Search results for: genomic

Authors: Anyaphat Srithanasuwan, Noppason Pangprasit, Montira Intanon, Phongsakorn Chuammitri, Witaya Suriyasathaporn, Ynte H. Schukken

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Streptococcus uberis is one of the most common mastitis-causing pathogens, with a wide range of intramammary infection (IMI) durations and pathogenicity. This study aimed to compare shared or unique virulence factor gene clusters distinguishing persistent and transient strains of S. uberis. A total of 139 S. uberis strains were isolated from three small-holder dairy herds with a high prevalence of S. uberis mastitis. The duration of IMI was used to categorize bacteria into two groups: transient and persistent strains with an IMI duration of less than 1 month and longer than 2 months, respectively. Six representative S. uberis strains, three from each group (transience and persistence) were selected for analysis. All transient strains exhibited multi-locus sequence types (MLST), indicating a highly diverse population of transient S. uberis. In contrast, MLST of persistent strains was available in an online database (pubMLST). Identification of virulence genes was performed using whole-genome sequencing (WGS) data. Differences in genomic size and number of virulent genes were found. For example, the BCA gene or alpha-c protein and the gene associated with capsule formation (hasAB), found in persistent strains, are important for attachment and invasion, as well as the evasion of the antimicrobial mechanisms and survival persistence, respectively. These findings suggest a genetic-level difference between the two strain types. Consequently, a comprehensive study of 139 S. uberis isolates will be conducted to perform an in-depth genetic assessment through WGS analysis on an Illumina platform.

Keywords: Streptococcus Uberis, mastitis, whole genome sequence, intramammary infection, persistent S. Uberis, transient s. Uberis

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Authors: Sreeja Shaw, Prosenjit Samanta, Asish Kumar Mukhopadhyay

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Cholera is still a global public health burden and is caused by Vibrio cholerae O1 and O139 serogroups. Evidence from recent outbreaks in Haiti and Yemen suggested that circulating V. cholerae O1 El Tor variant strains are continuously changing to cause more ruinous outbreaks worldwide, and most of them have emerged from the Indian subcontinents. Therefore, we studied the changing virulence characteristics along with the antibiotic resistance profile of V. cholerae O1strains isolated from seasonal outbreaks in three cholera endemic regions during 2018, Gujarat and Maharashtra in Western India (87 strains), and to compare those features with the isolates of West Bengal in Eastern India (48 strains) collected during the same period. All the strains from Western India were of Ogawa serotype, polymyxin B-sensitive, hemolytic, and contained a large fragment deletion in VSP-II genomic region similar with Yemen outbreak strains and carried more virulent Haitian genetic alleles of major virulence associated genes ctxB, tcpA, and rtxA. Conversely, 14.6% (7/48) of the strains from Eastern India were belong to the Inaba serotype, polymyxin B-resistant, non-hemolytic, harbored intact VSP-II region, classical ctxB, Haitian tcpA, and El Tor rtxA alleles. Interestingly, resistance to tetracycline and chloramphenicol was seen in isolates from both regions, which are not very common among V. cholerae O1 isolates in India. Therefore, this study indicated West Bengal as a diverse region where two different types of El Tor variant hypervirulent strains are co-existed, probably competing for their better environmental survival, which may result in severe irrepressible disease outcome in the future.

Keywords: cholera, vibrio cholerae, polymyxin B, Non-hemolytic, ctxB, tcpA, rtxA, VSP-II

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Authors: Hidayah Triana, Mahir S. Gani, Asmi Citra Malina, Hamka

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This study was conducted to determine genetic diversity of tiger groupers that are resistant to V. parahaemolyticus and tolerant to low extreme salinities. This research is useful to obtain superior broodstock of fish. Tiger grouper used were 6 to 8 cm obtained from Brackish Water Aquaculture Research Center Gondol (Bali). This study consists of four stages: preliminary stage was adaptation of fish exposed to several concentrations of V. parahaemolyticus (103, 104, 105, 106, and 107 CFU / ml); second stage was test of Lethal Concentration (LC50) of bacteria to fish; third stage was salinity tolerance test (low salinity 12, 14 and 16 ppt) and fourth stage was analysis of DNA profiles. For DNA profiles analysis, genomic DNA of fish were extracted for PCR using primers YNZ-22 and UBC-122 and visualized by electrophoresis method. The results showed that Lethal concentration of bacteria (LC50) to fish was 1,56x106 CFU/ml. Furthermore, survival rate of groupers exposed with low salinities (12, 14, 16 ppt) survival rates were found to be 54,17 %, 66,67 % and 79,16 % respectively. Average of DNA fragment (5 fragments) generated from primer UBC-122 in the group of fish resistant to V.parahaemolyticus and tolerant to low salinities was similar to group of susceptible to low salinities. Primer YNZ-22 generated more diverse of DNA fragments (8,0 and 5,8 fragments) both in the group of fish tolerant and susceptible to low salinities compared to primer UBC-122 (5,0 fragments). Size of DNA 1.5 kb resulted from primer YNZ-22. Primer YNZ-22 generated 4 (50 %) and 3 (42,8 %) polymorfic fragments in the group of fish tolerant and susceptible to low salinities, respectively. Four (4) monomorfic fragments were found both in the group of fish tolerant and susceptible to low salinities. Primer UBC-122 generated 6 (85,7 %) and 9 (90,0 %) polymorfic fragments in the fish tolerant and susceptible to low salinities, respectively.

Keywords: genetic diversity, epinephelus fuscoguttatus, V. parahaemolyticus, PCR-RAPD, low extreme salinity

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Authors: Faisal Nouroz, Mukaramin Mukaramin

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Of the transposable elements (TEs), the retrotransposons are the most copious elements identified from many sequenced genomes. They have played a major role in genome evolution, rearrangement, and expansions based on their copy and paste mode of proliferation. They are further divided into LTR and Non-LTR retrotransposons. The purpose of the current study was to identify the LTR REs in sequenced Phoenix dactylifera genome and to study their structural diversity. A total of 150 P. dactylifera BAC sequences with > 60kb sizes were randomly retrieved from National Center for Biotechnology Information (NCBI) database and screened for the presence of LTR retrotransposons. Seven bacterial artificial chromosomes (BAC) sequences showed full-length LTR Retrotransposons with 4 Copia and 3 Gypsy families having variable copy numbers in respective families. Reverse transcriptase (RT) domain was found as the most conserved domain among Copia and Gypsy superfamilies and was used to deduce evolutionary analysis. The amino acid residues among various RT sequences showed variability in their percentages indicating post divergence evolution. Amino acid Leucine was found in highest proportions followed by Lysine, while Methionine and Tryptophan were in lowest percentages. The phylogenetic analysis based on RT domains confirmed that although having most conserved RT regions, several evolutionary events occurred causing nucleotide polymorphisms and hence clustering of Gypsy and Copia superfamilies into their respective lineages. The study will be helpful in identification and annotation of these elements in other species and genera and their distribution patterns on chromosomes by fluorescent in situ hybridization techniques.

