Search results for: gene expression pattern

Authors: Faheema Khan

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To investigate the ability of sensitive and tolerant genotype of Glycine max to adapt to a saline environment in a field, we examined the growth performance, water relation and activities of antioxidant enzymes in relation to photosynthetic rate, chlorophyll a fluorescence, photosynthetic pigment concentration, protein and proline in plants exposed to salt stress. Ten soybean genotypes (Pusa-20, Pusa-40, Pusa-37, Pusa-16, Pusa-24, Pusa-22, BRAGG, PK-416, PK-1042, and DS-9712) were selected and grown hydroponically. After 3 days of proper germination, the seedlings were transferred to Hoagland’s solution (Hoagland and Arnon 1950). The growth chamber was maintained at a photosynthetic photon flux density of 430 μmol m−2 s−1, 14 h of light, 10 h of dark and a relative humidity of 60%. The nutrient solution was bubbled with sterile air and changed on alternate days. Ten-day-old seedlings were given seven levels of salt in the form of NaCl viz., T1 = 0 mM NaCl, T2=25 mM NaCl, T3=50 mM NaCl, T4=75 mM NaCl, T5=100 mM NaCl, T6=125 mM NaCl, T7=150 mM NaCl. The investigation showed that genotype Pusa-24, PK-416 and Pusa-20 appeared to be the most salt-sensitive. genotypes as inferred from their significantly reduced length, fresh weight and dry weight in response to the NaCl exposure. Pusa-37 appeared to be the most tolerant genotype since no significant effect of NaCl treatment on growth was found. We observed a greater decline in the photosynthetic variables like photosynthetic rate, chlorophyll fluorescence and chlorophyll content, in salt-sensitive (Pusa-24) genotype than in salt-tolerant Pusa-37 under high salinity. Numerous primers were verified on ten soybean genotypes obtained from Operon technologies among which 30 RAPD primers shown high polymorphism and genetic variation. The Jaccard’s similarity coefficient values for each pairwise comparison between cultivars were calculated and similarity coefficient matrix was constructed. The closer varieties in the cluster behaved similar in their response to salinity tolerance. Intra-clustering within the two clusters precisely grouped the 10 genotypes in sub-cluster as expected from their physiological findings.Salt tolerant genotype Pusa-37, was further analysed by 2-Dimensional gel electrophoresis to analyse the differential expression of proteins at high salt stress. In the Present study, 173 protein spots were identified. Of these, 40 proteins responsive to salinity were either up- or down-regulated in Pusa-37. Proteomic analysis in salt-tolerant genotype (Pusa-37) led to the detection of proteins involved in a variety of biological processes, such as protein synthesis (12 %), redox regulation (19 %), primary and secondary metabolism (25 %), or disease- and defence-related processes (32 %). In conclusion, the soybean plants in our study responded to salt stress by changing their protein expression pattern. The photosynthetic, biochemical and molecular study showed that there is variability in salt tolerance behaviour in soybean genotypes. Pusa-24 is the salt-sensitive and Pusa-37 is the salt-tolerant genotype. Moreover this study gives new insights into the salt-stress response in soybean and demonstrates the power of genomic and proteomic approach in plant biology studies which finally could help us in identifying the possible regulatory switches (gene/s) controlling the salt tolerant genotype of the crop plants and their possible role in defence mechanism.

Keywords: glycine max, salt stress, RAPD, genomic and proteomic variability

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Authors: Jafar Ahmadi, Zhohreh Asiaban, Sedigheh Fabriki Ourang

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Brassinosteroids (BRs) regulate cell elongation, vascular differentiation, senescence and stress responses. BRs signal through the BES1/BZR1 family of transcription factors, which regulate hundreds of target genes involved in this pathway. In this research a comprehensive genome-wide analysis was carried out in BES1/BZR1 gene family in Arabidopsis thaliana, Cucumis sativus, Vitis vinifera, Glycin max, and Brachypodium distachyon. Specifications of the desired sequences, dot plot and hydropathy plot were analyzed in the protein and genome sequences of five plant species. The maximum amino acid length was attributed to protein sequence Brdic3g with 374aa and the minimum amino acid length was attributed to protein sequence Gm7g with 163aa. The maximum Instability index was attributed to protein sequence AT1G19350 equal with 79.99 and the minimum Instability index was attributed to protein sequence Gm5g equal with 33.22. Aliphatic index of these protein sequences ranged from 47.82 to 78.79 in Arabidopsis thaliana, 49.91 to 57.50 in Vitis vinifera, 55.09 to 82.43 in Glycin max, 54.09 to 54.28 in Brachypodium distachyon 55.36 to 56.83 in Cucumis sativus. Overall, data obtained from our investigation contributes a better understanding of the complexity of the BES1/BZR1 gene family and provides the first step towards directing future experimental designs to perform systematic analysis of the functions of the BES1/BZR1 gene family.

Keywords: BES1/BZR1, brassinosteroids, phylogenetic analysis, transcription factor

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Authors: H. S. Saini, K. Vijayalakshmi, Rishi Sayal

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Network-based education has been growing rapidly in size and quality. Knowledge clustering becomes more important in personalized information retrieval for web-learning. A personalized-Learning service after the learners’ knowledge has been classified with clustering. Through automatic analysis of learners’ behaviors, their partition with similar data level and interests may be discovered so as to produce learners with contents that best match educational needs for collaborative learning. We present a specific mining tool and a recommender engine that we have integrated in the online learning in order to help the teacher to carry out the whole e-learning process. We propose to use sequential pattern mining algorithms to discover the most used path by the students and from this information can recommend links to the new students automatically meanwhile they browse in the course. We have Developed a specific author tool in order to help the teacher to apply all the data mining process. We tend to report on many experiments with real knowledge so as to indicate the quality of using both clustering and sequential pattern mining algorithms together for discovering personalized e-learning systems.

