Search results for: mitochondrial 16s rRNA gene

Authors: Ahlam Inayatullah Badrul Munir, M. Husaini A. Rahman, Mohd Sukri Hassan

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Real-time PCR is a molecular biology technique that is currently being widely used for halal services to differentiating between porcine and bovine DNA. The useful of technique become very important for student or workers (who works in the laboratory) to learn how the technique could be run smoothly without fail. Same concept with conventional PCR, real-time PCR also needed DNA template, primer, enzyme polymerase, dNTP, and buffer. The difference is in real-time PCR, have additional component namely fluorescent dye. The most common use of fluorescent dye in real-time PCR is SYBR green. The purpose of this study was to find out how sensitive, specific and efficient real-time PCR technique was combined with SYBR green method and specific primers of CYT b. The results showed that real-time PCR technique using SYBR Green, capable of detecting porcine and bovine DNA concentrations up to 0.0001 µl/ng. The level of efficiency for both types of DNA was 91% (90-110). Not only that in specific primer CYT b bovine primer could detect only bovine DNA, and porcine primer could detect only porcine primer. So, from the study could be concluded that real-time PCR technique that was combined with specific primer CYT b and SYBR green method, was sensitive, specific and efficient to detect porcine and bovine DNA.

Keywords: sensitivity, specificity, efficiency, real-time PCR, SYBR green, Cytochrome b, porcine DNA, bovine DNA

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Authors: S. Taghavi Kalajahi, A. Koerdt, T. Lund Skovhus

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Cathodic protection (CP) is an electrochemical method to control and manage corrosion in different industries and environments. CP which is widely used, especially in buried and sub-merged environments, which both environments are susceptible to microbiologically influenced corrosion (MIC). Most of the standards recommend performing CP using -800 mV, however, if MIC threats are high or sulfate reducing bacteria (SRB) is present, the recommendation is to use more negative potentials for adequate protection of the metal. Due to the lack of knowledge and research on the effectiveness of CP on MIC, to the author’s best knowledge, there is no information about what MIC threat is and how much more negative potentials should be used enabling adequate protection and not overprotection (due to hydrogen embrittlement risk). Recently, the development and cheaper price of molecular microbial methods (MMMs) open the door for more effective investigations on the corrosion in the presence of microorganisms, along with other electrochemical methods and surface analysis. In this work, using MMMs, the gene expression of SRB biofilm under different potentials of CP will be investigated. The specific genes, such as pH buffering, metal oxidizing, etc., will be compared at different potentials, enabling to determine the precise potential that protect the metal effectively from SRB. This work is the initial step to be able to standardize the recommended potential under MIC condition, resulting better protection for the infrastructures.

Keywords: cathodic protection, microbiologically influenced corrosion, molecular microbial methods, sulfate reducing bacteria

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Authors: Hai Xu, Yiwei Wang, Xi Bao, Bihua Deng, Pengcheng Li, Yu Lu

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T7 phage could be used as a perfect vector for peptides expression and haptens presentation. T7-3GnRH recombinant phage was constructed by inserting three copies of Gonadotrophin Releasing Hormone (GnRH) gene into the multiple cloning site of T7 Select 415-1b phage genome. The positive T7-3GnRH phage was selected by using polymerase chain reaction amplification, and the p10B-3GnRH fusion protein was verified by SDS-PAGE and Western-blotting assay. T7-3GnRH vaccine was made and immunized with 10¹⁰ pfu in 0.2 ml per dose in mice. Blood samples were collected at an interval in weeks, and anti-GnRH antibody and testosterone concentrations were detected by ELISA and radioimmunoassay, respectively. The results show that T7-3GnRH phage particles confer a high immunogenicity to the GnRH-derived epitope. Moreover, the T7-3GnRH vaccine induced higher level of anti-GnRH antibody than ImproVac^®. However, the testosterone concentrations in both immunized groups were at a similar level, and the testis developments were significantly inhibited compared to controls. These findings demonstrated that the anti-GnRH antibody could neutralize the endogenous GnRH to down regulate testosterone level and limit testis development, highlighting the potential value of T7-3GnRH in the immunocastration vaccine research.

Keywords: Gonadotrophin Releasing Hormone (GnRH), Immunocastration, T7 phage, Phage vaccine

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Authors: Ahmad Daryani, Yousef Dadimoghaddam, Mehdi Sharif, Ehsan Ahmadpour, Shahabeddin Sarvi, Baghar Hashemi

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Background: Excretory-secretory antigens (ESAs) of Toxoplasma gondii are one of the candidates for immunization against toxoplasmosis. For evaluation of immunization, we determined the kinetics of the distribution of Toxoplasma and parasite load in different tissues of mice immunized by ESAs. Methods: In this experimental study, 36 mice in case (n= 18) and control (n= 18) groups were immunized with ESAs and PBS, respectively. After 2 weeks, mice were challenged intraperitoneally with Toxoplasma virulent RH strain. Blood and different tissues (brain, spleen, liver, heart, kidney, and muscle) were collected daily after challenge (1, 2, 3 and last day before death). Parasite load was calculated using Real time QPCR targeted at the B1 gene. Results: ESAs as vaccine in different tissues showed various effects. However, infected mice which received the vaccine in comparison with control group, displayed a drastically decreasing in parasite burden, in their blood and tissues (P= 0.000). Conclusion: These results indicated that ESAs with reduction of parasite load in different tissues of host could be evaluable candidate for the development of immunization strategies against toxoplasmosis.

