Papers by Roop Singh Bora
Plasmid, 2019
Plasmids are important vectors for the transfer of genetic material among microbes. The transfer ... more Plasmids are important vectors for the transfer of genetic material among microbes. The transfer of plasmids causes transmission of genes involved in pathogenesis and survival, to the host bacteria leading to their evolution and adaptation to diverse environmental conditions. A large number of plasmids of varying sizes have been discovered and isolated from various microorganisms. Plasmids are also valuable tools to genetically manipulate microbes for various purposes including production of recombinant proteins. Escherichia coli is the most preferred microbe for production of recombinant proteins, due to rapid growth rate, cost-effectiveness, high yield of the recombinant proteins and easy scale-up process. Several plasmids have been designed to optimize the expression of heterologous proteins in E. coli. In order to circumvent the issues of protein refolding, the codon usage in E. coli, the absence of post-translational modifications, such as glycosylation and low recovery of functionally active recombinant proteins, various plasmids have been designed and constructed. This chapter summarizes the recent technological advancements that have extended the use of the E. coli expression system to produce more complex proteins, including glycosylated recombinant proteins and therapeutic antibodies.
Journal of Agriculture Biotechnology, 2017
Infectious diseases caused by MDR pathogens are one of the primary health concerns globally, affe... more Infectious diseases caused by MDR pathogens are one of the primary health concerns globally, affecting millions of people every year. Infectious diseases are responsible for high morbidity throughout the world, accounting for about a quarter of deaths around the globe. This growing problem has necessitated the exploration and development of novel antimicrobials such as natural products, plant phytochemicals and fabricated antibiotics with less or no side effects. Medicinal plants are known to be valuable resources for the development of novel antimicrobial drugs. Medicinal herbs are better equipped at metabolic Bio-engineering of active compounds than conventional laboratory approaches. Preliminary antibacterial assay by agar well assay of Allium Ampeloprasum on MDR pathogens MRSA and ESBL +ve E.coli have shown significant antibacterial activity. After clear zone of inhibition formation, the further evaluation of antimicrobial activity was performed by Transmission Electron Microsco...
Integrative Cancer Therapies, 2020
Background: Rhazya stricta has been used as a folkloric medicinal herb for treating various disea... more Background: Rhazya stricta has been used as a folkloric medicinal herb for treating various diseases such as diabetes, inflammatory disorders, and sore throat. Several studies have revealed the potential of this plant as an important source of phytochemicals with anticancer properties. Objective: The present study was designed to isolate a novel anticancer compound from Rhazya stricta and elucidate its mechanism of action using genomics approach. Methods: Rhazya stricta leaves extract was prepared, and several alkaloids were purified and characterized. These alkaloids were screened for their anticancer potential. One of the alkaloids, termed as isopicrinine, showed efficient cytotoxicity against MCF7 breast cancer cell line and was selected for further analysis. RNA-Seq transcription profiling was conducted to identify the affected genes and cellular pathways in MCF7 cells after treatment with isopicrinine alkaloid. Results: In vitro studies revealed that newly identified isopicrini...
Bioscience Journal, 2020
Halophilic bacteria are a microorganism that grows optimally in the presence of the very high con... more Halophilic bacteria are a microorganism that grows optimally in the presence of the very high concentration of sodium chloride. Halophiles are vital sources of various enzymes including hydrolases, which are very stable and catalytically highly efficient at high salt concentration and other extreme conditions such as high temperature, pH and presence of organic solvents. Several hydrolases such as amylases, proteases, and lipases have been obtained from halophilic bacteria and are commonly used for various industrial applications. We initiated a screening project to isolate and characterize the halophilic bacteria from the Red Sea, which is one of the saltiest bodies of water in the world. Water and soil samples, collected from the Red Sea coast, Jeddah, Saudi Arabia, were screened for isolation of halophilic bacteria. Ten bacterial isolates were obtained, which were characterized by biochemical tests and 16S rRNA gene sequencing. Hydrolase producing bacteria among the isolates wer...
Biotechnology & Biotechnological Equipment, 2018
The emergence of multidrug-resistant (MDR) microbes has become one of the major threat globally. ... more The emergence of multidrug-resistant (MDR) microbes has become one of the major threat globally. Infectious diseases are the second leading cause of death, two-third of which are caused by Gram-negative bacteria. The increasing number of multidrug resistant (MDR) microbes is quite alarming and has raised the necessity of development of new antibacterial drugs. Escherichia coli and Klebsiella have been reported among the top most resistance-developing pathogens. Ricinus communis is an important medicinal plant reported to possess antimicrobial phytochemicals such as a-pinene. The hexane treated crude ethanolic extract of R. communis was evaluated against Gram-negative bacteria E. coli and Klebsiella oxytoca. The agar well diffusion assay was used to determine the antibacterial activity. In the present study, we have shown experimentally that leaf extract of R. communis can induce the deterioration of the inner and outer cell membranes of E. coli and K. oxytoca and decrease their viability at a concentration of 50 mg/ml. Transmission electron microscopic results revealed cell membrane damage, cellular disintegration and release of cytoplasmic content, leading to cell death. To our knowledge, this is the first study of the antibacterial activity of R. communis against E. coli and K. oxytoca by Transmission electron microscopy. The ultramicroscopic observations showed that the phytochemical present in the leaf extract of R. communis could penetrate the bacterial cell, causing rupture of cell membranes and hence confirm the cytotoxic and antimicrobial property of R. communis.
