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.

The structure of the native cardiac thin filament at systolic Ca2+ levels.

Every heartbeat relies on cyclical interactions between myosin thick and actin thin filaments orchestrated by rising and falling Ca2+ levels. Thin filaments are comprised of two actin strands, each harboring equally separated troponin complexes, which bind Ca2+ to move tropomyosin cables away from the myosin binding sites and, thus, activate systolic contraction. Recently, structures of thin filaments obtained at low (pCa ∼9) or high (pCa ∼3) Ca2+ levels revealed the transition between the Ca2+-free and Ca2+-bound states. However, in working cardiac muscle, Ca2+ levels fluctuate at intermediate values between pCa ∼6 and pCa ∼7. The structure of the thin filament at physiological Ca2+ levels is unknown. We used cryoelectron microscopy and statistical analysis to reveal the structure of the cardiac thin filament at systolic pCa = 5.8. We show that the two strands of the thin filament consist of a mixture of regulatory units, which are composed of Ca2+-free, Ca2+-bound, or mixed (e.g., Ca2+ free on one side and Ca2+ bound on the other side) troponin complexes. We traced troponin complex conformations along and across individual thin filaments to directly determine the structural composition of the cardiac native thin filament at systolic Ca2+ levels. We demonstrate that the two thin filament strands are activated stochastically with short-range cooperativity evident only on one of the two strands. Our findings suggest a mechanism by which cardiac muscle is regulated by narrow range Ca2+ fluctuations.

Ca2+-induced movement of tropomyosin on native cardiac thin filaments revealed by cryoelectron microscopy.

Muscle contraction relies on the interaction of myosin motors with F-actin, which is regulated through a translocation of tropomyosin by the troponin complex in response to Ca2+ The current model of muscle regulation holds that at relaxing (low-Ca2+) conditions tropomyosin blocks myosin binding sites on F-actin, whereas at activating (high-Ca2+) conditions tropomyosin translocation only partially exposes myosin binding sites on F-actin so that binding of rigor myosin is required to fully activate the thin filament (TF). Here we used a single-particle approach to helical reconstruction of frozen hydrated native cardiac TFs under relaxing and activating conditions to reveal the azimuthal movement of the tropomyosin on the surface of the native cardiac TF upon Ca2+ activation. We demonstrate that at either relaxing or activating conditions tropomyosin is not constrained in one structural state, but rather is distributed between three structural positions on the surface of the TF. We show that two of these tropomyosin positions restrain actomyosin interactions, whereas in the third position, which is significantly enhanced at high Ca2+, tropomyosin does not block myosin binding sites on F-actin. Our data provide a structural framework for the enhanced activation of the cardiac TF over the skeletal TF by Ca2+ and lead to a mechanistic model for the regulation of the cardiac TF.

The shaker-1 mouse myosin VIIa deafness mutation results in a severely reduced rate of the ATP hydrolysis step.

Mutations in the MYO7A gene, encoding the motor protein myosin VIIa, can cause Usher 1B, a deafness/blindness syndrome in humans, and the shaker-1 phenotype, characterized by deafness, head tossing, and circling behavior, in mice. Myosin VIIa is responsible for tension bearing and the transduction mechanism in the stereocilia and for melanosome transport in the retina, in line with the phenotypic outcomes observed in mice. However, the effect of the shaker-1 mutation, a R502P amino acid substitution, on the motor function is unclear. To explore this question, we determined the kinetic properties and the effect on the filopodial tip localization of the recombinant mouse myosin VIIa-5IQ-SAH R502P (myoVIIa-sh1) construct. Interestingly, although residue 502 is localized to a region thought to be involved in interacting with actin, the kinetic parameters for actin binding changed only slightly for the mutant construct. However, the rate constant for ATP hydrolysis (k+H + k-H) was reduced by ∼200-fold from 12 s-1 to 0.05 s-1, making the hydrolysis step the rate-limiting step of the ATPase cycle in the presence and absence of actin. Given that wild-type mouse myosin VIIa is a slow, high-duty ratio, monomeric motor, this altered hydrolysis rate would reduce activity to extremely low levels. Indeed, the translocation to the filopodial tips was hampered by the diminished motor function of a dimeric construct of the shaker-1 mutant. We conclude that the diminished motor activity of this mutant is most likely responsible for impaired hearing in the shaker-1 mice.

