Modeling human spinal muscular atrophy mutations in Drosophila melanogaster

Spinal Muscular Atrophy (SMA) is a common neuromuscular disease that affects one in 6,000-8,000 young children; most of whom die before reaching the age of two years. One in fifty Americans is a carrier for SMA, making this genetic disease a serious health concern. More than 95% of patients with SMA carry deletions in the survival motor neuron 1 (SMN1) gene. The SMN protein is essential for survival and has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), which are core components of the spliceosome. Numerous additional functions, both housekeeping and tissue-specific, have been put forth in the literature, however, no convincing link has been made between any putative SMN function and the disease etiology. To address this question, we are studying the consequences of SMN loss in the Drosophila model system. We have generated a series of transgenic flies that exclusively express mutant forms of SMN derived from human SMA patients to uncouple the housekeeping and tissue-specific functions of SMN. Null mutants in Smn die as larvae, show significant motility defects, and have reduced levels of minor-class snRNAs. Surprisingly, despite these reductions, minor-class intron splicing in these mutants is unperturbed. In addition, we find that rescue of the null mutant with a WT SMN construct does not restore snRNA levels, but does rescue the motility defects and lethality of the Smn null flies. In addition, the majority of flies expressing an SMA point mutation, T205I, die as pupae but have a similar snRNA profile to the WT transgenic animals. These data suggest that the reduction in snRNAs in Smn mutants is not a major contributor to their lethality, and indicate that non-snRNP related functions of SMN may be critical to SMA pathology. We have generated twelve additional point mutations in Smn that mimic mutations identified in SMA patients. The phenotypes of these mutations reflect the range of severities seen in SMA patients, and will be important tools in identifying the functions of SMN that form the etiological basis for SMA.Doctor of Philosoph

Transcriptomic comparison of Drosophila snRNP biogenesis mutants reveals mutant-specific changes in pre-mRNA processing: implications for spinal muscular atrophy

Survival motor neuron (SMN) functions in the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) that catalyze pre-mRNA splicing. Here, we used disruptions in Smn and two additional snRNP biogenesis genes, Phax and Ars2, to classify RNA processing differences as snRNP-dependent or gene-specific in Drosophila. Phax and Smn mutants exhibited comparable reductions in snRNAs, and comparison of their transcriptomes uncovered shared sets of RNA processing changes. In contrast, Ars2 mutants displayed only small decreases in snRNA levels, and RNA processing changes in these mutants were generally distinct from those identified in Phax and Smn animals. Instead, RNA processing changes in Ars2 mutants support the known interaction of Ars2 protein with the cap-binding complex, as splicing changes showed a clear bias toward the first intron. Bypassing disruptions in snRNP biogenesis, direct knockdown of spliceosomal proteins caused similar changes in the splicing of snRNP-dependent events. However, these snRNP-dependent events were largely unaltered in three Smn mutants expressing missense mutations that were originally identified in human spinal muscular atrophy (SMA) patients. Hence, findings here clarify the contributions of Phax, Smn, and Ars2 to snRNP biogenesis in Drosophila, and loss-of-function mutants for these proteins reveal differences that help disentangle cause and effect in SMA model flies

Comparative study of oral ivermectin, topical permethrin and benzyl benzoate in the treatment of scabies

