Search results for: in-utero gene editing

Authors: Salina Y. Saddick

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Mild gestational hyperglycemia (MGH) is a very common complication of pregnancy that is characterized by intolerance to glucose. The association of angiotensin-converting enzyme (ACE) insertion/deletion (I/D) polymorphism to MGH has been previously reported. In this study, we evaluated the association between ACE polymorphism and the risk of MGH in a Saudi population. We conducted a case-control study in a population of 100 MGH patients and 100 control subjects. ACE gene polymorphism was analyzed by the novel approach of tetraprimer amplification refractory mutation system (ARMS)-polymerase chain reaction (PCR). The frequency of ACE polymorphism was not associated with either alleles or genotypes in MGH patients. Glucose concentration was found to be significantly associated with the MGH group. Our study suggests that ACE genotypes were not associated with ACE polymorphism in a Saudi population.

Keywords: MGH, ACE, insertion polymorphism, deletion polymorphism

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Authors: Agostinho Antunes

Abstract:

The completion of the human genome sequencing in 2003 opened a new perspective into the importance of whole genome sequencing projects, and currently multiple species are having their genomes completed sequenced, from simple organisms, such as bacteria, to more complex taxa, such as mammals. This voluminous sequencing data generated across multiple organisms provides also the framework to better understand the genetic makeup of such species and related ones, allowing to explore the genetic changes underlining the evolution of diverse phenotypic traits. Here, recent results from our group retrieved from comparative evolutionary genomic analyses of selected marine animal species will be considered to exemplify how gene novelty and gene enhancement by positive selection might have been determinant in the success of adaptive radiations into diverse habitats and lifestyles.

Keywords: comparative genomics, adaptive evolution, bioinformatics, phylogenetics, genome mining

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Authors: Ankush Kalra, Mirza Masroor, Usha Manaktala, B. C. Koner, T. K. Mishra

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Introduction: Pre-eclampsia affects 3-5% of pregnancies worldwide and increases maternal-fetal morbidity and mortality. Reduced placental perfusion induces the release of biomolecules by the placenta into maternal circulation causing endothelial dysfunction. Zinc dependent matrix metalloproteinase-2 (MMP-2) may be up-regulated and interact with circulating factors of oxidative stress and inflammation to produce endothelial dysfunction in pre-eclampsia. Aim: To study the functional genetic polymorphism of MMP-2 gene (g-1306 C>T) in pre-eclampsia and its effect on serum MMP-2 levels in these patients. Method: Hundred pre-eclampsia patients and hundred age and gestation period matched healthy pregnant women with their consent were recruited in the study. Serum MMP-2 levels in all subjects were estimated using standard ELISA kits. MMP-2 gene (g.- 1306 C>T) SNPs were genotyped using whole blood by ASO-PCR. Result: The pre-eclampsia patients had higher serum levels of MMP-2 compared to the healthy pregnant (p < 0.05). Also the MMP-2 genotype was associated with significant alteration in the serum MMP-2 concentration in these patients (p < 0.05). Conclusion: This study results suggest an association of MMP-2 genetic polymorphism and serum levels of MMP-2 to the path physiology of hypertensive disorder of pregnancy.

Keywords: allele specific oligonucleotide polymerase chain reaction (ASO-PCR), enzyme linked immunosorbent assay (ELISA), matrix metalloproteinase-2 (MMP-2), pre-eclampsia

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Authors: Jialin Ma, Jia Meng

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Genomic features, the genome-based coordinates, are commonly used for the representation of biological features such as genes, RNA transcripts and transcription factor binding sites. For the analysis of RNA-related genomic features, such as RNA modification sites, a common task is to correlate these features with transcript components (5'UTR, CDS, 3'UTR) to explore their distribution characteristics in terms of transcriptomic coordinates, e.g., to examine whether a specific type of biological feature is enriched near transcription start sites. Existing approaches for performing these tasks involve the manipulation of a gene database, conversion from genome-based coordinate to transcript-based coordinate, and visualization methods that are capable of showing RNA transcript components and distribution of the features. These steps are complicated and time consuming, and this is especially true for researchers who are not familiar with relevant tools. To overcome this obstacle, we develop a dedicated web app TARF, which represents web toolkit for annotating RNA-related genomic features. TARF web tool intends to provide a web-based way to easily annotate and visualize RNA-related genomic features. Once a user has uploaded the features with BED format and specified a built-in transcript database or uploaded a customized gene database with GTF format, the tool could fulfill its three main functions. First, it adds annotation on gene and RNA transcript components. For every features provided by the user, the overlapping with RNA transcript components are identified, and the information is combined in one table which is available for copy and download. Summary statistics about ambiguous belongings are also carried out. Second, the tool provides a convenient visualization method of the features on single gene/transcript level. For the selected gene, the tool shows the features with gene model on genome-based view, and also maps the features to transcript-based coordinate and show the distribution against one single spliced RNA transcript. Third, a global transcriptomic view of the genomic features is generated utilizing the Guitar R/Bioconductor package. The distribution of features on RNA transcripts are normalized with respect to RNA transcript landmarks and the enrichment of the features on different RNA transcript components is demonstrated. We tested the newly developed TARF toolkit with 3 different types of genomics features related to chromatin H3K4me3, RNA N6-methyladenosine (m6A) and RNA 5-methylcytosine (m5C), which are obtained from ChIP-Seq, MeRIP-Seq and RNA BS-Seq data, respectively. TARF successfully revealed their respective distribution characteristics, i.e. H3K4me3, m6A and m5C are enriched near transcription starting sites, stop codons and 5’UTRs, respectively. Overall, TARF is a useful web toolkit for annotation and visualization of RNA-related genomic features, and should help simplify the analysis of various RNA-related genomic features, especially those related RNA modifications.

