Biotechnology

Permanent URI for this collectionhttps://hdl.handle.net/20.500.14540/123

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An in-silico drug forage against trmd gene of salmonella typhimurium (LT2)
(2022) Nepal, Bisheshta; Pramod Aryal
Salmonella, that is multidrug resistant (MDR) poses a serious threat to people. An essential enzyme needed for cell growth and survival, TrmD is an S-adenosyl methionine (AdoMet or SAM)dependent methyl transferase that creates themethylationm1G37 in tRNA pro. It is distinct from its eukaryotic and archeal cousin Trm5. In-vitro tests of the effectiveness of Actinomycetes against Salmonella Typhimurium, homology modeling of the target protein, molecular docking, predictions of the ADME/T properties of particular antibacterial molecules, research into the druglikeness of particular ligands, and computational quantum mechanical modeling of particular hits are all objectives of this study. In order to comprehend the inhibitory mechanism and the spatial orientation of the ligands, as well as to identify important amino acid residues within the substrate-binding pocket that can be used for structure-based drug design, the study offers an insight into biomolecular interactions. The target protein (modeled using MODELLER) of Salmonella Typhimurium (LT2) was docked with a variety of natural products, kinase inhibitors, nucleoside mimics, and indole derivatives. After the compounds were narrowed, the compounds that were able to be effective against target protein and bind with the amino acid L138 in the active site were found to be one Natural Products; Antineoplaston, five kinase inhibitors 2-(4Fluorophenyl)-N-[2-(1H-indol-3-yl)ethyl] acetamide; N-benzyl-2-(2-methyl-1H-indol-3-yl)acetamide; 1-(1,3-Dimethylpyrazol-4-yl)-3-[(2R)-1-hydroxy-3-phenylpropan-2-yl]urea; ; 1-[(2S)-1Hydroxy-3-phenylpropan-2-yl]-3-(1-pyridin-4-ylethyl)urea;1-(1,5-Dimethylpyrazol-3-yl)-3-[(2S)1-hydroxy-3-phenylpropan-2-yl]urea three indole derivative (N-[(3,4-dimethoxyphenyl)methyl]3-(1H-indol-6-yl)propenamide;N-[2-(6-methoxy-1H-indol-3-yl)ethyl]-3-phenylpropanamide and2-(4-fluorophenyl)-N-[2-(5-methoxy-1H-indol-3-yl)ethyl]acetamide. Similarly, two nucleosidemimetics 7-[(3R)-1-Benzoylpyrrolidin-3-yl]-N-methylthieno[2,3-b] pyrazine-6-carboxamide and[3-(2-Methylpropyl)-1,2-oxazol-5-yl]-[(3S)-3-(1H-pyrazolo[3,4-b] pyridin-6-yl) piperidin-1-yl]methanone were found to be effective. Natural Product Antineoplaston's DFT calculation revealed that the substance was a stable and reactive molecule with drug-like properties. Keywords: ADMET, trmD, homology modelling, drug discovery, molecular docking, therapeutic alternative.
Therapeutic options of covid-19 management: computational approach targeting main protease (Mpro)
(2022) Subedi, Samiran; Pramod Aryal
Main protease (Mpro) enzyme of SARS-CoV-2 is involved in the digestion of viral polyproteins and considered as an attractive drug target for antiviral drug design. This study aims to carry out the molecular docking, density functional theory (DFT) studies, prediction of ADMET properties of selected potential antiviral molecules, study the efficacy of selected hits on predicted mutated Main protease (Mpro) enzyme, and the drug-drug interaction of potential hits. The study provides an insight into biomolecular interactions to understand the inhibitory mechanism and the spatial orientation of the ligands and further, identification of key amino acid residues within the substratebinding pocket that can be applied for structure-based drug design.Molecular docking of FDA approved drugs (1167) screened through stringent parameters and our formula for preference index identified tadalafil as competitive inhibitor of proteolytic function of main protease.In the docking studies, tadalafil exhibited superiority in binding with the crystal structure of Mproover the other selected molecules in this study, suggestedby the crucial roles of CYS145, HIS163, and GLU166 in the interaction within the activesite of COVID-19 Mpro. Further, the binding affinity in the predicted mutated structuresof main protease was also found to be higher than wild type suggesting its broad use. The thermodynamic properties and molecular orbital properties investigated for this compound followed the required drug like property of a probable drug. The original library of FDA compounds was docked with human cytochrome P450 3A4 and the inhibitory activity of drospirenone suggested its impeding caliber in clearance of tadalafil. Overall, the present in silico study indicated the direction to combat COVID19 using FDA-approved drugs as therapeutic alternative for viral inhibition or symptom management, which do not need much toxicity studies and could also serve as starting points for lead optimization in drug discovery. Keywords: ADMET, COVID-19, drug discovery, Mpro, molecular docking, therapeutic alternative
