Search results for: Gene Editing

Authors: Zilya R. Vershinina, Andrei K. Baymiev, Aleksei K. Baymiev, Aleksei V. Chemeris

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This paper reports optimization of characteristics of bioballistic transformation of spring soft wheat (Triticum aestivum L. cultivar Raduga) and getting of transgenic plants, carrying pea lectin gene. This gene will let to create new associative wheat symbiosis with nodule bacteria of field pea, which has growth encouraging, fungistatic and other useful characteristics.

Keywords: transgenic wheat, pea lectin, rhizobia root colonization, symbiosis

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Authors: Muhammad Azam, Micheal Olaolu Arowolo, Fei He, Mihail Popescu, Dong Xu

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The number of biological papers is growing quickly, which means that the number of biological pathway figures in those papers is also increasing quickly. Each pathway figure shows extensive biological information, like the names of genes and how the genes are related. However, manually annotating pathway figures takes a lot of time and work. Even though using advanced image understanding models could speed up the process of curation, these models still need to be made more accurate. To improve gene name recognition from pathway figures, we applied a Siamese network to map image segments to a library of pictures containing known genes in a similar way to person recognition from photos in many photo applications. We used a triple loss function and a triplet spatial pyramid pooling network by combining the triplet convolution neural network and the spatial pyramid pooling (TSPP-Net). We compared VGG19 and VGG16 as the Siamese network model. VGG16 achieved better performance with an accuracy of 93%, which is much higher than Optical Character Recognition (OCR) results.

Keywords: Biological pathway, image understanding, gene name recognition, object detection, Siamese network, Visual Geometry Group.

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Authors: Reena Murali, David Peter S.

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The small interfering RNA (siRNA) alters the regulatory role of mRNA during gene expression by translational inhibition. Recent studies show that upregulation of mRNA because serious diseases like cancer. So designing effective siRNA with good knockdown effects plays an important role in gene silencing. Various siRNA design tools had been developed earlier. In this work, we are trying to analyze the existing good scoring second generation siRNA predicting tools and to optimize the efficiency of siRNA prediction by designing a computational model using Artificial Neural Network and whole stacking energy (%G), which may help in gene silencing and drug design in cancer therapy. Our model is trained and tested against a large data set of siRNA sequences. Validation of our results is done by finding correlation coefficient of experimental versus observed inhibition efficacy of siRNA. We achieved a correlation coefficient of 0.727 in our previous computational model and we could improve the correlation coefficient up to 0.753 when the threshold of whole tacking energy is greater than or equal to -32.5 kcal/mol.

Keywords: Artificial Neural Network, Double Stranded RNA, RNA Interference, Short Interfering RNA.

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Authors: Roya Bakhtiar, Alireza Abdolmohammadi, Hadi Hajarian, Zahra Nikousefat, Davood, Kalantar-Neyestanaki

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The objective of the present study was to determine the polymorphism in the leptin (332G>A) and its association with biometric traits in Sanjabi sheep. For this purpose, blood samples from 96 rams were taken, and tail length, width tail, circumference tail, body length, body width, and height were simultaneously recorded. PCR was performed using specific primer to amplify 463 bp fragment including exon 3 of leptin gene, and PCR products were digested by Cail restriction enzymes. The 332G>A (at 332th nucleotide of exon 3 leptin gene) that caused an amino acid change from Arg to Gln was detected by Cail (CAGNNNCTG) endonuclease, as the endonuclease cannot cut this region if G nucleotide is located in this position. Three genotypes including GG (463), GA (463, 360and 103 bp) and GG (360 bp and 103 bp) were identified after digestion by enzyme. The estimated frequencies of three genotypes including GG, GA, and AA for 332G>A locus were 0.68, 0.29 and 0.03 and those were 0.18 and 0.82 for A and G alleles, respectively. In the current study, chi-square test indicated that 332G>A positions did not deviate from the Hardy–Weinberg (HW) equilibrium. The most important reason to show HW equation was that samples used in this study belong to three large local herds with a traditional breeding system having random mating and without selection. Shannon index amount was calculated which represent an average genetic variation in Sanjabi rams. Also, heterozygosity estimated by Nei index indicated that genetic diversity of mutation in the leptin gene is moderate. Leptin gene polymorphism in the 332G>A had significant effect on body length (P<0.05) trait, and individuals with GA genotype had significantly the higher body length compared to other individuals. Although animals with GA genotype had higher body width, this difference was not statistically significant (P>0.05). This non-synonymous SNP resulted in different amino acid changes at codon positions111(R/Q). As leptin activity is localized, at least in part, in domains between amino acid residues 106-1406, it is speculated that the detected SNP at position 332 may affect the activity of leptin and may lead to different biological functions. Based to our results, due to significant effect of leptin gene polymorphism on body size traits, this gene may be used a candidate gene for improving these traits.

Keywords: Body size, Leptin gene, PCR-RFLP, Sanjabi sheep.