Keywords: parasitic load, murine toxoplasmosis, immunization, excretory-secretory antigens, real time QPCR

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Authors: Ali Olfati, Parichehr Nouri

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Objective: In this study, we have investigated for the first time in the literature the efficacy of riboflavin [VB2] in preventing uterus As₂O₃ damage. Methods: Rats received 40 μg LHRHa for estrus synchronization. 48 pregnant Wistar rats were included. Four groups were formed with 7 rats in each group: Sham, 1.5 mg arsenic trioxide (As₂O₃/L) alone or in combination with VB2 [20 and 40 mg/L] in drinking water [for 21 days continuously]. Similar to maternal generation treatment, the F1-female generation was also arranged [for 35 days continuously until puberty]. Results: Data indicated that As₂O₃ reduced body weight and feed intake (p<0.05). Furthermore, the serum malondialdehyde levels in the As₂O₃ group were significantly higher than that of the control group (p<0.05). At the same time, total antioxidative status and the activities of glutathione peroxidase, superoxide dismutase, and catalase were reduced (p<0.05). Meanwhile, As₂O₃ remarkably increased the production of inflammatory markers [interleukin 6 and C-reactive protein](p<0.05). As₂O₃ administration induced uterus apoptosis-related genes by upregulating caspase-3, iNOS, and Bax genes and downregulating Bcl-2 gene of pubertal F1-female rats (p<0.05). Conclusion: Our observation indicated that VB2 therapy is potentially an effective strategy to modifying the detrimental effects of As₂O₃ in pubertal F1-female rats via suppresses oxidative damages.

Keywords: As₂O₃, inflammation, puberty, vitamin B2

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Authors: Daruliza Kernain, Shaharum Shamsuddin, See Too Wei Cun

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CTCF is a unique, highly conserved and ubiquitously expressed 11 zinc finger (ZF) transcriptional factor with multiple target sites. It is able to bind to various target sequences to perform different regulatory roles including promoter activation or repression, creating hormone-responsive gene silencing element, and functional block of enhancer-promoter interactions. The binding of CTCF to the essential binding site is through the combination of different ZF domain. On the other hand, BORIS for brother of the regulator of imprinted sites, which expressed only in the testis and certain cancer cell line is homology to CTCF 11 ZF domains. Since both transcriptional factors share the same ZF domains hence there is a possibility for both to bind to the same target sequences. In this study, the interaction of these two proteins to multi-functional Y-box DNA/RNA-binding factor, YB-1 was determined. The protein-protein interaction between CTCF/YB-1 and BORIS/YB-1 were discovered by Co-immuno-precipitation (CO-IP) technique through reciprocal experiment from RGBM total cell lysate. The results showed that both CTCF and BORIS were able to interact with YB-1 in Glioma RGBM cell line. To the best of our knowledge, this is the first findings demonstrating the ability of BORIS and YB-1 to form a complex in vivo.

Keywords: immunoprecipitation, CTCF/BORIS/YB-1, transcription factor, molecular medicine

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Authors: Sonam Kumari

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Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, possesses a remarkable feature of entering into and emerging from a persistent state. The mechanism by which Mtb switches from the dormant state to the replicative form is still poorly characterized. Proteome studies have given us an insight into the role of certain proteins in giving stupendous virulence to Mtb, but numerous dotsremain unconnected and unaccounted. The WhiB family of proteins is one such protein that is associated with developmental processes in actinomycetes.Mtb has seven such proteins (WhiB1 to WhiB7).WhiB proteins are transcriptional regulators; their conserved C-terminal HTH motif is involved in DNA binding. They regulate various essential genes of Mtbby binding to their promoter DNA. Biophysical Analysis of the effect of DNA binding on WhiB proteins has not yet been appropriately characterized. Interaction with DNA induces conformational changes in the WhiB proteins, confirmed by steady-state fluorescence and circular dichroism spectroscopy. ITC has deduced thermodynamic parameters and the binding affinity of the interaction. Since these transcription factors are highly unstable in vitro, their stability and solubility were enhanced by the co-expression of molecular chaperones. The present study findings help determine the conditions under which the WhiB proteins interact with their interacting partner and the factors that influence their binding affinity. This is crucial in understanding their role in regulating gene expression in Mtbandin targeting WhiB proteins as a drug target to cure TB.

Keywords: tuberculosis, WhiB proteins, mycobacterium tuberculosis, nucleic acid binding

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Authors: Katarína Koňariková, Georgios A. Perdikaris, Lucia Andrezálová, Zdeňka Ďuračková, Lucia Laubertová, Helena Gbelcová, Ingrid Žitňanová