Keywords: e-learning, cluster, personalization, sequence, pattern

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Authors: Rezvan Najafi, Korosh Heydari, Massoud Saidijam

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5-FU is a chemotherapeutic agent that has been used in colorectal cancer (CRC) treatment. However, it is usually associated with the acquired resistance, which decreases the therapeutic effects of 5-FU. miR-200c is involved in chemotherapeutic drug resistance, but its mechanism is not fully understood. In this study, the effect of inhibition of miR-200c in sensitivity of HCT-116 CRC cells to 5-FU was evaluated. HCT-116 cells were transfected with LNA-anti- miR-200c for 48 h. mRNA expression of miR-200c was evaluated using quantitative real- time PCR. The protein expression of phosphatase and tensin homolog (PTEN) and E-cadherin were analyzed by western blotting. Annexin V and propidium iodide staining assay were applied for apoptosis detection. The caspase-3 activation was evaluated by an enzymatic assay. The results showed LNA-anti-miR-200c inhibited the expression of PTEN and E-cadherin protein, apoptosis and activation of caspase 3 compared with control cells. In conclusion, these results suggest that miR-200c as a prognostic marker can overcome to 5-FU chemoresistance in CRC.

Keywords: colorectal cancer, miR-200c, 5-FU resistance, E-cadherin, PTEN

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Authors: Mekha Mathew, Varun P Gopi

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Wireless Capsule Endoscopy (WCE) has become a great device in Gastrointestinal (GI) tract diagnosis, which can examine the entire GI tract, especially the small intestine without invasiveness and sedation. Bleeding in the digestive tract is a symptom of a disease rather than a disease itself. Hence the detection of bleeding is important in diagnosing many diseases. In this paper we proposes a novel method for distinguishing bleeding regions from normal regions based on Contourlet transform and Local Binary Pattern (LBP). Experiments show that this method provides a high accuracy rate of 96.38% in CIE XYZ colour space for k-Nearest Neighbour (k-NN) classifier.

Keywords: Wireless Capsule Endoscopy, local binary pattern, k-NN classifier, contourlet transform

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Authors: W. Mohammedsaeed, A. J. Mcbain, S. M. Cruickshank, C. A. O’Neill

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Many studies have demonstrated the importance of probiotics and their potential therapeutic effects within the gut. Recently, the possible therapeutic effects of probiotics in other tissues have also begun to be investigated. Comparatively few studies have evaluated the use of topical probiotics in relation to the skin. In this study, we have conducted preliminary investigations into whether a well-known probiotic, Lactobacillus rhamnosus GG (LGG), can increase the rate of re-epithelialization in a model wound. Full-thickness skin was obtained from individuals undergoing elective cosmetic surgery. This skin was wounded using excisional punch and cultured using a serum-free medium, either in the presence or absence of L. rhamnosus GG lysate. Histological staining of the sections was performed with Haematoxylin& Eosin E to quantify “epithelial tongue length”. This is the length of the new epithelial ‘tongue’ that grows and covers the exposed dermis at the inner wound edges. The length of the new epithelial ‘tongue’ was compared in untreated section and section treated with and L. rhamnosus GG made using108CFU/ml bacterial cells. L. rhamnosus GG lysate enhanced significantly the re-epithelialisation of treated wounds compared with that of untreated wounds (P=0.005, n=3). Tongue length, at day 1 was 7.55μm 0.15, at day 3 it was 18.5μm 0.25 and at day 7 was 22.9μm 0.35. These results can be compared with untreated cultures in which tongue length was 3.25μm 0.35, day 3 was 9.65μm 0.25 and day 7 was 13.5μm 0.15 post-wounding. In ex-vivo proliferation and migration cells were measured by determining the expression of nuclear proliferation marker Ki-67 and the expression of Phosphorylated cortactin respectively demonstrated that L. rhamnosus GG significantly increased NHEK proliferation and migration rates relative to controls. However, the dominant mechanism was migration because in ex-vivo skin treated with the L. rhamnosus GG up-regulated the gene expression of the chemokine receptor and ligands CXCR2 and CXCL2 comparing with controls (P=0.02, P=0.03 respectively, n=3). High levels of CXCL2/CXCL2 have already been implicated in multiple aspects of stimulation of wound healing through activation of keratinocyte migration. These data demonstrate that lysates from Lactobacillus rhamnosus GG increase re-epithelialization by stimulation of keratinocyte migration. The current study identifies the partial mechanism that contribute to stimulating the wound-healing process ex vivo in response to L. rhamnosus GG lysate is an increase in the production of CXCL2/ CXCR2 in ex vivo models. The use of probiotic lysates potentially offers new options to develop treatments that could improve wound healing.

Keywords: Lactobacillus rhamnosus GG, wounds, migration, lysate

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Authors: Hasanah Md. Amin

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The existence of cyberspace and its growing contents is real and overwhelming. Visual as one of the properties of cyber contents is increasingly becoming more significant and popular among creator and user. The visual and aesthetic of the content is consistent with many similarities. Aesthetic, although universal, has slight differences across the world. Aesthetic power could impress, influence, and cause bias among the users. The content creator who knows how to manipulate this visuals and aesthetic expression can dominate the scenario and the user who is ‘expressive literate’ will gain much from the scenes. User who understands aesthetic will be rewarded with competence, confidence, and certainly, a personality enhanced experience in carrying out a task when participating in this chaotic but promising cyberworld. The aim of this article is to gain knowledge from related literature and research regarding User-Generated Content (UGC), which focuses on aesthetic expression by the Net generation. The objective of this preliminary study is to analyze the aesthetic expression linked to visual from the participatory cultural viewpoint looking for meaning, value, patterns, and characteristics.

Keywords: visual overloaded, user-generated content, net generation, visual arts

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Authors: Dana Gabriková, Martin Mistrík, Jarmila Bernasovská, Iveta Tóthová, Jana Kisková

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The Roma (Gypsies) is a transnational minority with a high degree of consanguineous marriages. Similar to other genetically isolated founder populations, the Roma harbor a number of unique or rare genetic disorders. This paper discusses about a rare form of Charcot-Marie-Tooth disease – type 4G (CMT4G), also called Hereditary Motor and Sensory Neuropathy type Russe, an autosomal recessive disease caused by mutation private to Roma characterized by abnormally increased density of non-myelinated axons. CMT4G was originally found in Bulgarian Roma and in 2009 two putative causative mutations in the HK1 gene were identified. Since then, several cases were reported in Roma families mainly from Bulgaria and Spain. Here we present a Slovak Roma family in which CMT4G was diagnosed on the basis of clinical examination and genetic testing. This case is a further proof of the role of the HK1 gene in pathogenesis of the disease. It confirms that mutation in the HK1 gene is a common cause of autosomal recessive CMT disease in Roma and should be considered as a common part of a diagnostic procedure.