C0 and C1 N-terminal Ig domains of myosin binding protein C exert different effects on thin filament activation.

Mutations in genes encoding myosin, the molecular motor that powers cardiac muscle contraction, and its accessory protein, cardiac myosin binding protein C (cMyBP-C), are the two most common causes of hypertrophic cardiomyopathy (HCM). Recent studies established that the N-terminal domains (NTDs) of cMyBP-C (e.g., C0, C1, M, and C2) can bind to and activate or inhibit the thin filament (TF). However, the molecular mechanism(s) by which NTDs modulate interaction of myosin with the TF remains unknown and the contribution of each individual NTD to TF activation/inhibition is unclear. Here we used an integrated structure-function approach using cryoelectron microscopy, biochemical kinetics, and force measurements to reveal how the first two Ig-like domains of cMyPB-C (C0 and C1) interact with the TF. Results demonstrate that despite being structural homologs, C0 and C1 exhibit different patterns of binding on the surface of F-actin. Importantly, C1 but not C0 binds in a position to activate the TF by shifting tropomyosin (Tm) to the "open" structural state. We further show that C1 directly interacts with Tm and traps Tm in the open position on the surface of F-actin. Both C0 and C1 compete with myosin subfragment 1 for binding to F-actin and effectively inhibit actomyosin interactions when present at high ratios of NTDs to F-actin. Finally, we show that in contracting sarcomeres, the activating effect of C1 is apparent only once low levels of Ca(2+) have been achieved. We suggest that Ca(2+) modulates the interaction of cMyBP-C with the TF in the sarcomere.

Omecamtiv Mecarbil modulates the kinetic and motile properties of porcine ß-cardiac myosin.

We determined the effect of Omecamtiv Mecarbil, a novel allosteric effector of cardiac muscle myosin, on the kinetic and "in vitro" motility properties of the porcine ventricular heavy meromyosin (PV-HMM). Omecamtiv Mecarbil increases the equilibrium constant of the hydrolysis step (M-ATP ⇄ M-ADP-Pi) from 2.4 to 6 as determined by quench flow, but the maximal rates of both the hydrolysis step and tryptophan fluorescence increase are unchanged by the drug. OM also increases the amplitude of the fast phase of phosphate dissociation (AM-ADP-Pi → AM-ADP + Pi) that is associated with force production in muscle by 4-fold. These results suggest a mechanism in which hydrolysis of M-ATP to M-ADP-Pi occurs both before and after the recovery stroke, but rapid acceleration of phosphate dissociation by actin occurs only on post-recovery stroke A-M-ADP-Pi. One of the more dramatic effects of OM on PV-HMM is a 14-fold decrease in the unloaded shortening velocity measured by the in vitro motility assay. The increase in flux through phosphate dissociation and the unchanged rate of ADP dissociation (AM-ADP → AM + ADP) by the drug produce a higher duty ratio motor in which a larger fraction of myosin heads are strongly bound to actin filaments. The increased internal load produced by a larger fraction of strongly attached crossbridges explains the reduced rate of in vitro motility velocity in the presence of OM and predicts that the drug will produce slower and stronger contraction of cardiac muscle.

Extending the PRISMA statement to equity-focused systematic reviews (PRISMA-E 2012): explanation and elaboration.