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Introduction: The continuous demand for new anti-cancer drugs has stimulated chemotherapeutic research based on the use of essential metalloelements with the aim to develop potential drugs with lower toxicity and higher antiproliferative activity against tumors. Copper(II) and its complexes play an important role as suitable species for antiproliferative tests. Objectives: The central objective of the current study was to investigate the potential in vitro anti-proliferative effects of N-salicylidene-L-glutamato copper (II) complexes and molecular mechanism of apoptosis induced by tested complexes. In our project we tested N-salicylidene-L-glutamato copper (II) complexes ZK1 - [Cu(N-salicylidene-L-glutamato)(H2O)2].H2O; MK0 - ([Cu2(N-sal-D,L-glu)2(isoquinoline)2].2H2O); MK1 - [Cu(N-salicylidene-5-methyl-L-glutamato)(H2O)].H2O; MK3 - transbis(ethanol)tetrakis(imidazol)Cu(II)(2+)bis(N-salicylidene-D,L-glutamato-N,O)-KO:KO´-(imidazol); MK5 - [Cu(N-salicylidene-D,L- glutamato)(2-methylimidazol] at concentration range 0.001-100 µmol/L against human colon carcinoma cell line HT-29 and human breast carcinoma cell line MCF-7. Methods: Viability was assessed by direct counting of 0.4% trypan blue dye-excluding cells after 24, 48 and 72 hour cultivations with or without copper complex and by MTT assay. To analyze the type of cell death and its mechanism induced by our copper complex we used different methods. To distinguish apoptosis from necrosis we used electrophoretic analysis, to study the activity of caspases 8 and 9 – luminometric analysis and caspase activity 3 colorimetric assay. Results: The observed anti-proliferative effect of the copper complexes appeared to be dose-, time- and cell line- dependent. Human colon carcinoma cells HT-29 appeared to be more sensitive to the complex MK0 ([Cu2(N-sal-D,L-glu)2(isoquinoline)2].2H2O) than to ZK1 ([Cu(N-salicylidene-L-glutamato)(H2O)2].H2O) and MK1 ([Cu(N-salicylidene-5-methyl-L-glutamato)(H2O)].H2O)). Human colon carcinoma cells HT-29 appeared to be more sensitive to the complex than human breast carcinoma cells MCF-7. IC50 decreased with time of incubation (24, 48 and 72h) for HT-29, but increased for MCF-7. By electrophoresis we found apoptotic cell death induced by our copper complexes in HT-29 at concentrations 1, 10, 50 and 100 µmol/L after 48h (ZK1) and 72h (MK0, MK1) and in MCF-7 we did not find apoptosis. We also studied molecular mechanism of apoptosis in HT-29 induced by copper complexes. We found active caspase 9 in HT-29 after ZK1 ([Cu(N-salicylidene-L-glutamato)(H2O)2].H2O) and MK1 ([Cu(N-salicylidene-5-methyl-L-glutamato)(H2O)].H2O)) influence and active caspase 8 after MK0 ([Cu2(N-sal-D,L-glu)2(isoquinoline)2].2H2O) influence. Conclusion: Our copper complexes showed cytotoxic activities against human colon carcinoma cells HT-29 and breast cancer cell line MCF-7 in vitro. Apoptosis was activated by mitochondrial pathway (intrinsic pathway) in case of ZK1 [Cu(N-salicylidene-L-glutamato)(H2O)2].H2O; MK1 [Cu(N-salicylidene-5-methyl-L-glutamato)(H2O)].H2O; MK3 - transbis(ethanol)tetrakis(imidazol)Cu(II)(2+)bis(N-salicylidene-D,L-glutamato-N,O)-KO:KO´-(imidazol) and MK5 - [Cu(N-salicylidene-D,L- glutamato)(2-methylimidazol] copper complexes and by death receptors (extrinsic pathway) in case of MK0 [Cu2(N-sal-D,L-glu)2(isoquinoline)2].2H2O copper complex in HT-29.

Keywords: apoptosis, copper complex, cancer, carcinoma cell line

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Authors: Caroline Kim

Abstract:

The designation autism spectrum disorder (ASD) groups complex disorders in the development of the brain. Autism is defined essentially as a condition in which an individual lacks a theory of mind. The theory of mind, in this sense, explains the ability of an individual to attribute feelings, emotions, or thoughts to another person. An autistic patient is characteristically unable to determine what an interlocutor is feeling, or to understand the beliefs of others. However, it is possible that autism cannot plausibly characterized as the lack of theory of mind in an individual. Genes, the bran, and its interplay with environmental factors may also cause autism. A mutation in a gene may be hereditary, or instigated by diseases such as mumps. Though an autistic patient may experience abnormalities in the cerebellum and the cortical regions, these are in fact only possible theories as to a biochemical explanation behind the disability. The prevailing theory identifying autism with lacking the theory of mind is supported by behavioral observation, but this form of observation is itself determined by socially constructed standards, limiting the possibility for empirical verification. The theory of mind infers that the beliefs and emotions of people are causally based on their behavior. This paper demonstrates the fallacy of this inference, critiquing its basis in socially constructed values, and arguing instead for a biochemical approach free from the conceptual apparatus of language and social expectation.

Keywords: autism spectrum disorder, sociology of psychology, social construction, the theory of mind

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Authors: Niranjan Koirala, Sumangala Darsandhari, Hye Jin Jung, Jae Kyung Sohng

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Sakuranetin, an antifungal agent and genkwanin, an anti-inflammatory agent, are flavonoids with several potential pharmaceutical applications. To produce such valuable flavonoids in large quantity, an Escherichia coli cell factory has been created. E. coli harboring O-methyltransferase (SaOMT2) derived from Streptomyces avermitilis was employed for regiospecific methylation of naringenin and apigenin. In order to increase the production via biotransformation, metK gene was overexpressed and the conditions were optimized. The maximum yield of sakuranetin and genkwanin under optimized conditions was 197 µM and 170 µM respectively when 200 µM of naringenin and apigenin were supplemented in the separate cultures. Furthermore, sakuranetin was purified in large scale and used as a substrate for in vitro glycosylation by YjiC to produce glucose and galactose derivatives of sakuranetin for improved solubility. We also found that unlike naringenin, sakuranetin effectively inhibits α-melanocyte stimulating hormone (α-MSH)-stimulated melanogenesis in B16F10 melanoma cells. In addition, genkwanin more potently inhibited angiogenesis than apigenin. Based on our findings, we speculate that these compounds warrant further investigation in vivo as potential new therapeutic anti-carcinogenic, anti-melanogenic and anti-angiogenic agents.

