Mechanisms underlying the phenotypic diversity in RYR1-associated Malignant Hyperthermia

Malignant hyperthermia (MH) is a potential fatal hereditary skeletal muscle disorder that occurs upon exposure to certain anaesthetic agents. Susceptibility is predominantly conferred by variants in the RYR1 gene encoding the type 1 ryanodine receptor (RYR1). A common feature observed in MH susceptible patients and in animal models, is a RYR1-leak dependent elevation in the intracellular Ca2+ concentration ([Ca2+]i) in the non-triggered state. However, it is not fully understood whether extracellular Ca2+ entry plays an important role in maintaining this elevated resting [Ca2+]i. It is also becoming increasingly evident that RYR1 variants can produce different MH phenotypes, for example the variant p.G2434R (c.7300G>A) is the major MH variant found globally, but has a weaker clinical phenotype relative to rarer variants such as p.R2454H (c.7361G>A). Live-cell calcium imaging was used to examine different aspects of the molecular mechanisms underlying RYR1-associated MH. The first was to use HEK293 cells to explore the caffeine sensitivity of novel potentially pathogenic RYR1 variants. Next skeletal muscle myotubes derived from MH patients with p.G2434R or p.R2454H, were found to have an enhanced sensitivity to caffeine, and a TRPC3/6 mediated increased sarcolemmal cationic influx relative to non-susceptible controls. There was no significant difference between the two MH associated RYR1 variants. Myotubes from a novel MH mouse model containing the equivalent variant to human p.G2434R, were also found to have an elevated sensitivity to RYR1 agonists. This model has further confirmed that the TRPC3/6 mediated enhanced cationic entry is conserved between the two species, and this entry can be reduced to control levels by blocking the RYR1-dependent leak. Taken together, the data presented in this thesis furthers our understanding of the molecular mechanisms that underlie the perturbed Ca2+ handling observed in MH tissue, and provides new avenues of research into MH and related RYR1 disorders as well as a potential target for novel therapies

The effects of hydrogen sulfide on HEK-293 cells and human primary bronchial fibroblasts

Hydrogen sulfide (H2S) is a key biologically relevant signalling molecule and has been recognised as the third endogenous gasotransmitter. H2S modulates various biological functions through complex mechanisms which may involve Transient Receptor Potential (TRP) channels such as TRPA1. TRPA1 is postulated to play a prominent role in pulmonary inflammation and airway hypersensitivity. H2S has been found to also modulate cell growth and survival, with high concentrations leading to reactive airway disease, acute respiratory failure and pulmonary fibrosis. N-Acetylcysteine (NAC) is a thiol precursor of L-Cysteine which elicits antioxidant effects and has been implicated in preventing the progression of pulmonary fibrosis. The aim of the study was to examine the effects of H2S on TRPA1 and human primary bronchial fibroblasts (HPBF).Initial experiments sought to examine the effects of NaHS on HEK293 cells transfected with TRPA1 (HEK-TRPA1) using fluorometric calcium assays. These assays were hampered by a direct reaction between the H2S donor NaHS and the fluorescent dye. However this has led to the identification of a putative new H2S sensor.Next, untransfected HPBF and HEK-TRPA1 were treated with NaHS or with NAC, and the effects assessed using a five-day growth assay. Chronic NaHS exposure induced a significant reduction in the growth of HEK-TRPA1 cells. Furthermore an acute 30 minute treatment with NaHS significantly reduced the growth of both HEK-TRPA1 cells and HPBFs, whereas NAC only inhibited growth of the latter.The NAC mediated inhibition in HPBF growth provides in vitro evidence for the potential anti-fibrotic actions of NAC in the lung. The inhibitory effects on growth at day five following a brief 30 minute NaHS exposure is a novel finding that requires further investigation to elucidate the mechanism of action. This could potentially provide a better understanding of the effects of H2S in modulating the progression from inflammation to fibrosis

Hashing Routing

A recent trend in ad hoc network routing is the reactive on-demand philosophy where routes are established only when required. Mostly work has been concentrated on routing aspect. Most of the protocols in this category are not incorporating proper security features. Security is one of the most important concepts in ad hoc networks. It has been observed that different protocols need different strategies for security. The study here proposes a theory in this article based on Hashing as a tool. This scheme can make most of the on demand protocols secure. The study should help in making protocols more robust against attacks and standardize parameters for security in routing protocols

Transient Receptor Potential Cation Channels and Calcium Dyshomeostasis in a Mouse Model Relevant to Malignant Hyperthermia