Keywords: RNA-related genomic features, annotation, visualization, web server

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Authors: M. Kouyate, M. Sangare, S. Samake, S. Keita, H. G. Kim, D. H. Geschwind

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Background & Objectives: Human genetic studies can be expensive, even unaffordable, in developing countries, partly due to the sequencing costs. Our aim is to pilot the use of bioinformatics tools to guide scientifically valid, locally relevant, and economically sound autism genetic research in Mali. Methods: The following databases, NCBI, HGMD, and LSDB, were used to identify hot point mutations. Phenotype, transmission pattern, theoretical protein expression in the brain, the impact of the mutation on the 3D structure of the protein) were used to prioritize selected autism genes. We used the protein database, Modeller, and clustal W. Results: We found Mef2c (Gly27Ala/Leu38Gln), Pten (Thr131IIle), Prodh (Leu289Met), Nme1 (Ser120Gly), and Dhcr7 (Pro227Thr/Glu224Lys). These mutations were associated with endonucleases BseRI, NspI, PfrJS2IV, BspGI, BsaBI, and SpoDI, respectively. Gly27Ala/Leu38Gln mutations impacted the 3D structure of the Mef2c protein. Mef2c protein sequences across species showed a high percentage of similarity with a highly conserved MADS domain. Discussion: Mef2c, Pten, Prodh, Nme1, and Dhcr 7 gene mutation frequencies in the Malian population will be very informative. PCR coupled with restriction enzyme digestion can be used to screen the targeted gene mutations. Sanger sequencing will be used for confirmation only. This will cut down considerably the sequencing cost for gene-to-gene mutation screening. The knowledge of the 3D structure and potential impact of the mutations on Mef2c protein informed the protein family and altered function (ex. Leu38Gln). Conclusion & Future Work: Bio-informatics will positively impact autism research in Mali. Our approach can be applied to another neuropsychiatric disorder.

Keywords: bioinformatics, endonucleases, autism, Sanger sequencing, point mutations

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Authors: Hamidreza Saberkari, Mousa Shamsi, Hossein Ahmadi, Saeed Vaali, , MohammadHossein Sedaaghi

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In the recent years, using signal processing tools for accurate identification of the protein coding regions has become a challenge in bioinformatics. Most of the genomic signal processing methods is based on the period-3 characteristics of the nucleoids in DNA strands and consequently, spectral analysis is applied to the numerical sequences of DNA to find the location of periodical components. In this paper, a novel ensemble algorithm for gene selection in DNA sequences has been presented which is based on the combination of Goertzel algorithm and anti-notch filter (ANF). The proposed algorithm has many advantages when compared to other conventional methods. Firstly, it leads to identify the coding protein regions more accurate due to using the Goertzel algorithm which is tuned at the desired frequency. Secondly, faster detection time is achieved. The proposed algorithm is applied on several genes, including genes available in databases BG570 and HMR195 and their results are compared to other methods based on the nucleotide level evaluation criteria. Implementation results show the excellent performance of the proposed algorithm in identifying protein coding regions, specifically in identification of small-scale gene areas.

Keywords: protein coding regions, period-3, anti-notch filter, Goertzel algorithm

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Authors: Aminah Salim, Idrees Ahmed Nasir, Abdul Qayyum Rao, Muhammad Ali, Muhammad Sohail Anjum, Ayesha Hameed, Bushra Tabassum, Anwar Khan, Arfan Ali, Mariyam Zameer, Tayyab Husnain

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Risk assessment of transgenic herbicide tolerant sugarcane having CEMB codon optimized cp4EPSPS gene was done in present study. Fifteen days old chicks taken from K&Ns Company were randomly assorted into four groups with eight chicks in each group namely control chicken group fed with commercial diet, non-transgenic group fed with non-experimental sugarcane and transgenic group fed with transgenic sugarcane with minimum and maximum level. Body weights, biochemical analysis for Urea, alkaline phosphatase, alanine transferase, aspartate transferase, creatinine and bilirubin determination and histological examination of chicks fed with four types of feed was taken at fifteen days interval and no significant difference was observed in body weight biochemical and histological studies of all four groups. Protein isolated from the serum sample was analyzed through dipstick and SDS-PAGE, showing the absence of transgene protein in the serum sample of control and experimental groups. Moreover the amplification of cp4EPSPS gene with gene specific primers of DNA isolated from chicks blood and also from commercial diet was done to determine the presence and mobility of any nucleotide fragment of the transgene in/from feed and no amplification was obtained in feed as well as in blood extracted DNA of any group. Also no mRNA expression of cp4EPSPS gene was obtained in any tissue of four groups of chicks. From the results it is clear that there is no deleterious or harmful effect of the CEMB codon optimized transgenic cp4EPSPS sugarcane on the chicks health.

Keywords: chicks, cp4EPSPS, glyphosate, sugarcane

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Authors: D. Tohlob, E. Abo Hashem, N. Ghareeb, M. Ghanem, R. Elfarahaty, S. A. Roberts, P. Pemberton, L. Mohiyiddeen, W. G. Newman

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Gonadotropin stimulation is used in females undergoing assisted reproductive treatment for ovulation induction, but ovarian response is variable and unpredictable in these women. More effective protocols and individualization of treatment are needed to increase the success rate of IVF/ICSI cycles. We genotyped seven variants reported in previous studies to be associated with ovarian response (number of ova retrieved and total gonadotropin dose) in women undergoing IVF treatment including FSHR variants Asn 680 Ser (c.2039 A > G), Thr 307 Ala (c. 919 > A), -29 G > A, HRG c.610 C > T gene, BMP15 -9 C > G, AMH Ile 49 Ser (c.146 G > T), and AMHR -489A˃G in 118 Egyptian females attending Mansoura Integrated Fertility Center in Egypt, these females were undergoing their first cycle of controlled ovarian hyper stimulation for IVF/ICSI treatment. They were analyzed by TaqMan allelic discrimination assay in Manchester Center of Genomic Medicine. We found no evidence of any significant difference (p value < 0.05) in the number of eggs retrieved or the gonadotropin dose used between individuals in all genotypes except for HRG c.610 C > T gene polymorphism where regression analysis gives a p value of 0.04 with a fewer eggs number in TT genotyped females. These results indicate that these variants do not provide sufficient clinically relevant data to individualize the treatment protocols.

Keywords: controlled ovarian hyperstimulation, gene variants, ovarian response, assisted reproduction

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Authors: Hieu Van Dong, Giang Thi Huong Tran, Giap Van Nguyen, Tung Duy Dao, Vuong Nghia Bui, Le Thi My Huynh, Yohei Takeda, Haruko Ogawa, Kunitoshi Imai

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Chicken anemia virus (CAV) has a ubiquitous and worldwide distribution in chicken production. Our group previously reported high seroprevalence of CAV in chickens in northern Vietnam. In the present study, 330 tissue samples collected from commercial and breeder chicken farms in eleven provinces in northern Vietnam were tested for the CAV infection. We found that 157 out of 330 (47.58%) chickens were positive with CAV genes by real-time PCR method. Nine CAV strains obtained from the different location and time were forwarded to the full-length sequence of CAV VP1 gene. Phylogenetic analysis of the Vietnamese CAV vp1 gene indicated that the CAVs circulating in northern Vietnam were divided into three distinct genotypes, II, III, and V, but not clustered with the vaccine strains. Among the three genotypes, genotype III was the major one widely spread in Vietnam, and that included three sub-genotypes, IIIa, IIIb, and IIIc. The Vietnamese CAV strains were closely related to the Chinese, Taiwanese, and USA strains. All the CAV isolates had glutamine at amino acid position 394 in the VP1 gene, suggesting that they might be highly pathogenic strains. One strain was defined to be genotype V, which had not been reported for Vietnamese CAVs. Additional studies are required to further evaluate the pathogenicity of CAV strains circulating in Vietnam.