Keywords: anti-carcinogenic, anti-melanogenic, glycosylation, methylation

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Authors: Ivar Zekker, Ergo Rikmann, Anni Mandel, Markus Raudkivi, Kristel Kroon, Liis Loorits, Andrus Seiman, Hannu Fritze, Priit Vabamäe, Toomas Tenno, Taavo Tenno

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The anaerobic ammonium oxidation (anammox) and nitritation-anammox (deammonification) processes are widely used for N-rich wastewater treatment nowadays. A deammonification moving bed biofilm reactor (MBBR) with a high maximum total nitrogen removal rate (TNRR) of 1.5 g N m-2 d-1 was started up and similarly high TNRR was sustained at low temperature of 15°C. During biofilm cultivation, temperature in MBBR was lowered by 0.5° C week-1 sustaining the high TNRR. To study the short-term effect of temperature on the TNRR, a series of batch-scale experiments performed showed sufficient TNRRs even at 9-15° C (4.3-5.4 mg N L-1 h-1, respectively). After biomass was adapted to lower temperature (15°C), the TNRR increase at lower temperature (15°C) was relatively higher (15-20%) than with biomass adapted to higher temperatures (17-18°C). Anammox qPCR showed increase of Candidatus Brocadia quantities from 5×103 to 1×107 anammox gene copies g-1 TSS despite temperature lowered to 15°C. Modeling confirmed causes of stable and unstable periods in the reactor and in batch test high Arrhenius constant of 29.7 kJ mol-1 of the process as high as at 100 mg NO2--N L-1 were determined. 

Keywords: deammonification, reject water, intermittent aeration, nitrite inhibition

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Authors: Petr Konvalina, Jan Moudry

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As organic farmers are searching foregoing crops for horticultural crops, there is possible to choice neglected wheat species and also have a new market and sale opportunities. Concerning wheat, there are landraces so called hulled wheat species (einkorn, emmer wheat, spelt) comprising parts of collections of the world gene banks. The advantage of this wheat species are small demands on growing conditions and also droughtiness in conditions of changing climate. Our paper aims at presenting the results of the study and the assessment of spring wheat forms, four einkorn cultivars, eight emmer wheat cultivars, seven spelt wheat cultivars in particular, as compared to modern bread wheat variety. Small-plot trials were established at two different localities within the Czech Republic and Austria in 2009 and 2012. The results of the trials show that some varieties were inclined to lodging. On the other hand, they were resistant to common wheat diseases (mildew, brown rust). Hulls served as barriers and obstacles against the DON grain contamination. The yield rate was lower. The grains were characterized by a high proportion of protein in grain (up to 18.1 %). However, they may be difficult to use for common baking. Moreover, new food products demonstrating a different technological quality of the hulled wheat species have to be launched on the market. They will be suitable for regional marketing.

Keywords: organic farming, hulled wheat species, einkorn, emmer, spelt

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Authors: F. M. El-Domyati, A. Atef, S. Edris, N. O. Gadalla, M. A. Al-Kordy, A. M. Ramadan, Y. M. Saad, H. S. Al-Zahrani, A. Bahieldin

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Transcriptome retrieved from SRA database of different tissues and treatments of C. roseus was assembled in order to detect tissue-specific transcription factors (TFs) and TFs possibly related to terpenoid indole alkaloids (TIA) pathway. A number of 290 TF-like transcripts along with 12 transcripts related to TIA biosynthetic pathway were divided in terms of co-expression in the different tissues, treatments and genotypes. Three transcripts encoding peroxidases 1 and 12 were downregulated in hairy root, while upregulated in mature leaf. Eight different transcripts of the TIA pathway co-expressed with TFs either functioning downstream tryptophan biosynthesis, e.g., tdc, str1 and sgd, or upstream vindoline biosynthesis, e.g., t16h, omt, nmt, d4h and dat. The results showed no differential expression of TF transcripts in hairy roots knocked down for tdc gene (TDCi) as compared to their wild type controls. There were several evidences of tissue-specific expression of TF transcripts in flower, mature leaf, root/hairy root, stem, seedling, hairy root and immature/mature leaves. Regulation included transcription factor families, e.g., bHLH, MYB and WRKY mostly induced by ABA and/or JA (or MeJA) and regulated during abiotic or biotic stress. The information of tissue-specific regulation and co-expression of TFs and genes in the TIA pathway can be utilized in manipulating alkaloid biosynthesis in C. roseus.

Keywords: SRA database, bHLH, MYB, WRKY, co-expression

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Authors: Zhan Dong, Qiu Jianrong, Li Qinghui, Zhang Lei

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The quantity and quality of the papers published on sport science from 2001 to 2013 had been counted and analysed and compared with the papers published on the journal from 1990 to 2000. The result showed that: 1. In the sports medicine field, the proportion of basic/application was abnormal. Basic research was far more than the application research. The papers on researching of imitating altitude training was the main part. Gene research made great progress.The research on sport injury and medical supervision were lower and lower. Research on sports prescription had made much progress, especially in the patients of heart infarction. 2. In building up people’s health field, the research on the old people had been more and more compared with the 10 years before, but it was not enough. 3. In the field of sports psychology, the research on disable people had been more compared with the 10 years before. Solved the problem of the sportmen before the game. 4. In the field of sports biomechanics, it showed that methods had made great progress compared with the 10 years before. Sport biomechanics combined with sports medicine, helped the sportsmen in good condition in the game. 5. In the exercise training field, the experts pay more attention to the outstanding sportsmen, and the researches emphasized that biology knowledge is the main basic for them to the research.

Keywords: sport medicine, sport injury, medical supervision

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Authors: Rim Frikha, Maha Ben Jema, Moez Elloumi, Tarek Rebai

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Background: Acute lymphoblastic leukemia (ALL); a common blood cancer characterized by the interaction between genetic and environmental factors. Methylenetetrahydrofolate reductase (MTHFR) is an essential folate metabolic enzyme in the processes of DNA synthesis and methylation. A common functional variant of the MTHFR gene, the A1298C, which induces disturbances in folate metabolism, may affect susceptibility to ALL. Objective: The present study aimed to assess the prevalence of MTHFR polymorphism A1298 > C in Tunisian children with ALL. Materials and Methods: A total of 28 Tunisian ALL children were enrolled in this study. Genomic DNA was extracted from whole venous blood collected in ethylenediaminetetraacetic acid (EDTA). Genotyping was carried out with restriction fragment length polymorphism (RFLP) using MboII restriction enzyme. Genotype distribution and allele frequency of MTHFR A1298C was calculated in ALL patients. Results: The A1298C variant of MTHFR was found in 11(19.6%) heterozygous and one homozygous patient (3.5%). Conclusions: This result highlights that A1298C polymorphism of MTHFR is common in Tunisian childhood ALL and suggests that this variant may have a potential role in leukemogenesis. Genotyping of large samples and different ethnicities are required to validate these findings.