BACKGROUND: The promotion of health equity, the absence of avoidable and unfair differences in health outcomes, is a global imperative. Systematic reviews are an important source of evidence for health decision-makers, but have been found to lack assessments of the intervention effects on health equity. The Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) is a 27 item checklist intended to improve transparency and reporting of systematic reviews. We developed an equity extension for PRISMA (PRISMA-E 2012) to help systematic reviewers identify, extract, and synthesise evidence on equity in systematic reviews. METHODS AND FINDINGS: In this explanation and elaboration paper we provide the rationale for each extension item. These items are additions or modifications to the existing PRISMA Statement items, in order to incorporate a focus on equity. An example of good reporting is provided for each item as well as the original PRISMA item. CONCLUSIONS: This explanation and elaboration document is intended to accompany the PRISMA-E 2012 Statement and the PRISMA Statement to improve understanding of the reporting guideline for users. The PRISMA-E 2012 reporting guideline is intended to improve transparency and completeness of reporting of equity-focused systematic reviews. Improved reporting can lead to better judgement of applicability by policy makers which may result in more appropriate policies and programs and may contribute to reductions in health inequities. To encourage wide dissemination of this article it is accessible on the International Journal for Equity in Health, Journal of Clinical Epidemiology, and Journal of Development Effectiveness web sites.

Regulation of actin-myosin interaction by conserved periodic sites of tropomyosin.

Cooperative activation of actin-myosin interaction by tropomyosin (Tm) is central to regulation of contraction in muscle cells and cellular and intracellular movements in nonmuscle cells. The steric blocking model of muscle regulation proposed 40 y ago has been substantiated at both the kinetic and structural levels. Even with atomic resolution structures of the major players, how Tm binds and is designed for regulatory function has remained a mystery. Here we show that a set of periodically distributed evolutionarily conserved surface residues of Tm is required for cooperative regulation of actomyosin. Based on our results, we propose a model of Tm on a structure of actin-Tm-myosin in the "open" (on) state showing potential electrostatic interactions of the residues with both actin and myosin. The sites alternate with a second set of conserved surface residues that are important for actin binding in the inhibitory state in the absence of myosin. The transition from the closed to open states requires the sites identified here, even when troponin + Ca(2+) is present. The evolutionarily conserved residues are important for actomyosin regulation, a universal function of Tm that has a common structural basis and mechanism.

Three-dimensional organization of troponin on cardiac muscle thin filaments in the relaxed state.

Muscle contraction is regulated by troponin-tropomyosin, which blocks and unblocks myosin binding sites on actin. To elucidate this regulatory mechanism, the three-dimensional organization of troponin and tropomyosin on the thin filament must be determined. Although tropomyosin is well defined in electron microscopy helical reconstructions of thin filaments, troponin density is mostly lost. Here, we determined troponin organization on native relaxed cardiac muscle thin filaments by applying single particle reconstruction procedures to negatively stained specimens. Multiple reference models led to the same final structure, indicating absence of model bias in the procedure. The new reconstructions clearly showed F-actin, tropomyosin, and troponin densities. At the 25 Å resolution achieved, troponin was considerably better defined than in previous reconstructions. The troponin density closely resembled the shape of troponin crystallographic structures, facilitating detailed interpretation of the electron microscopy density map. The orientation of troponin-T and the troponin core domain established troponin polarity. Density attributable to the troponin-I mobile regulatory domain was positioned where it could hold tropomyosin in its blocking position on actin, thus suggesting the underlying structural basis of thin filament regulation. Our previous understanding of thin filament regulation had been limited to known movements of tropomyosin that sterically block and unblock myosin binding sites on actin. We now show how troponin, the Ca(2+) sensor, may control these movements, ultimately determining whether muscle contracts or relaxes.

Modulation of thin filament activation of myosin ATP hydrolysis by N-terminal domains of cardiac myosin binding protein-C.

