Search results for: agarose stamps

Authors: Aliasghar Golmohammadian, Sona Rostampour Yasouri

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One of the most important intestinal pathogenic strains is E. coli O₁₅₇:H₇. This pathogenic bacterium is transmitted to humans through water and food. E. coli O₁₅₇:H₇ is the main cause of Hemorrhagic colitis (HC), Hemolytic Uremic Syndrome (HUS), Thrombotic Thrombocytopenic Purpura (TTP) and in some cases death. Since E. coli O₁₅₇:H₇ can be transmitted through the consumption of different foods, including vegetables, agricultural products, and fresh dairy products, this study aims to identify and isolate E. coli O₁₅₇:H₇ from wastewater by PCR technique. One hundred twenty samples of water and wastewater were collected by Falcom Sterile from Shahrood and Neka cities. The samples were checked for colony formation after appropriate centrifugation and cultivation in the specific medium of Sorbitol MacConkey Agar (SMAC) and other diagnostic media of E. coli O₁₅₇:H₇. Also, the plates were observed macroscopically and microscopically. Then, the necessary phenotypic tests were performed on the colonies, and finally, after DNA extraction, the PCR technique was performed with specific primers related to rfbE and stx2 genes. The number of 5 samples (6%) out of all the samples examined were determined positive by PCR technique with observing the bands related to the mentioned genes on the agarose gel electrophoresis. PCR is a fast and accurate method to identify the bacteria E. coli O₁₅₇:H₇. Considering that E. coli bacteria is a resistant bacteria and survives in water and food for weeks and months, the PCR technique can provide the possibility of quick detection of contaminated water. Moreover, it helps people in the community control and prevent the transfer of bacteria to healthy and underground water and agricultural and even dairy products.

Keywords: E. coli O₁₅₇:H₇, PCR, water, wastewater

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Authors: Emil F. Khisamutdinov

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Development of functional materials undergoing structural transformations in response to an external stimulus such as environmental changes (pH, temperature, etc.), the presence of particular proteins, or short oligonucleotides are of great interest for a variety of applications ranging from medicine to electronics. The dynamic operations of most nucleic acid (NA) devices, including circuits, nano-machines, and biosensors, rely on networks of NA strand displacement processes in which an external or stimulus strand displaces a target strand from a DNA or RNA duplex. The rate of strand displacement can be greatly increased by the use of “toeholds,” single-stranded regions of the target complex to which the invading strand can bind to initiate the reaction, forming additional base pairs that provide a thermodynamic driving force for transformation. Herein, we developed a highly robust nanoparticle shape transition, sequentially transforming DNA polygons from one shape to another using the toehold-mediated DNA strand displacement technique. The shape transformation was confirmed by agarose gel electrophoresis and atomic force microscopy. Furthermore, we demonstrate that our approach is applicable for RNA shape transformation from triangle to square, which can be detected by fluorescence emission from malachite green binding RNA aptamer. Using gel-shift and fluorescence assays, we demonstrated efficient transformation occurs at isothermal conditions (37°C) that can be implemented within living cells as reporter molecules. This work is intended to provide a simple, cost-effective, and straightforward model for the development of biosensors and regulatory devices in nucleic acid nanotechnology.

Keywords: RNA nanotechnology, bionanotechnology, toehold mediated DNA switch, RNA split fluorogenic aptamers

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Authors: Hanen Bouaziz-Ketata, Ghada Ben Salah, Hichem Ben Salah, Kamel Jamoussi, Najiba Zeghal

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The present study investigated the protective role of Hyparrhenia hirta on nitrate-induced liver damage. Experiments were carried out on adult rats divided into 3 groups, a control group and two treated groups. NaNO3 was administered daily by oral gavage at a dose of 400 mg/kg bw in treated groups either alone or coadministered with Hyparrhenia hirta methanolic extract via drinking water at a dose of 200 mg/kg bw for 50 days. Liver toxicity induced by NaNO3 was characterized by higher serum levels of glucose, total cholesterol and triglyceride and lower serum total protein than those of controls. Transaminases and lactate deshydrogenase activities in serum were elevated indicating hepatic cells’ damage after treatment with NaNO3. The hyperbilirubinemia and the increased serum gamma glutamyl transferase activities suggested the presence of cholestasis in NaNO3 exposed rats. In parallel, NaNO3 caused oxidant/antioxidant imbalance in the liver as reflected by the increased lipid peroxidation, the decreased total glutathione content and superoxide dismutase, catalase and glutathione peroxidase activities. Nitrate caused also a significant induction of DNA fragmentation as evidenced by the presence of a smear without ladder formation on agarose gel. Hyparrhenia hirta supplementation showed an improvement of all parameters cited above. We conclude that the present work provides ethnopharmacological relevance of Hyparrhenia hirta against the toxic effect of nitrate, suggesting its role as a potential antioxidant.

Keywords: Hyparrhenia hirta, liver, nitrate toxicity, oxidative stress, rat

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Authors: Chamilani Nikapitiya, Mahanama De Zoysa, Jehee Lee

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Most of the bacterial diseases are associated with high mortalities in aquaculture species and causing huge economic losses. Different approaches have been taken to prevent or control of bacterial diseases including use of vaccines, probiotics, chemotherapy, water quality management, etc. Antibiotics are widely applying as chemotherapy to control bacterial diseases, however, it has been shown that frequent use of antibiotics is favored to develop multi-drug resistance bacteria. Therefore, phages and phage encoded lytic proteins are known to be one of the most promising alternatives for antibiotics to avoid the emergence of antibiotic-resistant bacteria. We isolated and characterized the two lytic phages, namely pAh-1 and pAs-1 against pathogenic Aeromonas hydrophila and Aeromonas salmonicida, respectively. Morphological characteristics were analyzed by Transmission electron microscopy (TEM) and host strain specificities were tested with Aeromonas and other closely related bacterial strains. TEM analysis revealed that both pAh-1 and pAsm-1 are composed of an icosahedral head and a segmented tail, and we suggest that, they are new members of Myoviridae family. Genome sizes of isolated phages were estimated by restriction enzyme digestion of genomic DNA using selected endonucleases followed by agarose gel electrophoresis. Estimated genome size of pAh-1 and pAs-1 were approximately 64 Kbp and 120 Kbp, respectively. Both pAh-1 and pAs-1 have shown narrow host specificity. Moreover, protective effects of phage therapy against fish pathogenic A. hydrophila were investigated in zebrafish model. The survival rate was 40% higher when zebrafish received intra-peritoneal injection (i.p.) of pAh-1 were simultaneously challenge A. hydrophila (2 x 106 CFU/fish) compared to that without phage treatment. Overall results suggest that both pAh-1 and pAs-1 can be used as a potential phage therapy to control Aeromonas infections in aquaculture.

