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Mutation analysis of dystrophin gene using multiplex ligation dependent probe amplification (MLPA)
(2013) Shrestha, Kushal; Rohit Kumar
Duchenne muscular dystrophy (DMD), an allelic X-linked progressive muscle-wasting disease, and its allelic form Becker muscular dystrophy (BMD) are one of the most common single-gene disorders caused by mutations in the DMD gene (also dystrophin gene), the longest known human gene ranging 2.4 Mb, encoding a 427 kDa cytoskeletal protein called dystrophin. Due to the lack of reliable genetic diagnostic tool in Nepal the diagnosis of these genetic diseases are narrowed to phenotypic and clinical diagnoses. Besides, the lack of even the base line data of these genetic diseases has barred the progression of the further research of these genetic diseases in Nepal.This research has been carried out with the aim to introduce Multiplex ligation dependent probe amplification (MLPA) as one of the convenient molecular diagnostic tool in diagnosis of the genetic diseases as DMD/BMD. In this research, DNA was extracted from the blood samples of DMD/BMD patients and from the normal male and female to be taken as the reference samples. DNA samples so extracted were then amplified in the thermocycler by using the MLPA assay. The PCR products of the test samples and the reference samples so obtained were run on the capillary electrophoresis (CE) and the data were analysed. Using an algorithm of MLPA, 26 total samples were assayed. The capillary electrophoresis run (ABI-310 genetic analyzer) demonstrated that it could pick up the deletions in 14 of the 21 test samples considered. Consequently, MLPA was efficient in accurately confirming mutations in about 67% of all cases. Most prevalent exonic deletion regions were found to be confined in the exon 7-14, the proximal zone and 45-53, the first half of C-terminal domain. The reading frame (in-frame or out-frame) were determined by using the “DMD exonic deletions/duplications reading frame checker 1.9” as recommended by MRCHolland, which need to be confirmed by sequencing.No novel mutations were identified in this study. Overall, this approach confirmed mutations in 67% of the patients in our study which is compatible with the recent studies in Chinese and Indian population. Among the 21 test samples used MLPA could not diagnose the mutation in some of the samples which were clinically diagnosed as DMD/BMD. This result aware us that in order to know the exact point of mutation and to know exactly which of the exon is deleted or duplicated further sequencing should be done. But still the efficiency of this MLPA assay makes it a rapid, robust, efficient and reliable genetic tool in the diagnosis of genetic disorders. The systematic approach/algorithm used in this study offers the best possible less invasive and effective mutation analysis in the context of Nepal. Key words: Dystrophin gene, Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), Multiplex Ligation Dependent Probe Amplification (MLPA), capillary electrophoresis (CE) 1
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In Silico Drug Repurposing against Salmonella Typhimurium LT 2 Dam Protein
(2023) Maharjan, Suja; Pramod Aryal
The increasing prevalence of Multidrug-Resistant (MDR) pathogens has resulted in the failure of current antibiotics to effectively treat these infections. ComputerAided Drug Discovery CADD) has become a crucial tool in the drug discovery process recently. It has been demonstrated to be a successful method for screening lead compounds against target proteins within a short amount of time and with optimal resources. In the present study, a computational approach, CADD tools were employed to identify novel drug candidates against Salmonella enterica serovar Typhimurium LT2, targeting its essential gene, Dam. Virtual screening of various ligand libraries was conducted. From the initial library consisting of 21,000 compounds from natural products, 10,342 compounds from indole derivatives, 1,685 compounds from Kinase Inbibitors and 3,118 compounds from Nucleoside mimetics after ADME/Tox and druglikeness filters were narrowed down the number of compounds to 205 Natural Products, 462 Indole Derivatives, 6449 Kinase Inhibitors, and 654 Nucleoside Mimetics. The final screening from molecular docking and binding energy resulted in the identification of four lead compounds, Antineo plaston A10 and Cardamonin from natural products, 5-cyclopentaneamido-1-ethyl-N-(2 meth oxyethyl)-1H-indole-2-carboxamide from Indole Derivatives, 2[[anilino (oxo)methyl]amino]-4,5-dimeth oxybenzoic acid from Kinase Inhibitors and 3-[[[4-[2-(3,5-Dimethylpyrazol-1-yl)ethoxy]phenyl]methylamino]methyl]-1-(6methylpyrimidin-4-yl)pyrrolidin-3-ol from Nucleoside Mimetics were identified as potential leads. These compounds showed higher binding affinity with the target protein and lower binding efficiency for human hMAT1A protein compared to the reference compound S-Adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH). The stability and strength of protein-ligand binding were observed through protein-ligand interactions, Density Functional Theory (DFT), analysis of frontier molecular orbitals and vibrational spectra. The results suggest that these compounds may be potential candidates for further exploration against other MDR pathogens prioritized by the World Health Organization (WHO). Keywords: CADD, Multidrug-Resistant, Dam, essential gene, lead compounds