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Authors: Amer A. Hasan, Mehri Igci, Ersin Borazan, Rozhgar A. Khailany, Emine Bayraktar, Ahmet Arslan

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Gastric Cancer (GC) has high morbidity and fatality rate in various countries. It is still one of the most frequent and deadly diseases. Gastrokine1 (GKN1) and gastrokine2 (GKN2) genes are highly expressed in the normal stomach epithelium and play important roles in maintaining the integrity and homeostasis of stomach mucosal epithelial cells. In this study, 47 paired samples that were grouped according to the types of gastric cancer and the clinical characteristics of the patients, including gender and average of age. They were investigated with gene expression analysis and mutation screening by monitoring RT-PCR, SSCP and nucleotide sequencing techniques. Both GKN1 and GKN2 genes were observed significantly reduced found by (Wilcoxon signed rank test; p<0.05). As a result of gene screening, no mutation (no different genotype) was detected. It is considered that gene mutations are not the cause of gastrokines inactivation. In conclusion, the mRNA expression level of GKN1 and GKN2 genes statistically was decreased regardless the gender, age, or cancer type of patients. Reduced of gastrokine genes seem to occur at the initial steps of gastric cancer development.

Keywords: Diagnostic biomarker, gastric cancer, nucleotide sequencing, semi-quantitative RT-PCR.

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Authors: Rohayu Izanwati M. R., Muhamad Ridhwan M. R., Abbe Maleyki M. J., Ahmad Zubaidi A. L., Zahri M. K.

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PPARs function as regulators of lipid and lipoprotein metabolism. The aim of the study was to compare the lipid profile between two phases of fasting and to examine the frequency and relationship of peroxisome proliferator-activated receptor, PPARα gene polymorphisms to lipid profile in fasting respondents. We conducted a case-control study protocol, which included 21 healthy volunteers without gender discrimination at the age of 18 years old. 3 ml of blood sample was drawn before the fasting phase and during the fasting phase (in Ramadhan month). 1ml of serum for the lipid profile was analyzed by using the automated chemistry analyser (Olympus, AU 400) and the data were analysed using the Paired T-Test (SPSS ver.20). DNA was extracted and PCR was conducted utilising 6 sets of primer. Primers were designed within 6 exons of interest in PPARα gene. Genetic and metabolic characteristics of fasting respondents and controls were estimated and compared. Fasting respondents were significantly have lowered the LDL levels (p=0.03). There were no polymorphisms detected except in exon 1 with 5% of this population study respectively. The polymorphisms in exon 1 of the PPARα gene were found in low frequency. Regarding the 1375G/T and 1386G/T polymorphisms in the exon 1 of the PPARα gene, the T-allele in fasting phase had no association with the decreased LDL levels (Fisher Exact Test). However this association is more promising when the sample size is larger in order to elucidate the precise impact of the polymorphisms on lipid profile in the population. In conclusion, the PPARα gene polymorphisms do not appear to affect the LDL of fasting respondents.

Keywords: Fasting, LDL, Peroxisome proliferator activated receptor alpha (PPAR-α), Polymorphisms.

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Authors: Farahnaz Amini, Woo Yun Kin, Lazwani Kolandaiveloo

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Availability of different genetic tests after completion of Human Genome Project increases the physicians’ responsibility to keep themselves update on the potential implementation of these genetic tests in their daily practice. However, due to numbers of barriers, still many of physicians are not either aware of these tests or are not willing to offer or refer their patients for genetic tests. This study was conducted an anonymous, cross-sectional, mailed-based survey to develop a primary data of Malaysian physicians’ level of knowledge and perception of gene profiling. Questionnaire had 29 questions. Total scores on selected questions were used to assess the level of knowledge. The highest possible score was 11. Descriptive statistics, one way ANOVA and chi-squared test was used for statistical analysis. Sixty three completed questionnaires were returned by 27 general practitioners (GPs) and 36 medical specialists. Responders’ age ranges from 24 to 55 years old (mean 30.2 ± 6.4). About 40% of the participants rated themselves as having poor level of knowledge in genetics in general whilst 60% believed that they have fair level of knowledge; however, almost half (46%) of the respondents felt that they were not knowledgeable about available genetic tests. A majority (94%) of the responders were not aware of any lab or company which is offering gene profiling services in Malaysia. Only 4% of participants were aware of using gene profiling for detection of dosage of some drugs. Respondents perceived greater utility of gene profiling for breast cancer (38%) compared to the colorectal familial cancer (3%). The score of knowledge ranged from 2 to 8 (mean 4.38 ± 1.67). Non- significant differences between score of knowledge of GPs and specialists were observed, with score of 4.19 and 4.58 respectively. There was no significant association between any demographic factors and level of knowledge. However, those who graduated between years 2001 to 2005 had higher level of knowledge. Overall, 83% of participants showed relatively high level of perception on value of gene profiling to detect patient’s risk of disease. However, low perception was observed for both statements of using gene profiling for general population in order to alter their lifestyle (25%) as well as having the full sequence of a patient genome for the purpose of determining a patient’s best match for treatment (18%). The lack of clinical guidelines, limited provider knowledge and awareness, lack of time and resources to educate patients, lack of evidence-based clinical information and cost of tests were the most barriers of ordering gene profiling mentioned by physicians. In conclusion Malaysian physicians who participate in this study had mediocre level of knowledge and awareness in gene profiling. The low exposure to the genetic questions and problems might be a key predictor of lack of awareness and knowledge on available genetic tests. Educational and training workshop might be useful in helping Malaysian physicians incorporate genetic profiling into practice for eligible patients.