Keywords: transposable elements, Phoenix dactylifera, retrotransposons, phylogenetic analysis

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Authors: Najeeb Ullah Khan

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Background: The receptor activator NF-κβ ligand (RANKL) and Osteoprotegerin (OPG) polymorphisms have been associated with the progression of breast cancer to bone metastasis. Here, we aimed to investigate the association of RANKL and OPG gene polymorphism with breast to bone metastasis in the Pashtun population, Pakistan. Methods: Genomic DNA was obtained from all the study subjects (106 breast cancer, 58 breast to bone metastasis, and 51 healthy controls). RANKL (rs9533156) and OPG (rs2073618, rs3102735) polymorphisms were genotyped using Tetra-ARMS PCR. Results: Our results indicated that the frequencies of OPG (rs3102735) risk allele and genotypes carrying risk allele in breast cancer vs healthy control (C- p=0.005; CC- p=0.0208; TC- p=0.0181), bone metastasis vs healthy control (C- p=0.0211; CC- p=0.0153; TC- p=0.0775), and breast cancer vs breast to bone metastasis (C- p=0.0001; CC- p=0.0001; TC- p=0.001) were found significantly associated with disease risk. However, there was no significant association observed for OPG (rs2073618) risk allele and risk allele containing genotypes in all study groups. Similarly, RANKL (rs9533156) risk alleles and corresponding genotypes in breast cancer vs healthy control (C- p=0.0001; CC- p=0.0001; TC- p=0.0084), bone metastasis vs healthy control (C- p=0.0001; CC- p=0.0001; TC- p=0.5593), and breast cancer vs breast to bone metastasis (C- p=0.0185; CC- p=0.6077; TC- p=0.1436) showed significant association except for the risk allele carrying genotypes in breast cancer to bone metastasis (TC, p=0.1436; CC, p=0.6077). Conclusion: OPG (rs3102735) and RANKL (rs9533156) showed significant association with breast to bone metastasis, while OPG (rs2073618) didn’t show a significant association with breast to bone metastasis in Pashtun population of Pakistan. However, more investigation will be required to disseminate the results while gene sequencing or whole-exome sequencing.

Keywords: breast cancer, bone metastasis, OPG, RANKL, polymorphism

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Authors: Hamzeh Alipour, Masoumeh Bagheri, Tahereh Karamzadeh, Abbasali Raz, Kourosh Azizi

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Netrin-A, a protein identified for conducting commissural axons, has a similar role in angiogenesis. In addition, studies have shown that one of the netrin-A receptors is expressed in the growing cells of small capillaries. It will be interesting to study this new group of molecules because their role in wound healing will become clearer in the future due to angiogenesis. The greenbottle blowfly Luciliasericata (L. sericata) larvae are increasingly used in maggot therapy of chronic wounds. This aim of this was the identification of moleculareatures of Netrin-A in L. sericata larvae. Larvae were reared under standard maggotarium conditions. The nucleic acid sequence of L. sericataNetrin-A (LSN-A) was then identified using Rapid Amplification of cDNA Ends (RACE) and Rapid Amplification of Genomic Ends (RAGE). Parts of the Netrin-A gene, including the middle, 3′-, and 5′-ends were identified, TA cloned in pTG19 plasmid, and transferred into DH5ɑ Escherichia coli. Each part was sequenced and assembled using SeqMan software. This gene structure was further subjected to in silico analysis. The DNA of LSN-A was identified to be 2407 bp, while its mRNA sequence was recognized as 2115 bp by Oligo0.7 software. It translated the Netrin-A protein with 704 amino acid residues. Its molecular weight is estimated to be 78.6 kDa. The 3-D structure ofNetrin-A drawn by SWISS-MODEL revealed its similarity to the Netrin-1 of humans with 66.8% identity. The LSN-A protein conduces to repair the myelin membrane in neuronal cells. Ultimately, it can be an effective candidate in neural regeneration and wound healing. Furthermore, our next attempt is to deplore recombinant proteins for use in medical sciences.

Keywords: maggot therapy, netrin-A, RACE, RAGE, lucilia sericata

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Authors: Mamdouh Milad Adly Morkos

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Despite having the greatest rates of mortality and morbidity in the world, low- and middle-income (LMIC) nations trail high-income nations in terms of the number of clinical trials, the number of qualified researchers, and the amount of research information specific to their people. Health inequities and the use of precision medicine may be hampered by a lack of local genomic data, clinical pharmacology and pharmacometrics competence, and training opportunities. These issues can be solved by carrying out health care infrastructure development, which includes data gathering and well-designed clinical pharmacology training in LMICs. It will be advantageous if there is international cooperation focused at enhancing education and infrastructure and promoting locally motivated clinical trials and research. This paper outlines various instances where clinical pharmacology knowledge could be put to use, including pharmacogenomic opportunities that could lead to better clinical guideline recommendations. Examples of how clinical pharmacology training can be successfully implemented in LMICs are also provided, including clinical pharmacology and pharmacometrics training programmes in Africa and a Tanzanian researcher's personal experience while on a training sabbatical in the United States. These training initiatives will profit from advocacy for clinical pharmacologists' employment prospects and career development pathways, which are gradually becoming acknowledged and established in LMICs. The advancement of training and research infrastructure to increase clinical pharmacologists' knowledge in LMICs would be extremely beneficial because they have a significant role to play in global health

Keywords: electromagnetic solar system, nano-material, nano pharmacology, pharmacovigilance, quantum theoryclinical simulation, education, pharmacology, simulation, virtual learning low- and middle-income, clinical pharmacology, pharmacometrics, career development pathways

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Authors: Vijay Kumar Singh, Pooja Kushwaha, Prabhat Ranjan, Krishna Kumar Ojha, Jitendra Kumar