Keywords: chicken anemia virus, genotype, genetic characteristics, Vietnam

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Authors: Elsher Lawson-Boyd

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In the wake of the Human Genome Project, the life sciences have undergone some fascinating changes. In particular, conventional beliefs relating to gene expression are being challenged by advances in postgenomic sciences, especially by the field of epigenetics. Epigenetics is the modification of gene expression without changes in the DNA sequence. In other words, epigenetics dictates that gene expression, the process by which the instructions in DNA are converted into products like proteins, is not solely controlled by DNA itself. Unlike gene-centric theories of heredity that characterized much of the 20th Century (where the genes were considered as having almost god-like power to create life), gene expression in epigenetics insists on environmental ‘signals’ or ‘exposures’, a point that radically deviates from gene-centric thinking. Science and Technology Studies (STS) scholars have shown that epigenetic research is having vast implications for the ways in which chronic, non-communicable diseases are conceptualized, treated, and governed. However, to the author’s knowledge, there have not yet been any in-depth sociological engagements with neuroepigenetics that examine how the field is affecting mental health and trauma discourse. In this paper, the author discusses preliminary findings from a doctoral ethnographic study on neuroepigenetics, trauma, and embodiment. Specifically, this study investigates the kinds of causal relations neuroepigenetic researchers are making between experiences of trauma and the development of mental illnesses like complex post-traumatic stress disorder (PTSD), both throughout a human’s lifetime and across generations. Using qualitative interviews and nonparticipant observation, the author focuses on two public-facing research centers based in Melbourne: Florey Institute of Neuroscience and Mental Health (FNMH), and Murdoch Children’s Research Institute (MCRI). Preliminary findings indicate that a great deal of ambiguity characterizes this infant field, particularly when animal-model experiments are employed and the results are translated into human frameworks. Nevertheless, researchers at the FNMH and MCRI strongly suggest that adverse and traumatic life events have a significant effect on gene expression, especially when experienced during early development. Furthermore, they predict that neuroepigenetic research will have substantial implications for the ways in which mental illnesses like complex PTSD are diagnosed and treated. These preliminary findings shed light on why medical and health sociologists have good reason to be chiming in, engaging with and de-black-boxing ideations emerging from postgenomic sciences, as they may indeed have significant effects for vulnerable populations not only in Australia but other developing countries in the Global South.

Keywords: genetics, mental illness, neuroepigenetics, trauma

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Authors: Cristian Soitu, Alexander Feuerborn, Cyril Deroy, Alfonso Castrejon-Pita, Peter R. Cook, Edmond J. Walsh

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Microfluidics now stands as an academically mature technology after a quarter of a century research activities have delivered a vast array of proof of concepts for many biological workflows. However, translation to industry remains poor, with only a handful of notable exceptions – e.g. digital PCR, DNA sequencing – mainly because of biocompatibility issues, limited range of readouts supported or complex operation required. This technology exploits the domination of interfacial forces over gravitational ones at the microscale, replacing solid walls with fluid ones as building blocks for cell micro-environments. By employing only materials used by biologists for decades, the system is shown to be biocompatible, and easy to manufacture and operate. The method consists in displacing a continuous fluid layer into a pattern of isolated chambers overlaid with an immiscible liquid to prevent evaporation. The resulting fluid arrangements can be arrays of micro-chambers with rectangular footprint, which use the maximum surface area available, or structures with irregular patterns. Pliant, self-healing fluid walls confine volumes as small as 1 nl. Such fluidic structures can be reconfigured during the assays, giving the platform an unprecedented level of flexibility. Common workflows in cell biology are demonstrated – e.g. cell growth and retrieval, cloning, cryopreservation, fixation and immunolabeling, CRISPR-Cas9 gene editing, and proof-of-concept drug tests. This fluid-shaping technology is shown to have potential for high-throughput cell- and organism-based assays. The ability to make and reconfigure on-demand microfluidic circuits on standard Petri dishes should find many applications in biology, and yield more relevant phenotypic and genotypic responses when compared to standard microfluidic assays.

Keywords: fluid walls, micro-chambers, reconfigurable, freestyle

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Authors: Hannah R. Devens, Phillip L. Davidson, Dione Deaker, Kathryn E. Smith, Gregory A. Wray, Maria Byrne

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Marine invertebrate species with calcifying larvae are especially vulnerable to ocean acidification (OA) caused by rising atmospheric CO₂ levels. Acidic conditions can delay development, suppress metabolism, and decrease the availability of carbonate ions in the ocean environment for skeletogenesis. These stresses often result in increased larval mortality, which may lead to significant ecological consequences including alterations to the larval settlement, population distribution, and genetic connectivity. Importantly, many of these physiological and developmental effects are caused by genetic and molecular level changes. Although many studies have examined the effect of near-future oceanic pH levels on gene expression in marine invertebrates, little is known about the impact of OA on gene expression in a developmental context. Here, we performed mRNA-sequencing to investigate the impact of environmental acidity on gene expression across three developmental stages in the sea urchin Heliocidaris erythrogramma. We collected RNA from gastrula, early larva, and 1-day post-metamorphic juvenile sea urchins cultured at present-day and predicted future oceanic pH levels (pH 8.1 and 7.7, respectively). We assembled an annotated reference transcriptome encompassing development from egg to ten days post-metamorphosis by combining these data with datasets from two previous developmental transcriptomic studies of H. erythrogramma. Differential gene expression and time course analyses between pH conditions revealed significant alterations to developmental transcription that are potentially associated with pH stress. Consistent with previous investigations, genes involved in biomineralization and ion transport were significantly upregulated under acidic conditions. Differences in gene expression between the two pH conditions became more pronounced post-metamorphosis, suggesting a development-dependent effect of OA on gene expression. Furthermore, many differences in gene expression later in development appeared to be a result of broad downregulation at pH 7.7: of 539 genes differentially expressed at the juvenile stage, 519 of these were lower in the acidic condition. Time course comparisons between pH 8.1 and 7.7 samples also demonstrated over 500 genes were more lowly expressed in pH 7.7 samples throughout development. Of the genes exhibiting stage-dependent expression level changes, over 15% of these diverged from the expected temporal pattern of expression in the acidic condition. Through these analyses, we identify novel candidate genes involved in development, metabolism, and transcriptional regulation that are possibly affected by pH stress. Our results demonstrate that pH stress significantly alters gene expression dynamics throughout development. A large number of genes differentially expressed between pH conditions in juveniles relative to earlier stages may be attributed to the effects of acidity on transcriptional regulation, as a greater proportion of mRNA at this later stage has been nascent transcribed rather than maternally loaded. Also, the overall downregulation of many genes in the acidic condition suggests that OA-induced developmental delay manifests as suppressed mRNA expression, possibly from lower transcription rates or increased mRNA degradation in the acidic environment. Further studies will be necessary to determine in greater detail the extent of OA effects on early developing marine invertebrates.

