Search results for: cytochrome P450 oxidoreductase

Authors: R. M. Srivastava, Subbanna A.R.N.S, Md Abbas Ahmad, S. P.More, Shivashankar, B. Kalyanbabu

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The similar morphology associated with high genetic variability poses problems in phylogenetic studies of Helicoverpa armigera (Hubner). To identify genetic variation of North Western Himalayan population’s, partial (Mid to terminal region) cytochrome c oxidase subunit I (COX-1) gene was amplified and sequenced for three populations collected from Pantnagar, Almora, and Chinyalisaur. The alignment of sequences with other two populations, Nagpur representing central India population and Anhui, China representing complete COX-1 sequence revealed unanimity in middle region with eleven single nucleotide polymorphisms (SNPs) in Nagpur populations. However, the consensus is missing when approaching towards terminal region, which is associated with 15 each SNPs and pair base substitutions in Chinyalisaur populations. In minimum evolution tree, all the five populations were majorly separated into two clades, one comprising of only Nagpur population and the other with rest. Amongst, North Western populations, Chinyalisaur one is promising by farming a separate clade. The pairwise genetic distance ranges from 0.025 to 0.192 with the maximum between H. armigera populations of Nagpur and Chinyalisaur. This genetic isolation of populations can be attributed to a key role of topological barriers of weather and mountain ranges and temporal barriers due to cropping patterns.

Keywords: cytochrome c oxidase subunit I, northwestern Himalayan population, Helicoverpa armigera (Noctuidae: Lepidoptera), phylogenetic relationship, genetic variation

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Authors: Othman E. Othman, Agnés Germot, Daniel Petit, Abderrahman Maftah

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Threats to the biodiversity are increasing due to the loss of genetic diversity within the species utilized in agriculture. Due to the progressive substitution of the less productive, locally adapted and native breeds by highly productive breeds, the number of threatened breeds is increased. In these conditions, it is more strategically important than ever to preserve as much the farm animal diversity as possible, to ensure a prompt and proper response to the needs of future generations. Mitochondrial (mtDNA) sequencing has been used to explain the origins of many modern domestic livestock species. Studies based on sequencing of sheep mitochondrial DNA showed that there are five maternal lineages in the world for domestic sheep breeds; A, B, C, D and E. Because of the eastern location of Egypt in the Mediterranean basin and the presence of fat-tailed sheep breeds- character quite common in Turkey and Syria- where genotypes that seem quite primitive, the phylogenetic studies of Egyptian sheep breeds become particularly attractive. We aimed in this work to clarify the genetic affinities, biodiversity and phylogeny of five Egyptian sheep breeds using cytochrome B sequencing. Blood samples were collected from 63 animals belonging to the five tested breeds; Barki, Rahmani, Ossimi, Saidi and Sohagi. The total DNA was extracted and the specific primer allowed the conventional PCR amplification of the cytochrome B region of mtDNA (approximately 1272 bp). PCR amplified products were purified and sequenced. The alignment of Sixty-three samples was done using BioEdit software. DnaSP 5.00 software was used to identify the sequence variation and polymorphic sites in the aligned sequences. The result showed that the presence of 34 polymorphic sites leading to the formation of 18 haplotypes. The haplotype diversity in five tested breeds ranged from 0.676 in Rahmani breed to 0.894 in Sohagi breed. The genetic distances (D) and the average number of pairwise differences (Dxy) between breeds were estimated. The lowest distance was observed between Rahmani and Saidi (D: 1.674 and Dxy: 0.00150) while the highest distance was observed between Ossimi and Sohagi (D: 5.233 and Dxy: 0.00475). Neighbour-joining (Phylogeny) tree was constructed using Mega 5.0 software. The sequences of the 63 analyzed samples were aligned with references sequences of different haplogroups. The phylogeny result showed the presence of three haplogroups (HapA, HapB and HapC) in the 63 examined samples. The other two haplogroups described in literature (HapD and HapE) were not found. The result showed that 50 out of 63 tested animals cluster with haplogroup B (79.37%) whereas 7 tested animals cluster with haplogroup A (11.11%) and 6 animals cluster with haplogroup C (9.52%). In conclusion, the phylogenetic reconstructions showed that the majority of Egyptian sheep breeds belonging to haplogroup B which is the dominant haplogroup in Eastern Mediterranean countries like Syria and Turkey. Some individuals are belonging to haplogroups A and C, suggesting that the crosses were done with other breeds for characteristic selection for growth and wool quality.

Keywords: cytochrome B, diversity, phylogheny, Egyptian sheep breeds

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Authors: Hamsa T. Tayeb, Mohammad A. Arafah, Dana M. Bakheet, Duaa M. Khalaf, Agnieszka Tarnoska, Nduna Dzimiri

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Background: CYP2D6 is a member of the cytochrome P450 mixed-function oxidase system. The enzyme is responsible for the metabolism and elimination of approximately 25% of clinically used drugs, especially in breast cancer and psychiatric therapy. Different phenotypes have been described displaying alleles that lead to a complete loss of enzyme activity, reduced function (poor metabolizers – PM), hyperfunctionality (ultrarapid metabolizers–UM) and therefore drug intoxication or loss of drug effect. The prevalence of these variants may vary among different ethnic groups. Furthermore, the xTAG system has been developed to categorized all patients into different groups based on their CYP2D6 substrate metabolization. Aim of the study: To determine the prevalence of the different CYP2D6 variants in our population, and to evaluate their clinical relevance in personalized medicine. Methodology: We used the Luminex xMAP genotyping system to sequence 305 Saudi individuals visiting the Blood Bank of our Institution and determine which polymorphisms of CYP2D6 gene are prevalent in our region. Results: xTAG genotyping showed that 36.72% (112 out of 305 individuals) carried the CYP2D6_*2. Out of the 112 individuals with the *2 SNP, 6.23% had multiple copies of *2 SNP (19 individuals out of 305 individuals), resulting in an UM phenotype. About 33.44% carried the CYP2D6_*41, which leads to decreased activity of the CYP2D6 enzyme. 19.67% had the wild-type alleles and thus had normal enzyme function. Furthermore, 15.74% carried the CYP2D6_*4, which is the most common nonfunctional form of the CYP2D6 enzyme worldwide. 6.56% carried the CYP2D6_*17, resulting in decreased enzyme activity. Approximately 5.73% carried the CYP2D6_*10, consequently decreasing the enzyme activity, resulting in a PM phenotype. 2.30% carried the CYP2D6_*29, leading to decreased metabolic activity of the enzyme, and 2.30% carried the CYP2D6_*35, resulting in an UM phenotype, 1.64% had a whole-gene deletion CYP2D6_*5, thus resulting in the loss of CYP2D6 enzyme production, 0.66% carried the CYP2D6_*6 variant. One individual carried the CYP2D6_*3(B), producing an inactive form of the enzyme, which leads to decrease of enzyme activity, resulting in a PM phenotype. Finally, one individual carried the CYP2D6_*9, which decreases the enzyme activity. Conclusions: Our study demonstrates that different CYP2D6 variants are highly prevalent in ethnic Saudi Arabs. This finding sets a basis for informed genotyping for these variants in personalized medicine. The study also suggests that xTAG is an appropriate procedure for genotyping the CYP2D6 variants in personalized medicine.