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Present day high-throughput genomic technologies, NGS/microarrays, are producing large volume of data that require improved analysis methods to make sense of the data. The correlation between genes and samples has been regularly used to gain insight into many biological phenomena including, but not limited to, co-expression/co-regulation, gene regulatory networks, clustering and pattern identification. However, presence of outliers and violation of assumptions underlying Pearson correlation is frequent and may distort the actual correlation between the genes and lead to spurious conclusions. Here, we report a method to measure the strength of association between genes. The method assumes that the expression values of a gene are Bernoulli random variables whose outcome depends on the sample being probed. The method considers the two genes as uncorrelated if the number of sample with same outcome for both the genes (Ns) is equal to certainly expected number (Es). The extent of correlation depends on how far Ns can deviate from the Es. The method does not assume normality for the parent population, fairly unaffected by the presence of outliers, can be applied to qualitative data and it uses the binomial distribution to assess the significance of association. At this stage, we would not claim about the superiority of the method over other existing correlation methods, but our method could be another way of calculating correlation in addition to existing methods. The method uses binomial distribution, which has not been used until yet, to assess the significance of association between two variables. We are evaluating the performance of our method on NGS/microarray data, which is noisy and pierce by the outliers, to see if our method can differentiate between spurious and actual correlation. While working with the method, it has not escaped our notice that the method could also be generalized to measure the association of more than two variables which has been proven difficult with the existing methods.

Keywords: binomial distribution, correlation, microarray, outliers, transcriptome

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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: Thobela Conco, Sheena Kumari, Chika Nnadozie, Mahmoud Nasr, Thor A. Stenström, Mushal Ali, Arshad Ismail, Faizal Bux

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The discharge of antibiotics and its residues into the wastewater treatment plants (WWTP’s) create a conducive environment for the development of antibiotic resistant pathogens. This presents a risk of potential dissemination of antibiotic resistant pathogens and antibiotic resistance genes into the environment. It is, therefore, necessary to study the level of antibiotic resistance genes (ARG’s) among bacterial pathogens that proliferate in biological wastewater treatment systems. In the current study, metagenomic and meta-transcriptomic sequences of samples collected from the influents, secondary effluents and post chlorinated effluents of three wastewater treatment plants treating domestic and hospital effluents in Durban, South Africa, were analyzed for profiling of ARG’s among bacterial pathogens. Results show that a variety of ARG’s, mostly, aminoglycoside, β-lactamases, tetracycline and sulfonamide resistance genes were harbored by diverse bacterial genera found at different stages of treatment. A significant variation in diversity of pathogen and ARGs between the treatment plant was observed; however, treated final effluent samples from all three plants showed a significant reduction in bacterial pathogens and detected ARG’s. Both pre- and post-chlorinated samples showed the presence of mobile genetic elements (MGE’s), indicating the inefficiency of chlorination to remove of ARG’s integrated with MGE’s. In conclusion, the study showed the wastewater treatment plant efficiently caused the reduction and removal of certain ARG’s, even though the initial focus was the removal of biological nutrients.

Keywords: antibiotic resistance, mobile genetic elements, wastewater, wastewater treatment plants

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Authors: Kidane workelul, Li xu, Xiaoyang Pang, Jiaping Lv

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Dairy products are exceptionally ideal media for the growth of microorganisms because of their high nutritional content. There are several ways that milk might get contaminated throughout the milking process, including how the raw milk is transported and stored, as well as how long it is kept before being processed. Psychrotrophic bacteria are among the one which can deteriorate the quality of milk mainly their heat resistance proteas and lipase enzyme. For this research purpose 8 selected strains of Psychrotrophic bacteria (Entrococcus hirae, Pseudomonas fluorescens, Pseudomonas azotoformans, Pseudomonas putida, Exiguobacterium indicum, Pseudomonas paralactice, Acinetobacter indicum, Serratia liquefacients)are chosen and try to determine their characteristics based on the research methodology protocol. Thus, the 8 selected strains are cultured, plated incubate, extracted their genomic DNA and genome DNA was amplified, the purpose of the study was to identify their Psychrotrophic properties, lipase hydrolysis positive test, their optimal incubation temperature, designed primer using the noble strain P,flourescens conserved region area in target with lipA gene, optimized primer specificity as well as sensitivity and PCR detection for lipase positive strains using the design primers. Based on the findings both the selected 8 strains isolated from stored raw milk are Psychrotrophic bacteria, 6 of the selected strains except the 2 strains are positive for lipase hydrolysis, their optimal temperature is 20 to 30 OC, the designed primer specificity is very accurate and amplifies for those strains only with lipase positive but could not amplify for the others. Thus, the result is promising and could help in detecting the Psychrotrophic bacteria producing heat resistance enzymes (lipase) at early stage before the milk is processed and this will safe production loss for the dairy industry.

Keywords: dairy industry, heat-resistant, lipA, milk, primer and psychrotrophic

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Authors: Samaneh Nabavi, Hadi Shirzad, Somayeh Khanjani, Shirin Jalili

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Quantum dots (QDs) are semiconductor nanocrystals that exhibit intrinsic optical and electrical properties that are size dependent due to the quantum confinement effect. Quantum confinement is brought about by the fact that in bulk semiconductor material the electronic structure consists of continuous bands, and that as the size of the semiconductor material decreases its radius becomes less than the Bohr exciton radius (the distance between the electron and electron-hole) and discrete energy levels result. As a result QDs have a broad absorption range and a narrow emission which correlates to the band gap energy (E), and hence QD size. QDs can thus be tuned to give the desired wavelength of fluorescence emission.Due to their unique properties, QDs have attracted considerable attention in different scientific areas. Also, they have been considered for forensic applications in recent years. The ability of QDs to fluoresce up to 20 times brighter than available fluorescent dyes makes them an attractive nanomaterial for enhancing the visualization of latent fingermarks, or poorly developed fingermarks. Furthermore, the potential applications of QDs in the detection of nitroaromatic explosives, such as TNT, based on directive fluorescence quenching of QDs, electron transfer quenching process or fluorescence resonance energy transfer have been paid to attention. DNA analysis is associated tightly with forensic applications in molecular diagnostics. The amount of DNA acquired at a criminal site is inherently limited. This limited amount of human DNA has to be quantified accurately after the process of DNA extraction. Accordingly, highly sensitive detection of human genomic DNA is an essential issue for forensic study. QDs have also a variety of advantages as an emission probe in forensic DNA quantification.