Trap1: mitochondeial homolog of cancer chaperone Hsp90 in regulating mitochondrial fession
(2016) Shrestha, Mitesh; Tilak R. Shrestha
Cancer is a polygenic disease and emerges from the deregulation of both genetic and epigenetic mechanisms. Two hallmarks of cancer, deregulating cellular energetics and evading apoptosis can be directly associated with mitochondria since mitochondria remain the hot spot for both. Mitochondria exist in a dynamic state between 'fusion' and 'fission' and these states regulate several cellular functions including ATP production. High molecular weight heat shock protein, Hsp90 is identified as a 'cancer chaperone' due to its involvement in regulating the functions of several cancer related signal transduction molecules. However, its mitochondrial homologue, TRAP1 does not show cancer associated molecular interactions like Hsp90, but its enhanced expression in cancer cells did correlate with disease progression. To understand TRAP1 involvement in regulating the mitochondrial dynamics between fusion and fission, we examined TRAP1 over expression in comparison with knockdown. We found that TRAP1 over expression does not kill tumor cells, but induces mitochondrial fission-like structural reorganization. To assess whether or not these structural alterations are comparable with fission, we cloned and over expressed fission genes, Fis1, Drp1, and Mff in normal (SRA01 and HEK293T) and metastatic cancer cells (IMR-32) and examined for cell and mitochondrial morphology. TRAP1 over expression alone showed enhanced fission compared to individual fission protein expression suggesting that one or all of the fission proteins requires TRAP1 for their regulation. Tumor cells showed more sensitivity to TRAP1 over expression compared to normal cells indicating tumor selective functions. Preliminary studies with protein-protein interaction suggested that TRAP1 interacts with these proteins however subsequent experiments need to be performed for further confirmation with respect to type of fission protein and mapping of the interacting region. Keywords: Cancer, Mitochondrial dynamics, Heat shock proteins, Hsp90, TRAP1, Mitochondrial fission proteins
Molecular analysis of B-lactamase gene in multidrug resistant clinical isolates of pseudomonas aeruginosa
(2017) Sthapit, Krishna; Rajani Malla
The multi-drug resistant Pseudomonas aeruginosa which is one of the most prevalent opportunistic nosocomial pathogen is on the rise. Its major defense against β-lactam antibiotics is production of metallo-β-lactamases (MBLs) which degrade this group of antibiotics including carbapenems. Carbapenems are the drugs of choice for the treatment of P. aeruginosa infection but carbapenemases (MBLs) have emerged and have spread from this bacterium to Enterobacteriaceae. The finding of carbapenem resistance is a menacing development that challenges this “last resort antibiotic” and organisms harboring the enzyme may lead to therapeutic dead ends. The aim of the study is to determine the β-lactamase gene responsible for causing antimicrobial resistance and its prevalence in clinical isolates of P. aeruginosa in the context of Nepal. The clinical isolates of P. aeruginosa were collected from different hospitals of Kathmandu valley. The isolates were subjected to the biochemical test for confirmation and then to the antibiotic susceptibility test. ESBL production was tested by double disk synergy test. For MBL production, the carbapenem resistant isolates were screened out and then tested for the presence of MBL enzyme and its detection was done by EDTA combined disk test. Molecular identification of the responsible gene blaIMP, blaVIM and blaNDM causing MDR in those MBL producers was done by Polymerase Chain Reaction (PCR) using gene specific primers. Sequencing was then carried out. Of total 67 consecutive isolates of P. aeruginosa, 52 (77.61%) were MDR P. aeruginosa, none of them were ESBL producers and 30 (75%) of total 40 carbapenem resistant isolates showed positive result for MBL phenotypically. Additionally, the results of PCR method showed that 15 strains (37.5%) of carbapenem resistant isolates contained blaNDM (New Delhi Metallo-β-lactamase) gene. No other gene was found in the examined samples. Further sequence analysis showed the presence of blaNDM-1 in majority of sequenced isolates and 3 novel variants of this gene was also detected. The prevalence of the MBLs has been increasing worldwide, particularly among P. aeruginosa, leading to severe limitations in the therapeutic options. Thus, antimicrobial stewardship should be implemented to minimize the emergence of this β-lactamase producing pathogens. Molecular surveillance on a regular basis and proper resistance screening measures needs to be adopted to prevent spreading of these superbugs. Keywords: blaNDM, Carbapenem, Metallo-β-lactamases, Pseudomonas aeruginosa, Sequencing.