Keywords: Gene Profiling, Knowledge, Malaysia, Physician.

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Authors: Mohamed A. Mahfouz, M. A. Ismail

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Biclustering is a very useful data mining technique for identifying patterns where different genes are co-related based on a subset of conditions in gene expression analysis. Association rules mining is an efficient approach to achieve biclustering as in BIMODULE algorithm but it is sensitive to the value given to its input parameters and the discretization procedure used in the preprocessing step, also when noise is present, classical association rules miners discover multiple small fragments of the true bicluster, but miss the true bicluster itself. This paper formally presents a generalized noise tolerant bicluster model, termed as μBicluster. An iterative algorithm termed as BIDENS based on the proposed model is introduced that can discover a set of k possibly overlapping biclusters simultaneously. Our model uses a more flexible method to partition the dimensions to preserve meaningful and significant biclusters. The proposed algorithm allows discovering biclusters that hard to be discovered by BIMODULE. Experimental study on yeast, human gene expression data and several artificial datasets shows that our algorithm offers substantial improvements over several previously proposed biclustering algorithms.

Keywords: Machine learning, biclustering, bi-dimensional clustering, gene expression analysis, data mining.

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Authors: H. Merhni, A. Sbiti, I. Ratbi, A. Sefiani

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The proximal spinal muscular atrophy (SMA) is a group of neuromuscular disorders characterized by progressive muscle weakness due to the degeneration and loss of anterior motor neurons of the spinal cord. Depending on the age of onset of symptoms and their evolution, four types of SMA, varying in severity, result in a mutations of the SMN gene (survival of Motor neuron). We have analyzed the DNA of 295 patients referred to our genetic counseling; since January 1996 until October 2014; for suspected SMA. The homozygous deletion of exon 7 of the SMN gene was found in 133 patients; of which, 40.6% were born to consanguineous parents. In countries like Morocco, where the frequency of heterozygotes for SMA is high, genetic testing should be offered as first-line and, after careful clinical assessment, especially in newborns and infants with congenital hypotonia unexplained and prognosis compromise. The molecular diagnosis of SMA allows a quick and certainly diagnosis, provide adequate genetic counseling for families at risk and suggest, for couples who want prenatal diagnosis. The analysis of the SMN gene is a perfect example of genetic testing with an excellent cost/benefit ratio that can be of great interest in public health, especially in low-income countries. We emphasize in this work for the benefit of the generalization of molecular diagnosis of SMA by the technique of PCR-enzymatic digestion in other centers in Morocco.

Keywords: Exon7, PCR-digestion, SMA, SMN gene.

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Authors: M. Pandi, K. Premalatha

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The DNA microarray technology concurrently monitors the expression levels of thousands of genes during significant biological processes and across the related samples. The better understanding of functional genomics is obtained by extracting the patterns hidden in gene expression data. It is handled by clustering which reveals natural structures and identify interesting patterns in the underlying data. In the proposed work clustering gene expression data is done through an Advanced Nelder Mead (ANM) algorithm. Nelder Mead (NM) method is a method designed for optimization process. In Nelder Mead method, the vertices of a triangle are considered as the solutions. Many operations are performed on this triangle to obtain a better result. In the proposed work, the operations like reflection and expansion is eliminated and a new operation called spread-out is introduced. The spread-out operation will increase the global search area and thus provides a better result on optimization. The spread-out operation will give three points and the best among these three points will be used to replace the worst point. The experiment results are analyzed with optimization benchmark test functions and gene expression benchmark datasets. The results show that ANM outperforms NM in both benchmarks.

Keywords: Spread out, simplex, multi-minima, fitness function, optimization, search area, monocyte, solution, genomes.

