TNNT2 mutations in the tropomyosin binding region of TNT1 disrupt its role in contractile inhibition and stimulate cardiac dysfunction.

Muscle contraction is regulated by the movement of end-to-end-linked troponin-tropomyosin complexes over the thin filament surface, which uncovers or blocks myosin binding sites along F-actin. The N-terminal half of troponin T (TnT), TNT1, independently promotes tropomyosin-based, steric inhibition of acto-myosin associations, in vitro. Recent structural models additionally suggest TNT1 may restrain the uniform, regulatory translocation of tropomyosin. Therefore, TnT potentially contributes to striated muscle relaxation; however, the in vivo functional relevance and molecular basis of this noncanonical role remain unclear. Impaired relaxation is a hallmark of hypertrophic and restrictive cardiomyopathies (HCM and RCM). Investigating the effects of cardiomyopathy-causing mutations could help clarify TNT1's enigmatic inhibitory property. We tested the hypothesis that coupling of TNT1 with tropomyosin's end-to-end overlap region helps anchor tropomyosin to an inhibitory position on F-actin, where it deters myosin binding at rest, and that, correspondingly, cross-bridge cycling is defectively suppressed under diastolic/low Ca2+ conditions in the presence of HCM/RCM lesions. The impact of TNT1 mutations on Drosophila cardiac performance, rat myofibrillar and cardiomyocyte properties, and human TNT1's propensity to inhibit myosin-driven, F-actin-tropomyosin motility were evaluated. Our data collectively demonstrate that removing conserved, charged residues in TNT1's tropomyosin-binding domain impairs TnT's contribution to inhibitory tropomyosin positioning and relaxation. Thus, TNT1 may modulate acto-myosin activity by optimizing F-actin-tropomyosin interfacial contacts and by binding to actin, which restrict tropomyosin's movement to activating configurations. HCM/RCM mutations, therefore, highlight TNT1's essential role in contractile regulation by diminishing its tropomyosin-anchoring effects, potentially serving as the initial trigger of pathology in our animal models and humans.

Lysine acetylation of F-actin decreases tropomyosin-based inhibition of actomyosin activity.

Recent proteomics studies of vertebrate striated muscle have identified lysine acetylation at several sites on actin. Acetylation is a reversible post-translational modification that neutralizes lysine's positive charge. Positively charged residues on actin, particularly Lys326 and Lys328, are predicted to form critical electrostatic interactions with tropomyosin (Tpm) that promote its binding to filamentous (F)-actin and bias Tpm to an azimuthal location where it impedes myosin attachment. The troponin (Tn) complex also influences Tpm's position along F-actin as a function of Ca2+ to regulate exposure of myosin-binding sites and, thus, myosin cross-bridge recruitment and force production. Interestingly, Lys326 and Lys328 are among the documented acetylated residues. Using an acetic anhydride-based labeling approach, we showed that excessive, nonspecific actin acetylation did not disrupt characteristic F-actin-Tpm binding. However, it significantly reduced Tpm-mediated inhibition of myosin attachment, as reflected by increased F-actin-Tpm motility that persisted in the presence of Tn and submaximal Ca2+ Furthermore, decreasing the extent of chemical acetylation, to presumptively target highly reactive Lys326 and Lys328, also resulted in less inhibited F-actin-Tpm, implying that modifying only these residues influences Tpm's location and, potentially, thin filament regulation. To unequivocally determine the residue-specific consequences of acetylation on Tn-Tpm-based regulation of actomyosin activity, we assessed the effects of K326Q and K328Q acetyl (Ac)-mimetic actin on Ca2+-dependent, in vitro motility parameters of reconstituted thin filaments (RTFs). Incorporation of K328Q actin significantly enhanced Ca2+ sensitivity of RTF activation relative to control. Together, our findings suggest that actin acetylation, especially Lys328, modulates muscle contraction via disrupting inhibitory Tpm positioning.

Raman spectroscopic studies on screening of myopathies.