Keywords: gypsies, HK1, HSMN-Russe, rare disease

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Authors: Viktoriia Burkina, Vladimir Zlabek, Galia Zamaratskaia

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Cytochromes P450 (CYP) are important family of enzymes in Phase I metabolism. Environmental pollutants often act as inducers of the gene expression and activities CYP1A1 and CYP3A-like isoforms in fish. The activities are generally measured in the fish liver or gills, and less is known about tissue distribution of expression. In present study, the CYP1A1 and CYP3A-like activities were measured in rainbow trout liver, gill, intestine, heart, brain and gonads. The activities of CYP1A1 and CYP3A-like proteins were estimated as the rates of 7-ethoxyresorufin-O-deethylation (EROD) and benzyloxy-4-trifluoromethylcoumarin-O-debenzyloxylation (BFCOD), respectively. The CYP1A1 and CYP3A-like activities were detectable in all investigated fish tissues, with the highest activity in hepatic tissue followed by heart > brain > gill > intestine > gonads. To confirm the presence of CYP1A1 in different tissues, EROD activity was measured in presence of the selective inhibitors ellipticine (CYP1A1), ketoconazole (CYP3A), 8-methoxypsoralen (human CYP2A) and diallyl sulphide (CYP2E1). It was found that ellipticine, ketoconazole and 8-methoxypsoralen inhibited hepatic EROD activity by 88-98%. Ellipticine inhibited gill, intestine, and gonad EROD activity by 50%. In conclusion, EROD and BFCOD activities were detected in rainbow trout liver, gill, intestine, heart, brain and gonads. Further studies are needed to fully identify all CYP450 isoforms responsible for these activities. Acknowledgement: The study was financially supported by the Ministry of Education, Youth and Sports of the Czech Republic - projects „CENAKVA “(No. CZ.1.05/2.1.00/01.0024), “CENAKVA Center Development “(No. CZ.1.05/2.1.00/19.0380), “CENAKVA II “(No. LO1205 under the NPU I program), and "Development of USB - International mobility (No. CZ.02.2.69/0.0/0.0/16_027/0008364).

Keywords: BFCOD, EROD, fish, phase I metabolism, selective inhibitors

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Authors: Benjaporn Buranrat, Nootchanat Mairuae

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Cratoxylum formosum (Jack) Dyer (CF) has been used for the traditional medicines in South East Asian and Thailand. Normally, northeast Thai vegetables have proven cytotoxic to many cancer cells. Therefore, the present study aims to explore the molecular mechanisms underlying CF-induced cancer cell death and apoptosis on breast and liver cancer cells. The cytotoxicity and antiproliferative effects of CF on the human breast MCF-7 and liver HepG2 cancer cell lines were evaluated using sulforhodamine B assay and colony formation assay. Cell migration assay was measured using wound healing assay. The apoptosis induction mechanisms were investigated through reactive oxygen species formation, caspase 3 activity, and JC-1 activity. Gene expression by real-time PCR and apoptosis related protein levels by Western blot analysis. CF induced MCF-7 and HepG2 cell death by time- and dose-dependent manner. Furthermore, CF had the greater cytotoxic potency on MCF-7 more than HepG2 cells with IC50 values of 85.70+4.52 μM and 219.03±9.96 μM respectively, at 24 h. Treatment with CF also caused a dose-dependent decrease in colony forming ability and cell migration, especially on MCF-7 cells. CF induced ROS formation, increased caspase 3 activities, and decreased the mitochondrial membrane potential, and causing apoptotic body production and DNA fragmentation. CF significantly decreased expression of the cell cycle regulatory protein RAC1 and downstream proteins, cdk6. Additionally, CF enhanced p21 and reduced cyclin D1 protein levels. CF leaf extract induced cell death, apoptosis, antimigration in both of MCF-7 and HepG2 cells. CF could be useful for developing to anticancer drug candidate for breast and liver cancer therapy.

Keywords: cratoxylum formosum (jack) dyer, breast cancer, liver cancer, cell death

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Authors: Makhosi Buthelezi

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Mitochondrial DNA cytochrome c oxidase I (COI) gene analyses linked the South African groundnut leaf miner (GLM) to the Australian soya bean moth Aproaerema simplexella (Walker) and Indian Aproaerema modicella (Deventer). Thus, the genetic relatedness of GLM, A. simplexela, and A. modicella was examined by performing mitochondrial and nuclear (COI, cytochrome oxidase subunit II (COII), mitochondrial cytochrome b (CYTB), nuclear ribosomal 28S (28S) and intergenic spacer elongation factor-1 alpha ( EF-1 ALPHA) on 44 specimens collected from South Africa, four from Mozambique, and three each from single locations in India and Australia. Phylogenetic analyses were conducted using the Maximum Parsimony (MP) and Neighbour-Joining (NJ) methods. All of the datasets of the five DNA gene regions that were sequenced were also analyzed using the Basic Local Alignment Search Tool (BLAST) to find the closest matches for inclusion in the phylogenetic trees as outgroups and for purposes of information. In the phylogenetic trees for COI, COII, cytb and EF-1 ALPHA, a similar pattern was observed in the way that the sequences assembled into different groups; i.e., some sequences of A. simplexella from Australia were grouped separately from the others, but some Australian sequences grouped with those of the GLM from South Africa, India, and Mozambique. In the phylogenetic tree for 28S, all sequences from South Africa, Australia, India, and Mozambique grouped together and formed one group. For COI, genetic pairwise distance ranged from 0.97 to 3.60 %, for COII it ranged from 0.19% to 2.32%, for cytb it ranged from 0.25 to 9.77% and for EF-1 ALPHA it ranged 0.48 to 6.99%. Results of this study indicate that these populations are genetically related and presumably constitute a single species. Thus, further molecular and morphological studies need to be undertaken in order to resolve this apparent conundrum on the taxonomy of these populations.

Keywords: aproaerema modicella, aproaerema simplexella, mitochondrial DNA, nuclear DNA

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Authors: Assem El-Ansary

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This paper illustrates the event-oriented Behavioral Pattern Analysis (BPA) modeling approach using the Healthcare Multi-Agent System. The Event defined in BPA is a real-life conceptual entity that is unrelated to any implementation. The major contributions of this research are: The Behavioral Pattern Analysis (BPA) modeling methodology. The development of an interactive software tool (DECISION), which is based on a combination of the Analytic Hierarchy Process (AHP) and the ELECTRE Multi-Criteria Decision Making (MCDM) methods.