Keywords: Aeromonas infections, antibiotic resistance, bacteriophage, bio-control, lytic phage

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Authors: Leda Maria Costa Pereira, Maria Denise Lopes, Nucharin Songsasen

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Mammalian ovaries contain thousands of follicles that eventually degenerate or die after culture in vitro. Caspase-3 is a key enzyme that regulating cell death. Our objective was to examine the influence of anti-apoptotic drug Z-VAD-FMK (pan-caspase inhibitor) on in vitro viability of dog follicles within the ovarian cortex. Ovaries were obtained from prepubertal (age, 2.5–6 months) and adult (age, 8 months to 2 years) bitches and ovarian cortical fragments were recovered. The cortices were then incubated on 1.5% (w/v) agarose gel blocks within a 24-wells culture plate (three cortical pieces/well) containing Minimum Essential Medium Eagle - Alpha Modification (Alpha MEM) supplemented with 4.2 µg/ml insulin, 3.8 µg/ml transferrin, 5 ng/ml selenium, 2 mM L-glutamine, 100 µg/mL of penicillin G sodium, 100 µg/mL of streptomycin sulfate, 0.05 mM ascorbic acid, 10 ng/mL of FSH and 0.1% (w/v) polyvinyl alcohol in humidified atmosphere of 5% CO2 and 5% O2. The cortices were divided in six treatment groups: 1) 10 ng/mL EGF (EGF V0); 2) 10 ng/mL of EGF plus 1 mM Z-VAD-FMK (EGF V1); 3) 10 ng/mL of EGF and 10 mM Z-VAD-FMK (EGF V10); 4) 1 mM Z-VAD-FMK; 5) 10 mM Z-VAD-FMK and (6) no EGF and Z-VAD-FMK supplementation. Ovarian follicles within the tissues were processed for histology and assessed for follicle density, viability (based on morphology) and diameter immediately after collection (Control) or after 3 or 7 days of in vitro incubation. Comparison among fresh and culture treatment group was performed using ANOVA test. There were no differences (P > 0.05) in follicle density and viability among different culture treatments. However, there were differences in this parameter between culture days. Specifically, culturing tissue for 7 days resulted in significant reduction in follicle viability and density, regardless of treatments. We found a difference in size between culture days when these follicles were cultured using 10 mM Z-VAD-FMK or 10 ng/mL EGF (EGF V0). In sum, the finding demonstrated that Z-VAD-FMK at the dosage used in the present study does not provide the protective effect to ovarian tissue during in vitro culture. Future studies should explore different Z-VAD-FMK dosages or other anti-apoptotic agent, such as surviving in protecting ovarian follicles against cell death.

Keywords: anti apoptotic drug, bitches, follicles, Z-VAD-FMK

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Authors: Daina Jonkus, Solvita Petrovska, Dace Smiltina, Lasma Cielava

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The beta-lactoglobulin and kappa-casein are milk proteins which are important for milk composition. Cows with beta-lactoglobulin and kappa-casein gene BB genotypes have highest milk crude protein and fat content. The aim of the study was to determinate the frequencies of milk protein gene polymorphisms in local Latvian Brown (LB) cows breed and analyze the influence of beta-lactoglobulin and kappa-casein genotypes to milk productivity traits. 102 cows’ genotypes of milk protein genes were detected using Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP) and electrophoresis on 3% agarose gel. For beta-lactoglobulin were observed 2 types of alleles A and B and for kappa-casein 3 types: A, B and E. Highest frequency in beta-lactoglobulin gene was observed for B allele – 0.926. Molecular analysis of beta-lactoglobulin gene shows 86.3% of individuals are homozygous by B allele and animals are with genotypes BB and 12.7% of individuals are heterozygous with genotypes AB. The highest milk yield 4711.7 kg was for 1st lactation cows with AB genotypes, whereas the highest milk protein content (3.35%) and fat content (4.46 %) was for BB genotypes. Analysis of the kappa-casein locus showed a prevalence of the A allele – 0.750. The genetic variant of B was characterized by a low frequency – 0.240. Moreover, the frequency of E occurred in the LB cows’ population with very low frequency – 0.010. 54.9 % of cows are homozygous with genotypes AA, and only 4.9 % are homozygous with genotypes BB. 32.8 % of individuals are heterozygous with genotypes AB, and 2.0 % are with AE. The highest milk productivity was for 1st lactation cows with AB genotypes: milk yield 4620.3 kg, milk protein content 3.39% and fat content 4.53 %. According to the results, in local Latvian brown there are only 2.9% of cows are with BB-BB genotypes, which is related to milk coagulation ability and affected cheese production yield. Acknowledgment: the investigation is supported by VPP 2014-2017 AgroBioRes Project No. 3 LIVESTOCK.

Keywords: beta-lactoglobulin, cows, genotype frequencies, kappa-casein

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Authors: Abu Salim Mustafa

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Polymerase chain reaction (PCR) assays targeting genomic DNA segments have been established for the detection of Helicobacter pylori in clinical specimens. However, the data on comparative evaluations of various targets in detection of H. pylori are limited. Furthermore, the frequencies of vacA (s1 and s2) and cagA genotypes, which are suggested to be involved in the pathogenesis of H. pylori in other parts of the world, are not well studied in Kuwait. The aim of this study was to evaluate PCR assays for the detection and genotyping of H. pylori by targeting the amplification of DNA targets from four genomic segments. The genomic DNA were isolated from 72 clinical isolates of H. pylori and tested in PCR with four pairs of oligonucleotides primers, i.e. ECH-U/ECH-L, ET-5U/ET-5L, CagAF/CagAR and Vac1F/Vac1XR, which were expected to amplify targets of various sizes (471 bp, 230 bp, 183 bp and 176/203 bp, respectively) from the genomic DNA of H. pylori. The PCR-amplified DNA were analyzed by agarose gel electrophoresis. PCR products of expected size were obtained with all primer pairs by using genomic DNA isolated from H. pylori. DNA dilution experiments showed that the most sensitive PCR target was 471 bp DNA amplified by the primers ECH-U/ECH-L, followed by the targets of Vac1F/Vac1XR (176 bp/203 DNA), CagAF/CagAR (183 bp DNA) and ET-5U/ET-5L (230 bp DNA). However, when tested with undiluted genomic DNA isolated from single colonies of all isolates, the Vac1F/Vac1XR target provided the maximum positive results (71/72 (99% positives)), followed by ECH-U/ECH-L (69/72 (93% positives)), ET-5U/ET-5L (51/72 (71% positives)) and CagAF/CagAR (26/72 (46% positives)). The results of genotyping experiments showed that vacA s1 (46% positive) and vacA s2 (54% positive) genotypes were almost equally associated with VaCA+/CagA- isolates (P > 0.05), but with VacA+/CagA+ isolates, S1 genotype (92% positive) was more frequently detected than S2 genotype (8% positive) (P< 0.0001). In conclusion, among the primer pairs tested, Vac1F/Vac1XR provided the best results for detection of H. pylori. The genotyping experiments showed that vacA s1 and vacA s2 genotypes were almost equally associated with vaCA⁺/cagA^- isolates, but vacA s1 genotype had a significantly increased association with vacA⁺/cagA⁺ isolates.