Keywords: methylenetetrahydrofolate reductase, acute lymphoblastic leukemia, A1298C variant, prevalence

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Authors: Chengwei Wang, Shuqing Xu, Philipp M. Schlüter

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The genus Ophrys is remarkable for its mimicry, flower-lip closely resembling pollinator females in a species-specific manner. Therefore, floral traits associated with pollinator attraction, especially scent, are suitable models for investigating the molecular basis of adaption, speciation, and evolution. Within the two Ophrys species groups: O. sphegodes (S) and O. fusca (F), pollinator shifts among the same insect species have taken place. Preliminary data suggest that they involve a comparable hydrocarbon profile in their scent, which is mainly composed of alkanes and alkenes. Genes encoding stearoyl-acyl carrier protein desaturases (SAD) involved in alkene biosynthesis have been identified in the S group. This study aims to investigate the control and parallel evolution of ecologically significant alkene production in Ophrys. Owing to the central role those SAD genes play in determining positioning of the alkene double-bonds, a detailed understanding of their functional mechanism and of regulatory aspects is of utmost importance. We have identified 5 transcription factors potentially related to SAD expression in O. sphegodes which belong to the MYB, GTE, WRKY, and MADS families. Ultimately, our results will contribute to understanding genes important in the regulatory control of floral scent synthesis.

Keywords: floral traits, transcription factors, biosynthesis, parallel evolution

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Authors: Stephanie A. Schauder, Gosia Sylwestrzak

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Overweight and obese women report avoiding preventive care due to fear of weight-related bias from medical professionals. Gynecological exams, due to their sensitive and personally invasive nature, are especially susceptible to avoidance. This research investigates the association between body mass index (BMI) and screening rates for breast and cervical cancer using claims data from 1.3 million members of a large health insurance company. Because obesity is associated with increased cancer risk, screenings for these cancers should increase as BMI increases. However, this paper finds that the distribution of cancer screening rates by BMI take an inverted U-shape with underweight and obese members having the lowest screening rates. For cervical cancer screening, those in the target population with a BMI of 23 have the highest screening rate at 68%, while Obese Class III members have a screening rate of 50%. Those in the underweight category have a screening rate of 58%. This relationship persists even after controlling for health and demographic covariates in regression analysis. Interestingly, there is no association between BMI and BRCA (BReast CAncer gene) genetic testing. This is consistent with the narrative that stigma causes avoidance because genetic testing does not involve any assessment of a person’s body. More work must be done to determine how to increase cancer screening rates in those who may feel stigmatized due to their weight.

Keywords: cancer screening, cervical cancer, breast cancer, weight stigma, avoidance of care

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Authors: Ovidiu Vlaicu, Leontina Banica, Dan Otelea, Andrei-Jose Petrescu, Costin-Ioan Popescu

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Hepatitis C Virus (HCV) infects 170 million peoples worldwide. Major advances have been made recently in HCV standard of care with interferon-free therapy being already approved. Despite major progress in HCV therapy, the genotype associated treatment efficacy and toxicity still represent issues to address. To identify endogenous factors involved in different stages of HCV life cycle, we have developed a trans-packaging system for HCV subgenomic replicons lacking core protein gene. Huh7 cells were used to generate a packaging cell line expressing the core protein in an inducible manner. The core packaging cell line was able to trans-complemented various subgenomic replicons to secret infectious trans-complemented HCV particles (HCV-TCP). Further, we constructed subgenomic replicons with foreign epitopes suitable for immunoaffinity purification or fluorescence microscopy studies. We have shown that the insertion has not effects on the efficacy of trans-complementation yielding similar titers to the control subgenomic replicon. This system will be a valuable tool in studying pre- and post-assembly events in HCV life cycle and for the fast identification of HCV assembly inhibitors.

Keywords: assembly inhibitors, core protein, HCV, trans-complementation

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Authors: Iñaki Milton-Laskibar, Leixuri Aguirre, Maria P. Portillo