Myopathies are among the major causes of mortality in the world. There is no complete cure for this heterogeneous group of diseases, but a sensitive, specific, and fast diagnostic tool may improve therapy effectiveness. In this study, Raman spectroscopy is applied to discriminate between muscle mutants in Drosophila on the basis of associated changes at the molecular level. Raman spectra were collected from indirect flight muscles of mutants, upheld(1) (up(1)), heldup(2) (hdp(2)), myosin heavy chain(7) (Mhc(7)), actin88F(KM88) (Act88F(KM88)), upheld(101) (up(101)), and Canton-S (CS) control group, for both 2 and 12 days old flies. Difference spectra (mutant minus control) of all the mutants showed an increase in nucleic acid and ß-sheet and/or random coil protein content along with a decrease in α-helix protein. Interestingly, the 12th day samples of up(1) and Act88F(KM88) showed significantly higher levels of glycogen and carotenoids than CS. A principal components based linear discriminant analysis classification model was developed based on multidimensional Raman spectra, which classified the mutants according to their pathophysiology and yielded an overall accuracy of 97% and 93% for 2 and 12 days old flies, respectively. The up(1) and Act88F(KM88) (nemaline-myopathy) mutants form a group that is clearly separated in a linear discriminant plane from up(101) and hdp(2) (cardiomyopathy) mutants. Notably, Raman spectra from a human sample with nemaline-myopathy formed a cluster with the corresponding Drosophila mutant (up(1)). In conclusion, this is the first demonstration in which myopathies, despite their heterogeneity, were screened on the basis of biochemical differences using Raman spectroscopy.

Assessment of utilisation of government programmes and services by pregnant women in India.

BACKGROUND: Since the implementation of various maternal health programs, Maternal Mortality Ratio (MMR) has significantly declined in India through improvements in maternal health services. However, inequality persists at the regional and socio-economic levels. In light of this, the present study aims to assess the existing regional disparities in utilising various government initiatives for safe motherhood in India. METHODS: National-level datasets such as National Family and Health Surveys (NFHS-3 (2005-06); NFHS-4 (2015-16) and NFHS-5(2019-21); Health Management Information System (HMIS), 2019-20; Sample Registrar System (SRS), 2001-2018) were used in the study. In addition, composite Index and inequality measures (Range, Ratio, and Gini) were calculated to examine inequality. At the same time, the Pearson correlation was used to investigate the correlation between various components of maternal health services and Maternal Mortality Rate (MMR). RESULTS: The composite index score (0.65) reflects that India is still far behind the targets of the utilisation of maternal health care services. Within the utilisation of services, the Gini coefficient reveals that the least inequality was recorded in skilled birth assistance deliveries (0.03) and institutional deliveries (0.04). In contrast, the highest inequality was recorded in receiving Iron and Folic Acid (IFA) Tablets for 100 days (0.19) and four Antenatal Care (ANC) visits (0.13) among selected states. Based on the composite score for maternal health utilisation, Kerala, Tamil Nadu, Andhra Pradesh, Odisha, and Delhi were amongst the best performers, whereas Bihar, Jharkhand, Uttar Pradesh, and Assam were amongst the worst performers. CONCLUSION: This indicates that the government's single-minded focus on enhancing institutional deliveries and skilled health-assisted deliveries has detracted from other essential interventions related to maternal health. Therefore, the states with the utilisation of maternal services need to initiate immediate action to increase the ANC and Post-natal Care (PNC utilisation with more attention towards better implementation of existing ANC programmes by the government.

Myosin Transducer Inter-Strand Communication Is Critical for Normal ATPase Activity and Myofibril Structure.

The R249Q mutation in human ß-cardiac myosin results in hypertrophic cardiomyopathy. We previously showed that inserting this mutation into Drosophila melanogaster indirect flight muscle myosin yields mechanical and locomotory defects. Here, we use transgenic Drosophila mutants to demonstrate that residue R249 serves as a critical communication link within myosin that controls both ATPase activity and myofibril integrity. R249 is located on a ß-strand of the central transducer of myosin, and our molecular modeling shows that it interacts via a salt bridge with D262 on the adjacent ß-strand. We find that disrupting this interaction via R249Q, R249D or D262R mutations reduces basal and actin-activated ATPase activity, actin in vitro motility and flight muscle function. Further, the R249D mutation dramatically affects myofibril assembly, yielding abnormalities in sarcomere lengths, increased Z-line thickness and split myofibrils. These defects are exacerbated during aging. Re-establishing the ß-strand interaction via a R249D/D262R double mutation restores both basal ATPase activity and myofibril assembly, indicating that these properties are dependent upon transducer inter-strand communication. Thus, the transducer plays an important role in myosin function and myofibril architecture.