Keywords: CYP2D6, hormonal breast cancer, pharmacogenetics, polymorphism, psychiatric treatment, Saudi population

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Authors: Zain Ul Abdin, Mirza Aizaz Asim, Rao Sohail Ahmad Khan, Luqman Amrao, Fiaz Hussain, Hasooba Hira, Saqi Kosar Abbas

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The success of Integrated vector management programmes mainly depends on the correct identification of insect vector species involved in vector borne diseases. Based on molecular data the most important insect species involved as vectors for Zoonotic diseases in Pakistan were identified. The precise and accurate identification of such type of organism is only possible through molecular based techniques like “DNA barcoding”. Morphological species identification in insects at any life stage, is very challenging, therefore, DNA barcoding was used as a tool for rapid and accurate species identification in a wide variety of taxa across the globe and parallel studies revealed that DNA barcoding data can be effectively used in resolving taxonomic ambiguities, detection of cryptic diversity, invasion biology, description of new species etc. A comprehensive survey was carried out for the collection of insects (both adult and immature stages) in district Faisalabad, Pakistan and their DNA was extracted and mitochondrial cytochrome oxidase subunit I (COI-59) barcode sequences was used for molecular identification of immature and adult life stage.This preliminary research work opens new frontiers for developing sustainable insect vectors management programmes for saving lives of mankind from fatal diseases.

Keywords: zoonotic diseases, cytochrome oxidase, and insect vectors, CO1

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Authors: Minu Bharati, Priyanka Rai, Satarupa Dutta, Dhiraj Saha

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Culex quinquefasciatus Say, is a mosquito of immense public health concern due to its role in transmission of filariasis, which is an endemic disease in 20 states and union territories of India, putting about 600 million people at the risk of infection. The main strategies to control filaria in India include anti-larval measures in urban areas, Indoor Residual Spray (IRS) in rural areas and mass diethylcarbamazine citrate (DEC) administration. Larval destruction measures and IRS are done with the use of insecticides. In this study, Susceptibility/ Resistance to insecticides were assessed in Culex quinquefasciatus mosquitoes collected from eight densely populated areas of Siliguri subdivision, which has a high rate of filarial infection. To unveil the insecticide susceptibility status of Culex quinquefasciatus, bioassays were performed on field-caught mosquitoes against two major groups of insecticides, i.e. Synthetic Pyrethroids (SPs): 0.05% deltamethrin and 0.05% lambda-cyhalothrin and Organophosphates (OPs): 5% malathion and temephos using World Health Organisation (WHO) discriminating doses. The knockdown rates and knockdown times (KDT50) were also noted against deltamethrin, lambda-cyhalothrin and malathion. Also, activities of major detoxifying enzymes, i.e. α-carboxylesterases, β-carboxylesterases and cytochrome P450 (CYP450) monooxygenases were determined to find the involvement of biochemical mechanisms in resistance phenomenon (if any). The results obtained showed that, majority of the mosquito populations were moderately to severely resistant against both the SPs and one OP, i.e. temephos. Whereas, most of the populations showed 100% susceptibility to malathion. The knockdown rates and KDT50 in response to above-mentioned insecticides showed significant variation among different populations. Variability in activities of carboxylesterases and CYP450 monooxygenases were also observed with hints of their involvement in contribution towards insecticide resistance in some of the tested populations. It may be concluded that, Culex quinquefasciatus has started developing resistance against deltamethrin, lambda-cyhalothrin and temephos in Siliguri subdivision. Malathion seems to hold the greatest potentiality for control of these mosquitoes in this area as revealed through this study. Adoption of Integrated mosquito management (IMM) strategy should be the prime objective of the concerned authorities to delimit the insecticide resistance phenomenon and filariasis infections.

Keywords: Culex quinquefasciatus, detoxifying enzymes, insecticide resistance, knockdown rate

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Authors: Noraziah Nordin, Najihah Mohd Hashim, Nazia Abdul Majid, Mashitoh Abdul Rahman, Hamed Karimian, Hapipah Mohd Ali

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Enicosanthellum pulchrum belongs to family Annonaceae is also known as family of 'mempisang' in Malaysia. Liriodenine was isolated by prep-HPLC method. This method was first technique used for the isolation of this compound. The structure of the liriodenine was elucidated by 1D and 2D spectroscopy techniques. Liriodenine was tested on ovarian cancer cells line (CAOV-3) for MTT, AO/PI and cytotoxicity 3 assays. The MTT assay was performed to determine the cytotoxicity effect of lirodenine on CAOV-3 cells. The morphological changes on CAOV-3 cells were observed by AO/PI assay for the early and late stage of apoptosis, as well as necrosis. Meanwhile, the measurement of cell loss, nuclear morphology, DNA content, cell membrane permeability, mitochondrial membrane potential changes and cytochrome c release from mitochondria were detected through cytotoxicity 3 assay. The IC50 results showed liriodenine inhibits the growth of CAOV-3 cells after 24 h of treatment at 10.25 ± 1.06 µg/mL. After 48 and 72 h of treatments, the IC50 values were decreased to 7.65 ± 0:07 and 6.35 ± 1.62 µg/mL, respectively. The morphology changes can be seen on CAOV-3 with a production of cell membrane blebbing, cromatin condensation and apoptotic bodies with increasing time of treatment from 24 to 72 h. Evaluation of cytotoxicity 3 on CAOV-3 cells after treated with liriodenine, resulting loss of mitochondrial membrane potential and release of cytochrome c from mitochondria. The results demonstrated the capability of liriodenine as a promising anticancer agent, particularly on human ovarian cancer.

Keywords: Enicosanthellum pulchrum, ovarian cancer, apoptosis, cytotoxicity

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Authors: M. Y. Abubakar, Ipinjolu J. K., Yuzine B. Esa, Magawata I., Hassan W. A., Turaki A. A.

