Search results for: endogenous reference gene

Authors: Moustafa Elhamouly, Masayuki Shimada

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The production of competent oocytes is essential for reproductivity in mammals. Maintenance of mitochondrial efficiency is required to supply the ATP necessary for granulosa cell proliferation during the follicular development process. Treatment with Pyrroloquinoline quinone (PQQ) has been reported to increase the number of ovulated oocytes and pups per delivery in mice by maintaining healthy mitochondrial function. This study aimed to elucidate how PQQ maintains mitochondrial function during ovarian follicle growth. To do this, both in vitro and in vivo experiments were performed with granulosa cells from superovulated immature (3-week-old) mice that were pretreated with or without PQQ. The effects of PQQ on beta-oxidation, mitochondrial function, mitophagy, and mitochondrial biogenesis were examined. PQQ increased beta-oxidation-related genes and CPT1 protein content in granulosa cells and this was associated with a decreased phosphorylation of P38 signaling protein. Using the fatty acid oxidation assay on the flux analyzer, PQQ increased the reliance of beta-oxidation on the endogenous fatty acids and was associated with a mild UCP-dependant mitochondrial uncoupling, ATP production, mitophagy, and mitochondrial biogenesis. PQQ also increased the expression of endogenous antioxidant enzymes. Thus, PQQ induced beta-oxidation in growing granulosa cells relying on endogenous fatty acids. And reduced the Reactive oxygen species (ROS) production by inducing a mild mitochondrial uncoupling with keeping high mitochondrial function. Damaged mitochondria were recycled by the induced mitophagy and replaced by the increased mitochondrial biogenesis. Collectively, PQQ may enhance reproductivity by maintaining the efficiency of mitochondria to produce enough ATP required for normal folliculogenesis.

Keywords: granulosa cells, mitochondrial uncoupling, mitophagy, pyrroloquinoline quinone (PQQ), reactive oxygen species (ROS).

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Authors: Saad Bin Saeed, Fayzan Ahmed, Shahbaz Ahmed, Amjad Hussain

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The aim of this study is to evaluate dynamic log files (Dynalogs) at different dose rates by dose-volume histograms (DVH) and used as a (QA) procedure of IMRT. Seven patients of phase one head and neck cancer with similar OAR`s are selected randomly. Reference plans of dose rate 300 and 600 MU/Min with prescribed dose of 50Gy in 25 fractions for each patient is made. Dynalogs produced by delivery of reference plans processed by in-house MATLAB program which produces new field files contain actual positions of multi-leaf collimators (MLC`s) instead of planned positions in reference plans. Copies of reference plans are used to import new field files generated by MATLAB program and renamed as Dyn.plan. After dose calculations of Dyn.plans for different dose rates, DVH, and multiple linear regression tools are used to evaluate reference and Dyn.plans. The results indicate good agreement of correlation between different dose rate plans. The maximum dose difference among PTV and OAR`s are found to be less than 5% and 9% respectively. The study indicates the potential of dynalogs to be used as patient-specific QA of IMRT at different dose rate.

Keywords: IMRT, dynalogs, dose rate, DVH

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

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

Keywords: biological pathway, image understanding, gene name recognition, object detection, Siamese network, VGG

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Authors: R. A. Petre, S. E. Nichifor, A. Craifaleanu, I. Stroe

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The relative motion of a robotic arm formed by homogeneous bars of different lengths and masses, hinged to each other is investigated. The first bar of the mechanism is articulated on a platform, considered initially fixed on the surface of the Earth, while for the second case the platform is considered to be in rotation with respect to the Earth. For both analyzed cases the motion equations are determined using the Lagrangian formalism, applied in its traditional form, valid with respect to an inertial reference system, conventionally considered as fixed. However, in the second case, a generalized form of the formalism valid with respect to a non-inertial reference frame will also be applied. The numerical calculations were performed using a MATLAB program.

Keywords: Lagrange equations, relative motion, inertial reference frame, non-inertial reference frame

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Authors: Y. I. Tretyakova, S. G. Shulkina, T. Y. Kravtsova, A. A. Antipova, N. Y. Kolomeets