We have used enzyme kinetics to investigate the molecular mechanism by which the N-terminal domains of human and mouse cardiac MyBP-C (C0C1, C1C2, and C0C2) affect the activation of myosin ATP hydrolysis by F-actin and by native porcine thin filaments. N-Terminal domains of cMyBP-C inhibit the activation of myosin-S1 ATPase by F-actin. However, mouse and human C1C2 and C0C2 produce biphasic activating and inhibitory effects on the activation of myosin ATP hydrolysis by native cardiac thin filaments. Low ratios of MyBP-C N-terminal domains to thin filaments activate myosin-S1 ATP hydrolysis, but higher ratios inhibit ATP hydrolysis, as is observed with F-actin alone. These data suggest that low concentrations of C1C2 and C0C2 activate thin filaments by a mechanism similar to that of rigor myosin-S1, whereas higher concentrations inhibit the ATPase rate by competing with myosin-S1-ADP-Pi for binding to actin and thin filaments. In contrast to C0C2 and C1C2, the activating effects of the C0C1 domain are species-dependent: human C0C1 activates actomyosin-S1 ATPase rates, but mouse C0C1 does not produce significant activation or inhibition. Phosphorylation of serine residues in the m-linker between the C1 and C2 domains by protein kinase-A decreases the activation of thin filaments by huC0C2 at pCa > 8 but has little effect on the activation mechanism at pCa = 4. In sarcomeres, the low ratio of cMyBP-C to actin is expected to favor the activating effects of cMyBP-C while minimizing inhibition produced by competition with myosin heads.

Drosophila myosin-XX functions as an actin-binding protein to facilitate the interaction between Zyx102 and actin.

The class XX myosin is a member of the diverse myosin superfamily and exists in insects and several lower invertebrates. DmMyo20, the class XX myosin in Drosophila, is encoded by dachs, which functions as a crucial downstream component of the Fat signaling pathway, influencing growth, affinity, and gene expression during development. Sequence analysis shows that DmMyo20 contains a unique N-terminal extension, the motor domain, followed by one IQ motif, and a C-terminal tail. To investigate the biochemical properties of DmMyo20, we expressed several DmMyo20 truncated constructs containing the motor domain in the baculovirus/Sf9 system. We found that the motor domain of DmMyo20 had neither ATPase activity nor the ability to bind to ATP, suggesting that DmMyo20 does not function as a molecular motor. We found that the motor domain of DmMyo20 could specifically bind to actin filaments in an ATP-independent manner and enhance the interaction between actin filaments and Zyx102, a downstream component of DmMyo20 in the Fat signaling pathway. These results suggest that DmMyo20 functions as a scaffold protein, but not as a molecular motor, in a signaling pathway controlling cell differentiation.

Direct observation of the mechanochemical coupling in myosin Va during processive movement.

Myosin Va transports intracellular cargoes along actin filaments in cells. This processive, two-headed motor takes multiple 36-nm steps in which the two heads swing forward alternately towards the barbed end of actin driven by ATP hydrolysis. The ability of myosin Va to move processively is a function of its long lever arm, the high duty ratio of its kinetic cycle and the gating of the kinetics between the two heads such that ADP release from the lead head is greatly retarded. Mechanical studies at the multiple- and the single-molecule level suggest that there is tight coupling (that is, one ATP is hydrolysed per power stroke), but this has not been directly demonstrated. We therefore investigated the coordination between the ATPase mechanism of the two heads of myosin Va and directly visualized the binding and dissociation of single fluorescently labelled nucleotide molecules, while simultaneously observing the stepping motion of the fluorescently labelled myosin Va as it moved along an actin filament. Here we show that preferential ADP dissociation from the trail head of mouse myosin Va is followed by ATP binding and a synchronous 36-nm step. Even at low ATP concentrations, the myosin Va molecule retained at least one nucleotide (ADP in the lead head position) when moving. Thus, we directly demonstrate tight coupling between myosin Va movement and the binding and dissociation of nucleotide by simultaneously imaging with near nanometre precision.

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.

Effectiveness of road safety interventions: An evidence and gap map.