Keywords: development, gene expression, ocean acidification, RNA-sequencing, sea urchins

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Authors: Debasish Pradhan, Shaktiprasad Pradhan, Rakesh Kumar Pradhan, Gitanjali Tripathy

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Suppressors of cytokine signalling (SOCS1), a newly indentified antiapoptotic molecule is a downstream effector of the receptor tyrosine kinase-Ras signalling pathway. The current study has uncovered that SOCS1 may have wide and imperative capacities, particularly because of its close correlation with malignant tumors. To investigate the impact of SOCS1 on MDR, we analyzed the expression of P-gp and SOCS1 by immunohistochemistry and found there was a positive correlation between them. At that point, we effectively interfered with RNA translation by the contamination of siRNA of SOCS1 into MCF7/ADM breast cancer cell lines through a lentivirus, and the expression of the target gene was significantly inhibited. After RNAi, the drug resistance was reduced altogether and the expression of MDR1 mRNA and P-gp in MCF7/ADM cell lines demonstrated a significant decrease. Likewise, the expression of P53 protein increased in a statistically significant manner (p ≤ 0.01) after RNAi exposure. Moreover, flow cytometry analysis uncovers that cell cycle and anti-apoptotic enhancing capacity of cells changed after RNAi treatment. These outcomes proposed SOCS1 may take part in breast cancer MDR by managing MDR1 and P53 expression, changing cell cycle and enhancing the anti-apoptotic ability.

Keywords: breast cancer, multidrug resistance, SOCS1 gene, MDR1 gene, RNA interference

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Authors: Agostinho Antunes

Abstract:

The completion of the human genome sequencing in 2003 opened a new perspective into the importance of whole genome sequencing projects, and currently multiple species are having their genomes completed sequenced, from simple organisms, such as bacteria, to more complex taxa, such as mammals. This voluminous sequencing data generated across multiple organisms provides also the framework to better understand the genetic makeup of such species and related ones, allowing to explore the genetic changes underlining the evolution of diverse phenotypic traits. Here, recent results from our group retrieved from comparative evolutionary genomic analyses of selected marine animal species will be considered to exemplify how gene novelty and gene enhancement by positive selection might have been determinant in the success of adaptive radiations into diverse habitats and lifestyles.

Keywords: marine genomics, evolutionary bioinformatics, human genome sequencing, genomic analyses

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Authors: K. S. Vimal, D. Bodhini, K. Ramya, N. Lakshmipriya, R. M. Anjana, V. Sudha, J. A. Lovegrove, V. Mohan, V. Radha

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Gene-lifestyle interaction studies have been carried out in various populations. However, to date there are no studies in an Asian Indian population. Hence, we examined whether lifestyle factors such as diet and physical activity modify the association between fat mass and obesity–associated (FTO) gene variants and obesity and type 2 diabetes (T2D) in an Asian Indian population. We studied 734 unrelated T2D and 884 normal glucose-tolerant (NGT) participants randomly selected from the Chennai Urban Rural Epidemiology Study (CURES) in Southern India. Obesity was defined according to the World Health Organization Asia Pacific Guidelines (non-obese, BMI < 25 kg/m2; obese, BMI ≥ 25 kg/m2). Six single nucleotide polymorphisms (SNPs) in the FTO gene (rs9940128, rs7193144, rs8050136, rs918031, rs1588413 and rs11076023) identified from recent genome-wide association studies for T2D were genotyped by polymerase chain reaction-restriction fragment length polymorphism and direct sequencing. Dietary assessment was carried out using a validated food frequency questionnaire and physical activity was based upon the self-report. Interaction analyses were performed by including the interaction terms in the model. A joint likelihood ratio test of the main SNP effects and the SNP-diet/physical activity interaction effects was used in the linear regression analyses to maximize statistical power. Statistical analyses were performed using STATA version 13. There was a significant interaction between FTO SNP rs8050136 and carbohydrate energy percentage (Pinteraction=0.04) on obesity, where the ‘A’ allele carriers of the SNP rs8050136 had 2.46 times higher risk of obesity than those with ‘CC’ genotype (P=3.0x10-5) among individuals in the highest tertile of carbohydrate energy percentage. Furthermore, among those who had lower levels of physical activity, the ‘A’ allele carriers of the SNP rs8050136 had 1.89 times higher risk of obesity than those with ‘CC’ genotype (P=4.0x10-5). We also found a borderline interaction between SNP rs11076023 and carbohydrate energy percentage (Pinteraction=0.08) on T2D, where the ‘A’ allele carriers in the highest tertile of carbohydrate energy percentage, had 1.57 times higher risk of T2D than those with ‘TT’ genotype (P=0.002). There was also a significant interaction between SNP rs11076023 and physical activity (Pinteraction=0.03) on T2D. No further significant interactions between SNPs and macronutrient intake or physical activity on obesity and T2D were observed. In conclusion, this is the first study to provide evidence for a gene-diet and gene-physical activity interaction on obesity and T2D in an Asian Indian population. These findings suggest that the association between FTO gene variants and obesity and T2D is influenced by carbohydrate intake and physical activity levels. Greater understanding of how FTO gene influences obesity and T2D through dietary and exercise interventions will advance the development of behavioral intervention and personalised lifestyle strategies predicted to reduce the development of metabolic diseases in ‘A’ allele carriers of both SNPs in this Asian Indian population.

Keywords: dietary intake, FTO, obesity, physical activity, type 2 diabetes, Asian Indian.