Genetic structure of sub-ethic groups of newar population of Kathmandu valley
(2014) Awasthi, Nagendra prasad; Tilak R. Shrestha
Diversity in geographical region, physical environment, flora, and fauna including humans in Nepal has offered suitable platforms to study their origin and genetic relationship. There are very few studies on the populations of Nepal. To understand their genetic structure and affinities, for the first time, we studied Shakya (N = 19), Bajracharya (N = 20) and Udaaya (N=59) population of Newar ethnic group using high resolution Y Chromosomal, Mitochondrial and Autosomal DNA markers. About 10 ml of blood samples were collected from 98 healthy and unrelated individuals, inhabiting Kathmandu Valley with their informed written consent. DNA was extracted from the whole blood with the standard phenol-chloroform method and target regions were amplified using Polymerase chain reaction (PCR). Y-chromosomal Biallelic markers were used to dissect the paternal lineages and whole mitochondrial genome was sequenced and the variations were scored against the revised Cambridge Reference Sequence (rCRS) to trace the maternal lineages. Based on the variations, putative Haplogroups were assigned and were confirmed by coding region markers. Our mtDNA result indicates the dominance of Macrohaplogroup M (90%: includes M2, M3, M5, M9, M30, M33, M35, M38, Z, D, etc.) along with the branches of R (R6, R9) and U (U2 and U7) Haplogroups. Their maternal Genepool was found to harbour around 50% of South Asian, 25% East/Southeast Asian and 20% Central Asian specific Genepool. West Eurasian haplogroup were negligible (5%). Y-chromosome analysis revealed the presence of major haplogroups such as M117-O3a3, M17-R1a, M82-H1, M15-D1 and M124-R2. Overall, South Asian and East/Southeast Asian signature were found prominent among the Newars. The analyses of Autosomal markers (MYBPC3, LCT, SLC24A, EDAR, etc.) showed that the genetic structure of Newar population is more or less similar to the Indian Tibeto-Burman populations. In conclusion, the Newar population of Nepal was found to harbour prominent Genepool from South Asia, East/ Southeast Asia. Further, analysis on Y-STRs and high density Autosomal markers will help in tracing the precise origin and affinity of the Newar population of Nepal. Key Words: Biallelic marker, Haplogroup, Genepool, Autosomal Marker, Y-STR, rCRS
DNA Barcoding and Phylogenetic Analysis of Fishes of Pokhara Valley
(2014) Subedi, Kalpana; Tilak R. Shrestha
Despite extensive taxonomic studies, identification of fishes can be problematic often even to the experts due to various reasons. In this context, DNA barcoding can be a promising tool for species identification and biodiversity surveys through the use of short, standardized gene targets, ~652 bp of mitochondrial DNA. This tool can be more broadly applied if a comprehensive reference sequence library for all fish species can be constructed. Here, we make a small contribution to this grand challenge by barcoding some freshwater fishes from Pokhara. The standard barcode fragment of COI was used to barcode 14 individuals, representing 14 taxonomically recognized species in 13 genera, 7 families and 5 orders. A 99% sequence similarity threshold was employed as a matching criterion for specimen identification to the species level. After editing all obtained sequences using Codon Code Aligner 4.0 program, specimens and sequence data were archived and investigated using analytical tools available on BOLD and MEGA. The GC content was 44.97% on average. Mean genetic distance between families was 18.7%. The synonymous changes were much greater than the non-synonymous changes, especially in the 3rd codon position where variation is dominated. There were 174 conserved, 43 variable, 14 parsimony-informative and 29 singleton amino acid sites; while 367 conserved, 285 variable and 218 parsimony-informative sites were present out of 652 bp nucleotides. The NJ, ML and MP analysis indicated different clades corresponding to the recognized groupings; members of same families clustered together. Molecular species identification was in concordance with current taxonomical classification in all cases achieving success rate of ~94%. In addition to DNA barcodes, our study also provides supporting data in the form of specimen images, morphological characters, taxonomic bibliography, preserved vouchers and COI sequences. This work highlights the functional utility of barcodes for the discrimination of diverse ichthyofauna. We infer that DNA barcoding can be a valuable tool to increase accuracy, objectivity and comparability of taxonomic assessment in biodiversity studies. Finally, our study constituted an important contribution to the iBOL, providing barcode sequences for use in identification of the species by experts and non-experts, and allowing them to be available for use in other applications. Further research is needed to verify the deeper divergence within species and genera with larger sample size. Keywords: Mitochondria, Cytochrome oxidase, Cytochrome oxidase subunit I (COI) gene, Taxonomy, DNA sequencing, Species identification, GenBank, BOLD.
Preliminary study on the role of n-terminal regulatory domain of syntaxin 4 in mast cell exocytosis
(2014) Adhakari, Pratikshya; Rajani Malla
Not available
Immunohistochemistry for MHL1and MSH2: a methodfor identifying mismatch repairgenein Nepalese colorectal cancer patients
(2014) Bhattarai, Matrika; ilfalil Bin Alwi