Keywords: H. pylori, PCR, detection, genotyping

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Authors: AbdelRahman A. Faragalla, Mohamed H. Alqhtani, Mohamed M. M.Ahmed

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Saudi Arabia has currently been beset by a barrage of bizarre assemblages of subterranean termite fauna, inflicting heavy catastrophic havocs on human valued properties in various homes, storage facilities, warehouses, agricultural and horticultural crops including okra, sweet pepper, tomatoes, sorghum, date palm trees, citruses and many forest domains and green lush desert oases. The most pressing urgent priority is to use modern technologies to alleviate the painstaking obstacle of taxonomic identification of these injurious noxious pests that might lead to effective pest control in both infested agricultural commodities and field crops. Our study has indicated the use of DNA fingerprinting technologies, in order to generate basic information of the genetic similarity between 3 predominant families containing the most destructive termite species. The methodologies included extraction and DNA isolation from members of the major families and the use of randomly selected primers and PCR amplifications with the nucleotide sequences. GC content and annealing temperatures for all primers, PCR amplifications and agarose gel electrophoresis were also conducted in addition to the scoring and analysis of Random Amplification Polymorphic DNA-PCR (RAPDs). A phylogenetic analysis for different species using statistical computer program on the basis of RAPD-DNA results, represented as a dendrogram based on the average of band sharing ratio between different species. Our study aims to shed more light on this intriguing subject, which may lead to an expedited display of the kinship and relatedness of species in an ambitious undertaking to arrive at correct taxonomic classification of termite species, discover sibling species, so that a logistic rational pest management strategy could be delineated.

Keywords: DNA fingerprinting, Western Saudi Arabia, DNA primers, RAPD

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Authors: Neslihan Taskale Karatug, Mustafa Akcelik

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Some literature data show that misL protein play a role on host immune response formed against Salmonella Typhimurium. The aim of the present study is to learn the role of the protein in S. Typhimurium pathogenicity. To describe certain functions of the protein, primarily recombinant misL protein was produced and purified. PCR was performed using a primer set targeted to passenger domain of the misL gene on S. Typhimurium LT2 genome. Amplicon and pet28a vector were enzymatically cleaved with EcoRI and NheI. The digested DNA materials were purified with High Pure PCR Product Purification Kit. The ligation reaction was achieved with the pure products. After preparation of competent Escherichia coli Dh5α, ligation mix was transformed into the cell by electroporation. To confirm the existence of insert gene, recombinant plasmid DNA of Dh5α was isolated with high pure plasmid DNA kit. Proved the correctness of recombinant plasmid was electroporated to BL21. The cell was induced by IPTG. After induction, the presence of recombinant protein was checked by SDS-PAGE. The recombinant misL protein was purified using HisPur Ni-NTA spin colon. The pure protein was shown by SDS-PAGE and western blot immünoassay. The concentration of the protein was measured BCA Protein Assay kit. In the wake of ligation with digested products (2 kb misL and 5.4 kb pet28a) visualised on gel size of the band was about 7.4 kb and was named as pNT01. The pNT01 recombinant plasmid was transformed into Dh5α and colonies were chosen in selective medium. Plasmid DNA isolation from them was carried out. PCR was achieved on the pNT01 to check misL and 2 kb band was observed on the agarose gel. After electroporation of the plasmid and induction of the cell, 68 kDa misL protein was seen. Subsequent to the purification of the protein, only a band was observed on SDS-PAGE. Association of the pure protein with anti-his antibody was verified by the western blot assay. The concentration of the pure misL protein was determined as 345 μg/mL. Production of polyclonal antibody will be achieved by using the obtained pure recombinant misL protein as next step. The role of the protein will come out on the immune system together some assays.

Keywords: cloning, Escherichia coli, recombinant protein purification, Salmonella Typhimurium

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Authors: Ella Abaev, Shany Segal, Miri Gabay

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Background: Choking is a blockage of airways that prevents efficient breathing and air flow to the lungs. Choking may be partial or full and is an emergency situation. Complete or prolonged choking leads to apnea, lack of oxygen in the tissues of the body and brain, and can cause death. There are three mechanisms of choking: obstruction of internal respiratory tracts by food or object aspiration, any material that blocks or covers external air passages, external pressure on the neck or trapping between objects. Children's airways are narrower than that of adults and therefore the risk of choking is greater, due to the aspiration of food and other foreign bodies into the lungs. In the Child Development Center at Safra Children’s Hospital, Tel Hashomer in Israel are treated infants, toddlers, and children aged 0-18 years with various developmental disabilities. Due to the increase in reports of ‘almost an event’ of choking in the past year and the serious consequences of choking event, it was decided to give an emphasis to the issue. Incidence and methods: The number of reports of ‘almost an event’ or a choking event was examined at the center during the years 2013-2018 and a thorough research work was conducted on the subject in order to build a prevention program. Findings: Between 2013 and 2018 the center reported about ten cases of ‘almost choking events’. In the middle of 2018 alone three cases of ‘almost an event’ were reported. Objective: Providing knowledge leads to awareness raise, change of perception, change in behavior and prevention. The center employs more than 130 staff members from various sectors so that it is the work of multi-professional teams to promote the quality and safety of the treatment. The familiarity of the staff with risk factors, prevention guidelines, identification of choking signs, and treatment are most important and significant in determining the outcome of a choking event. Conclusions and recommendations: After in-depth research work was carried out in cooperation with the Risk Management Unit on the subject of choking, which include a description of the definitions, mechanisms, risk factors, treatment methods and extensive recommendations for prevention (e.g. using treatment and stimulation accessories with standards association stamps and adjustment of the type of food and the way it is served to match to the child's age and the ability to swallow). The expected stages of development and emphasis on the population of children with special needs were taken into account. The research findings will be published by the staff and parents of the patients, professional publications, and lectures and there is an expectation to decrease the number of choking events in the next years.

