Department Of Botany
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Item DNA Barcoding, Phylogeny and Phylogeography of Green Sea Weed Ulva from Indian Subcontinent(Central University of Punjab, 2019) Rani, Pooja; Bast, FelixItem 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 DNA barcoding and phylogeny based comparative evaluation of anti-cancer properties of Caulerpa (J V Lamouroux) spp. from Indian coasts(Central University of Punjab, 2019) Mehra, Richa; Bast, Felix and Singh, SandeepA total of 15 Caulerpa samples were collected from Indian coasts and identified based on morphological and molecular data inferred from ITS, 18S, tufA and rbcL. Seven different species viz. C. scalpelliformis, C. racemosa, C. sertularioides, C. verticillata, C. taxifolia, and C. corynephora; and their geographical isolates were identified. Barcode data for these species was generated using aforementioned molecular markers and used for phylogenetic assessment. Phylogenetic trees using Bayesian inference (BI) and Maximum Likelihood (ML) function were generated for each molecular marker. tufA was found to be most suitable marker for the genus Caulerpa, resolving the species into 17 different lineages, with 15 corresponding to already known sections and 2 new lineages. Besides, a database named DbIndAlgae of Indian algae was generated and all the morphological as well as molecular data generated in this study is uploaded on the database. In addition, the phycochemical analysis revealed the presence of alkaloids, terpenoids, steroids, tannins, saponins, flavonoids, and phenols in different Caulerpa species. The selective cytotoxicity of methanolic extracts of Caulerpa (CMEs) was evaluated on MDA-MB-231, T47-D and H1299 cells, and the results revealed significant cytotoxicity of all species. C. racemosa KNY-254 and C. taxifolia TEN-158 were found to be most potent on MDA-MB-231 cells with IC50 value of 0.226 ± 0.004 and 0.246 ± 0.009 µg/µL. The mitochondrial membrane perturbation was revealed by JC-1 and apoptotic cell death was confirmed by Annexin V/FITC staining. CMEs also induced ROS in MDA-MB-231 cells as depicted by DHE, and increased activity of SOD, decreased activity of gluthatione reductase. The CMEs also exhibit anti-invasion activity and inhibited up to 71% migration across the artificially scratched wound in MDA-MB-231, w.r.t. untreated control cells. Moreover, chemical probing of C. racemosa KNY-254 by LC-MS analysis revealed six previously reported and six unreported molecules. The molecular docking analysis revealed weak to moderate interactions with all of the protein targets viz. Bcl2, AMPK, mTOR, BID, PERK, IGF-1R, PI3K, PTP1B and Akt2, known to play important role in cancer cell signaling. Additionally, a moderately positive correlation amongst the phylogeny and anti-cancer activity was observed suggesting that phylogeny might provide cues for anti-cancer activity, subject to further validations.Item Transcriptomic investigations of gene networks in response to arsenic accumulation in Brassica juncea (L.) Czern & Coss(Central University of Punjab, 2019) Thakur, Sapna; Bhardwaj, PankajArsenic (As), a widespread toxic metalloid is class I carcinogen known to cause adverse health effects in human. In the present study, As accumulation potential and differential gene expression in B. juncea is investigated. The amount of arsenic accumulated varied in the range of 15.99 to 1138.70 mg/Kg on dry weight basis in five cultivars. A decrease in chlorophyll content and increase in membrane damage and enzymatic activities of antioxidants was observed with increase in As concentration in the B. juncea cultivars. Using maximum As accumulating cultivar (RLM514), a total of 10,870 significantly differentially expressed transcripts in response to As treatment were identified. Further, the pathway analysis revealed a large scale reprogramming of genes involving carbon metabolism (2.5%), plant hormone signaling (1.4%), and glutathione metabolism (0.6%). Moreover, a comparative account of Cd toxicity revealed a total of 11,294 transcripts to be significantly differentially expressed. The genes related to response to chemical, oxidative stress, transport, and secondary metabolism were upregulated whereas multicellular organismal development, developmental process, photosynthesis were downregulated by Cd treatment. Furthermore, 616 membrane transport proteins were found to be significantly differentially expressed. Cd-related transporters such as metal transporter (Nramp1), metal tolerance protein (MTPC2, MTP11), cadmiumtransporting ATPase, and plant cadmium resistance protein (PCR2, PCR6) were upregulated while cadmium/zinc- transporting ATPase (HMA2, HMA3, HMA4), highaffinity calcium antiporter (CAX1), and iron transport protein (IRT1) were downregulated by Cd treatment. Pathway analysis revealed signaling cascades including plant hormones signaling, MAPK signaling and Ca signaling was modulated suggesting their role in Cd-stress tolerance. The regulation overview using MapMan also revealed gene expression related to plant hormones, calcium regulation and MAP kinases were altered under Cd-stress.