Background: Efficacy of these modalities as shown by various investigations are inconsistent and ambiguous. Thus, evidence based effective treatment option is warranted. Aim of the study was to compare the efficacy of oral ivermectin, topical permethrin and benzyl benzoate in the treatment of uncomplicated scabies.Methods: Patients with confirmed diagnosis of scabies were included in this study. One hundred and ninety-five subjects were included in this investigation as per inclusion and exclusion criteria laid down. Equal numbers of patients were randomly allocated to one of the three treatment groups. Efficacy of three groups [oral ivermectin (Group A), topical permethrin (Group B) and benzyl benzoate (Group C)] of drugs was compared in terms of improvement in clinical grading of disease (%) and improvement in clinical grading of pruritus (%) during follow up visits.Results: Those subjects receiving topical permethrin, at 1st follow up 56.9% showed cure rate which increased to 89.2% at 2nd follow up with respect to clinical improvement in pruritus. Maximum relief in severity of pruritus at the end of 6th week was reported by 58(89.2%) patients receiving group B treatment modality followed by 52 patients (80%) in arm A. Regarding efficacy of three treatment groups in terms of improvement in severity of lesion at the end of 6 weeks, maximum number of patients 57(87.7%), receiving group B treatment reported improvement which is better than other two treatment groups.Conclusions: maximum number of patients receiving topical Permethrin treatment reported improvement better than other Oral Ivermectin therapy and topical benzyl benzoate. Oral ivermectin may serve a good alternative for managing scabies under certain conditions like poor compliance to topical scabicides

Nuclear Bodies: Random Aggregates of Sticky Proteins or Crucibles of Macromolecular Assembly?

The principles of self-assembly and self-organization are major tenets of molecular and cellular biology. Governed by these principles, the eukaryotic nucleus is composed of numerous subdomains and compartments, collectively described as nuclear bodies. Emerging evidence reveals that associations within and between various nuclear bodies and genomic loci are dynamic and can change in response to cellular signals. This review will discuss recent progress in our understanding of how nuclear body components come together, what happens when they form, and what benefit these subcellular structures may provide to the tissues or organisms in which they are found

SMA-Causing Missense Mutations in \u3cem\u3eSurvival motor neuron (Smn)\u3c/em\u3e Display a Wide Range of Phenotypes When Modeled in \u3cem\u3eDrosophila\u3c/em\u3e

Mutations in the human survival motor neuron 1 (SMN) gene are the primary cause of spinal muscular atrophy (SMA), a devastating neuromuscular disorder. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. Additional tissue-specific and global functions have been ascribed to SMN; however, their relevance to SMA pathology is poorly understood and controversial. Using Drosophila as a model system, we created an allelic series of twelve Smn missense mutations, originally identified in human SMA patients. We show that animals expressing these SMA-causing mutations display a broad range of phenotypic severities, similar to the human disease. Furthermore, specific interactions with other proteins known to be important for SMN\u27s role in RNP assembly are conserved. Intragenic complementation analyses revealed that the three most severe mutations, all of which map to the YG box self-oligomerization domain of SMN, display a stronger phenotype than the null allele and behave in a dominant fashion. In support of this finding, the severe YG box mutants are defective in self-interaction assays, yet maintain their ability to heterodimerize with wild-type SMN. When expressed at high levels, wild-type SMN is able to suppress the activity of the mutant protein. These results suggest that certain SMN mutants can sequester the wild-type protein into inactive complexes. Molecular modeling of the SMN YG box dimer provides a structural basis for this dominant phenotype. These data demonstrate that important structural and functional features of the SMN YG box are conserved between vertebrates and invertebrates, emphasizing the importance of self-interaction to the proper functioning of SMN. Author Summary Spinal Muscular Atrophy (SMA) is a prevalent childhood neuromuscular disease, which in its most common form causes death by the age of two. One in fifty Americans is a carrier for SMA, making this genetic disease a serious health concern. SMA is caused by loss of function mutations in the survival motor neuron 1 (SMN1) gene. SMN is an essential protein and has a well-characterized function in the assembly of small nuclear ribonucleoproteins (snRNPs), which are core components of the spliceosome. To elucidate the phenotypic consequences of disrupting specific SMN protein interactions, we have generated a series of SMA-causing point mutations, modeled in Drosophila melanogaster. Using this system, we have shown that key aspects of SMN structure and function are conserved between humans and flies. Intragenic complementation analyses reveal the potential for dominant negative interactions between wild-type and mutant SMN subunits, highlighting the essential nature of the YG box in formation of higher-order SMN multimers. These results provide a basis for future studies investigating therapy targeted at restoration of functional SMN oligomers

