Role of the BCL11A/B Homologue Chronophage (Cph) in Locomotor Behaviour of Drosophila melanogaster.

Functioning of the nervous system requires proper formation and specification of neurons as well as accurate connectivity and signalling between them. Locomotor behaviour depends upon these events that occur during neural development, and any aberration in them could result in motor disorders. Transcription factors are believed to be master regulators that control these processes, but very few linked to behaviour have been identified so far. The Drosophila homologue of BCL11A (CTIP1) and BCL11B (CTIP2), Chronophage (Cph), was recently shown to be involved in temporal patterning of neural stem cells but its role in post-mitotic neurons is not known. We show that knockdown of Cph in neurons during development results in animals with locomotor defects at both larval and adult stages. The defects are more severe in adults, with inability to stand, uncoordinated behaviour and complete loss of ability to walk, climb, or fly. These defects are similar to the motor difficulties observed in some patients with mutations in BCL11A and BCL11B. Electrophysiological recordings showed reduced evoked activity and irregular neuronal firing. All Cph-expressing neurons in the ventral nerve cord are glutamatergic. Our results imply that Cph modulates primary locomotor activity through configuration of glutamatergic neurons. Thus, this study ascribes a hitherto unknown role to Cph in locomotor behaviour of Drosophila melanogaster.

Genome sequencing and assembly of Indian golden silkmoth, Antheraea assamensis Helfer (Saturniidae, Lepidoptera).

Muga silkworm (Antheraea assamensis), one of the economically important wild silkmoths, is unique among saturniid silkmoths. It is confined to the North-eastern part of India. Muga silk has the highest value among the other silks. Unlike other silkmoths, A. assamensis has a low chromosome number (n = 15), and ZZ/ZO sex chromosome system. Here, we report the first high-quality draft genome of A. assamensis, assembled by employing the Illumina and PacBio sequencing platforms. The assembled genome of A. assamensis is 501.18 Mb long, with 2697 scaffolds and an N50 of 683.23 Kb. The genome encompasses 18,385 protein-coding genes, 86.29% of which were functionally annotated. Phylogenetic analysis of A. assamensis revealed its divergence from other Antheraea species approximately 28.7 million years ago. Moreover, an investigation into detoxification-related gene families, CYP450, GST, and ABC-transporter, revealed a significant expansion in A. assamensis as compared to the Bombyx mori. This expansion is comparable to Spodoptera litura, suggesting adaptive responses linked to the polyphagous behavior observed in these insects. This study provides valuable insights into the molecular basis of evolutionary divergence and adaptations in muga silkmoth. The genome assembly reported in this study will significantly help in the functional genomics studies on A. assamensis and other Antheraea species along with comparative genomics analyses of Bombycoidea insects.

Antibiotic alters host's gut microbiota, fertility, and antimicrobial peptide gene expression vis-à-vis ampicillin treatment on model organism Drosophila melanogaster.

Antibiotics are commonly used to treat infectious diseases; however, persistence is often expressed by the pathogenic bacteria and their long-term relative effect on the host have been neglected. The present study investigated the impact of antibiotics in gut microbiota (GM) and metabolism of host. The effect of ampicillin antibiotics on GM of Drosophila melanogaster was analyzed through deep sequencing of 16S rRNA amplicon gene. The dominant phyla consisted of Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria, Planctomycetes, Chloroflexi, Euryarchaeota, Acedobacteria, Verrucomicrobia, and Cyanobacteria. It was found that the composition of GM was significantly altered on administration of antibiotics. On antibiotic treatments, there were decline in relative abundance of Proteobacteria and Firmicutes, while there were increase in relative abundance of Chlorophyta and Bacteroidota. High abundance of 14 genera, viz., Wolbachia, Lactobacillus, Bacillus, Pseudomonas, Thiolamprovum, Pseudoalteromonas, Vibrio, Romboutsia, Staphylococcus, Alteromonas, Clostridium, Lysinibacillus, Litoricola, and Cellulophaga were significant (p ≤ 0.05) upon antibiotic treatment. Particularly, the abundance of Acetobacter was significantly (p ≤ 0.05) declined but increased for Wolbachia. Further, a significant (p ≤ 0.05) increase in Wolbachia endosymbiont of D. melanogaster, Wolbachia endosymbiont of Curculio okumai, and Wolbachia pipientis and a decrease in the Acinetobacter sp. were observed. We observed an increase in functional capacity for biosynthesis of certain nucleotides and the enzyme activities. Further, the decrease in antimicrobial peptide production in the treated group and potential effects on the host's defense mechanisms were observed. This study helps shed light on an often-overlooked dimension, namely the persistence of antibiotics' effects on the host.

