Search results for: deletion polymorphism

Authors: Ahmed M. Debela, Mayreli Ortiz , Ciara K. O´Sullivan

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The completion of the human genome Project (HGP) has paved the way for mapping the diversity in the overall genome sequence which helps to understand the genetic causes of inherited diseases and susceptibility to drugs or environmental toxins. Arrayed primer extension (APEX) is a microarray based minisequencing strategy for screening disease causing mutations. It is derived from Sanger DNA sequencing and uses fluorescently dideoxynucleotides (ddNTPs) for termination of a growing DNA strand from a primer with its 3´- end designed immediately upstream of a site where single nucleotide polymorphism (SNP) occurs. The use of DNA polymerase offers a very high accuracy and specificity to APEX which in turn happens to be a method of choice for multiplex SNP detection. Coupling the high specificity of this method with the high sensitivity, low cost and compatibility for miniaturization of electrochemical techniques would offer an excellent platform for detection of mutation as well as sequencing of DNA templates. We are developing an electrochemical APEX for the analysis of SNPs found in the MYH7 gene for group of cardiomyopathy patients. ddNTPs were labeled with four different redox active compounds with four distinct potentials. Thiolated oligonucleotide probes were immobilised on gold and glassy carbon substrates which are followed by hybridisation with complementary target DNA just adjacent to the base to be extended by polymerase. Electrochemical interrogation was performed after the incorporation of the redox labelled dedioxynucleotide. The work involved the synthesis and characterisation of the redox labelled ddNTPs, optimisation and characterisation of surface functionalisation strategies and the nucleotide incorporation assays.

Keywords: array based primer extension, labelled ddNTPs, electrochemical, mutations

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Authors: Vhuhwavho Gadisi, Rebecca Alowo, German Nkhonjera

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This article sets out to detail an investigation of groundwater management in the Juksei Catchment of South Africa through spatial mapping of key hydrological relationships, interactions, and parameters in catchments. The Department of Water Affairs (DWA) noted gaps in the implementation of the South African National Water Act 1998: article 16, including the lack of appropriate models for dealing with water quantity parameters. For this reason, this research conducted a drastic GIS-based groundwater assessment to improve groundwater monitoring system in the Juksei River basin catchment of South Africa. The methodology employed was a mixed-methods approach/design that involved the use of DRASTIC analysis, questionnaire, literature review and observations to gather information on how to help people who use the Juskei River. GIS (geographical information system) mapping was carried out using a three-parameter DRASTIC (Depth to water, Recharge, Aquifer media, Soil media, Topography, Impact of the vadose zone, Hydraulic conductivity) vulnerability methodology. In addition, the developed vulnerability map was subjected to sensitivity analysis as a validation method. This approach included single-parameter sensitivity, sensitivity to map deletion, and correlation analysis of DRASTIC parameters. The findings were that approximately 5.7% (45km2) of the area in the northern part of the Juksei watershed is highly vulnerable. Approximately 53.6% (428.8 km^2) of the basin is also at high risk of groundwater contamination. This area is mainly located in the central, north-eastern, and western areas of the sub-basin. The medium and low vulnerability classes cover approximately 18.1% (144.8 km2) and 21.7% (168 km2) of the Jukskei River, respectively. The shallow groundwater of the Jukskei River belongs to a very vulnerable area. Sensitivity analysis indicated that water depth, water recharge, aquifer environment, soil, and topography were the main factors contributing to the vulnerability assessment. The conclusion is that the final vulnerability map indicates that the Juksei catchment is highly susceptible to pollution, and therefore, protective measures are needed for sustainable management of groundwater resources in the study area.

Keywords: contamination, DRASTIC, groundwater, vulnerability, model

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Authors: Sara A. Al-Jaberi, Salma Ben-Salem, Meriam Messedi, Fatma Ayadi, Lihadh Al-Gazali, Bassam R. Ali

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Background: The chemokine receptor components play crucial roles in the immune system and some of them serve as co-receptors for the HIV virus. Several studies have documented those variants in chemokine receptors are correlated with susceptibility and resistance to infection with HIV virus. For example, mutations in the chemokine receptor 5 gene (CCR5) resulting in loss-of-function (such as the homozygous CCR5Δ32) confer high degree of resistance to HIV infection. Heterozygotes for these variants exhibit slow progression to AIDS. The prevalence of CCR5 polymorphisms varies among ethnic and geographical groups. For example, the CCR5 Δ32 variant is present in 10–15% of north Europeans but is rarely encountered among Africans. This study aims to identify the prevalence of some CCR5 variants in two geographically distant Arab populations (namely Emiratis and Tunisians). Methodology: The prevalence of CCR5 gene variants including CCR5Δ32, FS299, C101X, A29S and C178R has been determined using PCR and direct DNA sequencing. A total of 403 unrelated healthy individuals (253 Emiratis and 150 Tunisians) were genotyped for the CCR5Δ32 variant using PCR amplification and gel electrophoresis. In addition, 200 Emiratis have been screened for other SNPs using Sanger DNA sequencing. Results: Among Emiratis, the allele frequency of the CCR5Δ32 variant has been found to be 0.002. In addition, two variants L55Q and A159 were found at a frequency of 0.002.Moreover, the prevalence of the CCR5Δ32 variant in Tunisians was estimated to be 0.013 which is relatively higher than its frequency in Emiratis but lower than Europeans. Conclusion: We conclude that the allele frequency of the most critical CCR5 polymorphism (Δ32) is extremely low among Emiratis compared to other Arabs and North Europeans. In addition, very low allele frequencies of other CCR5 polymorphisms have been detected among Emiratis.