Keywords: children with special needs, choking, educational system, prevention guidelines

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Authors: Anna Cameron, Chunxia Zhao, Haofei Wang, Yun Liu, Guang Ze Yang

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Since the first publication in 1775, cancer research has built a comprehensive understanding of how cellular components of the tumor niche promote disease development. However, only within the last decade has research begun to establish the impact of non-cellular components of the niche, particularly the extracellular matrix (ECM). The ECM, a three-dimensional scaffold that sustains the tumor microenvironment, plays a crucial role in disease progression. Cancer cells actively deregulate and remodel the ECM to establish a tumor-promoting environment. Recent work has highlighted the need to further our understanding of the complexity of this cancer-ECM relationship. In vitro models use hydrogels to mimic the ECM, as hydrogel matrices offer biological compatibility and stability needed for long term cell culture. However, natural hydrogels are being used in these models verbatim, without tuning their biophysical characteristics to achieve pathophysiological relevance, thus limiting their broad use within cancer research. The biophysical attributes of these gels dictate cancer cell proliferation, invasion, metastasis, and therapeutic response. Evaluating the three most widely used natural hydrogels, Matrigel, collagen, and agarose gel, the permeability, stiffness, and pore-size of each gel were measured and compared to the in vivo environment. The pore size of all three gels fell between 0.5-6 µm, which coincides with the 0.1-5 µm in vivo pore size found in the literature. However, the stiffness for hydrogels able to support cell culture ranged between 0.05 and 0.3 kPa, which falls outside the range of 0.3-20,000 kPa reported in the literature for an in vivo ECM. Permeability was ~100x greater than in vivo measurements, due in large part to the lack of cellular components which impede permeation. Though, these measurements prove important when assessing therapeutic particle delivery, as the ECM permeability decreased with increasing particle size, with 100 nm particles exhibiting a fifth of the permeability of 10 nm particles. This work explores ways of adjusting the biophysical characteristics of hydrogels by changing protein concentration and the trade-off, which occurs due to the interdependence of these factors. The global aim of this work is to produce a more pathophysiologically relevant model for each tumor type.

Keywords: cancer, extracellular matrix, hydrogel, microfluidic

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Authors: Swati Gautam, Amita Jain, Shyampyari Jaiswar

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Objective: To elucidate the role of (ApaI&TaqI) VDR gene polymorphism in the pathogenesis of female genital tuberculosis (FGTB) cases. Background: Female genital TB represents about 15-20% of total extra-pulmonary TB (EPTB). Female subjects with vitamin D deficiency have been shown to be at higher risk of pulmonary TB as well as FGTB. In same context few functional polymorphism in vitamin D receptor (VDR) gene has been considered as an important genetic risk factor that modulate the development of FGTB. Therefore we aimed, to elucidate the role of (ApaI&TaqI) VDR gene polymorphism in the pathogenesis of FGTB. Study design: Case-Control study. Sample size: Cases (60) and Controls (60). Study site: Department of Obstetrics & Gynecology & Department of Microbiology, K.G.M.U. Lucknow, (UP). Inclusion criteria: Cases: Women with age group 20-35 years, premenstrual endometrial aspiration collected and included in the study, those were positive with acid-fast bacilli (AFB)/ TB-PCR/ LJ culture/ liquid culture. Controls: Women with age group 20-35 years having no history of ATT and all test negative for TB recruited as control. Exclusion criteria: -Women with endometriosis, polycystic ovaries (PCOD), positive on Chlamydia & gonorrhea, already on anti-tubercular therapy (ATT) excluded. Materials and Methods: Blood samples were collected in EDTA tubes from cases and controls stored at -20ºC. Genomic DNA extraction was carried out by salting-out method. Genotyping of VDR gene (ApaI&TaqI) polymorphism was performed by using single amplification refractory mutation system (ARMS) PCR technique. PCR products were analyzed by electrophoresis on 2% agarose gel. Statistical analysis was done by SPSS16.3 software & computing odds ratio (OR) with 95% CI. Results: Increased risk of female genital tuberculosis was observed in AA genotype (OR =1.1419-6.212 95% CI, P*<0.036) and A allele (OR =1.255-3.518, 95% CI, P* < 0.006) in FGTB as compared to controls. Moreover A allele was found more frequent in FGTB patients. No significant difference was observed in TaqI gene polymorphism of VDR gene. Conclusion: The ApaI polymorphism is significantly associated with etiology of FGTB and plays an important role as a genetic risk factor in FGTB women.

Keywords: ARMS, ATT, EPTB, FGTB, VDR

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Authors: Tayyaba Bari, Muhammad Hamza Anjum, Samra Kanwal, Fakhera Ikram

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Osteoarthritis, a degenerative condition, affects more than 213 million individuals globally. Since articular cartilage has no or limited vessels, therefore, after deteriorating, it is unable to rejuvenate. Traditional approaches for cartilage repair, like autologous chondrocyte implantation, microfracture and cartilage transplantation are often associated with postoperative complications and lead to further degradation. Decellularized human umbilical cord has gained interest as a viable treatment for cartilage repair. Decellularization removes all cellular contents as well as debris, leaving a biologically active 3D network known as extracellular matrix (ECM). This matrix is biodegradable, non-immunogenic and provides a microenvironment for homeostasis, growth and repair. UC derived bioink function as 3D scaffolding material, not only mediates cell-matrix interactions but also adherence, proliferation and propagation of cells for 3D organoids. This study comprises different physical, chemical and biological approaches to optimize the decellularization of human umbilical cord (UC) tissues followed by the solubilization of these tissues to bioink formation. The decellularization process consisted of two cycles of freeze thaw where the umbilical cord at -20˚C was thawed at room temperature followed by dissection in small sections from 0.5 to 1cm. Similarly decellularization with ionic and non-ionic detergents Sodium dodecyl sulfate (SDS) and Triton-X 100 revealed that both concentrations of SDS i.e 0.1% and 1% were effective in complete removal of cells from the small UC tissues. The results of decellularization was further confirmed by running them on 1% agarose gel. Histological analysis revealed the efficacy of decellularization, which involves paraffin embedded samples of 4μm processed for Hematoxylin-eosin-safran and 4,6-diamidino-2-phenylindole (DAPI). ECM preservation was confirmed by Alcian Blue, and Masson’s trichrome staining on consecutive sections and images were obtained. Sulfated GAG’s content were determined by 1,9-dimethyl-methylene blue (DMMB) assay, similarly collagen quantification was done by hydroxy proline assay. This 3D bioengineered scaffold will provide a typical atmosphere as in the extracellular matrix of the tissue, which would be seeded with the mesenchymal cells to generate the desired 3D ink for in vitro and in vivo cartilage regeneration applications.

