/fluorescent tracers by either incubation in the embryo medium (after dechorionation) or microinjection into the common cardinal vein. For morpholino treatments, final concentrations of 0.15 mM (nphs1MOex25) and 0.25 mM (nphs2MOex3) were used. For lrp2a and ocrl knockdowns, a p53 morpholino was also coinjected at the same concentration (0.24 mM). CRISPR-Cas9 depletion is described in the paper by Wu et al.12Wu R.S. Lam I.I. Clay H. et al.A rapid method for directed gene knockout for screening in G0 zebrafish.Dev Cell. 2018; 46: 112-125.e4Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar gRNAs were resuspended in nuclease-free water to a concentration of 20 μM. For the injection mix, 4 μM of each gRNA was combined with Cas9 (NEB #M0646) and Cas9 buffer. Embryos were grown to 4 days post fertilization (dpf); then 3 embryos per well were placed in 1 well of a 96-well dish. The E3 embryo medium was removed and replaced with 200 μl of fresh E3. μl of E3 medium was removed each well and placed in the well of a fresh 96-well of substrate the was then to each were then at for 1 and then assayed for luminescence a 3 in zebrafish embryos was as C. in to zebrafish 3: PubMed Scopus Google Scholar were using for zebrafish col4a3, and were by 4 zebrafish embryos were a and were an zebrafish embryos were in or at 4 embryos were then in after in were then a and for The used were and were collected a using a The were as 1 and were collected using the with were according to described S. et the of glomerular 2016; PubMed Scopus Google Scholar were a were in for were and to the of the glomerular basement membrane The of for each control col4a4 and For per were used and the was The of along this was and then this was by the The was and a was using for to develop a transgenic reporter to study endocytosis in the zebrafish pronephric proximal tubule. important of in the proximal tubule is the which has H. and Cell 2002; 3: PubMed Scopus Google Scholar function and processing is H. H. et protein is important for processing of in kidney proximal Nephrol. PubMed Google Scholar which that with varying to M. et of the Full Text Full Text PDF PubMed Scopus Google Scholar For our reporter we fused protein to A a protein that is in size that the size of the reporter protein its the glomerular and we fused the D3 domain of protein to the of NL to which has a size of to that the NL-D3 protein For this we NL-D3 in and used to the protein The NL-D3 was then used in uptake a of the and the of 4 large was by the and measuring and this that the with NL-D3 of uptake with control with NL-D3 control luminescence after incubation with NL in that NL-D3 is by and To develop a in reporter of proximal tubule we to the zebrafish used the Tol2 gene and methods in zebrafish.Dev Dyn. PubMed Scopus Google Scholar to generate transgenic zebrafish that NL-D3 under the control of the liver-specific fabp10a promoter To the under the control of the promoter embryo transgenic zebrafish in luminescence embryos A in luminescence was in NL-D3 reporter fish also the of NL-D3 in with the luminescence in the that liver-specific of NL-D3 in transgenic zebrafish in the robust of this reporter into the of NL-D3 transgenic zebrafish and not impact fish or analysis of NL-D3 zebrafish and To NL-D3 the glomerular and is by the pathway in the pronephric proximal tubule, we lrp2a gene that for using a described morpholino S. et of in the zebrafish Cell PubMed Scopus Google Scholar of a fluorescent dextran in proximal tubules was as or of lrp2a was to uptake of a fluorescent dextran in proximal tubules when with that these embryos proximal tubule For the NL-D3 proteinuria lrp2a at 4 were placed in 1 well of a 96-well the embryo medium was and embryos were further NL-D3 in the embryo medium was a that 3 embryos per well in the luminescence with 1 embryo per well also luminescence of the embryo medium lrp2a after 1 4 and for each This that the NL-D3 reporter is to proteinuria after as as 1 but the between and was at we used this in our luminescence the were to NL-D3 using a NL-D3 of lrp2a an in NL-D3 in the embryo medium gene in proximal tubule endocytosis is and Lowe which is associated with low-molecular-weight D. A. et in Lowe a proximal tubular J Nephrol. 3: PubMed Scopus Google L. The in Lowe and disease Nephrol. 2017; PubMed Scopus Google Scholar of the zebrafish ocrl using a et development in a zebrafish model for Lowe Genet. PubMed Scopus Google et al.The Lowe protein is for endocytosis in the zebrafish pronephric Genet. 