The association of cysteine to thiomersal attenuates its apoptosis-mediated cytotoxicity in zebrafish.

Thiomersal (TM) is an excellent preservative that is used in a wide variety of products, like pharmaceuticals, cosmetics, and vaccines, etc. Its usage has been in decline because of safety concerns. Since vaccine production is on the rise, its use may increase further in low-income and developing countries, as a cost-effective vaccine preservative. Further, Thiomersal is still being used as an essential component in various pharmaceutical preparations. In this light, the present study addresses its mechanism of toxicity in zebrafish and unveils a novel strategy for lessening its negative effects by conjugating cysteine to it, while retaining its antibacterial efficacy. We show that the mitochondrial membrane potential is destabilised by TM, leading to the induction of apoptosis. Interestingly, TM-cysteine conjugate (at a ratio of 1:1) showed no toxicity in zebrafish, whereas TM alone was highly toxic. Importantly, assaying for the bactericidal activity, tested using Escherichia coli (E. coli) and Methicillin-resistant Staphylococcus aureus (MRSA), revealed that the conjugate retains the antibacterial activity, demonstrating that the TM-cysteine conjugate is a safer alternative to TM as a vaccine preservative, and in all the other products that still use TM.

To RNA-binding and beyond: Emerging facets of the role of Rbfox proteins in development and disease.

The RNA-binding Fox-1 homologue (Rbfox) proteins represent an ancient family of splicing factors, conserved through evolution. All members share an RNA recognition motif (RRM), and a particular affinity for the GCAUG signature in target RNA molecules. The role of Rbfox, as a splice factor, deciding the tissue-specific inclusion/exclusion of an exon, depending on its binding position on the flanking introns, is well known. Rbfox often acts in concert with other splicing factors, and forms splicing regulatory networks. Apart from this canonical role, recent studies show that Rbfox can also function as a transcription co-factor, and affects mRNA stability and translation. The repertoire of Rbfox targets is vast, including genes involved in the development of tissue lineages, such as neurogenesis, myogenesis, and erythropoeiesis, and molecular processes, including cytoskeletal dynamics, and calcium handling. A second layer of complexity is added by the fact that Rbfox expression itself is regulated by multiple mechanisms, and, in vertebrates, exhibits tissue-specific expression. The optimum dosage of Rbfox is critical, and its misexpression is etiological to various disease conditions. In this review, we discuss the contextual roles played by Rbfox as a tissue-specific regulator for the expression of many important genes with diverse functions, through the lens of the emerging data which highlights its involvement in many human diseases. Furthermore, we explore the mechanistic details provided by studies in model organisms, with emphasis on the work with Drosophila. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications RNA Turnover and Surveillance > Regulation of RNA Stability RNA Processing > Splicing Regulation/Alternative Splicing.

Comparative in-silico analysis of microbial dysbiosis discern potential metabolic link in neurodegenerative diseases.