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Catfish (Heterobranchus spp.) is a major freshwater fish that are widely distributed in Nigeria waters and are gaining rapid aquaculture expansion. However, indiscriminate artificial crossbreeding of the species with others poses a threat to their biodiversity. There is a paucity of information about the genetic variability, hence this insight on the genetic variability is badly needed, not only for the species conservation but for aquaculture expansion. In this study, we tested the level of Genetic diversity, population differentiation and phylogenetic relationship analysis on 35 individuals of two populations of Heterobranchus bidorsalis and 29 individuals of three populations of Heterobranchus longifilis using the mitochondrial cytochrome c oxidase subunit I (mtDNA COI) gene sequence. Nucleotide sequences of 650 bp fragment of the COI gene of the two species were compared. In the whole 4 and 5 haplotypes were distinguished in the populations of H. bidorsalis & H. longifilis with accession numbers (MG334168 - MG334171 & MG334172 to MG334176) respectively. Haplotypes diversity indices revealed a range of 0.59 ± 0.08 to 0.57 ± 0.09 in H. bidorsalis and 0.000 to 0.001051 ± 0.000945 in H. longifilis population, respectively. Analysis of molecular variance (AMOVA) revealed no significant variation among H. bidorsalis population of the Niger & Benue Rivers, detected significant genetic variation was between the Rivers of Niger, Kaduna and Benue population of H. longifilis. Two main clades were recovered, showing a clear separation between H. bidorsalis and H. longifilis in the phylogenetic tree. The mtDNA COI genes studied revealed high gene flow between populations with no distinct genetic differentiation between the populations as measured by the fixation index (FST) statistic. However, a proportion of population-specific haplotypes was observed in the two species studied, suggesting a substantial degree of genetic distinctiveness for each of the population investigated. These findings present the description of the species character and accessions of the fish’s genetic resources, through gene sequence submitted in Genetic database. The data will help to protect their valuable wild resource and contribute to their recovery and selective breeding in Nigeria.

Keywords: AMOVA, genetic diversity, Heterobranchus spp., mtDNA COI, phylogenetic tree

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Authors: Umara Nissar Rafiqi, Irum Gul, Nazima Nasrullah, Monica Saifi, Malik Z. Abdin

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Background: Stevia rebaudiana, a member of Asteraceae family is an important medicinal plant and produces a commercially used non-caloric natural sweetener, which is also an alternate herbal cure for diabetes. Steviol glycosides are the main sweetening compounds present in these plants. Secondary metabolites are crucial to the adaption of plants to the environment and its overcoming stress conditions. In agricultural procedures, the abiotic stresses like salinity, high metal toxicity and drought, in particular, are responsible for the majority of the reduction that differentiates yield potential from harvestable yield. Salt stress and heavy metal toxicity lead to increased production of reactive oxygen species (ROS). To avoid oxidative damage due to ROS and osmotic stress, plants have a system of anti-oxidant enzymes along with several stress induced enzymes. This helps in scavenging the ROS and relieve the osmotic stress in different cell compartments. However, whether stress induced toxicity modulates the activity of these enzymes in Stevia rebaudiana is poorly understood. Aim: The present study focussed on the effect of salinity, heavy metal toxicity (lead and mercury) on physiological traits and transcriptional profiling of Stevia rebaudiana. Method: Stevia rebaudiana plants were collected from the Central Institute of Medicinal and Aromatic plants (CIMAP), Patnagar, India and maintained under controlled conditions in a greenhouse at Hamdard University, Delhi, India. The plants were subjected to different concentrations of salt (0, 25, 50 and 75 mM respectively) and heavy metals, lead and mercury (0, 100, 200 and 300 µM respectively). The physiological traits such as shoot length, root numbers, leaf growth were evaluated. The samples were collected at different developmental stages and analysed for transcription profiling by RT-PCR. Transcriptional studies in stevia rebaudiana involves important antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), cytochrome P450 monooxygenase (CYP) and stress induced aquaporin (AQU), auxin repressed protein (ARP-1), Ndhc gene. The data was analysed using GraphPad Prism and expressed as mean ± SD. Result: Low salinity and lower metal toxicity did not affect the fresh weight of the plant. However, this was substantially decreased by 55% at high salinity and heavy metal treatment. With increasing salinity and heavy metal toxicity, the values of all studied physiological traits were significantly decreased. Chlorosis in treated plants was also observed which could be due to changes in Fe:Zn ratio. At low concentrations (upto 25 mM) of NaCl and heavy metals, we did not observe any significant difference in the gene expressions of treated plants compared to control plants. Interestingly, at high salt concentration and high metal toxicity, a significant increase in the expression profile of stress induced genes was observed in treated plants compared to control (p < 0.005). Conclusion: Stevia rebaudiana is tolerant to lower salt and heavy metal concentration. This study also suggests that with the increase in concentrations of salt and heavy metals, harvest yield of S. rebaudiana was hampered.

Keywords: Stevia rebaudiana, natural sweetener, salinity, heavy metal toxicity

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Authors: Marina Passero, Tianhua Zhai, Zuyi (Jacky) Huang

