Intra-condensate demixing of TDP-43 inside stress granules generates pathological aggregates.

Cytosolic aggregation of the nuclear protein TDP-43 is associated with many neurodegenerative diseases, but the triggers for TDP-43 aggregation are still debated. Here, we demonstrate that TDP-43 aggregation requires a double event. One is up-concentration in stress granules beyond a threshold, and the other is oxidative stress. These two events collectively induce intra-condensate demixing, giving rise to a dynamic TDP-43 enriched phase within stress granules, which subsequently transitions into pathological aggregates. Mechanistically, intra-condensate demixing is triggered by local unfolding of the RRM1 domain for intermolecular disulfide bond formation and by increased hydrophobic patch interactions in the C-terminal domain. By engineering TDP-43 variants resistant to intra-condensate demixing, we successfully eliminate pathological TDP-43 aggregates in cells. We conclude that up-concentration inside condensates and simultaneous exposure to environmental stress could be a general pathway for protein aggregation, with intra-condensate demixing constituting a key intermediate step.

Preclinical mouse model of a misfolded PNLIP variant develops chronic pancreatitis.

OBJECTIVE: Increasing evidence implicates mutation-induced protein misfolding and endoplasm reticulum (ER) stress in the pathophysiology of chronic pancreatitis (CP). The paucity of animal models harbouring genetic risk variants has hampered our understanding of how misfolded proteins trigger CP. We previously showed that pancreatic triglyceride lipase (PNLIP) p.T221M, a variant associated with steatorrhoea and possibly CP in humans, misfolds and elicits ER stress in vitro suggesting proteotoxicity as a potential disease mechanism. Our objective was to create a mouse model to determine if PNLIP p.T221M causes CP and to define the mechanism. DESIGN: We created a mouse model of Pnlip p.T221M and characterised the structural and biochemical changes in the pancreas aged 1-12 months. We used multiple methods including histochemistry, immunostaining, transmission electron microscopy, biochemical assays, immunoblotting and qPCR. RESULTS: We demonstrated the hallmarks of human CP in Pnlip p.T221M homozygous mice including progressive pancreatic atrophy, acinar cell loss, fibrosis, fatty change, immune cell infiltration and reduced exocrine function. Heterozygotes also developed CP although at a slower rate. Immunoblot showed that pancreatic PNLIP T221M misfolded as insoluble aggregates. The level of aggregates in homozygotes declined with age and was much lower in heterozygotes at all ages. The Pnlip p.T221M pancreas had increased ER stress evidenced by dilated ER, increased Hspa5 (BiP) mRNA abundance and a maladaptive unfolded protein response leading to upregulation of Ddit3 (CHOP), nuclear factor-κB and cell death. CONCLUSION: Expression of PNLIP p.T221M in a preclinical mouse model results in CP caused by ER stress and proteotoxicity of misfolded mutant PNLIP.

Simulation with a colour indicator to help reduce contamination during airway management in COVID-19 times: An experience from a tertiary centre in India.

BACKGROUND AND AIMS: The coronavirus disease 2019 (COVID-19) pandemic has initiated modified protocols for aerosol-generating procedures. A simulation study using dye was conducted to highlight contamination at intubation and extubation and to encourage adherence to the new COVID-19 protocol among anaesthesia personnel in our institution. METHODS: A video demonstrating the new COVID-19 protocols was circulated in the Department of Anaesthesiology a week prior to the study. Thirty teams, each comprising an anaesthesia resident and a staff technician, were enroled. Each team was asked to demonstrate the steps of preparation, intubation and extubation on a mannequin in a COVID-19 scenario. Checklists were used to assess points of contamination and adherence to the protocols. Following debriefing, a repeat simulation was conducted. The use of a dye highlighted the points of contamination. The study subjects provided feedback on the usefulness of the session and practical difficulties encountered in adapting to the new protocols. RESULTS: The average contamination scores decreased by 3.4 (95% confidence interval (CI): 2.4-4.4, P < 0.001) in the post-debrief session. Adherence to the steps of the modified protocol improved by a score of 2.7 (CI: 3.6-1.83) among anaesthesiologists and by 4.3 (CI: 5.3-3.3) among technicians. Further, 93% felt that the use of the colour indicator reinforced awareness of the possible points of contamination. CONCLUSION: Simulation with a low-fidelity mannequin by using colour indicator for secretions is an effective teaching tool to reduce health hazards during airway management in COVID-19 times.

Apnoeic Oxygenation during Simulated Difficult Intubation in Obese Patients: Comparison of Buccal Ring, Adair and Elwyn Tube Versus Nasal Cannula: A Prospective Randomized Controlled Trial.

Background: Apnoeic oxygenation is an established method of increasing safe apnoea times during intubation and this is of more importance in obese patients. The usefulness of buccal Ring, Adair and Elwyn (RAE) oxygenation has been established in previous studies, however a head-to-head comparison with nasal cannula (NC) is lacking. Aim: The aim of this study was to compare apnoea time with buccal RAE (BR) versus NC in obese patients. Setting and Design: This was a prospective, nonblinded randomized controlled trial conducted in a tertiary hospital where fifty American Society of Anaesthesiologists Physical Status Class I and II, obese patients with body mass index ≥30, posted for elective surgery were included. Materials and Methods: Following adequate preoxygenation and standard induction of anaesthesia, a prolonged simulated difficult laryngoscopy was performed during which oxygen was provided via either BR or NC. The primary outcome was time to desaturation to <95% or 10 min, which ever occurred first. Other outcomes recorded were lowest saturation, time to resaturation and highest end tidal carbon di oxide. Statistical Analysis: Mean with standard deviation (SD) or median with inter quartile range were used for continuous variables and absolute number with percentage were used for categorical variables. The primary outcome was analyzed using Kaplan-Meier survival curves, and log-rank tests were applied. Results: Patient characteristics were similar in both arms. The mean apnoea time in seconds (SD) in the BR group, 375.3 (116.6) was higher than the NC group 316.1 (94.1), P = 0.054. From the Kapan Meier curves the probability of desaturating to <95% was earlier in the NC group than the BR group (P = 0.092). The other outcomes were similar in both groups. Conclusion: This is the first study that demonstrates that oxygenation via a BR is better than NC in providing apnoeic oxygenation in obese patients and can safely be used when NC are contraindicated.