Lynch syndrome (LS)(previously known asHereditary nonpolyposis colorectal cancer(HNPCC)syndrome)iscaused bymutations inmismatch repair (MMR) genes.LSis anautosomal dominant condition accounting for 2–5% of all colorectal carcinomas.Mismatch repair genes contain mainly four genes they are MLH1, MSH2, PMS2 andMSH6. Colorectal cancers with DNA mismatch repair gene mutationscharacteristically display a high rate of replication errors in simple repetitivesequences detectable as microsatellite instability (MSI). Monoclonal antibodiesagainst hMLH1and hMSH2are commercially available, those two DNA mismatchrepair proteins accounts for mostLScancers. This study pursued to investigate thepotential utility of these antibodies in determining the expression status of theseproteins in paraffin-embedded formalin-fixed tissue and to identify key technicalprotocol components associated with successful staining.Colorectalcancersample of 43 patients of Nepal wereexaminedand immunohistochemistrywas used todetermine which tumours lacked expressionMLH1 and MSH2 gene.Out of 43patient 18.6%(8 of 43)of the sampleshowed abnormal staining patternfor hMLH1antibodyand 11.6%(5 of 43)of sample showedabnormal staining patternfor hMSH2antibodyonly 9.30% (4 of 43 ) sampleshowedabnormal staining pattern for bothantibody.The key protocol point associated withsuccessful staining was an antigenretrieval step involving heat treatmentwith Tris-EDTAheated at 121 C for 2 minutes.Tumours with mismatch repair defect were frequently foundatthe age less than 50years (p<0.05) than tumours with no mismatch repair defect. This studydemonstrates the potential utility of immunohistochemistry in detecting LSprobands and identifies key technical components for successful staining.Immunohistochemistry can bedone in local laboratories to findmismatch repairproteinsdefect on theselected cases before referring for the expensive moleculartest. Keywords:Lynch syndrome,Hereditarynonpolyposis colorectal cancer,mismatchrepair defect, colorectal cancer, paraffin-embedded formalin-fixed tissue,Monoclonal antibodies,Antigen 0
Study the modulation of share machinery in macrophage in response to LPS
(2014) Bhatta, Tarka Raj; Rajani Malla
Human body is regularly under attack by various pathogens as it provides optimal environment for growth of pathogens. To defend against these pathogens and their toxic compounds, our immune system has developed an intricate method of recognition and responses. Recognition of antigens by T-cells requires uptake, processing and presentation of pathogen-derived antigens on MHC of APCs. While the pathways of antigen processing and presentation in APCs have been defined, not much is known about the molecular mechanisms and regulation of intracellular traffic involved in antigen processing and presentation pathways in response to pathogens. SNAREs are the proteins that play crucial role in vesicle trafficking in cells and therefore in all immune processes involving exocytosis of inflammatory mediators and uptake and killing of pathogens, also in transport, expression and downregulation of various receptors. So the main aim of our research was to study the modulation of expression of SNARE proteins against LPS challenges. MH-S cell line was choosen as the model for our study as it is a macrophage cell line which is capable of phagocytois and antigen presentation of various pathogens as well as exocytosis of various cytokines produced against the pathogens. We standardize RT-PCR, real-time PCR in term of various parameters and using those standardized condition the modulation of the SNARE proteins was studied in presence of LPS. In order to study the modulation of expression of SNAREs at protein level, western blotting was done. We found that expression of the SNAREs proteins studied was not modulated by LPS treatment pointing to some other mode of regulation of SNARE mediated intracellular traffic in response to LPS challenge in macrophages. Key words: LPS, SNAREs, RT-PCR, Real-time PCR, Western blotting, MH-S cell lines
Phytochemical analysis of some medicinal plants indigenous to Nepal and study of antioxidant, antimicrobial and cytotoxicity Phytochemical analysis of some medicinal plants indigenous to Nepal and study of antioxidant, antimicrobial and cytotoxicity Phytochemical analysis of some medicinal plants indigenous to Nepal and study of antioxidant, antimicrobial and cytotoxicity effect of their extracts on various cell lines.M. Sc. Thesis effect of their extracts on various cell lines
(2015) Thapa, Krishna; Rajani Malla
Plants and plant-based products are the bases of many of the modern pharmaceuticals aswell as traditional health care systemwe use today for various ailments especially thecountry like Nepal. Keeping therefore in view, the growing interest of the use of herbaldrugs, the present study was undertaken with a view to evaluate thephytochemical andpharmacological activities of thestem ofRheumaustraleandTinospora cordifoliaand fruitpart ofDatura stramonium. In this research plants were subjected to both chemical and biological assays. The resultsofphytochemical screening showed presence of all the necessary primary and secondarymetabolites except tannins and resins. The total flavonoid content in crudemethanolicextract of T.cordifolia was found to be highest i.e.9.350±0.286 mg QE/g. Similarly thehighest phenol content was also foundabout 39.2±2.173mg/g in methanolic extract ofT.cordifolia.The free radical-scavenging activity of the extracts was tested through DPPH-method and the results were compared with Ascorbic acid. During our experiment, it wasnoted that the IC value of the standard antioxidant Ascorbic acid was found to be41.69±2.309and the extracts exhibited a concentration-dependent antiradical activity byinhibiting DPPH- radical. Of the different extracts, crudemethanol extract of Tinosporacordifoliaexhibited the highest free radical scavenging activity of 87.5% followed by themethanolic extract ofDatura stramonium(85.39%) andRheumaustrale(80.67%). Whilethehexane and the chloroformcrude extractexhibited moderate level free radical scavengingactivity.CrudeMethanolic and chloroformextractof all the three plant demonstrated thesignificant activity onKlebsiella pneumoniaandStaphylococcus aureusrespectively as wellas on Saccharomyces cerevisae. In our investigation, the extracts that were obtaineddemonstrated very nominal cytotoxic effect on the macrophage cells at a givenconcentration of 0-300µg/ml using MTT assay signifying that plant extract render no risk tothe normal cell lines but provide platform to study cytotoxicity againstHelacell lines.Thecrude extract of T.cordifolia exhibited the pronounced effect on Hela cells in dosedependent manner especially the methanolic extract. Methanolic extract of T. cordifoliademonstrated the significant cytotoxicity (89.79±2.908) and lowest ICLower the IC 50 5o 50value(50.754±5.776).value higher the cytotoxicity effect.These findings suggest that medicinalplants taken under studyshowantibacterial and cytotoxic activity and show promisingfuture for further researches. Key words: Medicinal plants, Phytochemical screening, Antioxidant, DPPH, Flavonoid,Phenol,Cytotoxicity.
