Phosphatidylinositol-4,5-bisphosphate is required for KCNQ1/KCNE1 channel function but not anterograde trafficking

The slow delayed-rectifier potassium current (IKs) is crucial for human cardiac action potential repolarization. The formation of IKs requires co-assembly of the KCNQ1 α-subunit and KCNE1 β-subunit, and mutations in either of these subunits can lead to hereditary long QT syndrome types 1 and 5, respectively. It is widely recognised that the KCNQ1/KCNE1 (Q1/E1) channel requires phosphatidylinositol-4,5-bisphosphate (PIP2) binding for function. We previously identified a cluster of basic residues in the proximal C-terminus of KCNQ1 that form a PIP2/phosphoinositide binding site. Upon charge neutralisation of these residues we found that the channel became more retained in the endoplasmic reticulum, which raised the possibility that channel-phosphoinositide interactions could play a role in channel trafficking. To explore this further we used a chemically induced dimerization (CID) system to selectively deplete PIP2 and/or phosphatidylinositol-4-phosphate (PI(4)P) at the plasma membrane (PM) or Golgi, and we subsequently monitored the effects on both channel trafficking and function. The depletion of PIP2 and/or PI(4)P at either the PM or Golgi did not alter channel cell-surface expression levels. However, channel function was extremely sensitive to the depletion of PIP2 at the PM, which is in contrast to the response of other cardiac potassium channels tested (Kir2.1 and Kv11.1). Surprisingly, when using the CID system IKs was dramatically reduced even before dimerization was induced, highlighting limitations regarding the utility of this system when studying processes highly sensitive to PIP2 depletion. In conclusion, we identify that the Q1/E1 channel does not require PIP2 or PI(4)P for anterograde trafficking, but is heavily reliant on PIP2 for channel function once at the PM.: This work was funded by the British Heart Foundation (BHF). BHF grant numbers: [FS/12/21/ 29482 and RG/15/15/31742]. S.C.H is supported by a BHF Intermediate Basic Science Research Fellowship [FS/12/59/29756]. The work was facilitated by The National Institute for Health Research Barts Biomedical Research Centre

Social Class, Gender and Ethnic Differences in Subjects Taken at Age 14

In this paper we identify patterns of subject and qualification choices made at age 14. Much of the previous research on ‘subject choice’ has focussed on the later stages of educational trajectories, particularly Higher Education. However, the choices made at early branching points can limit pupils’ subsequent options, potentially contributing to educational inequalities. This paper identifies the patterns of General Certificate of Secondary Education (GCSE) subjects chosen by a cohort of young people born in 1989/1990. We make use of the Next Steps data (formerly the Longitudinal Study of Young People in England (LSYPE)) which is linked to the National Pupil Database. We develop an approach to measuring the academic selectivity of subjects and qualifications. We examine the roles of social class, parental education, income, gender and ethnicity in determining participation in these curriculum groupings. Using measures of prior attainment from age thirteen, we address the question of whether curriculum differentials simply reflect differences in prior attainment or whether they actually operate above and beyond existing inequalities. We find clear socio-economic, gender, ethnic and school-level differences in subjects studied which cannot be accounted for by prior attainment

LEARN: A multi-centre, cross-sectional evaluation of Urology teaching in UK medical schools

OBJECTIVE: To evaluate the status of UK undergraduate urology teaching against the British Association of Urological Surgeons (BAUS) Undergraduate Syllabus for Urology. Secondary objectives included evaluating the type and quantity of teaching provided, the reported performance rate of General Medical Council (GMC)-mandated urological procedures, and the proportion of undergraduates considering urology as a career. MATERIALS AND METHODS: LEARN was a national multicentre cross-sectional study. Year 2 to Year 5 medical students and FY1 doctors were invited to complete a survey between 3rd October and 20th December 2020, retrospectively assessing the urology teaching received to date. Results are reported according to the Checklist for Reporting Results of Internet E-Surveys (CHERRIES). RESULTS: 7,063/8,346 (84.6%) responses from all 39 UK medical schools were included; 1,127/7,063 (16.0%) were from Foundation Year (FY) 1 doctors, who reported that the most frequently taught topics in undergraduate training were on urinary tract infection (96.5%), acute kidney injury (95.9%) and haematuria (94.4%). The most infrequently taught topics were male urinary incontinence (59.4%), male infertility (52.4%) and erectile dysfunction (43.8%). Male and female catheterisation on patients as undergraduates was performed by 92.1% and 73.0% of FY1 doctors respectively, and 16.9% had considered a career in urology. Theory based teaching was mainly prevalent in the early years of medical school, with clinical skills teaching, and clinical placements in the later years of medical school. 20.1% of FY1 doctors reported no undergraduate clinical attachment in urology. CONCLUSION: LEARN is the largest ever evaluation of undergraduate urology teaching. In the UK, teaching seemed satisfactory as evaluated by the BAUS undergraduate syllabus. However, many students report having no clinical attachments in Urology and some newly qualified doctors report never having inserted a catheter, which is a GMC mandated requirement. We recommend a greater emphasis on undergraduate clinical exposure to urology and stricter adherence to GMC mandated procedures

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK.

BACKGROUND: A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. METHODS: This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. FINDINGS: Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0-75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4-97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8-80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3-4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. INTERPRETATION: ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials. FUNDING: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, Bill & Melinda Gates Foundation, Lemann Foundation, Rede D'Or, Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK

Background A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. Methods This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. Findings Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0–75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4–97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8–80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3–4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. Interpretation ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials

Charge-neutralising mutations in a PIP₂-binding region in the proximal C-terminus of KCNQ1 increase retention of the channel complex in the ER.