Keywords: chemokine receptors, CCR5Δ32, CCR5 polymorphisms, Emiratis, Arab populations

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Authors: Mohammad Mokhtari, Gires Usup, Zaidi Che Cob

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Bacteria communities as mediators of the biogeochemical process are the main component of the mangrove ecosystems. Crab burrows by increasing oxic-anoxic interfaces and facilitating the flux rate between sediment and tidal water affect biogeochemical properties of sediments. The effect of fiddler crab burrows on the density and diversity of bacteria were investigated to elucidate the effect of burrow on bacterial distribution. Samples collected from the burrow walls of three species of fiddler crabs including Uca paradussumieri, Uca rosea, and Uca forcipata. Sediment properties including grain size, temperature, Redox potential, pH, chlorophyll, water and organic content were measured from the burrow walls to assess the correlation between environmental variables and bacterial communities. Bacteria were enumerated with epifluorescence microscopy after staining with SYBR green. Bacterial DNA extracted from sediment samples and the community profiles of bacteria were determined with Terminal Restriction Fragment Length Polymorphism (T-RFLP). High endemism was observed among bacterial communities. Among the 152 observed OTU’s, 22 were found only in crab burrows. The highest bacterial density and diversity were recorded in burrow wall. The results of ANOSIM indicated a significant difference between the bacterial communities from the three species of fiddler crab burrows. Only 3% of explained bacteria variability in the constrained ordination model of CCA was contributed to depth, while much of the bacteria’s variability was attributed to coarse sand, pH, and chlorophyll content. Our findings suggest that crab burrows by affecting sediment properties such as redox potential, pH, water, and chlorophyll content induce significant effects on the bacterial communities.

Keywords: bioturbation, canonical corresponding analysis, fiddler crab, microbial ecology

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Authors: Annet Namusoke, Annet Namayanja, Peter Wasswa, Shakirah Nampijja

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Common bean cultivar G2333 which offers broad resistance for anthracnose has been widely used as a source of resistance in breeding for anthracnose resistance. The cultivar is pyramided with three genes namely CO-4, CO-5 and CO-7 and of these three genes, the CO-4 gene has been found to offer the broadest resistance. The main aim of this work was to identify and validate easily assayable PCR based co-dominant molecular markers for selection of the CO-4 gene in segregating populations derived from crosses of G2333 with RWR 1946 and RWR 2075, two commercial Andean cultivars highly susceptible to anthracnose. Marker sequences for the study were obtained by blasting the sequence of the COK-4 gene in the Phaseolus gene database. Primer sequence pairs that were not provided from the Phaseolus gene database were designed by the use of Primer3 software. PCR conditions were optimized and the PCR products were run on 6% HPAGE gel. Results of the polymorphism test indicated that out of 18 identified markers, only two markers namely BM588 and BM211 behaved co-dominantly. Phenotypic evaluation for reaction to anthracnose disease was done by inoculating 21days old seedlings of three parents, F1 and F2 populations with race 7 of Colletotrichum lindemuthianum in the humid chamber. DNA testing of the BM588 marker onto the F2 segregating population of the crosses RWR 1946 x G 2333 and RWR 2075 x G2333 further revealed that the marker BM588 co-segregated with disease resistance with co-dominance of two alleles of 200bp and 400bp, fitting the expected segregation ratio of 1:2:1. The BM588 marker was significantly associated with disease resistance and gave promising results for marker assisted selection of the CO-4 gene in the breeding lines. Activities to validate the BM211 marker are also underway.

Keywords: codominant, Colletotrichum lindemuthianum, MAS, Phaseolus vulgaris

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Authors: Reshu Saxena, R. K. Tripathi

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HIV-1 transmission and spread involves significant host-virus interaction. Potential targets for prevention of HIV-1 lies at the site of mucosal barriers. Thus a better understanding of how HIV-1 infects target cells at such sites and lead their invasion is required, with prime focus on the host determinants regulating HIV-1 spread. HIV-1 Nef is important for viral infectivity and pathogenicity. It promotes HIV-1 replication, facilitating immune evasion by interacting with various host factors and altering cellular pathways via multiple protein-protein interactions. In this study nef was sequenced from HIV-1 patients, and showed specific mutations revealing sequence variability in nef. To explore the difference in Nef functionality based on sequence variability we have studied the effects of HIV-1 Nef in human SupT1 T cell line and (THP-1) monocyte-macrophage cell lines through proteomics approach. 2D-Gel Electrophoresis in control and Nef-transfected SupT1 cells demonstrated several differentially expressed proteins with significant modulation of alpha-enolase. Through further studies, effects of Nef on alpha-enolase regulation were found to be cell lineage-specific, being stimulatory in macrophages/monocytes, inhibitory in T cells and without effect in HEK-293 cells. Cell migration and invasion studies were employed to determine biological function affected by Nef mediated regulation of alpha-enolase. Cell invasion was enhanced in THP-1 cells but was inhibited in SupT1 cells by wildtype nef. In addition, the modulation of enolase and cell invasion remained unaffected by a unique nef variant. These results indicated that regulation of alpha-enolase expression and invasive property of host cells by Nef is sequence specific, suggesting involvement of a particular motif of Nef. To precisely determine this site, we designed a heptapeptide including the suggested alpha-enolase regulating sequence of nef and a nef mutant with deletion of this site. Macrophages/monocytes being the major cells affected by HIV-1 at mucosal barriers, were particularly investigated by the nef mutant and peptide. Both the nef mutant and heptapeptide led to inhibition of enhanced enolase expression and increased invasiveness in THP-1 cells. Together, these findings suggest a possible mechanism of host invasion by HIV-1 through Nef mediated regulation of alpha-enolase and identifies a potential therapeutic target for HIV-1 entry at mucosal barriers.