Isolation and molecular characterization of bacillus thuringiensis from different habitats of Nepal and potato tuber moth (phthorimaea operculella)
(2015) Maharjan, Ashok; Jayashree Sijapati
Potato tuber moth (PTM), Phythorimae operculella (Zeller) is a pest of many solanaceous crops including potatoes. It is one of the major constraints to potato production worldwide. Farmers rely extensively on broad spectrum chemical pesticides to check the pests but this has serious side effects on human health, ecosystem and the beneficial insects. Therefore, for sustainable agriculture, the use of biopesticides is very critical. Biopesticides based on Bacillus thuringiensis (Bt) have been very popular and successful. Bt is a sporulating, Gram – positive facultative aerobic bacterium. Its principal characteristic is the synthesis of a crystalline inclusion containing the protein known as cry protein during sporulation. These proteins have insecticidal properties. This research was focused on the isolation and molecular characterization of Bt from wide range of habitat of Nepal. A total of 28 different samples were collected from Sauraha/Chitwan, Tulsipur/Dang, lalitpur, Beni/Myagdi, Ghasa/Myagdi, Bhairahawa/Rupandehi and Bandipur/Tanahun. Sodium acetate and quick isolation methods were used to isolate Bt. Gram staining was done to select Gram – positive bacteria. Bt was confirmed by Commassie Brilliant Blue (CBB) staining which stains the crystal into purple blue. A total of nine Bt strains were isolated. Most of them were bipyramidal. The types of cry gene of the isolates were determined by PCR using universal primers specific to cry1 and cry2 genes. Five of the isolated strains were positive for cry1 gene only and three strains were positive for both cry1 and cry2 genes. Cry1 subgrouping reveals the presence of cry1Aa gene in five strains, cry1Ab gene in seven strains and cry1Ac gene in six strains and absence of cry1B, cry1C and cry1D genes in all the isolated and reference strains. The bioassay of isolated strains against PTM showed their effectiveness. The lowest LDvalue was 6.67±3.024 µg/ml crude protein for strain d1 and highest LD 5050value was 36.71±5.68 µg/ml crude protein for reference strain B. thuringiensis kurstaki. These isolated strains showed high promise for biopesticides production. Key words: Bt, biopesticides, crystal proteins, PTM, bioassay.
Mutation Analysis and confirmation of beta thalassemia in Nepalese population
(2014) Lama, Raju; Tilak R. Shrestha
Beta (β) thalassemia is genetic disorder which passes from parents to their offspring in an autosomal recessive inheritance pattern. This disease is directly related to the haemoglobin chemical anatomy and functioning. This genetic disease leads to defective beta globin haemoglobin chain which means partial or complete loss of beta globin chain synthesis. This chemistry in RBCs make them vulnerable to lysis of cells themselves prematurely resulting in anaemia. Beta-thalassemia hence requires continuous blood transfusion along with other care to maintain the normal homeostasis of RBCs and other systems of body. This management procedure is costly, sensitive and tedious and became serious health problem in developing nation like Nepal. 107 subjects were selected of which 61 were already clinically distinguished cases and remaining were one of their immediate family members as carrier unaffected according to clinical data. These two groups were carefully recruited and evaluated by means of multiplex ARMS PCR. The multiplex ARMS PCR results were validated by direct sequencing. The group of 21 major mutations were investigated using allelespecific primerscategorised in 6 different panels in this study but only 9 mutations were revealed during study. Unaffected family member were analysed to find the link among them. The most common mutations were found as IVS 1-5(G-C) and Cd 26(G-A) with 23% followed by 619 deletion (20%), Cd 8/9(+G) 12%, Cd 16 (-C) 8%, Cd 41/42(-TTCT) 6%, IVS 1-1 (G-T) 4%, Cd 19 (A-G) 3% and Cd 17(A-T) 1% respectively. Heterozygous and homozygous mutation types were analysed using internal controls. The result of this study reveals that the mutational profile of Nepal resembles with two neighbouring countries China, India and other South Asian countries. Previously it was assumed that thalassemia is common in Terai region only but our study reflects its distribution all over Nepal in most of the ethnic groups. This technique has proved its value in β thalassemia studies and has been used widely for the analysis of β thalassemia worldwide. It is found reliable, simple, quicker and affordable. It is recommended to participate in the thalassemia screening programmes before marriage in endemic areas. And for the married couples to take prenatal diagnosis of foetus in high risk populations to predict and prevent or stop the frequency of the new patients in early future. Keywords: (β) thalassemia, autosomal recessive, Multiplex ARMS PCR, Sequencing, high risk population, prenatal diagnosis, Nepalese population.
Variation in biological actvities of methanolic extract of Nepalese swertia chirayita population
(2014) Bhattarai, Namita; DeepakRaj Pant
Swertia chirayitais a medicinal plant indigenous to temperate Himalaya. Itis known tocontain manybioactive compounds having pharmacological activities.An assessment ofphenolic content, flavonoid content, antioxidant property,and antimicrobialproperty ofmethanolic extracts from different populations of S. chirayitawas done in the presentwork. Furthremore, semiquantitative estimation of three marker chemicals namelyAmarogentin,SwertiamarinandMangiferinwas also done. Preliminary phytochemicalscreening showed the presence of saponin, alkaloid, phenols, flavonoids, glycoside,tannin, terpenoid and phytosterol in methanolic extracts from all populations.Evaluation of antimicrobial acitivity of methanolic extracts of different populatios of S.chirayita showed the antimicrobial activity against Escherichia coli, Salmonella typhii,Klebsiellapneumoniae, StaphylococcusaureusandEnterococcusfaecalis but no activityagainst Pseudomonas aeuregenosa.Similarly, antioxidant activity was shown by theextracts of all populations of chirayita. However the extracts from the populationscollected from Makawanpur showed the lowest IC50 value 42.07±3.39. Thin layerchromatography (TLC) showed the presence of all three marker compound in allsamples. Semiquantitative estimation of three marker compounds was done usingGelQuant.Net software. HighestAmarogentincontent (0.299 mg/gm DW) was found inwild populations from Lalitpur (Phulchowki)and lowest Amarogentincontent in wildpopulations from Jumla (0.005 mg/gm DW).The content of Swertiamarin was highest(0.15mg/gmDW) in wild populations of Lalitpur (Phulchowki)and lowest (0.017) incultivated populations from Ilam.Similarly, highest Mangiferin content (8.83 mg/gmDW) was found in wild populations from Tehrathum and the lowest content (0.23mg/gm DW)in wild samples from Lamjung. Key words:medicinal plant, Swertia chirayita, Methanolic extracts, TLC, antioxidantactivity, antimicrobial activityandmarker chemical compound.