SMA-Causing Missense Mutations in Survival motor neuron (Smn) Display a Wide Range of Phenotypes When Modeled in Drosophila

Mutations in the human survival motor neuron 1 (SMN) gene are the primary cause of spinal muscular atrophy (SMA), a devastating neuromuscular disorder. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. Additional tissue-specific and global functions have been ascribed to SMN; however, their relevance to SMA pathology is poorly understood and controversial. Using Drosophila as a model system, we created an allelic series of twelve Smn missense mutations, originally identified in human SMA patients. We show that animals expressing these SMA-causing mutations display a broad range of phenotypic severities, similar to the human disease. Furthermore, specific interactions with other proteins known to be important for SMN's role in RNP assembly are conserved. Intragenic complementation analyses revealed that the three most severe mutations, all of which map to the YG box self-oligomerization domain of SMN, display a stronger phenotype than the null allele and behave in a dominant fashion. In support of this finding, the severe YG box mutants are defective in self-interaction assays, yet maintain their ability to heterodimerize with wild-type SMN. When expressed at high levels, wild-type SMN is able to suppress the activity of the mutant protein. These results suggest that certain SMN mutants can sequester the wild-type protein into inactive complexes. Molecular modeling of the SMN YG box dimer provides a structural basis for this dominant phenotype. These data demonstrate that important structural and functional features of the SMN YG box are conserved between vertebrates and invertebrates, emphasizing the importance of self-interaction to the proper functioning of SMN

Effectiveness and cost-effectiveness of a Yoga-based Cardiac Rehabilitation (Yoga-CaRe) program following acute myocardial infarction: study rationale and design of a multi-centre randomized controlled trial

© 2019 Background: Cardiac rehabilitation (CR) is a standard treatment for secondary prevention of acute myocardial infarction (AMI) in high income countries (HICs), but it is inaccessible to most patients in India due to high costs and skills required for multidisciplinary CR teams. We developed a low-cost and scalable CR program based on culturally-acceptable practice of yoga (Yoga-CaRe). In this paper, we report the rationale and design for evaluation of its effectiveness and cost-effectiveness. Methods: This is a multi-center, single-blind, two-arm parallel-group randomized controlled trial across 22 cardiac care hospitals in India. Four thousand patients aged 18–80 years with AMI will be recruited and randomized 1:1 to receive Yoga-CaRe program (13 sessions supervised by an instructor and encouragement to self-practice daily) or enhanced standard care (3 sessions of health education) delivered over a period of three months. Participants will be followed 3-monthly till the end of the trial. The co-primary outcomes are a) time to occurrence of first cardiovascular event (composite of all-cause mortality, non-fatal myocardial infarction, non-fatal stroke and emergency cardiovascular hospitalization), and b) quality of life (Euro-QoL-5L) at 12 weeks. Secondary outcomes include need for revascularization procedures, return to pre-infarct activities, tobacco cessation, medication adherence, and cost-effectiveness of the intervention. Conclusion: This trial will alone contribute >20% participants to existing meta-analyses of randomized trials of CR worldwide. If Yoga-CaRe is found to be effective, it has the potential to save millions of lives and transform care of AMI patients in India and other low and middle income country settings

Development of a Yoga-Based Cardiac Rehabilitation (Yoga-CaRe) Programme for Secondary Prevention of Myocardial Infarction