A healthy gut flora contains a diverse and stable commensal group of microorganisms, whereas, in disease conditions, there is a shift toward pathogenic microbes, termed microbial dysbiosis. Many studies associate microbial dysbiosis with neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), Multiple sclerosis (MS), and Amyotrophic lateral sclerosis (ALS). Although, an overall comparative analysis of microbes and their metabolic involvement in these diseases is still lacking. In this study, we have performed a comparative analysis of microbial composition changes occurring in these four diseases. Our research showed a high resemblance of microbial dysbiosis signatures between AD, PD, and MS. However, ALS appeared dissimilar. The most common population of microbes to show an increase belonged to the phyla, Bacteroidetes, Actinobacteria, Proteobacteria, and Firmicutes. Although, Bacteroidetes and Firmicutes were the only phyla that showed a decrease in their population. The functional analysis of these dysbiotic microbes showed several potential metabolic links which can be involved in the altered microbiome-gut-brain axis in neurodegenerative diseases. For instance, the microbes with elevated populations lack pathways for synthesizing SCFA acetate and butyrate. Also, these microbes have a high capacity for producing L-glutamate, an excitatory neurotransmitter and precursor of GABA. Contrastingly, Tryptophan and histamine have a lower representation in the annotated genome of elevated microbes. Finally, the neuroprotective compound spermidine was less represented in elevated microbes' genomes. Our study provides a comprehensive catalog of potential dysbiotic microbes and their metabolic involvement in neurodegenerative disorders, including AD, PD, MS, and ALS.

Is the fundamental pathology in Duchenne's muscular dystrophy caused by a failure of glycogenolysis-glycolysis in costameres?

Duchenne muscular dystrophy (DMD) is the most common form of progressive childhood muscular dystrophy associated with weakness of limbs, loss of ambulation, heart weakness and early death. The mutations causing either loss-of-expression or function of the full-length protein dystrophin (Dp427) from the DMD gene are responsible for the disease pathology. Dp427 forms a part of the large dystroglycan complex, called DAPC, in the sarcolemma, and its absence derails muscle contraction. Muscle biopsies from DMD patients show an overactivation of excitation-contraction-coupling (ECC) activable calcium incursion, sarcolemmal ROS production, NHE1 activation, IL6 secretion, etc. The signalling pathways, like Akt/PBK, STAT3, p38MAPK, and ERK1/2, are also hyperactive in DMD. These pathways are responsible for post-mitotic trophic growth and metabolic adaptation, in response to exercise in healthy muscles, but cause atrophy and cell death in dystrophic muscles. We hypothesize that the metabolic background of repressed glycolysis in DMD, as opposed to excess glycolysis seen in cancers or healthy contracting muscles, changes the outcome of these 'growth pathways'. The reduced glycolysis has been considered a secondary outcome of the cytoskeletal disruptions seen in DMD. Given the cytoskeleton-crosslinking ability of the glycolytic enzymes, we hypothesize that the failure of glycogenolytic and glycolytic enzymes to congregate is the primary pathology, which then affects the subsarcolemmal cytoskeletal organization in costameres and initiates the pathophysiology associated with DMD, giving rise to the tissue-specific differences in disease progression between muscle, heart and brain. The lacunae in the regulation of the key components of the hypothesized metabolome, and the limitations of this theory are deliberated. The considerations for developing future therapies based on known pathological processes are also discussed.

Spatial and temporal requirement of Mlp60A isoforms during muscle development and function in Drosophila melanogaster.

Many myofibrillar proteins undergo isoform switching in a spatio-temporal manner during muscle development. The biological significance of the variants of several of these myofibrillar proteins remains elusive. One such myofibrillar protein, the Muscle LIM Protein (MLP), is a vital component of the Z-discs. In this paper, we show that one of the Drosophila MLP encoding genes, Mlp60A, gives rise to two isoforms: a short (279 bp, 10 kDa) and a long (1461 bp, 54 kDa) one. The short isoform is expressed throughout development, but the long isoform is adult-specific, being the dominant of the two isoforms in the indirect flight muscles (IFMs). A concomitant, muscle-specific knockdown of both isoforms leads to partial developmental lethality, with most of the surviving flies being flight defective. A global loss of both isoforms in a Mlp60A-null background also leads to developmental lethality, with muscle defects in the individuals that survive to the third instar larval stage. This lethality could be rescued partially by a muscle-specific overexpression of the short isoform. Genetic perturbation of only the long isoform, through a P-element insertion in the long isoform-specific coding sequence, leads to defective flight, in around 90% of the flies. This phenotype was completely rescued when the P-element insertion was precisely excised from the locus. Hence, our data show that the two Mlp60A isoforms are functionally specialized: the short isoform being essential for normal embryonic muscle development and the long isoform being necessary for normal adult flight muscle function.

Epilepsy Characteristics in Neurodevelopmental Disorders: Research from Patient Cohorts and Animal Models Focusing on Autism Spectrum Disorder.