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Authors: Ge Cui, Mei Fang Chien, Chihiro Inoue

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In this research, enrichment culture using an inorganic liquid medium collected soil contaminated with 1,2-dichlorobenzene (1,2-DCB) in Sendai, Japan, was added 1,2-DCB as the sole carbon source to create a stable consortium. The purpose of this research is to analysis dominant microorganisms in the stable consortium and enzyme system which play a role in the degradation of DCBs. The consortium is now at 30 generation and is still being cultured. By the result of PCR-DGGE and clone library, two bacteria are dominant. The bacteria named sk1 was isolated. 40mg/l of 1,2-DCB and 40mg/l of 1,4-DCB were completely degraded after 32 hours and 50 hours, respectively, but no degradation occurred in the case of 1,3-DCB. By PCR, tecA1 (α-subunit of DCB dioxygenase) gene which plays a role degrading DCB to DCB dihydrodiol, and tecB (dehydrogenase) gene which plays a role degrading DCB dihydrodiol to dichlorocatechol were amplified from strain sk1. Bacteria named sk100 was also isolated. 40mg/l of 1,2-DCB was completely degraded after 32 hours, but no degradation occurred in case of 1,3-DCB and 1,4-DCB. By the result of the catalytic core region of dioxygenase amplified by PCR, gene played a role degrading DCB was analyzed. The results of this study concluded that the isolated strains which have not been reported are able to degrade 1,2-DCB stably, and the characterization of degradation and the genomic analysis which is now in progress is helpful to have an overall view of this microbial degradation.

Keywords: DCB, 1, 2-DCB degrading strains, DCB dioxygenase, enrichment culture

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Authors: M. Marjaneh, M. Y. Narazah, H. Shahrul

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Array-based gene expression analysis is a powerful tool to profile expression of genes and to generate information on therapeutic effects of new anti-cancer compounds. Anti-apoptotic effect of thymoquinone was studied in MCF7 breast cancer cell line using gene expression profiling with cDNA micro array. The purity and yield of RNA samples were determined using RNeasyPlus Mini kit. The Agilent RNA 6000 Nano LabChip kit evaluated the quantity of the RNA samples. AffinityScript RT oligo-dT promoter primer was used to generate cDNA strands. T7 RNA polymerase was used to convert cDNA to cRNA. The cRNA samples and human universal reference RNA were labelled with Cy-3-CTP and Cy-5-CTP, respectively. Feature Extraction and GeneSpring software analysed the data. The single experiment analysis revealed involvement of 64 pathways with up-regulated genes and 78 pathways with down-regulated genes. The MAPK and p38-MAPK pathways were inhibited due to the up-regulation of PTPRR gene. The inhibition of p38-MAPK suggested up-regulation of TGF-ß pathway. Inhibition of p38 - MAPK caused up-regulation of TP53 and down-regulation of Bcl2 genes indicating involvement of intrinsic apoptotic pathway. Down-regulation of CARD16 gene as an adaptor molecule regulated CASP1 and suggested necrosis-like programmed cell death and involvement of caspase in apoptosis. Furthermore, down-regulation of GPCR, EGF-EGFR signalling pathways suggested reduction of ER. Involvement of AhR pathway which control cytochrome P450 and glucuronidation pathways showed metabolism of Thymoquinone. The findings showed differential expression of several genes in apoptosis pathways with thymoquinone treatment in estrogen receptor-positive breast cancer cells.

Keywords: cDNA microarray, thymoquinone, CARD16, PTPRR, CASP10

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Authors: Agnieszka H. Ludwig-Slomczynska, Michal T. Seweryn, Przemyslaw Kapusta, Ewelina Pitera, Katarzyna Cyganek, Urszula Mantaj, Lucja Dobrucka, Ewa Wender-Ozegowska, Maciej T. Malecki, Pawel Wolkow

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Obesity results from an imbalance between energy intake and its expenditure. Genome-Wide Association Study (GWAS) analyses have led to discovery of only about 100 variants influencing body mass index (BMI), which explain only a small portion of genetic variability. Analysis of gene epistasis gives a chance to discover another part. Since it was shown that interaction and communication between nuclear and mitochondrial genome are indispensable for normal cell function, we have looked for epistatic interactions between the two genomes to find their correlation with BMI. Methods: The analysis was performed on 366 T1DM patients using Illumina Infinium OmniExpressExome-8 chip and followed by imputation on Michigan Imputation Server. Only genes which influence mitochondrial functioning (listed in Human MitoCarta 2.0) were included in the analysis – variants of nuclear origin (MAF > 5%) in 1140 genes and 42 mitochondrial variants (MAF > 1%). Gene expression analysis was performed on GTex data. Association analysis between genetic variants and BMI was performed with the use of Linear Mixed Models as implemented in the package 'GENESIS' in R. Analysis of association between mRNA expression and BMI was performed with the use of linear models and standard significance tests in R. Results: Among variants involved in epistasis between mitochondria and nucleus we have identified one in mitochondrial transcription factor, TFB2M (rs6701836). It interacted with mitochondrial variants localized to MT-RNR1 (p=0.0004, MAF=15%), MT-ND2 (p=0.07, MAF=5%) and MT-ND4 (p=0.01, MAF=1.1%). Analysis of the interaction between nuclear variant rs6701836 (nuc) and rs3021088 localized to MT-ND2 mitochondrial gene (mito) has shown that the combination of the two led to BMI decrease (p=0.024). Each of the variants on its own does not correlate with higher BMI [p(nuc)=0.856, p(mito)=0.116)]. Although rs6701836 is intronic, it influences gene expression in the thyroid (p=0.000037). rs3021088 is a missense variant that leads to alanine to threonine substitution in the MT-ND2 gene which belongs to complex I of the electron transport chain. The analysis of the influence of genetic variants on gene expression has confirmed the trend explained above – the interaction of the two genes leads to BMI decrease (p=0.0308). Each of the mRNAs on its own is associated with higher BMI (p(mito)=0.0244 and p(nuc)=0.0269). Conclusıons: Our results show that nuclear-mitochondrial epistasis can influence BMI in T1DM patients. The correlation between transcription factor expression and mitochondrial genetic variants will be subject to further analysis.

Keywords: body mass index, epistasis, mitochondria, type 1 diabetes

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Authors: D. Mohammadzadeh S., J. Bolouri Bazaz

Abstract:

This paper presents a new prediction model for compression index of fine-grained soils using multi-gene genetic programming (MGGP) technique. The proposed model relates the soil compression index to its liquid limit, plastic limit and void ratio. Several laboratory test results for fine-grained were used to develop the models. Various criteria were considered to check the validity of the model. The parametric and sensitivity analyses were performed and discussed. The MGGP method was found to be very effective for predicting the soil compression index. A comparative study was further performed to prove the superiority of the MGGP model to the existing soft computing and traditional empirical equations.