Keywords: forensic science, quantum dots, DNA typing, explosive sensor, fingermark analysis

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Authors: Techale B., Hongxu Dong, Mihrete Getinet, Aregash Gabizew, Andrew H. Paterson, Kassahun Bantte

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The genetic architecture of drought tolerance is expected to involve multiple loci that are unlikely to all segregate for alternative alleles in a single bi-parental population. Therefore, the identification of quantitative trait loci (QTL) that are expressed in diverse genetic backgrounds of multiple bi-parental populations provides evidence about both background-specific and common genetic variants. The purpose of this study was to map QTL related to drought tolerance using three connected mapping populations of different genetic backgrounds to gain insight into the genomic landscape of this important trait in elite Ethiopian germplasm. The three bi-parental populations, each with 207 F₂:₃ lines, were evaluated using an alpha lattice design with two replications under two moisture stress environments. Drought tolerance related traits were analyzed separately for each population using composite interval mapping, finding a total of 105 QTLs. All the QTLs identified from individual populations were projected on a combined consensus map, comprising a total of 25 meta QTLs for seven traits. The consensus map allowed us to deduce locations of a larger number of markers than possible in any individual map, providing a reference for genetic studies in different genetic backgrounds. The mQTL identified in this study could be used for marker-assisted breeding programs in sorghum after validation. Only one trait, reduced leaf senescence, showed a striking bias of allele distribution, indicating substantial standing variation among present varieties that might be employed in improving drought tolerance of Ethiopian and other sorghums.

Keywords: Drought tolerance , Mapping populations, Meta QTL, QTL mapping, Sorghum

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Authors: A. B. M. S. Hossain, A. Abdelgadir, N. A. Ibrahim

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Fresh ripen olive cannot be consumed immediately after harvest due to the excessive bitterness having polyphenol as antioxidant. Industrial processing needs to be edible the fruit. The laboratory processing technique has been used to make it edible by using acid (vinegar, 5% acetic acid) and alkaline solvent (NaOH). Based on the treatment and consequence, innovative data have been found in this regard. The experiment was conducted to investigate biochemical content, nutritional and DNA characterization of olive fruit treated with alkaline (Sodium chloride anhydrous) and acidic solvent (5% acetic acid, vinegar). The treatments were used as control (no water), water control, 10% sodium chloride anhydrous (NaOH), vinegar (5% acetic acid), vinegar + NaOH and vinegar + NaOH + hot water treatment. Our results showed that inverted sugar and glucose content were higher in the vinegar and NaOH treated olive than in other treatments. Fructose content was the highest in vinegar + NaOH treated fruit. Nutrient contents NO3 K, Ca and Na were found higher in the treated fruit than the control fruit. Moreover, maximum K content was observed in the case of all treatments compared to the other nutrient content. The highest acidic (lower pH) condition (sour) was found in treated fruit. DNA yield was found higher in water control than acid and alkaline treated olives. DNA band was wider in the olive treated water control compared to the NaOH, vinegar, vinegar + NaOH and vinegar + NaOH + Hot water treatment. Finally, results suggest that vinegar + NaOH treated olive fruit was the best for fresh olive homemade processing after harvesting for edible purpose.

Keywords: olive, vinegar, sugars, DNA band, bioprocess biotechnology

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Authors: Peng Hou

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Natural products produced from marine bacteria serve as an immense reservoir for anti-infective drugs and therapeutic agents. Nowadays, heterologous expression of gene clusters of interests has been widely adopted as an effective strategy for natural product discovery. Briefly, the heterologous expression flowchart would be: biosynthetic gene cluster identification, pathway construction and expression, and product detection. However, gene cluster capture using traditional Transformation-associated recombination (TAR) protocol is low-efficient (0.5% positive colony rate). To make things worse, most of these putative new natural products are only predicted by bioinformatics analysis such as antiSMASH, and their corresponding natural products biosynthetic pathways are either not expressed or expressed at very low levels under laboratory conditions. Those setbacks have inspired us to focus on seeking new technologies to efficiently edit and refractor of biosynthetic gene clusters. Recently, two cutting-edge techniques have attracted our attention - the CRISPR-Cas9 and Gibson Assembly. By now, we have tried to pretreat Brevibacillus laterosporus strain genomic DNA with CRISPR-Cas9 nucleases that specifically generated breaks near the gene cluster of interest. This trial resulted in an increase in the efficiency of gene cluster capture (9%). Moreover, using Gibson Assembly by adding/deleting certain operon and tailoring enzymes regardless of end compatibility, the silent construct (~80kb) has been successfully refactored into an active one, yielded a series of analogs expected. With the appearances of the novel molecular tools, we are confident to believe that development of a high throughput mature pipeline for DNA assembly, transformation, product isolation and identification would no longer be a daydream for marine natural product discovery.

Keywords: biosynthesis, CRISPR-Cas9, DNA assembly, refactor, TAR cloning

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Authors: Umairah Natasya Binti Mohd Omeershffudin, Suresh Kumar

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Klebsiella pneumoniae, a Gram-negative enteric bacterium that causes nosocomial and urinary tract infections. Particular concern is the global emergence of multidrug-resistant (MDR) strains of Klebsiella pneumoniae. Characterization of antibiotic resistance determinants at the genomic level plays a critical role in understanding, and potentially controlling, the spread of multidrug-resistant (MDR) pathogens. In this study, drug-resistant Klebsiella pneumoniae strain 825795-1 was investigated with extensive computational approaches aimed at identifying novel drug targets among hypothetical proteins. We have analyzed 1099 hypothetical proteins available in genome. We have used in-silico genome subtraction methodology to design potential and pathogen-specific drug targets against Klebsiella pneumoniae. We employed bioinformatics tools to subtract the strain-specific paralogous and host-specific homologous sequences from the bacterial proteome. The sorted 645 proteins were further refined to identify the essential genes in the pathogenic bacterium using the database of essential genes (DEG). We found 135 unique essential proteins in the target proteome that could be utilized as novel targets to design newer drugs. Further, we identified 49 cytoplasmic protein as potential drug targets through sub-cellular localization prediction. Further, we investigated these proteins in the DrugBank databases, and 11 of the unique essential proteins showed druggability according to the FDA approved drug bank databases with diverse broad-spectrum property. The results of this study will facilitate discovery of new drugs against Klebsiella pneumoniae.