Keywords: HIV-1 Nef, nef variants, host-virus interaction, tissue invasion

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Authors: Martynova Alina, Lyubov Buzoleva

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Streptococcus pneumoniae is the causative agent of pneumonias and meningitids throught all the world. Having high genetic diversity, this microorganism can cause different clinical forms of pneumococcal infections and microbiologically it is really difficult diagnosed by routine methods. Also, epidemiological surveillance requires more developed methods of molecular typing because the recent method of serotyping doesn't allow to distinguish invasive and non-invasive isolates properly. Non-coding genome of bacteria seems to be the interesting source for seeking of highly distinguishable markers to discriminate the subspecies of such a variable bacteria as Streptococcus pneumoniae. Technically, we proposed scheme of discrimination of S.pneumoniae strains with amplification of non-coding region (SP_1932) with the following restriction with 2 types of enzymes of Alu1 and Mn1. Aim: This research aimed to compare different methods of typing and their application for molecular epidemiology purposes. Methods: we analyzed population of 100 strains of S.pneumoniae isolated from different patients by different molecular epidemiology methods such as pulse-field gel electophoresis (PFGE), restriction polymorphism analysis (RFLP) and multilolocus sequence typing (MLST), and all of them were compared with classic typing method as serotyping. The discriminative power was estimated with Simpson Index (SI). Results: We revealed that the most discriminative typing method is RFLP (SI=0,97, there were distinguished 42 genotypes).PFGE was slightly less discriminative (SI=0,95, we identified 35 genotypes). MLST is still the best reference method (SI=1.0). Classic method of serotyping showed quite weak discriminative power (SI=0,93, 24 genotypes). In addition, sensivity of RFLP was 100%, specificity was 97,09%. Conclusion: the most appropriate method for routine epidemiology surveillance is RFLP with non-coding region of Streptococcsu pneumoniae, then PFGE, though in some cases these results should be obligatory confirmed by MLST.

Keywords: molecular epidemiology typing, non-coding genome, Streptococcus pneumoniae, MLST

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Authors: C. A. Ologunde

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Escherichia coli have been a major microorganisms associated with, and isolated from feacal samples either in adult or children all over the world. Strains of these organisms are resistant to cephalosporins and fluoroquinolone (FQ) antimicrobial agents among hospitalized patients and FQs are the most frequently prescribed antimicrobial class in hospitals, and the level of resistant of E. coli to these antimicrobial agents is a risk factor that should be assessed. Hence, this study was conducted to determine the prevalence and risk factors for colonization with fluoroquinolone (FQ)-resistant E. coli in hospitalized patients in Ado-Ekiti. Rectal swabs were obtained from patients in hospitals in the study area and FQ-resistant E. coli were isolated and identified by means of Nalidixic acid multi-disk and a 1-step screening procedure. Species identification and FQ resistance were confirmed by automated testing (Vitek, bioMerieux, USA). Individual colonies were subjected to pulse-field gel electrophoresis (PAGE) to determine macro-restriction polymorphism after digestion of chromosomal DNA. FQ-resistant E. coli was detected in the stool sample of 37(62%) hospitalized patient. With multivariable analyses, the use of FQ before hospitalization was the only independent risk factor for FQ-resistant E. coli carriage and was consistent for FQ exposures for the 3-12 months of study. Pulsed-field gel electrophoresis of FQ-resistant E. coli identified conal spread of 1(one) strain among 18 patients. Loss (9 patients) or acquisition (10 residents) of FQ-resistant E. coli was documented and was associated with de novo colonization with genetically distinct strains. It was concluded that FQ-resistant E. coli carriage was associated with clonal spread. The differential effects of individual fluoroquinolone on antimicrobial drug resistance are an important area for future study, as hospitals manipulate their formularies with regard to use of individual fluoroquinolone, often for economic reasons.

