Search results for: 16S rDNA gene

Authors: Peter. M. Kusen, Georg Wandrey, Christopher Probst, Dietrich Kohlheyer, Jochen Buchs, Jorg Pietruszkau

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Light as a stimulus provides the capability to develop regulation techniques for customizable gene expression. A great advantage is the extremely flexible and accurate dosing that can be performed in a non invasive and sterile manner even for high throughput technologies. Therefore, light regulation in a multiwell microbioreactor system was realized providing the opportunity to control gene expression with outstanding complexity. A light-regulated gene expression system in Saccharomyces cerevisiae was designed applying the strategy of caged compounds. These compounds are photo-labile protected and therefore biologically inactive regulator molecules which can be reactivated by irradiation with certain light conditions. The “caging” of a repressor molecule which is consumed after deprotection was essential to create a flexible expression system. Thereby, gene expression could be temporally repressed by irradiation and subsequent release of the active repressor molecule. Afterwards, the repressor molecule is consumed by the yeast cells leading to reactivation of gene expression. A yeast strain harboring a construct with the corresponding repressible promoter in combination with a fluorescent marker protein was applied in a Photo-BioLector platform which allows individual irradiation as well as online fluorescence and growth detection. This device was used to precisely control the repression duration by adjusting the amount of released repressor via different irradiation times. With the presented screening platform the regulation of complex expression procedures was achieved by combination of several repression/derepression intervals. In particular, a stepwise increase of temporally-constant expression levels was demonstrated which could be used to study concentration dependent effects on cell functions. Also linear expression rates with variable slopes could be shown representing a possible solution for challenging protein productions, whereby excessive production rates lead to misfolding or intoxication. Finally, the very flexible regulation enabled accurate control over the expression induction, although we used a repressible promoter. Summing up, the continuous online regulation of gene expression has the potential to synchronize gene expression levels to optimize metabolic flux, artificial enzyme cascades, growth rates for co cultivations and many other applications addicted to complex expression regulation. The developed light-regulated expression platform represents an innovative screening approach to find optimization potential for production processes.

Keywords: caged-compounds, gene expression regulation, optogenetics, photo-labile protecting group

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Authors: Dedy Pratama, Akhmadu Muradi, Hilman Ibrahim, Raden Suhartono, Alexander Jayadi Utama, Patrianef Darwis, S. Dwi Anita, Luluk Yunaini, Kemas Dahlan

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Introduction: Vascular endothelial growth factor (VEGF) gene shows association with various angiogenesis conditions including Diabetic Foot Ulcer (DFU) disease. In this study, we performed this study to examine VEGF gene polymorphism associated with DFU. Methods: Case-control study of polymorphism of VEGF gene +405 C>G and -460 T>C, of diabetes mellitus (DM) type 2 with Diabetic Foot Ulcer (DFU) in Cipto Mangunkusumo National Hospital (RSCM) Jakarta from June to December 2016. Results: There were 203 patients, 102 patients with DFU and 101 patients without DFU. Forty-nine point 8 percent of total samples is male and 50,2% female with mean age 56,06 years. Distribution of the wild-type genotype VEGF +405 C>G wild type CC was found in 6,9% of respondents, the number of mutant heterozygote CG was 69,5% and mutant homozygote GG was 19,7%. Cumulatively, there were 6,9% wild-type and 85,2% mutant and 3,9% of total blood samples could not be detected on PCR-RFLP. Distribution of VEGF allele +405 C>G C alleles were 43% and G alleles were 57%. Distribution of genotype from VEGF gene -460 T>C is wild type TT 42,9%, mutant heterozygote TC 37,9% and mutant homozygote CC 13,3%. Cumulatively, there were 42,9% wild-type and 51% mutant type. Distribution of VEGF -460 T>C were 62% T allele and 38% C allele. Conclusion: In this study we found the distribution of alleles from VEGF +405 C>G is C 43% and G 57% and from VEGF -460 T>C; T 62% and C 38%. We propose that G allele in VEGF +405 C>G can act as a protective allele and on the other hands T allele in VEGF -460 T>C could be acted as a risk factor for DFU in diabetic patients.

Keywords: diabetic foot ulcer, diabetes mellitus, polymorphism, VEGF

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Authors: Natasha Petrovska, Mirjana Pavlovic, Maria M. Larrondo-Petrie

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Neural stem cells (NSCs) are multi-potent, self-renewing cells that generate new neurons. Three subtypes of NSCs can be separated regarding the stages of NSC lineage: quiescent neural stem cells (qNSCs), activated neural stem cells (aNSCs) and neural progenitor cells (NPCs), but their gene expression signatures are not utterly understood yet. Single-cell examinations have started to elucidate the complex structure of NSC populations. Nevertheless, there is a lack of thorough molecular interpretation of the NSC lineage heterogeneity and an increasing need for tools to analyze and improve the efficiency and correctness of single-cell sequencing data. Feature selection and ordering can identify and classify the gene expression signatures of these subtypes and can discover novel subpopulations during the NSCs activation and differentiation processes. The aim here is to review the implementation of the feature selection technique on NSC subtypes and the classification techniques that have been used for the identification of gene expression signatures.