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Authors: Chia-Ta Tsai, Wen-Lin Huang, Shinn-Jang Ho, Li-Sun Shu, Shinn-Ying Ho

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Prediction of bacterial virulent protein sequences can give assistance to identification and characterization of novel virulence-associated factors and discover drug/vaccine targets against proteins indispensable to pathogenicity. Gene Ontology (GO) annotation which describes functions of genes and gene products as a controlled vocabulary of terms has been shown effectively for a variety of tasks such as gene expression study, GO annotation prediction, protein subcellular localization, etc. In this study, we propose a sequence-based method Virulent-GO by mining informative GO terms as features for predicting bacterial virulent proteins. Each protein in the datasets used by the existing method VirulentPred is annotated by using BLAST to obtain its homologies with known accession numbers for retrieving GO terms. After investigating various popular classifiers using the same five-fold cross-validation scheme, Virulent-GO using the single kind of GO term features with an accuracy of 82.5% is slightly better than VirulentPred with 81.8% using five kinds of sequence-based features. For the evaluation of independent test, Virulent-GO also yields better results (82.0%) than VirulentPred (80.7%). When evaluating single kind of feature with SVM, the GO term feature performs much well, compared with each of the five kinds of features.

Keywords: Bacterial virulence factors, GO terms, prediction, protein sequence.

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Authors: Ahmad Ali Shahid, Muhammad Shakil Shaukat, Kamran Shehzad Bajwa, Abdul Qayyum Rao, Tayyab Husnain

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Agriculture is the backbone of economy of Pakistan and cotton is the major agricultural export and supreme source of raw fiber for our textile industry. To combat severe problems of insect and weed, combination of three genes namely Cry1Ac, Cry2A and EPSPS genes was transferred in locally cultivated cotton variety MNH-786 with the use of Agrobacterium mediated genetic transformation. The present study focused on the molecular screening of transgenic cotton plants at T3 generation in order to confirm integration and expression of all three genes (Cry1Ac, Cry2A and EPSP synthase) into the cotton genome. Initially, glyphosate spray assay was used for screening of transgenic cotton plants containing EPSP synthase gene at T3 generation. Transgenic cotton plants which were healthy and showed no damage on leaves were selected after 07 days of spray. For molecular analysis of transgenic cotton plants in the laboratory, the genomic DNA of these transgenic cotton plants were isolated and subjected to amplification of the three genes. Thus, seventeen out of twenty (Cry1Ac gene), ten out of twenty (Cry2A gene) and all twenty (EPSP synthase gene) were produced positive amplification. On the base of PCR amplification, ten transgenic plant samples were subjected to protein expression analysis through ELISA. The results showed that eight out of ten plants were actively expressing the three transgenes. Real-time PCR was also done to quantify the mRNA expression levels of Cry1Ac and EPSP synthase gene. Finally, eight plants were confirmed for the presence and active expression of all three genes at T3 generation.

Keywords: Agriculture, Cotton, Transformation, Cry Genes, ELISA and PCR.

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Authors: Chintanu K. Sarmah, Sandhya Samarasinghe, Don Kulasiri, Daniel Catchpoole

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Gene expression profiling is rapidly evolving into a powerful technique for investigating tumor malignancies. The researchers are overwhelmed with the microarray-based platforms and methods that confer them the freedom to conduct large-scale gene expression profiling measurements. Simultaneously, investigations into cross-platform integration methods have started gaining momentum due to their underlying potential to help comprehend a myriad of broad biological issues in tumor diagnosis, prognosis, and therapy. However, comparing results from different platforms remains to be a challenging task as various inherent technical differences exist between the microarray platforms. In this paper, we explain a simple ratio-transformation method, which can provide some common ground for cDNA and Affymetrix platform towards cross-platform integration. The method is based on the characteristic data attributes of Affymetrix- and cDNA- platform. In the work, we considered seven childhood leukemia patients and their gene expression levels in either platform. With a dataset of 822 differentially expressed genes from both these platforms, we carried out a specific ratio-treatment to Affymetrix data, which subsequently showed an improvement in the relationship with the cDNA data.

Keywords: Gene expression profiling, microarray, cDNA, Affymetrix, childhood leukaemia.

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Authors: M. Maimunah, G.A. Froemming, H. Nawawi, M.I. Nafeeza, O. Effat, M.R. Rohayu Izanwati, M.S. Mohamed Saifulaman

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Attachment of the circulating monocytes to the endothelium is the earliest detectable events during formation of atherosclerosis. The adhesion molecules, chemokines and matrix proteases genes were identified to be expressed in atherogenesis. Expressions of these genes may influence structural integrity of the luminal endothelium. The aim of this study is to relate changes in the ultrastructural morphology of the aortic luminal surface and gene expressions of the endothelial surface, chemokine and MMP-12 in normal and hypercholesterolemic rabbits. Luminal endothelial surface from rabbit aortic tissue was examined by scanning electron microscopy (SEM) using low vacuum mode to ascertain ultrastructural changes in development of atherosclerotic lesion. Gene expression of adhesion molecules, MCP-1 and MMP-12 were studied by Real-time PCR. Ultrastructural observations of the aortic luminal surface exhibited changes from normal regular smooth intact endothelium to irregular luminal surface including marked globular appearance and ruptures of the membrane layer. Real-time PCR demonstrated differentially expressed of studied genes in atherosclerotic tissues. The appearance of ultrastructural changes in aortic tissue of hypercholesterolemic rabbits is suggested to have relation with underlying changes of endothelial surface molecules, chemokine and MMP-12 gene expressions.

