School Of Basic And Applied Sciences
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Item Evaluation of amyloid beta (aβ)-induced Mitochondrial dysfunction: Neuroprotective role of Apurinic/apyrimidinic endonuclease (ape1) Via its interaction with cysteamine Dioxygenase (ado)(Central University of Punjab, 2020) Kaur,Navrattan; Mantha, Anil K.Oxidative stress and damage to mitochondrial DNA during the aging process can impair mitochondrial energy metabolism and ion homeostasis in neurons, ultimately leading to neurodegeneration. Themain pathway for repairing oxidative base lesions is base excision repair (BER), and such repair is crucial for neurons owing to high rate of oxygen metabolism. Apurinic/apyrimidinic endonuclease (APE1) is a protein of this pathway involved in DNA repair and also in the redox co-activating function of different transcription factors. Thus, manipulation of DNA repair mechanisms can be thought of as a putative approach to prevent neuronal loss in neurodegenerative disorders like Alzheimer’s disease (AD). Ginkgo biloba has been studied as a possible treatment for dementia and AD. The ginkgolides present in G. biloba possess antioxidant, neuroprotective and cholinergic activities. The aim of the study was to explore the repair and redox functions of APE1 and a detailed mechanism of association of APE1 with ADO (a thiol dioxygenase) and functional cross-talk between them has been studied. In the present study, we have standardized the differentiation of SH-SY5Y neuroblastoma cells into the cells possessing a mature neuron-like phenotype. The results of cell viability assay showed that differentiated cells are more sensitive/vulnerable to oxidative stress, which is elicited by Aβ. H2DCFDA and DAF- FM-based detection of ROS and RNS strongly advocates that under oxidative stress conditions elicited by Aβ, GB exerts ameliorating effect to render neuroprotection to the SH-SY5Y cells due to its antioxidant nature. Significant decrease in nNOS expression was seen, when cells were pre-treated with GB and then given Aβ treatment in whole cell, cytosol and nucleus. This shows that GB pre-treatment decreases the RNS (NO) levels due to its anti-oxidant property. Determination of DNA damage in terms of measurement of 8-oxo-dG was seen to be more pronounced in mitochondria. In response to DNA damage, pre-treatment with GB decreased the expression of DNA repair enzyme APE1 expression in mitochondria, showing that GB aids in lowering the oxidative stress generated by Aβ in the mitochondria. In the nuclear extracts, upon treatment with GB, there was a significant increase in ADO expression and Aβ treatment also increased the expression of ADO. Whereas, combination treatment of Aβ and GB led to lower expression of ADO. This points towards the possibility that ADO might be translocating to nucleus under oxidative stress and GB might be affecting APE1 – ADO interaction in lowering oxidative stress by the anti-oxidant action of GB, which was clearly observed by immunostaining using confocal microscopy. JC-1 assay points toward GB’s role in restoring the mitochondrial membrane potential against Aβ- challenge. Determination of apoptotic markers (Caspase 9 and AIF) showed that Aβ(25-35) induced oxidative stress caused initiation of apoptosis and GB treatment was able to rescue apoptosis. Our study elucidates activation of synaptic CaMKII and CREB exerting neuroprotective effects; and GB acting to restore the expression and active, phosphorylated state of CaMKII and CREB in presence of Aβ-induced oxidative stress in the SH-SY5Y neuroblastoma cells. This study points towards the use of phytochemicals like GB which will may prove to be beneficial for the enhancement of synaptic functionality and promote neuroprotection.Item Understanding the principles of protein-protein interactions: Designing novel means for virtual proteomics(Central University of Punjab, 2020) Kumar, Vicky; Kulharia, Mahesh and Munshi, AnjanaProteins are the basic functional units in the cellular world of life. They are nano- machines programmed to associate with other biomolecules in order to enact an array of molecular functions in response to biological events at cellular and system levels. Understanding the biomolecular phenomenon governing such associations may provide insights into the principles of protein chemistry that have a wide-range of applications. In the current work, two databases (PPInS and NRDB) in which the information of interacting protein chains from the experimentally determined protein- protein complexes (PPCs) for which structural information in terms of SCOP superfamily was available, is demarcated in the form of protein-protein interaction interfaces (PPIIs) were developed. The PPIIs contained in these databases were made