Keywords: feature selection, feature similarity, neural stem cells, genes, feature selection methods

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Authors: Aleksandr Nagay, Gulnoz Khamidullayeva

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It is known that an unbalanced intake of salt (NaCI), lifestyle and genetic predisposition to pathology is a key component of the risk and the development of essential hypertension (EH). Purpose: To study the relationship between salt-sensitivity and blood pressure (BP) on systolic (SBP) and diastolic (DBP) blood pressure, depending on the C825T polymorphism of GNB3 in individuals of Uzbek nationality with EH. Method: studied 148 healthy and 148 patients with EH with I-II degree (WHO/ISH, 2003) with disease duration 6,5±1,3 years. Investigation of the gene GNB3 was produced by PCR-RFLP method. Determination of salt-sensitivity was performed by the method of R. Henkin. Results: For a comparative analysis of BP, the groups with carriage of CТ and TT genotypes were combined. The analysis showed that carriers of CC genotype and low salt-sensitivity were determined by higher levels of SBP compared with carriers of CT and TT genotypes, and low salt-sensitivity of SBP: 166,2±4,3 against 158,2±9,1 mm Hg (p=0,000). A similar analysis on the values of DBP also showed significantly higher values of blood pressure in carriers of CC genotype DBP: 105,8±10,6 vs. 100,5±7,2 mm Hg, respectively (p=0,001). The average values of SBP and DBP in groups with carriers of CC genotype at medium or high salt-sensitivity in comparison with carriers of CT or TT genotype did not differ statistically SBP: 165,0±0,1 vs. 160,0±8,6 mm Hg (p=0,275) and DBP: 100,1±0,1 vs. 101,6±7,6 mm Hg (p=0,687), respectively. Conclusion: It is revealed that in patients with EH CC genotype of the gene GNB3 given salt-sensitivity has a negative effect on blood pressure profile. Since patients with EH with the CC genotype of GNB3 gene with low-salt taste sensitivity is determined by a higher level of blood pressure, both on SBP and DBP.

Keywords: salt sensitivity, essential hypertension EH, blood pressure BP, genetic predisposition

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Authors: Bououden Soraya

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This paper aims to study the existence of the change of coordinates which permits to transform a class of nonlinear dynamical systems into the so-called nonlinear observer canonical form (NOCF). Moreover, an algorithm to construct such a change of coordinates is given. Based on this form, we can design an observer with a linear error dynamic. This enables us to estimate the state of a nonlinear dynamical system. A concrete example (biological model) is provided to illustrate the feasibility of the proposed results.

Keywords: nonlinear observer canonical form, observer, design, gene regulation, gene expression

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Authors: Shahid Hussain Abro, Siamak Zohari, Lena H. M. Renström, Désirée S. Jansson, Faruk Otman, Karin Ullman, Claudia Baule

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Infectious bronchitis is an acute, highly contagious respiratory, nephropathogenic and reproductive disease of poultry that is caused by infectious bronchitis virus (IBV). The present study used a large data set of structural gene sequences, including newly generated ones and sequences available in the GenBank database to further analyze the diversity and to identify selective pressures and recombination spots. There were some deletions or insertions in the analyzed regions in isolates of the Italy-02 and D274 genotypes. Whereas, there were no insertions or deletions observed in the isolates of the Massachusetts and 4/91 genotype. The hypervariable nucleotide sequence regions spanned positions 152–239, 554–582, 686–737 and 802–912 in the S1 sub-unit of the all analyzed genotypes. The nucleotide sequence data of the E gene showed that this gene was comparatively unstable and subjected to a high frequency of mutations. The M gene showed substitutions consistently distributed except for a region between nucleotide positions 250–680 that remained conserved. The lowest variation in the nucleotide sequences of ORF5a was observed in the isolates of the D274 genotype. While, ORF5b and N gene sequences showed highly conserved regions and were less subjected to variation. Genes ORF3a, ORF3b, M, ORF5a, ORF5b and N presented negative selective pressure among the analyzed isolates. However, some regions of the ORFs showed favorable selective pressure(s). The S1 and E proteins were subjected to a high rate of mutational substitutions and non-synonymous amino acids. Strong signals of recombination breakpoints and ending break point were observed in the S and N genes. Overall, the results of this study revealed that very likely the strong selective pressures in E, M and the high frequency of substitutions in the S gene can probably be considered the main determinants in the evolution of IBV.

Keywords: IBV, avian infectious bronchitis, structural genes, genotypes, genetic diversity

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Authors: Aisling De Paor

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Scientific and technological developments are driving genetics and genetic technologies into the public sphere. Scientists are making genetic discoveries as to the make up of the human body and the cause and effect of disease, diversity and disability amongst individuals. Technological innovation in the field of genetics is also advancing, with the development of genetic testing, and other emerging genetic technologies, including gene editing (which offers the potential for genetic modification). In addition to the benefits for medicine, health care and humanity, these genetic advances raise a range of ethical, legal and societal concerns. From an ethical perspective, such advances may, for example, change the concept of humans and what it means to be human. Science may take over in conceptualising human beings, which may push the boundaries of existing human rights. New genetic technologies, particularly gene editing techniques create the potential to stigmatise disability, by highlighting disability or genetic difference as something that should be eliminated or anticipated. From a disability perspective, use (and misuse) of genetic technologies raise concerns about discrimination and violations to the dignity and integrity of the individual. With an acknowledgement of the likely future orientation of genetic science, and in consideration of the intersection of genetics and disability, this paper highlights the main concerns raised as genetic science and technology advances (particularly with gene editing developments), and the consequences for disability and human rights. Through the use of traditional doctrinal legal methodologies, it investigates the use (and potential misuse) of gene editing as creating the potential for a unique form of discrimination and stigmatization to develop, as well as a potential gateway to a form of new, subtle eugenics. This article highlights the need to maintain caution as to the use, application and the consequences of genetic technologies. With a focus on the law and policy position in Europe, it examines the need to control and regulate these new technologies, particularly gene editing. In addition to considering the need for regulation, this paper highlights non-normative approaches to address this area, including awareness raising and education, public discussion and engagement with key stakeholders in the field and the development of a multifaceted genetics advisory network.