Keywords: analysis, modeling methodology, software modeling, event-oriented, behavioral pattern, use cases, Healthcare Multi-Agent System

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Authors: Assem El-Ansary

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This paper illustrates the event-oriented Behavioral Pattern Analysis (BPA) modeling approach in developing an Intelligent Agent Reservation System (IARS). The Event defined in BPA is a real-life conceptual entity that is unrelated to any implementation. The major contributions of this research are developing the Behavioral Pattern Analysis (BPA) modeling methodology, and developing an interactive software tool (DECISION) which is based on a combination of the Analytic Hierarchy Process (AHP) and the ELECTRE Multi-Criteria Decision Making (MCDM) methods.

Keywords: analysis, intelligent agent, reservation system, modeling methodology, software modeling, event-oriented, behavioral pattern, use cases

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Authors: Shan-Ying Wu, Sheng-Hui Lan, Xi-Zhang Lin, Ih-Jen Su, Ting-Fen Tsai, Chia-Jui Yen, Tsung-Hsueh Lu, Fu-Wen Liang, Huey-Jen Su, Chun-Li Su, Hsiao-Sheng Liu

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In hepatocelluar carcinoma (HCC), dysregulated expression of cyclin D1 and impaired autophagy has been reported separately. However, the relationship between them has not been explored. In this study, we demonstrated that autophagy was inversely correlated with cyclin D1 expression in 147 paired HCC patient specimens. HCC specimen with highly expression of cyclin D1 shows correlation with poor overall survival rate. Furthermore, induction of autophagy by amiodarone (antiarrhythmic drug) in Hep 3B cells, cyclin D1 was recruited into autophagosomes demonstrated by immune-gold labeling of cyclin D1 after extraction of autophagosomes. We further demonstrated that autophagy suppresses Hep 3B cell proliferation, and further analysis revealed that cell cycle was arrested at G1 phase. The interaction between LC3 (maker of autophagy) and cyclin D1 was increased after autophagy induction. In addition, ubiquitinated-cyclin D1 was also increased after autophagy induction, which is selectively degraded by autophagosome through binding with SQSTM1/p62 (an adaptor protein). In vivo study showed that amiodarone induced autophagy suppresses liver tumor formation in xenograft mouse and orthotopic rat model through decreasing cyclin D1 expression and inhibition of cell proliferation. Altogether, we reveal a novel mechanism that ubiquitinated cyclin D1 degraded by autophagic pathway by p62 and amiodarone is a promising drug for targeting cyclin D1 in liver cancer therapy.

Keywords: autophagy, cyclin D1, hepatocellular carcinoma, amiodarone

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Authors: Yousuf M. Al Suleimani, Robin Hiley

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The endogenous phospholipid lysophosphatidylinositol (LPI) was recently identified as a novel ligand for the G protein-coupled receptor 55 (GPR55) and an inducer of intracellular Ca2+ [Ca2+]i release. This study attempts to characterize Ca2+ signals provoked by LPI in HEK293 cells engineered to stably express human GPR55 and to test cannabinoid ligand activity at GPR55. The study shows that treatment with LPI stimulates a sustained, oscillatory Ca2+ release. The response is characterized by an initial rapid rise, which is mediated by the Gαq-PLC-IP3 pathway, and this is followed by prolonged oscillations that require RhoA activation. Ca2+ oscillations are initiated by intracellular mechanisms and extracellular Ca2+ is only required to replenish Ca2+ lost from the cytoplasm. Analysis of cannabinoid ligand activity at GPR55 revealed no clear effect of the endocannabinoid anandamide, however, rimonabant and the CB1 receptor antagonist AM251 evoked GPR55-mediated [Ca2+]i. Thus, LPI is likely to be a key plasma membrane mediator of signaling events and changes in gene expression through GPR55 activation.

Keywords: lysophosphatidylinositol, calcium, GPR55, cannabinoid

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Authors: Fatma M. Benrabha

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The aim of the study was to determine the type and pattern of antibiotic susceptibility of the pathogenic microorganisms causing chronic suppurative otitis media (CSOM), which could lead to better therapeutic decisions and consequently avoidance of appearance of resistance to specific antibiotics. Most frequently isolated agents were Pseudomonas aeruginosa 28.5%; followed by Staphylococcus aureus 18.2%; proteus mirabilis 13.9%; Providencia stuartti 6.7%; Bacteroides melaninogenicus, Aspergillus sp., candida sp., 4.2% each; and other microorganisms were represented in 3-0.2%. Drug sensitivities pattern of Pseudomonas aeruginosa showed that ciprofloxacin was active against the majority of isolates (93.9%) followed by ceftazidime 86.2%, amikacin 76.2% and gentamicin 40.8%. However, Staphylococcus aureus isolates were resistant to penicillin 72.7%, erythromycin 28.6%, cephalothin 18.2%, cloxacillin 8.3% and ciprofloxacin was active against 96.2% of isolates. The resistance pattern of proteus mirabilis were 55.6% to ampicillin, 47.1% to carbencillin, 29.4% to cephalothin, 14.3% to gentamicin and 4.8% to amikacin while 100% were sensitive to ciprofloxacin. We conclude that ciprofloxacin is the best drug of choice in treatment of CSOM caused by the common microorganisms.

Keywords: otitis media, chronic suppurative otitis media (CSOM), microorganism, drug sensitivity

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Authors: Haveesha Buddhdev

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The most important right for a human being in a society is the freedom of expression as stated by Article 18 and 19 of the Universal Declaration of Human rights pledged by member states of United Nations. Will it be fair to expect him/her to be of sound mental health if this right is taken away? Religion as a primary social institution controls many rights, freedoms and duties of people in a society. It does so by imposing certain values and beliefs on people which would either enhance quality of life or curb their freedom adversely thus affecting individual mental health. This paper aims to study the positive and negative role that religion plays in influencing one’s freedom of expression. This paper will focus on reviewing existing studies on the positive and negative impacts of religion on mental health. It will also contain data collected by the researcher about the impacts of religion on freedom of expression which will be obtained by surveying a sample of 30 adolescents and young adults. The researcher will use a Likert scale for these purpose, with response options ranging from strongly disagree to strongly agree and quantify it accordingly. Descriptive statistics would be used to analyse the data. Such research would help to identify possible problems faced by adolescents and young adults when it comes to religio-cultural ethos and also facilitate further researches to study the role that religion plays in mental health.

