E3 ubiquitin ligase Deltex facilitates the expansion of Wingless gradient and antagonizes Wingless signaling through a conserved mechanism of transcriptional effector Armadillo/ß-catenin degradation.

The Wnt/Wg pathway controls myriads of biological phenomena throughout the development and adult life of all organisms across the phyla. Thus, an aberrant Wnt signaling is associated with a wide range of pathologies in humans. Tight regulation of Wnt/Wg signaling is required to maintain proper cellular homeostasis. Here, we report a novel role of E3 ubiquitin ligase Deltex in Wg signaling regulation. Drosophila dx genetically interacts with wg and its pathway components. Furthermore, Dx LOF results in a reduced spreading of Wg while its over-expression expands the diffusion gradient of the morphogen. We attribute this change in Wg gradient to the endocytosis of Wg through Dx which directly affects the short- and long-range Wg targets. We also demonstrate the role of Dx in regulating Wg effector Armadillo where Dx down-regulates Arm through proteasomal degradation. We also showed the conservation of Dx function in the mammalian system where DTX1 is shown to bind with ß-catenin and facilitates its proteolytic degradation, spotlighting a novel step that potentially modulates Wnt/Wg signaling cascade.

Regulation of Notch signaling by non-muscle myosin II Zipper in Drosophila.

The Notch pathway is an evolutionarily conserved signaling system that is intricately regulated at multiple levels and it influences different aspects of development. In an effort to identify novel components involved in Notch signaling and its regulation, we carried out protein interaction screens which identified non-muscle myosin II Zipper (Zip) as an interacting partner of Notch. Physical interaction between Notch and Zip was further validated by co-immunoprecipitation studies. Immunocytochemical analyses revealed that Notch and Zip co-localize within same cytoplasmic compartment. Different alleles of zip also showed strong genetic interactions with Notch pathway components. Downregulation of Zip resulted in wing phenotypes that were reminiscent of Notch loss-of-function phenotypes and a perturbed expression of Notch downstream targets, Cut and Deadpan. Further, synergistic interaction between Notch and Zip resulted in highly ectopic expression of these Notch targets. Activated Notch-induced tumorous phenotype of larval tissues was enhanced by over-expression of Zip. Notch-Zip synergy resulted in the activation of JNK pathway that consequently lead to MMP activation and proliferation. Taken together, our results suggest that Zip may play an important role in regulation of Notch signaling.

Nanofibers of N,N,N-trimethyl chitosan capped bimetallic nanoparticles: Preparation, characterization, wound dressing and in vivo treatment of MDR microbial infection and tracking by optical and photoacoustic imaging.

Recent advancements in wound care have led to the development of interactive wound dressings utilizing nanotechnology, aimed at enhancing healing and combating bacterial infections while adhering to established protocols. Our novel wound dressings consist of N,N,N-trimethyl chitosan capped gold­silver nanoparticles (Au-Ag-TMC-NPs), with a mean size of 108.3 ± 8.4 nm and a zeta potential of +54.4 ± 1.8 mV. These optimized nanoparticles exhibit potent antibacterial and antifungal properties, with minimum inhibitory concentrations ranging from 0.390 µg ml-1 to 3.125 µg ml-1 and also exhibited promising zones of inhibition against multi-drug resistant strains of S. aureus, E. coli, P. aeruginosa, and C. albicans. Microbial transmission electron microscopy reveals substantial damage to cell walls and DNA condensation post-treatment. Furthermore, the nanoparticles demonstrate remarkable inhibition of microbial efflux pumps and are non-hemolytic in human blood. Incorporated into polyvinyl alcohol/chitosan nanofibers, they form Au-Ag-TMC-NPs-NFs with diameters of 100-350 nm, facilitating efficient antimicrobial wound dressing. In vivo studies on MDR microbial-infected wounds in mice showed 99.34 % wound healing rate within 12 days, corroborated by analyses of wound marker protein expression levels and advanced imaging techniques such as ultrasound/photoacoustic imaging, providing real-time visualization and blood flow assessment for a comprehensive understanding of the dynamic wound healing processes.

Identification and molecular characterization of two recurrent missense mutations in the RS1 gene in two families with X-linked retinoschisis from North India.