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The research aimed to assess the functional significance of tumor necrosis factor-alpha (TNF-α) gene polymorphism at the -308G/A (rs1800629) region and vascular endothelial growth factor A (VEGFA) gene polymorphism at the -634G/C (rs 2010963) region in the development of ulcerative colitis (UC), focusing on patients from the Perm region, Russia. We examined 70 UC patients and 50 healthy donors during the active phase of the disease. Our focus was on TNF-α and VEGF concentration in the blood serum, as well as TNF-α and VEGFA gene polymorphisms at the -308G/А and -634G/C regions, respectively. We found that TNF-α and VEGF levels were significantly higher in patients with severe UC and high endoscopic activity compared to those with milder forms of the disease and low endoscopic activity. These tests could serve as additional non-invasive markers for assessing mucosal damage in the large intestine of UC patients. The frequency of allele variations in the TNF-α gene -308G/A (rs1800629) revealed a significantly higher occurrence of the unfavorable homozygote AA in UC patients compared to donors. Additionally, the major allele G and the allele pair GG were more frequent in patients with mild to moderate disease and 1-2 degree of endoscopic activity than in those with severe UC and 3-4 degree of endoscopic activity (χ2=14.19; p=0.000). We also observed a mutant allele A and the unfavorable homozygote AA associated with severe progressive UC. The occurrence of the mutant allele increased the risk of severe UC by 5 times (OR 5.03; CI 12.07-12.21). We did not find any significant differences in the frequency of the CC homozygote (χ2=1.02; p=0.6; OR=1.32) and the mutant allele C of the VEGFA gene -634G/C (rs 2010963) (χ2=0.01; p=0.913; OR=0.97) between groups of UC patients and healthy individuals. However, we detected that the mutant allele C and the unfavorable homozygote CC of the VEGFA gene were associated with more severe endoscopic changes in the colonic mucosa of UC patients (χ2=25,76; р=0,000; OR=0,15). The presence of the mutant allele increased the risk of severe UC by 6 times (OR 6,78; CI 3,13–14,7). We found a direct correlation between TNF-α and VEGFA gene polymorphisms, increased production of the same factors, disease severity, and endoscopic activity (р=0.000). Therefore, the presence of the mutant allele A and homozygote AA of the TNF-α gene at the -308G/A region and the mutant allele C and homozygote CC of the VEGFA gene at the -634G/C region are associated with risks related to an unfavorable clinical course of UC, frequent recurrences, and rapid progression. These findings should be considered when making prognoses regarding the clinical course of the disease and selecting treatment strategies. The presence of the homozygote AA in the TNF-α gene (rs1800629) is considered a sign of genetic predisposition to UC.

Keywords: gene polymorphism, TNF-α, ulcerative colitis, VEGF

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Authors: I. Boroňová, J. Bernasovská, J. Kmec, E. Petrejčíková

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Dilated cardiomyopathy (DCM) is a primary myocardial disease, it is characterized by progressive systolic dysfunction due to cardiac chamber dilatation and inefficient myocardial contractility with estimated prevalence of 37 in 100 000 people. It is the most frequent cause of heart failure and cardiac transplantation in young adults. About one-third of all patients have a suspected familial disease indicating a genetic basis of DCM. Many candidate gene studies in humans have tested the association of single nucleotide polymorphisms (SNPs) in various genes coding for proteins with a known cardiovascular function. In our study we present the results of ZBTB17 gene rs10927875 polymorphism genotyping in relation to dilated cardiomyopathy in Slovak population. The study included 78 individuals, 39 patients with DCM and 39 healthy control persons. The mean age of patients with DCM was 50.7±11.5 years; the mean age of individuals in control group was 51.3±9.8 years. Risk factors detected at baseline in each group included age, sex, body mass index, smoking status, diabetes and blood pressure. Genomic DNA was extracted from leukocytes by a standard methodology and screened for rs10927875 polymorphism in intron of ZBTB17 gene using Real-time PCR method (Step One Applied Biosystems). The distribution of investigated genotypes for rs10927875 polymorphism in the group of patients with DCM was as follows: CC (89.74%), CT (10.26%), TT (0%), and the distribution in the control group: CC (92.31%), CT (5.13%), and TT (2.56%). Using the chi-square (χ2) test we compared genotype and allele frequencies between patients and controls. There was no difference in genotype or allele frequencies in ZBTB17 gene rs10927875 polymorphism between patients and control group (χ2=3.028, p=0.220; χ2=0.264, p=0.608). Our results represent an initial study, it can be considered as preliminary and first of its kind in Slovak population. Further studies of ZBTB17 gene polymorphisms of more numerous files and additional functional investigations are needed to fully understand the role of genetic associations.

Keywords: dilated cardiomyopathy, SNP polymorphism, ZBTB17 gene, bioscience

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Authors: Sujit K. Basak

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This paper presents a comparison of the reference management software between Zotero and Mendeley and the results were drawn by comparing the two software’s. The novelty of this paper is the comparative analysis of the software and it has shown that Mendeley can import more information from the Google Scholar for the researchers. This finding can help to know researchers to use the reference management software.

Keywords: analysis, comparative analysis, zotero, researchers, Mendeley

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Authors: Huong M. T. Nguyen, Hoa A. P. Nguyen, Mai P. T. Nguyen, Ngoc D. Ngo, Van T. Ta, Hai T. Le, Chi V. Phan

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Wilson’s disease (WD) is an autosomal recessive disorder of the copper metabolism, which is caused by a mutation in the copper-transporting P-type ATPase (ATP7B). The mechanism of this disease is the failure of hepatic excretion of copper to bile, and leads to copper deposits in the liver and other organs. The ATP7B gene is located on the long arm of chromosome 13 (13q14.3). This study aimed to investigate the gene mutation in the Vietnamese patients with WD, and make a presymptomatic diagnosis for their familial members. Forty-three WD patients and their 65 siblings were identified as having ATP7B gene mutations. Genomic DNA was extracted from peripheral blood samples; 21 exons and exon-intron boundaries of the ATP7B gene were analyzed by direct sequencing. We recognized four mutations ([R723=; H724Tfs*34], V1042Cfs*79, D1027H, and IVS6+3A>G) in the sum of 20 detectable mutations, accounting for 87.2% of the total. Mutation S105* was determined to have a high rate (32.6%) in this study. The hotspot regions of ATP7B were found at exons 2, 16, and 8, and intron 14, in 39.6 %, 11.6 %, 9.3%, and 7 % of patients, respectively. Among nine homozygote/compound heterozygote siblings of the patients with WD, three individuals were determined as asymptomatic by screening mutations of the probands. They would begin treatment after diagnosis. In conclusion, 20 different mutations were detected in 43 WD patients. Of this number, four novel mutations were explored, including [R723=; H724Tfs*34], V1042Cfs*79, D1027H, and IVS6+3A>G. The mutation S105* is the most prevalent and has been considered as a biomarker that can be used in a rapid detection assay for diagnosis of WD patients. Exons 2, 8, and 16, and intron 14 should be screened initially for WD patients in Vietnam. Based on risk profile for WD, genetic testing for presymptomatic patients is also useful in diagnosis and treatment.