Background: Road Traffic injuries (RTI) are among the top ten leading causes of death in the world resulting in 1.35 million deaths every year, about 93% of which occur in low- and middle-income countries (LMICs). Despite several global resolutions to reduce traffic injuries, they have continued to grow in many countries. Many high-income countries have successfully reduced RTI by using a public health approach and implementing evidence-based interventions. As many LMICs develop their highway infrastructure, adopting a similar scientific approach towards road safety is crucial. The evidence also needs to be evaluated to assess external validity because measures that have worked in high-income countries may not translate equally well to other contexts. An evidence gap map for RTI is the first step towards understanding what evidence is available, from where, and the key gaps in knowledge. Objectives: The objective of this evidence gap map (EGM) is to identify existing evidence from all effectiveness studies and systematic reviews related to road safety interventions. In addition, the EGM identifies gaps in evidence where new primary studies and systematic reviews could add value. This will help direct future research and discussions based on systematic evidence towards the approaches and interventions which are most effective in the road safety sector. This could enable the generation of evidence for informing policy at global, regional or national levels. Search Methods: The EGM includes systematic reviews and impact evaluations assessing the effect of interventions for RTI reported in academic databases, organization websites, and grey literature sources. The studies were searched up to December 2019. Selection Criteria: The interventions were divided into five broad categories: (a) human factors (e.g., enforcement or road user education), (b) road design, infrastructure and traffic control, (c) legal and institutional framework, (d) post-crash pre-hospital care, and (e) vehicle factors (except car design for occupant protection) and protective devices. Included studies reported two primary outcomes: fatal crashes and non-fatal injury crashes; and four intermediate outcomes: change in use of seat belts, change in use of helmets, change in speed, and change in alcohol/drug use. Studies were excluded if they did not report injury or fatality as one of the outcomes. Data Collection and Analysis: The EGM is presented in the form of a matrix with two primary dimensions: interventions (rows) and outcomes (columns). Additional dimensions are country income groups, region, quality level for systematic reviews, type of study design used (e.g., case-control), type of road user studied (e.g., pedestrian, cyclists), age groups, and road type. The EGM is available online where the matrix of interventions and outcomes can be filtered by one or more dimensions. The webpage includes a bibliography of the selected studies and titles and abstracts available for preview. Quality appraisal for systematic reviews was conducted using a critical appraisal tool for systematic reviews, AMSTAR 2. Main Results: The EGM identified 1859 studies of which 322 were systematic reviews, 7 were protocol studies and 1530 were impact evaluations. Some studies included more than one intervention, outcome, study method, or study region. The studies were distributed among intervention categories as: human factors (n = 771), road design, infrastructure and traffic control (n = 661), legal and institutional framework (n = 424), post-crash pre-hospital care (n = 118) and vehicle factors and protective devices (n = 111). Fatal crashes as outcomes were reported in 1414 records and non-fatal injury crashes in 1252 records. Among the four intermediate outcomes, speed was most commonly reported (n = 298) followed by alcohol (n = 206), use of seatbelts (n = 167), and use of helmets (n = 66). Ninety-six percent of the studies were reported from high-income countries (HIC), 4.5% from upper-middle-income countries, and only 1.4% from lower-middle and low-income countries. There were 25 systematic reviews of high quality, 4 of moderate quality, and 293 of low quality. Authors' Conclusions: The EGM shows that the distribution of available road safety evidence is skewed across the world. A vast majority of the literature is from HICs. In contrast, only a small fraction of the literature reports on the many LMICs that are fast expanding their road infrastructure, experiencing rapid changes in traffic patterns, and witnessing growth in road injuries. This bias in literature explains why many interventions that are of high importance in the context of LMICs remain poorly studied. Besides, many interventions that have been tested only in HICs may not work equally effectively in LMICs. Another important finding was that a large majority of systematic reviews are of low quality. The scarcity of evidence on many important interventions and lack of good quality evidence-synthesis have significant implications for future road safety research and practice in LMICs. The EGM presented here will help identify priority areas for researchers, while directing practitioners and policy makers towards proven interventions.

Reporting of equity in observational epidemiology: A methodological review.