Low-dose Succinylcholine to Facilitate Laryngeal Mask Airway Insertion: A Comparison of Two Doses.

BACKGROUND AND AIMS: Around the world, the use of the laryngeal mask airway (LMA) is becoming more common for different surgeries accounting for it being the dominant choice of airway in numerous surgeries. Although propofol is known to blunt the laryngeal reflexes often patient movement, coughing, and gagging occur on insertion. This study aims to identify the optimum dose of succinylcholine required to facilitate LMA insertion comparing placebo, 0.1 mg/kg and 0.25 mg/kg of succinylcholine. Further objectives were to compare (a) the overall insertion conditions of the LMA, (b) the number of insertion attempts, (c) the amount of propofol consumption, and (d) the hemodynamics in the three groups. SETTING AND DESIGN: This is a prospective, double-blinded, randomized control trial of 283 patients randomized into three groups-placebo, 0.1 mg/kg and 0.25 mg/kg of succinylcholine. It was done in the day case theatres of a tertiary hospital in Southern India. SUBJECTS AND METHODS: Patients were induced with 2 mg/kg of propofol, after 2 µg/kg of fentanyl. The study drug was given after loss of consciousness. After 60 s, a classic LMA was inserted by the standard method by a single investigator. Jaw relaxation, coughing, gagging, movement, laryngospasm, ease of insertion, number of attempts, propofol usage, and hemodynamics were assessed. STATISTICAL ANALYSIS: Statistical methods used were analysis of variance with Bonferroni's t-test, Chi-square test, and Fisher's test. P < 0.05 was considered statistically significant. RESULTS: Jaw relaxation was significantly better in the 0.25 mg/kg succinylcholine group. There was no significant difference in coughing and gagging in the groups, but patient movement was more in the placebo group. Two patients in the placebo group experienced partial laryngospasm. Overall insertion conditions were significantly better in the 0.25 mg/kg group compared to the other two groups. Propofol consumption was significantly more in the placebo group. CONCLUSIONS: The study concludes that 0.25 mg/kg succinylcholine facilitates insertion of the LMA.

Association between a prolonged corrected QT interval and outcomes in patients in a medical Intensive Care Unit.

INTRODUCTION: Patients admitted into a medical Intensive Care Unit (ICU) have varying illnesses and risk factors. An electrocardiogram (ECG) is a useful tool to assess the cardiac status. The aim of the study was to determine the prevalence of QT prolongation of the ECG in patients admitted to a medical ICU in a tertiary hospital, to assess outcomes in terms of mortality, cardiovascular events, and duration of ICU stay. MATERIALS AND METHODS: Prospective observational study, 6 months duration, assessing the prevalence of prolonged corrected QT interval (QTc) at admission into a medical ICU. A QTc calculated by Bazett's formula, of >440 ms for males and >460 ms for females was considered prolonged. Details of illness, clinical and lab parameters were monitored. RESULTS: The total number of patients screened was 182. There was a high prevalence of prolonged QTc (30%) on admission to the ICU. This reduced to 19% on day 3 (P = 0.011). In patients with a prolonged QTc the odds ratio of adverse outcome from ICU was 3.17 (confidence interval [CI]: 1.52-6.63) (P = 0.001) and of adverse outcome for hospital stay was 2.27 (CI: 1.11-4.66) (P = 0.014). In the study, 35% of all patients received drugs with QT prolonging action. Of patients with a prolonged QTc at admission 18 (35%) received a QT prolonging drug. CONCLUSIONS: We found that prolonged QTc is common (30%) in our medical ICU at admission and a large proportion (35%) received drugs capable of prolonging QT interval. These patients with QTc prolongation have a higher odds ratio for adverse outcomes.

Safety and efficacy of vemurafenib in end stage renal failure.

BACKGROUND: Serine-threonine inhibitors, such as vemurafenib, are being used increasingly in cancer treatment, and the toxicity and therapeutic benefit need to be balanced carefully both before and during treatment. CASE PRESENTATION: A patient with metastatic melanoma and end stage renal failure who was on peritoneal dialysis was treated with the serine-threonine kinase inhibitor, vemurafenib. After 5 months of treatment, a substantial response to vemurafenib was observed using imaging, but when he developed a prolonged QTc interval (common toxicity criteria (CTC) grade 3), treatment was interrupted. Vemurafenib was restarted at a reduced dose when the QTc interval returned to normal. The patient has had a significant response to vemurafenib and continued on treatment for 12 months after beginning the therapy. CONCLUSION: This is the first reported case of end stage renal failure in a patient who is taking vemurafenib. Although the patient developed QTc prolongation, it appears to be asymptomatic, and was managed with dose reduction. This case highlights the need for closer QTc monitoring at the start and during treatment.