Epilepsy, a heterogeneous group of brain-related diseases, has continued to significantly burden society and families. Epilepsy comorbid with neurodevelopmental disorders (NDDs) is believed to occur due to multifaceted pathophysiological mechanisms involving disruptions in the excitation and inhibition (E/I) balance impeding widespread functional neuronal circuitry. Although the field has received much attention from the scientific community recently, the research has not yet translated into actionable therapeutics to completely cure epilepsy, particularly those comorbid with NDDs. In this review, we sought to elucidate the basic causes underlying epilepsy as well as those contributing to the association of epilepsy with NDDs. Comprehensive emphasis is put on some key neurodevelopmental genes implicated in epilepsy, such as MeCP2, SYNGAP1, FMR1, SHANK1-3 and TSC1, along with a few others, and the main electrophysiological and behavioral deficits are highlighted. For these genes, the progress made in developing appropriate and valid rodent models to accelerate basic research is also detailed. Further, we discuss the recent development in the therapeutic management of epilepsy and provide a briefing on the challenges and caveats in identifying and testing species-specific epilepsy models.

Neuronal role of taxi is imperative for flight in Drosophila melanogaster.

Extensive studies in Drosophila have led to the elucidation of the roles of many molecular players involved in the sensorimotor coordination of flight. However, the identification and characterisation of new players can add novel perspectives to the process. In this paper, we show that the extant mutant, jumper, is a hypermorphic allele of the taxi/delilah gene, which encodes a transcription factor. The defective flight of jumper flies results from the insertion of an I-element in the 5'-UTR of taxi gene, leading to an over-expression of the taxi. We also show that the molecular lesion responsible for the taxi1 allele results from a 25 bp deletion leading to a shift in the reading frame at the C-terminus of the taxi coding sequence. Thus, the last 20 residues are replaced by 32 disparate residues in taxi1. Both taxi1, a hypomorphic allele, and the CRISPR-Cas9 knock-out (taxiKO) null allele, show a defective flight phenotype. Electrophysiological studies show taxi hypermorphs, hypomorphs, and knock out flies show abnormal neuronal firing. We further show that neuronal-specific knock-down or over-expression of taxi cause a defect in the brain's inputs to the flight muscles, leading to reduced flight ability. Through transcriptomic analysis of the taxiKO fly head, we have identified several putative targets of Taxi that may play important roles in flight. In conclusion, from molecularly characterising jumper to establishing Taxi's role during Drosophila flight, our work shows that the forward genetics approach still can lead to the identification of novel molecular players required for neuronal transmission.

Evaluation of cytogenotoxic potential and embryotoxicity of KRS-Cauvery River water in zebrafish (Danio rerio).

In the Cauvery River (CR), indiscriminate discharge of waste causes unexplained skeletal deformity in some fish species present in the water. To investigate this phenomenon, we analyzed the biological, physical, and chemical parameters present in the water and then evaluated the toxicity effects on the zebrafish (Danio rerio) model. The zebrafish were treated with KRS-CR water samples collected from three stations (fast-flowing water [X], slow-flowing [Y], and stagnant [Z] water), before and after filtration. Firstly, we detected microscopic organisms (MO) such as Cyclops, Daphnia, Spirogyra, Spirochaeta, and total coliform (Escherichia coli), which are bioindicators of water pollution present in the samples. All physicochemical parameters analyzed, including heavy metals before and after filtration of the water with Millipore filter paper (0.45 µm), were within the acceptable limits set by standard organizations, except for decreased dissolved oxygen (DO), and increased biochemical oxygen demand (BOD), and chemical oxygen demand (COD), which are indicators of hypoxic water conditions, as well as the presence of microplastics (polybutene (< 15 µm), polyisobutene (≤ 20 µm), and polymethylpentene (≤3 mm)) and cyclohexyl in CR water samples. Zebrafish embryos treated with the water samples, both before and after filtration exerts the same cytogenotoxic effects by inducing increased reactive oxygen species (ROS) production, which triggers subcellular organelle dysfunctions, DNA damage, apoptosis, pericardial edema, skeletal deformities, and increased mortality. As a result, we observed that both water samples and zebrafish larvae had significantly less oxygen using SEM and EDS. Our findings show that KRS-CR water can induce cytogenotoxic and embryotoxic defects in zebrafish due to hypoxic water conditions triggered by the microplastics influx. The present study would provide valuable insights for health hazards evaluation and future river water treatment strategies.