Keywords: ATP7B gene, mutation detection, presymptomatic diagnosis, Vietnamese Wilson’s disease

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Authors: Mudawamah Mudawamah, Muhammad Z. Fadli, Gatot Ciptadi, Aulanni’am

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Canary breeders have spread throughout Indonesian regions for the low-middle society and become an income source for them. The interesting phenomenon of the canary market is the feather colours become one of determining factor for the price. The advantages of this research were contributed to the molecular database as a base of selection and mating for the Indonesia canary breeder. The research method was experiment with the genome obtained from canary blood isolation. The genome did the PCR amplification with PMEL marker followed by sequencing. Canaries were used 24 heads of light and dark colour feathers. Research data analyses used BioEdit and Network 4.6.0.0 software. The results showed that all samples were amplification with PMEL gene with 500 bp fragment length. In base sequence of 40 was found Cytosine(C) in the light colour canaries, while the dark colour canaries was obtained Thymine (T) in same base sequence. Sequence results had 286-415 bp fragment and 10 haplotypes. The conclusions were the PMEL gene (gene of white pigment) was likely to be used PMEL gene to detect molecular genetic variation of dark and light colour feather.

Keywords: canary, haplotype, PMEL, sequence

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Authors: Mengying Wang, Dongjing Liu, Holger Schwender, Ping Wang, Hongping Zhu, Tao Wu, Terri H Beaty

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Background: Maternal smoking is a recognized risk factor for nonsyndromic cleft lip with or without cleft palate (NSCL/P). It has been reported that the effect of maternal smoking on oral clefts is mediated through genes that influence nicotine dependence. The polymorphisms of cholinergic receptor nicotinic alpha (CHRNA) and beta (CHRNB) subunits genes have previously shown strong associations with nicotine dependence. Here, we attempted to investigate whether the above genes are associated with clefting risk through testing for potential gene-gene (G×G) and gene-environment (G×E) interaction. Methods: We selected 120 markers in 14 genes associated with nicotine dependence to conduct transmission disequilibrium tests among 806 Chinese NSCL/P case-parent trios ascertained in an international consortium which conducted a genome-wide association study (GWAS) of oral clefts. We applied Cordell’s method using “TRIO” package in R to explore G×G as well as G×E interaction involving environmental tobacco smoke (ETS) based on conditional logistic regression model. Results: while no SNP showed significant association with NSCL/P after Bonferroni correction, we found signals for G×G interaction between 10 pairs of SNPs in CHRNA3, CHRNA5, and CHRNB4 (p<10-8), among which the most significant interaction was found between RS3743077 (CHRNA3) and RS11636753 (CHRNB4, p<8.2×10-12). Linkage disequilibrium (LD) analysis revealed only low level of LD between these markers. However, there were no significant results for G×ETS interaction. Conclusion: This study fails to detect association between nicotine dependence genes and NSCL/P, but illustrates the importance of taking into account potential G×G interaction for genetic association analysis in NSCL/P. This study also suggests nicotine dependence genes should be considered as important candidate genes for NSCL/P in future studies.

Keywords: Gene-Gene Interaction, Maternal Smoking, Nicotine Dependence, Non-Syndromic Cleft Lip with or without Cleft Palate

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Authors: Moneesh Thakur, Hridayesh Prasad, Nikitasha Bora, Parimal Roy Choudhary, A. K. Samanta, Sanjeev Kumar

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Canine demodicosis is a common parasitic condition which involves dog skin. Demodicosis in dogs is due the prominent growth of Demodex. Out of various canine Demodex spp., Demodex canis is the most often involved species. Canine demodicosis can occur as either a localized or generalized form of demodicosis severely affect the dogs and in non-treated dogs may cause death. This study was planned with the aim to screen and characterize the 18S rRNA gene of isolated Demodex canis. A total of 1200 dogs were screened during this study period. The skin scrapings of all the suspected dogs were examined under a microscope at 100X magnification for the presence of Demodex canis. The skin scrapings positive for Demodex canis were examined using PCR for confirmation. A total of 35 dogs were confirmed a positive result for D. canis based on 18S rRNA gene amplification by PCR. Further, the 18S rRNA gene of isolated Demodex canis was cloned and sequenced for genome analysis. On the sequence analysis, it was found that isolated sequence (GenBank Accession No. MK177513) had close similarity (99.7%) to that of D. canis genotype of China (Accession No. MG372254).