Enhancement of xylose transport in saccharomyces cerevisiae by transformation of GXF1, a xylose transporter gene, from candida intermedia
(2014) Regmi, Priti; Tribikram Bhattarai
Rising of energy costs and the increased awareness of global warming have inspired production of renewable, biomass-derived chemicals and fuels. Plant biomass is potentially an inexhaustible source of bioenergy. To produce the fuel for the motor vehichles, industrial bioethanol production from cheap renewable lignocellulosic substrates has been regarded as an important attempt. However, efficient production of bioethanol is attributed to the ability of microbial cell to utilize the abundant glucose and pentose sugar present in lignocellulosic biomass. Because of the lack of xylose transporter in wild S. cerevisiae, they are unable to utilize xylose sugar which is the second most abundant sugar in lignocellulosic biomass. Several successful researches have been performed in Sachharomyces cerevisiae to induce efficient xylose transport, and xylose fermentation. One of the limiting steps in xylose fermentation is the xylose transportation step. Different xylose transporter encoding gene are naturally occurring in several species, the integration of those xylose transporting genes in Sachharomyces cerevisiae would led to the transportation of xylose inside the cells. GXF1 is the glucose/xylose faciliator occurring in fungus Candida intermedia which transports the xylose by facilitated diffusion. The yeast cloned with GXF1 xylose transporter, in the present study, has been shown for improved xylose growth kinetics (specific growth rate, µ = 0.02 hr -1 and xylose transporting phenotypes compared to the control cells. However, very less amount of ethanol production 0.09mg/ml is found which might be due to lack of xylose metabolizing pathways in wild yeasts. The aim of this resarch is the heterologous expression of GXF1 in Sachharomyces cerevisiae for efficient xylose transport inside the cell for the production of bioethanol. Good xylose transport kinetics has been shown by the expression of GXF1, however, ethanol production is quite impossible by just expression of transporter gene, as yeast lacks xylose metabolizing pathways too. Hence further improvement of the strain with the xylose metabolizing pathways is believed to uplift the production of bioethanol from cheap lignocellulosic substrates. Keywords: Transformation, Sachharomyces cerevisiae, Candida intermedia, GXF1, xylose transporter, Bioethanol.
Nano-biochemical Approach for ‘One Pot’ Reduction of Atmospheric N 2 and CO 2 for Bio-fuel and Bio-fertilizer
(2018) Adhikari, Saraswoti; Pramod Aryal
Taming the global warming and air pollution is one of the major issues till date and alternative reduced nitrogen carbon sources for developing bio-fertilizer and bio-fuel, respectively, is highly sought. Carbon dioxide reduction without mimicking the plant photosynthesis, serving the reactions in dark would be of love as it can overcome the burden of exposing light sources in vessel. Bacteria were isolated from Nepalease prehistoric nature soils (rhizosphere of tree fern) that included extracellular electron transfer bacteria Geobacter sps, diazotrophic Azotobacter sps, Pseudomonas sps and diazotrophic autotroph Azospirillium sps on the basis of Carbon Catabolite Repression (CCR). Syntrophic growth of microbial consortium was developed for reducing nitrogen (Nessler’s test for ammonium) and carbon (Molisch’s test). The reduced carbon was quantified by DNS method. Ethanol was detected (Dichromate oxidation method) by fermenting with commercially available yeast. The residue after ethanol production can be used as Nitrogen fertilizer since these diazotrophs can fix atmospheric N 2 upon application in soil. In addition, the CO2 reduction potential will increase soil carbon content supporting the growth of other microbes in amending soil. Biogenic silica nanoparticles also synthesized from rice husk char for augmenting reduction rate in syntrophic growth. Synthesized silica nanoparticles were characterized by XRD, FTIR and SEM. Since silica is the most abundant minerals, functionalization of Si with ammonium group as a nitrogenous fertilizer to substitute urea was done. The developed technology could be further developed to mitigate global warming through nitrogen fixation to substitute urea and non-photosynthetic CO2 reduction for bio-fuel and others. Keywords: Bacterial consortium, Syntrophic growth, CO2 reduction, Nitrogen fixation, Electrotrophs and Electricity, Biogenic Silica nanoparticle, Bio-ethanol, Bio-fertilizer.