Keywords: E. coli, fluoroquinolone, risk factors, feacal carriage, hospitalized patients, Ado-Ekiti

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Authors: Elham Sharif, Nasser Rizk, Sirin Abu Aqel, Ofelia Masoud

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Background: Previous studies have investigated the association of rs1544410, rs7975232 and rs731236 polymorphisms in vitamin D receptor gene and its impact on diseases such as cancer, diabetes and hypertension in different ethnic backgrounds. Aim: The aim of this study is to investigate the association between VDR polymorphisms using three SNP’s (rs1544410, rs7975232 and rs731236) and the severity of the significant lesion in coronary arteries among angiographically diagnosed CAD. Methods: A prospective-retrospective study was conducted on 192 CAD patients enrolled from the cardiology department-Heart Hospital HMC, grouped in 96 subjects with significant stenosis and 96 with non-significant stenosis with a mean age between 30 and 75 years old. Genotyping was performed for the following SNPs rs1544410, rs7975232 and rs731236 using TaqMan assay by the Real Time PCR, ABI 7500 in Health Sciences Labs at Qatar University Biomedical Research Center. Results: The results showed that both groups have matched age and gender distribution but patients with the significant stenosis have significantly higher; BMI (p=0.047); smoking status (p=0.039); FBS (p= 0.031); CK-MB (p=0.025) and Troponin (p=0.002) than the patients with non–significant lesion. Among the traditional risk factors, smoking increases the odds of the severe stenotic lesion in CAD patients by 1.984, with 95% CI between 1.024 – 7.063, with p= 0.042.HWE showed deviations of the rs1544410 and rs731236 among the study subjects. The most frequent genotype in distribution of rs7975232 is the AA among the significant stenosis patients, while the heterozygous AC was the frequent genotype in distribution among the non-significant stenosis group. The carriers of CC genotype in rs7975232 increased the risk of having significant coronary arteries stenotic lesion by 1.83 with 95% CI (1.020 – 3.280), p=0.043. No association was found between the rs7975232 with vitamin D and VDBP. Conclusion: There is a significant association between rs7975232 and the severity of CAD lesion. The carrier of CC genotype in rs7975232 increased the risk of having significant coronary arteries atherosclerotic lesion especially in patients with smoking history independent of vitamin D.

Keywords: vitamin D, vitamin D receptor, polymorphism, coronary harat disease

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Authors: Salman Naveed, Nitant Gandhi, Grant Billings, Zachary Jones, B. Todd Campbell, Michael Jones, Sachin Rustgi

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Cotton (Gossypium spp.) is the primary source of natural fiber in the U.S. and a major crop in the Southeastern U.S. Despite constant efforts to increase the cotton fiber yield, the yield gain has stagnated. Therefore, we undertook a novel approach to improve the cotton fiber yield by altering its growth habit from perennial to annual. In this effort, we identified genotypes with high-expression alleles of five floral induction and meristem identity genes (FT, SOC1, FUL, LFY, and AP1) from an upland cotton mini-core collection and crossed them in various combinations to develop cotton lines with annual growth habit, optimal flowering time and enhanced productivity. To facilitate the characterization of genotypes with the desired combinations of stacked alleles, we identified markers associated with the gene expression traits via genome-wide association analysis using a 63K SNP Array (Hulse-Kemp et al. 2015 G3 5:1187). Over 14,500 SNPs showed polymorphism and were used for association analysis. A total of 396 markers showed association with expression traits. Out of these 396 markers, 159 mapped to genes, 50 to untranslated regions, and 187 to random genomic regions. Biased genomic distribution of associated markers was observed where more trait-associated markers mapped to the cotton D sub-genome. Many quantitative trait loci coincided at specific genomic regions. This observation has implications as these traits could be bred together. The analysis also allowed the identification of candidate regulators of the expression patterns of these floral induction and meristem identity genes whose functions will be validated via virus-induced gene silencing.

Keywords: cotton, GWAS, QTL, expression traits

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Authors: Anida Causevic-Ramosevac, Sabina Semiz

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The aim of this study was to investigate the association of four single nucleotide polymorphisms (SNPs) - rs673548, rs693 in ApoB gene, rs1800775 in CETP gene and rs4846914 in GALNT2 gene with parameters of type 2 diabetes (T2D) and diabetic dyslipidemia in the population of Bosnia and Herzegovina (BH). Materials and methods: Our study involved 352 patients with T2D and 156 healthy subjects. Biochemical and anthropometric parameters were measured in all participants. DNA was extracted from the peripheral blood for the purpose of genetic testing. Polymorphisms in ApoB (rs673548, rs693), CETP (rs1800775) and GALNT2 (rs4846914) genes were analyzed by using Sequenom IPLEX platform. Results: Our results demonstrated significant associations for rs180075 polymorphism in CETP gene with levels of fasting insulin (p = 0.020; p = 0.027; p = 0.044), triglycerides (p = 0.046) and ALT (p = 0.031) activity in control group. In group of diabetic patients, results showed a significant association of rs673548 in ApoB gene with levels of fasting insulin (p = 0.008), HOMA-IR (p = 0.013), VLDL-C (p = 0.037) and CRP (p = 0.029) and rs693 in ApoB gene with BMI (p = 0.025), systolic blood pressure (p = 0.027), fasting insulin (p = 0.037) and HOMA-IR (p = 0.023) levels. Significant associations were also observed for rs1800775 in CETP gene with triglyceride (p = 0.023) levels and rs4846914 in GALNT2 gene with HbA1C (p = 0.013) and triglyceride (p = 0.043) levels. Conclusion: In conclusion, this is the first study that examined the impact of variations of candidate genes on a wide range of metabolic parameters in BH population. Our results suggest an association of variations of ApoB, CETP and GALNT2 genes with specific markers of T2D and dyslipidemia. Further studies would be needed in order to confirm these genetic effects in other ethnic groups as well.