Keywords: new prediction model, compression index soil, multi-gene genetic programming, MGGP

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Authors: Chao Wang

Abstract:

Under rapid innovation of information technology, the media plays a very important role in the dissemination of information, and it has a totally different analogy generations face. However, the involvement of narrative images provides more possibilities of narrative text. "Images" through the process of aperture, a camera shutter and developable photosensitive processes are manufactured, recorded and stamped on paper, displayed on a computer screen-concretely saved. They exist in different forms of files, data, or evidence as the ultimate looks of events. By the interface of media and network platforms and special visual field of the viewer, class body space exists and extends out as thin as sebum layer, extremely soft and delicate with real full tension. The physical space of sebum layer of confuses the fact that physical objects exist, needs to be established under a perceived consensus. As at the scene, the existing concepts and boundaries of physical perceptions are blurred. Sebum layer physical simulation shapes the “Topical-Similarity" immersing, leading the contemporary social practice communities, groups, network users with a kind of illusion without the presence, i.e. a non-real illusion. From the investigation and discussion of literatures, digital movies editing manufacture and produce the variability characteristics of time (for example, slices, rupture, set, and reset) are analyzed. Interactive eBook has an unique interaction in "Waiting-Greeting" and "Expectation-Response" that makes the operation of image narrative structure more interpretations functionally. The works of digital editing and interactive technology are combined and further analyze concept and results. After digitization of Interventional Imaging and interactive technology, real events exist linked and the media handing cannot be cut relationship through movies, interactive art, practical case discussion and analysis. Audience needs more rational thinking about images carried by the authenticity of the text.

Keywords: sebum layer, topical and similarity, interactive technology, image narrative

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Authors: Ali Aydin, Mert Sudagidan, Aysen Coban, Alparslan Kadir Devrim

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Pseudomonas spp. (specifically, P. fluorescens and P. fragi) are considered the principal spoilage microorganisms of refrigerated poultry meats. The higher the level psychrophilic spoilage Pseudomonas spp. on carcasses at the end of processing lead to decrease the shelf life of the refrigerated product. The aim of the study was the identification of psychrophilic Pseudomonas spp. having proteolytic and lipolytic activities from poultry meats by 16S rRNA and rpoB gene sequencing, investigation of protease and lipase related genes and determination of proteolytic activity of Pseudomonas spp. In the of isolation procedure, collected chicken meat samples from local markets and slaughterhouses were homogenized and the lysates were incubated on Standard method agar and Skim Milk agar for selection of proteolytic bacteria and tributyrin agar for selection of lipolytic bacteria at +4 °C for 7 days. After detection of proteolytic and lipolytic colonies, the isolates were firstly analyzed by biochemical tests such as Gram staining, catalase and oxidase tests. DNA gene sequencing analysis and comparison with GenBank revealed that 126 strong enzyme Pseudomonas spp. were identified as predominantly P. fluorescens (n=55), P. fragi (n=42), Pseudomonas spp. (n=24), P. cedrina (n=2), P. poae (n=1), P. koreensis (n=1), and P. gessardi (n=1). Additionally, protease related aprX gene was screened in the strains and it was detected in 69/126 strains, whereas, lipase related lipA gene was found in 9 Pseudomonas strains. Protease activity was determined using commercially available protease assay kit and 5 strains showed high protease activity. The results showed that psychrophilic Pseudomonas strains were present in chicken meat samples and they can produce important levels of proteases and lipases for food spoilage to decrease food quality and safety.

Keywords: Pseudomonas, chicken meat, protease, lipase

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Authors: Jarmila Bernasovska, Pavel Ružbarský, Ivan Bernasovsky, Regina Lohajová Behulová

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The paper presents the results of the application of molecular genetics methods in sport research, with special emphasis on the most advanced methods and trends in diagnosing of motoric predispositions for the sake of identifying talented children. Genetic tests differ in principle from the traditional motoric tests, because the DNA of an individual does not change during life. Genetics is important in determining the capacity of an individual and for professional sport level. Genetic information can be used for individual genetic predispositions in early childhood. The phenotypes are influenced by a combination of genetic and environmental factors. The aim of the presented study was to examine physical condition, coordination skills, motoric docility and to determine the frequency of ACTN3 (R577X) gene in Romany children from Eastern Slovakia and compared their motoric performance with non-Romany children. This paper is not looking just for a performance, but also its association to genetic predispositions in relation to ACTN3 gene and its R577X polymorphism. Genotype data were obtained from 175 Romany children from 6 to 15 years old and 218 non-Romany children at the same age from Eastern Slovakia. Biological material for genetic analyses comprised samples of buccal swabs. Genotypes were determined using Real Time High resolution melting PCR method (Rotor Gene 6000 Corbett and LightCycler 480 Roche). Romany children of analyzed group legged to non-Romany children at the same age in all the compared tests. The % distribution of R and X alleles in children was different from controls. The frequency of XX genotype was 11,45% which is comparable to a frequency of an Indian population. Data were analysed with the ANOVA statistical programme and parametric and nonparametric tests. This work was supported by grants APVV-0716-10, ITMS 26220120023 and ITMS 26220120041.

Keywords: ACTN3 gene, R577X polymorphism, Romany children, sport performance, Slovakia

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Authors: Emanuela Chiarella, Annamaria Aloisio, Nisticò Clelia, Maria Mesuraca

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ZNF521 is a stem cell-associated transcription co-factor, that plays a crucial role in the homeostatic regulation of the stem cell compartment in the hematopoietic, osteo-adipogenic, and neural system. In normal hematopoiesis, primary human CD34+ hematopoietic stem cells display typically a high expression of ZNF521, while its mRNA levels rapidly decrease when these progenitors progress towards erythroid, granulocytic, or B-lymphoid differentiation. However, most acute myeloid leukemias (AMLs) and leukemia-initiating cells keep high ZNF521 expression. In particular, AMLs are often characterized by chromosomal translocations involving the Mixed Lineage Leukemia (MLL) gene, which MLL gene includes a variety of fusion oncogenes arisen from genes normally required during hematopoietic development; once they are fused, they promote epigenetic and transcription factor dysregulation. The chromosomal translocation t(9;11)(p21-22;q23), fusing the MLL gene with AF9 gene, results in a monocytic immune phenotype with an aggressive course, frequent relapses, and a short survival time. To better understand the dysfunctional transcriptional networks related to genetic aberrations, AML gene expression profile datasets were queried for ZNF521 expression and its correlations with specific gene rearrangements and mutations. The results showed that ZNF521 mRNA levels are associated with specific genetic aberrations: the highest expression levels were observed in AMLs involving t(11q23) MLL rearrangements in two distinct datasets (MILE and den Boer); elevated ZNF521 mRNA expression levels were also revealed in AMLs with t(7;12) or with internal rearrangements of chromosome 16. On the contrary, relatively low ZNF521 expression levels seemed to be associated with the t(8;21) translocation, that in turn is correlated with the AML1-ETO fusion gene or the t(15;17) translocation and in AMLs with FLT3-ITD, NPM1, or CEBPα double mutations. Invitro, we found that the enforced co-expression of ZNF521 in cord blood-derived CD34+ cells induced a significant proliferative advantage, improving MLL-AF9 effects on the induction of proliferation and the expansion of leukemic progenitor cells. Transcriptome profiling of CD34+ cells transduced with either MLL-AF9, ZNF521, or a combination of the two transgenes highlighted specific sets of up- or down-regulated genes that are involved in the leukemic phenotype, including those encoding transcription factors, epigenetic modulators, and cell cycle regulators as well as those engaged in the transport or uptake of nutrients. These data enhance the functional cooperation between ZNF521 and MA9, resulting in the development, maintenance, and clonal expansion of leukemic cells. Finally, silencing of ZNF521 in MLL-AF9-transformed primary CD34+ cells inhibited their proliferation and led to their extinction, as well as ZNF521 silencing in the MLL-AF9+ THP-1 cell line resulted in an impairment of their growth and clonogenicity. Taken together, our data highlight ZNF521 role in the control of self-renewal and in the immature compartment of malignant hematopoiesis, which, by altering the gene expression landscape, contributes to the development and/or maintenance of AML acting in concert with the MLL-AF9 fusion oncogene.