<p><b>A</b>. Representative confocal images of the localisation of WT and mutant KCNQ1-GFP channel complexes in CHO-K1 cells. The left panel shows the GFP-tagged KCNQ1 subunit (GFP), the middle panel shows DsRed2-ER, an ER marker, and the right panel shows the merged images. In the merged image panel, the presence of yellow indicates colocalisation between KCNQ1-GFP and DsRed2-ER. Top row: WT KCNQ1-GFP with KCNE1. Lower rows: Localisation of the mutant KCNQ1 channels investigated (all n = 25, except KCNQ1(R360A-GFP), where n = 24). Scale bar indicates 20 μm. <b>B</b>. Quantified data showing the proportion of ER colocalisation of the WT and mutant KCNQ1-GFP subunits (in all cases KCNE1 was co-expressed). Data are presented as mean ± S.E.M. * indicates significant difference (<i>P</i> <0.05) from control (KCNQ1-GFP + KCNE1) value.</p

Depletion of PI(4)P at the Golgi does not affect the cell-surface or total expression level of KCNQ1.

<p><b>A</b> and <b>B</b>. Representative on-cell [4°C fixed] and in-cell western assays, enabling quantification of the cell-surface and total cellular expression, respectively. The VSV-E1-Q1 construct was co-expressed in HEK293 cells with pcDNA3.1, or with Tgn38-FRB and PJ-SAC or PJ-DEAD. Cells expressing Tgn38-FRB with either PJ-SAC or PJ-DEAD were incubated in the absence of 5 μM rapamycin (Rap), or in the presence of Rap for 1 hour or 24 hours as indicated. Each condition was performed in triplicate. Mean data, normalised to VSV-E1-Q1 + pcDNA3.1 values (VSV Control), from three independent experiments for cell-surface and total channel expression are shown in <b>C</b> and <b>D</b>, respectively. Data presented as mean ± S.E.M. * indicates significant difference (<i>P</i> <0.05) from control (VSV-E1-Q1 + pcDNA3.1) values.</p

Measuring and manipulating the cell-surface and total expression level of KCNQ1.

<p><b>A</b> and <b>B</b>. Representative on-cell [37°C live] and in-cell western assays, enabling quantification of the cell-surface and total channel expression of the VSV-KCNE1-KCNQ1 (VSV-E1-Q1) channel, respectively. The VSV-E1-Q1 construct was co-expressed in HEK293 cells with either pcDNA3.1, SAR1-H79G or KCNQ1-E261D. Cells expressing VSV-E1-Q1 with pcDNA3.1 were incubated with either 10 μM wortmannin (WTM) for 1 hour or 5 μM brefeldin A (Brf A) for 24 hours before the start of the assay. Each condition was performed in triplicate. Mean data, normalised to VSV-E1-Q1 + pcDNA3.1 values (VSV Control), from three independent experiments for cell-surface and total channel expression are shown in <b>C</b> and <b>D</b>, respectively. Data presented as mean ± S.E.M. * indicates significant difference (<i>P</i> <0.05) from control (VSV-E1-Q1 + pcDNA3.1) value.</p

The expression of lipid-depleting dimerization constructs dramatically reduces <i>I</i>_Ks prior to rapamycin addition.

<p><b>A</b>. Representative traces of currents recorded from HEK-<i>I</i>_Ks cells (control), and HEK-<i>I</i>_Ks cells transiently expressing PJ-DEAD, PJ-SAC, PJ-INPP5E or PJ with LYN₁₁-FRB. Mean CD <b>(B)</b> and PTCD <b>(C)</b> of currents from HEK-<i>I</i>_Ks cells transiently expressing PJ (n = 11), PJ-SAC (n = 23), PJ-INPP5E (n = 24) or PJ-DEAD (n = 16) with LYN₁₁-FRB. <b>D</b>. Representative traces of the effect of PJ expression alone (without LYN₁₁-FRB) on <i>I</i>_Ks in HEK-<i>I</i>_Ks cells. <b>E</b>. Effect of PJ expression alone (without LYN₁₁-FRB) on <i>I</i>_Ks PTCD in HEK-<i>I</i>_Ks cells (PJ-expressing cells, n = 12; control (untransfected cells), n = 12). Data are presented as mean ± S.E.M. * indicates significant difference (<i>P</i> <0.05) from control (untransfected cells) value.</p

The localisation of the PIP₂ sensor, Tubby-YFP, in HEK293 cells before and after the rapamycin-induced recruitment of PJ or PJ-DEAD to the PM.

<p>The rapamycin (Rap)-induced dimerization of PJ and PJ-DEAD with PM-localised LYN₁₁-FRB was investigated in transiently transfected HEK293 cells. Tubby-YFP localises to the PM in the presence of a sufficient PIP₂ concentration. <b>A</b> and <b>B</b>. Top panel: Before rapamycin addition. Lower panels: Increasing time after addition of rapamycin showing the increased PM signal of PJ and PJ-DEAD. Upon recruitment of PJ to the PM, Tubby-YFP redistributes from the PM to the cytosol (<b>A</b>). Upon recruitment of PJ-DEAD to the PM, Tubby-YFP remains PM-localised (<b>B</b>). Scale bar indicates 15 μm. <b>C–F</b>. Quantified line (<b>C</b> and <b>E</b>) and box plots (<b>D</b> and <b>F</b>) from the indicated red lines and boxed areas in <b>A</b> and <b>B</b> (located in the top right hand merged panels), highlighting translocation of Tubby-YFP to the cytosol when PJ (<b>C</b> and <b>D</b>) but not PJ-DEAD (<b>E</b> and <b>F</b>) is recruited to the PM. Red arrows indicate rapamycin addition.</p