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Alzheimer’s disease has become a major public health issue, as indicated by the increasing populations of Americans living with Alzheimer’s disease. After decades of extensive research in Alzheimer’s disease, only seven drugs have been approved by Food and Drug Administration (FDA) to treat Alzheimer’s disease. Five of these drugs were designed to treat the dementia symptoms, and only two drugs (i.e., Aducanumab and Lecanemab) target the progression of Alzheimer’s disease, especially the accumulation of amyloid-b plaques. However, controversial comments were raised for the accelerated approvals of either Aducanumab or Lecanemab, especially with concerns on safety and side effects of these two drugs. There is still an urgent need for further drug discovery to target the biological processes involved in the progression of Alzheimer’s disease. Excessive cholesterol has been found to accumulate in the brain of those with Alzheimer’s disease. Cholesterol can be synthesized in both the blood and the brain, but the majority of biosynthesis in the adult brain takes place in astrocytes and is then transported to the neurons via ApoE. The blood brain barrier separates cholesterol metabolism in the brain from the rest of the body. Various proteins contribute to the metabolism of cholesterol in the brain, which offer potential targets for Alzheimer’s treatment. In the astrocytes, SREBP cleavage-activating protein (SCAP) binds to Sterol Regulatory Element-binding Protein 2 (SREBP2) in order to transport the complex from the endoplasmic reticulum to the Golgi apparatus. Cholesterol is secreted out of the astrocytes by ATP-Binding Cassette A1 (ABCA1) transporter. Lipoprotein receptors such as triggering receptor expressed on myeloid cells 2 (TREM2) internalize cholesterol into the microglia, while lipoprotein receptors such as Low-density lipoprotein receptor-related protein 1 (LRP1) internalize cholesterol into the neuron. Cytochrome P450 Family 46 Subfamily A Member 1 (CYP46A1) converts excess cholesterol to 24S-hydroxycholesterol (24S-OHC). Cholesterol has been approved for its direct effect on the production of amyloid-beta and tau proteins. The addition of cholesterol to the brain promotes the activity of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), secretase, and amyloid precursor protein (APP), which all aid in amyloid-beta production. The reduction of cholesterol esters in the brain have been found to reduce phosphorylated tau levels in mice. In this work, a computational pipeline was developed to identify the protein targets involved in cholesterol regulation in brain and further to identify chemical compounds as the inhibitors of a selected protein target. Since extensive evidence shows the strong correlation between brain cholesterol regulation and Alzheimer’s disease, a detailed literature review on genes or pathways related to the brain cholesterol synthesis and regulation was first conducted in this work. An interaction network was then built for those genes so that the top gene targets were identified. The involvement of these genes in Alzheimer’s disease progression was discussed, which was followed by the investigation of existing clinical trials for those targets. A ligand-protein docking program was finally developed to screen 1.5 million chemical compounds for the selected protein target. A machine learning program was developed to evaluate and predict the binding interaction between chemical compounds and the protein target. The results from this work pave the way for further drug discovery to regulate brain cholesterol to combat Alzheimer’s disease.

Keywords: Alzheimer’s disease, drug discovery, ligand-protein docking, gene-network analysis, cholesterol regulation

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Authors: Sehrish Iftikhar, Ahmad Ali Shahid, Kiran Nawaz, Waheed Anwar

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Discovery and development of new compounds require intense studies in chemistry, biochemistry. Numerous experiments under laboratory-, greenhouse- and field conditions can be performed to select suitable candidates and to understand their full potential. Novel fungicides are fundamental to combat plant diseases. Fusarium solani is important plant pathogen. New broad spectrum foliar fungicides against complex II were designed in this study. Complex II, namely succinate dehydrogenase (SDH), or succinate quinone oxidoreductase (SQR) is a multi-subunit enzyme at the crossroads of TCA and ETC at the inner mitochondrial membrane. The need for new and innovative fungicides is driven by resistance management, regulatory hurdles and increasing customer expectations amongst others. Fungicidal activity was assessed for the effect on mycelial growth and spore germination of the fungi using fungicide amended media assay. In mycelial growth assay compounds C10 and C6 were highly active against all the isolates. The compounds C1 and C10 were found most potent in spore germination test. It fully proved that the SDHIs designed in this paper displayed as good inhibitory effects against Fusarium solani.

Keywords: Wilt, Fusarium, SDH, antifungal

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Authors: Hanouf A. S. Al Nuwaysir, Nadine Moubayed, Abir Ben Bacha, Islem Abid

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Consumption of waste water continues to cause significant problems for human health in both developed and developing countries. Many regulations have been implied by different world authorities controlling water quality for the presence of coliforms used as standard indicators of water quality deterioration and historically leading health protection concept. In this study, the European directive for the detection of Escherichia coli, ISO 9308-1, was applied to examine and monitor coliforms in water samples collected from Wadi Hanifa and neighboring wells, Riyadh governorate, kingdom of Saudi Arabia, which is used for irrigation and industrial purposes. Samples were taken from different locations for 8 months consecutively, chlorine concentration ranging from 0.1- 0.4 mg/l, was determined using the DPD FREE CHLORINE HACH kit. Water samples were then analyzed following the ISO protocol which relies on the membrane filtration technique (0.45µm, pore size membrane filter) and a chromogenic medium TTC, a lactose based medium used for the detection and enumeration of total coliforms and E.coli. Data showed that the number of bacterial isolates ranged from 60 to 300 colonies/100ml for well and surface water samples respectively; where higher numbers were attributed to the surface samples. Organisms which apparently ferment lactose on TTC agar plates, appearing as orange colonies, were selected and additionally cultured on EMB and MacConkey agar for a further differentiation among E.coli and coliform bacteria. Two additional biochemical tests (Cytochrome oxidase and indole from tryptophan) were also investigated to detect and differentiate the presence of E.coli from other coliforms, E. coli was identified in an average of 5 to 7colonies among 25 selected colonies.On the other hand, a more rapid, specific and sensitive analytical molecular detection namely single colony PCR was also performed targeting hha gene to sensitively detect E.coli, giving more accurate and time consuming identification of colonies considered presumptively as E.coli. Comparative methodologies, such as ultrafiltration and direct DNA extraction from membrane filters (MoBio, Grermany) were also applied; however, results were not as accurate as the membrane filtration, making it a technique of choice for the detection and enumeration of water coliforms, followed by sufficiently specific enzymatic confirmatory stage.

Keywords: coliform, cytochrome oxidase, hha primer, membrane filtration, single colony PCR

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Authors: Ankita Kushwaha, M. P. Singh

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Background: In the present investigation the efficiency of laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) from Pleurotus citrinopileatus was assessed for the decolorization of azo dyes. Aim: Enzyme production, characterization and kinetics of a partially purified laccase from Pleurotus citrinopileatus were determined for its application in bioremediation of azo dyes. Methods & Results: Laccase has been partially purified by using 80% ammonium sulphate solution. Total activity, total protein, specific activity and purification fold for partially purified laccase were found to be 40.38U, 293.33mg/100ml, 0.91U/mg and 2.84, respectively. The pH and temperature optima of laccase were 5.0 and 50ºC, respectively, while the enzyme was most stable at pH 4.0 and temperature 30ºC when exposed for one hour. The Km of the partially purified laccase for substrates guaiacol, DMP (2,6-dimethoxyphenol) and syringaldazine (3,5-dimethoxy-4-hydroxybenzaldehyde azine) were 60, 95 and 26, respectively. This laccase has been tested for the use in the bioremediation of azo dyes in the absence of mediator molecules. Two dyes namely congo red and bromophenol blue were tested. Discussion: It was observed that laccase enzyme was very effective in the decolorization of these two dyes. More than 80% decolorization was observed within half an hour even in the absence of mediator and their lower Km value indicates that efficiency of the enzyme is very high. The results were promising due to quicker decolorization in the absence of mediators showing that it can be used as a valuable biocatalyst for quick bioremediation of azo dyes. Conclusion: The enzymatic properties of laccase from P. citrinopileatus should be considered for a potential environmental (biodegradation and bioremediation) or industrial applications.