Biotechnology & Biotechnological Equipment, 2019
Ricinus communis is a traditional medicinal plant which has been utilized for centuries for treat... more Ricinus communis is a traditional medicinal plant which has been utilized for centuries for treatment of various conditions. Due to the presence of diverse phytochemicals, Ricinus is an outstanding natural resource to discover new drugs for various diseases such as diabetes, cancer, arthritis, ulcer and asthma. In this study, we performed whole-genome gene expression profiling using RNA-Seq to determine the differentially expressed genes in a mammalian cell line after exposure to Ricinus leaf extract and elucidate their pharmacological effects in order to support its ethnomedicinal uses. Various genes involved in cancer, inflammation, atherosclerosis and diabetes were found to be differentially regulated after exposure to sub-lethal concentrations of the Ricinus extract in MCF7 cells. An important gene involved in cancer progression and metastasis, that is, PIK3R3 (Phosphatidylinositol 3-kinase regulatory subunit gamma), was downregulated in MCF7 cells after treatment with Ricinus extract. PIK3R3 is an important component of the PI3K/AKT signalling pathway which is essential for cell proliferation, angiogenesis, inhibition of apoptosis and metastasis to distant organs. The Ricinus extract downregulated the expression of DPP4 (Dipeptidyl peptidase-4) and upregulated the expression of PPAR-c (Peroxisome proliferator-activated receptor gamma) which are crucial in controlling blood glucose levels. Expression of TNFAIP6 (Tumor necrosis factor-inducible gene 6), which is shown to mediate anti-inflammatory and protective effects, was increased after treatment with Ricinus extract. We also analyzed the genes which might also confer toxicity. Our gene expression profiling data corroborate the potential therapeutic benefits of Ricinus communis plant.
Cancer Research, 2017
Rhazya stricta is a unique medicinal plant source for many indole alkaloids, non- alkaloids, flav... more Rhazya stricta is a unique medicinal plant source for many indole alkaloids, non- alkaloids, flavonoids, triterpenes and other unknown molecules with tremendous potential for therapeutic applications against many diseases. In the present article, we generated computational data on predictive properties and activity across two key therapeutic areas of cancer and obesity, and corresponding cheminformatics studies were carried out to examine drugable properties of these alkaloids. Computed physiochemical properties of the 78 indole alkaloids from Rhazya stricta plant using industry-standard scientific molecular modeling software and their predictive anti-cancer activities from reliable web-source technologies indicate their plausible therapeutic applications. Their predictive ADME properties are further indicative of their drug-like-ness. We believe that the top-ranked molecules with anti-cancer activity are clearly amenable to chemical modifications for creating potent, safe and effic...
Cancer Research, 2017
Despite extremely high morbidity and mortality of >90% from metastasis, it remains one of the ... more Despite extremely high morbidity and mortality of >90% from metastasis, it remains one of the most challenging process for targeted therapeutic interventions. Identification and characterization of chemically active and bioavailable compounds from medicinal plants targeting the cell-signaling pathways of Cancer Stem Cells (CSCs), tumor cells and their microenvironment that abet metastasis is of great clinical significance. Hence, MCF-7; a breast cancer cell line, was treated with fractionated alkaloidal extracts from Rhazya stricta with previously documented anti-cancer activity in lung and pancreatic cancer cell lines. RNA-seq. analysis was performed for samples treated with compounds that actively inhibited cell migration in scratch assay to further substantiate their anti-metastatic role at the cellular and molecular level. Genomic data corroborated the presence of anti-metastatic compounds that actively down-regulated genes involved in promoting metastasis (CDC6), cell migrat...