Keywords: pneumonia, drug target, hypothetical protein, subtractive genomics

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Authors: Anjan Hazra, Nirjhar Dasgupta, Chandan Sengupta, Sauren Das

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Tea (Camellia sinensis) has received substantial attention from the scientific world time to time, not only for its commercial importance, but also for its demand to the health-conscious people across the world for its extensive use as potential sources of antioxidant supplement. These health-benefit traits primarily rely on some regulatory networks of different metabolic pathways. Development of microsatellite markers from the conserved genomic regions is being worthwhile for studying the genetic diversity of closely related species or self-pollinated species. Although several SSR markers have been reported, in tea the trait-specific Simple Sequence Repeats (SSRs) are yet to be identified, which can be used for marker assisted breeding technique. MicroRNAs are endogenous, noncoding, short RNAs directly involved in regulating gene expressions at the post-transcriptional level. It has been found that diversity in miRNA gene interferes the formation of its characteristic hair pin structure and the subsequent function. In the present study, the precursors of small regulatory RNAs (microRNAs) has been fished out from tea Expressed Sequence Tag (EST) database. Furthermore, the simple sequence repeat motifs within the putative miRNA precursor genes are also identified in order to experimentally validate their existence and function. It is already known that genic-SSR markers are very adept and breeder-friendly source for genetic diversity analysis. So, the potential outcome of this in-silico study would provide some novel clues in understanding the miRNA-triggered polymorphic genic expression controlling specific metabolic pathways, accountable for tea quality.

Keywords: micro RNA, simple sequence repeats, tea quality, trait specific marker

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Authors: Abu Salim Mustafa

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Rv3873 is a relatively large size protein (371 amino acids in length) and its gene is located in the immunodominant genomic region of difference (RD)1 that is present in the genome of Mycobacterium tuberculosis but deleted from the genomes of all the vaccine strains of Bacillus Calmette Guerin (BCG) and most other mycobacteria. However, when tested for cellular immune responses using peripheral blood mononuclear cells from tuberculosis patients and BCG-vaccinated healthy subjects, this protein was found to be a major stimulator of cell mediated immune responses in both groups of subjects. In order to further identify the sequence of immunodominant epitopes and explore their Human Leukocyte Antigen (HLA)-restriction for epitope recognition, 24 peptides (25-mers overlapping with the neighboring peptides by 10 residues) covering the sequence of Rv3873 were synthesized chemically using fluorenylmethyloxycarbonyl chemistry and tested in cell mediated immune responses. The results of these experiments helped in the identification of an immunodominant peptide P9 that was recognized by people expressing varying HLA-DR types. Furthermore, it was also predicted to be a promiscuous binder with multiple epitopes for binding to HLA-DR, HLA-DP and HLA-DQ alleles of HLA-class II molecules that present antigens to T helper cells, and to HLA-class I molecules that present antigens to T cytotoxic cells. In addition, the evaluation of peptide P9 using an immunogenicity predictor server yielded a high score (0.94), which indicated a greater probability of this peptide to elicit a protective cellular immune response. In conclusion, P9, a peptide with multiple epitopes and ability to bind several HLA class I and class II molecules for presentation to cells of the cellular immune response, may be useful as a peptide-based vaccine against tuberculosis.

Keywords: mycobacterium tuberculosis, PPE68, peptides, vaccine

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Authors: Athicha Cherdpunt, Patompong Satapornpong

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The medical cannabis is made up of components also known as cannabinoids, which consists of two ingredients which are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Interestingly, the Cannabinoid can be used for many treatments such as chemotherapy, including nausea and vomiting, cachexia, anorexia nervosa, spinal cord injury and disease, epilepsy, pain, and many others. However, the adverse drug reactions (ADRs) of THC can cause sedation, anxiety, dizziness, appetite stimulation and impairments in driving and cognitive function. Furthermore, genetic polymorphisms of CYP2C9, CYP2C19 and CYP3A4 influenced the THC metabolism and might be a cause of ADRs. Particularly, CYP2C19*17 allele increases gene transcription and therefore results in ultra-rapid metabolizer phenotype (UM). The aim of this study, is to investigate the frequency of CYP2C19*17 alleles in Thai patients who have been treated with medical cannabis. We prospectively enrolled 60 Thai patients who were treated with medical cannabis and clinical data from College of Pharmacy, Rangsit University. DNA of each patient was isolated from EDTA blood, using the Genomic DNA Mini Kit. CYP2C19*17 genotyping was conducted using the real time-PCR ViiA7 (ABI, Foster City, CA, USA). 30 patients with medical cannabis-induced ADRs group, 20 (67%) were female, and 10 (33%) were male, with an age range of 30-69 years. On the other hand, 30 patients without medical cannabis-induced ADRs (control group) consist of 17 (57%) female and 13 (43%) male. The most ADRs for medical cannabis treatment in the case group were dry mouth and dry throat (77%), tachycardia (70%), nausea (30%) and arrhythmia(10%). Accordingly, the case group carried CYP2C19*1/*1 (normal metabolizer) approximately 93%, while 7% patients carrying CYP2C19*1/*17 (ultra rapid metabolizers) exhibited in this group. Meanwhile, we found 90% of CYP2C19*1/*1 and 10% of CYP2C19*1/*17 in control group. In this study, we identified the frequency of CYP2C19*17 allele in Thai population which will support the pharmacogenetics biomarkers for screening and avoid ADRs of medical cannabis treatment.

Keywords: CYP2C19, allele frequency, ultra rapid metabolizer, medical cannabis

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Authors: Sarah Almuhayya, Andrew Mcbain, Gavin Humphreys

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Background. The microbiome of the upper respiratory tract (URT) has received less research attention than other body sites. This study aims to investigate the microbial ecology of the human URT with a focus on the antagonism between the corynebacteria and staphylococci. Methods. Mucosal swabs were collected from the anterior nares and nasal turbinates of 20 healthy adult subjects. Genomic DNA amplification targeting the (V4) of the 16Sr RNA gene was conducted and analyzed using QIIME. Nasal swab isolates were cultured and identified using near full-length sequencing of the 16S rRNA gene. Isolates identified as corynebacteria or staphylococci were typed using (rep-PCR). Antagonism was determined using an agar-based inhibition assay. Results. Four major bacterial phyla (Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria) were identified from all volunteers. The typing of cultured staphylococci and corynebacteria suggested that intra-individual strain diversity was limited. Analysis of generated nasal microbiota profiles suggested an inverse correlation in terms of relative abundance between staphylococci and corynebacteria. Despite the apparent antagonism between these genera, it was limited when investigated on agar. Of 1000 pairwise interactions, observable zones of inhibition were only reported between a single strain of C.pseudodiphtheriticum and S.aureus. Imaging under EM revealed this effect to be bactericidal with clear lytic effects on staphylococcal cell morphology. Conclusion. Nasal microbiota is complex, but culturable staphylococci and corynebacteria were limited in terms of clone type. Analysis of generated nasal microbiota profiles suggested an inverse correlation in terms of relative abundance between these genera suggesting an antagonism or competition between these taxonomic groups.