Keywords: Ultrastructure of luminal endothelial surface, Macrophage metalloelastase (MMP-12), Real-time PCR.

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Authors: Mehrdad Hashemi, Mitra Behrooz Aghdam, Reza Mahdian, Ahmad Reza Kamyab

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Cytogenetic analysis still remains the gold standard method for prenatal diagnosis of trisomy 21 (Down syndrome, DS). Nevertheless, the conventional cytogenetic analysis needs live cultured cells and is too time-consuming for clinical application. In contrast, molecular methods such as FISH, QF-PCR, MLPA and quantitative Real-time PCR are rapid assays with results available in 24h. In the present study, we have successfully used a novel MGB TaqMan probe-based real time PCR assay for rapid diagnosis of trisomy 21 status in Down syndrome samples. We have also compared the results of this molecular method with corresponding results obtained by the cytogenetic analysis. Blood samples obtained from DS patients (n=25) and normal controls (n=20) were tested by quantitative Real-time PCR in parallel to standard G-banding analysis. Genomic DNA was extracted from peripheral blood lymphocytes. A high precision TaqMan probe quantitative Real-time PCR assay was developed to determine the gene dosage of DSCAM (target gene on 21q22.2) relative to PMP22 (reference gene on 17p11.2). The DSCAM/PMP22 ratio was calculated according to the formula; ratio=2 -ΔΔCT. The quantitative Real-time PCR was able to distinguish between trisomy 21 samples and normal controls with the gene ratios of 1.49±0.13 and 1.03±0.04 respectively (p value <0.001). These results represent the presence of 3 copies of target gene in DS samples Vs 2 copies in normal controls. The results of quantitative Real-time PCR were in complete agreement with results of cytogenetic analysis. This study confirms previous reports regarding successful implementation of quantitative Real-time PCR for detection of trisomy 21. However, the assay has been improved by using MGB probes and more accurate data analysis. This assay, in particular, when performed in combination with another molecular assay such as QF-PCR or MLPA, can be used as a reliable technique for rapid prenatal diagnosis of trisomy 21.

Keywords: Trisomy 21, Real-time PCR, MGB-TaqMan Probes, Gene Dosage.

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Authors: Tom Snir, Eitan Rubin

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Mendelian Disease Genes represent a collection of single points of failure for the various systems they constitute. Such genes have been shown, on average, to encode longer proteins than 'non-disease' proteins. Existing models suggest that this results from the increased likeli-hood of longer genes undergoing mutations. Here, we show that in saturated mutagenesis experiments performed on model organisms, where the likelihood of each gene mutating is one, a similar relationship between length and the probability of a gene being lethal was observed. We thus suggest an extended model demonstrating that the likelihood of a mutated gene to produce a severe phenotype is length-dependent. Using the occurrence of conserved domains, we bring evidence that this dependency results from a correlation between protein length and the number of functions it performs. We propose that protein length thus serves as a proxy for protein cardinality in different networks required for the organism's survival and well-being. We use this example to argue that the collection of Mendelian Disease Genes can, and should, be used to study the rules governing systems vulnerability in living organisms.

Keywords: Systems Biology, Protein Length

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Authors: Ngo Thanh Xuan, Mai Thi Hang, Vu Nguyen Thanh

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A. niger XP isolated from Vietnam produces very low amount of acidic phytase with optimal pH at 2.5 and 5.5. The phytase production of this strain was successfully improved through gene cloning and expression. A 1.4 - kb DNA fragment containing the coding region of the phyA gene was amplified by PCR and inserted into the expression vector pPICZαA with a signal peptide α- factor, under the control of AOX1 promoter. The recombined plasmid was transformed into the host strain P. pastoris KM71H and X33 by electroporation. Both host strains could efficiently express and secret phytase. The multicopy strains were screened for over expression of phytase. All the selected multicopy strains of P. pastoris X33 were examined for phytase activity, the maximum phytase yield of 1329 IU/ml was obtained after 4 days of incubation in medium BMM. The recombinant protein with MW of 97.4 KW showed to be the only one protein secreted in the culture broth. Multicopy transformant P. pastoris X33 supposed to be potential candidate for producing the commercial preparation of phytase.

Keywords: Aspergillus niger XP, cloning, over expression, Pichia pastoris, phyA , phytase.

