Understanding the mechanism of cardiac gene remodelling via PPAR-α signalling in cardiac hypertrophy

identified and unveiled the functional relevance of differentially expressed miRNAs in ISO-treated PPAR α-/- mice. As of now, limited literature was available about miRNA abundance and their altered expression in cardiac hypertrophy models in the absence of PPAR α protein. So for the very first time, using PPAR α-/- mice, we showed that several miRNAs are differentially expressed that have never been reported in context to cardiac hypertrophy. Additionally, pathway analysis, indicated that the dysregulated miRNAs target genes were enriched mostly in signal transduction pathways. Overall, our findings suggest the miRNAs expression profile of miRNAs like mmu-miR3102-5p; mmu-miR-30a-5p; mmu-miR-30c-5p; mmu-miR-466i-5p is significantly altered in response to cardiac hypertrophy in PPAR-/- mice that eventually impacts the apoptotic pathway in a negative manner

Role Of Astrocytes On Neuronal Health In Models Of Alzheimer’s Disease

Alzheimer’s disease (AD), one of the major forms of dementia in elderly population, is characterized by amyloid beta (Aβ) plaque along with commonly procured tau hyperphosphorylation. Here, we set to recognize the molecules which are secreted by Aβ1-42 treated astrocytes at very early stage and their probable role in conferring neuroprotection along with improvement in cognitive behavior and pathogenesis of AD. Astrocyte activation has been marked by various specific marker proteins like GFAP, Vimentin, S100β. The conditioned medium obtained from the astrocytes cultured from neonatal pups has been used to check the differential expression of various cytokines in Aβ42 treated astrocytes as compared to control cells. From there soluble intercellular adhesion molecule-1 (sICAM-1) has been noted as a potential candidate. We observed that sICAM-1 protects the cortical neurons from death influenced by Aβ42 oligomers. It attenuates the PARP cleavage caused by Aβ42 and increases the amount of anti-apoptotic proteins such as Bcl-2 and Bcl-xL, along with decrease in the amount of pro-apoptotic protein Bim. TUNEL assay performed both in cortex and hippocampus of Aβ42 infused rat brain and 5xFAD mice brain showed that rrICAM-1 treatment reduced the number of TUNEL positive cells in vivo. Several behavioral experiments namely open field test, contextual and cue dependent fear conditioning, passive avoidance tests, novel object recognition and elevated plus maze that can be related to multiple kinds of memory and learning have displayed that rat recombinant ICAM-1 on being injected within the system intraperitoneally, restored the malfunctioning in the rat behavior caused due to bilateral Aβ42 infusion. We took further interest in checking the underlying disease modifying mechanism rendered by ICAM-1 both in vitro and in vivo systems which might be considered being a therapeutic target for neuroprotection. Mechanistically we observed that ICAM-1 administration decreases NF-κB protein level in AD pathology suggesting that ICAM-1 might play a crucial role in manipulating the stability of NF-κB through multiple degradation process. Also PDTC mediated inhibition of NF-κB protein in 5xFAD transgenic mice brain showed an improvement in cognitive behaviors including learning and memory in mice. Therapeutic approach towards prevalent neurodegenerative diseases like AD is one of the major concerns in the scientific world. Our work suggests that ICAM-1 protein could be said as a potential therapeutic agent that promotes neuronal protection along with recovery in cognitive functioning complemented with clearance of Aβ in Aβ42 infused rats and 5xFAD mice model of AD

Studies On The Role Of Mitochondrial Dysfunction In Amyloid- β Mediated Neurodegeneration