2015; PubMed Scopus Google Scholar a in fluorescent dextran uptake in the proximal tubule and induced NL-D3 proteinuria also the of that proximal tubules and proteinuria. and are drugs that are that kidney by proximal tubule and they used to the zebrafish proximal tubule L. et renal in a novel system to study a J PubMed Scopus Google Scholar The analysis of NL-D3 into the embryo medium that of proteinuria by with Treating embryos with the of further the proteinuria to that a is in the NL-D3 reporter. A was with a an in proteinuria, and a an these the NL-D3 transgenic fish as a tool for proteinuria to proximal tubular dysfunction after genetic or further the of the of glomerular specific genes to the NL-D3 reporter also be used to proteinuria associated with glomerular dysfunction. and for proteins and that the in these genes which is by proteinuria. depletion of and in zebrafish has in human patients with in and et and are between the zebrafish pronephros and PubMed Scopus Google S. I.A. of the pronephric filtration in zebrafish and the domain protein PubMed Scopus Google Scholar Using morpholino to used by et et and are between the zebrafish pronephros and PubMed Scopus Google Scholar we clearance of a dextran the and that these glomerular dysfunction. also NL-D3 in the embryo medium zebrafish depleted of either or with control that the NL-D3 reporter as a reporter of both glomerular and proximal tubular dysfunction analysis of glomerular development in the zebrafish pronephros suggests that a filtration is established at 4 Z. C. et of the pronephric glomerulus development in 2016; PubMed Scopus Google Scholar tubule this, with a and segmentation by post fertilization I.A. Majumdar A. Hentschel H. et al.Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function.Development. 1998; 125: 4655-4667Crossref PubMed Google Scholar,8Wingert R.A. Selleck R. Yu J. et al.The cdx genes and retinoic acid control the positioning and segmentation of the zebrafish pronephros.PLoS Genet. 2007; 3: 1922-1938Crossref PubMed Scopus (247) Google I.A. Zebrafish kidney Cell PubMed Scopus Google Scholar the NL-D3 reporter be used at of development to proteinuria associated with proximal tubule dysfunction and ocrl or glomerular dysfunction that proteinuria is in the reporter between and in lrp2a or ocrl of the same embryos between and a in proteinuria in lrp2a but embryos depleted of ocrl or not proteinuria that the NL-D3 line can proteinuria associated with tubular dysfunction but glomerular dysfunction and tubular dysfunction are not The potential for the NL-D3 reporter line to glomerular dysfunction is of given the of kidney associated with glomerular is Alport which is by in or in genes that to the which is to the The is to to the it to the by J. Alport its the glomerular filtration and for future Scopus Google Scholar, et renal and renal in with Alport Int. Full Text Full Text PDF PubMed Scopus Google Scholar, D. L. et gene to Alport glomerular disease.Kidney Int. Full Text Full Text PDF PubMed Scopus (54) Google Scholar in or to reduced or in the affecting its with Alport with by proteinuria and a in kidney function to kidney The of type in the zebrafish glomerulus has not Zebrafish type which are of the type genes in in to the of for in 4 zebrafish a when the glomerulus is Z. C. et of the pronephric glomerulus development in 2016; PubMed Scopus Google Scholar of and were which is given that type gene is for and R. M. et al.The structural genes for 1 and of human type are and an promoter Full Text PDF PubMed Google R. The genes for the and of human basement membrane type are and by a promoter of J. PubMed Scopus Google Scholar and were detected in the in the and in the for was in embryos In these also pronephric and cardinal with and and col4a4 were of for and col4a4 were in the and the as well as in the of and for in the and Using in we were to of in the using This is in the of type that the and 1 can with 1 J. M. et of type in and human kidney and in renal and J PubMed Scopus Google Scholar, M.G. et of a distinct type with kidney and of its gene to the of Alport Natl Acad Sci U S A. PubMed Scopus Google Scholar, A. et of the glomerular basement that the of is by the Full Text Full Text PDF PubMed Scopus Google Scholar Zebrafish are to human type H. in the zebrafish the type 2007; PubMed Scopus Google Scholar and it that the zebrafish