Biochemistry And Microbial Sciences - Master Dissertation

Permanent URI for this collectionhttps://kr.cup.edu.in/handle/32116/24

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Antibacterial and Anticancer property of bioactive secondary metabolites from Actinomycetes isolated from different regions of Himachal Pradesh
(Central University of Punjab, 2018) Chambiyal, Gourav; Kushawaha Pramod K.
The prime objective of the present work is to isolate, characterize and purify soil microbes and to know about their antibacterial and anticancer properties. Soil samples were obtained from different locations in Dharamsala, Himachal Pradesh, India. Serial dilutions of the samples were made and plated on starch casein agar. Screening, purification and further tests were performed on the isolates to find the potential colonies. Biochemical and morphological characterization was done as well. Following characterization, isolates were used to extract bioactive secondary metabolites which can be anti-bacterial and anti-cancer. Secondary screening for antibacterial properties of isolates was evaluated by Agar well diffusion method. Further, the isolates were subjected to the GC-MS and MTT cytotoxic assay. Totally 7 isolates of actinomycetes were isolated and out of them only 4 were tested for antagonistic activity against 4 pathogenic microorganisms. Isolates M1,and M3 of dilution 10-1 were active, while M2 and M4 showed less activity against the pathogenic microorganisms. All actinomycetes isolates showed antibacterial activity against S. enterica and P. putida while they showed less activity against S. aureus and E.coli. These isolates showed antibacterial and anti-cancer activities and may be used for the growth of new antibiotics for pharmaceutical or agricultural purposes.
Nutrient Analysis of Two Chickpea Cultivars Grown in Local Soil and Treated with Plant Growth Promoting Bacteria from Bathinda Region
(Central University of Punjab, 2018) Dogra, Nitin; Wusirika, Ramakrishna
The bacteria found near plant roots are known to affect plant and soil health. Several important characteristics, such as biological nitrogen fixation, phosphate solubilization and production of siderophores and phytohormones are together called plant growth promoting (PGP) traits. In the current study, the plant growth promoting activity of five bacterial strains, Pseudomonas citronellis (PC), Pseudomonas sp. RA6, S2, Serratia marcescens CDP-13 and Symbion-K (Frateuria aurantia) were observed on chickpea varieties PBG1 and PBG5, grown in pots containing soil collected from Central University of Punjab city campus. The plants were harvested after 30 days and shoot length, root length, plant biomass and nutrient content of shoots were evaluated.
Role of Plant Growth Promoting Bacteria in Improving the Quality of Marginal Soil for Multiple Purposes
(Central University of Punjab, 2018) Kumari, Ritu; Wusirika, Ramakrishna
Degrading quality of soil by the presence of xenobiotics or change in the natural environment of soil is of great concern. One of them is heavy metal pollution of soil. With increasing population, sustainable food production is a challenge in this climate changing and water deteriorating environment. Marginal soil has been defined from different perspectives. In general, marginal land can be defined as arid and inhospitable land that has low inherent productivity. Marginal land is a good source for the production of biofuels. This can be achieved with the help of Plant Growth Promoting Bacteria (PGPB). This project provides a review of marginal soil and how PGPB can be used to improve its quality, in phytoremediation, and in the production of bioenergy and food.
Identification of natural inhibitors of proteins involved in the pathology of Parkinson's disease
(Central University of Punjab, 2018) Mahapatra, Prareeta; Kumar, Shashank
Parkinson's disease (PD) is a progressive neurodegenerative disorder caused due to the lack of dopamine in the brain. Different drug therapies are available for PD showing excellent efficiency, but most of them are cost intensive and with side effects. All these issues have brought natural products in attention. The present study was designed to identify the potent anti-Parkinson phytochemicals. Proteins that are involved in Parkinson's disease were targeted. In the present study, methylated flavonoids were selected for studies including molecular docking against protein involved in Parkinson's disease such as Murine Keap 1 (5CGJ), brain permeable Polo-like kinase (4I5P), Methionyl tRNA synthetase(1PFU) and Roco-4- kinase( 4F0F).To predict the drug-likeness property of the phytochemicals, Lipinski's rules of five, Caco-2, CMC-like rule and MDCK value were used. By prediction of ADME, drug-likeness properties and toxicity properties of the phytochemicals it can be stated that most of the phytochemicals have the potential to cross the Blood Brain Barrier and have good ROS quenching potential also.