X-linked retinoschisis (XLR) is a rare medical condition that involves in the splitting of neurosensory layers and the impairment of vision in the retina. In majority of the XLR cases, pathogenic variants in Retinoschisin 1 (RS1) gene have been implicated in males with an early age of onset during early childhood. In the present study, we have recruited two North Indian families having multiple affected male members, who were diagnosed with XLR. The entire protein-coding region of RS1 was screened by PCR-Sanger sequencing and two recurrent pathogenic variants (p.I81N and p.R102Q) were unraveled. The in vitro study of these variants demonstrated the aggregation of mutant RS1 within the endoplasmic reticulum. Furthermore, mutant forms of this protein showed significant intracellular retention, which was evident by the absence of retinoschisin protein fractions in the extracellular media. These inferences were also supported by extensive bioinformatics analysis of the mutants, which showed dramatic conformational changes in the local structure of retinoschisin. Thus, our study suggests that the identified pathogenic variants interfere with proper protein folding, leading to anomalous structural changes ultimately resulting in intracellular retention of retinoschisin within the retina.

Notch signalling: multifaceted role in development and disease.

Notch pathway is an evolutionarily conserved signalling system that operates to influence an astonishing array of cell fate decisions in different developmental contexts. Notch signalling plays important roles in many developmental processes, making it difficult to name a tissue or a developing organ that does not depend on Notch function at one stage or another. Thus, dysregulation of Notch signalling is associated with many developmental defects and various pathological conditions, including cancer. Although many recent advances have been made to reveal different aspects of the Notch signalling mechanism and its intricate regulation, there are still many unanswered questions related to how the Notch signalling pathway functions in so many developmental events. The same pathway can be deployed in numerous cellular contexts to play varied and critical roles in an organism's development and this is only possible because of the complex regulatory mechanisms of the pathway. In this review, we provide an overview of the mechanism and regulation of the Notch signalling pathway along with its multifaceted functions in different aspects of development and disease.

Spoonbill positively regulates JNK signalling mediated apoptosis in Drosophila melanogaster.

A-kinase anchoring protein (AKAP) comprises a family of scaffold proteins, which decides the subcellular localisation of a combination of signalling molecules. Spoonbill (Spoon) is a putative A-kinase anchoring protein in Drosophila. We have earlier reported that Spoon suppresses ribonuclear foci formed by trinucleotide repeat expanded transcripts associated with Spinocerebellar Ataxia 8 neurodegeneration in Drosophila. However, the role of Spoonbill in cellular signalling was unexplored. In this report, we have unravelled a novel function of Spoon protein in the regulation of the apoptotic pathway. The Drosophila TNFα homolog, Eiger, induces apoptosis via activation of the JNK pathway. We have shown here that Spoonbill is a positive regulator of the Eiger-induced JNK signalling. Further genetic interaction studies show that the spoon interacts with components of the JNK pathway, TGF-ß activated kinase 1 (Tak1 - JNKKK), hemipterous (hep - JNKK) and basket (bsk - JNK). Interestingly, Spoonbill alone can also induce ectopic activation of the JNK pathway in a context-specific manner. To understand the molecular mechanism underlying Spoonbill-mediated modulation of the JNK pathway, the interaction between Spoon and Drosophila JNK was assessed. basket encodes the only known JNK in Drosophila. This serine/threonine-protein kinase phosphorylates Jra/Kay, which transcriptionally regulate downstream targets like Matrix metalloproteinase 1 (Mmp1), puckered (puc), and proapoptotic genes hid, reaper and grim. Interestingly, we found that Spoonbill colocalises and co-immunoprecipitates with the Basket protein in the developing photoreceptor neurons. Hence, we propose that Spoon plays a vital role in JNK-induced apoptosis. Furthermore, stress-induced JNK activation underlying Parkinson's Disease was also examined. In the Parkinson's Drosophila model of neurodegeneration, depletion of Spoonbill leads to a partial reduction of JNK pathway activation, along with improvement in adult motor activity. These observations suggest that the putative scaffold protein Spoonbill is a functional and physical interacting partner of the Drosophila JNK protein, Basket. Spoon protein is localised on the outer mitochondrial membrane (OMM), which may perhaps provide a suitable subcellular niche for activation of Drosophila Basket protein by its kinases which induce apoptosis.