Keywords: PCR, phylogenetic analysis, cloning and sequening, Demodex canis

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Authors: Jyoti Singh, Swati Dubey, Mukta Singh, R. P. Singh

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The freshwater microalgae Chlorella sp. is alluring considerable interest as a source for biofuel production due to its fast growth rate and high lipid content. Under nitrogen limited conditions, they can accumulate significant amounts of lipids. Thus, it is important to gain insight into the molecular mechanism of their lipid metabolism. In this study under nitrogen limited conditions, regular pattern of growth characteristics lipid accumulation and gene expression analysis of key regulatory genes of lipid biosynthetic pathway were carried out in microalgae Chlorella sp 3. Our results indicated that under nitrogen limited conditions there is a significant increase in the lipid content and lipid productivity, achieving 44.21±2.64 % and 39.34±0.66 mg/l/d at the end of the cultivation, respectively. Time-course transcript patterns of lipid biosynthesis genes i.e. acetyl coA carboxylase (accD) and diacylglycerol acyltransferase (dgat) showed that during late log phase of microalgae Chlorella sp.3 both the genes were significantly up regulated as compared to early log phase. Moreover, the transcript level of the dgat gene is two-fold higher than the accD gene. The results suggested that both the genes responded sensitively to the nitrogen limited conditions during the late log stage, which proposed their close relevance to lipid biosynthesis. Further, this transcriptome data will be useful for engineering microalgae species by targeting these genes for genetic modification to improve microalgal biofuel quality and production.

Keywords: biofuel, gene, lipid, microalgae

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Authors: Amir Bastani

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The result of the research in the field of human behavioural genetics demonstrates a genetic contribution of behavioural differences in aggression, violence, drug and substance abuse, antisocial personality disorder and other related traits. As the field of human behavioural genetics progresses and achieves credibility, the criminal accused continue to use its types of evidence into the criminal law. One of the most important genetic factors which controls certain neurotransmitters like dopamine and serotonin is the Monoamine Oxidase Acid A (MAOA) gene, known as the 'warrior gene'. The high-profile study by Caspi and colleagues in 2002 showed that the combination between one type of variation of the MAOA gene and childhood maltreatment noticeably predisposes a person to antisocial behaviour. Moreover, further scientific research shows that individuals with the MAOA gene have to some degree difficulties in controlling their impulses. Based on the evidence of MAOA, some criminal accused claimed difficulties in self-control. In the first case – the famous case of Mobley – the court rejected the MAOA evidence on the ground of the lack of scientific support. In contrast, in other cases after the Mobley trial, courts accepted the evidence of MAOA. In this paper, the issue of lack of impulse control produced by the MAOA gene and cases which relied on the MAOA evidence and successfully being accepted will be reviewed in detail. Finally, the anticipation of the paper for the future use of the MAOA evidence in criminal cases will be presented.

Keywords: genetic defence, criminal responsibility, MAOA, self-control

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Authors: Tanveer Hussain, Misbah Hussain, Masroor Ellahi Babar, Muhammad Traiq Pervez, Fiaz Hussain, Sana Zahoor, Rashid Saif

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Interleukin 2 (IL-2) is a cytokine which is produced by activated T cells, play important role in immune response against antigen. It act in both autocrine and paracrine manner. It can stimulate B cells and various other phagocytic cells like monocytes, lymphokine-activated killer cells and natural killer cells. Acting in autocrine fashion, IL-2 protein plays a crucial role in proliferation of T cells. IL-2 triggers the release of pro and anti- inflammatory cytokines by activating several pathways. In present study, exon 1 of IL-2 gene of four local Pakistani breeds (Dera Din Panah, Beetal, Nachi and Kamori) from two provinces was amplified by using reported Ovine IL-2 primers, yielding PCR product of 501 bp. The sequencing of all samples was done to identify the polymorphisms in amplified region of IL-2 gene. Analysis of sequencing data resulted in identification of one novel nucleotide substitution (T→A) in amplified non-coding region of IL-2 gene. Comparison of IL-2 gene sequence of all four breeds with other goat breeds showed high similarity in sequence. While phylogenetic analysis of our local breeds with other mammals showed that IL-2 is a variable gene which has undergone many substitutions. This high substitution rate can be due to the decreased or increased changed selective pressure. These rapid changes can also lead to the change in function of immune system. This pioneering study of Pakistani goat breeds urge for further studies on immune system of each targeted breed for fully understanding the functional role of IL-2 in goat immunity.

Keywords: interleukin 2, mutational analysis, phylogeny, goat breeds, Pakistan

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Authors: Sundru Manjulata Devi, Prakash M. Halami

