Troponin Structural Dynamics in the Native Cardiac Thin Filament Revealed by Cryo Electron Microscopy.

Cardiac muscle contraction occurs due to repetitive interactions between myosin thick and actin thin filaments (TF) regulated by Ca2+ levels, active cross-bridges, and cardiac myosin-binding protein C (cMyBP-C). The cardiac TF (cTF) has two nonequivalent strands, each comprised of actin, tropomyosin (Tm), and troponin (Tn). Tn shifts Tm away from myosin-binding sites on actin at elevated Ca2+ levels to allow formation of force-producing actomyosin cross-bridges. The Tn complex is comprised of three distinct polypeptides - Ca2+-binding TnC, inhibitory TnI, and Tm-binding TnT. The molecular mechanism of their collective action is unresolved due to lack of comprehensive structural information on Tn region of cTF. C1 domain of cMyBP-C activates cTF in the absence of Ca2+ to the same extent as rigor myosin. Here we used cryo-EM of native cTFs to show that cTF Tn core adopts multiple structural conformations at high and low Ca2+ levels and that the two strands are structurally distinct. At high Ca2+ levels, cTF is not entirely activated by Ca2+ but exists in either partially or fully activated state. Complete dissociation of TnI C-terminus is required for full activation. In presence of cMyBP-C C1 domain, Tn core adopts a fully activated conformation, even in absence of Ca2+. Our data provide a structural description for the requirement of myosin to fully activate cTFs and explain increased affinity of TnC to Ca2+ in presence of active cross-bridges. We suggest that allosteric coupling between Tn subunits and Tm is required to control actomyosin interactions.

Effect of the N-terminal extension in myosin essential light chain A1 on the mechanism of actomyosin ATP hydrolysis.

Myosin essential light chains A1 and A2 are identical isoforms except for an extension of ∼40 amino acids at the N terminus of A1 that binds F-actin. The extension has no bearing on the burst hydrolysis rate (M-ATP → M-ADP-Pi) as determined by chemical quench flow (100 µM isoenzyme). Whereas actomyosin-S1A2 steady state MgATPase (low ionic strength, 20 °C) is hyperbolically dependent on concentration: Vmax 7.6 s-1, Kapp 6.4 µM (F-actin) and Vmax 10.1 s-1, Kapp 5.5 µM (native thin filaments, pCa 4), the relationship for myosin-S1A1 is bimodal; an initial rise at low concentration followed by a decline to one-third the Vmax of S1A2, indicative of more than one rate-limiting step and A1-enforced flux through the slower actomyosin-limited hydrolysis pathway. In double-mixing stopped-flow with an indicator, Ca(II)-mediated activation of Pi dissociation (regulatedAM-ADP-Pi → regulatedAM-ADP + Pi) is attenuated by A1 attachment to thin filaments (pCa 4). The maximum accelerated rates of Pi dissociation are: 81 s-1 (S1A1, Kapp 8.9 µM) versus 129 s-1 (S1A2, Kapp 58 µM). To investigate apomyosin-S1-mediated activation, thin filaments (EGTA) are premixed with a given isomyosin-S1 and double-mixing is repeated with myosin-S1A1 in the first mix. Similar maximum rates of Pi dissociation are observed, 44.5 s-1 (S1A1) and 47.1 s-1 (S1A2), which are lower than for Ca(II) activation. Overall, these results biochemically demonstrate how the longer light chain A1 can contribute to slower contraction and higher force and the shorter version A2 to faster contraction and lower force, consistent with their distribution in different types of striated muscle.

Impact of Abrupt Interruption of Home Psychotropic Medications at ICU Admission.