Nitrogen fixing biofertilizer for carbon storage
(2018) Tamang, Anju; Pramod Aryal
Nitrogen is primarily required by plants for their growth and dinitrogen as it exists has very stable triple bond which requires relatively high amount of energy to cleave making it unusable by plants. Therefore, despite its availability in large excess; are still unavailable for the crops and has to undergo biological nitrogen fixation so as to be absorbed by them. To meet the nutritional demand of plants nitrogen (N) is employed in the form of fertilizers in agricultural field. Long-term use of synthetic chemical fertilizers has resulted in soil acidification, poor soil aggregate stability and low levels of micronutrients. Further, it aggravates the environmental pollution through denitrification (N2 and N2 O) and volatilization (NH3 ) potentially contributing in global warming, stratospheric ozone layer depletion and acid deposition. Hence, alternative for these are sought in the form of biofertilizer that minimizes the potential hazardous effects to environment. Further, steep increase in production and utilization of polymers has resulted in significant burden on solid waste management followed by rapid depletion of fossil fuels used in production of such polymers. Moreover, synthetic biodegradable plastics are actually not truly biodegradable as they are labelled imposing harsh impacts on ecological degradation. Therefore, green alternative for these are bioplastics and biocomposites which when discarded in soil and landfills are usually acted upon by microbes such as bacteria, fungi, algae generating CO2 , CH4 , cellular components and other products. PHB in particular is polymer of industrial importance and has found to contain properties like biodegradability, thermoplasticity along with other traits similar to petrochemical plastics of recent usage. The preliminary identification of isolates via biochemical tests were followed by molecular characterization that involved PCR amplification, gel electrophoresis and DNA sequence analysis. When subjected to PCR amplification, the universal primer used yielded an amplicon of the expected size ~1500 bp. The sample A17 showed 98% similarity with the gene cluster sequence of Azotobacter species available from GenBank database while P8 depicted 99% concurrence with Pseudomonas species. The sequence obtained by DNA sequencing was further characterized by multiple sequencing alignment. The clustering approach called neighbor joining (NJ) method was used for the reconstruction of phylogenetic trees that yielded unrooted tree. The isolates of A. vinelandii and P. fluorescens were presented as nitrogen fixing species spectrophotometrically and as PHB producer using Flow cytometry that utilizes FlowJo software to quantify the PHB. The amount of fixed nitrogen was found to be as high as 21.8395 gm/L for A17 while the value of 1.8199 gm/L was achieved for P8 for 144 hours of incubation. PHB was quantified under different condition and maximum production was recorded at nitrogen limiting condition. The bacterial PHB content can directly be correlated with median fluorescence intensity expressed in AFU, Arbitrary Fluorescence Units. For P8 maximum value of 688.7505 AFU has been achieved with median 57.4 where 99.9% of the population produced PHB. Similarly for A17 maximum value of 545.1923 AFU of PHB was obtained with the median value of 30.0 where 93.6% of the total population were actively producing PHB. The isolates which possess the capability to fix the nitrogen along with its ability to store the carbon source as PHB was obtained. The amount of fixed nitrogen and PHB produced by the isolates established them as potent candidate for both biofertlizer as well as biopolymer producer. Keywords: Biofertilizer, biopolymer, nitrogen fixation, PHB, spectrophotometry, Flow cytometry
Trinucleotide repeat length distribution and mitochondrial DNA haplogroup in sub-ethnic group of newar population of Nepal
(2017) K.C., Medha; Tilak R. Shrestha
Tandem nucleotide repeats are repetitive DNA in which two or more contiguous, approximate copies of a pattern of nucleotide occur in a DNA sequence. Trinucleotide repeats are a form of tandem repeat which are caused by an expansion of a segment of DNA that contains a repeat of 3 nucleotides (Triplet repeat). Variable number of triplet repeats are constituted in a healthy individual but there is a threshold beyond which a high number of repeats causes disease. This threshold varies in different disorders. Trinucleotide repeat expansions are highly polymorphic and sometimes called dynamic or unstable mutation because the number of repeats increases as the gene passes from parents to offspring. Expansion of disease arises from existence of large normal of normal repetitions (Large Normal alleles). Frequency of large normal alleles’ estimation is the indirect measure of the prevalence of the disease. To investigate the normal allele range of trinucleotide repeat disorder (SCA1, SCA2, SCA3, SCA7, SCA8, SCA12, DM1, DRPLA, FXTAS and HD) blood samples from 55 healthy unrelated individual belonging to Newar sub‐ethnic group, the Maharjan of Nepal were examined. PCR products were subjected to capillary electrophoresis (ABI 3130xl Genetic Analyser) for fragment analysis. In the studied population the normal range of 10 different loci were SCA1, 22 to 36 (CAG)n ; SCA2, 19 to 26 (CAG)n; SCA3, 14 to 36 (CAG)n ; SCA7, 6 to 14 (CAG)n; SCA8, 11 to 32 (CTG)n; SCA12, 9 to 18 (CAG)n; HD, 12 to 28 (CAG)n; FXTAS , 18 to 34 (CGG)n; DRPLA, 7 to 22 (CAG)n; DM1, 1 to 28 (CTG)n . Variability of human mitochondrial DNA has provided valuable data about the genetic history of human. Analysis of the frequency, variation and distribution of mitochondrial DNA haplogroup have been used to evaluate genetic structure of various population residing in different geographical region. Second part of this dissertation concentrates on the mitochondrial DNA haplogroup determination of Newar sub‐ethnic group, the Maharjan of Nepal. Mitochondrial D‐loop is the non‐coding region which has two hypervariable regions that exhibit very high mutation rates, thus, can distinguish recently diverged population. In this study, D‐loop region was sequenced and Individual lineages were constructed on the basis of mtDNA mutations. 5 major haplogroups with different frequencies were observed in mtDNA haplogroup viz. M (58.2%), N (18.2%), H (3.6%), R (12.7%), U (7.3%). Our results indicated presence of South Asian specific haplogroup M, including M3, M5, Z, G etc. in high frequency along with branches R (R9), U (U7, U8). These results indicated Maharjan gene pool was found to harbour almost 57.56% of South Asian, 19.99% of East Asian, 18.85% of Western Eurasian and 3.6% of Central Asia specific gene pool. Key words: Trinucleotide repeat disorder, Haplogroup, Gene pool, D‐Loop