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The immemorial use of fermented foods from vegetables, dairy and other biological sources are of great demand in India because of their health benefits. However, the diversity of Lactobacillus plantarum group (LPG) of vegetable origin has not been revealed yet, particularly with reference to their probiotic functionalities. In the present study, the different species of probiotic Lactobacillus plantarum group (LPG) i.e., L. plantarum subsp. plantarum MTCC 5422 (from fermented cereals), L. plantarum subsp. argentoratensis FG16 (from fermented bamboo shoot) and L. paraplantarum MTCC 9483 (from fermented gundruk) (as characterized by multiplex recA PCR assay) were considered to investigate their relative expression of MUB domains of mub gene (mucin binding protein) by Real time PCR. Initially, the allelic variation in the mub gene was assessed and found to encode three different variants (Type I, II and III). All the three types had 8, 9 and 10 MUB domains respectively (as analysed by Pfam database) and were found to be responsible for adhesion of bacteria to the host intestinal epithelial cells. These domains either get inserted or deleted during speciation or evolutionary events and lead to divergence. The reverse transcriptase qPCR analysis with mubLPF1+R1 primer pair supported variation in amplicon sizes with 300, 500 and 700 bp among different LPG strains. The relative expression of these MUB domains significantly unregulated in the presence of 1% mucin in overnight grown cultures. Simultaneously, the mub gene expressed efficiently by 7 fold in the culture L. paraplantarum MTCC 9483 with 10 MUB domains. An increase in the expression levels for L. plantarum subsp. plantarum MTCC 5422 and L. plantarum subsp. argentoratensis FG16 (MCC 2974) with 9 and 8 repetitive domains was around 4 and 2 fold, respectively. The detection and expression of an integrase (int) gene in the upstream region of mub gene reveals the excision and integration of these repetitive domains. Concurrently, an in vitro adhesion assay to mucin and exclusion of pathogens (such as Listeria monocytogenes and Micrococcus leuteus) was investigated and observed that the L. paraplantarum MTCC 9483 with more adhesion domains has more ability to adhere to mucin and inhibited the growth of pathogens. The production and expression of plantaricin-like bacteriocin (plnNC8 type) in MTCC 9483 suggests the pathogen inhibition. Hence, the expression of MUB domains can act as potential biomarkers in the screening of a novel probiotic LPG strain with adherence property. The present study provides a platform for an easy, rapid, less time consuming, low-cost methodology for the detection of potential probiotic bacteria. It was known that the traditional practices followed in the preparation of fermented bamboo shoots/gundruk/cereals of Indian foods contain different kinds of neutraceuticals for functional food and novel compounds with health promoting factors. In future, a detailed study of these food products can add more nutritive value, consumption and suitable for commercialization.

Keywords: adhesion gene, fermented foods, MUB domains, probiotics

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Authors: Othman E. Othman, Hassan R. Darwish, Amira M. Nowier

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Lactoferrin (LF) is a multifunctional protein involved in economically production traits like milk protein composition and skeletal structure in small ruminants including sheep and goat. So, LF gene - with its genetic polymorphisms associated with production traits - is considered a candidate genetic marker used in marker-assisted selection in goats. This study aimed to identify the different alleles and genotypes of this gene in three Egyptian goat breeds using PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) and DNA sequencing. Genomic DNA was extracted from 120 animals belonging to Barki, Zaraibi, and Damascus goat breeds. Using specific primers, PCR amplified 247-bp fragments from exon 2 of LF goat gene. The PCR products were subjected to Single-Strand Conformation Polymorphism (SSCP) technique. The results showed the presence of two genotypes GG and AG in the tested animals. The frequencies of both genotypes varied among the three tested breeds with the highest frequencies of GG genotype in all tested goat breeds. The sequence analysis of PCR products representing these two detected genotypes declared the presence of an SNP (single nucleotide polymorphisms) substitution (G/A) among G and A alleles of this gene. The association between different LF genotypes and milk composition as well as body measurement was estimated. The comparison showed that the animals possess AG genotypes are superior over those with GG genotypes for different parameters of milk protein compositions and skeletal structures. This finding declared that allele A of LF gene is considered the promising marker for the productive traits in goat. In conclusion, the Egyptian goat breeds will be needed to enhance their milk protein composition and growth trait parameters through the increasing of allele A frequency in their herds depending on the superior production traits of this allele in goats.

Keywords: lLactoferrin gene, PCR-SSCP, SNPs, Egyptian goat

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Authors: Kittitara Chunlakittiphan, Patompong Satapornpong

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Introduction: The variation of genetics affects how our body responds to pharmaceuticals elucidates the correlation between long-term use of medical cannabis and adverse drug reactions (ADRs). Medical cannabis is regarded as the treatment for chronic pain, cancer pain, acute pain, psychological disorders, multiple sclerosis and migraine management. However, previous studies have shown that delta-9-Tetrahydrocannabinol (THC), an ingredient found in cannabis, was the cause of ADRs in CB1 receptors found in humans. Previous research suggests that distributions of the cannabinoid type 1 (CB1) receptor gene and pharmacogenetic markers, which vary amongst different populations, might affect incidences of ADRs. Although there is an evident need to investigate the level of the CB1 receptor gene (rs806365), studies on the distribution of CB1-pharmacogenetics markers in Thai patients are limited. Objective: Therefore, the aim of this study is to investigate the distribution of the rs806365 polymorphism in Thai patients who have been treated with medical cannabis. Materials and Methods: We enrolled 31 Thai patients with THC-induced ADRs and 34 THC-tolerant controls to take part in this study. All patients with THC-induced ADRs were accessed through a review of medical records by physicians. EDTA blood of 3ml was collected to obtain the CNR1 gene (rs806365) and genotyping of this gene was conducted using the real-time PCR ViiA7 (ABI, Foster City, CA, USA) following the manufacturer’s instruction. Results: The sample consisted of 65 patients (40/61.54%) were females and (25/38.46%) were males, with an age range of 19-87 years, who have been treated with medical cannabis. In this study, the most common THC-induced ADRs were dry mouth and/or dry throat, tachycardia, nausea, and arrhythmia. Across the whole sample, we found that 52.31% of Thai patients carried a heterozygous variant (rs806365, CT allele). Moreover, the number of rs806365 (CC, homozygous variant) carriers totaled seventeen people (26.15%) amongst the subjects of Thai patients treated with medical cannabis. Furthermore, 17 out of 22 patients (77.27%) who experienced severe ADRs: Tachycardia and/or arrhythmia, carried an abnormal rs806365 gene (CT and CC alleles). Conclusions: The results propose that the rs806365 gene is widely distributed amongst the Thai population and there is a link between this gene and vulnerability to developing THC-induced ADRs after being treated with medical cannabis. Therefore, it is necessary to screen for the rs806365 gene before using medical cannabis to treat a patient.