Deltex modulates Dpp morphogen gradient formation and affects Dpp signaling in Drosophila.

Deltex (Dx) is a context-dependent regulator of Notch signaling that can act in a non-canonical fashion by facilitating the endocytosis of the Notch receptor. In an RNAi-based modifier screen of kinases and phosphatases, we identified Thickveins (Tkv), the receptor of Decapentaplegic (Dpp), as one of the interactors of Dx. Dpp, a Drosophila homolog of TGF-ß and bone morphogenetic proteins, acts as a morphogen to specify cell fate along the anterior-posterior axis of the wing. Tight regulation of Dpp signaling is thus indispensable for its proper functioning. Here, we present Dx as a novel modulator of Dpp signaling. We show evidence for the very first time that dx genetically interacts with dpp and its pathway components. Immunocytochemical analysis revealed that Dx colocalizes with Dpp and its receptor Tkv in Drosophila third-instar larval tissues. Furthermore, Dx was also seen to modulate the expression of dpp and its target genes, and we attribute this modulation to the involvement of Dx in the endocytosis and trafficking of Dpp. This study thus presents a whole new avenue of Dpp signaling regulation via the cytoplasmic protein Dx. This article has an associated First Person interview with the first author of the paper.

Somatic Clonal Analyses Using FLP/FRT and MARCM System to Understand Notch Signaling Mechanism and Its Regulation.

Notch signaling regulates an array of developmental decisions and has been implicated in a multitude of diseases, including cancer over the past a few decades. The simplicity and versatility of the Notch pathway in Drosophila make it an ardent system to study Notch biology, its regulation, and functions. In this chapter, we highlight the use of two powerful techniques, namely, FLP/FRT and MARCM in the study of Notch signaling. These mosaic analysis techniques are powerful tools to analyze gene functions in different biological processes. The section briefly explains the principle and the protocols with suitable examples.

Recent Updates on the Genetics of Amyotrophic Lateral Sclerosis and Frontotemporal Dementia.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) primarily affect the motor and frontotemporal areas of the brain, respectively. These disorders share clinical, genetic, and pathological similarities, and approximately 10-15% of ALS-FTD cases are considered to be multisystemic. ALS-FTD overlaps have been linked to families carrying an expansion in the intron of C9orf72 along with inclusions of TDP-43 in the brain. Other overlapping genes (VCP, FUS, SQSTM1, TBK1, CHCHD10) are also involved in similar functions that include RNA processing, autophagy, proteasome response, protein aggregation, and intracellular trafficking. Recent advances in genome sequencing have identified new genes that are involved in these disorders (TBK1, CCNF, GLT8D1, KIF5A, NEK1, C21orf2, TBP, CTSF, MFSD8, DNAJC7). Additional risk factors and modifiers have been also identified in genome-wide association studies and array-based studies. However, the newly identified genes show higher disease frequencies in combination with known genes that are implicated in pathogenesis, thus indicating probable digenetic/polygenic inheritance models, along with epistatic interactions. Studies suggest that these genes play a pleiotropic effect on ALS-FTD and other diseases such as Alzheimer's disease, Ataxia, and Parkinsonism. Besides, there have been numerous improvements in the genotype-phenotype correlations as well as clinical trials on stem cell and gene-based therapies. This review discusses the possible genetic models of ALS and FTD, the latest therapeutics, and signaling pathways involved in ALS-FTD.

Whole exome sequencing identifies a novel splice-site mutation in IMPG2 gene causing Stargardt-like juvenile macular dystrophy in a north Indian family.

We report on the genetic analysis of a north Indian family affected with Stargardt-like juvenile macular dystrophy. Considering an autosomal recessive inheritance of macular dystrophy in the recruited family, whole exome sequencing was employed in two affected siblings and their mother. We have identified a novel splice-site variant NC_000003.11(NM_016247.3):c.1239 + 1G > T, co-segregating in the affected siblings, in the Interphotoreceptor Matrix Proteoglycan 2 (IMPG2) gene. The identified variant is present immediately after exon 11, and is predicted to disrupt the wild-type donor splice-site of IMPG2 transcripts. We confirmed the splice-site changes in the IMPG2 transcripts using minigene functional assay. Although a number of studies on IMPG2 have demonstrated its involvement in retinitis pigmentosa and vitelliform macular dystrophy, this is the first report of a splice-site variant in IMPG2 that is responsible for Stargardt-like juvenile macular dystrophy.