Background: Abrupt discontinuation of home psychotropic medications is common among critically ill patients but may precipitate clinically significant withdrawal. Objective: To determine the percent of patients with interruptions in home psychotropic medications upon intensive care unit (ICU) admission and to identify outcomes associated with these interruptions. Methods: This was an institutional review board-approved, single-center, retrospective study of critically ill patients with a history of mental illness taking an antipsychotic or antidepressant medication. The primary outcome was the percent of patients with interruption in at least one home psychotropic medication for ≥24 hours upon ICU admission. Secondary outcomes included time to psychotropic re-initiation, percent of home psychotropic medications restarted in the ICU, ICU length of stay (LOS), delirium, withdrawal-related complications, need for acute antipsychotics or benzodiazepines, and reasons for psychotropic interruption. Results: Among 183 patients, 93 (50.8%) had interruptions in home psychotropic therapy for ≥24 hours upon ICU admission. Mean time to reinitiation of at least one psychotropic agent was 1.4 days, and 16.4% of patients did not have any home psychotropics restarted. Patients with psychotropic interruption had a longer ICU LOS (P = 0.01) and greater incidence of ICU delirium (P < 0.01). Withdrawal-related complications were similar between groups. Acute antipsychotic use was greater in patients with psychotropic interruption (P < 0.01). Acute benzodiazepine use was not different between groups (P = 0.87). Most patients did not have a documented reason for therapy interruption. Conclusion and Relevance: Unless contraindicated, clinicians should attempt to restart home psychotropic medications as soon as possible in critically ill patients.

A survey of practice in the management of haemolysis, icterus and lipaemia in blood specimens in the United Kingdom and Republic of Ireland.

BACKGROUND: Haemolysis, icterus and lipaemia (HIL) are common interferants in laboratory medicine, potentially impacting patient care. This survey investigates HIL management in medical laboratories across the UK and Republic of Ireland (ROI). METHODS: A survey was sent to members of key professional organisations for laboratory medicine in the UK and ROI. Questions related to the detection, monitoring, quality control, and management of HIL. RESULTS: In total, responses from 124 laboratories were analysed, predominantly from England (52%) and ROI (36%). Most responses were from public hospitals with biochemistry services (90%), serving primary care (91%), inpatients (91%), and outpatients (89%). Most laboratories monitored H (98%), I (88%), and L (96%) using automated indices (93%), alone or in combination with visual inspection.Manufacturer-stated cut-offs were used by 83% and were applied to general chemistries in 79%, and immunoassays in 50%. Where HIL cut-offs are breached, 64% withheld results, while 96% reported interference to users. HIL were defined using numeric scales (70%) and ordinal scales (26%). HIL targets exist in 35% of laboratories, and 54% have attempted to reduce HIL. Internal Quality Control for HIL was lacking in 62% of laboratories, and just 18% of respondents have participated in External Quality Assurance. Laboratories agree manufacturers should: standardise HIL reporting (94%), ensure comparability between platforms (94%), and provide information on HIL cross-reactivity (99%). Respondents (99%) showed interest in evidence-based, standardised HIL cut-offs. CONCLUSIONS: Most respondents monitor HIL, although the wide variation in practice may differentially affect clinical care. Laboratories seem receptive to education and advice on HIL management.

Myosin with hypertrophic cardiac mutation R712L has a decreased working stroke which is rescued by omecamtiv mecarbil.

Hypertrophic cardiomyopathies (HCMs) are the leading cause of acute cardiac failure in young individuals. Over 300 mutations throughout ß-cardiac myosin, including in the motor domain, are associated with HCM. A ß-cardiac myosin motor mutation (R712L) leads to a severe form of HCM. Actin-gliding motility of R712L-myosin is inhibited, despite near-normal ATPase kinetics. By optical trapping, the working stroke of R712L-myosin was decreased 4-fold, but actin-attachment durations were normal. A prevalent hypothesis that HCM mutants are hypercontractile is thus not universal. R712 is adjacent to the binding site of the heart failure drug omecamtiv mecarbil (OM). OM suppresses the working stroke of normal ß-cardiac myosin, but remarkably, OM rescues the R712L-myosin working stroke. Using a flow chamber to interrogate a single molecule during buffer exchange, we found OM rescue to be reversible. Thus, the R712L mutation uncouples lever arm rotation from ATPase activity and this inhibition is rescued by OM.