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Introduction: Energy restriction is an effective approach in preventing liver steatosis. However, due to social and economic reasons among others, compliance with this treatment protocol is often very poor, especially in the long term. Resveratrol, a natural polyphenolic compound that belongs to stilbene group, has been widely reported to imitate the effects of energy restriction. Objective: To analyze the effects of resveratrol under normoenergetic feeding conditions and under a mild energy restriction on liver fat accumulation and hepatic fatty acid oxidation. Methods: 36 male six-week-old rats were fed a high-fat high-sucrose diet for 6 weeks in order to induce steatosis. Then, rats were divided into four groups and fed a standard diet for 6 additional weeks: control group (C), resveratrol group (RSV, resveratrol 30 mg/kg/d), restricted group (R, 15 % energy restriction) and combined group (RR, 15 % energy restriction and resveratrol 30 mg/kg/d). Liver triacylglycerols (TG) and total cholesterol contents were measured by using commercial kits. Carnitine palmitoyl transferase 1a (CPT 1a) and citrate synthase (CS) activities were measured spectrophotometrically. TFAM (mitochondrial transcription factor A) and peroxisome proliferator-activator receptor alpha (PPARα) protein contents, as well as the ratio acetylated peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α)/Total PGC1α were analyzed by Western blot. Statistical analysis was performed by using one way ANOVA and Newman-Keuls as post-hoc test. Results: No differences were observed among the four groups regarding liver weight and cholesterol content, but the three treated groups showed reduced TG when compared to the control group, being the restricted groups the ones showing the lowest values (with no differences between them). Higher CPT 1a and CS activities were observed in the groups supplemented with resveratrol (RSV and RR), with no difference between them. The acetylated PGC1α /total PGC1α ratio was lower in the treated groups (RSV, R and RR) than in the control group, with no differences among them. As far as TFAM protein expression is concerned, only the RR group reached a higher value. Finally, no changes were observed in PPARα protein expression. Conclusions: Resveratrol administration is an effective intervention for liver triacylglycerol content reduction, but a mild energy restriction is even more effective. The mechanisms of action of these two strategies are different. Thus resveratrol, but not energy restriction, seems to act by increasing fatty acid oxidation, although mitochondriogenesis seems not to be induced. When both treatments (resveratrol administration and a mild energy restriction) were combined, no additive or synergic effects were appreciated. Acknowledgements: MINECO-FEDER (AGL2015-65719-R), Basque Government (IT-572-13), University of the Basque Country (ELDUNANOTEK UFI11/32), Institut of Health Carlos III (CIBERobn). Iñaki Milton is a fellowship from the Basque Government.

Keywords: energy restriction, fat, liver, oxidation, resveratrol

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Authors: Naser Shagerdi Esmaeli, Mohsen Hamidpour, Parisa Hasankhani Tehrani

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Background: The TP53 mutation is frequently detected in acute myeloid leukemia (AML) patients with complex karyotype (CK), but the stability of this mutation during the clinical course remains unclear. Material and Methods: In this study, TP53 mutations were identified in 7% of 500 patients with de novo AML and 58.8% of patients with CK in Tabriz, Iran. TP53 mutations were closely associated with older age, lower white blood cell (WBC) and platelet counts, FAB M6 subtype, unfavorable-risk cytogenetics, and CK, but negatively associated with NPM1 mutation, FLT3/ITD and DNMT3A mutation. Result: Multivariate analysis demonstrated that TP53 mutation was an independent poor prognostic factor for overall survival and disease-free survival among the total cohort and the subgroup of patients with CK. A scoring system incorporating TP53 mutation and nine other prognostic factors, including age, WBC counts, cytogenetics, and gene mutations, into survival analysis proved to be very useful to stratify AML patients. Sequential study of 420 samples showed that TP53 mutations were stable during AML evolution, whereas the mutation was acquired only in 1 of the 126 TP53 wild-type patients when therapy-related AML originated from different clone emerged. Conclusion: In conclusion, TP53 mutations are associated with distinct clinic-biological features and poor prognosis in de novo AML patients and are rather stable during disease progression.

Keywords: acute myloblastic leukemia, TP53, FLT3/ITD, Iran

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Authors: Youn Young Shim, Ji Hye Kim, Jae Youl Cho, Martin J. T. Reaney

Abstract:

Flaxseed (Linum usitatissimum L.) is gaining popularity in the food industry as a superfood due to its health-promoting properties. The flax plant synthesizes an array of biologically active cyclic peptides or linusorbs (LOs, a.k.a. cyclolinopeptides) from three or more ribosome-derived precursors. [1–9-NαC]-linusorb B3 and [1–9-NαC]-linusorb B2, suppress immunity, induce apoptosis in human epithelial cancer cell line (Calu-3) cells, and inhibit T-cell proliferation, but the mechanism of LOs action is unknown. Using gene expression analysis in nematode cultures and human cancer cell lines, we have observed that LOs exert their activity, in part, through induction of apoptosis. Specific LOs’ properties include: 1) distribution throughout the body after flaxseed consumption; 2) induce heat shock protein (HSP) 70A production as an indicator of stress and address the issue in Caenorhabditis elegans (exposure of nematode cultures to [1–9-NαC]-linusorb B3 induced a 30% increase in production of the HSP 70A protein); 3) induce apoptosis in Calu-3 cells; and 4) modulate regulatory genes in microarray analysis. These diverse activities indicate that LOs might induce apoptosis in cancer cells or act as versatile platforms to deliver a variety of biologically active molecules for cancer therapy.

Keywords: flaxseed, linusorb, cyclic peptide, orbitides, heat shock protein, apoptosis, anti-cancer

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Authors: Mi-Hye Hwang, Young Min Son, Kichan Lee, Bang-Hun Hyun, Byeong Yeal Jung

Abstract:

Botulism is a paralytic disease of human beings and animals caused by neurotoxin produced by Clostridium botulinum. The neurotoxins are genetically distinguished into 8 types, A to H. Ingestion of performed toxin, usually types B, C, and D, have been shown to produce diseases in most cases of cattle botulism. Vaccination is the best measure to prevent cattle botulism. However, the commercially available toxoid-based vaccines are difficult and hazardous to produce. We produced recombinant protein using gene of heavy chain domain of botulinum toxin B of which binds to cellular receptor of neuron cells and used as immunogen. In this study, we evaluated the safety and efficacy of botulism vaccine composed of recombinant types B. Safety test was done by National Regulation for Veterinary Biologicals. For efficacy test, female ICR mice (5 weeks old) were subcutaneously injected, intraperitoneally challenged, and examined the survival rates compared with vaccination and non-vaccination group. Mouse survival rate of recombinant types B vaccine was above 80%, while one of non-vaccination group was 0%. A vaccine composed of recombinant types B was safe and efficacious in mouse. Our results suggest that recombinant heavy chain receptor binding domain can be used as an effective vaccine candidate for type B botulism.