Chemistry Central Journal, 2017
Rhazya stricta is a unique medicinal plant source for many indole alkaloids, non-alkaloids, flavo... more Rhazya stricta is a unique medicinal plant source for many indole alkaloids, non-alkaloids, flavonoids, triterpenes and other unknown molecules with tremendous potential for therapeutic applications against many diseases. In the present article, we generated computational data on predictive properties and activity across two key therapeutic areas of cancer and obesity, and corresponding cheminformatics studies were carried out to examine druggable properties of these alkaloids. Computed physiochemical properties of the 78 indole alkaloids from R. stricta plant using industrystandard scientific molecular modeling software and their predictive anti-cancer activities from reliable web-source technologies indicate their plausible therapeutic applications. Their predictive ADME properties are further indicative of their drug-like-ness. We believe that the top-ranked molecules with anti-cancer activity are clearly amenable to chemical modifications for creating potent, safe and efficacious compounds with the feasibility of generating new chemical entities after pre-clinical and clinical studies.
BioMed Research International, 2016
Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from develop... more Increase in the incidence of Insulin Dependent Diabetes Mellitus (IDDM) among people from developed and developing countries has created a large global market for insulin. Moreover, exploration of new methods for insulin delivery including oral or inhalation route which require very high doses would further increase the demand of cost-effective recombinant insulin. Various bacterial and yeast strains have been optimized to overproduce important biopharmaceuticals. One of the approaches we have taken is the production of recombinant human insulin along with C-peptide in yeastPichia pastoris. We procured a cDNA clone of insulin from Origene Inc., USA. Insulin cDNA was PCR amplified and cloned into yeast vector pPICZ-α. Cloned insulin cDNA was confirmed by restriction analysis and DNA sequencing. pPICZ-α-insulin clone was transformed intoPichia pastoris SuperMan5strain. Several Zeocin resistant clones were obtained and integration of insulin cDNA inPichiagenome was confirmed by PCR usi...
Biological Systems: Open Access, 2015
Medicinal plants assumed principle role in folkloric medicine throughout history. They have been ... more Medicinal plants assumed principle role in folkloric medicine throughout history. They have been the subject of many recent studies for the evaluation of what have been ascribed to them of medicinal properties by means of modern techniques. For example Rhazya stricta decne of the Apocynaceae family is a widely distributed plant in Saudi Arabia. Extract of its leaves is prescribed in folkloric medicine for the treatment of various disorders such as diabetes, sore throat, helminthiasis, inflammatory conditions and rheumatism. The extract contains mainly alkaloids, glycosides, flavonoids, tannins and triterpenes. Several studies on rats and mice reported that the leaves extract causes sedation, analgesia, decreases motor activity and has anti-depressant, anti-oxidant activity, complex effect on brain endogenous monoamine oxidase activity and central-mediated hypotension. Moreover, some studies ascribed anticancer activities to indole alkaloids of Rhazya stricta. The genotoxicity of Rhazya stricta leaves was demonstrated by Baeshen and colleagues in a battery of tests. We also demonstrated various therapeutic properties of Rhazya stricta for the treatment of cancer, insulin insensitivity, MDRs (multi-drug resistant organisms), cardiovascular diseases, obesity and some other ailments. We are currently having collaboration with some international institutes in the United States and Europe for extensive studies on the whole genome of Rhazya stricta which may lead to future Natural Products Genomics and PDT (Phytodynamic Therapy).
Cytogenetics and cell genetics
The Journal of Toxicological Sciences, 2008
Drug-induced hepatotoxicity is one of the most common adverse events associated with predict the ... more Drug-induced hepatotoxicity is one of the most common adverse events associated with predict the safety of a new molecule. To examine genes involved in hepatotoxicity, we have used oligo-ed and 248 downregulated) within the liver of APAP treated mice as compared to control. In comparison to AMAP treated mice, 62 genes were upregulated and 70 genes were downregulated in mice liver after ciated with stress response, cell cycle, growth inhibition, cell death, structural components, cell signal-the molecular basis of drug-induced hepatotoxicity that will lead to rational development of safer drugs, particularly in pre-clinical stages.
Infectious Disorders - Drug Targets, 2013
Gene, 1999
A mouse acetyl-CoA transporter (Acatn) cDNA was isolated by PCR cloning. Mouse Acatn exhibited 92... more A mouse acetyl-CoA transporter (Acatn) cDNA was isolated by PCR cloning. Mouse Acatn exhibited 92% homology with human sequence on the basis of amino-acid sequence. The predicted gene product of Acatn is a 61 kDa hydrophobic protein with six to 10 transmembrane domains. Transfection of mouse Acatn cDNA into HeLa/GT3+ cells resulted in significant increase in the amount of 9-O-acetylated gangliosides, suggesting that Acatn does play an important role in the acetylation of gangliosides. Northern blot analysis of Acatn mRNA suggested that transcript of Acatn is widely distributed in various adult tissues. Expression of Acatn was found to be developmentally regulated, with high expression levels during early embryonic stages, and then there was a subsequent decrease in expression levels in the later embryonic stages.