Keywords: azo dyes, decolorization, laccase, P.citrinopileatus

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Authors: Jiang Xu, Daoping Wang, Qun Li, Yinghong Pan

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2,4-Epibrassinolide (EBR), which is a kind of plant hormone Brassinosteroids (BRs), is widely studied and applied in the global scale but the proteomic characteristics of EBR alleviating low temperature stress in rice seedling leaves are still not clear. In this study, seeding rice of Nipponbare were treated with EBR and distilled water, then stressed at 4℃ or 26 ℃, and analyzed by mass spectrometry analysis, verified by parallel reaction monitoring technique (PRM). The results showed that 5778 proteins were identified in total and 4834 proteins were identified with quantitative information. Among them, 401 up-regulated and 220 down-regulated proteins may be related to EBR alleviating low temperature stress in rice seedling leaves. The molecular functions of most of up-regulated proteins are RNA binding and hydrolase activity and are mainly enriched in the pathways of carbon metabolism, folic acid synthesis, and amino acid biosynthesis. The down-regulated proteins are mainly related to catalytic activity and oxidoreductase activity and are mainly enriched in the pathways of limonene and pinene degradation, riboflavin metabolism, porphyrin and chlorophyll metabolism, and other metabolic pathways. PRM validation and literature analysis showed that NADP-malic acidase, peroxidase, 3-phosphoglycerate dehydrogenase, enolase, glyceraldehyde-3- phosphate dehydrogenase and pyruvate kinase are closely related to the effect of EBR on low temperature stress. These results also suggested that BRs could relieve the effect of low temperature stress on rice seed germination in many ways.

Keywords: 2, 4-Epibrassinolid, low temperature stress, proteomic analysis, rice

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Authors: Mikhail S. Zastrozhin, Valery V. Smirnov, Dmitry A. Sychev, Ludmila M. Savchenko, Evgeny A. Bryun, Mark O. Nechaev

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Cytochrome P-450 isoenzyme 3A4 takes part in the biotransformation of medical drugs. The activity of CYP isoenzymes depends on genetic (polymorphisms of genes which encoded it) and phenotypic factors (a kind of food, a concomitant drug therapy). The aim of the study was to evaluate a carbamazepine effect on the CYP3A4 activity in patients with alcohol addiction. The study included 25 men with alcohol dependence, who received haloperidol during the exacerbation of the addiction. CYP3A4 activity was assessed by urinary 6-beta-hydroxycortisol/cortisol ratios measured by high performance liquid chromatography with mass spectrometry. The study modeled a graph and an equation of the logarithmic regression, that reflects the dependence of CYP3A4 activity on a dose of carbamazepine: y = 5,5 * 9,1 * 10-5 * x2. The study statistically significant demonstrates the effect of carbamazepine on CYP2D6 isozyme activity in patients with alcohol addiction.

Keywords: CYP3A4, biotransformation, carbamazepine, alcohol abuse

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Authors: N. Boussoualim, H. Trabsa, I. Krache, S. Aouachria, S. Boumerfeg, L. Arrar, A. Baghiani

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The aim of this study consisted in evaluating the antioxidant in vivo properties, anti-hemolytic and XOR inhibitory effect of Globularia alypum L. (GA) extracts. GA was submitted to extraction and fractionation to give crude (CrE), chloroformique (ChE), ethyle acetate (EAE) and aqueos (AqE) extracts. Total polyphenols contents of GA extracts were determined; EAE is the most rich in polyphenols (157,74±5,27 mg GAE/mg of extract). GA Extracts inhibited XO in a concentration-dependent manner, the EAE showed the highest inhibitory properties on the XOR activity (IC50=0,083±0,001 mg/ml), followed by CrE and ChE. The antioxidant activities of the CrE, EAE, and AqE were tested by an in vivo assay in mice, the plasma ability to inhibit DPPH radical was measured, The CrE was found to exhibit the greatest scavenger activity with 48.41±2.763%, followed by AqE and EAE (40.54±7.51% and 41.79±1.654%, respectively). Total antioxidant capacity of red blood cells was measured, from the kinetics of hemolysis obtained. The calculated HT50 reveal an extension of time for half hemolysis in all treated groups compared with the control group. CrE increase significantly HT50 (112,8±2,427). The hemolysis is lagged, indicating that endogenous antioxidants in the erythrocytes can trap radicals to protect them against free-radical-induced hemolysis. Antimicrobial activities of the extracts were determined by the disc diffusion method. Test microorganisms were; 4 Gram positive, 7 gram negative bacteria, most active extracts were EAE and CrE. We deduce a great relationship between the effect on the extracts antibacterial effect and their contents in flavonoid.

Keywords: Globularia alypum, Xanthine oxidoreductase, in vivo-antioxidant activity, hemolysis, polyphenol

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Authors: Omar S. Sharaf

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The mitochondrial 12s rRNA (mt-12s rDNA) gene for pig-specific was developed to detect material from pork species in different products collected from Jordanian market. The amplification PCR products of 359 bp and 531 bp were successfully amplified from the cyt b gene of pig the amplification product using mt-12S rDNA gene were successfully produced a single band with a molecular size of 456 bp. In the present work, the PCR amplification of mtDNA of cytochrome b has been shown as a suitable tool for rapid detection of pig DNA. 100 samples from different dairy, gelatin and chocolate based products and 50 samples from baby food formula were collected and tested to a presence of any pig derivatives. It was found that 10% of chocolate based products, 12% of gelatin and 56% from dairy products and 5.2% from baby food formula showed single band from mt-12S rDNA gene.

Keywords: halal food, baby infant formula, chocolate based products, PCR, Jordan

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Authors: B. M. Md-Zain, N. A. Rahman, M. A. B. Abdul-Latiff, W. M. R. Idris

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We conducted molecular research to portray phylogenetic relationships of Malaysian primates particularly in the genus of Macaca. We have sequenced cytochrome C oxidase subunit I (COI) of mitochondrial DNA of several individuals from M. fascicularis and M. arctoides. PCR amplifications were performed and COI DNA sequences were aligned using ClustalW. Phylogenetic trees were constructed using distance analyses by employing neighbor-joining algorithm (NJ). We managed to sequence 700 bp of COI DNA sequences. The tree topology showed that M. fascicularis did not clump based on phyleogeography division in Peninsular Malaysia. Individuals from Negeri Sembilan merged together with samples from Perak and Penang into one clade. In addition, phylogenetic analyses indicated that M. arctoides was classified into sinica group instead of fascicularis group supported by genetic distance data. COI gene is an effective locus to clarify phylogenetic position of M. arctoides but not in discriminating M. fascicularis population in Peninsular Malaysia.