Keywords: disability, gene-editing, genetics, law, regulation

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Authors: Chao Sima, Shanaz Ghandhi, Sally A. Amundson, Michael L. Bittner, David J. Brenner

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In a large-scale radiologic emergency, potentially affected population need to be triaged efficiently using various biomarkers where personal dosimeters are not likely worn by the individuals. It has long been established that radiation injury can be estimated effectively using panels of genetic biomarkers. Furthermore, the rate of radiation, in addition to dose of radiation, plays a major role in determining biological responses. Therefore, a better and more accurate triage involves estimating both the dose level of the exposure and the length of time of that exposure. To that end, a large in vivo study was carried out on mice with internal emitter caesium-137 (¹³⁷Cs). Four different injection doses of ¹³⁷Cs were used: 157.5 μCi, 191 μCi, 214.5μCi, and 259 μCi. Cohorts of 6~7 mice from the control arm and each of the dose levels were sacrificed, and blood was collected 2, 3, 5, 7 and 14 days after injection for microarray RNA gene expression analysis. Using a generalized linear model with penalized maximum likelihood, a panel of 244 genes was established and both the doses of injection and the number of days after injection were accurately predicted for all 155 subjects using this panel. This has proven that microarray gene expression can be used effectively in radiation biodosimetry in predicting both the dose levels and the length of exposure time, which provides a more holistic view on radiation exposure and helps improving radiation damage assessment and treatment.

Keywords: caesium-137, gene expression microarray, multivariate responses prediction, radiation biodosimetry

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Authors: Jake Gonzalez, Tommy Dang

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This paper proposes a temporal graph approach to visualize and analyze the evolution of gene relationships and nomenclature over time. An interactive web-based tool implements this temporal graph, enabling researchers to traverse a timeline and observe coupled dynamics in network topology and naming conventions. Analysis of a real human genomic dataset reveals the emergence of densely interconnected functional modules over time, representing groups of genes involved in key biological processes. For example, the antimicrobial peptide DEFA1A3 shows increased connections to related alpha-defensins involved in infection response. Tracking degree and betweenness centrality shifts over timeline iterations also quantitatively highlight the reprioritization of certain genes’ topological importance as knowledge advances. Examination of the CNR1 gene encoding the cannabinoid receptor CB1 demonstrates changing synonymous relationships and consolidating naming patterns over time, reflecting its unique functional role discovery. The integrated framework interconnecting these topological and nomenclature dynamics provides richer contextual insights compared to isolated analysis methods. Overall, this temporal graph approach enables a more holistic study of knowledge evolution to elucidate complex biology.

Keywords: temporal graph, gene relationships, nomenclature evolution, interactive visualization, biological insights

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Authors: Catherine Felce, Lior Pachter

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Current methods for building phylogenetic trees from gene expression data consider mean expression levels. With single-cell technologies, we can leverage more information about cell dynamics by considering the entire distribution of gene expression across cells. Using biophysical modeling, we propose a method for constructing phylogenetic trees from scRNA-seq data, building on Felsenstein's method of continuous characters. This method can highlight genes whose level of expression may be unchanged between species, but whose rates of transcription/decay may have evolved over time.

Keywords: phylogenetics, single-cell, biophysical modeling, transcription

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Authors: Kumaran Narayanan, Andrew N. Osahor

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E. coli has been engineered by our group and by others as a vector to deliver DNA into cultured human and animal cells. However, so far conditions to improve gene delivery using this vector have not been investigated, resulting in a major gap in our understanding of the requirements for this vector to function optimally. Our group recently published novel data showing that simple addition of the DNA transfection reagent Lipofectamine increased the efficiency of the E. coli vector by almost 3-fold, providing the first strong evidence that further optimization of bactofection is possible. This presentation will discuss advances that demonstrate the effects of several intracellular strategies that improve the efficiency of this vector. Conditions that promote endosomal escape of internalized bacteria to evade lysosomal destruction after entry in the cell, a known obstacle limiting this vector, are elucidated. Further, treatments that increase bacterial lysis so that the vector can release its transgene into the mammalian environment for expression will be discussed. These experiments will provide valuable new insight to advance this E. coli system as an important class of vector technology for genetic correction of human disease models in cells and whole animals.

Keywords: DNA, E. coli, gene expression, vector

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Authors: Fuad M. Alkoot

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Different experimental technologies such as microarray sequencing have been proposed to generate high-resolution genetic data, in order to understand the complex dynamic interactions between complex diseases and the biological system components of genes and gene products. However, the generated samples have a very large dimension reaching thousands. Therefore, hindering all attempts to design a classifier system that can identify diseases based on such data. Additionally, the high overlap in the class distributions makes the task more difficult. The data we experiment with is generated for the identification of autism. It includes 142 samples, which is small compared to the large dimension of the data. The classifier systems trained on this data yield very low classification rates that are almost equivalent to a guess. We aim at reducing the data dimension and improve it for classification. Here, we experiment with applying a multistage PCA on the genetic data to reduce its dimensionality. Results show a significant improvement in the classification rates which increases the possibility of building an automated system for autism detection.