Understanding Immune-etiology of Psoriasis: An autoimmune disease
(Central University of Punjab, 2018) Kumari, Anjna; Jain, Manju
Psoriasis is a chronic inflammatory autoimmune disease. Disease etiology is understood in terms of altered crosstalk between skin keratinocytes and immune cell infiltrates, specifically T cells leading to the development of characteristic psoriatic skin lesions. T cell alterations in skin lesions as well as in the peripheral blood of psoriatic patients have been shown to be associated with the disease condition. With a major research focus on keratinocyte abnormalities, more studies are required to understand the immune-etiology of disease. There are fewer reports with inconsistence findings on T cell changes in psoriatic patients. Towards this end, we performed a study using blood samples from psoriasis patients and healthy controls to access alterations in peripheral blood mononuclear cell and T cell count along with phenotyping of blood cells in terms of CD4+ Th and CD8+ Tc cells and expression pattern of T cell-associated cytokines in plasma samples. Furthermore, TREC analysis was done to understand possible origin of T cell alterations associated with psoriasis.
Expression, Purification and Kinetic Characterization of Lactate Dehydrogenase of Lactic Acid Bacteria and ProB of Bacillus subtilis
(Central University of Punjab, 2018) Gunjan; Verma, Malkhey
Lactate dehydrogenase is very well known for its role in glucose metabolism in Lactic Acid Bacteria (LAB). These bacteria are widely used in the industrial and research areas. LAB use NADH as a cofactor to produce lactate or lactic acid from glucose through fermentation, whereas ProB is an enzymatic protein which catalyzes the conversion of L-glutamate to ?-glutamyl phosphate at the expense of ATP in proline biosynthesis. Both the recombinant proteins are expressed in the E. coli BL 21 strain using pET plasmids. Proteins are purified using affinity chromatography and purity is established by a single band in SDS Page. Proteins were quantified by Bradford assay. In this study, the proteins selected are Lactate Dehydrogenase and ProB. In the absence of FBP, Pi is an activator of L. lactis LDH at pH 6. This effect can be interpreted by considering the computed binding affinities of Pi to the catalytic and allosteric binding sites of the enzymes modelled in protonation states corresponding to pH 6 and pH 7. In this study, we can find out the delicate interplay among the effects of Pi, FBP, and pH that results in different regulatory effects on the LDH of LAB and ProB of B. subtilis.
Production and Optimization of Lovastatin by Microbial Fermentation using Aspergillus terreus
(Central University of Punjab, 2018) Raj, Rohit; Verma, Malkhey
In the recent times, the fungus Aspergillus (A.) terreus has been highly popularized regarding its domination for the production of the "crackerjack" drugs known as statins, particularly lovastatin. The aim of this research was the production of lovastatin which is a known cholesterol-lowering drug, through microbial fermentation using A. terreus. Besides, it also aimed to analyze certain bioactive chemical products and evaluation of such antibacterial and antifungal products, if any produced. Bioactives (chemical compounds often referred as secondary metabolites) were analyzed using the Gas Chromatography-Mass Spectroscopy technique (GC-MS) technique. A. terreus is known to produce a vast variety of important secondary metabolites with high biological activities. The extraction of the natural statins such as lovastatin or mevastatin from A. terreus is seen as one of the major breakthrough in the field of Industrial Microbiology/Fermentation Technology. Here we report the Aspergillus terreus NBRC (IFO) 31217 (Strain I) and ATCC 11877 (Strain II) don't produce lovastatin but they produce important bioactive compounds of high commercial value like Isovaline (C5H11NO2) and Silane etc.