Alzheimer’s disease (AD) is one of the major causes of dementia worldwide. AD is characterized clinically by progressive cognitive decline, and pathologically by the presence of extracellular senile plaques composed primarily of amyloid-β peptide (Aβ) and intracellular neurofibrillary tangles made up mainly of hyper phosphorylated tau.AD is a progressive disease and may takes more than twenty years to develop prominent clinical symptoms such as memory loss. It is important to unfold early molecular mechanisms that contribute to development of the disease.Aberrant accumulation of amyloid-β (Aβ) in brain is the major trigger for pathogenesis in AD. It is hence essential to understand how Aβ attains such toxic levels in the brain parenchyma. Aging is one of the major risk factors of AD. DNA damage plays an important role both in aging and AD. We studied the effect of both toxic and sub lethal dose of DNA damage on neurons. Treating differentiated SH-SY5Y cells with a toxic dose of Camptothecin(CPT) induced ROS, mitochondrial dysfunction and apoptosis. Increased expression of BID which is a BH3-only pro-apoptotic protein was observed under similar treatment. Down regulation of BID provided transient protection from toxic dose of CPT in neurons. Next we shifted our focus to study the stress response in neuron induced by sub lethal dose of DNA damaging agent CPT and Doxorubicin (Dox) without inducing cell death.We detected that a very subtle and tolerable amount of DNA damage, related to aging, increased intraneuronal Aβ1-42 production both in cultured neuron and in the cortex of rodent brain.Strikingly, we also observed elevated levels of mitochondrial fusion and of its major driver protein, MFN2. Hyperfusion of mitochondria may be seen as an adaptive stress response resulting from the induction of ER stress since we detected upregulated phosphorylation of both PERK, an ii | P a g e important arm of unfolded protein response and an ER-stress marker, and its substrate eukaryotic initiation factor 2 α (eIF2α), together constituting a signaling responsible for protective mitochondrial remodeling. This adaptive remodeling of mitochondria resulted in an increase in mitochondrial oxygen consumption rate and ATP production. At later time points this elongated structure of mitochondria shifts towards fission. Mitochondrial fission is observed in AD brain but the phenomenon that is leading to this shift is not well studied in the disease model. Reports suggest that eIF2α phosphorylation can increase BACE1 activity, the rate limiting enzyme in Aβ production. In our model, we show that inhibiting PERK, decreased Aβ1-42 level while direct BACE1 inhibition, reduced the mitochondrial fusion.Moreover, we found increased MFN2 expression in younger 5xFAD transgenic mice when Aβ plaques and neurodegeneration were absent. Down regulation of MFN2 decreased CPT induced increase in Aβ1-42in neuron. Thus, this study indicates that mild DNA damage leads to increased Aβ1-42 production via ER stress and may also direct mitochondrial elongation as an initial adaptive/protective response. We propose that age-related subtle genomic DNA damage may trigger enhanced intraneuronal Aβ1- 42 production in an apparently healthy neuron way before the appearance of clinical symptoms in AD. Mitochondrial genome can bear signatures for AD. Mutation in mitochondrial genome can compromise its function and also can be considered as biomarker for early diagnosis of the disease. In the next part of our work we have analysed point mutations in mtDNA from AD patient’s whole blood. We identified SNPs which are common and unique to all the samples. The nature and distribution of mutations were also studied. To our surprise the mutations were not due to oxidation, but it was due to replication error. The SNPs were dispersed in coding region not in D loop of mtDNA. Defective replication of mtDNA could be a result of ineffective replication carried by POLG. Further work in this direction will help to understand the reason of developing these mutations in AD patients’ whole blood. Altogether this work explores an unconventional role of mitochondria in AD. The connection of mitochondria in subtle DNA damage associated stress response and increase in Aβ1-42 production in neuronsis a novel finding and may lead to better understanding of the pathogenesis of AD

Multifunctional behavior of bis-acylhydrazone: Real-time detection of moisture in organic solvents, halochromism and aggregation induced emission

A versatile novel indenopyrazine/indenoquinoxaline appended acylhydrazones (1 and 2) have been designed and synthesized successfully. Compounds 1 and 2 are designed such that, it comprises of acylhydrazone, which is responsible for moisture detection via deprotonation of the original molecule, pyrazine, pyridine and hydrazone unit which is responsible for halochromism via protonation and deprotonation, further the integrated twisted molecular structure results in the aggregation-induced emission features. Successive treatment of Fˉ and moisture to compound 1 and 2 produce reversible colorimetric responses that are easily visualized by the naked eye. Further, the corresponding mechanism was effectively confirmed by 1H NMR spectral analysis. The inherent halochromic features of appended unique pyrazine and pyridine core in compounds 1 and 2 were studied by the sequential addition of trifluoroacetic acid (TFA) and triethylamine (TEA) which is authenticated by reversible colorimetric changes as well as absorption spectral studies. Compound 1 adopts a twisted scissor-like structure and due to multiple weak interactions results in an interesting supramolecular network. Furthermore, both compound 1 and 2 exhibits the aggregation-induced emission features in DMF/water mixture, which was expansively confirmed through DLS particle analysis and TEM images. The integration of three distinct features into a single molecule are scarce