Keywords: cercopithecine, long-tailed macaque, Macaca fascicularis, Macaca arctoides

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Authors: Bahadir Batar, Elif Serdal, Berna Erdal, Hasan Ogul

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Micro-ribonucleic acids (miRNAs) are small short non-coding ribonucleic acid molecules about 22 nucleotides long. miRNAs play a key role in response to chemotherapeutic agents. WW domain-containing oxidoreductase (WWOX) gene encodes a tumor suppressor protein. Loss or reduction of Wwox protein is observed in many breast cancer cases. WWOX protein deficiency is increased in triple-negative breast cancer (TNBC). TNBC is a heterogeneous, highly aggressive, and difficult to treat tumor type. WWOX loss contributes to resistance to cisplatin therapy in patients with TNBC. Here, the aim of the study was to investigate the potential role of miRNAs in cisplatin therapy resistance of WWOX-deficient TNBC cells. This was a cell culture study. miRNA expression profiling was analyzed by LightCycler 480 system. miRNA Set Enrichment Analysis tool was used to integrate experimental data with literature-based biological knowledge to infer a new hypothesis. Increased miR-182 and decreased miR-214 were significantly correlated with cisplatin resistance in WWOX-deficient TNBC cells. miR-182 and miR-214 may involve in cisplatin resistance of WWOX-deficient TNBC cells by deregulating the DNA repair, apoptosis, or protein kinase B signaling pathways. These data highlight the mechanism by which WWOX regulates cisplatin resistance of TNBC and the potential use of WWOX as a predictor biomarker for cisplatin resistance.

Keywords: cisplatin, microRNA, triple-negative breast cancer, WWOX

Procedia PDF Downloads 131

Authors: Marcel Verwey, Anna M. Joubert, Elsie M. Nolte, Wolfgang Dohle, Barry V. L. Potter, Anne E. Theron

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A tetrahydroisoquinolinone (THIQ) core can be used to mimic the A,B-ring of colchicine site-binding microtubule disruptors such as 2-methoxyestradiol in the design of anti-cancer agents. Steroidomimeric microtubule disruptors were synthesized by introducing C'2 and C'3 of the steroidal A-ring to C'6 and C'7 of the THIQ core and by introducing a decorated hydrogen bond acceptor motif projecting from the steroidal D-ring to N'2. For this in vitro study, four non-steroidal THIQ-based analogues were investigated and comparative studies were done between the non-sulphamoylated compound STX 3450 and the sulphamoylated compounds STX 2895, STX 3329 and STX 3451. The objective of this study was to investigate the modes of cell death induced by these four THIQ-based analogues in A549 lung carcinoma epithelial cells and metastatic breast adenocarcinoma MDA-MB-231 cells. Cytotoxicity studies to determine the half maximal growth inhibitory concentrations were done using spectrophotometric quantification via crystal violet staining and lactate dehydrogenase (LDH) assays. Microtubule integrity and morphologic changes of exposed cells were investigated using polarization-optical transmitted light differential interference contrast microscopy, transmission electron microscopy and confocal microscopy. Flow cytometric quantification was used to determine apoptosis induction and the effect that THIQ-based analogues have on cell cycle progression. Signal transduction pathways were elucidated by quantification of the mitochondrial membrane integrity, cytochrome c release and caspase 3, -6 and -8 activation. Induction of autophagic cell death by the THIQ-based analogues was investigated by morphological assessment of fluorescent monodansylcadaverine (MDC) staining of acidic vacuoles and by quantifying aggresome formation via flow cytometry. Results revealed that these non-steroidal microtubule disrupting analogues inhibited 50% of cell growth at nanomolar concentrations. Immunofluorescence microscopy indicated microtubule depolarization and the resultant mitotic arrest was further confirmed through cell cycle analysis. Apoptosis induction via the intrinsic pathway was observed due to depolarization of the mitochondrial membrane, induction of cytochrome c release as well as, caspase 3 activation. Potential involvement of programmed cell death type II was observed due to the presence of acidic vacuoles and aggresome formation. Necrotic cell death did not contribute significantly, indicated by stable LDH levels. This in vitro study revealed the induction of the intrinsic apoptotic pathway as well as possible involvement of autophagy after exposure to these THIQ-based analogues in both MDA-MB-231- and A549 cells. Further investigation of this series of anticancer drugs still needs to be conducted to elucidate the temporal, mechanistic and functional crosstalk mechanisms between the two observed programmed cell deaths pathways.

Keywords: apoptosis, autophagy, cancer, microtubule disruptor

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Authors: Nadeem Bashir, Ghulam Mustafa Peerzada

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The present study pertains to the nonlinear behavior of acetaminophen based uncatalyzed Belousov-Zhabotinsky (BZ) oscillator and its dynamics in the presence of Ferroin as the catalyst. The role of free metal ions as catalysts was examined and the results compared with corresponding complexed catalysts. Free metal ions were found to be sluggish with respect to the evolution of the oscillatory regime as compared to complexed ones. Effect of change of the ligand moiety of the catalyst complex on the oscillatory parameters was monitored. Since ethanol potentiates the hepatotoxicity caused by acetaminophen in-vivo, it is thought to understand this interaction by virtue of causing perturbation of the acetaminophen based oscillator with different concentrations of the ethanol with and without ferroin as the catalyst. Another dimension to the ethanol effect was added by perturbation of the system with ethanol at different stages of the reaction so as to get an idea whether it is acetaminophen or some reactive intermediate generated in the reaction system which reacts with ethanol. Further, the ferroin-catalyzed oscillator is taken as a prototype inorganic model of the acetaminophen-ethanol syndrome, as ferroin and HOBr were inorganic replacements to Cyt P450 and NADPH in the alcohol metabolism.