Regulation of Notch signaling by E3 ubiquitin ligases.

Notch signaling is an evolutionarily conserved pathway that is widely used for multiple cellular events during development. Activation of the Notch pathway occurs when the ligand from a neighboring cell binds to the Notch receptor and induces cleavage of the intracellular domain of Notch, which further translocates into the nucleus to activate its downstream genes. The involvement of the Notch pathway in diverse biological events is possible due to the complexity in its regulation. In order to maintain tight spatiotemporal regulation, the Notch receptor, as well as its ligand, undergoes a series of physical and biochemical modifications that, in turn, helps in proper maintenance and fine-tuning of the signaling outcome. Ubiquitination is the post-translational addition of a ubiquitin molecule to a substrate protein, and the process is regulated by E3 ubiquitin ligases. The present review describes the involvement of different E3 ubiquitin ligases that play an important role in the regulation and maintenance of proper Notch signaling and how perturbation in ubiquitination results in abnormal Notch signaling leading to a number of human diseases.

Maheshvara regulates JAK/STAT signaling by interacting and stabilizing hopscotch transcripts which leads to apoptosis in Drosophila melanogaster.

Maheshvara (mahe), an RNA helicase that is widely conserved across taxa, regulates Notch signaling and neuronal development in Drosophila. In order to identify novel components regulated by mahe, transcriptome profiling of ectopic mahe was carried out and this revealed striking upregulation of JAK/STAT pathway components like upd1, upd2, upd3, and socs36E. Further, significant downregulation of the pathway components in mahe loss-of-function mutant as well as upon lowering the level of mahe by RNAi, supported and strengthened our transcriptome data. Parallelly, we observed that mahe, induced caspase-dependent apoptosis in photoreceptor neurons, and this phenotype was significantly modulated by JAK/STAT pathway components. RNA immunoprecipitation unveiled the presence of JAK/STAT tyrosine kinase hopscotch (hop) transcripts in the complex immunoprecipitated with Mahe, which ultimately resulted in stabilization and elevation of hop transcripts. Additionally, we also observed the surge in activity of downstream transcription factor Stat92E, which is indicative of activation of the JAK/STAT signaling, and this in turn led to apoptosis via upregulation of hid. Taken together, our data provide a novel regulation of JAK/STAT pathway by RNA helicase Maheshvara, which ultimately promotes apoptosis.

Signaling cross-talk during development: Context-specific networking of Notch, NF-κB and JNK signaling pathways in Drosophila.

Multicellular organisms depend on a handful of core signaling pathways that regulate a variety of cell fate choices. Often these relatively simple signals integrate to form a large and complex signaling network to achieve a distinct developmental fate in a context-specific manner. Various pathway-dependent and independent events control the assembly of signaling complexes. Notch pathway is one such conserved signaling mechanism that integrates with other signaling pathways to exhibit a context-dependent pleiotropic output. To understand how Notch signaling provides a spectrum of distinct outputs, it is important to understand various regulatory switches involved in mediating signaling cross-talk of Notch with other pathways. Here, we review our current understanding as to how Notch signal integrates with JNK and NF-κB signaling pathways in Drosophila to regulate various developmental events such as sensory organ precursor formation, innate immunity, dorsal closure, establishment of planar cell polarity as well as during proliferation and tumor progression. We highlight the importance of conserved signaling molecules during these cross-talks and debate further possibilities of novel switches that may be involved in mediating these cross-talk events.

Deltex positively regulates Toll signaling in a JNK independent manner in Drosophila.