available on a web server for public use. These were analysed w.r.t. physicochemical and geometrical characteristics of PPI sites. With the belief that designing of computational tools with prediction ability must be trained and tested on real instances of the phenomenon for which it is designed, the analytical information obtained from the analysis of PPIIs from NRDB was incorporated in development of a computational tool, Anveshan, for prediction of putative protein-protein interaction (PPI) sites. The training and test datasets for Anveshan development were obtained from the PPInS. PPInS is a high-performance database of PPIIs in which atomic-level information of the molecular interactions amongst various protein chains in PPCs together with their evolutionary information in Structural Classification of Proteins (SCOPe release 2.06), is made available. Total 32,468 PDB files representing X-ray crystallized multimeric PPCs with structural resolution better than 2.5 Å were shortlisted to demarcate the PPIIs. Total 111,857 PPIIs with approximately 32.24 million atomic contact pairs were generated and made available on a web server, named PPInS, (http://www.cup.edu.in:99/ppins/home.php) for on-site analysis and downloading purpose. A non-redundant database (NRDB) of PPInS containing 2,265 PPIIs with over 1.8 million ACPs corresponding to the 1,931 PPCs was also designed by removing structural redundancies at the level of SCOP superfamily (SCOP release 1.75) was also designed to provide the foundation to the development of Anveshan. All the PPIIs and PPIPs involved in both these databases were analysed w.r.t. residues interface propensity (RIP), hydrophobic content, solvation free energy, compactness of interacting residue’ neighbourhood, planarity, and depth index. The PPIIs were also examined in the context of sequence similarity shared by the protein chains involved in the PPII formation which revealed the presence of homodimers in abundance in PDB. Therefore, prior to analysing the PPIIs w.r.t to other parameters, PPIIs from both the databases were categorized in three PPII classes depicting the low-sequence similarity (LSS), moderate-sequence similarity (MSS), and high- sequence similarity (HSS) between the protein chains involved in PPIIs. Analysis pertaining to RIP showed the presence of aliphatic and aromatic residues on interaction sites in abundance and the least occurrence of charged residues (except Arg). Physicochemical and structural analysis of PPIPs, initially, showed a significant difference between their parametric scores w.r.t. all three PPII classes from PPInS and NRDB. However, on removing less than 1% statistical outliers from each PPII class, the parametric scores from all three classes of PPInS and NRDB converged to a statistical indistinguishable common sub-range and followed the similar distribution trends. This indicates that the principles of molecular recognition among proteins are not driven by their sequence similarity and reinforces the importance of geometrical and electrostatic complementarity as the main determinants for PPIs. The parametric score obtained by analysing 4,530 PPIPs from NRDB w.r.t. their RIP, their hydrophobic content and the amount of solvation free energy associated with them provided the basis for the implementation of Anveshan. By applying Anveshan on another dataset of 4,290 PPIPs from 2,145 PPIIs, the optimal range of these parametric scores and protein-probe van der Waals energy of interaction was determined. Subsequently, taking the optimal range of PPIP parametric scores and threshold for protein-probe van derWaals energy of interaction into the consideration, the Anveshan was tested on a blind dataset of 554 protein chains. Predicting 10 sites for each protein chain and taking the best-predicted patch into account, Anveshan was successful in predicting 69.67% sites correctly with at least 50% accuracy in both precision and coverage separately. On predicting only one PPI site for each protein chain, sites predicted by Anveshan on an average covered 21.91% of actual sites in them. Analysing the sites predicted by SPPIDER, it was found that 22.7% of actual sites were covered in predicted sites. However, on predicting two sites for each protein chain, the percentage coverage of actual sites in the sites predicted by Anveshan exceeded two- fold (i.e. 41.81%), thus making Anveshan a superior approach.Item Synthesis and Biological Evaluation of Inhibitors of Topoisomerases and Histone Deacetylase for In Vitro Anticancer Activity(Central University of Punjab, 2019) Joshi, Gaurav; Kumar, Raj & Singh, SandeepTopoisomerases (Topos) and histone deacetylases (HDACs) are validated oncotherapeutic targets due to their involvement in most of the cellular events such as initiation, proliferation, and