Investigating the role of BEST1 protein in Best vitelliform macular dystrophy using a novel, disease-specific human embryonic stem cell line

Best vitelliform macular dystrophy (BVMD) is a rare form of macular dystrophy involving degeneration of the retinal pigment epithelium (RPE). It is characterised by accumulation of fluid and lipofuscin in the macula region, and consequently shares similarities with one of the leading causes of blindness, age-related macular degeneration. BVMD is known to be caused by mutations in the BEST1 gene, which encodes for the Bestrophin-1 (BEST1) protein. Over 100 BVMD-causing BEST1 mutations have been reported including the F305S mutation. The aim of this thesis was to characterise a new and unreported human embryonic stem (ES) cell line containing the F305S BEST1 mutation to better understand the molecular pathology of BVMD. The first experimental chapter focused on characterising the pluripotent properties of the F305S-mutant BEST1 human ES cell line and its capacity to differentiate into RPE. The results revealed the F305S-mutant BEST1 human ES cells were highly similar to normal human ES cells, and that RPE derived from both normal and F305S-mutant BEST1 human ES cells displayed defining RPE properties including: pigmentation, polygonal morphology and expression of important RPE genes and proteins. A key finding was that BEST1 protein in the F305S-mutant BEST1 RPE was shown to be localised predominantly within the cells compared to BEST1 in the normal RPE, which was localised predominantly at the plasma membrane. In the second experimental chapter, whole-cell patch clamp electrophysiology showed that RPE derived from the F305S-mutant BEST1 human ES cells had similar voltage-gated sodium and potassium currents compared to RPE derived from normal human ES cells. As heterologous overexpression systems suggest BEST1 protein may regulate chloride (Cl-) and calcium (Ca2+) movements, the third experimental chapter used whole-cell patch clamp to compare Cl- and Ca2+ currents in RPE derived from normal and F305S-mutant human ES cells. These data revealed reduced Cl- and Ca2+ conductance in the F305S-mutant BEST1 RPE compared to normal human ES cell-derived RPE. These data suggest that in human RPE BEST1 can i) act as a Ca2+-activated Cl- channel, and ii) regulate intracellular Ca2+. Importantly, while these data are consistent with data from heterologous overexpression systems, they are also biologically more relevant as they were obtained from human RPE expressing endogenous levels of BEST1 protein without BEST1 overexpression. Overall, the data presented in this thesis begin to define the molecular consequences of BEST1 mutation on both Ca2+ and Cl- conductance in human RPE. The data also indicate further investigation of human ES cell-derived RPE will provide new molecular insights and potentially identify different treatment options for BVMD and age-related macular degeneration

Bronchiectasis in India:results from the European Multicentre Bronchiectasis Audit and Research Collaboration (EMBARC) and Respiratory Research Network of India Registry