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 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 OXIDATIVE STRESS INDUCED CELL PROLIFERATION AND DNA REPAIR MECHANISMS IN GLIOBLASTOMA CELLS: ROLE OF ENPP2 AND APE1(Central University of Punjab, 2018) Cholia, Ravi Parkash; Mantha, Anil K. and Kumar, RajGlioblastoma Multiforme (GBM) is a grade IV, most frequent, and invasive devastating brain tumor with poor prognosis, even with the advancement of multimodal therapies, patients have survival period of less than 15 months. GBM is a multifactorial disease with oxidative stress as a key accelerating player. In the present study, rat glioma C6 and human glioblastoma U-87 MG cell lines were exposed to non-cytotoxic concentrations i.e. 10 -35) peptide, 10 U/ml GO, and 50 M H2O2, respectively. Further, the ROS levels were measured via NBT and H2DCFDA assays. Our genome encounters exogenous and intracellular oxidants which result in the DNA damage; small DNA base lesions such as apurinic/apyrimidinic (AP) sites are generated following the oxidants exposure and repairing of these AP sites is the prerequisite to maintaining the genomic integrity. In the present study, it was observed that APE1 being a redox-sensitive protein, with the moderate level of oxidative stress [induced -35) peptide, GO, and H2O2] resulted in the elevation of APE1 expression as measured using Western blotting, RT-PCR, and (repair) activity was boosted after the treatment of oxidants. Oxidative stress also resulted in the secretion of APE1 extracellularly. Additionally, in this study dysregulated expression of BER-pathway enzymes were observed after the treatment of non-cytotoxic concentrations of the oxidants. Cancer shows higher metabolic properties as compared to the normal cells. Pyruvate Kinase M2 (PKM2) one of the isoform of pyruvate kinase (PK), is a key enzyme in the glycolytic pathway, which catalyses the terminal step of the glycolysis, converts phosphoenolpyruvate (PEP) into pyruvate. PKM2 also perform nonglycolytic functions via enhancing the expression of cyclin D, c-myc, and contributing towards the aggressiveness of GBM. In the present study, oxidative stress resulted in up-regulation of PKM2 level, as analyzed using Western blotting and majorly in the cytosolic regions as identified by immunocytochemistry. Ectonucleotide pyrophosphatase/phosphordiestrase2 (ENPP2) is the secretary protein, known to be involved in a variety of processes like embryonic development, blood vessel formation during development, inflammation, favoring PKM2 dimeric form, and progression of cancer through its enzymatic product LPA. ENPP2 is highly expressed in the GBM, and LPA receptors are also predominate in GBM and play a role in its growth and development. In the present study, elevated expression and activity of ENPP2 was observed after the treatment of non-cytotoxic doses of oxidants in C6 and U-87 MG cells as analyzed using Western blotting and immunocytochemistry. In addition, LPA treatment resulted in the induction of migratory potential of C6 and U-87 MG cells. LPA treatment also up-regulated the key transcription factors such as c-jun, p-c-jun, NF- B, and HIF-1- advocating for their involvement in the survival of GBM cells. LPA treatment resulted in the timedependent increase in the PKM2 and ENPP2 expression and subcellular translocation in the C6 and U-87 MG cells. However, LPA treatment resulted in the elevation of nuclear APE1 expression after 48 hr incubation period. Oxidants - 35) peptide, GO, and H2O2 treatment enhanced the secretory levels of ENPP2 in the extracellular media up to 48 hr, suggesting the protective role of ENPP2 against the oxidative stress. Co-localization of APE1, PKM2, and ENPP2 were observed in the C6 and U87 MG cells when treated with -35) peptide, GO, and H2O2 treatment suggesting the role of oxidative stress in the cross talk interaction of three proteins towards the aggressiveness of GBM. In addition, anti-APE1 inhibitors were synthesized activity, and one of the screened molecule GR5G-b showed ani-proliferative property along with dysregulated APE1 level and repair function; and also displayed potential in cell cycle arrest as analyzed by flow cytometry. Taken together, it can be concluded that oxidative stress enhances the aggressiveness of GBM cells via up-regulating the key proteins (APE1, PKM2, and ENPP2) and altering the functions associated as studied in C6 and U-87 MG cell lines. Further studies focusing towards blocking of their activities by designing, help in development of new therapeutic interventions for GBM.Item Screening of natural compounds for receptor tyrosine kinase inhibitors: in silico and in vitro investigation in cancer cell lines(Central University of Punjab, 2016) Singh, Pushpendra; Bast, Felix