Characterization of Hexaploid Wheat Protein on the Basis of their Date of Release in Indian sub- continent
(Central University of Punjab, 2018) Negi, Arti; Dhiman Monisha
Wheat is the third most grown cereal worldwide. The storage proteins of wheat represent an important source of food and energy and are also involved in the determination of bread wheat quality. These gluten proteins are categorized as prolamins, composed of monomeric gliadin (single chain polypeptides) and polymeric glutenin (multiple polypeptide chains).Unfortunately consumption of these gluten protein is known to be linked with range of clinical disorders e.g. celiac disease, wheat allergy and wheat intolerance. The main objective of the present work is to elaborate a detailed knowledge of the variability of proteins and protein fractions of the wheat varieties based on their origin. 25 wheat varieties procured from Indian Institute of Wheat and Barley Research were evaluated for analysis of total wheat protein and gluten protein by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS- PAGE) and cluster analysis was done based on SDS-PAGE gels to investigate variation among varieties. Multiple sequence alignment of ?-gliadin protein of different genes was done and region of highest variability among them is reported. This study shows that 25 varieties of wheat of different origin vary in their total wheat protein as well as gluten content. However the results of cluster analysis of gluten showed low degree of heterogeneity among the varieties.
Bioinformatic Analysis Of Four Proteins Differentially Expressed In A Colon Cancer Cell Line Treated With Rice Callus Suspension Culture
(Central University of Punjab, 2018) Banerjee, Aditi; Wusirika, Ramakrishna
Plant callus extracts have been shown to have effective anticancer activity. In a previous study, cancer cells (colon cancer cell line SW620), was treated with rice callus suspension culture and certain proteins were found to be differentially regulated (9 upregulated proteins and 3 downregulated proteins). Bioinformatic analysis was done for three upregulated and one downregulated proteins to elucidate the molecular mechanism of cancer progression in the RCSC treated cells. NCBI, STRING, KEGG, PDB and miRNA database searches were performed for the four proteins. Information about the gene size, chromosomal location, protein structure, protein interactions, and pathways was extracted and analyzed. Based on the previously mentioned attributes, possible role of upregulated proteins (Liprin?1, OFD1, SPINK5) and downregulated protein (Vinculin) in progression of cancer was predicted.
Estimation of heavy metals in human blood samples- To evaluate immunotoxic correlation with health risk in local population of Malwa region of Punjab
(Central University of Punjab, 2018) Padhi, Padhi; Jain Manju
Elements play a significant role in the human body for growth, development, and metabolism. But there are certain heavy metals which adversely affect the various processes of the biological system. A certain amount of different heavy metals is tolerable but above the optimum level, these have a drastic effect on the body. In this work, we evaluated the concentrations of specific heavy metals - Copper, Arsenic, Cadmium, and Lead in human blood. The study area chosen is Malwa region of Punjab specifically Bathinda, as the area is reported to have heavy metal contamination in groundwater and soil which enter the food chain. There is no single study demonstrating the presence of heavy metals in blood samples of subjects in the study area and its possible correlation with high incidence of various diseases reported in the region. In our work, we have analyzed the concentration of four heavy metals- Copper, Arsenic, Cadmium and Lead in whole blood samples collected from susceptible and diseased individuals from study area using ICP- MS. Our preliminary data show that the concentration of arsenic and the lead was higher in the diseased group compared to the susceptibility group. Presence of these two heavy metals in blood found to be above the permissible limits can be the possible reason for high disease incidence in the study area.