Keywords: nasal, microbiota, S.aureus, microbioal interaction

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Authors: Juan Antonio Mondragon Sanchez, Ruben Santamaria

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The DNA G-quadruplex is a four-stranded DNA structure formed by stacked planes of four base paired guanines (G-quartet). Guanine rich DNA sequences appear in many sites of genomic DNA and can potential form G-quadruplexes, such as those occurring at 3'-terminus of the human telomeric DNA. The formation and stabilization of a G-quadruplex by small ligands at the telomeric region can inhibit the telomerase activity. In turn, the ligands can be used to down regulate oncogene expression making G-quadruplex an attractive target for anticancer therapy. Many G-quadruplex ligands have been proposed with a planar core to facilitate the pi–pi stacking and electrostatic interactions with the G-quartets. However, many drug candidates are impossibilitated to discriminate a G-quadruplex from a double helix DNA structure. In this context, it is important to investigate the site topology for the interaction of a G-quadruplex with a ligand. In this work, we determine the free energy surface of a G-quadruplex-ligand to study the binding modes of the G-quadruplex (TG4T) with the daunomycin (DM) drug. The complex TG4T-DM is studied using classical molecular dynamics in combination with metadynamics simulations. The metadynamics simulations permit an enhanced sampling of the conformational space with a modest computational cost and obtain free energy surfaces in terms of the collective variables (CV). The free energy surfaces of TG4T-DM exhibit other local minima, indicating the presence of additional binding modes of daunomycin that are not observed in short MD simulations without the metadynamics approach. The results are compared with similar calculations on a different structure (the mutated mu-G4T-DM where the 5' thymines on TG4T-DM have been deleted). The results should be of help to design new G-quadruplex drugs, and understand the differences in the recognition topology sites of the duplex and quadruplex DNA structures in their interaction with ligands.

Keywords: g-quadruplex, cancer, molecular dynamics, metadynamics

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Authors: Mohammad K. Parvez, Mohammed S. Al-Dosari

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Hepatitis E virus (HEV) is an emerging RNA virus that causes acute and chronic liver disease with a global mortality rate of about 2%. Despite milestone developments in understanding of HEV biology, there is still lack of a robust culture system or animal model. Therefore, in a novel approach, two recombinant-baculoviruses (vBac-ORF2 and vBac-ORF3) that could overexpress HEV ORF2 (structural/capsid) and ORF3 (nonstructural/regulatory) proteins, respectively were constructed. The established HEV-SAR55 (genotype 1) replicon that contained GFP gene, in place of ORF2/ORF3 sequences was in vitro transcribed, and GFP production in RNA transfected S10-3 cells was scored by FACS. Enhanced infectivity, if any, of nascent virions produced by exogenously-supplied ORF2 and viral RNA by co-expression of ORF3 was tested on naïve HepG2 cells. Co-transduction with vBac-ORF2/vBac-ORF3 (108 pfu/microL) produced high amounts of native ORF2/ORF3 in approximately 60% of S10-3 cells, determined by immunofluorescence microscopy and Western analysis. FACS analysis showed about 9% GFP positivity of S10-3 cells on day6 post-transfection (i.e, day5 post-transduction). Further, FACS scoring indicated that lysates from S10-3 cultures receiving the RNA plus vBac-ORF2 were capable of producing HEV particles with about 4% infectivity in HepG2 cells. However, lysates of cultures co-transduced with vBac-ORF3, were found to further enhance virion infectivity by approximately 17%. This supported a previously proposed role of ORF3 as a minor-structural protein in HEV virion assembly and infectivity. In conclusion, the present model for efficient genomic RNA packaging and production of infectious virions could be a valuable tool to study various aspects of HEV molecular biology, in vitro.

Keywords: chronic liver disease, hepatitis E virus, ORF2, ORF3, replicon

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Authors: Wafa Toumi, Alessandro Ripalti, Luigi Ricciardiello, Dalila Gargouri, Jamel Kharrat, Abderraouf Cherif, Ahmed Bouhafa, Slim Jarboui, Mohamed Zili, Ridha Khelifa

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Background & aims: Colorectal cancer (CRC) is one of the most common malignancies throughout the world. Several risk factors, both genetic and environmental, including viral infections, have been linked to colorectal carcinogenesis. A few studies report the detection of human polyomavirus JC (JCV) DNA and transformation antigen (T-Ag) in a fraction of the colorectal tumors studied and suggest an association of this virus with CRC. In order to investigate whether such an association of JCV with CRC will hold in a different epidemiological setting, we looked for the presence of JCV DNA and T-Ag expression in a group of Tunisian CRC patients. Methods: Fresh colorectal mucosa biopsies were obtained from 17 healthy volunteers and from both colorectal tumors and adjacent normal tissues of 47 CRC patients. DNA was extracted from fresh biopsies or from formalin-fixed, paraffin-embedded tissue sections using the Invitrogen Purelink Genomic DNA mini Kit. A simple PCR and a nested PCR were used to amplify a region of the T-Ag gene. The obtained PCR products revealed a 154 bp and a 98 bp bands, respectively. Specificity was confirmed by sequencing of the PCR products. T-Ag expression was determined by immunohistochemical staining using a mouse monoclonal antibody (clone PAb416) directed against SV40 T-Ag that cross reacts with JCV T-Ag. Results: JCV DNA was found in 12 (25%) and 22 (46%) of the CRC tumors by simple PCR and by nested PCR, respectively. All paired adjacent normal mucosa biopsies were negative for viral DNA. Sequencing of the DNA amplicons obtained confirmed the authenticity of T-Ag sequences. Immunohistochemical staining showed nuclear T-Ag expression in all 22 JCV DNA- positive samples and in 3 additional tumor samples which appeared DNA-negative by PCR. Conclusions: These results suggest an association of JCV with a subpopulation of Tunisian colorectal tumors.