Background: Observational studies can inform how we understand and address persisting health inequities through the collection, reporting and analysis of health equity factors. However, the extent to which the analysis and reporting of equity-relevant aspects in observational research are generally unknown. Thus, we aimed to systematically evaluate how equity-relevant observational studies reported equity considerations in the study design and analyses. Methods: We searched MEDLINE for health equity-relevant observational studies from January 2020 to March 2022, resulting in 16 828 articles. We randomly selected 320 studies, ensuring a balance in focus on populations experiencing inequities, country income settings, and coronavirus disease 2019 (COVID-19) topic. We extracted information on study design and analysis methods. Results: The bulk of the studies were conducted in North America (n = 95, 30%), followed by Europe and Central Asia (n = 55, 17%). Half of the studies (n = 171, 53%) addressed general health and well-being, while 49 (15%) focused on mental health conditions. Two-thirds of the studies (n = 220, 69%) were cross-sectional. Eight (3%) engaged with populations experiencing inequities, while 22 (29%) adapted recruitment methods to reach these populations. Further, 67 studies (21%) examined interaction effects primarily related to race or ethnicity (48%). Two-thirds of the studies (72%) adjusted for characteristics associated with inequities, and 18 studies (6%) used flow diagrams to depict how populations experiencing inequities progressed throughout the studies. Conclusions: Despite over 80% of the equity-focused observational studies providing a rationale for a focus on health equity, reporting of study design features relevant to health equity ranged from 0-95%, with over half of the items reported by less than one-quarter of studies. This methodological study is a baseline assessment to inform the development of an equity-focussed reporting guideline for observational studies as an extension of the well-known Strengthening Reporting of Observational Studies in Epidemiology (STROBE) guideline.

Influence of lever structure on myosin 5a walking.

Using electron microscopy and image processing, we have observed myosin 5a modified with lever arms of different lengths (four, six, and eight calmodulin-binding IQ domains) and orientations walking along actin filaments. Step lengths were dependent on lever length: 8IQ > 6IQ > 4IQ, which is consistent with myosin 5a having evolved to walk straight along actin. Lead heads were mostly in the prepowerstroke state, tethered there by the trail head. However, improved image processing showed that in 5-10% of molecules the lead motor was in the postpowerstroke state. This is a unique attached state of myosin, where the motor domain has completed its powerstroke at the expense of severe lever distortion, but with little cargo movement. Postpowerstroke lead heads were seen in both wild-type and modified lever molecules, mostly where there was least strain. These data allow the strain dependence of the equilibrium between pre- and postpowerstroke conformations to be measured. Slow rates of ADP dissociation observed from lead heads of these molecules can be explained by the unfavorable equilibrium between the pre- and postpowerstroke conformations preceding ADP loss.

Effectiveness of interventions for improving social inclusion outcomes for people with disabilities in low- and middle-income countries: A systematic review.