Keywords: PCA, gene expression, dimensionality reduction, classification, autism

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Authors: Harunori Kawabe, Hideyuki Aoshima, Koji Murakami, Minoru Kawakami, Yuka Nakano, David D. Ordinario, C. W. Crawford, Iri Sato-Baran

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With the recent advances in gene editing technologies allowing the rewriting of genetic sequences, additional market growth in the global floriculture market beyond previous trends is anticipated through increasingly sophisticated plant breeding techniques. As a prerequisite for gene editing, the gene sequence of the target plant must first be identified. This necessitates the genetic analysis of plants with unknown gene sequences, the extraction of RNA, and comprehensive expression analysis. Consequently, a technology capable of consistently and effectively extracting high-purity DNA and RNA from plants is of paramount importance. Although model plants, such as Arabidopsis and tobacco, have established methods for DNA and RNA extraction, floricultural species such as roses present unique challenges. Different techniques to extract DNA and RNA from various floricultural species were investigated. Upon sampling and grinding the petals of several floricultural species, it was observed that nucleic acid extraction from the ground petal solutions of low viscosity was straightforward; solutions of high viscosity presented a significant challenge. It is postulated that the presence of substantial quantities of polysaccharides and polyphenols in the plant tissue was responsible for the inhibition of nucleic acid extraction. Consequently, attempts were made to extract high-purity DNA and RNA by improving the CTAB method and combining it with commercially available nucleic acid extraction kits. The quality of the total extracted DNA and RNA was evaluated using standard methods. Finally, the effectiveness of the extraction method was assessed by determining whether it was possible to create a library that could be applied as a suitable template for a next-generation sequencer. In conclusion, a method was developed for consistent and accurate nucleic acid extraction from high-viscosity floricultural samples. These results demonstrate improved techniques for DNA and RNA extraction from flowers, help facilitate gene editing of floricultural species and expand the boundaries of research and commercial opportunities.

Keywords: floriculture, gene editing, next-generation sequencing, nucleic acid extraction

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Authors: Mehdi Nasr-Esfahani, Leila Mohammad Bagheri, Ava Nasr-Esfahani

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Root and collar rot disease caused by Phytophthora capsici (Leonian) is one of the most serious diseases in pepper, Capsicum annuum L. In this study, a diverse collection of 37 commercial edible and ornamental pepper genotypes infected with P. capsici were investigated for biomass parameters and enzymatic activity of peroxidase or peroxide reductases (EC), superoxide dismutase (SOD), polyphenol oxidase (PPOs), catalase (CAT) and phenylalanine ammonia-lyase (PAL). Seven candidate DEG genes were also evaluated on resistant and susceptible pepper cultivars, through measuring product formation, using spectrophotometry and real-time polymerase chain reaction. All the five enzymes and seven defense-gene candidates were up-regulated in all inoculated pepper accessions to P. capsici. But, the enzymes and DEG genes were highly expressed in resistant cv. 19OrnP-PBI, 37ChillP-Paleo, and “23CherryP-Orsh". The expression level of enzymes were 1.5 to 5.6-fold higher in the resistant peppers, than the control non-inoculated genotypes. Also, the transcriptional levels of related candidate DEG genes were 3.16 to 5.90-fold higher in the resistant genotypes. There was a direct and high correlation coefficient between resistance, bio-mass parameters, enzymatic activity, and resistance gene expression. The related enzymes and candidate genes expressed herein will provide a basis for further gene cloning and functional verification studies, and also will aid in an understanding of the regulatory mechanism of pepper resistance to P. capsici.

Keywords: AP2/ERF, cDNA, enzymes, MIP gene, q-RTPCR, XLOC

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Authors: Seyed Reza Aghili, Tahereh Shokohi, Shirin Sadat Hashemi Fesharaki, Mohammad Ali Boroumand, Bahar Salmanian

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Introduction: Intravenous catheter-associated fungal infection as nosocomial infection continue to be a deep problem among hospitalized patients, decreasing quality of life and adding healthcare costs. The capacity of catheters in the spread of candidemia in heart failure patients is obvious. The aim of this study was to evaluate the prevalence and genetic identification of Candida species in heart disorder patients. Material and Methods: This study was conducted in Tehran Hospital of Cardiology Center (Tehran, Iran, 2014) during 1.5 years on the patients hospitalized for at least 7 days and who had central or peripheral vein catheter. Culture of catheters, blood and skin of the location of catheter insertion were applied for detecting Candida colonies in 223 patients. Identification of Candida species was made on the basis of a combination of various phenotypic methods and confirmed by sequencing the ITS1-5.8S-ITS2 region amplified from the genomic DNA using PCR and the NCBI BLAST. Results: Of the 223 patients samples tested, we identified totally 15 Candida isolates obtained from 9 (4.04%) catheter cultures, 3 (1.35%) blood cultures and 2 (0.90%) skin cultures of the catheter insertion areas. On the base of ITS region sequencing, out of nine Candida isolates from catheter, 5(55.6%) C. albicans, 2(22.2%) C. glabrata, 1(11.1%) C. membranifiaciens and 1 (11.1%) C. tropicalis were identified. Among three Candida isolates from blood culture, C. tropicalis, C. carpophila and C. membranifiaciens were identified. Non-candida yeast isolated from one blood culture was Cryptococcus albidus. One case of C. glabrata and one case of Candida albicans were isolated from skin culture of the catheter insertion areas in patients with positive catheter culture. In these patients, ITS region of rDNA sequence showed a similarity between Candida isolated from the skin and catheter. However, the blood samples of these patients were negative for fungal growth. We report two cases of catheter-related candidemia caused by C. membranifiaciens and C. tropicalis on the base of genetic similarity of species isolated from blood and catheter which were treated successfully with intravenous fluconazole and catheter removal. In phenotypic identification methods, we could only identify C. albicans and C. tropicalis and other yeast isolates were diagnosed as Candida sp. Discussion: Although more than 200 species of Candida have been identified, only a few cause diseases in humans. There is some evidence that non-albicans infections are increasing. Many risk factors, including prior antibiotic therapy, use of a central venous catheter, surgery, and parenteral nutrition are considered to be associated with candidemia in hospitalized heart failure patients. Identifying the route of infection in candidemia is difficult. Non-albicans candida as the cause of candidemia is increasing dramatically. By using conventional method, many non-albicans isolates remain unidentified. So, using more sensitive and specific molecular genetic sequencing to clarify the aspects of epidemiology of the unknown candida species infections is essential. The positive blood and catheter cultures for candida isolates and high percentage of similarity of their ITS region of rDNA sequence in these two patients confirmed the diagnosis of intravenous catheter-associated candidemia.

