Virender Singh

Therapeutic implication of L-phenylalanine aggregation mechanism and its modulation by D-phenylalanine in phenylketonuria

Self-assembly of phenylalanine is linked to amyloid formation toxicity in phenylketonuria disease. We are demonstrating that L-phenylalanine self-assembles to amyloid fibrils at varying experimental conditions and transforms to a gel state at saturated concentration. Biophysical methods including nuclear magnetic resonance, resistance by alpha-phenylglycine to fibril formation and preference of protected phenylalanine to self-assemble show that this behaviour of L-phenylalanine is governed mainly by hydrophobic interactions. Interestingly, D-phenylalanine arrests the fibre formation by L-phenylalanine and gives rise to flakes. These flakes do not propagate further and prevent fibre formation by L-phenylalanine. This suggests the use of D-phenylalanine as modulator of L-phenylalanine amyloid formation and may qualify as a therapeutic molecule in phenylketonuria.

Glycogen synthase protects neurons from cytotoxicity of mutant huntingtin by enhancing the autophagy flux

Healthy neurons do not store glycogen while they do possess the machinery for the glycogen synthesis albeit at an inactive state. Neurons in the degenerating brain, however, are known to accumulate glycogen, although its significance was not well understood. Emerging reports present contrasting views on neuronal glycogen synthesis; a few reports demonstrate a neurotoxic effect of glycogen while a few others suggest glycogen to be neuroprotective. Thus, the specific role of glycogen and glycogen synthase in neuronal physiology is largely unexplored. Using cellular and animal models of Huntington’s disease, we show here that the overexpression of cytotoxic mutant huntingtin protein induces glycogen synthesis in the neurons by activating glycogen synthase and the overexpressed glycogen synthase protected neurons from the cytotoxicity of the mutant huntingtin. Exposure of neuronal cells to proteasomal blockade and oxidative stress also activate glycogen synthase to induce glycogen synthesis and to protect against stress-induced neuronal death. We show that the glycogen synthase plays an essential and inductive role in the neuronal autophagic flux, and helps in clearing the cytotoxic huntingtin aggregate. We also show that the increased neuronal glycogen inhibits the aggregation of mutant huntingtin, and thus could directly contribute to its clearance. Finally, we demonstrate that excessive autophagy flux is the molecular basis of cell death caused by the activation of glycogen synthase in unstressed neurons. Taken together, our results thus provide a novel function for glycogen synthase in proteolytic processes and offer insight into the role of glycogen synthase and glycogen in both survival and death of the neurons.

NMR Spectroscopy-based Metabolomics of Drosophila Model of Huntington’s Disease Suggests Altered Cell Energetics

Huntingtons disease (HD) is a neurodegenerative disorder induced by aggregation of the pathological form of Huntingtin protein that has expanded polyglutamine (polyQ) repeats. In the Drosophila model, for instance, expression of transgenes with polyQ repeats induces HD-like pathologies, progressively correlating with the increasing lengths of these repeats. Previous studies on both animal models and clinical samples have revealed metabolite imbalances during HD progression. To further explore the physiological processes linked to metabolite imbalances during HD, we have investigated the 1D 1H NMR spectroscopy-based metabolomics profile of Drosophila HD model. Using multivariate analysis (PCA and PLS-DA) of metabolites obtained from methanolic extracts of fly heads displaying retinal deformations due to polyQ overexpression, we show that the metabolite imbalance during HD is likely to affect cell energetics. Six out of the 35 metabolites analyzed, namely, nicotinamide adenine dinucleotide (NAD), lactate, pyruvate, succinate, sarcosine, and acetoin, displayed segregation with progressive severity of HD. Specifically, HD progression was seen to be associated with reduction in NAD and increase in lactate-to-pyruvate ratio. Furthermore, comparative analysis of fly HD metabolome with those of mouse HD model and HD human patients revealed comparable metabolite imbalances, suggesting altered cellular energy homeostasis. These findings thus raise the possibility of therapeutic interventions for HD via modulation of cellular energetics.

Conceptualizing open agile software development life cycle (OASDLC) model

Purpose – Software development life cycle (SDLC) has always been the core methodology for any software engineer that depicts the entire development process which an organization is bound to utilize to achieve successful software. The purpose of this paper is to bring forth a conceptual model after analysing the best practices in SDLC, and extracting the best out of agile methodologies and the open source software, thereby bringing forward an optimised structure. Design/methodology/approach – The OASDLC is hypothesized specifically for “Brihaspati” project and is formulated keeping in mind the gaps and limitations posed by existing SDLC models. OASDLC is further put to test for achieving lower costs and efforts involved. The tests are further substantiated by means of hypothesis validation through execution of a survey based research. Findings – It has been observed that the present conceptual model further optimizes the efforts involved while adopting such a practice. Originality/value – This paper propos...

Calmidazolium Chloride and Its Complex with Serum Albumin Prevent Huntingtin Exon1 Aggregation

Huntingtons disease (HD) is a genetic disorder caused by a CAG expansion mutation in Huntingtin gene leading to polyglutamine (polyQ) expansion in the N-terminus side of Huntingtin (Httex1) protein. Neurodegeneration in HD is linked to aggregates formed by Httex1 bearing an expanded polyQ. Initiation and elongation steps of Httex1 aggregation are potential target steps for the discovery of therapeutic molecules for HD, which is currently untreatable. Here we report Httex1 aggregation inhibition by calmidazolium chloride (CLC) by acting on the initial aggregation event. Because it is hydrophobic, CLC was adsorbed to the vial surface and could not sustain an inhibition effect for a longer duration. The use of bovine serum albumin (BSA) prevented CLC adsorption by forming a BSA-CLC complex. This complex showed improved Httex1 aggregation inhibition by interacting with the aggregation initiator, the NT17 part of Httex1. Furthermore, biocompatible CLC-loaded BSA nanoparticles were made which reduced the polyQ aggregates in HD-150Q cells.

Osmolytes modulate polyglutamine aggregation in a sequence dependent manner

Osmolytes stabilize protein structure and suppress protein aggregation. The mechanism of how osmolytes impact polyglutamine (polyQ) aggregation implicated in Huntingtons disease was studied. By using a reverse‐phase chromatography assay, we show that methylamines‐trimethylamine N‐oxide and betaine are generic in enhancing polyQ aggregation, while a disaccharide trehalose and an amino acid citrulline moderately retard polyQ aggregation in a sequence specific manner. Despite the altered kinetics, the fundamental nucleation mechanism of polyQ aggregation and the nature of end stage aggregates remains unaffected. These results highlight the importance of using osmolytes as modulatory agents of polyQ aggregation.