Keywords: umbilical cord, 3d printing, bioink, tissue engineering, cartilage regeneration

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Authors: Sontana Mimapan, Phattarawadee Wattanasuntorn, Phanom Saijit

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Aflatoxin contamination in maize, one of several agriculture crops grown for livestock feeding, is still a problem throughout the world mainly under hot and humid weather conditions like Thailand. In this study Aspergillus flavus (A. Flavus), the key fungus for aflatoxin production especially aflatoxin B1 (AFB1), isolated from naturally infected maize were identified and characterized according to colony morphology and PCR using ITS, Beta-tubulin and calmodulin genes. The strains were analysed for the presence of four aflatoxigenic biosynthesis genes in relation to their capability to produce AFB1, Ver1, Omt1, Nor1, and aflR. Aflatoxin production was then confirmed using immunoaffinity column technique. A loop-mediated isothermal amplification (LAMP) was applied as an innovative technique for rapid detection of target nucleic acid. The reaction condition was optimized at 65C for 60 min. and calcein flurescent reagent was added before amplification. The LAMP results showed clear differences between positive and negative reactions in end point analysis under daylight and UV light by the naked eye. In daylight, the samples with AFB1 producing A. Flavus genes developed a yellow to green color, but those without the genes retained the orange color. When excited with UV light, the positive samples become visible by bright green fluorescence. LAMP reactions were positive after addition of purified target DNA until dilutions of 10⁻⁶. The reaction products were then confirmed and visualized with 1% agarose gel electrophoresis. In this regards, 50 maize samples were collected from dairy farms and tested for the presence of four aflatoxigenic biosynthesis genes using LAMP technique. The results were positive in 18 samples (36%) but negative in 32 samples (64%). All of the samples were rechecked by PCR and the results were the same as LAMP, indicating 100% specificity. Additionally, when compared with the immunoaffinity column-based aflatoxin analysis, there was a significant correlation between LAMP results and aflatoxin analysis (r= 0.83, P < 0.05) which suggested that positive maize samples were likely to be a high- risk feed. In conclusion, the LAMP developed in this study can provide a simple and rapid approach for detecting AFB1 producing A. Flavus genes from maize and appeared to be a promising tool for the prediction of potential aflatoxigenic risk in livestock feedings.

Keywords: Aflatoxin B1, Aspergillus flavus genes, maize, loop-mediated isothermal amplification

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Authors: Hamzeh Alipour, Masoumeh Bagheri, Abbasali Raz, Javad Dadgar Pakdel, Kourosh Azizi, Aboozar Soltani, Mohammad Djaefar Moemenbellah-Fard

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Background: Maggot debridement therapy is an appropriate, effective, and controlled method using sterilized larvae of Luciliasericata (L.sericata) to treat wounds. Netrin-A is an enzyme in the Laminins family which secreted from salivary gland of L.sericata with a central role in neural regeneration and angiogenesis. This study aimed to production of new recombinant Netrin-A protein of Luciliasericata larvae by baculovirus expression vector system (BEVS) in SF9. Material and methods: In the first step, gene structure was subjected to the in silico studies, which were include determination of Antibacterial activity, Prion formation risk, homology modeling, Molecular docking analysis, and Optimization of recombinant protein. In the second step, the Netrin-A gene was cloned and amplified in pTG19 vector. After digestion with BamH1 and EcoR1 restriction enzymes, it was cloned in pFastBac HTA vector. It was then transformed into DH10Bac competent cells, and the recombinant Bacmid was subsequently transfected into insect Sf9 cells. The expressed recombinant Netrin-A was thus purified in the Ni-NTA agarose. This protein evaluation was done using SDS-PAGE and western blot, respectively. Finally, its concentration was calculated with the Bradford assay method. Results: The Bacmid vector structure with Netrin-A was successfully constructed and then expressed as Netrin-A protein in the Sf9 cell lane. The molecular weight of this protein was 52 kDa with 404 amino acids. In the in silico studies, fortunately, we predicted that recombinant LSNetrin-A have Antibacterial activity and without any prion formation risk.This molecule hasa high binding affinity to the Neogenin and a lower affinity to the DCC-specific receptors. Signal peptide located between amino acids 24 and 25. The concentration of Netrin-A recombinant protein was calculated to be 48.8 μg/ml. it was confirmed that the characterized gene in our previous study codes L. sericata Netrin-A enzyme. Conclusions: Successful generation of the recombinant Netrin-A, a secreted protein in L.sericata salivary glands, and because Luciliasericata larvae are used in larval therapy. Therefore, the findings of the present study could be useful to researchers in future studies on wound healing.

Keywords: blowfly, BEVS, gene, immature insect, recombinant protein, Sf9

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Authors: Masauso Moses Phiri