survival of cancer cells. Widespread research has undergone to design and discover small molecule inhibitors of each protein which has led to the development of several drugs that are making their presence felt in clinic. Considering the issues of stability, toxicity, reported crosstalk(s) and resitance of existing pharmacophores, we herein report the discovery of target-based molecules pertaining to pyrazolo[1,5-c]quinazolines, 2-aryl quinolines and imidazo[1,2- a]quinoxaline scaffolds as inhibitors of TopoI or dual TopoI and II designed rationally via in silico tools. The chemical space of scaffolds was further exploited to design and synthesise dual/multi inhibitors of Topo-HDAC by connecting pharmacophoric features of HDAC inhibitors via a linker. Detailed biological evaluation of synthetics was performed using multiple cancer cell lines as well as normal cells/cell lines. Utilizing MTT, dye exclusion, redox potential, cell cycle and annexin V vs PI assays in 2D as well as 3D cultures, we established their preferential cytotoxic potential. Signaling responsible for anticancer mechanism was delineated using western immunoblotting and qPCR assays. Further, in vitro assays v for topoisomerases (DNA relaxation and catenation), and/or HDAC1 revealed target specificity of synthetics. In addition, we also demonstrated a novel bioreductive methodology, specific to cancer cells, exploiting cancer microenvironment leading to delivery of molecularly targeted agents as topo(s) inhibitors.Item STRUCTURAL AND ELECTROCHEMICAL PROPERTIES OF INTERCALATED AND DISPERSED TYPE POLYMER NANOCOMPOSITE FILMS(Central University of Punjab, 2019) Arya, Anil; Sharma, A. L.The renewable and green source of energy now becomes the burning topic for worldwide research among the scientists. The demand for such energy resources is increasing day by day and it becomes the lifeblood of modern society. Global warming, finite fossil-fuel supplies, and city pollution conspire to make the use of environmentfriendly energy sources. Due to the enlarged dependency of a modern human being on energy resources in every sector and a limited supply of fossil fuels, leads to two main problematic consequences: (1) vulnerability of nation-states to fossil-fuel imports and (2) CO2 emissions that are acidifying our oceans and creating global warming. The controlled environment/climate has drawn the attention of the scientific community towards development and replacement of fossil fuels by an alternative/efficient energy resources. The prospective renewable energy resources are solar, tidal, hydro, wind energy etc. Next challenge comes to store it and could be supplied as per the demand. The said challenge could be overcome through the electrochemical storage/conversion devices (ESCDs) like supercapacitor, Lithium-ion batteries (LIB) and fuel cells. Especially, LIB having the ability to the portability of stored energy and to deliver it as and when required without gaseous exhaust, unlike fossil fuels. A secondary battery converts chemical energy into electrical energy and vice versa. Its structure is composed of a positive electrode as a cathode, a negative electrode as an anode, and electrolyte. Simultaneous movement of ions and electrons occurs in the battery system; ions flow through the electrolyte while electrons are generated at the anode and flow towards the cathode via an external circuit. The heart of the battery is the electrolyte as it is sandwiched between both electrodes and participate in charging/discharging. Although all the three components affect the overall cell performance, the electrolyte is dominating in nature and deciding the specific capacity, energy density, working voltage and the lifespan of the battery. Various types of electrolytes are liquid electrolytes, semi-gel and gel electrolytes. However, safety issues with lithium-metal anodes, the reaction of volatile/flammable organic solvents and the leakage of electrolytes have hindered the commercialization of any lithium-ion battery based on such electrolyte. The drawbacks associated with the battery comprising of above-mentioned electrolyte pushes us to develop new generation solid state polymer nanocomposite films (PNC films) which could possess inherent safety and good compatibility with electrodes as compared with liquid, semi-gel and gel electrolytes. v PNC films have numerous advantages like they are light in weight, flexible, have interfacial compatibility, no leakage issue and are very processable. Most importantly, they are very safe. SPEs are prepared by dissolving lithium salts in a high-molecularweight polymer matrix. The polymer acts as the host for the transmission of lithium ions through the motion of polymer segments. Solid polymer based electrolytes appear to be