Keywords: AML, human zinc finger protein 521 (hZNF521), mixed lineage leukemia gene (MLL) AF9 (MLLT3 or LTG9), cord blood-derived hematopoietic stem cells (CB-CD34+)

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Authors: Syed M. Shahid, Rozeena Shaikh, Syeda N. Nawab, Abid Azhar

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Diabetes mellitus (DM) is a prevalent non-communicable disease worldwide. DM may lead to many vascular complications like hypertension, nephropathy, retinopathy, neuropathy and foot infections. Pathogenesis of diabetic nephropathy (DN) is implicated by the polymorphisms in genes encoding the specific components of renin angiotensin aldosterone system (RAAS) which include angiotensinogen (AGT), angiotensin-II receptor and angiotensin converting enzyme (ACE) genes. This study was designed to explore the possible association of AG (M268T) polymorphism in the patients of diabetes and nephropathy in Pakistan. Study subjects included 100 controls, 260 diabetic patients without renal insufficiency and 190 diabetic nephropathy patients with persistent albuminuria. Fasting blood samples were collected from all the subjects after getting institutional ethical approval and informed consent. The biochemical estimations, PCR amplification and direct sequencing for the specific region of AGT gene was carried out. A significantly high frequency of TT genotype and T allele of AGT (M268T) was observed in the patients of diabetes with nephropathy as compared to controls and diabetic patients without any known renal impairment. The TT genotype and T allele of AGT (M268T) polymorphism may be considered as a genetic risk factor for the development and progression of nephropathy in diabetes. Further cross sectional population studies would be of help to establish and confirm the observed possible association of AGT gene variations with development of nephropathy in diabetes.

Keywords: RAAS, AGT (M268T), diabetes, nephropathy

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Authors: Mahbuba Rahman, Sabri Boughorbel, Scott Presnell, Charlie Quinn, Darawan Rinchai, Damien Chaussabel, Nico Marr

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Collections of large-scale datasets made available in public repositories can be used to identify and fill gaps in biomedical knowledge. But first, these data need to be made readily accessible to researchers for analysis and interpretation. Here a collection of transcriptome datasets was made available to investigate the functional programming of human hematopoietic cells in early life. Thirty two datasets were retrieved from the NCBI Gene Expression Omnibus (GEO) and loaded in a custom, interactive web application called the Gene Expression browser (GXB), designed for visualization and query of integrated large-scale data. Multiple sample groupings and gene rank lists were created based on the study design and variables in each dataset. Web links to customized graphical views can be generated by users and subsequently be used to graphically present data in manuscripts for publication. The GXB tool also enables browsing of a single gene across datasets, which can provide information on the role of a given molecule across biological systems. The dataset collection is available online. As a proof-of-principle, one of the datasets (GSE25087) was re-analyzed to identify genes that are differentially expressed by regulatory T cells in early life. Re-analysis of this dataset and a cross-study comparison using multiple other datasets in the above mentioned collection revealed that PMCH, a gene encoding a precursor of melanin-concentrating hormone (MCH), a cyclic neuropeptide, is highly expressed in a variety of other hematopoietic cell types, including neonatal erythroid cells as well as plasmacytoid dendritic cells upon viral infection. Our findings suggest an as yet unrecognized role of MCH in immune regulation, thereby highlighting the unique potential of the curated dataset collection and systems biology approach to generate new hypotheses which can be tested in future mechanistic studies.

Keywords: early-life, GEO datasets, PMCH, interactive query, systems biology

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Authors: Nan Deng, Zhenqiu Liu

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Thousands of patients with metastatic tumors were diagnosed with cancers of unknown primary sites each year. The inability to identify the primary cancer site may lead to inappropriate treatment and unexpected prognosis. Nowadays, a large amount of genomics and transcriptomics cancer data has been generated by next-generation sequencing (NGS) technologies, and The Cancer Genome Atlas (TCGA) database has accrued thousands of human cancer tumors and healthy controls, which provides an abundance of resource to differentiate cancer types. Meanwhile, deep convolutional neural networks (CNNs) have shown high accuracy on classification among a large number of image object categories. Here, we utilize 25 cancer primary tumors and 3 normal tissues from TCGA and convert their RNA-Seq gene expression profiling to color images; train, validate and test a CNN classifier directly from these images. The performance result shows that our CNN classifier can archive >80% test accuracy on most of the tumors and normal tissues. Since the gene expression pattern of distant metastases is similar to their primary tumors, the CNN classifier may provide a potential computational strategy on identifying the unknown primary origin of metastatic cancer in order to plan appropriate treatment for patients.

Keywords: bioinformatics, cancer, convolutional neural network, deep leaning, gene expression pattern

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Authors: K. R. Thabethe, G. A. Adefolaju, M. J. Hosie

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Cervical cancer is the third most commonly diagnosed cancer globally and it is one of three AIDS defining malignancies. Highly active antiretroviral therapy (HAART) is a combination of three or more antiretroviral drugs and has been shown to play a significant role in reducing the incidence of some AIDS defining malignancies, although its effect on cervical cancer is still unclear. The aim of this study was to investigate the relationship between cervical cancer and HAART. This was achieved by studying the expression of two signalling molecules expressed in cervical cancer; MUC1 and P65. Following the 24 hour treatment of a cervical cancer cell line, HCS-2, with drugs which are commonly used as part of HAART at their clinical plasma concentrations, real-time qPCR and immunofluorescence were used in order to study gene and protein expression. A one way ANOVA followed by a Tukey Kramer Post Hoc test was conducted using JMP 11 software on both sets of data. The drug classified as a protease inhibitor (PI) (i.e. LPV/r) reduced MUC1 and P65 gene and protein expression more than the other drug tested. PIs are known to play a significant role in cell death, therefore the cells were thought to be more susceptible to cell death following treatment with PIs. In conclusion, the drugs used, especially the PI showed some anticancer effects by facilitating cell death through decreased gene and protein expression of MUC1 and P65 and present promising agents for cancer treatment.