BACKGROUND: Bronchiectasis is a common but neglected chronic lung disease. Most epidemiological data are limited to cohorts from Europe and the USA, with few data from low-income and middle-income countries. We therefore aimed to describe the characteristics, severity of disease, microbiology, and treatment of patients with bronchiectasis in India. METHODS: The Indian bronchiectasis registry is a multicentre, prospective, observational cohort study. Adult patients ( 6518 years) with CT-confirmed bronchiectasis were enrolled from 31 centres across India. Patients with bronchiectasis due to cystic fibrosis or traction bronchiectasis associated with another respiratory disorder were excluded. Data were collected at baseline (recruitment) with follow-up visits taking place once per year. Comprehensive clinical data were collected through the European Multicentre Bronchiectasis Audit and Research Collaboration registry platform. Underlying aetiology of bronchiectasis, as well as treatment and risk factors for bronchiectasis were analysed in the Indian bronchiectasis registry. Comparisons of demographics were made with published European and US registries, and quality of care was benchmarked against the 2017 European Respiratory Society guidelines. FINDINGS: From June 1, 2015, to Sept 1, 2017, 2195 patients were enrolled. Marked differences were observed between India, Europe, and the USA. Patients in India were younger (median age 56 years [IQR 41-66] vs the European and US registries; p<0\ub70001]) and more likely to be men (1249 [56\ub79%] of 2195). Previous tuberculosis (780 [35\ub75%] of 2195) was the most frequent underlying cause of bronchiectasis and Pseudomonas aeruginosa was the most common organism in sputum culture (301 [13\ub77%]) in India. Risk factors for exacerbations included being of the male sex (adjusted incidence rate ratio 1\ub717, 95% CI 1\ub703-1\ub732; p=0\ub7015), P aeruginosa infection (1\ub729, 1\ub710-1\ub750; p=0\ub7001), a history of pulmonary tuberculosis (1\ub720, 1\ub707-1\ub734; p=0\ub7002), modified Medical Research Council Dyspnoea score (1\ub732, 1\ub725-1\ub739; p<0\ub70001), daily sputum production (1\ub716, 1\ub703-1\ub730; p=0\ub7013), and radiological severity of disease (1\ub703, 1\ub701-1\ub704; p<0\ub70001). Low adherence to guideline-recommended care was observed; only 388 patients were tested for allergic bronchopulmonary aspergillosis and 82 patients had been tested for immunoglobulins. INTERPRETATION: Patients with bronchiectasis in India have more severe disease and have distinct characteristics from those reported in other countries. This study provides a benchmark to improve quality of care for patients with bronchiectasis in India. FUNDING: EU/European Federation of Pharmaceutical Industries and Associations Innovative Medicines Initiative inhaled Antibiotics in Bronchiectasis and Cystic Fibrosis Consortium, European Respiratory Society, and the British Lung Foundation

Novel ocular therapies from new cell sources

Stem cells and their derivatives are being used as a means of understanding ocular disease progression and as a source for transplantation

Co-expression of calcium and hERG potassium channels reduces the incidence of proarrhythmic events.

AIMS: Cardiac electrical activity is extraordinarily robust. However, when it goes wrong it can have fatal consequences. Electrical activity in the heart is controlled by the carefully orchestrated activity of more than a dozen different ion conductances. Whilst there is considerable variability in cardiac ion channel expression levels between individuals, studies in rodents have indicated that there are modules of ion channels whose expression co-vary. The aim of this study was to investigate whether meta-analytic co-expression analysis of large-scale gene expression data sets could identify modules of co-expressed cardiac ion channel genes in human hearts that are of functional importance. METHODS AND RESULTS: Meta-analysis of 3653 public human RNA-seq datasets identified a strong correlation between expression of CACNA1C (L-type calcium current, ICaL) and KCNH2 (rapid delayed rectifier K+ current, IKr), which was also observed in human adult heart tissue samples. In silico modeling suggested that co-expression of CACNA1C and KCNH2 would limit the variability in action potential duration seen with variations in expression of ion channel genes and reduce susceptibility to early afterdepolarizations, a surrogate marker for pro-arrhythmia. We also found that levels of KCNH2 and CACNA1C expression are correlated in human induced pluripotent stem cell derived cardiac myocytes and the levels of CACNA1C and KCNH2 expression were inversely correlated with the magnitude of changes in repolarization duration following inhibition of IKr. CONCLUSIONS: Meta-analytic approaches of multiple independent human gene expression datasets can be used to identify gene modules that are important for regulating heart function. Specifically, we have verified that there is co-expression of CACNA1C and KCNH2 ion channel genes in human heart tissue, and in silico analyses suggest that CACNA1C-KCNH2 co-expression increases the robustness of cardiac electrical activity. TRANSLATIONAL PERSPECTIVE: Here, we show, using meta-analysis of multiple independent human gene expression datasets, that there is co-expression of KCNH2-CACNA1C in human heart tissue which was then confirmed in human cardiac myocytes derived from induced pluripotent stem cells. Both in silico and functional studies show that the co-expression of CACNA1C and KCNH2 increases the robustness of cardiac electrical signalling. Our data also suggest that those patients who express higher levels of CACNA1C and KCNH2 are likely to be more susceptible to arrhythmias when exposed to drugs that block IKr, the major cause of drug-induced cardiac arrhythmias

Pharmacological activation of IKr in models of long QT Type 2 risks overcorrection of repolarization