Keywords: cultural Ethos, freedom of expression, adolescent mental health, social science

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Authors: Jae Woong Sull, Sun Ha Jee

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High-density lipoprotein (HDL) cholesterol levels are associated with decreased risk of coronary artery disease. Several genome-wide association studies (GWAS) for HDL cholesterol levels have implicated cholesterol ester transfer protein (CETP) as possibly causal. We tested for the association between single nucleotide polymorphisms (SNPs) in CETP gene and HDL cholesterol levels in Korean population. Subjects were selected from the Korean Metabolic Syndrome Research Initiative study in the Bundang-Gu area. A total of 2,304 individuals from Bundang-Gu were recruited in 2008. Other subjects were selected from the Severance Hospital (N=4,294). SNP rs6499861 in the CETP gene was associated with mean HDL cholesterol levels (effect per allele -2.044 mg/dL, p=7.23×10-7). Subjects with the CG/GG genotype had a 1.46 -fold (range 1.24–1.72-fold) higher risk of having abnormal HDL cholesterol levels (<40 mg/dL) than subjects with the CC genotype. When analyzed by gender, the association of CETP was stronger in women than in men. When analyzed by physical activity behavior, the association with CETP was much stronger in male subjects with low physical activity (OR=1.54, 95% CI: 1.23-1.92, P=0.0001) than in male subjects with high physical activity. This study clearly demonstrates that genetic variants in CETP influence HDL cholesterol levels in Korean adults.

Keywords: CETP, HDL cholesterol, physical activity, polymorphisms

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Authors: Naseem Fatima, A. N. Srivastava, Tasleem Raza, Vijay Kumar

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Introduction: Carcinoma gallbladder is third most common gastrointestinal lethal disease with the highest incidence and mortality rate among women in Northern India. Scientists have found several risk factors that make a person more likely to develop gallbladder cancer; among these risk factors, deregulation of miRNAs has been demonstrated to be one of the most crucial factors. The changes in the expression of specific miRNA genes result in the control of inflammation, cell cycle regulation, stress response, proliferation, differentiation, apoptosis and invasion thus mediate the process in tumorgenesis. The aim of this study was to investigate the role of MiRNA-335 and may as a molecular marker in early detection of gallbladder cancer in suspected cases. Material and Methods: A total of 20 consecutive patients with gallbladder cancer aged between 30-75 years were registered for the study. Total RNA was extracted from tissue by using the mirVANA MiRNA isolation Kit according to the manufacturer’s protocol. The MiRNA- 335 and U6 snRNA-specific cDNA were reverse-transcribed from total RNA using Taqman microRNA reverse-transcription kit according to the manufacturer’s protocol. TaqMan MiRNA probes hsa-miR-335 and Taqman Master Mix without AmpEase UNG, Individual real-time PCR assays were performed in a 20 μL reaction volume on a Real-Time PCR system (Applied Biosystems StepOnePlus™) to detect MiRNA-335 expression in tissue. Relative quantification of target MiRNA expression was evaluated using the comparative cycle threshold (CT) method. The correlation was done in between cycle threshold (CT Value) of target MiRNA in gallbladder cancer with respect to non-cancerous Cholelithiasis gallbladder. Each sample was examined in triplicate. The Newman-Keuls Multiple Comparison Test was used to determine the expression of miR-335. Results: MiRNA335 was found to be significantly downregulated in the gallbladder cancer tissue (P<0.001), when compared with non-cancerous Cholelithiasis gallbladder cases. Out of 20 cases, 75% showed reduced expression of MiRNA335, were at last stage of disease with low overall survival rate and remaining 25% were showed up-regulated expression of MiRNA335 with high survival rate. Conclusion: The present study showed that reduced expression of MiRNA335 is associated with the advancement of the disease, and its deregulation may provide important clues to understanding it as a prognostic marker and opportunities for future research.

Keywords: carcinoma gallbladder, downregulation, MiRNA-335, RT-PCR assay

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Authors: Dipanshu Sur, Ratnabali Chakravorty

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Background: Polycystic ovary syndrome (PCOS) is the most common metabolic abnormality such as changes in lipid profile, diabetes, hypertension and metabolic syndrome occurring in young women of reproductive age. Low vitamin D levels were found to be associated with the development of obesity and insulin resistance in women with PCOS. Variants on vitamin D receptor (VDR) gene have also been related to metabolic comorbidities in general population. Aim: The aim of this case-control study was to investigate whether the VDR gene polymorphisms are associated with susceptibility to PCOS. Methods: Women with PCOS and a control group, all aged 16-40 years, were enrolled. Genotyping of VDR Fok-I (rs2228570), VDR Apa-I (rs7975232) as well as GC (rs2282679), DHCR7 (rs12785878) SNPs between groups were determined by using direct sequencing. Serum 25-hydroxyvitamin D [25(OH)] levels were measured by ELISA. Results: Mean serum 25(OH)D in the PCOS and control samples were 19.08±7 and 23.27±6.03 (p=0.048) which were significantly lower in PCOS patients compared with controls. CC genotype of the VDR Apa-I SNP was same frequent in PCOS (25.6%) and controls (25.6%) (OR: 0.9995; 95%CI: 0.528 to 1.8921; p= 0.9987). The CC genotype was also significantly associated with both lower E2 (p=0.031) and Androstenedione levels (p=0.062). We observed a significant association of GC polymorphism with 25(OH)D levels. PCOS women carrying the GG genotype (in GC genes) had significantly higher risk for vitamin D deficiency than women carrying the TT genotype. Conclusions: In conclusion, data from this study indicate that vitamin D levels are lower, and vitamin D deficiency more frequent, in PCOS than in controls. The present findings suggest that the Apa-I, Fok-I polymorphism of the VDR gene is associated with PCOS and seems to modulate ovarian steroid secretion. Further studies are needed to better clarify the biological mechanisms by which the polymorphism influences PCOS risk.