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Physicochemical and Microbial Profiling of Kefir from Cow and Buffalo Milk; Implications for Probiotic Use
(2024) Soni, Sushmita; Jarina Joshi
Kefir, a traditional fermented dairy product, is valued for its unique taste, thickness, and health benefits attributed to its rich microbial diversity. This study aimed to investigate the processing, physiochemical properties, and probiotic profile of kefir, particularly focusing on its therapeutic potential. The introduction highlights kefir's historical and contemporary significance, including its increasing popularity as a functional food. The problem statement addresses the rising prevalence of digestive issues and the need for effective dietary interventions like kefir. Kefir, with its high probiotic content, offers potential health benefits, including improved gastrointestinal health and immune function. The specific objectives include producing kefir using traditional fermentation methods, identifying the microbial strains present, and characterizing their probiotic properties. The study utilized traditional kefir fermentation methods with kefir grains. Physiochemical analyses of kefir and metagenomic profiling of lactic acid bacteria and yeasts from kefir were conducted. This study investigated the isolation, identification, and probiotic characterization of lactic acid bacteria (LAB) and yeast from kefir, identifying strains such as Lactobacillus helveticus, Lactobacillus rhamnosus, Lactobacillus fermentum, Kazachstania martinaie, and Pichia chibdodasensis. The isolates exhibited distinct morphological, biochemical, and genetic characteristics, confirmed through PCR amplification of the 16S rRNA and 18S rRNA genes. Among the LAB, Lactobacillus helveticus showed the highest acid tolerance at lower pH levels (2.5 and 3), while L. rhamnosus performed best at mildly acidic to neutral conditions (pH 5.6). L. helveticus also demonstrated superior bile salt tolerance, autoaggregation (65%), coaggregation (30%), and hydrophobicity (40%). All strains tolerated lower salt concentrations (2% and 5%) well, with L. rhamnosus showing the highest overall salt tolerance. L. fermentum exhibited the highest phenol tolerance, while L. rhamnosus and L. fermentum formed biofilms, unlike L. helveticus. Lastly, L. helveticus displayed the highest bile salt hydrolase activity (+++), followed by moderate activity in L. rhamnosus and L. fermentum (++). L. helveticus showed superior antibiotic susceptibility, and notable antimicrobial activity, particularly against E. coli and Staphylococcus aureus. In contrast, L. rhamnosus exhibited effectiveness against Pseudomonas aeruginosa. These findings indicate L. helveticus as a promising probiotic candidate, suggesting its application in enhancing gastrointestinal health. Buffalo milk kefir differs notably from cow dairy milk kefir in pH, acidity, Degree Brix, and HPLC-measured concentrations of organic acids and sugars. Buffalo milk kefir shows higher pH (4.7 ± 0.082), lower acidity (1.37 ± 0.024%), and higher lactose content (32.92 ± 0.088 mg/ml) compared to cow dairy milk kefir, which has lower pH (4.5 ± 0.082), higher acidity (1.96 ± 0.033%), and lower lactose content (18.02 ± 0.061 mg/ml). Significant yeast growth, essential for ethanol production, was observed in buffalo milk kefir, contributing to its distinctive flavor and aroma. The findings confirmed kefir's rich probiotic content and beneficial physiochemical properties. These findings support the broader use of kefir in dietary interventions aimed at improving overall health. Further research should focus on standardizing kefir production methods and conducting clinical trials to substantiate its health benefits. Additionally, promoting kefir consumption could help address common digestive health issues, especially in regions with limited access to healthcare. Keywords : Kefir, fermented dairy product, probiotics, therapeutic potential, digestive health, lactic acid bacteria (LAB), yeasts, antimicrobial activity, functional food, dietary interventions, HPLC, lactic acid.