Keywords: cervical cancer, haart, MUC1, P65

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Authors: Dalia. G. Aseel

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Begomoviruses are economically important plant viruses that infect dicotyledonous plants and exclusively transmitted by the whitefly Bemisia tabaci. Here, replicative form was isolated from Okra, Cotton, Tomato plants and whitefly infected with Begomoviruses. Using coat protein specific primers (AV1), the viral infection was verified with amplicon at 450 bp. The sequence of OLCuV-AV1 gene was recorded and received an accession number (FJ441605) from Genebank. The phylogenetic tree of OLCuV was closely related to Okra leaf curl virus previously isolated from Cameroon and USA with nucleotide sequence identity of 92%. The protein purification was carried out using His-Tag methodology by using Affinity Chromatography. The purified protein was separated on SDS-PAGE analysis and an enriched expected size of band at 30 kDa was observed. Furthermore, RAPD and SDS-PAGE were used to detect genetic variability between different hosts of okra leaf curl virus (OLCuV), cotton leaf curl virus (CLCuV), tomato yellow leaf curl virus (TYLCuV) and the whitefly vector. Finally, the present study would help to understand the relationship between the whitefly and different economical crops in Egypt.

Keywords: okra leaf curl virus, AV1 gene, sequencing, phylogenetic, cloning, purified protein, genetic diversity and viral proteins

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Authors: Alina Smalinskiene Janina Petkeviciene, Jurate Klumbiene, Vilma Kriaucioniene, Vaiva Lesauskaite

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The worldwide prevalence of obesity has been increasing dramatically in the last few decades, and Lithuania is no exception. In 2012, every fifth adult (19% of men and 20.5 % of women) was obese and every third was overweight Association studies have highlighted the influence of SNPs in obesity, with particular focus on FTO rs9939609. Thus far, no data on the possible association of this SNP to obesity in the adult Lithuanian population has been reported. Here, for the first time, we demonstrate an association between the FTO rs9939609 homozygous AA genotype and increased BMI when compared to homozygous TT. Furthermore, a positive association was determined between the FTO rs9939609 variant and risk of metabolic syndrome. Background: This study aimed to examine the associations between the fat mass and obesity associated (FTO) gene rs9939609 variant with obesity and metabolic syndrome in Lithuanian adult population. Materials and Methods: A cross-sectional health survey was carried out in randomly selected municipalities of Lithuania. The random sample was obtained from lists of 25–64 year-old inhabitants. The data from 1020 subjects were analysed. The rs9939609 SNP of the FTO gene was assessed using TaqMan assays (Applied Biosystems, Foster City, CA, USA). The Applied Biosystems 7900HT Real-Time Polymerase Chain Reaction System was used for detecting the SNPs. Results: The carriers of the AA genotype had the highest mean values of BMI and waist circumference (WC) and the highest risk of obesity. Interactions ‘genotype x age’ and ‘genotype x physical activity’ in determining BMI and WC were shown. Neither lipid and glucose levels, nor blood pressure were associated with the rs9939609 independently of BMI. In the age group of 25-44 years, association between the FTO genotypes and metabolic syndrome was found. Conclusion: The FTO rs9939609 variant was significantly associated with BMI and WC, and with the risk of obesity in Lithuanian population. The FTO polymorphism might have a greater influence on weight status in younger individuals and in subjects with a low level of physical activity.

Keywords: obesity metabolic syndrome, FTO gene, polymorphism, Lithuania

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Authors: T. C. C. Soo, S. Bhassu

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Unlike the common presence of both innate and adaptive immunity in vertebrates, crustaceans, in particular, shrimps, have been discovered to possess only innate immunity. This further emphasizes the importance of innate immunity within shrimps in pathogenic resistance. Under the study of pathogenic immune challenge, different shrimp species actually exhibit varying degrees of immune resistance towards the same pathogen. Furthermore, even within the same shrimp species, different batches of challenged shrimps can have different strengths of immune defence. Several important pathways are activated within shrimps during pathogenic infection. One of them is JAK-STAT pathway that is activated during bacterial, viral and fungal infections by which STAT(Signal Transducer and Activator of Transcription) gene is the core element of the pathway. Based on theory of Central Dogma, the genomic information is transmitted in the order of DNA, RNA and protein. This study is focused in uncovering the important evolutionary patterns present within the DNA (non-coding region) and RNA (coding region). The three shrimp species involved are Macrobrachium rosenbergii, Penaeus monodon and Litopenaeus vannamei which all possess commercial significance. The shrimp species were challenged with a famous penaeid shrimp virus called white spot syndrome virus (WSSV) which can cause serious lethality. Tissue samples were collected during time intervals of 0h, 3h, 6h, 12h, 24h, 36h and 48h. The DNA and RNA samples were then extracted using conventional kits from the hepatopancreas tissue samples. PCR technique together with designed STAT gene conserved primers were utilized for identification of the STAT coding sequences using RNA-converted cDNA samples and subsequent characterization using various bioinformatics approaches including Ramachandran plot, ProtParam and SWISS-MODEL. The varying levels of immune STAT gene activation for the three shrimp species during WSSV infection were confirmed using qRT-PCR technique. For one sample, three biological replicates with three technical replicates each were used for qRT-PCR. On the other hand, DNA samples were important for uncovering the structural variations within the genomic region of STAT gene which would greatly assist in understanding the STAT protein functional variations. The partially-overlapping primers technique was used for the genomic region sequencing. The evolutionary inferences and event predictions were then conducted through the Bayesian Inference method using all the acquired coding and non-coding sequences. This was supplemented by the construction of conventional phylogenetic trees using Maximum likelihood method. The results showed that adaptive evolution caused STAT gene sequence mutations between different shrimp species which led to evolutionary divergence event. Subsequently, the divergent sites were correlated to the differing expressions of STAT gene. Ultimately, this study assists in knowing the shrimp species innate immune variability and selection of disease resistant shrimps for breeding purpose. The deeper understanding of STAT gene evolution from the perspective of both purifying and adaptive approaches not only can provide better immunological insight among shrimp species, but also can be used as a good reference for immunological studies in humans or other model organisms.

Keywords: gene evolution, JAK-STAT pathway, immunology, STAT gene

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