Keywords: Belousov-Zhabotinsky reaction, ferroin, Paracetamol-Ethanol syndrome, kinetics

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Authors: Naso Isaiah Thanavisuth

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Medical cannabis can be used for treatment including pain, multiple sclerosis, Parkinson's disease, and cancer. However, medical cannabis leads to adverse effects (AEs), which is delta-9-tetrahydrocannabinol (THC). In previous studies, the major of THC metabolism enzymes are CYP2C9. Especially, the variation of CYP2C9 gene consist of CYP2C9*2 on exon 3 and CYP2C9*3 on exon 7 to decrease enzyme activity. Notwithstanding, there is no data describing whether the variant of CYP2C9 genes are apharmacogenetics marker for the prediction of THC-induced AEs in Thai patients. We want to investigate the association between CYP2C9 gene and THC-induced AEs in Thai patients. We enrolled 39 Thai patients with medical cannabis treatment who were classified by clinical data. The CYP2C9*2 and *3 genotyping were conducted using the TaqMan real time PCR assay. All Thai patients who received the medical cannabis consist of twenty-four (61.54%) patients were female, and fifteen (38.46%) were male, with age range 27- 87 years. Moreover, the most AEs in Thai patients who were treated with medical cannabis between cases and controls were tachycardia, arrhythmia, dry mouth, and nausea. Particularly, thirteen (72.22%) medical cannabis-induced AEs were female and age range 33 – 69 years. In this study, none of the medical cannabis groups carried CYP2C9*2 variants in Thai patients. The CYP2C9*3 variants (*1/*3, intermediate metabolizer, IM) and (*3/*3, poor metabolizer, PM) were found, three of thirty-nine (7.69%) and one of thirty-nine (2.56%), respectively. Although, our results indicate that there is no found the CYP2C9*2. However, the variation of CYP2C9 allele might serve as a pharmacogenetics marker for screening before initiating the therapy with medical cannabis for the prevention of medical cannabis-induced AEs.

Keywords: CYP2C9, medical cannabis, adverse effects, THC, P450

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Authors: Maryam Zahedi Fard, Nazia Abdul Majid, Hapipah Mohd Ali, Mahmood Ameen Abdulla

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New quinazoline schiff base 3-(5-bromo-2-hydroxy-3-methoxybenzylideneamino)-2-(5-bromo-2-hydroxy-3-methoxyphenyl)-2,3-dihydroquinazolin-4(1H)-one was investigated for anticancer activity against MCF-7 human breast cancer cell line with involved mechanism of apoptosis. The compound demonstrated a remarkable antiproliferative effect, with an IC50 value of 3.41 ± 0.34, after 72 hours of treatment. Morphological apoptotic features in treated MCF-7 cells were observed by AO/PI staining. Furthermore, treated MCF-7 cells subjected to apoptosis death, as exhibited by perturbation of mitochondrial membrane potential and cytochrome c release as well as increase in ROS generation. We also found activation of caspases 3/7 and -9. Moreover, acute toxicity test demonstrated the nontoxic nature of the compound in mice. Our results showed the selected compound significantly induce apoptosis in MCF-7 cells via intrinsic pathway, which might be considered as a potent candidate for further in vivo and clinical breast cancer studies.

Keywords: antiproliferative effect, MCF-7 human breast cancer cell line, apoptosis, caspases

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Authors: C. I. Boshoff, S. Steenkamp-Jonker

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Cystic echinococcosis (CE), caused by Echinococcus granulosus is an important parasitic infection in livestock worldwide, with severe zoonotic potential. It is important to understand the variability of Echinococcus granulosus, as genotype variations may influence lifecycle patterns, development rate, and transmission. Cystic Echinococcus samples were collected from domestic animals in Eastern Cape Province, South Africa. A molecular study was performed on 14 hydatid cysts obtained from caprine, ovine and bovine livers in order to determine the Echinococcus granulosus strain present in these hosts. The sequencing of the mitochondrial cytochrome C oxidase subunit I (coxI) gene of the hydatid cysts produced sequences of 400 bp for each sample analysed. These sequences were aligned with those present in GenBank and a phylogenetic tree was constructed. Based on coxI genotype the isolates could be grouped into E. granulosus sensu stricto. The findings of the study represent a pilot molecular study on Echinococcus from domestic animals undertaken in South Africa.

Keywords: Echinococcus granulosus, genotypes, livestock, South Africa

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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: Thanusha D. Abeywickrama, Inoka C. Perera, Genji Kurisu

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Tuberculosis, considered being as the ninth leading cause of death worldwide, cause from a single infectious agent M. tuberculosis and the drug resistance nature of this bacterium is a continuing threat to the world. Therefore TB preventing treatment is expanding, where this study designed to analyze the regulatory mechanism of GntR transcriptional regulator gene Rv0792c, which lie between several genes codes for some hypothetical proteins, a monooxygenase and an oxidoreductase. The gene encoding Rv0792c was cloned into pET28a and expressed protein was purified to near homogeneity by Nickel affinity chromatography. It was previously reported that the protein binds within the intergenic region (BS region) between Rv0792c gene and monooxygenase (Rv0793). This resulted in binding of three protein molecules with the BS region suggesting tight control of monooxygenase as well as its own gene. Since monooxygenase plays a key role in metabolism, this gene may have a global regulatory role. The natural ligand for this regulator is still under investigation. In relation to the Rv0792 protein structure, a Circular Dichroism (CD) spectrum was carried out to determine its secondary structure elements. Percentage-wise, 17.4% Helix, 21.8% Antiparallel, 5.1% Parallel, 12.3% turn and 43.5% other were revealed from CD spectrum data under room temperature. Differential Scanning Calorimetry (DSC) was conducted to assess the thermal stability of Rv0792, which the melting temperature of protein is 57.2 ± 0.6 °C. The graph of heat capacity (Cp) versus temperature for the best fit was obtained for non-two-state model, which concludes the folding of Rv0792 protein occurs through stable intermediates. Peak area (∆HCal ) and Peak shape (∆HVant ) was calculated from the graph and ∆HCal / ∆HVant was close to 0.5, suggesting dimeric nature of the protein.

Keywords: CD spectrum, DSC analysis, GntR transcriptional regulator, protein structure

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Authors: Irina S. Burashnikova, Dmitriy A. Sychev, Ruslan Y. Kazakov

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Сytochrome P450 CYP2D6 activity affects antipsychotic therapy safety. CYP2D6*4 polymorphism frequency varies among different ethnic groups. We studied CYP2D6*4 polymorphism frequency in Tatar and Russian schizophrenic patients and association of CYP2D6*4 polymorphism and extrapyramidal disorders (EPD) frequency in schizophrenic patients on haloperidol monotherapy in daily doses up to 20 mg. Results: Heterozygous CYP2D6*4 allele carrier frequency among Tatars was lower (23.8% vs 32.4% in Russians), but the differences did not reach statistical significance. CYP2D6*4 allele frequency among Tatars was also lower (11.9% vs 24.3% in Russians), but the difference was not quite significant (p=0.0592). Average daily haloperidol dose in the group without EPD was significantly higher than in the group with EPD (11.35±4.6 vs 13.87±3.3 mg, p=0.0252), but average daily haloperidol dose/weight ratios in the compared groups had no significant differences. Statistically significant association between EPD development and heterozygous CYP2D6*1/*4 genotype and CYP2D6*4 allele carrier frequency was revealed among all schizophrenic patients and among those of Tatar nationality. Further well designed pharmacogenetic studies in different Russian regions are needed to improve psychotropic therapy safety and to establish evidence-based indications for pharmacogenetic testing in clinical practice.