Keywords: vitamin D receptor, polymorphism, vitamin D, polycystic ovary syndrome

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Authors: Meng-Hui Chen, Chen-Yu Kao, Chia-Yu Hsu, Pei-Chann Chang

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The main idea in this paper is using sequential pattern mining to find the information which is helpful for finding high performance solutions. By combining this information, it is defined as blocks. Using the blocks to generate artificial chromosomes (ACs) could improve the structure of solutions. Estimation of Distribution Algorithms (EDAs) is adapted to solve the combinatorial problems. Nevertheless many of these approaches are advantageous for this application, but only some of them are used to enhance the efficiency of application. Generating ACs uses patterns and EDAs could increase the diversity. According to the experimental result, the algorithm which we proposed has a better performance to solve the permutation flow-shop problems.

Keywords: combinatorial problems, sequential pattern mining, estimationof distribution algorithms, artificial chromosomes

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Authors: Gizem Buldum, Alexander Bismarck, Athanasios Mantalaris

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Cellulose is the most abundant biopolymer on earth and it is currently being utilised in a multitude of industrial applications. Over the last 30 years, attention has been paid to the bacterial cellulose (BC), since BC exhibits unique physical, chemical and mechanical properties when compared to plant-based cellulose, including high purity and biocompatibility. Although Acetobacter xylinum is the most efficient producer of BC, it’s long doubling time results in insufficient yields of the cellulose production. This limits widespread and continued use of BC. In this study, E. coli BL21 (DE3) or E. coli HMS cells are selected as host organisms for the expression of bacterial cellulose synthase operon (bcs) of A.xylinum. The expression system is created based on pET-Duet1 and pCDF plasmid vectors, which carry bcs operon. The results showed that all bcs genes were successfully transferred and expressed in E.coli strains. The expressions of bcs proteins were shown by SDS and Native page analyses. The functionality of the bcs operon was proved by congo red binding assay. The effect of culturing temperature and the inducer concentration (IPTG) on cell growth and plasmid stability were monitored. The percentage of plasmid harboring cells induced with 0.025 mM IPTG was obtained as 85% at 22˚C in the end of 10-hr culturing period. It was confirmed that the high output cellulose production machinery of A.xylinum can be transferred into other organisms.

Keywords: bacterial cellulose, biopolymer, recombinant expression system, production

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Authors: Omneya M. Helmy, Mona T. Kashef

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Aminoglycosides are used in treating a wide range of infections caused by both Gram-negative and Gram positive bacteria. The presence of 16S rRNA methyl transferases (16S-RMTase) is among the newly discovered resistance mechanisms that confer high resistance to clinically useful aminoglycosides. Cephalosporins are the most commonly used antimicrobials in Egypt; therefore, this study was conducted to determine the isolation frequency of 16S rRNA methyl transferases among third generation cephalosporin-resistant clinical isolates in Egypt. One hundred and twenty three cephalosporin resistant Gram-negative clinical isolates were screened for aminoglycoside resistance by the Kirby Bauer disk diffusion method and tested for possible production of 16S-RMTase. PCR testing and sequencing were used to confirm the presence of 16S-RMTase and the associated antimicrobial resistance determinants, as well as the genetic region surrounding the armA gene. Out of 123 isolates, 66 (53.66%) were resistant to at least one aminoglycoside antibiotic. Only one Escherichia coli isolate (E9ECMO) which was totally resistant to all tested aminoglycosides, was confirmed to have the armA gene in association with blaTEM-1, blaCTX-M-15, blaCTX-M-14 and aac(6)-Ib genes. The armA gene was found to be carried on a large A/C plasmid. Genetic mapping of the armA surrounding region revealed, for the first time, the association of armA with aac(6)-Ib on the same transposon. In Conclusion, the isolation frequency of 16S-RMTase was low among the tested cephalosporin-resistant clinical samples. However, a novel composite transposon has been detected conferring high-level aminoglycosides resistance.

Keywords: aminoglcosides, armA gene, β lactmases, 16S rRNA methyl transferases

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Authors: Garima Chaudhary

Abstract:

The gender of an individual in forensic DNA analysis is normally accessed by using the STR multiplexes with the incorporated gender based marker amelogenin or in other words by presence or absence of Y-Chromosome, but it may not be true in all the cases. We hereby report an interesting case of a phenotypic female carrying a male karyotype (46XY). In the alleged murder case, the deceased female with XY genotype was noticed. The expression of 18 Y-linked genes was studied to measure the extent of expression. Expression at 4 loci was observed that might have caused the misinterpretation in forensic casework. This clinical situation of the deceased in this case was diagnosed as testicular feminization syndrome, which characterize a female phenotype with a male karyotype (46, XY). Most of these cases have SRY (testis determining factor). The genetic explanation of this phenomenon is not very clear. Here, we are discussing the impact of such situations of genetic discrepancy in forensic interpretation of results. In the presented murder case of a phenotypic female, sexual assault was also suspected. For confirmation vaginal swabs and micro slides were also sent to us for DNA examination. After DNA analysis using STR markers, Y-chromosome was detected in the samples which supporting the suspicion of sexual assault before murder. When the reference blood sample of the deceased was analyzed, it was found to be case of testicular feminization syndrome. Interesting inferences were made from the results obtained.

Keywords: DNA profiling, forensic case study, Y chromosome, females

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Authors: Precious Barnes, Abraham Mensah, Leonard Derkyi-Kwarteng, Benjamin Amoani, George Adjei, Ernest Adankwah, Faustina Pappoe, Kwabena Dankwah, Daniel Amoako-Sakyi, Samuel Victor Nuvor, Dorcas Obiri-Yeboah, Ewura Seidu Yahaya, Patrick Kafui Akakpo, Roland Osei Saahene

Abstract:

Context: Breast cancer is a prevalent and aggressive type of cancer among African women, with high mortality rates in Ghana. Nuclear factor kappa B (NF-kB) is a transcription factor that has been associated with tumor progression in breast cancer. However, there is a lack of published data on NF-kB in breast cancer patients in Ghana or other African countries. Research Aim: The aim of this study was to assess the prognostic significance of NF-kB (p65) expression and its association with various clinicopathological features in breast cancer patients at the Cape Coast Teaching Hospital in Ghana. Methodology: A total of 90 formalin-fixed breast cancer tissues and 15 normal breast tissues were used in this study. The expression level of NF-kB (p65) was examined using immunohistochemical techniques. Correlation analysis between NF-kB (p65) expression and clinicopathological features was performed using SPSS version 25. Findings: The study found that NF-kB (p65) was expressed in 86.7% of breast cancer tissues. There was a significant relationship between NF-kB (p65) expression and tumor grade, proliferation index (Ki67), and molecular subtype. High-level expression of NF-kB (p65) was more common in tumor grade 3 compared to grade 1, and Ki67 > 20 had higher expression of NF-kB (p65) compared to Ki67 ≤ 20. Triple-negative breast cancer patients had the highest overexpression of NF-kB (p65) compared to other molecular subtypes. There was no significant association between NF-kB (p65) expression and other clinicopathological parameters. Theoretical Importance: This study provides important insights into the expression of NF-kB (p65) in breast cancer patients in Ghana, particularly in relation to tumor grade and proliferation index. The findings suggest that NF-kB (p65) could serve as a potential biological marker for cancer stage, progression, prognosis and as a therapeutic target. Data Collection and Analysis Procedures: Formalin-fixed breast cancer tissues and normal breast tissues were collected and analyzed using immunohistochemical techniques. Correlation analysis between NF-kB (p65) expression and clinicopathological features was performed using SPSS version 25. Question Addressed: This study addressed the question of the prognostic significance of NF-kB (p65) expression and its association with clinicopathological features in breast cancer patients in Ghana. Conclusion: This study, the first of its kind in Ghana, demonstrates that NF-kB (p65) is highly expressed among breast cancer patients at the Cape Coast Teaching Hospital, especially in triple-negative breast cancer patients. The expression of NF-kB (p65) is associated with tumor grade and proliferation index. NF-kB (p65) could potentially serve as a biological marker for cancer stage, progression, prognosis, and as a therapeutic target.

Keywords: breast cancer, Ki67, NF-kB (p65), tumor grade

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Authors: Carlo Stephen O. Moneva, Sharon Rose M. Tabugo

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The present study aims to assess polymorphism in the prolactin (C576A) gene and determine the influence of different prolactin (PRL) genotypes to milk yield performance in crossbred Anglo-Nubian dairy goats raised from Awang, Opol, Misamis Oriental and Talay, Dumaguete City, Negros Oriental. Genomic DNA was extracted from hair follicles and Polymerase Chain Reaction – Restriction Fragment Length Polymorphism (PCR-RFLP) was performed for the genotyping of the C576A polymorphism located in exon 5 of goats’ prolactin gene using Eco241 restriction enzyme. Genotypic and allelic frequencies of 0.56 for AA, 0.44 for AB, 0.78 for A, and 0.22 for B were recorded. Observed heterozygosity values were higher than the expected heterozygosity. All populations followed the Hardy–Weinberg principle at p>0.05, except for dairy goats from Farm A located in Opol, Misamis Oriental. A two-way factorial (2 x 4) in a Randomized Complete Block Design was used to be able to evaluate the relationship between genotypes and milk yield performance. PRL genotypes and parity were used as main factors and farm as the blocking factor. AB genotype goats produced significantly higher average daily milk yield and total milk production than AA genotype (p<0.05), an indication that the polymorphism in the caprine PRL (C576A) gene influenced milk yield performance in the population of crossbred Anglo-Nubian goats from Opol, Misamis Oriental and Dumaguete City, Negros Oriental. However, these results have to be validated in other dairy goat breeds.

Keywords: polymorphism, prolactin, milk yield, Anglo-Nubian, PCR-RFLP

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Authors: Z. Chekini, P. Afsharian, F. Ramezanali, A. A. Akhlaghi, R. Aflatoonian

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Macrophage migration inhibitory factor (MIF) is a key pro-inflammatory cytokine that involves in pathophysiological events of endometriosis. We aimed to evaluate the association between mRNA expression levels and polymorphisms of MIF in endometriosis. Seventy endometriosis patients and 70 volunteer fertile women were recruited. RFLP was applied to determine -173G/C polymorphism. ORF polymorphisms and -794(CATT)5-8 were detected by sequencing. Q-PCR was used for expression study of 14 ectopic tissues of patients. Homozygote of CATT5 was observed only in controls. The CATT5/G haplotype related to controls (p=0.094, OR=0.61). Expression level of MIF with -794(CATT)6,7/-173GC was significantly more than the other haplotypes (p=0.00). We identified four SNPs including: +254rs2096525 (p=0.843), +626rs33958703 (p=0.029), +656rs2070766 (p=0.703) and +509rs182012324 (p=1.00). In conclusion, increased repeat of CATT and presence of C allele in promoter of MIF were significantly associated with mRNA level in patients. It seems that +509rs182012324 and +626rs33958703 SNPs were significantly correlated with susceptibility to endometriosis.

Keywords: endometriosis, haplotype, macrophage migration inhibitory factor, polymorphism

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Authors: Bianca Peterson, Tomasz J. Sańko, Carlos C. Bezuidenhout, Johnnie Van Den Berg

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Busseola fusca (Fuller) (Lepidoptera: Noctuidae), the maize stem borer, is a major pest in sub-Saharan Africa. It causes economic damage to maize and sorghum crops and has evolved non-recessive resistance to genetically modified (GM) maize expressing the Cry1Ab insecticidal toxin. Since B. fusca is a non-model organism, very little genomic information is publicly available, and is limited to some cytochrome c oxidase I, cytochrome b, and microsatellite data. The biology of B. fusca is well-described, but still poorly understood. This, in combination with its larval-specific behavior, may pose problems for limiting the spread of current resistant B. fusca populations or preventing resistance evolution in other susceptible populations. As part of on-going research into resistance evolution, B. fusca larvae were collected from Bt and non-Bt maize in South Africa, followed by RNA isolation (15 specimens) and sequencing on the Illumina HiSeq 2500 platform. Quality of reads was assessed with FastQC, after which Trimmomatic was used to trim adapters and remove low quality, short reads. Trinity was used for the de novo assembly, whereas TransRate was used for assembly quality assessment. Transcript identification employed BLAST (BLASTn, BLASTp, and tBLASTx comparisons), for which two libraries (nucleotide and protein) were created from 3.27 million lepidopteran sequences. Several transcripts that have previously been implicated in Cry toxin resistance was identified for B. fusca. These included aminopeptidase N, cadherin, alkaline phosphatase, ATP-binding cassette transporter proteins, and mitogen-activated protein kinase. MEGA7 was used to align these transcripts to reference sequences from Lepidoptera to detect mutations that might potentially be contributing to Cry toxin resistance in this pest. RSEM and Bioconductor were used to perform differential gene expression analysis on groups of B. fusca larvae challenged and unchallenged with the Cry1Ab toxin. Pairwise expression comparisons of transcripts that were at least 16-fold expressed at a false-discovery corrected statistical significance (p) ≤ 0.001 were extracted and visualized in a hierarchically clustered heatmap using R. A total of 329,194 transcripts with an N50 of 1,019 bp were generated from the over 167.5 million high-quality paired-end reads. Furthermore, 110 transcripts were over 10 kbp long, of which the largest one was 29,395 bp. BLAST comparisons resulted in identification of 157,099 (47.72%) transcripts, among which only 3,718 (2.37%) were identified as Cry toxin receptors from lepidopteran insects. According to transcript expression profiles, transcripts were grouped into three subclusters according to the similarity of their expression patterns. Several immune-related transcripts (pathogen recognition receptors, antimicrobial peptides, and inhibitors) were up-regulated in the larvae feeding on Bt maize, indicating an enhanced immune status in response to toxin exposure. Above all, extremely up-regulated arylphorin genes suggest that enhanced epithelial healing is one of the resistance mechanisms employed by B. fusca larvae against the Cry1Ab toxin. This study is the first to provide a resource base and some insights into a potential mechanism of Cry1Ab toxin resistance in B. fusca. Transcriptomic data generated in this study allows identification of genes that can be targeted by biotechnological improvements of GM crops.