Screening of Novel Genes Involvedin Biofilm Mediated resistance to Antibiotics in MDR Pseudomonas aeruginasa
(2022) Manandhar, Padam Ratna; Suresh Subedi
The role of biofilm in the pathogenesis of some chronic human infection is now widely accepted. Pseudomonas aeruginosa is a model organism for biofilm study, which seems to have complex regulatory circuit that controls biofilm formation. Biofilm forming physiology of P. aeruginosa should have some genetic implication. In this study, the potential of biofilm formation in P. aeruginosa, was assessed and susceptibility to selected carbapenem antibiotic was determined. The potential regulatory gene was identified using homology searches and used in the study for determining the role of this gene in biofilm formation using PCR amplification and cloning. Assessment of biofilm formation by 8 confirmed Pseudomonas isolates were done using normal polystyrene microtitre plate (non-tissue coated), using crystal violet staining and read at 551nm. The AST was carried following Clinical and Laboratory Standard Institute protocol in Muller Hilton Agar plate using 10 mcg disc of imipenem and meropenem antibiotics. BLASTp program identified PA1961 gene as putative regulatory gene of the family LysR- type transcriptional regulator in P. aeruginosa for which primer was designed using PrimerBLAST tool of NCBI. PCR was used to determine whether presence or absence of the gene was related to biofilm formation physiology. Out of 8 isolates, two different isolates, 1 isolate showed consistent biofilm formation using cut-off (Mean OD of negative control + 3X SD of negative control) and 1 isolate showed antibiotic resistance to both IPM and MRP, making 12.5%(12.5% biofilm former and 12.5% antibiotic resistance), which is considerably low as compared to previous studies. Next we aimed to draw if there is any relationship of biofilm formation with antibiotic resistance. We identified PA1961 gene using homology search and expected its presence only in biofilm forming carbapenem resistant isolates. In contrary the gene was found to be present in all isolates, including the positive control (PAO1) and the result was inconclusive in drawing direct relationship of biofilm formation with antibiotic resistance in Pseudomonas. We could have done protein analysis for the same gene to be sure of this genes role in biofilm formation. But since it was a surface protein, protein quantification would not provide be done. Further study with the increase in number of fresh isolates, stringent control (inclusion of negative control strain) and robust bioinformatics analysis will provide conclusive result for the identification of novel genes involved in biofilm mediated antibiotic resistance in MDR pathogens. Keywords: Biofilm, Antibiotic resistance, Carbapenem, P. aeruginosa, LysR-type transcriptional regulator, Antibiotic susceptibility test.
Utilization of endophytic bacteria as a biological control agent of fungal pathogens and its plant growth promoting activities solanum lycopersicum
(2018) Adhikari, Manju; Rajani Malla
Bacterial endophytes are a class of microorganisms that are found in the inner part of plant, especially in the plant tissues without showing any detrimental effects on the plant growth and development. They are distributed widely and are found to be aiding plants growth and development by overcoming various biotic and abiotic stresses. The main aim of this study was to isolate endophytic bacteria as biological control agents against fungal pathogens and its utilization as plant growth promoters. For this, ten endophytic bacteria were isolated from the leaves of different tomato plants from JNU campus. Preliminary screening of all the isolates was carried out against tomato plant pathogens Alternaria alternata, ITCC 6134. The most promising strain was further subjected to molecular characterization by 16s rRNA analysis. The liquid Chromatography Mass Spectrometry technique showed the biological active compounds present in the bacterial exract of BBB3 which might have played role in inhibiting fungal growth. Different tests for enzymic activity like cellulase, phosphatase and heavy metal resistance (Mercury and Cadmium) were done. The in vivo plant growth promoting activities on tomato seedling and seeds were carried out in a greenhouse to observe the efficacy of isolated endophyte directly. The isolated bacteria coded BBB3 among ten showed the efficient fungal inhibition as it suppressed the pathogen invitro. BBB3 also showed the degradation of cellulose media, phosphate solubilization activity and resistance to a high concentration of Mercury and Cadmium. The greenhouse evaluations of the endophyte showed significant (p≤0.05) biological control efficacy, higher growth and yield components of tomato plant. Furthermore, the metabolomics profile of the active compound could be exploited for large scale production of natural and environmental friendly biological control agents for the replacement of synthetic chemical agents and directing towards sustainable agriculture. Keywords:Endophytes, Heavy metal resistance, sustainable, plant growth promoter
Development of efficient E. coli Host strain for Expression of Functionally active Human Cytochrome P450,2A6 by Endogenous increased Biosynthesis of 5- Aminole Vulinic Acid (ALA)
(2012) Mali, Sujina; Tribikram Bhattarai
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