Keywords: antipsychotic, CYP2D6 polymorphism, ethnic differences of CYP2D6*4 allele frequency, extrapyramidal side effects/disorder, schizophrenia, pharmacogenetics, Russians, Tatars

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Authors: Mohsina Akhter

Abstract:

Cancer has become a wide spread disease around the globe and takes many lives every year. Different treatments are being practiced but all have potential side effects with somewhat less specificity towards target sites. Pseudomonas aeruginosa is known to secrete a protein azurin with special anti-cancer function. It has unique cell penetrating peptide comprising of 18 amino acids that have ability to enter cancer cells specifically. Reported function of Azurin is to stabilize p53 inside the tumor cells and induces apoptosis through Bax mediated cytochrome c release from mitochondria. At laboratory scale, we have made recombinant azurin through cloning rpTZ57R/T-azu vector into E.coli strain DH-5α and subcloning rpET28-azu vector into E.coli BL21-CodonPlus (DE3). High expression was ensured with IPTG induction at different concentrations then optimized high expression level at 1mM concentration of IPTG for 5 hours. Purification has been done by using Ni+2 affinity chromatography. We have concluded that azurin can be a remarkable improvement in cancer therapeutics if it produces on a large scale. Azurin does not enter into the normal cells so it will prove a safe and secure treatment for patients and prevent them from hazardous anomalies.

Keywords: azurin, pseudomonas aeruginosa, cancer, therapeutics

Procedia PDF Downloads 311

Authors: Rizka Ardhiyana, Liesbetini Haditjaroko, Sri Mulijani, Reki Ashadi Wicaksono, Raafqi Ranasasmita

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Designing a sensitive, specific and easy to use method to detect pork contamination in the food industry remains a major challenge. In the current study, we developed a sensitive thiol-bond AuNP-Probe biosensor that will change color when detecting pork DNA in the Cytochrome B region. The interaction between the biosensors and DNA sample is measured by spectrophotometer at 540 nm. The biosensor is made by reducing gold with trisodium citrate to produce gold nanoparticle with 39.05 nm diameter. The AuNP-Probe biosensor (gold nanoprobe) achieved 16.04 ng DNA/µl limit of detection and 53.48 ng DNA/µl limit of quantification. The linearity (R2) between color absorbance changes and DNA concentration is 0.9916. The biosensor has a good specificty as it does not cross-react with DNA of chicken and beef. To verify specificity towards the target sequence, PCR was tested to the target sequence and reacted to the PCR product with the biosensor. The PCR DNA isolate resulted in a 2.7 fold higher absorbance compared to pork-DNA isolate alone (without PCR). The sensitivity and specificity of the method show the promising application of the thiol-bond AuNP biosensor in pork-detection.

Keywords: biosensor, DNA probe, gold nanoparticle (AuNP), pork meat, qPCR

Procedia PDF Downloads 359

Authors: Jiahui Song, Ravindra Joshi

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High-intensity, nanosecond, pulsed electric fields have been shown to be useful non-thermal tools capable of producing a variety of specific cellular responses. While reversible and temporary changes are often desired based on electromanipulation, irreversible effects can also be important objectives. These include elimination of tumor cells and bacterial decontamination. A simple model-based rate-equation treatment of the various cellular biochemical processes was used to qualitatively predict the pulse number-dependent caspase activation and cell survival trends. The model incorporated the caspase-8 associated extrinsic pathway, the delay inherent in its activation, cytochrome c release, and the internal feedback mechanism between caspase-3 and Bid. Results were roughly in keeping with the experimental cell-survival data. A pulse-number threshold was predicted followed by a near-exponential fall-off. The intrinsic pathway was shown to be much weaker as compared to the extrinsic mechanism for electric pulse induced cell apoptosis. Also, delays of about an hour are predicted for detectable molecular concentration increases following electrical pulsing.

Keywords: apoptosis, cell survival, model, pathway

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Authors: V. Konjik, J. Schwartz, R. Sandhoff, M. Mack

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The rising number of multi-resistant pathogens demands the development of new antibiotics in order to reduce the lethal risk of infections. Here, we investigate roseoflavin, a vitamin B2 analogue which is produced by Streptomyces davawensis and Streptomyces cinnabarinus. We consider roseoflavin to be a 'Trojan horse' compound. Its chemical structure is very similar to riboflavin but in fact it is a toxin. Furthermore, it is a clever strategy with regard to the delivery of an antibiotic to its site of action but also with regard to the production of this chemical: The producer cell has only to convert a vitamin (which is already present in the cytoplasm) into a vitamin analog. Roseoflavin inhibits the activity of Flavin depending proteins, which makes up to 3.5 % of predicted proteins in organisms sequenced so far. We sequentially knocked out gene clusters and later on single genes in order to find the ones which are involved in the roseoflavin biosynthesis. Consequently, we identified the gene rosB, coding for the protein carrying out the first step of roseoflavin biosynthesis, starting form Flavin mononucleotide. Here we show, that the protein RosB has so far unknown features. It is per se an oxidoreductase, a decarboxylase and an aminotransferase, all rolled into one enzyme. A screen of cofactors revealed needs of oxygen, NAD+, thiamine and glutamic acid to carry out its function. Surprisingly, thiamine is not only needed for the decaboxylation step, but also for the oxidation of 8-demethyl-8-formyl Flavin mononucleotide. We had managed to isolate three different Flavin intermediates with different oxidation states, which gave us a mechanistic insight of RosB functionality. Our work points to a so far new function of thiamine in Streptomyces davawensis. Additionally, RosB could be extremely useful for chemical synthesis. Careful engineering of RosB may allow the site-specific replacement of methyl groups by amino groups in polyaromatic compounds of commercial interest. Finally, the complete clarification of the roseoflavin biosynthesis opens the possibility of engineering cost-effective roseoflavin producing strains.

Keywords: antibiotic, flavin analogue, roseoflavin biosynthesis, vitamin B2

Procedia PDF Downloads 243