a to our to by in further analysis is to is in the zebrafish pronephric To for the presence of type protein in the we immunofluorescence with a type 4 larvae of type was in the as well as in the distinct glomerular of and col4a4 in the zebrafish, we to these genes using a CRISPR-Cas9 method that in embryos R.S. Lam I.I. Clay H. et al.A rapid method for directed gene knockout for screening in G0 zebrafish.Dev Cell. 2018; 46: 112-125.e4Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar This induced in and col4a4 when by of by Supplementary and analysis However, the single analysis the findings of Wu et R.S. Lam I.I. Clay H. et al.A rapid method for directed gene knockout for screening in G0 zebrafish.Dev Cell. 2018; 46: 112-125.e4Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar of in 4 gRNAs were not with this 1 gRNA in of the 3 in the site for of and gRNAs in it is that a of our and are of in Alport C. et of and of Alport 2020; PubMed Scopus Google Scholar analysis that for and col4a4 were reduced when each gene was using gRNAs analysis of the glomerular filtration by defects in the glomerular of the of and see Supplementary of that and col4a4 and was glomerular in human patients with Alport and knockout of Alport D. et a model for Alport PubMed Scopus Google and functional defects in of for Alport Cell PubMed Scopus Google Scholar to the and zebrafish model of proteinuria, which is a of Alport of or col4a4 by CRISPR-Cas9 in clearance of a dextran glomerular dysfunction in these determined or col4a4 proteinuria in our transgenic NL-D3 reporter This analysis that of either or col4a4 in zebrafish induced proteinuria for for col4a4 when with embryos In of either or col4a4 defects in the glomerular filtration and induced proteinuria, the of in Alport genes in both knockout and human that the zebrafish NL-D3 transgenic reporter line can be used for glomerular dysfunction proteinuria is a key clinical as in Alport we to glomerular filtration using to the potential for this system for col4a4 were used as a model of Alport for these In col4a4 captopril was used 3 to to and we assayed for proteinuria 4 to is an and has used in zebrafish to et in zebrafish is associated with and the pathway and J 2015; PubMed Scopus Google we that also that captopril zebrafish embryos not but proteinuria in col4a4 also used to the in zebrafish embryos 3 to that a to reduced glomerular thereby glomerular In zebrafish a analysis that at a concentration of μM was to of col4a4 with this concentration of proteinuria in these to a that was but col4a4 Using the NL-D3 reporter we also detected the of zebrafish is a that by that μM not proteinuria in NL-D3 μM in an in proteinuria Similarly, injection of or a in proteinuria in the NL-D3 reporter is a that injection water into the and has to glomerular S. et membrane defects in glomerular disease.Pediatr Nephrol. PubMed Scopus Google Scholar Thus, we that the injection of an in the glomerulus that proteinuria. In of this, we clearance of a dextran In NL-D3 we detected an in luminescence is given in the clinical to the of a concentration of into zebrafish embryos was to glomerular an in clearance of both a dextran and NL-D3 proteinuria which was these that the NL-D3 reporter line can proteinuria associated with in and glomerular also the potential of this new tool in kidney function Here, we describe a new high-throughput tool for the quantification of proteinuria in the zebrafish that the NL-D3 transgenic reporter can in both glomerular and proximal tubular into the impact of and that in proteinuria can be the of our NL-D3 it is to in future and the NL-D3 reporter to with the that the reporter is a transgenic system, and in of the be To the impact of we used 3 embryos per well of 1 embryo per these measures the in embryos was of the The of the NL system also that embryos or are the embryo medium these not be To the impact of we also used 96-well to luminescence in also that the NL-D3 reporter is at tubular dysfunction by lrp2a depletion to NL-D3 proteinuria between and However, glomerular dysfunction by depletion of not in NL-D3 proteinuria at this This is to the glomerulus not 4 and we that the use of the NL-D3 reporter be to for glomerular studies. we not NL-D3 proteinuria in ocrl between and between 4 and This highlights the to at a of to that are when using the NL-D3 proteinuria reporter system. that the NL-D3 protein to the glomerular and be to be in the proximal tubule megalin-mediated However, the glomerulus also as a to NL-D3 given that we genetic or of glomerular filtration an in the presence of NL-D3 in the embryo medium. filtration of NL-D3 the glomerular function to which was to be to the filter, but has a A. H. of proteins in using J Google Scholar suggests that the and the of are important for the size of the glomerular L. Unnersjö-Jess D. Höhne M. et al.A molecular mechanism explaining albuminuria in kidney disease.Nat Metab. 