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Authors: Maria E. Manioudaki, Panayiota Poirazi

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Yeast cells live in a constantly changing environment that requires the continuous adaptation of their genomic program in order to sustain their homeostasis, survive and proliferate. Due to the advancement of high throughput technologies, there is currently a large amount of data such as gene expression, gene deletion and protein-protein interactions for S. Cerevisiae under various environmental conditions. Mining these datasets requires efficient computational methods capable of integrating different types of data, identifying inter-relations between different components and inferring functional groups or 'modules' that shape intracellular processes. This study uses computational methods to delineate some of the mechanisms used by yeast cells to respond to environmental changes. The GRAM algorithm is first used to integrate gene expression data and ChIP-chip data in order to find modules of coexpressed and co-regulated genes as well as the transcription factors (TFs) that regulate these modules. Since transcription factors are themselves transcriptionally regulated, a three-layer regulatory cascade consisting of the TF-regulators, the TFs and the regulated modules is subsequently considered. This three-layer cascade is then modeled quantitatively using artificial neural networks (ANNs) where the input layer corresponds to the expression of the up-stream transcription factors (TF-regulators) and the output layer corresponds to the expression of genes within each module. This work shows that (a) the expression of at least 33 genes over time and for different stress conditions is well predicted by the expression of the top layer transcription factors, including cases in which the effect of up-stream regulators is shifted in time and (b) identifies at least 6 novel regulatory interactions that were not previously associated with stress-induced changes in gene expression. These findings suggest that the combination of gene expression and protein-DNA interaction data with artificial neural networks can successfully model biological pathways and capture quantitative dependencies between distant regulators and downstream genes.

Keywords: gene modules, artificial neural networks, yeast, stress

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Authors: D. Vainauska, S. Kozireva, A. Karpovs, M. Chistyakovs, M. Baryshev

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Liposomal magnetofection is a simple, highly efficient technology for cell transfection, demonstrating better outcome than a number of other common gene delivery methods. However, aggregate complexes distribution over the cell surface is non-uniform due to the gradient of the permanent magnetic field. The aim of this study was to estimate the efficiency of liposomal magnetofection for prostate carcinoma PC3 cell line using newly designed device, “DynaFECTOR", ensuring magnetofection in a dynamic gradient magnetic field. Liposomal magnetofection in a dynamic gradient magnetic field demonstrated the highest transfection efficiency for PC3 cells – it increased for 21% in comparison with liposomal magnetofection and for 42% in comparison with lipofection alone. The optimal incubation time under dynamic magnetic field for PC3 cell line was 5 minutes and the optimal rotation frequency of magnets – 5 rpm. The new approach also revealed lower cytotoxic effect to cells than liposomal magnetofection.

Keywords: Dynamic gradient magnetic field, gene delivery, liposomal magnetofection, prostate cancer cell line

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Authors: M. Omer Faruk, A. M. A. M. Zonaed Siddiki, M. Fazal Karim, Md. Masuduzzaman, S. Chowdhury, Md. Shafiqul Islam, M. Alamgir Hossain

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The dog tapeworms Echinococcus granulosus develop hydatid cysts in various organs in human and domestic animals worldwide including Bangladesh. The aim of this study was to identify and characterize the genotype of E. granulosus isolated from cattle using 12S rRNA and Cytochrome oxidase 1 (COX 1) genes. A total of 43 hydatid cyst samples were collected from 390 examined cattle samples derived from slaughterhouses. Among them, three cysts were fertile. Genomic DNA was extracted from germinal membrane and/or protoscoleces followed by PCR amplification of mitochondrial 12S rRNA and Cytochrome oxidase 1 gene fragments. The sequence data revealed existence of G1 (64.28%) and possible G3 (21.43%) genotypes for the first time in Bangladesh. The study indicates that common sheep strain G1 is the dominant subtype of E. granulosus in Chittagong region of Bangladesh. This will increase our understanding of the epidemiology of hydatidosis in the southern part of the country and will be useful to plan suitable control measures in the long run.

Keywords: Echinococcus granulosus, molecular characterization, cattle, Bangladesh.

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Authors: Ines Hamdi, Mohamed Ben Ahmed

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The understanding of the system level of biological behavior and phenomenon variously needs some elements such as gene sequence, protein structure, gene functions and metabolic pathways. Challenging problems are representing, learning and reasoning about these biochemical reactions, gene and protein structure, genotype and relation between the phenotype, and expression system on those interactions. The goal of our work is to understand the behaviors of the interactions networks and to model their evolution in time and in space. We propose in this study an ontological meta-model for the knowledge representation of the genetic regulatory networks. Ontology in artificial intelligence means the fundamental categories and relations that provide a framework for knowledge models. Domain ontology's are now commonly used to enable heterogeneous information resources, such as knowledge-based systems, to communicate with each other. The interest of our model is to represent the spatial, temporal and spatio-temporal knowledge. We validated our propositions in the genetic regulatory network of the Aarbidosis thaliana flower

Keywords: Ontological model, spatio-temporal modeling, Genetic Regulatory Networks (GRNs), knowledge representation.