Transgenic Silkworms Overexpressing Relish and Expressing Drosomycin Confer Enhanced Immunity to Multiple Pathogens.

The sericulture industry faces substantial economic losses due to severe pathogenic infections caused by fungi, viruses, and bacteria. The development of transgenic silkworms against specific pathogens has been shown to enhance disease resistance against a particular infection. A single gene or its products that can confer protection against multiple pathogens is required. In an attempt to develop silkworms with enhanced immunity against multiple pathogens, we generated transgenic silkworm lines with an overexpressed NF-kB transcription factor, Relish 1, under two different promoters. Separately, a potent anti-fungal gene, Drosomycin, was also expressed in transgenic silkworms. Both Relish 1 and Drosomycin transgenic silkworms had single copy genomic integration, and their mRNA expression levels were highly increased after infection with silkworm pathogens. The overexpression of the Relish 1 in transgenic silkworms resulted in the upregulation of several defense-related genes, Cecropin B, Attacin, and Lebocin, and showed enhanced resistance to Nosema bombycis (microsporidian fungus), Nucleopolyhedrovirus (BmNPV), and bacteria. The Drosomycin expressing transgenic silkworms showed elevated resistance to N. bombycis and bacteria. These findings demonstrate the role of Relish 1 in long-lasting protection against multiple pathogens in silkworms. Further, the successful introduction of a foreign gene, Drosomycin, also led to improved disease resistance in silkworms.

Before the "cytokine storm": Boosting efferocytosis as an effective strategy against SARS-CoV-2 infection and associated complications.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is responsible for the ongoing COVID-19 pandemic, and causes many health complications, including major lung diseases. Besides investigations into the virology of SARS-CoV-2, understanding the immunological routes underlying the clinical manifestations of COVID-19 is important for developing effective therapeutic interventions. The clearance of SARS-CoV-2-infected apoptotic cells by professional efferocytes, through a process termed as 'efferocytosis', is essential for maintaining tissue homeostasis, and reducing the chances of health complications caused by SARS-CoV-2 infection. In this review, we focus on the cellular events leading to engagement of the SARS-CoV-2 with type 2 alveolar cells, and how SARS-COV-2 infection impairs the macrophage anti-inflammatory programming. We also discuss accounts of impaired efferocytosis, and the "cytokine storm" which occur concomitantly with the SARS-CoV-2 infection. Finally, we propose how targeting impaired efferocytosis, due to the SARS-CoV-2 infection, may be a beneficial therapeutic strategy to combat COVID-19, and its complications.

Rbfox1 is required for myofibril development and maintaining fiber type-specific isoform expression in Drosophila muscles.

Protein isoform transitions confer muscle fibers with distinct properties and are regulated by differential transcription and alternative splicing. RNA-binding Fox protein 1 (Rbfox1) can affect both transcript levels and splicing, and is known to contribute to normal muscle development and physiology in vertebrates, although the detailed mechanisms remain obscure. In this study, we report that Rbfox1 contributes to the generation of adult muscle diversity in Drosophila Rbfox1 is differentially expressed among muscle fiber types, and RNAi knockdown causes a hypercontraction phenotype that leads to behavioral and eclosion defects. Misregulation of fiber type-specific gene and splice isoform expression, notably loss of an indirect flight muscle-specific isoform of Troponin-I that is critical for regulating myosin activity, leads to structural defects. We further show that Rbfox1 directly binds the 3'-UTR of target transcripts, regulates the expression level of myogenic transcription factors myocyte enhancer factor 2 and Salm, and both modulates expression of and genetically interacts with the CELF family RNA-binding protein Bruno1 (Bru1). Rbfox1 and Bru1 co-regulate fiber type-specific alternative splicing of structural genes, indicating that regulatory interactions between FOX and CELF family RNA-binding proteins are conserved in fly muscle. Rbfox1 thus affects muscle development by regulating fiber type-specific splicing and expression dynamics of identity genes and structural proteins.