Keywords: rs806365, THC-induced adverse drug reactions, CB1 receptor, Thai population

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Authors: Manaphol Kulpraneet, Anirut Limtrakul, Surangrat Srisurapanon, Piyatida Tangteerawatana

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Tuberculosis (TB) remains a global health care disease world-wide. Control of the global TB epidemic has been impaired by the lack of an effective vaccine, by the emergence of drug resistant forms of Mycobacterium tuberculosis and by lack of sensitive and rapid diagnostics. Cytokines play a major role in defense against M. tuberculosis infection. Polymorphisms in the genes encoding various cytokines have been associated with tuberculosis susceptibility. Polymorphisms of the regulatory cytokine gene, the interleukin (IL)-10 is associated with the risk of tuberculosis (TB) in different populations. However, IL-10 gene polymorphism and susceptibility to TB in Thai is still unknown. The purpose of this study was to evaluate whether the common IL-10 promoter gene polymorphisms are associated with TB in Thai population. Forty eight patients with newly diagnosed pulmonary tuberculosis were studied. DNA samples were extracted from leukocytes and used to investigate -1087A/G, -819C/T, -252C/A (rs1800896, rs1800871, rs1800872) in IL-10 gene using restriction fragment length polymorphism (PCR-RFLP) methods. In this study, the genotype and allele frequencies of IL-10-1087A/G, -819C/T, -252C/A polymorphism did not significantly different between TB patients and healthy controls ((genotype: p=0.38, p=0.92, p=1; allele: p=0.57, p=0.77, p=0.89, respectively). The lack of association between common IL-10 promoter polymorphisms and TB susceptibility in this study may provide clue for better understanding of IL-10-1087A/G, -819C/T, -252C/A polymorphism and TB susceptibility in Thai population, which might facilitate the rationale design of vaccines. However, further studies in large scales population are required for confirmation.

Keywords: IL-10, cytokines, single nucleotide polymorphism (SNP), tuberculosis

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Authors: Riffat Iqbal, Sidra Ihsan, Andleeb Batool, Maryam Mukhtar

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Glaucoma is a gathering of optic neuropathies described by dynamic degeneration of retinal ganglionic cells. It is clinically and innately heterogenous illness containing a couple of particular forms each with various causes and severities. Primary open-angle glaucoma (POAG) is the most generally perceived kind of glaucoma. This study investigated the genetic association of single nucleotide polymorphisms (SNPs; rs10483727 and rs33912345) at the SIX1/SIX6 locus with primary open-angle glaucoma (POAG) in the Pakistani population. The SIX6 gene plays an important role in ocular development and has been associated with morphology of the optic nerve. A total of 100 patients clinically diagnosed with glaucoma and 100 control individuals of age over 40 were enrolled in the study. Genomic DNA was extracted by organic extraction method. The SNP genotyping was done by (i) PCR based restriction fragment length polymorphism (RFLP) and sequencing method. Significant genetic associations were observed for rs10483727 (risk allele T) and rs33912345 (risk allele C) with POAG. Hence, it was concluded that Six6 gene is genetically associated with pathogenesis of Glaucoma in Pakistan.

Keywords: genotyping, Pakistani population, primary open-angle glaucoma, SIX6 gene

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Authors: Reena Murali, David Peter S.

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

Keywords: artificial neural network, double stranded RNA, RNA interference, short interfering RNA

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Authors: Hajar Hosseini Khorami

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Porphyrins are organic, aromatic compounds found in heme, cytochrome, cobalamin, chlorophyll , and many other natural products with essential roles in biological processes that their cationic forms have been used as groups of favorable non-viral vectors recently. Cationic porphyrins are self-chromogenic reagents with a high capacity for modifications, great interaction with DNA and protection of DNA from nuclease during delivery of it into a cell with low toxicity. In order to have high efficient gene transfection into the cell while causing low toxicity, genetically manipulations of the non-viral vector, cationic porphyrin, would be useful. In this study newly modified cationic porphyrin derivative, 5, 10-dihexyl-15, 20bis (N-methyl-4-pyridyl) porphyrin was applied. Cytotoxicity of synthesized cationic porphyrin on Chinese Hamster Ovarian (CHO) cells was evaluated by using MTT assay. This cationic derivative is dose-dependent, with low cytotoxicity at the ranges from 100 μM to 0.01μM. It was uptake by cells at high concentration. Using direct non-viral gene transfection method and different concentration of cationic porphyrin were tested on transfection of CHO cells by applying derived transfection reagent with X-tremeGENE HP DNA as a positive control. However, no transfection observed by porphyrin derivative and the parameters tested except for positive control. Results of this study suggested that applying different protocol, and also trying other concentration of cationic porphyrins and DNA for forming a strong complex would increase the possibility of efficient gene transfection by using cationic porphyrins.