Aims: Current treatment for congenital long QT syndrome Type 2 (cLQTS2), an electrical disorder that increases the risk of life-threatening cardiac arrhythmias, is aimed at reducing the incidence of arrhythmia triggers (beta-blockers) or terminating the arrhythmia after onset (implantable cardioverter-defibrillator). An alternative strategy is to target the underlying disease mechanism, which is reduced rapid delayed rectifier current (IKr) passed by Kv11.1 channels. Small molecule activators of Kv11.1 have been identified but the extent to which these can restore normal cardiac signalling in cLQTS2 backgrounds remains unclear. Here, we examined the ability of ICA-105574, an activator of Kv11.1 that impairs transition to the inactivated state, to restore function to heterozygous Kv11.1 channels containing either inactivation enhanced (T618S, N633S) or expression deficient (A422T) mutations. Methods and results: ICA-105574 effectively restored Kv11.1 current from heterozygous inactivation enhanced or expression defective mutant channels in heterologous expression systems. In a human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of cLQTS2 containing the expression defective Kv11.1 mutant A422T, cardiac repolarization, estimated from the duration of calcium transients in isolated cells and the rate corrected field potential duration (FPDc) in culture monolayers of cells, was significantly prolonged. The Kv11.1 activator ICA-105574 was able to reverse the prolonged repolarization in a concentration-dependent manner. However, at higher doses, ICA-105574 produced a shortening of the FPDc compared to controls. In vitro and in silico analysis suggests that this overcorrection occurs as a result of a temporal redistribution of the peak IKr to much earlier in the plateau phase of the action potential, which results in early repolarization. Conclusion: Kv11.1 activators, which target the primary disease mechanism, provide a possible treatment option for cLQTS2, with the caveat that there may be a risk of overcorrection that could itself be pro-arrhythmic

Real-time monitoring of peptide grafting onto chitosan films using capillary electrophoresis

International audienceChitosan, being antimicrobial and biocompatible, is attractive as a cell growth substrate. To improve cell attachment, arginine-glycine-aspartic acid-serine (RGDS) peptides were covalently grafted to chitosan films, through the widely used coupling agents 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC-HCl) and N-hydroxysuccinimide (NHS), via the carboxylic acid function of the RGDS molecule. The grafting reaction was monitored, for the first time, in real time using free-solution capillary electrophoresis (CE). This enabled fast separation and determination of the peptide and all other reactants in one separation with no sample preparation. Covalent RGDS peptide grafting onto the chitosan film surface was demonstrated using solid-state NMR of swollen films. CE indicated that oligomers of RGDS, not simply RGDS, were grafted on the film, with a likely hyperbranched structure. To assess the functional properties of the grafted films, cell growth was compared on control and peptide-grafted chitosan films. Light microscopy and polymerase chain reaction (PCR) analysis demonstrated greatly improved cell attachment to RGDS-grafted chitosan films

Post-abortion care services in Zambian health facilities: a qualitative study of users’ experiences and perceptions

Abstract Background Despite attempts to increase Universal Health Coverage, availability, accessibility, acceptability, and quality-related challenges remain barriers to receiving essential services by women who need them. We aimed to explore the experiences and perceptions of women receiving post-abortal care services in Zambia, within a human-rights framework. Methods A qualitative case study was conducted between August and September 2021 in Lusaka and Copperbelt provinces of Zambia. Fifteen (15) women seeking post-abortion care services were` interviewed using audio recorders; transcribed data was analyzed using thematic analysis. We report women’s experiences and perceptions of the healthcare system, their experiences of abortion, and healthcare-seeking behaviour. We used the availability, accessibility, acceptability, and quality (AAAQ) framework to understand how women claimed their right to healthcare as they sought and utilized post-abortion care services. Results Women who experienced spontaneous abortions delayed seeking health care by viewing symptoms as ‘normal pregnancy symptoms’ and not dangerous. Women also delayed seeking care because they feared the negative attitudes from their communities and the health care providers towards abortion in general, despite it being legal in Zambia. Some services were considered costly, impeding their right to access quality care. Conclusions Women delayed seeking care compounded by fear of negative attitudes from the community and healthcare providers. To ensure the provision and utilization of quality all abortion-related healthcare services, there is a need to increase awareness of the availability and legality of safe abortion services, the importance of seeking healthcare early for any abortion-related discomfort, and the provision and availability of free services at all levels of care should be emphasized