Keywords: botulism, livestock, vaccine, recombinant protein, toxin

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Authors: Giang Tran Thi Huong, Hieu Dong Van, Tung Dao Duy, Saadanov Iskender, Isakeev Mairambek, Tsutomu Omatsu, Yukie Katayama, Tetsuya Mizutani, Yuki Ozeki, Yohei Takeda, Haruko Ogawa, Kunitoshi Imai

Abstract:

Newcastle disease virus (NDV) is a contagious viral disease of the poultry industry and other birds throughout the world. At present, very little is known about molecular epidemiological data regarding the causes of ND outbreak in commercial poultry farms in Kyrgyzstan. In the current study, the NDV isolated from the one out of three samples from the unvaccinated flock was confirmed as NDV. Phylogenetic analysis indicated that this NDV strain is clustered in the Class II subgenotype VIId, and closely related to the Chinese NDV isolate. Phylogenetic analyses revealed that the isolated NDV strain has an origin different from the 4 NDV strains previously identified in Kyrgyzstan. According to the mean death time (MDT: 61.1 h) and a multibasic amino acid (aa) sequence at the F0 proteolytic cleavage site (¹¹²R-R-Q-K-R-F¹¹⁷), the NDV isolate was determined as mesogenic strain. Several mutations in the neutralizing epitopes (notably, ³⁴⁷E→K) and the global head were observed in the hemagglutinin-neuraminidase (HN) protein of the current isolate. The present study represents the molecular characterization of the coding gene region of NDV in Kyrgyzstan. Additionally, further study will be investigated on the antigenic characterization using monoclonal antibody.

Keywords: Kyrgyzstan, Newcastle disease, genotype, genome characterization

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Authors: Bin Liu

Abstract:

MicroRNAs (miRNAs) are small non-coding RNA molecules, functioning in transcriptional and post-transcriptional regulation of gene expression. The discrimination of the real pre-miRNAs from the false ones (such as hairpin sequences with similar stem-loops) is necessary for the understanding of miRNAs’ role in the control of cell life and death. Since both their small size and sequence specificity, it cannot be based on sequence information alone but requires structure information about the miRNA precursor to get satisfactory performance. Kmers are convenient and widely used features for modeling the properties of miRNAs and other biological sequences. However, Kmers suffer from the inherent limitation that if the parameter K is increased to incorporate long range effects, some certain Kmer will appear rarely or even not appear, as a consequence, most Kmers absent and a few present once. Thus, the statistical learning approaches using Kmers as features become susceptible to noisy data once K becomes large. In this study, we proposed a Gapped k-mer approach to overcome the disadvantages of Kmers, and applied this method to the field of miRNA prediction. Combined with the structure status composition, a classifier called imiRNA-GSSC was proposed. We show that compared to the original imiRNA-kmer and alternative approaches. Trained on human miRNA precursors, this predictor can achieve an accuracy of 82.34 for predicting 4022 pre-miRNA precursors from eleven species.

Keywords: gapped k-mer, imiRNA-GSSC, microRNA precursor, support vector machine

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Authors: Aqeela Ashraf, Muhammad Imran, Tahir Yaqub, Muhammad Tayyab, Yung Fu Chang

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For the identification of bovine mastitic pathogen, an economical, rapid and sensitive molecular diagnostic assay is developed by PCR multiplexing of gene and pathogenic species specific DNA sequences. The multiplex PCR assay is developed for detecting nine important bacterial pathogens causing mastitis Worldwide. The bacterial species selected for this study are Streptococcus agalactiae, Streptococcus dysagalactiae, Streptococcus uberis, Staphylococcus aureus, Escherichia coli, Staphylococcus haemolyticus, Staphylococcus chromogenes Mycoplasma bovis and Staphylococcus epidermidis. A single reaction assay was developed and validated by 27 reference strains and further tested on 276 bacterial strains obtained from culturing mastitic milk. The multiplex PCR assay developed here is further evaluated by applying directly on genomic DNA isolated from 200 mastitic milk samples. It is compared with bacterial culturing method and proved to be more sensitive, rapid, economical and can specifically identify 9 bacterial pathogens in a single reaction. It has detected the pathogens in few culture negative mastitic samples. Recognition of disease is the foundation of disease control and prevention. This assay can be very helpful for maintaining the udder health and milk monitoring.

Keywords: multiplex PCR, bacteria, mastitis, milk

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Authors: Ji Hye Im, Nguyen T. T. Phuong, Keon Wook Kang

Abstract:

Estrogens are important for the development and growth of estrogen receptor (ER)-positive breast cancer, for which anti-estrogen therapy is one of the most effective treatments. However, its efficacy can be limited by either de novo or acquired resistance. Aromatase is a key enzyme for the biosynthesis of estrogens, and inhibition of this enzyme leads to profound hypoestrogenism. Here, we found that the basal expression and activity of aromatase were significantly increased in tamoxifen (TAM)-resistant human breast cancer (TAMR-MCF-7) cells compared to control MCF-7 cells. We further revealed that aromatase immunoreactivity in tumor tissues was increased in recurrence group after TAM therapy compared to non-recurrence group after TAM therapy. Phosphorylation of Akt, extracellular signal-regulated kinase (ERK), and p38 kinase were all increased in TAMR-MCF-7 cells. Inhibition of phosphoinositide 3-kinase (PI3K) suppressed the transactivation of the aromatase gene and its enzyme activity. Furthermore, we have also shown that PI3K/Akt-dependent cAMP-response element binding protein (CREB) activation was required for the enhanced expression of aromatase in TAMR-MCF-7 cells. Our findings suggest that aromatase expression is up-regulated in TAM-resistant breast cancer via PI3K/Akt-dependent CREB activation.