Keywords: epithelial healing, Lepidoptera, resistance, transcriptome

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Authors: Mandeep Singh Azad.Dibyendu Chakraborty, Vikas Vohra

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Introduction: Poonch is one of the remotest districts of the Jammu and Kashmir (UT) and situated on international borders. This native poultry population in these areas is quite hardy and thrives well in adverse climatic conditions. Till date, no local breed from this area (Jammu Province) has been characterized thus present study was undertaken with the main objectives of molecular characterization of ChB6 gene in local native chicken of Poonch region located at international borders between India and Pakistan. The chicken B-cell marker (ChB6) gene has been proposed as a candidate gene in regulating B-cell development. Material and Method: RNA was isolated by Blood RNA Purification Kit (HiPura) and Trizol method from whole blood samples. Positive PCR products with size 1110 bp were selected for further purification, sequencing and analysis. The amplified PCR product was sequenced by Sangers dideoxy chain termination method. The obtained sequence of ChB6 gene of Poonchi chicken were compared by MEGAX software. BioEdit software was used to construct phylogenic tree, and Neighbor Joining method was used to infer evolutionary history. In order to compute evolutionary distance Maximum Composite Likelihood method was used. Results: The positively amplified samples of ChB6 genes were then subjected to Sanger sequencing with “Primer Walking. The sequences were then analyzed using MEGA X and BioEdit software. The sequence results were compared with other reported sequence from different breed of chicken and with other species obtained from the NCBI (National Center for Biotechnology Information). ClustalW method using MEGA X software was used for multiple sequence alignment. The sequence results of ChB6 gene of Poonchi chicken was compared with Centrocercus urophasianus, G. gallus mRNA for B6.1 protein, G. gallus mRNA for B6.2, G. gallus mRNA for B6.3, Gallus gallus B6.1, Halichoeres bivittatus, Miniopterus fuliginosus Ferringtonia patagonica, Tympanuchus phasianellus. The genetic distances were 0.2720, 0.0000, 0.0245, 0.0212, 0.0147, 1.6461, 2.2394, 2.0070 and 0.2363 for ChB6 gene of Poonchi chicken sequence with other sequences in the present study respectively. Sequencing results showed variations between different species. It was observed that AT content were higher then GC content for ChB6 gene. The lower AT content suggests less thermostable. It was observed that there was no sequence difference within the Poonchi population for ChB6 gene. The high homology within chicken population indicates the conservation of ChB6 gene. The maximum difference was observed with Miniopterus fuliginosus (Eastern bent-wing bat) followed by Ferringtonia patagonica and Halichoeres bivittatus. Conclusion: Genetic variation is the essential component for genetic improvement. The results of immune related gene Chb6 shows between population genetic variability. Therefore, further association studies of this gene with some prevalent diseases in large population would be helpful to identify disease resistant/ susceptible genotypes in the indigenous chicken population.

Keywords: ChB6, sequencing, ClustalW, genetic distance, poonchi chicken, SNP

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Authors: Mohammad Faheem, M. Tabish Rehman, Mohd Danishuddin, Asad U. Khan

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The worldwide dissemination of CTX-M type β-lactamases is a threat to human health. Previously, we have reported the spread of blaCTX-M-15 gene in different clinical strains of Enterobacteriaceae from the hospital settings of Aligarh in north India. In view of the varying resistance pattern against cephalosporins and other β-lactam antibiotics, we intended to understand the correlation between MICs and catalytic activity of CTX-M-15. In this study, steady-state kinetic parameters and MICs were determined on E. coli DH5α transformed with blaCTX-M-15 gene that was cloned from Enterobacter cloacae (EC-15) strain of clinical background. The effect of conventional β-lactamase inhibitors (clavulanic acid, sulbactam and tazobactam) on CTX-M-15 was also studied. We have found that tazobactam is the best among these inhibitors against CTX-M-15. The inhibition characteristic of tazobactam is defined by its very low IC50 value (6 nM), high affinity (Ki = 0.017 µM) and better acylation efficiency (k+2/K9 = 0.44 µM-1s-1). It forms an acyl-enzyme covalent complex, which is quite stable (k+3 = 0.0057 s-1). Since increasing resistance has been reported against conventional b-lactam antibiotic-inhibitor combinations, we aspire to design a non-b-lactam core containing b-lactamase inhibitor. For this, we screened ZINC database and performed molecular docking to identify a potential non-β-lactam based inhibitor (ZINC03787097). The MICs of cephalosporin antibiotics in combination with this inhibitor gave promising results. Steady-state kinetics and molecular docking studies showed that ZINC03787097 is a reversible inhibitor which binds non-covalently to the active site of the enzyme through hydrogen bonds and hydrophobic interactions. Though, it’s IC50 (180 nM) is much higher than tazobactam, it has good affinity for CTX-M-15 (Ki = 0.388 µM). This study concludes that ZINC03787097 compound can be used as seed molecule to design more efficient non-b-lactam containing b-lactamase inhibitor that could evade pre-existing bacterial resistance mechanisms.

Keywords: ESBL, non-b-lactam-b-lactamase inhibitor, bioinformatics, biomedicine

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