Evaluation of developmental toxicity and genotoxicity of aqueous seed extract of Croton tiglium L. using zebrafish.

Croton tiglium L. has been used in Ayurvedic and Chinese herbal medicinal formulations from ancient times. Although its seeds are widely prescribed as traditional medicine, there is a dearth of information, regarding its toxic effects, and the mechanisms underlying its toxicity. This study aims to investigate the developmental toxicity and genotoxicity of the aqueous seed extract of C. tiglium L. (AECT) in zebrafish. We have examined the effects of AECT on the early embryonic development of zebrafish. Zebrafish embryos, treated with different concentrations of the AECT, suffered embryonic lethality and displayed various developmental defects. The 96 h-LC50 of AECT was found to be 162.78 µg/ml. Interestingly, the developmental abnormalities observed, such as pericardial edema (PE), yolk sac edema (YSE), spinal curvature (SC), and delayed hatching, varied in severity, in a dose-dependent manner. Zebrafish embryos, treated with different concentrations of AECT, exhibited exaggerated cell death in the anatomical regions of brain, heart, and trunk. Our data suggest that the phenomenon of apoptosis is probably responsible for both embryonic lethality and developmental toxicity in zebrafish embryos. Furthermore, the genotoxic potential of the AECT, in vivo, was evaluated using micronucleus assay and comet assay, on the peripheral blood of zebrafish. The results suggest that AECT has the potential to cause genotoxicity in the peripheral blood of zebrafish.

Matrilineal analysis of mutations in the DMD gene in a multigenerational South Indian cohort using DMD gene panel sequencing.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disorder characterised by progressive irreversible muscle weakness, primarily of the skeletal and the cardiac muscles. DMD is characterised by mutations in the dystrophin gene, resulting in the absence or sparse quantities of dystrophin protein. A precise and timely molecular detection of DMD mutations encourages interventions such as carrier genetic counselling and in undertaking therapeutic measures for the DMD patients. RESULTS: In this study, we developed a 2.1 Mb custom DMD gene panel that spans the entire DMD gene, including the exons and introns. The panel also includes the probes against 80 additional genes known to be mutated in other muscular dystrophies. This custom DMD gene panel was used to identify single nucleotide variants (SNVs) and large deletions with precise breakpoints in 77 samples that included 24 DMD patients and their matrilineage across four generations. We used this panel to evaluate the inheritance pattern of DMD mutations in maternal subjects representing 24 DMD patients. CONCLUSION: Here we report our observations on the inheritance pattern of DMD gene mutations in matrilineage samples across four generations. Additionally, our data suggest that the DMD gene panel designed by us can be routinely used as a single genetic test to identify all DMD gene variants in DMD patients and the carrier mothers.

Fluorescence based rapid optical volume screening system (OVSS) for interrogating multicellular organisms.

Continuous monitoring of large specimens for long durations requires fast volume imaging. This is essential for understanding the processes occurring during the developmental stages of multicellular organisms. One of the key obstacles of fluorescence based prolonged monitoring and data collection is photobleaching. To capture the biological processes and simultaneously overcome the effect of bleaching, we developed single- and multi-color lightsheet based OVSS imaging technique that enables rapid screening of multiple tissues in an organism. Our approach based on OVSS imaging employs quantized step rotation of the specimen to record 2D angular data that reduces data acquisition time when compared to the existing light sheet imaging system (SPIM). A co-planar multicolor light sheet PSF is introduced to illuminate the tissues labelled with spectrally-separated fluorescent probes. The detection is carried out using a dual-channel sub-system that can simultaneously record spectrally separate volume stacks of the target organ. Arduino-based control systems were employed to automatize and control the volume data acquisition process. To illustrate the advantages of our approach, we have noninvasively imaged the Drosophila larvae and Zebrafish embryo. Dynamic studies of multiple organs (muscle and yolk-sac) in Zebrafish for a prolonged duration (5 days) were carried out to understand muscle structuring (Dystrophin, microfibers), primitive Macrophages (in yolk-sac) and inter-dependent lipid and protein-based metabolism. The volume-based study, intensity line-plots and inter-dependence ratio analysis allowed us to understand the transition from lipid-based metabolism to protein-based metabolism during early development (Pharyngula period with a critical transition time, [Formula: see text] h post-fertilization) in Zebrafish. The advantage of multicolor lightsheet illumination, fast volume scanning, simultaneous visualization of multiple organs and an order-less photobleaching makes OVSS imaging the system of choice for rapid monitoring and real-time assessment of macroscopic biological organisms with microscopic resolution.