attractive as they can compensate for the volume changes of electrodes by elastic and plastic deformation. A PEO-based SPE is the most preferred polymer host in the research system due to its flexible backbone and ability to solvate lithium ions, with the coordination number dependent upon the salt concentration and identity of the anion. The main advantage of a PEO is its high solvation power. Hence it can form a complex easily with many alkali salts and provides a direct path for cation migration due to the presence of the ether group in the polymer backbone. But the low conductivity value (10–10 S cm−1 ) and poor mechanical properties of PEOs at ambient temperatures limit their use in devices. Many approaches have been explored to improve the ionic conductivity in order to suppress the concentration polarization and desirable electrochemical properties such as polymer blending, cross-linking, the addition of nano-sized fillers etc. Out of these approaches at host polymer level blending seems more appropriate and justified. Further to scale the relevant properties parameters by minimizing the concentration polarization, two novel approach i.e. (i) nanofiller dispersed polymer nanocomposites, and (ii) intercalated polymer nanocomposites have been adopted. The idea behind using nanofiller was the expectation to get percolation pathways composed of inorganic fillers grains through a flexible polymeric matrix. Such a phenomenon could lead to an increase in ionic conductivity followed, possibly, by an enhancement of the cation transport number while preserving mechanical properties and flexibility of the composite electrolyte prepared in the thin film configuration. The second one is also a thought-provoking approach and plays key role in (i) suppressing the concentration polarization by avoiding anion mobility, (ii) enhancement of the ion migration by allowing the cation coordinated polymer chain confinement in clay galleries, and (iii) negative surface charge on clay acts as Lewis acid and competes with Li+ cation to form complex with polymer which reduces ion coupling. An attempt has been made to understand the role of nanofiller and nanoclay in dispersed and intercalated blend polymer nanocomposites prepared by solution cast technique. A strong correlation exists between crystallinity, conductivity, free ion area, the number density of charge carriers, ion mobility, diffusion coefficient, activation energy, and glass transition temperature. Polymer-intercalated polymer nanocomposites display remarkable higher ionic conductivity, broad voltage stability window, high specific capacity and open-circuit voltage than the dispersed based polymer nanocomposites. Here we provide a cumulative account of an efficient polymer nanocomposite materials to identify their importance in the energy storage/conversion devices.Item Studies on adaptive environmental responses in Himalayan Rhododendron arboreum(Central University of Punjab, 2019) Choudhary, Shruti; Bhardwaj, PankajTemperate plants acclimatize to survive freezing temperatures, which are otherwise prerequisite in the initiation/transition of a developmental phase. The dominance of Rhododendron arboreum under a highly fluctuating Himalayan environment makes it enticing for genetic structure and functional analysis. In the present study, transcript, small RNA and metabolome libraries from flowers and foliar tissues of reproductive and vegetative seasons were analyzed. The high-quality paired-end reads were assembled into 157,427 non-redundant transcripts and categorized functionally based on gene ontology, pathway, and transcription factor database. The screening for molecular markers identified 35,419 SSR and 811 high-quality SNPs. A comparison of transcript profiles for the vegetative and flowering season tissues revealed that 12,577 unigenes with fluctuating expression were responsible for seasonal adaptations. Additional to the gene interaction networks, 421 ions obtained from LC-MS were annotated to distinct pathways, especially secondary metabolites. Thirdly, 466 conserved and novel miRNAs, 442 precursors, and 27,139 targets were predicted and the miRNAs modulating circadian clock and reproductive development were discussed further. Other than the genes, miRNAs, and compounds held for an active metabolism, signaling, development, and their regulations, supplementary responses to abiotic/biotic stimuli were induced. A multifaceted response not only sponsored the climatic encounters but brought the shift from vegetative to reproductive growth. The genome-wide profiling and the spatiotemporal variation in mRNA and miRNA expression, as well as the nontargeted metabolome, will enhance the understanding of development and tolerance strategies in high altitude tree species.Item DNA