FEBS Letters, 2000
The acetyl-CoA transporter gene (Acatn) encodes a hydrophobic, multitransmembrane protein that is... more The acetyl-CoA transporter gene (Acatn) encodes a hydrophobic, multitransmembrane protein that is involved in the process of O-acetylation of sialic acid residues on gangliosides. O-Acetylated gangliosides have been found to play important roles in tissue development and organization during early embryonic stages. We have cloned the gene for mouse acetyl-CoA transporter. The gene spans approximately 20 kb and is composed of seven exons and six introns. A single transcription initiation site, 371 bp upstream of the ATG start codon, was identified. The promoter region was found to lack a TATA box. However, several potential transcription factor binding motifs such as AP1, AP2, C/EBPK K, C/EBPL L, HSF, GATA2 and MZF1 were identified in the promoter region.
British Journal of Pharmacology, 2009
Background and purpose: Muraglitazar, a dual PPARa/g agonist, caused a robust increase in body we... more Background and purpose: Muraglitazar, a dual PPARa/g agonist, caused a robust increase in body weight in db/db mice. The purpose of the study was to see if this increase in weight was due to oedema and/or adipogenesis. Experimental approach: The affinity of muraglitazar at PPARa/g receptors was characterized using transactivation assays. Preadipocyte differentiation, expression of genes for adipogenesis (aP2), fatty acid oxidation (ACO) and sodium reabsorption (ENaCg and Na þ , K þ-ATPase); haemodilution parameters and serum electrolytes were measured to delineate the role of muraglitazar in causing weight gain vis a vis rosiglitazone. Key Results: Treatment with muraglitazar (10 mg kg À1) for 14 days significantly reduced plasma glucose and triglycerides. Reduction in plasma glucose was significantly greater than after similar treatment with rosiglitazone (10 mg kg À1). A marked increase in weight was also observed with muraglitazar that was significantly greater than with rosiglitazone. Muraglitazar increased aP2 mRNA and caused adipocyte differentiation in 3T3-L1 cells similar to rosiglitazone. It also caused a marked increase in ACO mRNA in the liver of the treated mice. Expression of mRNA for ENaCg and Na þ , K þ-ATPase in kidneys was upregulated after either treatment. Increased serum electrolytes and decreased RBC count, haemoglobin and haematocrit were observed with both muraglitazar and rosiglitazone. Conclusions and implications: Although muraglitazar has a better glucose lowering profile, it also has a greater potential for weight gain than rosiglitazone. In conclusion, muraglitazar causes both robust adipogenesis and oedema in a 14-day treatment of db/db mice as observed in humans.
Biotechnology Letters, 2006
cDNAs encoding for five mAChR subtypes (M1-M5) were cloned under different promoters in various e... more cDNAs encoding for five mAChR subtypes (M1-M5) were cloned under different promoters in various eukaryotic vectors and each subtype was expressed in different mammalian cell lines. CHO-K1 cell line was the best for generating stable cell lines expressing muscarinic receptors. Immunofluorescence and flow cytometry revealed that expression of M1-M5 was primarily localized on the cell membrane. Western blotting and radio-ligand binding studies revealed that expression of each receptor was stable at higher passages.
Biotechnology Letters, 2006
The cDNAs encoding for three subtypes of adrenergic receptors, alpha1A-, alpha1B- and alpha1D-ARs... more The cDNAs encoding for three subtypes of adrenergic receptors, alpha1A-, alpha1B- and alpha1D-ARs, were cloned and expressed in HEK 293 cells. Expression of alpha1A- and alpha1B-AR subtypes in HEK 293 cells was stable even with increased passages but that of alpha1D-AR was not. Cellular localization studies using immunofluorescence and flow cytometry revealed that expression of alpha1A- and alpha1B-ARs was primarily localized on the cell membrane whereas expression of alpha1D-AR was predominantly intracellular. Our studies clearly demonstrated that the culturing of the recombinant cell lines expressing alpha1D-AR in charcoal/dextran treated fetal bovine serum (FBS) resulted in targeting of alpha1D-AR to the cell membrane and thus, significantly improving its stability and availability for ligand binding studies.
Biotechnology and Applied Biochemistry, 2008
cDNA encoding PDE10A (phosphodiesterase 10A) was cloned and a stable recombinant HEK-293 (human e... more cDNA encoding PDE10A (phosphodiesterase 10A) was cloned and a stable recombinant HEK-293 (human embryonic kidney-293) cell line expressing high levels of PDE10A was generated. Transient transfection of pCRE-Luc plasmid, harbouring the luciferase reporter gene under the control of CRE (cAMP-response element)-binding sequence, into the stable recombinant cell line, followed by treatment with PDE10 inhibitor, resulted in a dose-dependent increase in luciferase activity. This method provides a simple and sensitive cell-based assay for screening of PDE10 inhibitors for development of novel therapeutics for the treatment of neurological disorders.
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Papers by Roop Singh Bora