Keywords: ApoB, CETP, dyslipidemia, GALNT2, type 2 diabetes

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Authors: Naso Isaiah Thanavisuth

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Introduction: Medical cannabis can be used for treatment, including anorexia, pain, inflammation, multiple sclerosis, Parkinson's disease, epilepsy, cancer, and metabolic syndrome-related disorders. However, medical cannabis leads to adverse effects (AEs), which is delta-9-tetrahydrocannabinol (THC). In previous studies, the major of THC metabolism enzymes are CYP2C9. Especially, the variation of CYP2C9 gene consist of CYP2C9*2 on exon 3 (C430T) (Arg144Cys) and CYP2C9*3 on exon 7 (A1075C) (Ile359Leu) to decrease enzyme activity. Notwithstanding, there is no data describing whether the variant of CYP2C9 genes are a pharmacogenetics marker for prediction of THC-induced AEs in Thai patients. Objective: We want to investigate the association between CYP2C9 gene and THC-induced AEs in Thai patients. Method: We enrolled 39 Thai patients with medical cannabis treatment consisting of men and women who were classified by clinical data. The quality of DNA extraction was assessed by using NanoDrop ND-1000. The CYP2C9*2 and *3 genotyping were conducted using the TaqMan real time PCR assay (ABI, Foster City, CA, USA). Results: All Thai patients who received the medical cannabis consist of twenty four (61.54%) patients who were female and fifteen (38.46%) were male, with age range 27- 87 years. Moreover, the most AEs in Thai patients who were treated with medical cannabis between cases and controls were tachycardia, arrhythmia, dry mouth, and nausea. Particularly, thirteen (72.22%) medical cannabis-induced AEs were female and age range 33 – 69 years. In this study, none of the medical cannabis groups carried CYP2C9*2 variants in Thai patients. The CYP2C9*3 variants (*1/*3, intermediate metabolizer, IM) and (*3/*3, poor metabolizer, PM) were found, three of thirty nine (7.69%) and one of thirty nine (2.56%) , respectively. Conclusion: This is the first study to confirm the genetic polymorphism of CYP2C9 and medical cannabis-induced AEs in the Thai population. Although, our results indicates that there is no found the CYP2C9*2. However, the variation of CYP2C9 allele might serve as a pharmacogenetics marker for screening before initiating the therapy with medical cannabis for prevention of medical cannabis-induced AEs.

Keywords: CYP2C9, medical cannabis, adverse effects, THC, P450

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Authors: Minwuyelet Maru, Solomon Habtemariam, Endalamaw Gadissa, Abraham Aseffa

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Background: Tuberculosis is a major public health problem in Ethiopia. The burden of TB is aggravated by emergence and expansion of drug resistant tuberculosis and different lineages of Mycobacterium tuberculosis (M. tuberculosis) have been reported in many parts of the country. Describing strains of Mycobacterial isolates and drug susceptibility pattern is necessary. Method: Sputum samples were collected from smear positive pulmonary TB patients age >= 7 years between October 1, 2012 to September 30, 2013 and Mycobacterial strains isolated on Loweensten Jensen (LJ) media. Each strain was characterized by deletion typing and Spoligotyping. Drug sensitivity testing was determined with the indirect proportion method using Middle brook 7H10 media and association to determine possible risk factors to drug resistance was done. Result: A total of 144 smear positive pulmonary tuberculosis patients were enrolled. The age of participants ranged from 7 to 78 with mean age of 29.22 (±10.77) years. In this study 82.2% (n=97) of the isolates were sensitive to the four first line anti-tuberculosis drugs and resistance to any of the four drugs tested was 17.8% (n=21). A high frequency of any resistance was observed in isoniazid, 13.6%, (n=16) followed by streptomycin, 11.8% (n=14). No significant association of isoniazid resistance with HIV, sex and history of previous TB treatment was observed but there was significant association with age, high between 31-35 years of age (p=0.01). Majority, 89.9% (n=128) of participants were new cases and only 11.1% (n=16) had history of previous TB treatment. No MDR-TB from new cases and 2 MDRTB (13.3%) was isolated from re-treatment cases which was significantly associated with previous TB treatment (p<0.01). Thirty two different types of spoligotype patterns were identified and 74.1% were grouped in to 13 clusters. The dominant strains were SIT 25, 18.1% (n=21), SIT 53, 17.2% (n=20) and SIT 149, 8.6% (n=10). Lineage 4 is the predominant lineage followed by lineage 3 and lineage 7 comprising 65.5% (n=76), 28.4% (n=33) and 6% (n=7) respectively. Majority of strains from lineage 3 and 4 were SIT 25 (63.6%) and SIT 53 (26.3%) whereas SIT 343 was the dominant strain from lineage 7 (71.4%). Conclusion: Wide spread of lineage 3 and lineage 4 of the modern lineage and high number of strain cluster indicates high ongoing transmission. The high proportion resistance to any of the first line anti-tuberculosis drugs may be a potential source in the emergence of MDR-TB. Wide spread of SIT 25 and SIT 53 having a tendency of ease transmission and presence of higher resistance of isoniazid in working and mobile age group, 31-35 years of age may increase risk of drug resistant strains transmission.