Keywords: TAMR-MCF-7, CREB, estrogen receptor, aromatase

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Authors: Yina A. Cifuentes Triana, Andrés M. Pinzón Velásco, Marío E. Velásquez Lozano

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In this work the sequencing and genome characterization of a natural isolate of Saccharomyces cerevisiae yeast (strain 202-3), identified with potential for the production of second generation ethanol from sugarcane bagasse hydrolysates is presented. This strain was selected because its capability to consume xylose during the fermentation of sugarcane bagasse hydrolysates, taking into account that many strains of S. cerevisiae are incapable of processing this sugar. This advantage and other prominent positive aspects during fermentation profiles evaluated in bagasse hydrolysates made the strain 202-3 a candidate strain to improve the production of second-generation ethanol, which was proposed as a first step to study the strain at the genomic level. The molecular characterization was carried out by genome sequencing with the Illumina HiSeq 2000 platform paired end; the assembly was performed with different programs, finally choosing the assembler ABYSS with kmer 89. Gene prediction was developed with the approach of hidden Markov models with Augustus. The genes identified were scored based on similarity with public databases of nucleotide and protein. Records were organized from ontological functions at different hierarchical levels, which identified central metabolic functions and roles of the S. cerevisiae strain 202-3, highlighting the presence of four possible new proteins, two of them probably associated with the positive consumption of xylose.

Keywords: cellulosic ethanol, Saccharomyces cerevisiae, genome sequencing, xylose consumption

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Authors: Azza Gaber Farag, Yasser Atta Shehata, Sara Elsayed Elghazouly, Mustafa Elsayed Elshaib, Nesreen Gamal Elden Elhelbawy

Abstract:

Background: Androgen receptor (AR) polymorphism in cytosine adenineguanine (CAG) repeat has an effect on the functional capacity of AR in males. However, little researches in this field are available regarding female sexual function. Aim: To investigate the possible link between polymorphism in the CAG repeat of AR gene and female sexual function in a sample of the Egyptian population. Materials and methods: 500 Egyptian married females completed a questionnaire regarding sociodemographic, reproductive, and sexual data. AR CAG repeat length was analyzed for those having female sexual dysfunctions (FSD) using real-time PCR. Results: The most sensitive domain to AR CAG repeat length was the orgasm domain that showed significant positive correlations with short allele (p=0.001), long allele (p=.015), biallellic mean (p=.000), and X weighted biallelic mean (p=.000). The satisfaction domain had significant positive correlations with the biallelic mean (p=.035), and the X weighted biallelic mean (p=. 032). However, the pain domain was of significant negative correlations with AR polymorphism of short allele (p=.002), biallelic mean (p=.013), and X weighted biallelic mean (p = . 011). Conclusions: AR polymorphism could represent a non-negligible aspect in female sexual function. The lower AR CAG repeat polymorphism was of significant impact on FSD, affecting mainly female orgasm followed by pain disorders that finally reflected On her sexual satisfaction.

Keywords: female sexual dysfunction, androgen receptor, CAG repeat polymorphism, androgen

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Authors: Sanxiong Fu, Yanyan Lv, Song Chen, Wei Zhang, Cunkou Qi

Abstract:

Ethylene response factor proteins are known to play an important role in regulating a variety of stress responses in plants, but their exact functions in submergence stress are not completely understood. In this study, we isolated BnERF from Brassica napus L. to study the function of BnERF in submergence tolerance. The expression of BnERF gene in Brassica napus L. and the expression of antioxidant enzyme genes in transgenic Arabidopsis were analyzed by Quantitative RT-PCR. It was found that expression of BnERF is apparently induced by submergence in Brassica napus L. and overexpression of BnERF in Arabidopsis increases the tolerance level to submergence and oxidative stress. Histochemical method detected lower level of H2O2, O2•− and malondialdehyde (MDA) in the transgenic Arabidopsis. Compared to wild type, transgenic lines also have higher soluble sugar content and higher activity of antioxidant enzymes, which helps protect the plants against the oxidative damage caused by submergence. It was concluded that BnERF can increase the tolerance of plants to submergence stress and BnERF might be involved in regulating soluble sugar content and the antioxidant system in the defense against submergence stress.

Keywords: antioxidant enzyme, Arabidopsis, ethylene response factor, submergence

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Authors: Yen-Ni Teng, Hsing-Yi Chen, Hsien-An Pan, Yung-Ming Lin, Hany A. Omar, Jui-Hsiang Hung

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Leucine-rich repeats and WD repeat domain containing 1 (LRWD1) is highly expressed in the testes of healthy males. On the other hand, LRWD1 is significantly down-regulated in the testicular tissues of patients with severe spermatogenic defects. In our study, the downregulation of LRWD1 expression by shRNA caused a significant reduction of cell growth and mitosis and a noteworthy increase in the cell microtubule atrophy rate. Here, we used EMBOSS CpG plot analysis to explore the promoter region of LRWD1 gene. We found that CpG islands are located between positions -253 to +5 nucleotides upstream from the LRWD1 transcription start site. Luciferase reporter assay revealed that the hypermethylation of the LRWD1 promoter reduced the transcription activity in cells. In addition, quantitative methylation-specific PCR and immunostaining showed that the methylation inhibitor, 5-Aza-2'-deoxycytidine, increased LRWD1 promoter activity, LRWD1 mRNA, protein expression and cell viability. Whereas, the methylation activator, S-adenosylmethionine, caused opposite effects. The overexpression of p53 and Nrf2 in NT2/D1 cells increased LRWD1 promoter activity while 5-fluorodeoxyuridine decreased it. In conclusion, this study highlights evidence that the methylation status of LRWD1 promoter is associated with LRWD1 expression. Since the expression level of LRWD1 plays an important role in spermatogenesis, the methylation status of LRWD1 may serve as a novel molecular diagnostic or therapeutic approach in male's infertility.

Keywords: LRWD1, DNA methylation, p53, Nrf2

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