2020; 2: 461-474Crossref PubMed Scopus (59) Google Scholar,3Lawrence M.G. Altenburg M.K. Sanford R. et al.Permeation of macromolecules into the renal glomerular basement membrane and capture by the tubules.Proc Natl Acad Sci U S A. 2017; 114: 2958-2963Crossref PubMed Scopus (78) Google Scholar Thus, we that these of NL-D3 simple the glomerular in the healthy However, when we see in our of Alport the and are and NL-D3 is to the filter, the reabsorption in the tubules and to the proteinuria we In our we a new model of Alport that of the disease in human The in the glomerulus of Alport zebrafish were not as as in human this is to the embryos days depletion of or col4a4 in zebrafish is a model of the of Alport that and col4a4 are in the zebrafish glomerulus by in were to in the glomerulus by in which be to the as However, we cannot that zebrafish are distinct in their of this is as specific human and zebrafish when their are H. in the zebrafish the type 2007; PubMed Scopus Google Scholar their and of the human suggests that and are to with 1 A. et of the glomerular basement that the of is by the Full Text Full Text PDF PubMed Scopus Google Scholar will be to is in the pronephric glomerulus in zebrafish, and it is then an of which type in the pronephric at this stage into the of type and their in For this we used a gRNA to in R.S. Lam I.I. Clay H. et al.A rapid method for directed gene knockout for screening in G0 zebrafish.Dev Cell. 2018; 46: 112-125.e4Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar which is not to to each gene to 4 and the of However, the can to H. in the zebrafish the type 2007; PubMed Scopus Google Scholar and the of and col4a4 suggests that a of Alport zebrafish will be to and these be used to the in A line also be used for a to chemicals that ameliorate proteinuria associated with Alport In our the development of a reporter for proteinuria in the zebrafish system. the of this system in of and the feasibility of high-throughput we that this new proteinuria reporter will be a addition to the of kidney the The the in the for with zebrafish The used in this study were with and the of to and for their with the The also the in the in the of and for their and the for to the for the were in under the of Rachel with Supplementary

BACKGROUND: In the light of recent changes in the structure and provision of out-of-hours service in the UK, there is a need to re-assess the quality of care. One way to assess the quality of care is through patient experience. OBJECTIVES: This study aimed to explore patient expectations and help-seeking behaviour, in order to understand their relationship with satisfaction and experience of out-of-hours care. METHODS: 30 semistructured telephone interviews were carried out with users of the general practitioner out-of-hours service in Gwent, South Wales. The interviews explored users' experiences of using the service. A thematic analysis of transcripts was carried out using NUD*IST software. Comparison of data within and across codes facilitated the identification of explanatory constructs. Double coding of a sample of transcripts and discussion of emerging themes by members of the research team ensured the reliability of findings. RESULTS: Most (n = 25, 83%) respondents reported satisfaction with the overall service received but a few (n = 5, 17%) were dissatisfied. Patients generally had specific expectations of their consultation and there was a mismatch between patients' expectations of the service and what the service actually provides in some specific user groups. Unmet expectations resulted in subsequent, and in some cases, multiple consultations. CONCLUSIONS: Users' views and expectations may be used to inform service design and improve services, but the data also indicated a need to address user expectations of services, for example by enhanced information provision. Any such interventions to improve patient experience of out-of-hours care will need to be evaluated.