Assessment of Extract of Syzygium cumini Against Doxorubicin Induced Cardiotoxicity
(Central University of Punjab, 2018) Chayan, Mukherjee; Monisha Dhiman
For the past four decades, doxorubicin (DOX) has been used to treat cancer, mainly solid tumours and haematological malignancies. However, clinical community is greatly concerned regarding the administration of this as DOX treatment is commonly associated with dose-dependent cardiotoxicity. Attempts at alleviating drug generated cardiac damage using an extract from different parts of plants with radical scavenging property are a promising area of research. Hydroalcoholic extract derived from fruit pulp of Syzygiumcumini which has a significant antiradical scavenging effect. This study aims to assess the effect of parallel administration of SC fruit pulp extract (SC) on mitigating or preventing DOX induced cardiotoxicity in vitro using H9c2 cardiomyoblast cell lines. Addition of SC fruit pulp extract and DOX were performed for both treatment and control sets on H9c2 cells. SC fruit pulp extract showed strong ABTS cation radical scavenging activity in a dose dependent manner. MTT assay was used to study the cytotoxic effect of SC fruit pulp extract and DOX. ROS levels were estimated using NBT assay and DHE assay. The results showed that DOX has significant cytotoxic effect in a dose dependent manner while SC fruit pulp extract did not display any significant cytotoxicity on H9c2 cells. The DOX induced ROS production was found to be significantly reduced in SC fruit pulp extract treated cells. Results of the current study also suggest that the treatment of SC fruit pulp extract along with DOX, displayed cardioprotective potential in H9c2 cells by: 1) reducing lipid peroxidation; 2) decreasing extracellular nitric oxide (NO); 3) decreasing the expression of the protein p47phox and iNOS/NOS-2. These results clearly suggest that treatment of SC fruit pulp extract along with DOX reduces the DOX induced toxicity and hence can be a promising therapeutic intervention in managing DOX mediated cardiotoxicity.
Isolation And Determination Of Antibiotic Susceptibility Of Enterococcus Faecalis From Malwa Region
(Central University of Punjab, 2018) Garg, Dimple; Baranwal,Somesh
About 20 isolates of Enterococcus faecalis is isolated from six different places of Malwa region. They include isolate from Animal waste soil, Agriculture soil, Sewage soil and public place soil also. Isolation of Enterococcus faecalis from environment causes concern for diseases i.e. Endocarditis, UTI, neonatal infections due to maintaince of unhygienic conditions. The biochemical tests are performed and measure as presumptive results for Enterococcus faecalis. Antibiotic susceptibility test is performed to observe the susceptibility of strains for different antibiotics. Six different types of antibiotics such as Ampicillin, Piperacillin, Tetracycline, Rifampin, Streptomycin, and Gentamycin are used for Antibiotic Susceptibility test. Antibiotic Susceptibility test analyse that Gentamycin and Streptomycin are found more resistant towards Enterococcus faecalis than others in Malwa region. The susceptibility of strains for antibiotics are different due to the IS elements which are horizontal transfer to the other Enterococcus faecalis also formed biofilm which makes it more antibiotic resistant due to slower growth rate, metabolic activities and poor penetration rate.
Identification of Novel Natural Inhibitors Of Proteins Involved In Cancer Cell Stemness
(Central University of Punjab, 2018) Malik, Rebati; Kumar, Shashank
Cancer stem cells (CSCs) are a small subpopulation of cells identified in a variety of tumors that are capable of self-renewal, differentiation and have the unique property to evade radiotherapy and chemotherapy. CSCs are a very likely cause of resistance to current cancer treatments, as well as relapse in cancer patients. Compared to differentiated tumor cells, CSCs have some important distinguishing feature that confers chemoresistance in these cells. Different proteins such as Bcl-2 (2O21), CXCR4 (3ODU), CHK1 (4FSZ), MTH1 (5ANV), VEGFR2 (1Y6A) and Carbonic anhydrase II (5SZ2) have been reported to involve in cancer cell stemness. Now day's natural products are popular remedies against various diseases including cancer. These products have been reported for their low/non-toxicity and cost-effectiveness. The phytochemical terpenoids, biggest class of naturally occurring compounds derived from five-carbon isoprene unit. They play an important role in binding to the above signaling proteins which are involved in cancer stem cells. Therefore, we studied receptor-based molecular docking of natural terpenoids against target proteins.