Exome sequencing and functional studies in zebrafish identify WDR8 as the causative gene for isolated Microspherophakia in Indian families.

Isolated Microspherophakia (MSP) is an autosomal recessive disorder characterized by a smaller than normal spherical lens. Till date, LTBP2 is the only gene shown to cause MSP. We used homozygosity mapping and whole-exome sequencing and identified a homozygous mutation, c.1148C > T (p.Pro383Leu), in the WDR8 (or WRAP73) gene in two Indian MSP families. In vitro experiments showed that the missense mutation renders the protein unstable. WDR8 is a centriolar protein that has important roles in centrosomal assembly, spindle pole formation and ciliogenesis. Co-immunoprecipitation experiments from HeLa cells indicated that the mutation interferes with the interaction of WDR8 with its binding partners. In zebrafish, both morpholino-mediated knockdown and CRISPR/Cas knockout of wdr8 resulted in decreased eye and lens size. The lack of wdr8 affected cell cycle progression in the retinal cells, causing a reduction in cell numbers in the retina and lens. The reduction in eye size and the cell cycle defects were rescued by exogenous expression of the human wild-type WDR8. However, the human mutant WDR8 (p.Pro383Leu) was unable to rescue the eye defects, indicating that the missense mutation abrogates WDR8 protein function. Thus, our zebrafish results suggested that WDR8 is the causative gene for MSP in these Indian families.

Marf-mediated mitochondrial fusion is imperative for the development and functioning of indirect flight muscles (IFMs) in drosophila.

Dynamic changes in mitochondrial shape and size are vital for mitochondrial health and for tissue development and function. Adult Drosophila indirect flight muscles contain densely packed mitochondria. We show here that mitochondrial fusion is critical during early muscle development (in pupa) and that silencing of the outer mitochondrial membrane fusion gene, Marf, in muscles results in smaller mitochondria that are functionally defective. This leads to abnormal muscle development resulting in muscle dysfunction in adult flies. However, post-developmental silencing of Marf has no obvious effects on mitochondrial and muscle phenotype in adult flies, indicating the importance of mitochondrial fusion during early muscle development.

A Dominant C150Y Mutation in FHL1 Induces Structural Alterations in LIM2 Domain Causing Protein Aggregation In Human and Drosophila Indirect Flight Muscles.

FHL1-related myopathies are rare X-linked dominant myopathies. Though clinically classified into several subgroups, spinal and scapuloperoneal muscle involvement are common to all. In this study, we identified c.449G > A, p.C150Y mutation by clinical exome sequencing in two patients from same family (son and mother) of Indian origin who presented with multiple contractures. Muscle biopsy showed numerous intracytoplasmic aggregates intensely stained on HE and MGT. The strong reactions to M-NBT revealed aggregates to be reducing bodies and positively labeled to anti-FHL1 antibody. Ultrastructurally, Z-band streaming and granular and granulofilamentous material were seen. Further, the translational evidence of mutant peptide was confirmed using mass spectrometric analysis. To establish p.C150Y as the cause for protein aggregation, in vivo studies were carried out using transgenic Drosophila model which highlighted Z-band abnormalities and protein aggregates in indirect flight muscles with compromised physiological function. Thus, recapitulating the X-linked human disease phenotype. Additionally, the molecular dynamics simulation analysis unraveled the drastic change in α-helix of LIM2, the region immediately next to site of C150Y mutation that could be the plausible cause for protein aggregation. To the best of our knowledge, this is the first study of p.C150Y mutation in FHL1 identified in Indian patients with in vivo and in silico analysis to establish the cause for protein aggregation in muscle.