Keywords: catheter-associated infections, heart failure patient, molecular genetic sequencing, ITS region of rDNA, Candidemia

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Authors: Priya Arora, Ashutosh Mishra

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Gene identification, towards the pursuit of mutated genes, leading to Parkinson’s disease, puts forward a challenge towards proactive cure of the disorder itself. Computational analysis is an effective technique for exploring genes in the form of protein sequences, as the theoretical and manual analysis is infeasible. The limitations and effectiveness of a particular computational method are entirely dependent on the previous data that is available for disease identification. The article presents a sequence-based classification method for the identification of genes responsible for Parkinson’s disease. During the initiation phase, the physicochemical properties of amino acids transform protein sequences into a feature vector. The second phase of the method employs Jaccard distances to select negative genes from the candidate population. The third phase involves artificial neural networks for making final predictions. The proposed approach is compared with the state of art methods on the basis of F-measure. The results confirm and estimate the efficiency of the method.

Keywords: disease gene identification, Parkinson’s disease, physicochemical properties of amino acid, protein sequences

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Authors: Swati Gautam, Amita Jain, Shyampyari Jaiswar

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Objective: To elucidate the role of (ApaI&TaqI) VDR gene polymorphism in the pathogenesis of female genital tuberculosis (FGTB) cases. Background: Female genital TB represents about 15-20% of total extra-pulmonary TB (EPTB). Female subjects with vitamin D deficiency have been shown to be at higher risk of pulmonary TB as well as FGTB. In same context few functional polymorphism in vitamin D receptor (VDR) gene has been considered as an important genetic risk factor that modulate the development of FGTB. Therefore we aimed, to elucidate the role of (ApaI&TaqI) VDR gene polymorphism in the pathogenesis of FGTB. Study design: Case-Control study. Sample size: Cases (60) and Controls (60). Study site: Department of Obstetrics & Gynecology & Department of Microbiology, K.G.M.U. Lucknow, (UP). Inclusion criteria: Cases: Women with age group 20-35 years, premenstrual endometrial aspiration collected and included in the study, those were positive with acid-fast bacilli (AFB)/ TB-PCR/ LJ culture/ liquid culture. Controls: Women with age group 20-35 years having no history of ATT and all test negative for TB recruited as control. Exclusion criteria: -Women with endometriosis, polycystic ovaries (PCOD), positive on Chlamydia & gonorrhea, already on anti-tubercular therapy (ATT) excluded. Materials and Methods: Blood samples were collected in EDTA tubes from cases and controls stored at -20ºC. Genomic DNA extraction was carried out by salting-out method. Genotyping of VDR gene (ApaI&TaqI) polymorphism was performed by using single amplification refractory mutation system (ARMS) PCR technique. PCR products were analyzed by electrophoresis on 2% agarose gel. Statistical analysis was done by SPSS16.3 software & computing odds ratio (OR) with 95% CI. Results: Increased risk of female genital tuberculosis was observed in AA genotype (OR =1.1419-6.212 95% CI, P*<0.036) and A allele (OR =1.255-3.518, 95% CI, P* < 0.006) in FGTB as compared to controls. Moreover A allele was found more frequent in FGTB patients. No significant difference was observed in TaqI gene polymorphism of VDR gene. Conclusion: The ApaI polymorphism is significantly associated with etiology of FGTB and plays an important role as a genetic risk factor in FGTB women.

Keywords: ARMS, ATT, EPTB, FGTB, VDR

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Authors: Abdullah Alaklabi, Ibrahim A. Arif, Sameera O. Bafeel, Ahmad H. Alfarhan, Anis Ahamed, Jacob Thomas, Mohammad A. Bakir

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Diospyros mespiliformis (Hochst. ex A.DC.; Ebenaceae) is a large deciduous medicinal plant. This plant species is currently listed as endangered in Saudi Arabia. Molecular identification of this plant species based on short sequence regions (571 and 664 bp) of plastid rbcL (ribulose-1, 5-biphosphate carboxylase) gene was investigated in this study. The endangered plant specimens were collected from Al-Baha, Saudi Arabia (GPS coordinate: 19.8543987, 41.3059349). Phylogenetic tree inferred from the rbcL gene sequences showed that this species is very closely related with D. brandisiana. The close relationship was also observed among D. bejaudii, D. Philippinensis and D. releyi (≥99.7% sequence homology). The partial rbcL gene sequence region (571 bp) that was amplified by rbcL primer-pair rbcLaF-rbcLaR failed to discriminate D. mespiliformis from the closely related plant species, D. brandisiana. In contrast, primer-pair rbcL1F-rbcL724R yielded longer amplicon, discriminated the species from D. brandisiana and demonstrated nucleotide variations in 3 different sites (645G>T; 663A>C; 710C>G). Although D. mespiliformis (EU980712) and D. brandisiana (EU980656) are very closely related species (99.4%); however, studied specimen showed 100% sequence homology with D. mespiliformis and 99.6% with D. brandisiana. The present findings showed that rbcL short sequence region (664 bp) of plastid rbcL gene, amplified by primer-pair rbcL1F-rbcL724R, can be used for authenticating samples of D. mespiliforformis and may provide help in authentic identification and management process of this medicinally valuable endangered plant species.