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Authors: Alexander Kaplunovsky, Vladimir Khailenko, Alexander Bolshoy, Shara Atambayeva, AnatoliyIvashchenko

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Eukaryotic protein-coding genes are interrupted by spliceosomal introns, which are removed from the RNA transcripts before translation into a protein. The exon-intron structures of different eukaryotic species are quite different from each other, and the evolution of such structures raises many questions. We try to address some of these questions using statistical analysis of whole genomes. We go through all the protein-coding genes in a genome and study correlations between the net length of all the exons in a gene, the number of the exons, and the average length of an exon. We also take average values of these features for each chromosome and study correlations between those averages on the chromosomal level. Our data show universal features of exon-intron structures common to animals, plants, and protists (specifically, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Cryptococcus neoformans, Homo sapiens, Mus musculus, Oryza sativa, and Plasmodium falciparum). We have verified linear correlation between the number of exons in a gene and the length of a protein coded by the gene, while the protein length increases in proportion to the number of exons. On the other hand, the average length of an exon always decreases with the number of exons. Finally, chromosome clustering based on average chromosome properties and parameters of linear regression between the number of exons in a gene and the net length of those exons demonstrates that these average chromosome properties are genome-specific features.

Keywords: Comparative genomics, exon-intron structure, eukaryotic clustering, linear regression.

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Authors: Sang-Ho Baik

Abstract:

D-erythro-cyclohexylserine (D chiral unnatural β-hydroxy amino acid expected for the synthesis of drug for AIDS treatment. To develop a continuous bioconversion system with whole cell biocatalyst of D-threonine aldolase (D genes for the D-erythro-CHS production, D-threonine aldolase gene was amplified from Ensifer arboris 100383 by direct PCR amplication using two degenerated oligonucleotide primers designed based on genomic sequence of Shinorhizobium meliloti Sequence analysis of the cloned DNA fragment revealed one open-reading frame of 1059 bp and 386 amino acids. This putative D-TA gene was cloned into NdeI and EcoRI (pEnsi His-tag sequence or BamHI (pEnsi-DTA[2]) sequence of the pET21(a) vector. The expression level of the cloned gene was extremely overexpressed by E. coli BL21(DE3) transformed with pEnsi-DTA[1] compared to E. coli BL21(DE3) transformed with pEnsi-DTA[2]. When the cells expressing the wild used for D-TA enzyme activity, 12 mM glycine was successfully detected in HPLC analysis. Moreover, the whole cells harbouring the recombinant D-TA was able to synthesize D-erythro of 0.6 mg/ml in a batch reaction.

Keywords: About four key words or phrases in alphabetical order, separated by commas.

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Authors: Huihai Wu, Xiaohui Liu

Abstract:

Using Dynamic Bayesian Networks (DBN) to model genetic regulatory networks from gene expression data is one of the major paradigms for inferring the interactions among genes. Averaging a collection of models for predicting network is desired, rather than relying on a single high scoring model. In this paper, two kinds of model searching approaches are compared, which are Greedy hill-climbing Search with Restarts (GSR) and Markov Chain Monte Carlo (MCMC) methods. The GSR is preferred in many papers, but there is no such comparison study about which one is better for DBN models. Different types of experiments have been carried out to try to give a benchmark test to these approaches. Our experimental results demonstrated that on average the MCMC methods outperform the GSR in accuracy of predicted network, and having the comparable performance in time efficiency. By proposing the different variations of MCMC and employing simulated annealing strategy, the MCMC methods become more efficient and stable. Apart from comparisons between these approaches, another objective of this study is to investigate the feasibility of using DBN modeling approaches for inferring gene networks from few snapshots of high dimensional gene profiles. Through synthetic data experiments as well as systematic data experiments, the experimental results revealed how the performances of these approaches can be influenced as the target gene network varies in the network size, data size, as well as system complexity.

Keywords: Genetic regulatory network, Dynamic Bayesian network, GSR, MCMC.

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Authors: A. Molee, N. Duanghaklang, P. Mernkrathoke

Abstract:

The objective was to determine the single gene and interaction effect of composite genotype of beta-kappa casein and DGAT1 gene on milk yield (MY) and milk composition, content of milk fat (%FAT), milk protein (%PRO), solid not fat (%SNF), and total solid (%TS) in crossbred Holstein cows. Two hundred and thirty- one cows were genotyped with PCR-RFLP for DGAT1 and composite genotype data of beta-kappa casein from previous work were used. Two model, (1), and (2), was used to estimate single gene effect, and interaction effect on the traits, respectively. The significance of interaction effects on all traits were detected. Most traits have consistent pattern of significant when model (1), and (2) were compared, except the effect of composite genotype of betakappa casein on %FAT, and the effect of DGAT1 on MY, which the significant difference was detected in only model (1).The results suggested that when the optimum of all traits was necessary, interaction effect should be concerned.