Keywords: tuberculosis, drug susceptibility, strain diversity, lineage, Ethiopia, spoligotyping

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Authors: J. Vinay , Kusumbati Besra, Niharika Pattnaik, Shivaram Prasad Singh, Manjusha Dixit

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Gallbladder cancer (GBC) is rare but highly malignant cancer; its prevalence is more in certain geographical regions and ethnic groups, which include the Northern and Eastern states of India. Previous studies in India have reported genetic predisposition as one of the risk factors in GBC pathogenesis. Although the matrix metalloproteinase-14 (MMP14) is a well-known modulator of the tumor microenvironment and tumorigenesis and TCGA data also suggests its upregulation yet, its role in the genetic predisposition for GBC is completely unknown. We elucidated the role of MMP14 promoter variants as genetic risk factors and their implications in expression modulation. We screened MMP14 promoter variants association with GBC using Sanger’s sequencing in approximately 300 GBC and 300 control subjects and 26 GBC tissue samples of Indian ethnicity. The immunohistochemistry was used to check the MMP14 protein expression in GBC tissue samples. The role of promoter variants on expression levels was elucidated using a luciferase reporter assay. The variants rs1004030 (p-value = 0.0001) and rs1003349 (p-value = 0.0008) were significantly associated with gallbladder cancer. The luciferase assay in two different cell lines, HEK-293 (p = 0.0006) and TGBC1TKB (p = 0.0036) showed a significant increase in relative luciferase activity in the presence of risk alleles for both the single nucleotide polymorphisms (SNPs). Similarly, genotype-phenotype correlation in patients samples confirmed that the presence of risk alleles at rs1004030 and rs1003349 increased MMP14 expression. Overall, this study unravels the genetic association of MMP14 promoter variants with gallbladder cancer, which may contribute to pathogenesis by increasing its expression.

Keywords: gallbladder cancer, matrix metalloproteinase-14, single nucleotide polymorphism, case control study, genetic association study

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Authors: Mads Schultz, Galice Hoarau, Marvin Choquet

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Members of the genus Calanus are key species in the North Atlantic and Arctic marine ecosystems due to their vast abundance and their ability to accumulate high amounts of lipid. As a link between primary producers and higher trophic levels, the temporal presence of each Calanus species is important in a time of changing communities and northward distribution shifts. This study focused on the temporal niches of the sympatric species Calanus helgolandicus, Calanus finmarchicus, Calanus glacialis, and Calanus hyperboreus in Skjerstad fjord, a Norwegian fjord (67˚14’N, 14 ˚44’E). Three depth intervals were sampled monthly over a year, targeting copepodite stages of the genus Calanus. Species determination was carried out genetically using insertion/deletion markers. In addition, during the reproductive season (Jan-May), weekly samples of the upper 50 meters of the water column targeting nauplii and 5 depth intervals targeting copepodites were collected. Nauplii samples were sorted into two groups (NI-NIII and NIV-NVI), and species were genetically identified. Specimens from stage CIV to adults from each depth interval of copepodite sampling were photographed in order to generate a supporting timeline of visual traits, including gonad maturation stage, presence of stomach content, and total lipid content. The most abundant species were Calanus finmarchicus and Calanus glacialis, followed by Calanus hyperboreus. These species were present in the water column throughout the year, whereas Calanus helgolandicus, the least abundant species, was only present during the summer and autumn period. Each species showed distinct temporal niches, with Calanus finmarchicus occupying the upper 50 meters longer than any of the other species. Calanus hyperboreus dominates in abundance early in the spring but are outnumbered by Calanus glacialis and Calanus finmarchicus after spring bloom sets in. In Skjerstad fjord, Calanus hyperboreus is a clear capital breeder with a long period of nauplii presence before the spring bloom. Calanus glacialis and Calanus finmarchicus both utilize income breeding, with Calanus glacialis developing to the larger nauplii stages quicker than Calanus finmarchicus, but also having a shorter reproduction period. Indeed, the “traditional Arctic” species Calanus hyperboreus and Calanus glacialis appear to end their reproduction period earlier than the North Atlantic Calanus finmarchicus.

Keywords: calanus, depth distribution, reproduction, stage composition, temporal niches

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Authors: Kamran Jawed, Anu Jose Mattam, Zia Fatma, Saima Wajid, Malik Z. Abdin, Syed Shams Yazdani