Keywords: Diospyros mespiliformis, endangered plant, identification partial rbcL

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Authors: Waqas Shafqat Chattha, Muhammad Iqbal, Amir Shakeel

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Transcriptional factors are proteins that play a vital role in regulating the transcription of target genes in different biological processes and are being widely studied in different plant species. In the current era of genomics, plant genomes sequencing has directed to the genome-wide identification, analyses and categorization of diverse transcription factor families and hence provide key insights into their structural as well as functional diversity. The DNA-binding with One Finger (DOF) proteins belongs to C2-C2-type zinc finger protein family. DOF proteins are plant-specific transcription factors implicated in diverse functions including seed maturation and germination, phytohormone signalling, light-mediated gene regulation, cotton-fiber elongation and responses of the plant to biotic as well as abiotic stresses. In this context, a genome-wide in-silico analysis of DOF TF family in diploid cotton species i.e. Gossypium raimondii has enabled us to identify 55 non-redundant genes encoding DOF proteins renamed as GrDofs (Gossypium raimondii Dof). Gene distribution studies have shown that all of the GrDof genes are unevenly distributed across 12 out of 13 G. raimondii chromosomes. The gene structure analysis illustrated that 34 out of 55 GrDof genes are intron-less while remaining 21 genes have a single intron. Protein sequence-based phylogenetic analysis of putative 55 GrDOFs has divided these proteins into 5 major groups with various paralogous gene pairs. Molecular evolutionary studies aided with the conserved domain as well as gene structure analysis suggested that segmental duplications were the principal contributors for the expansion of Dof genes in G. raimondii.

Keywords: diploid cotton , G. raimondii, phylogenetic analysis, transcription factor

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Authors: Gehan Moubarz, Atef M. F. Mohammed, Inas A. Saleh, Heba Mahdy-Abdallah, Amal Saad-Hussein

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This study estimates the association between respiratory outcomes among employees of a secondary aluminum plant and airborne pollutants. Additionally, it looks into the relationship between pulmonary dysfunction in workers and XRCC1 gene polymorphisms. 110 exposed workers and 58 non-exposed workers participated in the study. Measurements have been conducted on SO₂, NO₂, and particulate particles. Pulmonary function was tested. Eosinophil cationic protein (ECP), C-reactive protein (CRP), matrix metalloproteinase-1 (MMP-1), interleukin 6 (IL6), GM-CSF, X-Ray Repair Cross Complementing 1 (XRCC1) protein, and genotyping of XRCC1 gene polymorphisms were examined. Results: The annual average concentrations of (PM₂.₅, PM₁₀, TSP, SO₂, and NO₂) were lower than the permissible limit. The areas around ovens, evaporators, and cold rolling mills exhibited the highest amounts. The majority of employees in these departments had impaired lung function. With longer exposure times, the exposed group's FEV1% and FVC% considerably reduced. The exposed workers had considerably higher XRCC1 levels. The evaluated inflammatory biomarkers showed no statistically significant difference. Conclusion: Aluminum workers are at risk of developing respiratory disorders. The level of serum XRCC1 may act as a biomarker that might be very useful for detecting susceptible workers.

Keywords: aluminum industry, particulate matter, SO₂, NO₂, lung function, XRCC1 gene polymorphism, XRCC1 protein, inflammatory biomarkers

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Authors: John Saaka

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The increasing world population poses a threat to food security. To meet current and future food demands, sweetpotato stand a good chance because of its recent food security roles. Concerted efforts are needed for both regional and local level varietal development. Heterosis exploiting breeding scheme (HEBS) is one of the options used to improve yield in some crop species and could be a good approach for sweetpotato improvement in Ghana by establishing heterotic gene pools within a population. To achieve this, 22 parental lines were collected from different sources and put in a full diallel arrangement. A total of 149 families, 20 individual cuttings per family, were taken to the field, including ‘checks’ and parental lines for experimentation in a 1m X 0.3m planting order according to the Westcott design. Results from this study led to the characterization of the selected parents into three main heterotic gene pools based on their suitability for use as male, female or both, respectively. This study serves as a baseline for further characterization of the rest of the germplasm in the Ghanaian sweetpotato breeding program.

Keywords: sweetpotato, heterosis, germplasm, food security

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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, Cox1, 12S rRNA, molecular characterization, Bangladesh

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

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

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

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Authors: Kadeejah Alsolami, Zainab Alrefay, Husaam Awad

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Obesity and vitamin D deficiency have both been related to cardiovascular disease. The present work aimed to investigate the possible protective effect of vitamin D on cardiac apoptosis in a rat model of dietary-induced obesity. Methods: 30 male Wistar rats included in this study. They were allocated into 4 groups: Control (n=5), animal were fed standard diet for 3 months: Control + vitamin D (VD) (n=5),animals were fed a standard diet with 400IU VD/kg for 3 months: hypercaloric diets group (n=10), animals were fed a high fat diet for 3 months: hypercaloric diet with VD group (n=10), animals were fed a high fat diet with 400IU VD/kg for 3 months. At the beginning of the experiment, the weight and length were measured to assess body mass index (BMI) and repeated every 45 days. Food intake and body weight were monitored throughout the study period. Then rats were sacrificed and heart tissues collected for Quantitative Real-time polymerase chain reaction (qRT-PCR). qRT-PCR used to detect different genetic markers of apoptosis (anti-apoptotic gene (BCL2), a pro-apoptotic gene(BAX), pro-apoptotic genes (FAS, FAS-L), tumour necrosis factor (TNF), mitogen-activated protein kinases (MAPK). Results: FAS and FAS-L gene expression were significantly upregulated in rats fed with high fat diet. And FAS-L gene expression was significantly upregulated in all groups on comparison with control. Whereas Bax gene expression was significantly downregulated in rats fed with high-fat diet supplied with vitamin D. TNF was significantly upregulated in rats fed with high-fat diet treated with vitamin D. MAPK was significantly upregulated in rats fed with high fat diet group, and in rats fed with high-fat diet supplied with vitamin D. Conclusion: The cardiac apoptotic pathways were more activated in rats fed with high-fat than lean rats. And vitamin D protect the heart from the cardiac mitochondrial-dependent apoptotic pathway.