Keywords: composite genotype of beta-kappa casein, DGAT1gene, Milk composition, Milk yield

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Authors: C. Kridtayopas, W. Danvilai, P. Sopannarath, A. Kayan, W. Loongyai

Abstract:

Both Betong chicken (KU Line) and Thai Native chickens were the high quality of the meat and low carcass fat compared to broiler chickens. The objective of this study was to determine the growth performance, carcass characteristic, meat quality and association of polymorphism in the ApoVLDL-II gene with fat accumulation in the female broiler, Thai Native and Betong (KU line) chickens at 4-14 weeks. The chickens were used and reared under the same environment and management (100 chicks per breed). The results showed that body weight (BW) of broiler chickens was significantly higher than Thai Native and Betong (KU line) chickens (P < 0.01) through all the experiment. At 4-8 weeks of age, feed conversion ratio (FCR) of broiler chickens was significantly better than Thai Native and Betong (KU line) chickens (P < 0.01), then increased at week 8-14. The percentage of breast, abdominal fat and subcutaneous fat of broiler chickens was significantly greater than Thai Native and Betong (KU line) chickens (P < 0.01). However, Thai Native chickens showed the highest percentage of liver (P < 0.01) when compared to other breeds. In addition, the percentage of wing of Thai Native and Betong (KU line) chickens were significantly (P < 0.01) higher than broiler chickens. Meat quality was also determined and found that, pH of breast meat left from slaughter 45 minutes (pH45) and 24 hours (pH24) of broiler was significantly higher than Thai Native and Betong (KU line) (P < 0.01) whereas the percentage of drip loss, thawing loss, cooking loss and shear force was not significantly different between breeds. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was used to genotype the polymorphism in the ApoVLDL-II gene in the broiler, Thai Native and Betong (KU line) chickens. The results found that, the polymorphism in the ApoVLDL-II gene at VLDL6 loci was not associated with fat accumulation in those studied population.

Keywords: ApoVLDL-II Gene, Betong (KU line) chickens, broiler chickens, carcass characteristic, growth performance, meat quality, Thai Native Chickens.

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Authors: Ekaterina Y. Lukianova-Hleb, Dmitri O. Lapotko

Abstract:

Cell processing techniques for gene and cell therapies use several separate procedures for gene transfer and cell separation or elimination, because no current technology can offer simultaneous multi-functional processing of specific cell sub-sets in heterogeneous cell systems. Using our novel on-demand nonstationary intracellular events instead of permanent materials, plasmonic nanobubbles, generated with a short laser pulse only in target cells, we achieved simultaneous multifunctional cell-specific processing with the rate up to 50 million cells per minute.

Keywords: Delivery, cell separation, graft, laser, plasmonic nanobubble, cell therapy, gold nanoparticle.

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Authors: Atnaf Tiruneh Mulugeta, Mohammed Ali Hussein, Zelleke Habtamu

Abstract:

Thirty six genotypes (8 parents and 28 F1 diallel crosses) were grown in randomized complete block design during 2006 at Mandura, North western Ethiopia. The experiment was executed to study the inheritance of two primary yield component traits: number of seeds per pod and 1000 seed weight. Statistical significant difference was observed between genotypes, parents, and crosses for these traits. The mean square due to GCA was significant for the two traits. However, SCA mean square was significant only for number of seeds per pod. Thus both additive and non-additive types of gene actions were important in the inheritance of number of seeds per pod. Significant b1 component was obtained for this trait. The b2 and b3 components, however, were not significant, suggesting the absence of gene asymmetry. From Wr/Vr graph, inheritance of seeds per pod was governed by partial dominance with additive gene action.

Keywords: Diallel crosses, General combining ability, Phaseolus vulgaris L., Specific combining ability

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Authors: R. Majidzadeh Heravi, M. Sankian, H. Kermanshahi, M. R. Nassiri, A. Heravi Moussavi, S. A. Lari, A. R. Varasteh

Abstract:

The use of probiotics engineered to express specific enzymes has been the subject of considerable attention in poultry industry because of increased nutrient availability and reduced cost of enzyme supplementation. Phytase enzyme is commonly added to poultry feed to improve digestibility and availability of phosphorus from plant sources. To construct a probiotic with potential of phytate degradation, phytase gene (appA) from E. coli was cloned and transformed into two probiotic bacteria Lactobacillus salivarius and Lactococcus lactis. L. salivarous showed plasmid instability, unable to express the gene. The expression of appA gene in L. lactis was analyzed by detecting specific RNA and zymography assay. Phytase enzyme was isolated from cellular extracts of recombinant L. lactis, showing a 46 kDa band upon the SDS-PAGE analysis. Zymogram also confirmed the phytase activity of the 46 kDa band corresponding to the enzyme. An enzyme activity of 4.9U/ml was obtained in cell extracts of L. lactis. The growth of native and recombinant L. lactis was similar in the presence of two concentrations of ox bile.

Keywords: Lactobacillus salivarus, Lactococcus lactis, recombinant, phytase, poultry.

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Authors: Jafar Ahmadi, Zhohreh Asiaban, Sedigheh Fabriki Ourang

Abstract:

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

Keywords: BES1/BZR1, Brassinosteroids, Phylogenetic analysis, Transcription factor.

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