barcode-based identification and comparative anti-cancer effects of different species of brown seaweed Sargassum C. Agardh of Indian coasts(Central University of Punjab, 2018) Bhushan, Satej; Bast, Felix & Singh, SandeepSargassum C. Agardh is a ubiquitous, multicellular brown seaweed that represents the most species-rich genus of the brown algal order Fucales, with more than 500 species reported worldwide. The present study aimed to identify different Sargassum isolates from India by DNA barcoding of mitochondrial (cox3), chloroplast (rbcL), and nuclear (18S) regions and further phylogenetic analyses. Total of 17 geographical isolates were collected across Indian coasts. Phylogeny reconstruction using Bayesian Inference was done which suggested congruency with known taxonomic hierarchy of Sargassum. Total of five different species were identified (S. portierianum, S. cymosum, S.aquifolium, S. ilicifolium, S. polycystum). In addition, comparative evaluation of anti-cancer potential of all the isolates was carried out and putative relationship between phylogeny and anticancer potential was established. MTT assay with 3 different cell lines showed cytotoxicity with IC50 as low as 0.167 ± 0.01, 0.243 ± 0.007, 0.25 ± 0.03 µg/µL in MDA-MB-231 (Breast Cancer), T-47D (Breast Cancer), H1299 (Lung Cancer) cells respectively, while no toxicity was observed with human peripheral blood mononuclear cells (hPBMCs). I was also able to isolate one lead aliphatic compound (SA1) whch was identified to be a polysaccharide using NMR spectroscopy. Similar to the extract, purified compound SA1 also showed anticancer activity. Further evaluations revealed that SA1 as well as the extracts interfere with the antioxidant defence components of cancer cells (SOD, Catalase, and GR) which results in the induction of mitochondrial death pathway at G1 phase (for extracts) as well as at G2M phase (for SA1). Extracts as well as SA1 were also able to inhibit cancer cell migration at sub IC50 doses. In addition, sub IC50 treatments lead to decreased colony formation compared to the control. Overall, our results show that these extracts as well as SA1 are able to target multiple properties of cancerItem Design, Synthesis and Evaluation of Indole Based Compounds as Putative Anticancer Agents(Central University of Punjab, 2018) Singla, Ramit; Jaitak, VikasIn the course of efforts to develop new chemotherapeutic agent for targeting breast cancer, indole-benzimidazole, indole-xanthendione, indole-chromene carbonitrile and indole-dihydropyridine derivatives were computationally designed and synthesized. All the compounds were first analyzed for antiproliferative activity using ER-α responsive T47D breast cancer cells line and cytotoxicity using hPBMC. Further, all the synthesized compounds were also evaluated for ER-α binding affinity. Lead compounds 5f and 8f of series 1 and 2; 10e and 10f of series 3, 11c and 12d of series 4 and 5 were found to be most active at both cellular and receptor level hence were biologically evaluated for gene expression studies for targeting ER-α. Cell imaging experiment clearly suggest that compounds were able to cross cell membrane and accumulate thus causing cytotoxicity. Semiquantitative RT-PCR and Western blotting experiments further supported that lead compounds altered the expression of mRNA and protein of ER-α, thereby preventing the further transactivation and signaling pathway in T47D cells line. Structural investigation from induced fit simulation study suggest that lead compounds binds in a conformation similar to bazedoxifene by extensive hydrogen bonding and Van der Waals forces. All these results indicate that compounds 5f, 8f, 10e, 10f, 11c and 12d represents new putative anticancer agents and can be proved promising in the discovery of antiestrogens for the management of breast cancer.Item GANODERMA LUCIDUM, A POTENTIAL THERAPEUTIC MODULATOR IN CANCER SIGNALING: IN SILICO AND IN VITRO ANALYSIS(Central University of Punjab, 2018) Gill, Balraj Singh; Kumar , Sanjeev and Kumar, VinodItem ANALYSIS OF MICRORNA SIGNATURES AS BIOMARKER TO INVESTIGATE INTERLINK BETWEEN TYPE 2 DIABETES AND BREAST CANCER(Central University of Punjab, 2018) Sharma, Prateek; Kumar, SanjeevType 2 diabetes and breast cancer are two heterogeneous, multifactorial, chronic health problems involving several overlapping risk factors. Studies have suggested that type 2 diabetes is associated with 10-20% excessive relative risk of breast cancer. Evidence indicates link between type 2 diabetes and breast cancer, through insulin resistance and hyperinsulinemia. Numerous substantial evidence pointing towards the potential efficacy of antidiabetic metformin as anticancer therapeutics. MicroRNAs are endogenous, small non-coding RNA molecules regulating protein-coding