Keywords: apoptosis, heart, obesity, Vitamin D

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Authors: Mariedel Autriz, Angel Lambio, Renato Vega, Severino Capitan, Rita Laude

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The study was conducted to associate genetic polymorphism of the leptin gene T3469C with reproductive performance in purebred sows. DNA were isolated from hair follicles of 29 Landrace and 24 Large White sows. Amplification of the leptin gene was done followed by Hinf1digestion to determine the base at the T3469C site. Electrophoresis of the digestion products revealed that there were 25 Landrace and 15 Large White sows with the TT genotype while there were 3 Landrace and 6 Large White TC. There was 1 CC for Landrace and 3 for Large White. Significant genotype associations were observed for total litter size born and total born alive. Significant breed differences, on the other hand, was observed for gestation length and average birth weight. Significant breed by genotype interaction was observed in litter size total born and litter size born alive.

Keywords: genetic polymorphism, leptin, swine, T3469C

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Authors: Malik SS, Masood N, Mubarik S, Khadim TM

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Introduction: Xeroderma pigmentosum group G (XPG) gene plays a crucial role in the correction of UV-induced DNA damage through nucleotide excision repair pathway. Single nucleotide polymorphisms in XPG gene have been reported to be associated with different cancers. Current case-control study was designed to evaluate the relationship between one of the most frequently found XPG (rs1047768 T>C) polymorphism and breast cancer risk. Methodology: A total of 200 individuals were screened for this polymorphism including 100 pathologically confirmed breast cancer cases and age-matched 100 controls. Genotyping was carried out using Tetra amplification-refractory mutation system (ARMS) PCR and results were confirmed by gel electrophoresis. Results: Conditional logistic regression analysis showed significant association between TC genotype (OR: 8.9, CI: 2.0 – 38.7) and increased breast cancer risk. Although homozygous CC genotype was more frequent in patients as compared to controls, but it was statistically non-significant (OR: 3.9, CI: 0.4 – 35.7). Conclusion: In conclusion, XPG (rs1047768 T>C) polymorphism may contribute towards increased risk of breast cancer but other polymorphisms may also be evaluated to elucidate their role in breast cancer.

Keywords: XPG, breast cancer, NER, ARMS-PCR

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Authors: Jasbir Singh, Devender Kumar, Davender Redhu, Surender Kumar, Vandana Bhardwaj

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Single Nucleotide polymorphism (SNP) of proteosomal gene complex is involved in the pathogenesis of hepatitis B Virus (HBV) infection. Some of such proteosomal gene complex are large multifunctional proteins (LMP) and antigen associated transporters that help in antigen presentation. Both are involved in intracellular processing and presentation of viral antigens in association with Major Histocompatability Complex (MHC) Class I molecules. A total of hundred each of hepatitis B virus infected and control samples from northern India were studied. Genomic DNA was extracted from all studied samples and PCR-RFLP method was used for genotyping at different positions of LMP genes. Genotypes at a given position were inferred from the pattern of bands and genotype frequencies and haplotype frequencies were also calculated. Homozygous SNP {A>C} was observed at codon 145 of LMP7 gene and having a protective role against HBV as there was statistically significant high distribution of this SNP among controls than cases. Heterozygous SNP {A>C} was observed at codon 145 of LMP7 gene and made individuals more susceptible to HBV infection as there was statistically significant high distribution of this SNP among cases than control. SNP {T>C} was observed at codon 60 of LMP2 gene but statistically significant differences were not observed among controls and cases. For codon 145 of LMP7 and codon 60 of LMP2 genes, four haplotypes were constructed. Haplotype I (LMP2 ‘C’ and LMP7 ‘A’) made individuals carrying it more susceptible to HBV infection as there was statistically significant high distribution of this haplotype among cases than control. Haplotype II (LMP2 ‘C’ and LMP7 ‘C’) made individuals carrying it more immune to HBV infection as there was statistically significant high distribution of this haplotype among control than cases. Thus it can be concluded that homozygous SNP {A>C} at codon 145 of LMP7 and Haplotype II (LMP2 ‘C’ and LMP7 ‘C’) has a protective role against HBV infection whereas heterozygous SNP {A>C} at codon 145 of LMP7 and Haplotype I (LMP2 ‘C’ and LMP7 ‘A’) made individuals more susceptible to HBV infection.

Keywords: Hepatitis B Virus, single nucleotide polymorphism, low molecular weight proteins, transporters associated with antigen presentation

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Authors: Ali Bayram, Burak Uz, Remzi Yiğiter

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The aim of the present study was to investigate the expression levels of homo sapiens nuclear factor of kappa light polypeptide gene enhancer in B-cells 1, transcript variant 1 (NFKB1*1) mRNA in the peripheral blood of patients with Parkinson to elucidate the role in the pathogenesis of Parkinson disease (PD). The study group comprised 50 patients with the diagnosis of PD who have applied to Gaziantep University Faculty of Medicine, and Department of Neurology. 50 healthy individuals without any neuro degenerative disease are included as controls. Ribonucleic acid (RNA) was obtained from blood samples of patient and control groups. Complementary deoxyribonucleic acid (cDNA) was obtained from RNA samples using reverse transcription polymerase chain reaction (RT-PCR) technique. The gene expression of NFKB1*1 in patient/control groups were observed to decrease significantly, and the differences between groups with the Mann-Whitney method within 95% confidence interval (p<0.05) were analyzed. This salient finding provide a clue for our hypothesis that reduced activity of NFKB1*1 gene might play a role, at least partly, in the pathophysiology of PD.