Toll pathway is the center for the function of immune system in both Drosophila and mammals. Toll pathway in Drosophila gets activated upon binding of the ligand Spätzle to the receptor, Toll, triggering a series of proteolytic cascade culminating into the activation of the NF-κB factors Dorsal and/or Dif (Dorsal-related immunity factor). Inappropriate activation of the Toll pathway is often associated with systemic inflammation phenotype in the absence of infection, and thus, it is important to understand the regulation of Toll signaling. Deltex (Dx) is a context-dependent regulator of Notch signaling and has been linked with cell-mediated immunity in the mammalian system lately. However, the unambiguous role of Dx in humoral and cell-mediated immunity is yet to be explored. Our study unravels the novel role of Dx in Toll pathway activation. Gain of function of dx in Drosophila larvae results in increased melanotic mass formation and increased lamellocyte production. Our results also reveal the nuclear accumulation of transcription factors Dorsal and Dif and expression of Toll-associated antimicrobial peptides (AMP) in Dx over-expression background. Further, we also tried to elucidate the role of Dx in JNK-independent Toll activation. Here we present Dx as a novel candidate in the regulation of Toll pathway.

Deltex cooperates with TRAF6 to promote apoptosis and cell migration through Eiger-independent JNK activation in Drosophila.

JNK signaling is a highly conserved signaling pathway that regulates a broad spectrum of cellular processes including cell proliferation, migration, and apoptosis. In Drosophila, JNK signaling is activated by binding of the tumor necrosis factor (TNF) Eiger to its receptor Wengen, and a conserved signaling cascade operates that culminates into activation of dual phosphatase Puckered thereby triggering apoptosis. The tumor necrosis factor receptor (TNFR) associated factor 6 (TRAF6) is an adaptor protein, which transduces the signal from TNFRs and Toll-like receptor/interleukin-1 receptor superfamily to induce a wide spectrum of cellular responses. TRAF6 also acts as the adaptor protein that mediates Eiger/JNK signaling in Drosophila. In a genetic interaction study, deltex (Dx) was identified as a novel interactor of TRAF6. Dx is well known to regulate Notch signaling in a context-dependent manner. Our data suggest that combinatorial action of Dx and TRAF6 enhances the Dx-induced wing nicking phenotype by inducing caspase-mediated cell death. Co-expression of Dx and TRAF6 also results in enhanced invasive behavior and perturbs the normal morphology of cells. The cooperative action of Dx and TRAF6 is attributed to JNK activation, which also leads to ectopic wingless (Wg) and decapentaplegic (Dpp) expression. Our results also reveal that the endocytic pathway component Rab7 may play a pivotal role in the regulation of Dx-TRAF6-mediated activation of JNK signaling. Here, we present the fact that Dx and TRAF6 together activate JNK signaling in an Eiger-independent mechanism.

A Forward Genetic Approach to Mapping a P-Element Second Site Mutation Identifies DCP2 as a Novel Tumor Suppressor in Drosophila melanogaster.

The use of transposons to create mutations has been the cornerstone of Drosophila genetics in the past few decades. Second-site mutations caused by transpositions are often devoid of transposons and thereby affect subsequent analyses. In a P-element mutagenesis screen, a second site mutation was identified on chromosome 3, wherein the homozygous mutants exhibit classic hallmarks of tumor suppressor mutants, including brain tumor and lethality; hence the mutant line was initially named as lethal (3) tumorous brain [l(3)tb]. Classical genetic approaches relying on meiotic recombination and subsequent complementation with chromosomal deletions and gene mutations mapped the mutation to CG6169, the mRNA decapping protein 2 (DCP2), on the left arm of the third chromosome (3L). Thus the mutation was renamed as DCP2l(3)tb Fine mapping of the mutation further identified the presence of a Gypsy-LTR like sequence in the 5'UTR coding region of DCP2, along with the expansion of the adjacent upstream intergenic AT-rich sequence. The mutant phenotypes are rescued by the introduction of a functional copy of DCP2 in the mutant background, thereby establishing the causal role of the mutation and providing a genetic validation of the allelism. With the increasing repertoire of genes being associated with tumor biology, this is the first instance of mRNA decapping protein being implicated in Drosophila tumorigenesis. Our findings, therefore, imply a plausible role for the mRNA degradation pathway in tumorigenesis and identify DCP2 as a potential candidate for future explorations of cell cycle regulatory mechanisms.

Maheshvara, a Conserved RNA Helicase, Regulates Notch Signaling in Drosophila melanogaster.