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Frequent glucose monitoring is essential to the management of diabetes. Plasmonic enzyme-based glucose biosensors have the advantages of greater specificity, simplicity and rapidity. The aim of this study was to develop a rapid plasmonic colorimetric glucose biosensor based on biocatalytic enlargement of AuNS guided by GOx. Gold nanoparticles of 18 nm in diameter were synthesized using the citrate method. Using these as seeds, a modified seeded method for the synthesis of monodispersed gold nanostars was followed. Both the spherical and star-shaped nanoparticles were characterized using ultra-violet visible spectroscopy, agarose gel electrophoresis, dynamic light scattering, high-resolution transmission electron microscopy and energy-dispersive X-ray spectroscopy. The feasibility of a plasmonic colorimetric assay through growth of AuNS by silver coating in the presence of hydrogen peroxide was investigated by several control and optimization experiments. Conditions for excellent sensing such as the concentration of the detection solution in the presence of 20 µL AuNS, 10 mM of 2-(N-morpholino) ethanesulfonic acid (MES), ammonia and hydrogen peroxide were optimized. Using the optimized conditions, the glucose assay was developed by adding 5mM of GOx to the solution and varying concentrations of glucose to it. Kinetic readings, as well as color changes, were observed. The results showed that the absorbance values of the AuNS were blue shifting and increasing as the concentration of glucose was elevated. Control experiments indicated no growth of AuNS in the absence of GOx, glucose or molecular O₂. Increased glucose concentration led to an enhanced growth of AuNS. The detection of glucose was also done by naked-eye. The color development was near complete in ± 10 minutes. The kinetic readings which were monitored at 450 and 560 nm showed that the assay could discriminate between different concentrations of glucose by ± 50 seconds and near complete at ± 120 seconds. A calibration curve for the qualitative measurement of glucose was derived. The magnitude of wavelength shifts and absorbance values increased concomitantly with glucose concentrations until 90 µg/mL. Beyond that, it leveled off. The lowest amount of glucose that could produce a blue shift in the localized surface plasmon resonance (LSPR) absorption maxima was found to be 10 – 90 µg/mL. The limit of detection was 0.12 µg/mL. This enabled the construction of a direct sensitivity plasmonic colorimetric detection of glucose using AuNS that was rapid, sensitive and cost-effective with naked-eye detection. It has great potential for transfer of technology for point-of-care devices.

Keywords: colorimetric, gold nanostars, glucose, glucose oxidase, plasmonic

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Authors: Benyam Zenebe, Tesfaye Sisay, Gurja Belay, Workabeba Abebe

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Background: The prevalence and antibiogram of pathogenic E. coli strains, which cause diarrhea vary from region to region, and even within countries in the same geographical area. In Ethiopia, diagnostic approaches to E. coli induced diarrhea in children less than five years of age are not standardized. The aim of this study was to determine the involvement of pathogenic E. coli strains in child diarrhea and determine the antibiograms of the isolates in children less than 5 years of age with diarrhea at Addis Ababa University College of Health Sciences TikurAnbessa Specialized Hospital, Addis Ababa, Ethiopia. Methods: A purposive study that included 98 diarrheic children less than five years of age was conducted at Addis Ababa University College of Health Sciences, TikurAnbessa Specialized Hospital, Addis Ababa, Ethiopia to detect pathogenic E. coli biotypes. Stool culture was used to identify presumptive E. coliisolates. Presumptive isolates were confirmed by biochemical tests, and antimicrobial susceptibility tests were performed on confirmed E. coli isolates by the disk diffusion method. DNA was extracted from confirmed isolates by a heating method and subjected to Polymerase Chain Reaction or the presence of virulence genes. Amplified PCR products were analyzed by agarose gel electrophoresis. Data were collected on child demographics and clinical conditions using administered questionnaires. The prevalence of E. coli strains from the total diarrheic children, and the prevalence of pathogenic strains from total E. coli isolates along with their susceptibility profiles; the distribution of pathogenic E.coli biotypes among different age groups and between the sexes were determined by using descriptive statistics. Result: Out of 98 stool specimens collected from diarrheic children less than 5 years of age, 75 presumptive E. coli isolates were identified by culture; further confirmation by biochemical tests showed that only 56 of the isolates were E. coli; 29 of the isolates were found in male children and 27 of them in female children. Out of the 58 isolates of E. coli, 25 pathotypes belonging to different classes of pathogenic strains: STEC, EPEC, EHEC, EAEC were detected by using the PCR technique. Pathogenic E. coli exhibited high rates of antibiotic resistance to many of the antibiotics tested. Moreover, they exhibited multiple drug resistance. Conclusion: This study found that the isolation rate of E. coli and the involvement of antibiotic-resistant pathogenic E. coli in diarrheic children is prominent, and hence focus should be given on the diagnosis and antimicrobial sensitivity testing of pathogenic E. coli at Addis Ababa University College of Health Sciences TikurAnbessa Specialized Hospital. Among antibiotics tested, Cefotitan could be a drug of choice to treat E. coli.

Keywords: antibiotic susceptibility profile, children, diarrhea, E. coli, pathogenic

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Authors: Evgeny A. Ermakov, Lyudmila P. Smirnova, Valentina N. Buneva

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Schizophrenia associated with dysregulation of neurotransmitter processes in the central nervous system and disturbances in the humoral immune system resulting in the formation of antibodies (Abs) to the various components of the nervous tissue. Abs to different neuronal receptors and DNA were detected in the blood of patients with schizophrenia. Abs hydrolyzing DNA were detected in pool of polyclonal autoantibodies in autoimmune and infectious diseases, such catalytic Abs were named abzymes. It is believed that DNA-hydrolyzing abzymes are cytotoxic, cause nuclear DNA fragmentation and induce cell death by apoptosis. Abzymes with DNAase activity are interesting because of the mechanism of formation and the possibility of use as diagnostic markers. Therefore, in our work we have set following goals: to determine the level anti-DNA Abs in the serum of patients with schizophrenia and to study DNA-hydrolyzing activity of IgG of patients with schizophrenia. Materials and methods: In our study there were included 41 patients with a verified diagnosis of paranoid or simple schizophrenia and 24 healthy donors. Electrophoretically and immunologically homogeneous IgGs were obtained by sequential affinity chromatography of the serum proteins on protein G-Sepharose and gel filtration. The levels of anti-DNA Abs were determined using ELISA. DNA-hydrolyzing activity was detected as the level of supercoiled pBluescript DNA transition in circular and linear forms, the hydrolysis products were analyzed by agarose electrophoresis followed by ethidium bromide stain. To correspond the registered catalytic activity directly to the antibodies we carried out a number of strict criteria: electrophoretic homogeneity of the antibodies, gel filtration (acid shock analysis) and in situ activity. Statistical analysis was performed in ‘Statistica 9.0’ using the non-parametric Mann-Whitney test. Results: The sera of approximately 30% of schizophrenia patients displayed a higher level of Abs interacting with single-stranded (ssDNA) and double-stranded DNA (dsDNA) compared with healthy donors. The average level of Abs interacting with ssDNA was only 1.1-fold lower than that for interacting with dsDNA. IgG of patient with schizophrenia were shown to possess DNA hydrolyzing activity. Using affinity chromatography, electrophoretic analysis of isolated IgG homogeneity, gel filtration in acid shock conditions and in situ DNAse activity analysis we proved that the observed activity is intrinsic property of studied antibodies. We have shown that the relative DNAase activity of IgG in patients with schizophrenia averaged 55.4±32.5%, IgG of healthy donors showed much lower activity (average of 9.1±6.5%). It should be noted that DNAase activity of IgG in patients with schizophrenia with a negative symptoms was significantly higher (73.3±23.8%), than in patients with positive symptoms (43.3±33.1%). Conclusion: Anti-DNA Abs of patients with schizophrenia not only bind DNA, but quite efficiently hydrolyze the substrate. The data show a correlation with the level of DNase activity and leading symptoms of patients with schizophrenia.