Keywords: colorectal cancer, immunohistochemistry, Polyomavirus JC, PCR

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Authors: V. Karuppaiah, J. C. Padaria, C. Srivastava

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The tobacco caterpillar, Spodoptera litura Fab (Lepidoptera: Noctuidae) is a polyphagous pest various field and horticulture crops in India. Pest had virtually developed resistance to all commonly used insecticides. Enhanced detoxification is the prime mechanism that is dictated by detoxification different enzymes and carboxylesterase is one of the major enzyme responsible development of resistance. In India, insecticide resistance studies on S. litura are mainly deployed on detoxification enzymes activity and investigation at gene level alteration i.e. at nucleotide level is very merger. In the present study, we collected the S. litura larvae from three different cauliflower growing belt viz., IARI, New Delhi (Delhi), Palari, Sonepat (Haryana) and Varanasi (Uttar Pradesh) to study the role of carboxylesterase activity and its gene level variation The CarE activity was measured using UV-VIS spectrophotometer with 3rd instar larvae of S. litura. The elevated activity of CarE was observed in Sonepat strain (28.09 ± 0.09 µmol/min/mg of protein) followed by Delhi (26.72 ± 0.04 µmol/min/mg of protein) and Varanasi strain (10.00 ± 0.44 µmol/min/mg of protein) of S. litura. The genomic DNA was isolated from 3rd instar larvae and CarE gene was amplified using a primer sequence, F:5’tccagagttccttgtcaggcac3’; R:5’ctgcatcaagcatgtctc3. CarE gene, about 500bp was partially amplified, sequenced and submitted to NCBI (Accession No. KF835886, KF835887 and KF835888). The sequence data revealed polymorphism at nucleotide level in all the three strains and gene found to have 88 to 97% similarity with previous available nucleotide sequences of S. litura, S. littoralis and S. exiqua. The polymorphism at the nucleotide level could be a reason for differential activity of carboxylesterase enzymes among the strains. However, investigation at gene expression level would be useful to analyze the overproduction of carboxylesterase enzyme.

Keywords: carboxylesterase, CarE gene, nucleotide polymorphism, insecticide resistance, spodoptera litura

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Authors: Suvalux Chaichuchote, Ratchadaporn Thonghem

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Pleurotus mushroom is one of popular edible mushrooms in Thailand. It is much favored by consumers due to its delicious taste and high nutrition. It is commonly used as an ingredient in several dishes. The commercially cultivated strain grown in most farms is the Pleurotus sp., Hed Bhutan, that is widely distributed to mushroom farms throughout the country and can be cultivated almost all year round. However, it demands different cultivated strains from mushroom growers, therefore, the improving mushroom strains should be done to their benefits. In this study, we used a di-mon mating method to hybrid production from Hed Bhutan (P-3) as dikaryon material and monokaryotic mycelium were isolated from basidiospores of other three Pleurotus sp. by single spore isolation. The 3 hybrids: P-3XSA-6, P-3XSB-24 and P-3XSE-5 were recognized from the 12 hybridized successfully. They were appropriate hybridized in terms of fruiting body performance in the three time cycles of cultivation such as the number of days until growing, time for pinning, color and shape of fruiting bodies and yield. For genetic study, genomic DNAs of both Hed Bhutan (P-3) and three hybrids were extracted. A couple of primer ITS1 and ITS4 were used to amplify the gene coding for ITS1, ITS2 and 5.8S rRNA. The similarities between these amplified genes and databases of DNA revealed that Hed Bhutan (P-3) was the Pleurotus pulmonarius as well as P-3XSA-6, P-3XSB-24 and P-3XSE-5 hybrids. Furthermore, Hed Bhutan (P3) and three hybrids were distributed to 3 small-scale farms, with mushroom farming experience, in the countryside. To address this, one hundred and twenty mushroom bags of each strain were supplied to them. The findings, by interview, indicated two mushroom farmers were satisfied with P-3XSA-6 hybrid and P-3XSB-24 hybrid, thanks to their simultaneous fruiting time and good yield. While the other was satisfied with P-3XSB-24 hybrid due to its good yield and P-3XSE-5 hybrids thanks to its gradually fruiting body, benefiting in frequent harvest. Overall, farmers adopted all hybrids to grow as commercially cultivated strains as well as Hed Bhutan (P-3) strain.

Keywords: dikaryon, monokaryon, pleurotus, strain improvement

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Authors: Tanyaporn Chairoch

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Introduction: Cannabis is a drug to treat patients with many diseases such as Multiple sclerosis, Alzheimer’s disease, and Epilepsy, where theycontain many active compounds such as delta-9 tetrahydrocannabinol (THC) and cannabidiol (CBD). Especially, THC is the primary psychoactive ingredient in cannabis and binds to cannabinoid 1 (CB1) receptors. Moreover, CB1 is located on the neocortex, hippocampus, basal ganglia, cerebellum, and brainstem. In previous study, we found the association between the variant of CB1recptors gene (rs2023239) and decreased effect of nicotine reinforcement in patients. However, there are no data describing whether the distribution of CB1 receptor gene is a genetic marker for Thai patients who are treated with cannabis. Objective: Thus, the aim of this study we want to investigate the frequency of the CB1 receptor gene in Thai patients. Materials and Methods: All of sixty Thai patients received the medical cannabis for treatment who were recruited in this study. DNA will be extracted from EDTA whole blood by Genomic DNA Mini Kit. The genotyping of CNR1 gene (rs 2023239) was genotyped by the TaqMan real time PCR assay (ABI, Foster City, CA, USA).and using the real-time PCR ViiA7 (ABI, Foster City, CA, USA). Results: We found thirty-eight (63.3%) Thai patients were female, and twenty-two (36.70%) were male in this study with median age of 45.8 (range19 – 87 ) years. Especially, thirty-two (53.30%) medical cannabis tolerant controls were female ( 55%) and median age of52.1 (range 27 – 79 ) years. The most adverse effects for medical cannabis treatment was tachycardia. Furthermore, the number of rs 2023239 (TT) carriers was 26 of 27 (96.29%) in medical cannabis-induced adverse effects and 32 of 33 (96.96%) in tolerant controls. Additionally, rs 2023239 (CT) variant was found just only one of twenty-seven (3.7%) in medical cannabis-induced adverse effects and 1 of 33 (3.03%) in tolerant controls. Conclusions: The distribution of genetic variant in CNR1 gene might serve as a pharmacogenetics markers for screening before initiating the therapy with medical cannabis in Thai patients.

Keywords: cannabis, pharmacogenetics, CNR1 gene, thai patient

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Authors: Marzia Bilkiss, Muhammad J. A. Shiddiky, Mostafa K. Masud, Prabhakaran Sambasivam, Ido Bar, Jeremy Brownlie, Rebecca Ford

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A greater achievement in the Integrated Disease Management (IDM) to prevent the loss would result from early diagnosis and quantitation of the causal pathogen species for accurate and timely disease control. This could significantly reduce costs to the growers and reduce any flow on impacts to the environment from excessive chemical spraying. Necrotrophic fungal disease botrytis grey mould, caused by Botrytis cinerea and Botrytis fabae, significantly reduce temperate legume yield and grain quality during favourable environmental condition in Australia and worldwide. Several immunogenic and molecular probe-type protocols have been developed for their diagnosis, but these have varying levels of species-specificity, sensitivity, and consequent usefulness within the paddock. To substantially improve speed, accuracy, and sensitivity, advanced nanoparticle-based biosensor approaches have been developed. For this, two sets of primers were designed for both Botrytis cinerea and Botrytis fabae which have shown the species specificity with initial sensitivity of two genomic copies/µl in pure fungal backgrounds using multiplexed quantitative PCR. During further validation, quantitative PCR detected 100 spores on artificially infected legume leaves. Simultaneously an electro-catalytic assay was developed for both target fungal DNA using functionalised magnetic nanoparticles. This was extremely sensitive, able to detect a single spore within a raw total plant nucleic acid extract background. We believe that the translation of this technology to the field will enable quantitative assessment of pathogen load for future accurate decision support of informed botrytis grey mould management.