Identification of Curcuma longa phytochemicals as a novel inhibitor of proteins involved in Allergic Rhinitis
(Central University of Punjab, 2018) Das, Swagata; Kumar, Shashank
Histamine and other chemical mediators play crucial role in the disease development condition of allergic rhinitis. Allergic rhinitis is a mild or severe allergic condition due to the interaction of allergens with the IgE antibodies leading to release of chemical mediators like histamine, leukotrienes, prostaglandins etc. Various drug therapies have been elucidated based on the target proteins or enzymes involved. Certain specific proteins like histamine H1 receptor (3rze), histidine decarboxylase (4e1o), leukotriene C4 synthase (3hkk), 5-lipoxygenase (3o8y) alongwith non specific proteins like adenylate kinase (2c9y), phospholipase C (4qj4) are mainly targeted. Commonly prescribed drugs are antihistamines and leukotriene receptor antagonists, which generally reduces the symptoms occurring due to the release of the chemical mediators. Yet, there are persistent and prevalent conditions whereby the release and accumulation of histamine is misinterpreted as allergy instead of a totally different condition called histamine intolerance resulting in histamine accumulation due to defected or mutated enzymes related in its metabolism. Now days natural products are popular remedies against a number of diseases and allergic rhinitis is no exception. These products have been significantly reported due to the low/non-toxicity and cost effectiveness. Curcuma longa or turmeric is a common medicinal herb with iv enormous medicinal properties including anti-inflammatory properties. Various phytoconstituents of turmeric were identified and considered for receptor-based molecular docking. The target proteins and their interactions with each of phytoconstituent present in turmeric were studied.
Role of Helicobacter pylori Enriched Media in Inducing Oxidative Stress in Human Cell lines
(Central University of Punjab, 2018) Samal, Pallavi; Dhiman, Monisha
Helicobacter pylori is a gram-negative, helical, microaerophilic bacterium which colonizes the human gastrointestinal tract. Vacuolating cytotoxin A (VacA) is one of the major virulent factors. Reactive oxygen species (ROS) and Reactive nitrogen species (RNS) produced by the immune and epithelial cells damage the host cell thereby resulting in a persistent infection. The prolonged infection results in chronic inflammation, oxidative stress and DNA damage. The microbe affects the major macromolecules of the host tissues lipids, proteins and DNA which leads to lipid peroxidation, protein oxidation and DNA fragmentation hence making the oxidative stress a deleterious damage. Role of H. pylori enriched media (HPEM) in inducing oxidative stress in two human cell lines AGS (human gastric cell line) and THP-1(human monocytic cell line) was studied in present work. The AGS cells and THP-1 cells was treated with various concentrations of HPEM and oxidative stress was evaluated by examining the levels of protein carbonyls, TBARS (thiobarbituric acid reactive species) and nitric oxide by spectophotometric and Western blotting methods. The oxidative stress induced by HPEM showed damaging effects on the cell membrane, protein and produced significantly high nitric oxide (NO) when compared with the untreated controls. From the present work it can be concluded that HPEM exposure to THP-1 and AGS cells enhanced the oxidative stress which leads to cellular damage and is ultimately responsible for the severe H. pylori associated fatal complications during its pathogenesis.
In Silico Identification of Novel Natural Inhibitors Of Carbohydrate Metabolic Pathway In Cancer Cells
(Central University of Punjab, 2018) Dash, Swastika; Kumar, Shashank
Carbohydrate metabolism in cancer cells is linked to the 'Warburg Effect' which states that, under aerobic conditions, cancer cells metabolize approximately ten fold more glucose to lactate in a given time than normal cells; typically altered glycolytic pathway regulation. This has made the blocking of glycolytic pathway enzymes, a fascinating strategy to find treatment for cancer. This project addresses in a comprehensive manner the main glycolytic enzymes accounting for high-rate glycolysis in cancer cells. In addition, highlights of inhibitors that can be used to target the particular enzymes to decrease proliferation have also been done. Furthermore, besides the known inhibitors, receptor-based molecular docking of certain methylated flavonoids was performed with the proteins (isozymes of carbohydrate metabolic pathway enzymes) to find the lead inhibitors. The proteins used in the study are GLUT1 (4PYP), Hexokinase2 (2NZT), Phosphofructokinase2 (2AXN), Pyruvate kinaseM2 (3GQY), Lactate dehydrogenase A (4AJP) and Enolase2 (5IDZ). The dock scores were in the range of -5.88 to -9.68 against different target proteins. The methylated flavonoids 2-(3,4-dihydroxyphenyl)-3,5-dihydroxy-7-methoxy-4H-chromen- 4-one, 5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-6,8-dimethoxy-4H-chromen-4- one, 2-(3,4-dimethylphenyl)-5,7-dimethyl-4H-chromen-4-one and 6-hydroxy-3,5,7,8- tetramethoxy-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one showed better dock scores for the target proteins in comparison to the standard inhibitors. Thus these methylated flavonoids might be considered promising leads for further development of glycolytic pathway inhibitors in cancer cells.