gene expression and participate in nearly all the events of life. These small RNA molecules can have diagnostic or prognostic value, as microRNA expression profiles reflect disease origin, stage and other pathological factors. We hypothesized that there might be several microRNAs which commonly function in the “origin of type 2 diabetes to progression towards breast cancer.” Such common microRNAs can act via the related signalling pathways which may provide the critical insight into the better understanding of these diseases. The present study is aimed to investigate the interlinking between type 2 diabetes and breast cancer through microRNA signatures. Methods: In vitro cell experiments (using breast cancer cell lines MCF-7, MDA-MB-231, & T47D and pancreatic beta insulinoma cell lines MIN6 and RIN-5F) referred as MTT proliferation, trypan blue exclusion test, NBT assay, colony formation analysis, and scratch assay. Reactive oxygen species (ROS) assays (DCFH-DA and DHE) along with fluorescence microscopy (DAPI staining, Acridine orange + Ethidium bromide dual staining, JC1 staining) were used for apoptotic parameters. Insulin release in pancreatic beta cell lines was measured by ELISA. mRNA expression levels of Bax, Bcl-2, MMP-2, MMP-9, SOD 1, SOD 2, SOD 3, were quantified by qRT-PCR. Four common microRNAs- let 7a, miR-21, miR-155, miR-375 expression profiling in both breast cancer cell lines and pancreatic cell lines was performed by relative quantification real time analysis. Results: Insulin acts as a potential mitogenic factor accelerating the proliferation of breast cancer cells. On the other hand, metformin inhibits growth, proliferation and v clonogenic potential of breast carcinoma cells. ROS levels in breast cancer cells were significantly reduced by metformin by up-regulating SOD isoforms expression. Insulin increased the ROS to a very small limit. Metformin activates apoptosis by inducing mitochondrial dysfunction, upregulating Bax and downregulating Bcl-2. Migration is strongly suppressed by metformin by regulating matrix metalloproteinase (MMP-2 and MMP-9). Oncogenic miR-21 and miR-155 were downregulated by metformin, significantly correlated with reduced metastasis. The results of our study suggest that both MIN6 and RIN-5F cells show a significant differential pattern of proliferation, insulin secretion, and microRNA expression pattern. RIN-5F beta cells were found to be highly refractory to glucose-stimulated insulin secretion. However, metformin negatively regulates glucose-stimulated insulin release in both MIN6 and RIN-5F. In MIN6 cells, levels of microRNA-375 and let-7a were significantly up- & down-regulated by metformin at normal-glucose and high glucose culture conditions respectively whereas in RIN-5F both were significantly down-regulated. Conclusions: Our data supports that metformin plays a pivotal role in the modulation of the antioxidant system including SOD machinery. Our results indicate that metformin inhibit breast cancer cell proliferation by inducing apoptosis via mitochondrial signalling. Furthermore, emerging view from this study is that microRNAs (let-7a, mir-21, miR-155 and miR- 375) are involved in the process of disease (type 2 diabetes and breast cancer) development, and there is the potential utility of microRNAs as effective biomarker for diagnostic and prognostic application in type 2 diabetes and breast cancer.Item ORGANOPHOSPHATE PESTICIDES PESTER Aβ- INDUCED GENOTOXIC RESPONSES IN CULTURED NEURONAL CELLS: APE1/Ref-1 MEDIATED INTERVENTION(Central University of Punjab, 2018) Sarkar, Bibekananda; Mantha, Anil K. and Mittal, SunilAmyloid beta ( ) peptide deposition is the primary cause of neurodegeneration in reasons deposition, but the actual cause is not apparent. Several reports point towards the role of pesticides in the AD pathogenesis, especially organophosphate pesticides (OPPs) that also act as acetylcholinesterase inhibitors (AChEIs) and are reported to be neurotoxic in nature at sub-lethal doses. Monocrotophos (MCP) and Chlorpyrifos (CP) are the most widely used OPPs with highest production and consumption throughout the world. - induced oxidative stress associated with the neurodegeneration in AD has been assessed -35) peptide. Natural compounds like curcumin have been well documented for their ameliorating powers against various neurodegenerative disease models. The cell survival assay showed that MCP and CP kill the neuronal cells in both dose- and time-dependently. Nitro blue tetrazolium (NBT) based assay for determination of intracellular reactive oxygen species (ROS) demonstrated that MCP and CP produce significant oxidative stress in IMR-32 and SH-SY -35) increased oxidative stress in IMR-32 and SH-SY5Y cells, whereas curcumin reduced ROS levels significantly (p