Background: Until recently, the mechanism for the malignant hyperthermia crisis has been attributed solely to sustained massive Ca2+ release from the sarcoplasmic reticulum on exposure to triggering agents. This study tested the hypothesis that transient receptor potential cation (TRPC) channels are important contributors to the Ca2+ dyshomeostasis in a mouse model relevant to malignant hyperthermia. Methods: This study examined the mechanisms responsible for Ca2+ dyshomeostasis in RYR1-p.G2435R mouse muscles and muscle cells using calcium and sodium ion selective microelectrodes, manganese quench of Fura2 fluorescence, and Western blots. Results: RYR1-p.G2435R mouse muscle cells have chronically elevated intracellular resting calcium and sodium and rate of manganese quench (homozygous greater than heterozygous) compared with wild-type muscles. After exposure to 1-oleoyl-2-acetyl-sn-glycerol, a TRPC3/6 activator, increases in intracellular resting calcium/sodium were significantly greater in RYR1-p.G2435R muscles (from 153 ± 11 nM/10 ± 0.5 mM to 304 ± 45 nM/14.2 ± 0.7 mM in heterozygotes P < 0.001] and from 251 ± 25 nM/13.9 ± 0.5 mM to 534 ± 64 nM/20.9 ± 1.5 mM in homozygotes [P < 0.001] compared with 123 ± 3 nM/8 ± 0.1 mM to 196 ± 27 nM/9.4 ± 0.7 mM in wild type). These increases were inhibited both by simply removing extracellular Ca2+ and by exposure to either a nonspecific (gadolinium) or a newly available, more specific pharmacologic agent (SAR7334) to block TRPC6- and TRPC3-mediated cation influx into cells. Furthermore, local pretreatment with SAR7334 partially decreased the elevation of intracellular resting calcium that is seen in RYR1-p.G2435R muscles during exposure to halothane. Western blot analysis showed that expression of TRPC3 and TRPC6 were significantly increased in RYR1-p.G2435R muscles in a gene–dose–dependent manner, supporting their being a primary molecular basis for increased sarcolemmal cation influx. Conclusions: Muscle cells in knock-in mice expressing the RYR1-p.G2435R mutation are hypersensitive to TRPC3/6 activators. This hypersensitivity can be negated with pharmacologic agents that block TRPC3/6 activity. This reinforces the working hypothesis that transient receptor potential cation channels play a critical role in causing intracellular calcium and sodium overload in malignant hyperthermia–susceptible muscle, both at rest and during the malignant hyperthermia crisis

Prospective observational cohort study on grading the severity of postoperative complications in global surgery research

Background The Clavien–Dindo classification is perhaps the most widely used approach for reporting postoperative complications in clinical trials. This system classifies complication severity by the treatment provided. However, it is unclear whether the Clavien–Dindo system can be used internationally in studies across differing healthcare systems in high- (HICs) and low- and middle-income countries (LMICs). Methods This was a secondary analysis of the International Surgical Outcomes Study (ISOS), a prospective observational cohort study of elective surgery in adults. Data collection occurred over a 7-day period. Severity of complications was graded using Clavien–Dindo and the simpler ISOS grading (mild, moderate or severe, based on guided investigator judgement). Severity grading was compared using the intraclass correlation coefficient (ICC). Data are presented as frequencies and ICC values (with 95 per cent c.i.). The analysis was stratified by income status of the country, comparing HICs with LMICs. Results A total of 44 814 patients were recruited from 474 hospitals in 27 countries (19 HICs and 8 LMICs). Some 7508 patients (16·8 per cent) experienced at least one postoperative complication, equivalent to 11 664 complications in total. Using the ISOS classification, 5504 of 11 664 complications (47·2 per cent) were graded as mild, 4244 (36·4 per cent) as moderate and 1916 (16·4 per cent) as severe. Using Clavien–Dindo, 6781 of 11 664 complications (58·1 per cent) were graded as I or II, 1740 (14·9 per cent) as III, 2408 (20·6 per cent) as IV and 735 (6·3 per cent) as V. Agreement between classification systems was poor overall (ICC 0·41, 95 per cent c.i. 0·20 to 0·55), and in LMICs (ICC 0·23, 0·05 to 0·38) and HICs (ICC 0·46, 0·25 to 0·59). Conclusion Caution is recommended when using a treatment approach to grade complications in global surgery studies, as this may introduce bias unintentionally

The surgical safety checklist and patient outcomes after surgery: a prospective observational cohort study, systematic review and meta-analysis

© 2017 British Journal of Anaesthesia Background: The surgical safety checklist is widely used to improve the quality of perioperative care. However, clinicians continue to debate the clinical effectiveness of this tool. Methods: Prospective analysis of data from the International Surgical Outcomes Study (ISOS), an international observational study of elective in-patient surgery, accompanied by a systematic review and meta-analysis of published literature. The exposure was surgical safety checklist use. The primary outcome was in-hospital mortality and the secondary outcome was postoperative complications. In the ISOS cohort, a multivariable multi-level generalized linear model was used to test associations. To further contextualise these findings, we included the results from the ISOS cohort in a meta-analysis. Results are reported as odds ratios (OR) with 95% confidence intervals. Results: We included 44 814 patients from 497 hospitals in 27 countries in the ISOS analysis. There were 40 245 (89.8%) patients exposed to the checklist, whilst 7508 (16.8%) sustained ≥1 postoperative complications and 207 (0.5%) died before hospital discharge. Checklist exposure was associated with reduced mortality [odds ratio (OR) 0.49 (0.32–0.77); P\u3c0.01], but no difference in complication rates [OR 1.02 (0.88–1.19); P=0.75]. In a systematic review, we screened 3732 records and identified 11 eligible studies of 453 292 patients including the ISOS cohort. Checklist exposure was associated with both reduced postoperative mortality [OR 0.75 (0.62–0.92); P\u3c0.01; I2=87%] and reduced complication rates [OR 0.73 (0.61–0.88); P\u3c0.01; I2=89%). Conclusions: Patients exposed to a surgical safety checklist experience better postoperative outcomes, but this could simply reflect wider quality of care in hospitals where checklist use is routine

Critical care admission following elective surgery was not associated with survival benefit: prospective analysis of data from 27 countries

This was an investigator initiated study funded by Nestle Health Sciences through an unrestricted research grant, and by a National Institute for Health Research (UK) Professorship held by RP. The study was sponsored by Queen Mary University of London