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Worldwide demand of natural and sustainable fuels and chemicals have encouraged researchers to develop microbial platform for synthesis of short chain fatty acids as they are useful precursors to replace petroleum-based fuels and chemicals. In this study, we evaluated the role of fatty acid synthesis and β-oxidation cycle of Escherichia coli to produce butyric acid, a 4-carbon short chain fatty acid, with the help of three thioesterases, i.e., TesAT from Anaerococcus tetradius, TesBF from Bryantella formatexigens and TesBT from Bacteroides thetaiotaomicron. We found that E. coli strain transformed with gene for TesBT and grown in presence of 8 g/L glucose produced maximum butyric acid titer at 1.46 g/L, followed by that of TesBF at 0.85 g/L and TesAT at 0.12 g/L, indicating that these thioesterases were efficiently converting short chain fatty acyl-ACP intermediate of fatty acid synthesis pathway into the corresponding acid. The titer of butyric acid varied significantly depending upon the plasmid copy number and strain genotype. Deletion of genes for fatty acyl-CoA synthetase and acyl-CoA dehydrogenase, which are involved in initiating the fatty acid degradation cycle, and overexpression of FadR, which is a dual transcriptional regulator and exerts negative control over fatty acid degradation pathway, reduced up to 30% of butyric acid titer. This observation suggested that β-oxidation pathway is working synergistically with fatty acid synthesis pathway in production of butyric acid. Moreover, accelerating the fatty acid elongation cycle by overexpressing acetyl-CoA carboxyltransferase (Acc) and 3-hydroxy-acyl-ACP dehydratase (FabZ) or by deleting FabR, the transcription suppressor of elongation, did not improve the butyric acid titer, rather favored the long chain fatty acid production. Finally, a balance between cell growth and butyric acid production was achieved with the use of phosphorous limited growth medium and 14.3 g/L butyric acid, and 17.5 g/L total free fatty acids (FFAs) titer was achieved during fed-batch cultivation. We have engineered an E. coli strain which utilizes the intermediate of both fatty acid synthesis and degradation pathway, i.e. butyryl-ACP and -CoA, to produce butyric acid from glucose. The strategy used in this study resulted in highest reported titers of butyric acid and FFAs in engineered E. coli.

Keywords: butenoic acid, butyric acid, Escherichia coli, fed-batch fermentation, short chain fatty acids, thioesterase

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Authors: Weaam Aldeeban, Majd Aljamali, Lama A. Youssef

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Background & Objective: Warfarin is considered a problematic drug due to its narrow therapeutic window and wide inter-individual response variations, which are attributed to demographic, environmental, and genetic factors, particularly single nucleotide polymorphism (SNPs) in the genes encoding VKORC1 and CYP2C9 involved in warfarin's mechanism of action and metabolism, respectively. CYP2C9*2rs1799853 and CYP2C9*3rs1057910 alleles are linked to reduced enzyme activity, as carriers of either or both alleles are classified as moderate or slow metabolizers, and therefore exhibit higher sensitivity of warfarin compared with wild type (CYP2C9*1*1). Our study aimed to assess the frequency of *1, *2, and *3 alleles in the CYP2C9 gene in a cohort of Syrian patients receiving a maintenance dose of warfarin for different indications, the impact of genotypes on warfarin dosing, and the frequency of adverse effects (i.e., bleedings). Subjects & Methods: This retrospective cohort study encompassed 94 patients treated with warfarin. Patients’ genotypes were identified by sequencing the polymerase chain reaction (PCR) specific products of the gene encoding CYP2C9, and the effects on warfarin therapeutic outcomes were investigated. Results: Sequencing revealed that 43.6% of the study population has the *2 and/or *3 SNPs. The mean weekly maintenance dose of warfarin was 37.42 ± 15.5 mg for patients with the wild-type allele (CYP2C9*1*1), whereas patients with one or both variants (*2 and/or *3) demanded a significantly lower dose (28.59 ±11.58 mg) of warfarin, (P= 0.015). A higher percentage (40.7%) of patients with allele *2 and/or *3 experienced hemorrhagic accidents compared with only 17.9% of patients with the wild type *1*1, (P = 0.04). Conclusions: Our study proves an association between *2 and *3 genotypes and higher sensitivity to warfarin and a tendency to bleed, which necessitates lowering the dose. These findings emphasize the significance of CYP2C9 genotyping prior to commencing warfarin therapy in order to achieve optimal and faster dose control and to ensure effectiveness and safety.

Keywords: warfarin, CYP2C9, polymorphisms, Syrian, hemorrhage

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Authors: Yahia Massinissa, Yahia Mouloud

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Cystic fibrosis (CF), the most common lethal genetic disease in the Europe population, is caused by mutations in the transmembrane conductance regulator gene (CFTR). It affects most organs including an epithelial tissue, base of hydroelectrolytic transepithelial transport, notably that aerial ways, the pancreas, the biliary ways, the intestine, sweat glands and the genital tractus. The gene whose anomalies are responsible of the cystic fibrosis codes for a protein Cl channel named CFTR (cystic fibrosis transmembrane conductance regulator) that exercises multiple functions in the cell, one of the most important in control of sodium and chlorine through epithelia. The deficient function translates itself notably by an abnormal production of viscous secretion that obstructs the execrator channels of this target organ: one observes then a dilatation, an inflammation and an atrophy of these organs. It also translates itself by an increase of the concentration in sodium and in chloride in sweat, to the basis of the sweat test. In order to do a phenotypical and genotypical diagnosis at a part of the Algerian population, our survey has been carried on 16 patients with evocative symptoms of the cystic fibrosis at that the clinical context has been confirmed by a sweat test. However, anomalies of the CFTR gene have been determined by electrophoresis in gel of polyacrylamide of the PCR products (polymerase chain reaction), after enzymatic digestion, then visualized to the ultraviolet (UV) after action of the ethidium bromide. All mutations detected at the time of our survey have already been identified at patients attained by this pathology in other populations of the world. However, the important number of found mutation with regard to the one of the studied patients testifies that the origin of this big clinical variability that characterizes the illness in the consequences of an enormous diversity of molecular defects of the CFTR gene.