Social distancing as risk reduction strategy during COVID-19 pandemic: A study of Delhi-NCT, India.

COVID-19 pandemic impacted countries all over the world calling as urgent need to enhance the capacity of individuals and communities to respond in such disease outbreaks. Public health interventions, such as social distancing could help to protect many vulnerable people and reduce secondary transmissions within the community. This research was undertaken as a longitudinal study to identify and comprehend the preferences given to different preventive measures adopted by individuals to ensure protection against the spread of coronavirus. An online survey was conducted with a representative sample of 176 stakeholders to identify practices and behaviour adopted by the key stakeholders working in the domains of water, health and disaster risk reduction to curb the spread of COVID-19 in Delhi-National Capital Territory of India (Delhi-NCT). Findings of this study shown preferred willingness to adopt sanitisation measures, often by cleaning their hands with soap and water, and restricting any movement inside and outside of the house. Three-fourth of the respondents considered washing their hands after returning from outside an important measure to limit the spread of the disease. Moreover, two-third of the people stopped ordering food from outside as a prevention strategy. Furthermore, only about 36% of the respondents showed interest in stocking up on grocery items as their most preferred choice, reflecting that this was considered to be the least important of all choices. Research outcomes of this study will help policymakers to better understand mitigation options that are used in the early and later stages of the lockdown to improve resilience from such viruses. Further, it will assist decision-makers to understand the range of individual-level practices adopted by stakeholders to mitigate disease transmission at the community level and inform the government's planning efforts in the future.

Acoustotactic response of mosquitoes in untethered flight to incidental sound.

Mosquitoes are vectors for some of the most devastating diseases on the planet. Given the centrality of acoustic sensing in the precopulatory behavior of these vectors, the use of an exogenous acoustic stimulus offers the potential of interfering with the courtship behavior of these insects. Previous research on the acoustotactic response of mosquitoes has been conducted on tethered preparations using low-intensity sound stimuli. To quantify differences in acoustotactic responses between mosquitos of distinct sex and species, we examined the effects of incidental sound stimuli on the flight behavior of free-flying male vs. female Aedes aegypti and Anopheles gambiae mosquitoes. The key variables were sound frequency (100-1000 Hz) and intensity (67-103 dB, measured at 12.5 cm from the source), and the acoustotactic response was measured in terms of the relative increase in flight speed in response to the stimulus. The data show, for the first time, significant sex- and species-specific differences in acoustotactic responses. A. aegypti exhibited a greater response to sound stimulus compared to An. gambiae, and the response also extended over a larger range of frequencies. Furthermore, the males of both species displayed a greater acoustotactic response than females, with An. gambiae females exhibiting minimal response to sound.

A role for actin flexibility in thin filament-mediated contractile regulation and myopathy.

Striated muscle contraction is regulated by the translocation of troponin-tropomyosin strands over the thin filament surface. Relaxation relies partly on highly-favorable, conformation-dependent electrostatic contacts between actin and tropomyosin, which position tropomyosin such that it impedes actomyosin associations. Impaired relaxation and hypercontractile properties are hallmarks of various muscle disorders. The α-cardiac actin M305L hypertrophic cardiomyopathy-causing mutation lies near residues that help confine tropomyosin to an inhibitory position along thin filaments. Here, we investigate M305L actin in vivo, in vitro, and in silico to resolve emergent pathological properties and disease mechanisms. Our data suggest the mutation reduces actin flexibility and distorts the actin-tropomyosin electrostatic energy landscape that, in muscle, result in aberrant contractile inhibition and excessive force. Thus, actin flexibility may be required to establish and maintain interfacial contacts with tropomyosin as well as facilitate its movement over distinct actin surface features and is, therefore, likely necessary for proper regulation of contraction.

Overexpression of miRNA-9 Generates Muscle Hypercontraction Through Translational Repression of Troponin-T in Drosophila melanogaster Indirect Flight Muscles.