Keywords: cationic porphyrins, gene delivery, non-viral vectors, transfection reagents

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Authors: Ngoc Tu Truong, Nuong T. Bui, Ben Rao, Ya L. Shen

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Quorum sensing is a phenomenon present in many gram-negative bacteria that allows bacterial communication and controlled expression of a large suite of genes through quorum sensing signals - N-acyl homoserine lactones (AHLs). In order to investigate the ability of the rhlR/rhlI quorum sensing system in Pseudomonas aeruginosa to express the ß-Galactosidase reporter gene, an engineered E. coli strain EpHL02, was genetically engineered. This engineered E. coli strain EpHL02 responded to the presence of the N-butanoyl homoserine lactone and N-hexanoyl homoserine lactone to express the ß-Galactosidase reporter gene at a concentration limit of 5x10⁻⁸ M. This was also found to be comparable to AHLs extraction from Serratia marcescens H31. Moreover, we examined this ability of this engineered E. coli strain for respond of AHLs from extractions of Pseudomonas aeruginosa ATCC9027. The results demonstrated that the rhlR/rhlI quorum sensing system can express the ß-Galactosidase reporter gene by using the N-butanoyl homoserine lactone, N-hexanoyl homoserine lactone and AHLs from extractions of Serratia marcescens H31 and Pseudomonas aeruginosa ATCC9027 in the engineered E. coli strain EpHL02.

Keywords: N-butanoyl homoserine lactone, C4-HSL, N-hexanoyl homoserine lactone, C6-HSL, Pseudomonas aeruginosa, quorum sensing, Serratia marcescens, ß-galactosidase reporter gene

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Authors: Saad Mahmud Khan, Sarah El Hajj Chehadeh, Mehera Abdulrahman, Wael Osman, Habiba Al Safar

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Obesity is a non-communicable disease that is widely prevalent with approximately 600 million people classified as obese worldwide. Its etiology is multifactorial and involves a complex interplay between genes and the environment. Over the past few decades, obesity rates among the Emirati population have been increasing. The aim of this study was to investigate the association of candidate gene single nucleotide polymorphisms (SNPs), namely the fat mass and obesity associated (FTO) gene SNP rs9939609 and Vitamin D Receptor (VDR) gene SNP rs1544410, with obesity in the UAE population. Methods: This is a case-control study in which 414 individuals were enrolled during their routine visit to endocrinology clinics in Abu Dhabi, United Arab Emirates between the period of June 2012 and December 2013. Several biochemical tests and clinical assessments along with a lifestyle questionnaire for each participant were completed at the clinic. Genomic DNA was extracted from saliva samples of 201 obese, 114 overweight and 99 normal subjects. Genotyping for the variants was performed using TaqMan assay. Results: The mean Body Mass Index (BMI) ± SD for the obese, overweight, and normal subjects was 35.76 ± 4.54, 27.53 ± 1.45 and 22.69 ± 1.84 kg/m2, respectively. Increasing BMI values were associated with an increase in values for systolic blood pressure, diastolic blood pressure, HbA1c, and triglycerides. The SNP rs9939609 in the FTO gene was found to be significantly associated with the BMI (p=0.028), with the minor allele A having a clear additive effect on BMI values. No significant association was detected between BMI and rs1544410 of the VDR gene. Conclusions: Our study findings indicate that the minor allele A of the rs9939609 has a significant association with increasing BMI values. In addition, our findings support the fact that increasing BMI is associated with increasing risks of other comorbidities such as higher blood pressure, poorer glycemic control and higher triglycerides.

Keywords: body mass index, FTO gene, obesity, rs9939609, United Arab Emirates

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Authors: Libo Jiang, Huan Li, Rongling Wu

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Ordinary differentiation equations (ODE) have proven to be powerful for reconstructing precise and informative gene regulatory networks (GRNs) from dynamic gene expression data. However, joint modeling and analysis of all genes, essential for the systematical characterization of genetic interactions, are challenging due to high dimensionality and a complex pattern of genetic regulation including activation, repression, and antitermination. Here, we address these challenges by unifying variable selection and game theory through ODE. Each gene within a GRN is co-expressed with its partner genes in a way like a game of multiple players, each of which tends to choose an optimal strategy to maximize its “fitness” across the whole network. Based on this unifying theory, we designed and conducted a real experiment to infer salt tolerance-related GRNs for Euphrates poplar, a hero tree that can grow in the saline desert. The pattern and magnitude of interactions between several hub genes within these GRNs were found to determine the capacity of Euphrates poplar to resist to saline stress.

Keywords: gene regulatory network, ordinary differential equation, game theory, LASSO, saline resistance

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Authors: Vishal V. Choudhari, Gaurao A. Dongre, S. P. Diwan

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A number of reference current generation have been developed for analysis of shunt active power filter to mitigate the load compensation. Depending upon the type of load the technique has to be chosen. In this paper, six reference current generation techniques viz. instantaneous reactive power theory(IRP), Synchronous reference frame theory(SRF), Perfect harmonic cancellation(PHC), Unity power factor method(UPF), Self-tuning filter method(STF), Predictive filtering method(PFM) are compared for different operating conditions. The harmonics are introduced because of non-linear loads in the system. These harmonics are eliminated using above techniques. The results and performance of system simulated on MATLAB/Simulink platform. The system is experimentally implemented using DS1104 card of dSPACE system.