Keywords: biosensor, botrytis grey mould, sensitive, species specific

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Authors: Dharmendra Pratap

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Large Cardamom (Amomum subulatum), family Zingiberaceae is an aromatic spice crop and has rich medicinal value. Large Cardamom is as synonymous to Sikkim as Tea is to Darjeeling. Since Sikkim alone contributes up to 88% of India's large cardamom production which is the world leader by producing over 50% of the global yield. However, the production of large cardamom has declined almost to half since last two decade. The economic losses have been attributed to two viral diseases namely, chirke and Foorkey. Chirke disease is characterized by light and dark green streaks on leaves. The affected leaves exhibit streak mosaic, which gradually coalesce, turn brown and eventually dry up. Excessive sprouting and formation of bushy dwarf clumps at the base of mother plants that gradually die characterize the foorkey disease. In our surveys in Sikkim–Darjeeling hill area during 2012-14, 40-45% of plants were found to be affected with foorkey disease and 10-15% with chirke. Mechanical and aphid transmission study showed banana as an alternate host for both the disease. For molecular identification, total genomic DNA and RNA was isolated from the infected leaf tissues and subjected to Rolling circle amplification (RCA) and RT-PCR respectively. The DNA concatamers produced in the RCA reaction were monomerized by different restriction enzymes and the bands corresponding to ~1 kb genomes were purified and cloned in the respective sites. The nucleotide sequencing results revealed the association of Nanovirus with the foorkey disease of large cardamom. DNA1 showed 74% identity with Replicase gene of FBNYV, DNA2 showed 77% identity with the NSP gene of BBTV and DNA3 showed 74% identity with CP gene of BBTV. The finding suggests the presence of a new species of nanovirus associated with foorkey disease of large cardamom in Sikkim and Darjeeling hills. The details of their epidemiology and other factors would be discussed.

Keywords: RCA, nanovirus, large cardamom, molecular virology and microbiology

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Authors: Tahira Hussain, Ilhom Rahamkulov, Muhammad Aasim, Ugur Pirlak, Emre Aksoy, Mehmet Emin Caliskan, Allah Bakhsh

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RNA interference (RNAi) has proved its usefulness in functional genomic research on insects recently and is considered potential strategy in crop improvement for the control of insect pests. The different insect pests incur significant losses to potato yield worldwide, Colorado Potato Beetle (CPB) being most notorious one. The present study focuses to knock down highly specific 20-hydroxyecdysone hormone-receptor complex interaction by using RNAi approach to silence Ecdysone receptor (EcR) gene of CPB in transgenic potato plants expressing dsRNA of EcR gene. The partial cDNA of Ecdysone receptor gene of CPB was amplified using specific primers in sense and anti-sense orientation and cloned in pRNAi-GG vector flanked by an intronic sequence (pdk). Leaf and internodal explants of Lady Olympia, Agria and Granola cultivars of potato were infected with Agrobacterium strain LBA4404 harboring plasmid pRNAi-CPB, pRNAi-GFP (used as control). Neomycin phosphotransferase (nptII) gene was used as a plant selectable marker at a concentration of 100 mg L⁻¹. The primary transformants obtained have shown proper integration of T-DNA in plant genome by standard molecular analysis like polymerase chain reaction (PCR), real-time PCR, Sothern blot. The transgenic plants developed out of these cultivars are being evaluated for their efficacy against larvae as well adults of CPB. The transgenic lines are expected to inhibit expression of EcR protein gene, hindering their molting process, hence leading to increased potato yield.

Keywords: plant mediated RNAi, molecular strategy, ecdysone receptor, insect metamorphosis

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Authors: I. Boroňová, J.Bernasovská, J. Kľoc, Z. Tomková, E. Petrejčíková, D. Gabriková, S. Mačeková

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Osteoporosis is a complex health disease characterized by low bone mineral density, which is determined by an interaction of genetics with metabolic and environmental factors. Current research in genetics of osteoporosis is focused on identification of responsible genes and polymorphisms. TNFRSF11B gene plays a key role in bone remodeling. The aim of this study was to investigate the genotype and allele distribution of A163G (rs3102735) osteoprotegerin gene promoter and G1181C (rs2073618) osteoprotegerin first exon polymorphisms in the group of 180 unrelated postmenopausal women with diagnosed osteoporosis and 180 normal controls. Genomic DNA was isolated from peripheral blood leukocytes using standard methodology. Genotyping for presence of different polymorphisms was performed using the Custom Taqman®SNP Genotyping assays. Hardy-Weinberg equilibrium was tested for each SNP in the groups of participants using the chi-square (χ2) test. The distribution of investigated genotypes in the group of patients with osteoporosis were as follows: AA (66.7%), AG (32.2%), GG (1.1%) for A163G polymorphism; GG (19.4%), CG (44.4%), CC (36.1%) for G1181C polymorphism. The distribution of genotypes in normal controls were follows: AA (71.1%), AG (26.1%), GG (2.8%) for A163G polymorphism; GG (22.2%), CG (48.9%), CC (28.9%) for G1181C polymorphism. In A163G polymorphism the variant G allele was more common among patients with osteoporosis: 17.2% versus 15.8% in normal controls. Also, in G1181C polymorphism the phenomenon of more frequent occurrence of C allele in the group of patients with osteoporosis was observed (58.3% versus 53.3%). Genotype and allele distributions showed no significant differences (A163G: χ2=0.270, p=0.605; χ2=0.250, p=0.616; G1181C: χ2= 1.730, p=0.188; χ2=1.820, p=0.177). Our results represents an initial study, further studies of more numerous file and associations studies will be carried out. Knowing the distribution of genotypes is important for assessing the impact of these polymorphisms on various parameters associated with osteoporosis. Screening for identification of “at-risk” women likely to develop osteoporosis and initiating subsequent early intervention appears to be most effective strategy to substantially reduce the risks of osteoporosis.

Keywords: osteoporosis, real-time PCR method, SNP polymorphisms

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