MicroRNAs (miRNAs) are small noncoding endogenous RNAs, typically 21-23 nucleotides long, that regulate gene expression, usually post-transcriptionally, by binding to the 3'-UTR of target mRNA, thus blocking translation. The expression of several miRNAs is significantly altered during cardiac hypertrophy, myocardial ischemia, fibrosis, heart failure, and other cardiac myopathies. Recent studies have implicated miRNA-9 (miR-9) in myocardial hypertrophy. However, a detailed mechanism remains obscure. In this study, we have addressed the roles of miR-9 in muscle development and function using a genetically tractable model system, the indirect flight muscles (IFMs) of Drosophila melanogaster Bioinformatics analysis identified 135 potential miR-9a targets, of which 27 genes were associated with Drosophila muscle development. Troponin-T (TnT) was identified as major structural gene target of miR-9a. We show that flies overexpressing miR-9a in the IFMs have abnormal wing position and are flightless. These flies also exhibit a loss of muscle integrity and sarcomeric organization causing an abnormal muscle condition known as "hypercontraction." Additionally, miR-9a overexpression resulted in the reduction of TnT protein levels while transcript levels were unaffected. Furthermore, muscle abnormalities associated with miR-9a overexpression were completely rescued by overexpression of TnT transgenes which lacked the miR-9a binding site. These findings indicate that miR-9a interacts with the 3'-UTR of the TnT mRNA and downregulates the TnT protein levels by translational repression. The reduction in TnT levels leads to a cooperative downregulation of other thin filament structural proteins. Our findings have implications for understanding the cellular pathophysiology of cardiomyopathies associated with miR-9 overexpression.

Transcriptome analysis of IFM-specific actin and myosin nulls in Drosophila melanogaster unravels lesion-specific expression blueprints across muscle mutations.

Muscle contraction is a highly fine-tuned process that requires the precise and timely construction of large protein sub-assemblies to form sarcomeres. Mutations in many genes encoding constituent proteins of this macromolecular machine result in defective functioning of the muscle tissue. However, the pathways underlying muscle degeneration, and manifestation of myopathy phenotypes are not well understood. In this study, we explored transcriptional alterations that ensue from the absence of the two major muscle proteins - myosin and actin - using the Drosophila indirect flight muscles. Our aim was to understand how the muscle tissue responds as a whole to the absence of either of the major scaffold proteins, whether the responses are generic to the tissue; or unique to the thick versus thin filament systems. Our results indicated that muscles respond by altering gene transcriptional levels in multiple systems active in muscle remodelling, protein degradation and heat shock responses. However, there were some responses that were filament-specific signatures of muscle degeneration, like immune responses, metabolic alterations and alterations in expression of muscle structural genes and mitochondrial ribosomal genes. These general and filament-specific changes in gene expression may be of relevance to human myopathies.

Comparing two epidemiologic surveillance methods to assess underestimation of human stampedes in India.

BACKGROUND: Two separate but complementary epidemiologic surveillance methods for human stampedes have emerged since the publication of the topic in 2009. The objective of this study is to estimate the degree of underreporting in India. METHOD: The Ngai Search Method was compared to the Roy Search Method for human stampede events occurring in India between 2001 and 2010. RESULTS: A total of 40 stampedes were identified by both search methods. Using the Ngai method, 34 human stampedes were identified. Using a previously defined stampede scale: 2 events were class I, 21 events were class II, 8 events were class III, and 3 events were class IV. The median deaths were 5.5 per event and median injuries were 13.5 per event. Using the Roy method, 27 events were identified, including 9 events that were not identified by the Ngai method. After excluding events based on exclusion criteria, six additional events identified by the Roy's method had a median of 4 deaths and 30 injuries. In multivariate analysis using the Ngai method, religious (6.52, 95%CI 1.73-24.66, p=0.006) and political (277.09, 95%CI 5.12-15,001.96, p=0.006) events had higher relative number of deaths. CONCLUSION: Many causes accounting for the global increase in human stampede events can only be elucidated through systematic epidemiological investigation. Focusing on a country with a high recurrence of human stampedes, we compare two independent methods of data abstraction in an effort to improve the existing database and to identify pertinent risk factors. We concluded that our previous publication underestimated stampede events in India by approximately 18% and an international standardized database to systematically record occurrence of human stampedes is needed to facilitate understanding of the epidemiology of human stampedes.