Cardiac rehabilitation (CR) after myocardial infarction is highly effective. It is unavailable in public hospitals in India due to limited resources. Our objective was to develop a scalable model of CR for India based on yoga, which could also appeal to some groups with low uptake of CR (e.g., ethnic minorities, women, and older people) globally. The intervention was developed using a structured process. A literature review and consultations with yoga experts, CR experts, and postmyocardial infarction patients were conducted to systematically identify and shortlist appropriate yoga exercises and postures, breathing exercises, meditation and relaxation practices, and lifestyle changes, which were incorporated into a conventional CR framework. The draft intervention was further refined based on the feedback from an internal stakeholder group and an external panel of international experts, before being piloted with yoga instructors and patients with myocardial infarction. A four-phase yoga-based CR (Yoga-CaRe) programme was developed for delivery by a single yoga instructor with basic training. The programme consists of a total of 13 instructor-led sessions (2 individual and 11 group) over a 3-month period. Group sessions include guided practice of yoga exercises and postures, breathing exercises, and meditation and relaxation practices, and support for the lifestyle change and coping through a moderated discussion. Patients are encouraged to self-practice daily at home and continue long-term with the help of a booklet and digital video disc (DVD). Family members/carers are encouraged to join throughout. In conclusion, a novel yoga-based CR programme has been developed, which promises to provide a scalable CR solution for India and an alternative choice for CR globally. It is currently being evaluated in a large multicentre randomised controlled trial across India

Sensor-based precision nutrient and irrigation management enhances the physiological performance, water productivity, and yield of soybean under system of crop intensification

Sensor-based decision tools provide a quick assessment of nutritional and physiological health status of crop, thereby enhancing the crop productivity. Therefore, a 2-year field study was undertaken with precision nutrient and irrigation management under system of crop intensification (SCI) to understand the applicability of sensor-based decision tools in improving the physiological performance, water productivity, and seed yield of soybean crop. The experiment consisted of three irrigation regimes [I1: standard flood irrigation at 50% depletion of available soil moisture (DASM) (FI), I2: sprinkler irrigation at 80% ETC (crop evapo-transpiration) (Spr 80% ETC), and I3: sprinkler irrigation at 60% ETC (Spr 60% ETC)] assigned in main plots, with five precision nutrient management (PNM) practices{PNM1-[SCI protocol], PNM2-[RDF, recommended dose of fertilizer: basal dose incorporated (50% N, full dose of P and K)], PNM3-[RDF: basal dose point placement (BDP) (50% N, full dose of P and K)], PNM4-[75% RDF: BDP (50% N, full dose of P and K)] and PNM5-[50% RDF: BDP (50% N, full P and K)]} assigned in sub-plots using a split-plot design with three replications. The remaining 50% N was top-dressed through SPAD assistance for all the PNM practices. Results showed that the adoption of Spr 80% ETC resulted in an increment of 25.6%, 17.6%, 35.4%, and 17.5% in net-photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), and intercellular CO2 concentration (Ci), respectively, over FI. Among PNM plots, adoption of PNM3 resulted in a significant (p=0.05) improvement in photosynthetic characters like Pn (15.69 µ mol CO2 m−2 s−1), Tr (7.03 m mol H2O m−2 s−1), Gs (0.175 µmol CO2 mol−1 year−1), and Ci (271.7 mol H2O m2 s−1). Enhancement in SPAD (27% and 30%) and normalized difference vegetation index (NDVI) (42% and 52%) values were observed with nitrogen (N) top dressing through SPAD-guided nutrient management, helped enhance crop growth indices, coupled with better dry matter partitioning and interception of sunlight. Canopy temperature depression (CTD) in soybean reduced by 3.09–4.66°C due to adoption of sprinkler irrigation. Likewise, Spr 60% ETc recorded highest irrigation water productivity (1.08 kg ha−1 m−3). However, economic water productivity (27.5 INR ha−1 m−3) and water-use efficiency (7.6 kg ha−1 mm−1 day−1) of soybean got enhanced under Spr 80% ETc over conventional cultivation. Multiple correlation and PCA showed a positive correlation between physiological, growth, and yield parameters of soybean. Concurrently, the adoption of Spr 80% ETC with PNM3 recorded significantly higher grain yield (2.63 t ha−1) and biological yield (8.37 t ha−1) over other combinations. Thus, the performance of SCI protocols under sprinkler irrigation was found to be superior over conventional practices. Hence, integrating SCI with sensor-based precision nutrient and irrigation management could be a viable option for enhancing the crop productivity and enhance the resource-use efficiency in soybean under similar agro-ecological regions