Keywords: anti-DNA antibodies, abzymes, DNA hydrolysis, schizophrenia

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Authors: Boudjelal Amel

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Arisarum vulgare, a member of the Araceae family, is an herbaceous perennial widely distributed in the Mediterranean region. A. vulgare is recognized for its medicinal properties and holds significant traditional importance in Algeria for the treatment of various human ailments, including pain, infections, inflammation, digestive disorders, skin problems, eczema, cancer, wounds, burns and gynecological diseases. Despite its extensive traditional use, scientific exploration of A. vulgare remains limited. The study aims to investigate for the first time the therapeutic potential of A. vulgare ethanolic extract obtained by ultrasound-assisted extraction. The chemical composition of the extract was determined by LC-MS/MS analysis. For in vitro phytopharmacological evaluation, several assays, including DPPH, ABTS, FRAP and reducing power, were employed to evaluate the antioxidant activity. The antibacterial activity was assessed againt Escherichia coli, Salmonella typhimurium, Staphylococus aureus, Enterococcus feacium by disk diffusion and microdilution methods. The possible inhibitory activity of ethanolic extract was analyzed against the cholinesterases enzymes (AChE and BChE). The DNA protection activity of A. vulgare ethanolic extract was estimated using the agarose gel electrophoresis method. The capacities of the extract to protect plasmid DNA (pBR322) from the oxidizing effects of H2O2 and UV treatment were evaluated by their DNA-breaking forms. The in vivo wound healing potential of a traditional ointment containing 5% of A. vulgare ethanolic extract was also investigated. The LC-MS/MS profiling of the extract revealed the presence of various bioactive compounds, including naringenin, chlorogenic, vanillic, cafeic, coumaric acids, trans-cinnamic and trans ferrulic acids. The plant extract presented considerable antioxidant potential, being the most active for Reducing power (0,07326±0.001 mg/ml) and DPPH (0.14±0.004 mg/ml). The extract showed the highest inhibition zone diameter against Enterococcus feacium (36±0.1 mm). The ethanolic extract of A. vulgare suppressed the growth of Staphylococus aureus, Escherichia coli and Salmonella typhimurium according to the MIC values. The extract of the plant significantly inhibited both AChE and BChE enzymes. DNA protection activity of the A. vulgare extract was determined as 90.41% for form I and 51.92% for form II. The in vivo experiments showed that 5% ethanolic extract ointment accelerated the wound healing process. The topical application of the traditional formulation enhanced wound closure (95,36±0,6 %) and improved histological parameters in the treated group compared to the control groups. The promising biological properties of Arisarum vulgare revealed that the plant could be appraised as a potential origin of bioactive molecules having multifunctional medicinal uses.

Keywords: arisarum vulgare, LC-MS/MS, antioxidant activity, antimicrobial activity, cholinesterases enzymes inhibition, dna-damage activity, in vivo wound healing

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Authors: Okolie Pius Ifeanyi, Emerenini Emilymary Chima

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Aims: The study investigated the diversity and identities of Lactic Acid Bacteria (LAB) isolated from different fresh fruits using Molecular Nested PCR analysis and the efficacy of cell free supernatants from Lactic Acid Bacteria (LAB) isolated from fresh fruits for in vitro control of some tomato pathogens. Study Design: Nested PCR approach was used in this study employing universal 16S rRNA gene primers in the first round PCR and LAB specific Primers in the second round PCR with the view of generating specific Nested PCR products for the LAB diversity present in the samples. The inhibitory potentials of supernatant obtained from LAB isolates of fruits origin that were molecularly characterized were investigated against some tomato phytopathogens using agar-well method with the view to develop biological agents for some tomato disease causing organisms. Methodology: Gram positive, catalase negative strains of LAB were isolated from fresh fruits on Man Rogosa and Sharpe agar (Lab M) using streaking method. Isolates obtained were molecularly characterized by means of genomic DNA extraction kit (Norgen Biotek, Canada) method. Standard methods were used for Nested Polymerase Chain Reaction (PCR) amplification targeting the 16S rRNA gene using universal 16S rRNA gene and LAB specific primers, agarose gel electrophoresis, purification and sequencing of generated Nested PCR products (Macrogen Inc., USA). The partial sequences obtained were identified by blasting in the non-redundant nucleotide database of National Center for Biotechnology Information (NCBI). The antimicrobial activities of characterized LAB against some tomato phytopathogenic bacteria which include (Xanthomonas campestries, Erwinia caratovora, and Pseudomonas syringae) were obtained by using the agar well diffusion method. Results: The partial sequences obtained were deposited in the database of National Centre for Biotechnology Information (NCBI). Isolates were identified based upon the sequences as Weissella cibaria (4, 18.18%), Weissella confusa (3, 13.64%), Leuconostoc paramensenteroides (1, 4.55%), Lactobacillus plantarum (8, 36.36%), Lactobacillus paraplantarum (1, 4.55%) and Lactobacillus pentosus (1, 4.55%). The cell free supernatants of LAB from fresh fruits origin (Weissella cibaria, Weissella confusa, Leuconostoc paramensenteroides, Lactobacillus plantarum, Lactobacillus paraplantarum and Lactobacillus pentosus) can inhibits these bacteria by creating clear zones of inhibition around the wells containing cell free supernatants of the above mentioned strains of lactic acid bacteria. Conclusion: This study shows that potentially LAB can be quickly characterized by molecular methods to specie level by nested PCR analysis of the bacteria isolate genomic DNA using universal 16S rRNA primers and LAB specific primer. Tomato disease causing organisms can be most likely biologically controlled by using extracts from LAB. This finding will reduce the potential hazard from the use of chemical herbicides on plant.