Extraction And Identification Of Secondary Metabolites Produced By Soil Actinomycetes And Their Biochemical Characterization
(Central University of Punjab, 2018) Singh, Jahnavi Kumari; Kushawaha, Pramod
The worldwide use of antibiotics has rapidly increased since the discovery of penicillin in 1928 by Alexander Fleming, for treating terrible bacterial diseases. But in due course of time, over-consumption of antibiotics has led to an enormous selective pressure on bacteria, which has forced them to adapt to the antibiotics and hence gain development of resistance. In the past years, the excessive use of antibiotics has led to significant evolution in microorganisms where naturally occurring genes resistant to the antibiotic have been spreading fast, thus, the need for new antibiotics has increased. In the stated work, samples were collected from the mountainous sites of Himachal Pradesh and were analyzed for the presence of secondary metabolites. The bacterial species were characterized by biochemical tests and metabolites produced by them were identified.
Identification of some novel natural inhibitors of proteins in the life cycle of the malarial parasite
(Central University of Punjab, 2018) Kumari, Priyanka; Kumar,Shashank
Antimalarial phytoconstituents were identified and analysed.We studied receptor based molecular docking of protein drug targets( Plasmepsin 2 (1lf2),HSP90 (3k60), PfATPase ( 1u5n), Human orotidine 5 - decarboxylase domain (2v30), and Plasmodium orotidine 5- decarboxylase domain (3n3m) against the natural phytochemical .The dock score were in range of -6.1 to -10.7 for antimalarial targets respectively. Some ligands showed better dock score than standard inhibitors of the respected proteins from the range (-6.9 to -10.7).
Comparison of Pyruvate Kinase of different species of Lactic Acid Bacteria
(Central University of Punjab, 2018) Sahoo, Debasis; Verma Malkhey
Pyruvate kinase (PK) is an important allosterically regulated enzyme that connects Glycolysis from the primary energy to cellular metabolism. It catalyzes the last step of Glycolysis by producing one mole of energy in the form of ATP. On the other hand, Lactic acid bacteria (LAB) under anaerobic condition produce energy by Glycolysis process that strengthens the vital role of PK in their cellular metabolism. The recombinant proteins of PKs expression were performed by inserting the PKs genes of LAB strains in the pET 30 plasmid and transformed into the E. coli BL21 (DE3) cells. After cell cultivation, recombinant protein expression was induced. The targeted protein was purified by Affinity chromatography and quantified by Bradford quantitative assay. Protein expression was compared by the SDS-PAGE. Here in this study, the main targeted protein is Pyruvate kinase and its activity is compared among Enterococcus faecalis, Lactococcus lactis, Lactococcus plantarum and Streptococcus pyogenes.
To Study the Alteration in Thymic Functionality in Experimental Visceral Leishmaniasis
(Central University of Punjab, 2018) Mehara, Arjun Kumar; Jain,Manju
Visceral Leishmaniasis (VL) is a neglected tropical disease and is potentially-fatal. Species belonging to L.donovani complex are the causative agent of VL in humans. Host immunity against VL critically depends on T cell based cell mediated immune response. VL is associated with lymphopenia such that progression of VL involves depletion of T cells. However, the origin and mechanism of T cell alterations in peripheral blood is not clearly understood. So we have tried to understand the origin of T cell changes in context of thymic functions which is the site of T cell development based on T cell Receptor Excision Circles analysis in experimental murine VL model. The result shows higher copy number of T cell Receptor Excision Circles (TRECs) in peripheral blood and a trend towards increase in developing thymocytes from thymi of infected mice compared to control uninfected group. The results imply that the peripheral T cell repertoires comprise a significant fraction of unexpanded naïve T cells. TREC change in thymocytes is statistically not significant implying no change in thymic output of naïve T cells. Thus our findings show that leishmania parasite can modulate T cell arm of immunity by altering the proliferation capacity of recent thymic naïve T cell emigrants.