Gene expression is regulated at multiple steps after generation of primary RNA transcripts, including mRNA processing, stability, and transport, along with co- and post-transcriptional regulation. These processes are controlled via the involvement of a multitude of RNA binding proteins (RBPs). Innumerable human diseases have been associated with altered expression of RNA binding proteins. In this chapter we have focused on Maheshvara (mahe) which encodes a putative DEAD box RNA helicase protein in Drosophila. We have recently reported that mahe plays an important role in regulation of Notch signaling. Fine tuning of Notch signaling is required at multiple steps and it's misregulation leads to a variety of human diseases. Additionally, mutation in DDX59, a human homolog of mahe results in broad neurological phenotypes associated with orofaciodigital syndrome. Drosophila mahe mutants show abnormal peripheral and central nervous system development that resemble neuropathology of patients having mutation in DDX59 gene. This chapter will help in advancing the knowledge as to how mahe regulates Notch signaling and nervous system development.

Regulation of Notch Signaling in Drosophila melanogaster: The Role of the Heterogeneous Nuclear Ribonucleoprotein Hrp48 and Deltex.

Notch signaling is an evolutionarily conserved pathway that plays a central role in a number of cellular events during metazoan development. Due to its involvement in numerous developmental events, Notch signaling requires tight spatial and temporal regulation. Deltex is a cytoplasmic protein that physically binds to the Notch and regulates its signaling activity in a context-dependent manner. However, the biology of Deltex in regulation of Notch signaling is not well explored. For a better understanding of Deltex activity in the regulatory circuit of Notch pathway, a co-IP-based screening was performed. Hrp48, an RNA-binding protein, was identified as an interacting partner of Deltex in that screening. Interaction of these two proteins seemed to regulate the Notch signaling outcome in the epithelial tissue. Additionally, it was found that coexpression of Deltex and Hrp48 can lead to cell death as well as JNK activation. Considering the fact of well conserved nature of Notch as well as both of these two proteins, namely, Hrp48 and Deltex, this interaction can be helpful to understand the regulation of Notch signaling both in development and disease condition.

Regulation of Notch signaling by the chromatin-modeling protein Hat-trick.

Notch signaling plays a pleiotropic role in a variety of cellular processes, including cell fate determination, differentiation, proliferation and apoptosis. The increasingly complex regulatory mechanisms of Notch signaling account for the many functions of Notch during development. Using a yeast two-hybrid screen, we identified the Drosophila DNA-binding protein Hat-trick (Htk) to be an interacting partner of Notch-intracellular domain (Notch-ICD); their physical interaction was further validated by co-immunoprecipitation experiments. htk genetically interacts with Notch pathway components in trans-heterozygous combinations. Loss of htk function in htk mutant somatic clones resulted in the downregulation of Notch targets, whereas its overexpression caused ectopic expression of Notch targets, without affecting the level of the Notch protein. In the present study, immunocytochemical analyses demonstrate that Htk and overexpressed Notch-ICD colocalize in the same nuclear compartment. Here, we also show that Htk cooperates with Notch-ICD and Suppressor of Hairless to form an activation complex and binds to the regulatory sequences of Notch downstream targets such as Enhancer of Split complex genes, to direct their expression. Together, our results suggest a novel mode of regulation of Notch signaling by the chromatin-modeling protein Htk.

Synergistic interaction of Deltex and Hrp48 leads to JNK activation.

The communication among the cells plays a seminal role in metazoan development by coordinating multiple cellular processes that, in turn, helps in the maintenance of biological homeostasis. Our previous study demonstrated that Dx and Hrp48 together downregulate Notch signaling and induce cell death in Drosophila. To understand the signaling events behind the Dx and Hrp48-induced cell death in a greater detail, we performed a set of genetic experiments followed by immunocytochemical analyses. Our data revealed that Dx along with Hrp48 induced JNK activation and consequently cell death in the eye tissue. Additionally, using genetic and molecular approaches, we identified the domain of Dx protein responsible for its synergistic activity with Hrp48. Altogether, our analyses suggest that coexpression of Dx and Hrp48 activates JNK pathway to induce cell death in eye disc of Drosophila melanogaster.