Keywords: nested pcr, molecular characterization, 16s rRNA gene, lactic acid bacteria

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Authors: Tatiana Butkova, Nikolai Kibrik, Kristina Malsagova, Alexander Izotov, Alexander Stepanov, Anna Kaysheva

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Relevance. The genetic risk of developing schizophrenia is determined by two factors: single nucleotide polymorphisms and gene copy number variations. The search for serological markers for early diagnosis of schizophrenia is driven by the fact that the first five years of the disease are accompanied by significant biological, psychological, and social changes. It is during this period that pathological processes are most amenable to correction. The aim of this study was to analyze single nucleotide polymorphisms (SNPs) that are hypothesized to potentially influence the onset and development of the endogenous process. Materials and Methods It was analyzed 73 single nucleotide polymorphism variants. The study included 48 patients undergoing inpatient treatment at "Psychiatric Clinical Hospital No. 1" in Moscow, comprising 23 females and 25 males. Inclusion criteria: - Patients aged 18 and above. - Diagnosis according to ICD-10: F20.0, F20.2, F20.8, F21.8, F25.1, F25.2. - Voluntary informed consent from patients. Exclusion criteria included: - The presence of concurrent somatic or neurological pathology, neuroinfections, epilepsy, organic central nervous system damage of any etiology, and regular use of medication. - Substance abuse and alcohol dependence. - Women who were pregnant or breastfeeding. Clinical and psychopathological assessment was complemented by psychometric evaluation using the PANSS scale at the beginning and end of treatment. The duration of observation during therapy was 4-6 weeks. Total DNA extraction was performed using QIAamp DNA. Blood samples were processed on Illumina HiScan and genotyped for 652,297 markers on the Infinium Global Chips Screening Array-24v2.0 using the IMPUTE2 program with parameters Ne=20,000 and k=90. Additional filtration was performed based on INFO>0.5 and genotype probability>0.5. Quality control of the obtained DNA was conducted using agarose gel electrophoresis, with each tested sample having a volume of 100 µL. Results. It was observed that several SNPs exhibited gender dependence. We identified groups of single nucleotide polymorphisms with a membership of 80% or more in either the female or male gender. These SNPs included rs2661319, rs2842030, rs4606, rs11868035, rs518147, rs5993883, and rs6269.Another noteworthy finding was the limited combination of SNPs sufficient to manifest clinical symptoms leading to hospitalization. Among all 48 patients, each of whom was analyzed for deviations in 73 SNPs, it was discovered that the combination of involved SNPs in the manifestation of pronounced clinical symptoms of schizophrenia was 19±3 out of 73 possible. In study, the frequency of occurrence of single nucleotide polymorphisms also varied. The most frequently observed SNPs were rs4849127 (in 90% of cases), rs1150226 (86%), rs1414334 (75%), rs10170310 (73%), rs2857657, and rs4436578 (71%). Conclusion. Thus, the results of this study provide additional evidence that these genes may be associated with the development of schizophrenia spectrum disorders. However, it's impossible cannot rule out the hypothesis that these polymorphisms may be in linkage disequilibrium with other functionally significant polymorphisms that may actually be involved in schizophrenia spectrum disorders. It has been shown that missense SNPs by themselves are likely not causative of the disease but are in strong linkage disequilibrium with non-functional SNPs that may indeed contribute to disease predisposition.

Keywords: gene polymorphisms, genotyping, single nucleotide polymorphisms, schizophrenia.

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Authors: Swati Gautam, Salman Akhtar, S. P. Jaiswar, Amita Jain

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Background: FGTB is now a major global health problem mostly in developing countries including India. In humans, Mycobacterium Tuberculosis (M.tb) is a causating agent of infection. High index of suspicion is required for early diagnosis due to asymptomatic presentation of FGTB disease. In macrophages Toll Like Receptor-2 (TLR-2) is one which mediated host’s immune response to M.tb. The expression of TLR-2 on macrophages is important to determine the fate of innate immune responses to M.tb. TLR-2 have two work. First its high expression on macrophages worsen the outer of infection and another side, it maintains M.tb to its dormant stage avoids activation of M.tb from latent phase. Single Nucleotide Polymorphism (SNP) of TLR-2 gene plays an important role in susceptibility to TB among different populations and subsequently, in the development of infertility. Methodology: This Case-Control study was done in the Department of Obs and Gynae and Department of Microbiology at King George’s Medical University, U.P, Lucknow, India. Total 300 subjects (150 Cases and 150 Controls) were enrolled in the study. All subjects were enrolled only after fulfilling the given inclusion and exclusion criteria. Inclusion criteria: Age 20-35 years, menstrual-irregularities, positive on Acid-Fast Bacilli (AFB), TB-PCR, (LJ/MGIT) culture in Endometrial Aspiration (EA). Exclusion criteria: Koch’s active, on ATT, PCOS, and Endometriosis fibroid women, positive on Gonococal and Chlamydia. Blood samples were collected in EDTA tubes from cases and healthy control women (HCW) and genomic DNA extraction was carried out by salting-out method. Genotyping of TLR2 genetic variants (Arg753Gln and Arg677Trp) were performed by using single amplification refractory mutation system (ARMS) PCR technique. PCR products were analyzed by electrophoresis on 1.2% agarose gel and visualized by gel-doc. Statistical analysis of the data was performed using the SPSS 16.3 software and computing odds ratio (OR) with 95% CI. Linkage Disequiliribium (LD) analysis was done by SNP stats online software. Results: In TLR-2 (Arg753Gln) polymorphism significant risk of FGTB observed with GG homozygous mutant genotype (OR=13, CI=0.71-237.7, p=0.05), AG heterozygous mutant genotype (OR=13.7, CI=0.76-248.06, p=0.03) however, G allele (OR=1.09, CI=0.78-1.52, p=0.67) individually was not associated with FGTB. In TLR-2 (Arg677Trp) polymorphism a significant risk of FGTB observed with TT homozygous mutant genotype (OR= 0.020, CI=0.001-0.341, p < 0.001), CT heterozygous mutant genotype (OR=0.53, CI=0.33-0.86, p=0.014) and T allele (OR=0.463, CI=0.32-0.66, p < 0.001). TT mutant genotype was only found in FGTB cases and frequency of CT heterozygous more in control group as compared to FGTB group. So, CT genotype worked as protective mutation for FGTB susceptibility group. In haplotype analysis of TLR-2 genetic variants, four possible combinations, i.e. (G-T, A-C, G-C, and A-T) were obtained. The frequency of haplotype A-C was significantly higher in FGTB cases (0.32). Control group did not show A-C haplotype and only found in FGTB cases. Conclusion: In conclusion, study showed a significant association with both genetic variants of TLR-2 of FGTB disease. Moreover, the presence of specific associated genotype/alleles suggest the possibility of disease severity and clinical approach aimed to prevent extensive damage by disease and also helpful for early detection of disease.

Keywords: ARMS, EDTA, FGTB, TLR

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