Parents know best: transgenerational predator recognition through parental effects.

In highly biodiverse systems, such as coral reefs, prey species are faced with predatory threats from numerous species. Recognition of predators can be innate, or learned, and can help increase the chance of survival. Research suggests that parental exposure to increased predatory threats can affect the development, behaviour, and ultimately, success of their offspring. Breeding pairs of damselfish (Acanthochromis polyacanthus) were subjected to one of three olfactory and visual treatments (predator, herbivore, or control), and their developing embryos were subsequently exposed to five different chemosensory cues. Offspring of parents assigned to the predator treatment exhibited a mean increase in heart rate two times greater than that of offspring from parents in herbivore or control treatments. This increased reaction to a parentally known predator odour suggests that predator-treated parents passed down relevant threat information to their offspring, via parental effects. This is the first time transgenerational recognition of a specific predator has been confirmed in any species. This phenomenon could influence predator-induced mortality rates and enable populations to adaptively respond to fluctuations in predator composition and environmental changes.

Preanalytical errors in medical laboratories: a review of the available methodologies of data collection and analysis.

Preanalytical errors have previously been shown to contribute a significant proportion of errors in laboratory processes and contribute to a number of patient safety risks. Accreditation against ISO 15189:2012 requires that laboratory Quality Management Systems consider the impact of preanalytical processes in areas such as the identification and control of non-conformances, continual improvement, internal audit and quality indicators. Previous studies have shown that there is a wide variation in the definition, repertoire and collection methods for preanalytical quality indicators. The International Federation of Clinical Chemistry Working Group on Laboratory Errors and Patient Safety has defined a number of quality indicators for the preanalytical stage, and the adoption of harmonized definitions will support interlaboratory comparisons and continual improvement. There are a variety of data collection methods, including audit, manual recording processes, incident reporting mechanisms and laboratory information systems. Quality management processes such as benchmarking, statistical process control, Pareto analysis and failure mode and effect analysis can be used to review data and should be incorporated into clinical governance mechanisms. In this paper, The Association for Clinical Biochemistry and Laboratory Medicine PreAnalytical Specialist Interest Group review the various data collection methods available. Our recommendation is the use of the laboratory information management systems as a recording mechanism for preanalytical errors as this provides the easiest and most standardized mechanism of data capture.

Monitoring and reporting of preanalytical errors in laboratory medicine: the UK situation.

BACKGROUND: Most errors in the clinical laboratory occur in the preanalytical phase. This study aimed to comprehensively describe the prevalence and nature of preanalytical quality monitoring practices in UK clinical laboratories. METHODS: A survey was sent on behalf of the Association for Clinical Biochemistry and Laboratory Medicine Preanalytical Working Group (ACB-WG-PA) to all heads of department of clinical laboratories in the UK. The survey captured data on the analytical platform and Laboratory Information Management System in use; which preanalytical errors were recorded and how they were classified and gauged interest in an external quality assurance scheme for preanalytical errors. RESULTS: Of the 157 laboratories asked to participate, responses were received from 104 (66.2%). Laboratory error rates were recorded per number of specimens, rather than per number of requests in 51% of respondents. Aside from serum indices for haemolysis, icterus and lipaemia, which were measured in 80% of laboratories, the most common errors recorded were booking-in errors (70.1%) and sample mislabelling (56.9%) in laboratories who record preanalytical errors. Of the laboratories surveyed, 95.9% expressed an interest in guidance on recording preanalytical error and 91.8% expressed interest in an external quality assurance scheme. CONCLUSIONS: This survey observes a wide variation in the definition, repertoire and collection methods for preanalytical errors in the UK. Data indicate there is a lot of interest in improving preanalytical data collection. The ACB-WG-PA aims to produce guidance and support for laboratories to standardize preanalytical data collection and to help establish and validate an external quality assurance scheme for interlaboratory comparison.