Supercritical carbon dioxide extracts of small cardamom and yellow mustard seeds have fasting hypoglycaemic effects: diabetic rat, predictive iHOMA2 models and molecular docking study

In the present investigation, the supercritical carbon dioxide (SC-CO2) extracts of small cardamom (SC) and yellow mustard (YM) seeds have been investigated for their efficacies in combating type 2 diabetes in streptozotocin-induced Wistar albino rats. Fasting blood glucose (FBG) levels in the rats were monitored on days 8, 15 and 21. On day 15, FBG level reduced appreciably by 31·49 % in rats treated with SC seed extract and by 32·28 % in rats treated with YM seed extract, comparable to metformin (30·70 %) and BGR-34 (a commercial polyherbal drug) (31·81 %) administered rats. Either extract exhibited desirable effects on hepatic glucose-6-phosphatase, glucose-6-phosphate dehydrogenase (G6PD) and catalase activities in controlling diabetes. A molecular docking exercise was conducted to identify specific compounds in the extracts which possessed augmenting effect on G6PD. The results revealed that all the bioactive compounds in the extracts have binding affinities with the enzyme and contributed to the antidiabetic efficacies of the extracts as G6PD augmenters. The effects of the extracts on insulin sensitivity and glucose uptake were investigated using non-invasive modelling by iHOMA2 software. This in vitro approach indicated that extract administration resulted in increased both insulin sensitivity of the liver and glucose uptake in the gut. The findings of the present study attest these SC-CO2 extracts of the spices as safe alternatives of metformin and BGR-34 in combating type 2 diabetes and could be safely subjected to clinical studies. These extracts could also be employed in designing proactive food supplements in mitigating the metabolic disorder

Role of leishmanial glyceraldehyde3-phosphate dehydrogenase in host-parasite interaction

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a classic glycolytic enzyme and is involved in the reversible oxidative phosphorylation of glyceraldehyde-3-phosphate in the presence of inorganic phosphate and nicotinamide adenine dinucleotide (NAD). However, emerging evidence indicates that GAPDH is a multifunctional protein implicated in diverse functions independent of its role in energy metabolism; GAPDH participates in numerous cellular functions, in addition to glycolytic effects, contributes to nuclear tRNA export, DNA replication and repair, endocytosis, exocytosis, cytoskeletal organization, iron metabolism, carcinogenesis, and cell death. It also acts as an mRNA-binding protein, controlling posttranscriptional gene regulation. In the case of Leishmania, apart from glycolysis, no other function of GAPDH is known. Specific post-translational modifications, protein-protein interactions, and subsequent changes in the intracellular distribution of GAPDH in leishmanial species remain unknown. Considering all these facts together, we tried to find out the role of GAPDH in Leishmania other than its role in glycolysis. To understand the non-glycolytic function of LmGAPDH, we have generated control (CT), overexpressed (OE), half-knockout (HKO) and complement (CM) cell lines. HKO cells exhibited reduced virulence compared to control cells when infected with macrophages and BALB/c mice, showing that LmGAPDH plays an important role in Leishmania infection and disease progression. We have demonstrated that LmGAPDH is localized within the extracellular vesicles (EVs) released by Leishmania during infection and, by different molecular biology techniques, established that EV mediated LmGAPDH suppresses the production of pro-inflammatory cytokines like Tumor Necrosis Factor α (TNF-α), which seems to have a primordial role in the process of controlling infection. In vitro protein translation and mRNA binding assays indicate that LmGAPDH binding to the AU-rich 3'-UTR region of TNF-α mRNA is the primary cause for the limitation of its production. Together, these findings confirm that the LmGAPDH found in EVs inhibits TNF-α expression in macrophages via posttranscriptional repression during infection. So, in this project, we attempted to uncover a novel mechanism by which the Leishmania parasite suppresses host immune response, which is critical for developing new drugs and therapeutic strategies against the disease. Here we have illustrated our research in three separate chapters; Chapter1 elucidate mainly the cloning, expression and localization of LmGAPDH from Leishmania major. Chapter2 deals with the functional characterization of LmGAPDH contained within the extracellular vesicles from Leishmania major and its essentiality in disease development and progression. Chapter3 unravels a novel mechanism by which LmGAPDH contained in the extracellular vesicles modulate host immune response through post-transcriptional regulation of TNF-α expression