Background: People with disabilities-more than a billion people worldwide-are frequently excluded from social and political life, and often experience stigmatising attitudes and behaviours from people without disabilities. This stigma, coupled with inaccessible environments and systems and institutional barriers (e.g., lack of inclusive legislation), may result in discrimination against people with disabilities (and their families) to the degree that they are not able to enjoy their rights on an equal basis with others. Objectives: This review examines the effectiveness of interventions for improving social inclusion outcomes (acquisition of skills for social inclusion, broad-based social inclusion, and improved relationships) for people with disabilities in low- and middle-income countries (LMICs). Search Methods: We searched academic and online databases, carried out citation tracking of included studies, and contacted experts to ensure our search was as comprehensive as possible. We also ran the searches with search terms specific to social inclusion review using Open Alex in EPPI reviewer. Selection Criteria: We included all studies which reported on impact evaluations of interventions to improve social inclusion outcomes for people with disabilities in LMIC. Data Collection and Analysis: We used review management software EPPI Reviewer to screen the search results. Two review authors independently extracted the data from each study report, including for the confidence in study findings appraisal. Data and information were extracted regarding available characteristics of participants, intervention characteristics and control conditions, research design, sample size, risk of bias and outcomes, and results. Random-effects inverse variance weighted meta-analytic methods were used to synthesise standardised mean differences for the outcomes. Main Results: We identified 37 experimental and quasi-experimental studies. Studies were conducted in 16 countries, with the majority of the included studies (n = 13) from South Asia and nine each from East Asia, the Pacific, the Middle East, and North Africa. Most studies targeted children with disabilities (n = 23), and 12 targeted adults with disabilities. Most focused on people with intellectual disabilities (n = 20) and psychosocial disabilities (n = 13). Regarding intervention content, most (n = 17) of the included programmes aimed to improve the social and communication skills of people with disabilities through social skills training programmes. Ten studies aimed at providing personal assistance and support and evaluated the effects of a parent training programme on the interactive skills of parents of children and their children with disabilities. We calculated effect sizes from experimental and quasi-experimental studies for outcomes on skills for social inclusion, relationships of people with disabilities with family and community members, and broad-based social inclusion among people with disabilities. A meta-analysis of 16 studies indicates an overall positive, statistically significant and large effect of the interventions for skills for social inclusion with standardised mean difference (SMD) = 0.87, confidence interval (CI) = 0.57 to 1.16, k = 26, I 2 = 77%, p < 0.001). For relationships across 12 studies, we find a positive but moderate effect (SMD = 0.61, CI = 0.41 to 0.80, k = 15, I 2 = 64%, p < 0.01). As for the overall effect on broad-based social inclusion, we find the average effect size was large, and there was significant dispersion across studies (SMD = 0.72, CI = 0.33 to 1.11, k = 2, I 2 = 93%, p < 0.01). Despite the significant and large effects estimated by the studies, some limitations must be noted. Although there was a consensus on the direction of the effects, the studies presented considerable heterogeneity in the size of the effects. A majority (n = 27) of studies were assessed to be of low confidence related to methodological limitations, so the findings must be interpreted with caution. Tests for publication bias show that the effect sizes of social skills (p < 0.01) and social inclusion (p = 0.01) are all likely to be inflated by the existence of the publication bias. Authors' Conclusions: The review's findings suggest that various interventions to improve the social inclusion of people with disabilities have a significant positive effect. Interventions such as social and communication training and personal assistance led to significant improvement in the social behaviour and social skills of people with disabilities. Studies targeting broad-based social inclusion showed a large and significant positive effect. A moderate effect was reported from interventions designed to improve relationships between people with disabilities and their families and communities. However, the findings of this review must be interpreted cautiously, given the low confidence in study methods, severe heterogeneity and significant publication bias. The available evidence focused primarily on individual-level barriers such as interventions for improving social or communications skills of people with disabilities and not the systemic drivers of exclusions such as addressing societal barriers to inclusion, such as stigma reduction, and interventions to strengthen legislation, infrastructure, and institutions.

PROTOCOL: Treatment for depressive disorder among adults: An evidence and gap map of systematic reviews.

This is the protocol for a Campbell evidence and gap map. The objective of the map is to map available systematic reviews on the effectiveness of treatments for depressive disorders among adults. Specifically, this EGM includes studies on the effectiveness of treatments across a range of outcome domains.

PROTOCOL: An evidence and gap map of studies of implementation issues for interventions for those affected by and at risk of homelessness in high-income countries.

This is the protocol for a Campbell systematic review. The objectives are as follows. The proposed evidence and gap map will present relevant process evaluations and other studies of barriers and facilitators, both qualitative and quantitative, for eligible homelessness interventions to highlight the issues arising in the implementation of these interventions. Specifically, the objectives of the map are to: (i) develop a clear taxonomy of interventions and implementation issues (e.g., barriers and facilitators-factors which works as barriers to hinder successful implementation of policies and programmes and factors which facilitate the intervention and therefore support its implementation) related to homelessness in high-income countries; (ii) map available systematic reviews and primary studies of the implementation issues of interventions for those experiencing homelessness and those at risk of homelessness, with an overview provided in a summary report; (iii) provide a searchable database of included studies accessible to research users via CHI website.