Keywords: Parkinson’s Disease, NFKB1, mRNA expression, RT-PCR

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Authors: Al-Mutary M., Alhimaidi A., Al-Ghadi M. Iwamoto D., Javed Ahmad. Abdulaziz A. Al-Khedhairy

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The aim of the present study was to evaluate the effect of ovine follicular fluid supplementation during IVM of sheep oocytes on the resumption of meiosis, glutathione (GSH) content and expression of Bax, Bcl-2, and HSPB1 genes. Sheep ovaries were collected from Riyadh slaughterhouse, KSA. Oocytes were aspirated from 3-6 mm follicles. Ovine oocytes were cultured in maturation medium with 0% (control), 10%, 20%, 40% of ovine follicular fluid for 24 h. Results indicated that the rate of oocyte maturation was significantly (P≤0.05) decreased in 40% OFF (36.87%) versus the control (61.3%), 10% OFF (63.95%) and 20% OFF (64.08%). Supplementation of 10% OFF to IVM medium induced an intra-oocyte GSH concentration significantly higher than that found in ovine oocytes cultured with 20% OFF and 40% OFF and similar to the GSH content in oocytes cultured without FF. Real time polymerase chain reaction analysis for gene expression revealed no differences in Bax, Bcl-2, HSPB1 genes between control and 10% OFF group, whereas they were strongly expressed in 20% OFF and 40% OFF (P < 0.05) when compared to the control and 10% OFF. In conclusion the addition of 10% OFF to the IVM culture of sheep oocytes is recommended to support cytoplasmic maturation and increase oocytes competence.

Keywords: IVM, oocyte maturation, gene expression, follicular fluid

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Authors: Yi-Hao Wong, Li-Li Chan, Chee-Onn Leong, Stephen Ambu, Joon-Wah Mak, Priyasashi Sahu

Abstract:

Background: Acanthamoeba is a genus of amoebae which lives as a free-living in nature or as a human pathogen that causes severe brain and eye infections. Virulence potential of Acanthamoeba is not constant and can change with growth conditions. DNA methylation, an epigenetic process which adds methyl groups to DNA, is used by eukaryotic cells, including several human parasites to control their gene expression. We used qPCR, siRNA gene silencing, and RNA sequencing (RNA-Seq) to study DNA-methyltransferase gene family (DNMT) in order to indicate the possibility of its involvement in programming Acanthamoeba virulence potential. Methods: A virulence-attenuated Acanthamoeba isolate (designation: ATCC; original isolate: ATCC 50492) was subjected to mouse passages to restore its pathogenicity; a virulence-reactivated isolate (designation: AC/5) was generated. Several established factors associated with Acanthamoeba virulence phenotype were examined to confirm the succession of reactivation process. Differential gene expression of DNMT between ATCC and AC/5 isolates was performed by qPCR. Silencing on DNMT gene expression in AC/5 isolate was achieved by siRNA duplex. Total RNAs extracted from ATCC, AC/5, and siRNA-treated (designation: si-146) were subjected to RNA-Seq for comparative transcriptomic analysis in order to identify the genome-wide effect of DNMT in regulating Acanthamoeba gene expression. qPCR was performed to validate the RNA-Seq results. Results: Physiological and cytophatic assays demonstrated an increased in virulence potential of AC/5 isolate after mouse passages. DNMT gene expression was significantly higher in AC/5 compared to ATCC isolate (p ≤ 0.01) by qPCR. si-146 duplex reduced DNMT gene expression in AC/5 isolate by 30%. Comparative transcriptome analysis identified the differentially expressed genes, with 3768 genes in AC/5 vs ATCC isolate; 2102 genes in si-146 vs AC/5 isolate and 3422 genes in si-146 vs ATCC isolate, respectively (fold-change of ≥ 2 or ≤ 0.5, p-value adjusted (padj) < 0.05). Of these, 840 and 1262 genes were upregulated and downregulated, respectively, in si-146 vs AC/5 isolate. Eukaryotic orthologous group (KOG) assignments revealed a higher percentage of downregulated gene expression in si-146 compared to AC/5 isolate, were related to posttranslational modification, signal transduction and energy production. Gene Ontology (GO) terms for those downregulated genes shown were associated with transport activity, oxidation-reduction process, and metabolic process. Among these downregulated genes were putative genes encoded for heat shock proteins, transporters, ubiquitin-related proteins, proteins for vesicular trafficking (small GTPases), and oxidoreductases. Functional analysis of similar predicted proteins had been described in other parasitic protozoa for their survival and pathogenicity. Decreased expression of these genes in si146-treated isolate may account in part for Acanthamoeba reduced pathogenicity. qPCR on 6 selected genes upregulated in AC/5 compared to ATCC isolate corroborated the RNA sequencing findings, indicating a good concordance between these two analyses. Conclusion: To the best of our knowledge, this study represents the first genome-wide analysis of DNA methylation and its effects on gene expression in Acanthamoeba spp. The present data indicate that DNA methylation has substantial effect on global gene expression, allowing further dissection of the genome-wide effects of DNA-methyltransferase gene in regulating Acanthamoeba pathogenicity.

Keywords: Acanthamoeba, DNA methylation, RNA sequencing, virulence

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