Keywords: SAPF, power quality, THD, IRP, SRF, dSPACE module DS1104

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Authors: Piotr J. Balwierz, Mikhail Pachkov, Phil Arnold, Andreas J. Gruber, Mihaela Zavolan, Erik van Nimwegen

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Understanding the key players and interactions in the regulatory networks that control gene expression and chromatin state across different cell types and tissues in metazoans remains one of the central challenges in systems biology. Our laboratory has pioneered a number of methods for automatically inferring core gene regulatory networks directly from high-throughput data by modeling gene expression (RNA-seq) and chromatin state (ChIP-seq) measurements in terms of genome-wide computational predictions of regulatory sites for hundreds of transcription factors and micro-RNAs. These methods have now been completely automated in an integrated webserver called ISMARA that allows researchers to analyze their own data by simply uploading RNA-seq or ChIP-seq data sets and provides results in an integrated web interface as well as in downloadable flat form. For any data set, ISMARA infers the key regulators in the system, their activities across the input samples, the genes and pathways they target, and the core interactions between the regulators. We believe that by empowering experimental researchers to apply cutting-edge computational systems biology tools to their data in a completely automated manner, ISMARA can play an important role in developing our understanding of regulatory networks across metazoans.

Keywords: gene expression analysis, high-throughput sequencing analysis, transcription factor activity, transcription regulation

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Authors: Ying Zhao, Xingyan Bin

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Semi-supervised clustering algorithms have been shown effective to improve clustering process with even limited supervision. However, semi-supervised hierarchical clustering remains challenging due to the complexities of expressing constraints for agglomerative clustering algorithms. This paper proposes novel semi-supervised agglomerative clustering algorithms to build a hierarchy based on a known reference tree. We prove that by enforcing distance constraints defined by a reference tree during the process of hierarchical clustering, the resultant tree is guaranteed to be consistent with the reference tree. We also propose a framework that allows the hierarchical tree generation be aware of levels of levels of the agglomerative tree under creation, so that metric weights can be learned and adopted at each level in a recursive fashion. The experimental evaluation shows that the additional cost of our contraint-based semi-supervised hierarchical clustering algorithm (HAC) is negligible, and our combined semi-supervised HAC algorithm outperforms the state-of-the-art algorithms on real-world datasets. The experiments also show that our proposed methods can improve clustering performance even with a small number of unevenly distributed labeled data.

Keywords: semi-supervised clustering, hierarchical agglomerative clustering, reference trees, distance constraints

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Authors: Sabin Aslam, Sultan Habibullah Khan, James G. Thomson, Abhaya M. Dandekar

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Losses due to viral diseases are posing a serious threat to crop production. A quick breakdown of resistance to viruses like Cotton Leaf Curl Virus (CLCuV) demands the application of a proficient technology to engineer durable resistance. Gene stacking has recently emerged as a potential approach for integrating multiple genes in crop plants. In the present study, recombinase technology has been used for site-specific gene stacking. A target vector (pG-Rec) was designed for engineering a predetermined specific site in the plant genome whereby genes can be stacked repeatedly. Using Agrobacterium-mediated transformation, the pG-Rec was transformed into Coker-312 along with Nicotiana tabacum L. cv. Xanthi and Nicotiana benthamiana. The transgene analysis of target lines was conducted through junction PCR. The transgene positive target lines were used for further transformations to site-specifically stack two genes of interest using Bxb1 and PhiC31 recombinases. In the first instance, Cas9 driven by multiplex gRNAs (for Rep gene of CLCuV) was site-specifically integrated into the target lines and determined by the junction PCR and real-time PCR. The resulting plants were subsequently used to stack the second gene of interest (AVP3 gene from Arabidopsis for enhancing cotton plant growth). The addition of the genes is simultaneously achieved with the removal of marker genes for recycling with the next round of gene stacking. Consequently, transgenic marker-free plants were produced with two genes stacked at the specific site. These transgenic plants can be potential germplasm to introduce resistance against various strains of cotton leaf curl virus (CLCuV) and abiotic stresses. The results of the research demonstrate gene stacking in crop plants, a technology that can be used to introduce multiple genes sequentially at predefined genomic sites. The current climate change scenario highlights the use of such technologies so that gigantic environmental issues can be tackled by several traits in a single step. After evaluating virus resistance in the resulting plants, the lines can be a primer to initiate stacking of further genes in Cotton for other traits as well as molecular breeding with elite cotton lines.

Keywords: cotton, CRISPR/Cas9, gene stacking, genome editing, recombinases

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

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Gastric cancer (GC) has high morbidity and fatality rate in various countries and is still one of the most frequent and deadly diseases. Novel mitogenic and motogenic Gastrokine1 (GKN1) and Gastrokine 2 (GKN2) genes that are highly expressed in the normal stomach epithelium and plays an important role in maintaining the integrity and homeostasis of stomach mucosal epithelial cells. Significant loss of copy number and mRNA transcript of GKN1 and GKN2 gene expression were frequently observed in all types of gastric cancer. In this study, 47 paired samples that were grouped according to the types of gastric cancer and the clinical characteristics of the patients, including gender and average of age were investigated with gene expression analysis and mutation screening by monetering RT-PCR, SSCP and nucleotide sequencing techniques. Both GKN1 and GKN2 genes were observed significantly reduced found by (Wilcoxon signed rank test; p<0.05). As a result of gene screening, no mutation (no different genotype) was detected. It is considered that gene mutations are not the cause of inactivation of gastrokines. In conclusion, the mRNA expression level of GKN1 and GKN2 genes statistically was decreased regardless the gender, age or cancer type of patients. Reduced of gastrokine genes seems to occur at the initial steps of cancer development. In order to understand the investigation between gastric cancer and diagnostic biomarker; further analysis is necessary.

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

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

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

Keywords: body size, Leptin gene, PCR-RFLP, Sanjabi sheep

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