Keywords: cystic fibrosis, CFTR gene, polymorphism, algerian population, sweat test, genotypical diagnosis

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Authors: Manar Makhoul, Buthainah Alsalamah, Salam Lawand, Hassan Azzam

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The major storage proteins in endosperm of 33 cultivated and wild barley genotypes (H.vulgare, H. spontaneum, H. bulbosum, H. murinum, H. marinum) were analyzed to demonstrate the variation in the hordein polypeptides encoded by multigene families in grains. The SDS-PAGE revealed 13 and 17 alleles at the Hor1 and the Hor2 loci respectively, with frequencies from 0.83 to 14 and 0.56 to 13.41% respectively, while seven alleles at the Hor3 locus with frequencies from 3.63 to 30.91% were recognized. The phylogenetic analysis indicated to relevance of the polymorphism in hordein patterns as successful tool in identifying the individual genotypes and discriminating the species according to genome type. We also reported in this research complete nucleotide sequence B-hordein genes of seven wild and cultivated barley genotypes. A 152bp upstream sequence of B-hordein promoter contained a TATA box, CATC box, AAAG motif, N-motif and E-motif. In silico analysis of B-Hordein sequences demonstrated that the coding regions were not interrupted by any intron, and included the complete ORF which varied between 882 and 906 bp, and encoded mature proteins with 293-301 residues characterized by high contents of glutamine (29%), and proline (18%). Comparison of the predicted polypeptide sequences with the published ones suggested that all S-rich prolamins genes are descended from common ancestor. The sequence started at N-terminal with a signal peptide, and then followed directly by two domains; a repetitive one based on the repetition of the repeat unit PQQPFPQQ and C-terminal domain. Also, it was found that positions of the eight cysteine residues were highly conserved in all the B-hordein sequences, but Hordeum bulbosum had additional unpaired one. The phylogenetic tree of B-hordein polypeptide separated the genotypes in distinct seven subgroups. In general, the high homology between B-hordeins and LMW glutenin subunits suggests similar bread-making influences for these B-hordeins.

Keywords: hordeum, phylogenetic tree, sequencing, storage protein

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Authors: Gargi Prasad, Ashiho A. Mao, Deepu Vijayan, S. Mandal

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The main objective of the present study was to optimize and develop an efficient protocol for in vitro propagation of a medicinally important orchid Cephalantheropsis obcordata (Lindl.) Ormerod along with genetic stability analysis of regenerated plants. This plant has been traditionally used in Chinese folk medicine and the decoction of whole plant is known to possess anticancer activity. Nodal segments used as explants were inoculated on Murashige and Skoog (MS) medium supplemented with various concentrations of isopentenyl adenine (2iP). The rooted plants were successfully acclimatized in the greenhouse with 100% survival rate. Inter-simple sequence repeats (ISSR) markers were used to assess the genetic fidelity of in vitro raised plants and the mother plant. It was revealed that monomorphic bands showing the absence of polymorphism in all in vitro raised plantlets analyzed, confirming the genetic uniformity among the regenerants. Phytochemical analysis was done to compare the antioxidant activities and HPLC fingerprinting assay of 80% aqueous ethanol extract of the leaves and stem of in vitro and in vivo grown C. obcordata. The extracts of the plants were examined for their antioxidant activities by using free radical 1, 1-diphenyl-2-picryl hydrazyl (DPPH) scavenging method, 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging ability, reducing power capacity, estimation of total phenolic content, flavonoid content and flavonol content. A simplified method for the detection of ascorbic acid, phenolic acids and flavonoids content was also developed by using reversed phase high-performance liquid chromatography (HPLC). This is the first report on the micropropagation, genetic integrity study and quantitative phytochemical analysis of in vitro regenerated plants of C. obcordata.

Keywords: Cephalantheropsis obcordata, genetic fidelity, ISSR markers, HPLC

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Authors: Tanakrit Doltanakarn

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Introduction: These days, cannabis is being accepted in many countries due to the fact that cannabis could be use in medical. The medical cannabis is used to treat and reduce the pain many diseases. For example, neuropathic pain, Parkinson, autism disorders, cancer pain reduce the adverse effect of chemotherapy, diabetes, and migraine. Active ingredients in cannabis that modulate patients' perceptions of their conditions include Δ9‐tetrahydrocannabinol (THC), cannabidiol (CBD), flavonoids, and terpenes. However, there is an adverse effect of cannabis, cardiovascular effects, psychosis, schizophrenia, mood disorder, and cognitive alternation. These effects are from the THC and CBD ingredients in the cannabis. The metabolize processes of delta-9 THC to 11-OH-delta 9 -THC (inactive form), THC were cause of adverse effects. Interestingly, the distributions of CYP2C9 gene (CYP2C9*2 and CYP2C9*3, poor metabolizer) that might affect incidences of adverse effects in patients who treated with medical cannabis. Objective: The aim of this study we want to investigate the association between genetic polymorphism of CYP2C9 frequency and Thai patients who treated with medical cannabis. Materials and Methods:We recruited sixty-five unrelated Thai patients from the College of Pharmacy, Rangsit University. DNA were extracted using Genomic DNA Mini Kit. Genotyping of CYP2C9*2 (430C>T, rs1799853) and CYP2C9*3 (1075A>C, rs1057910) were genotyped by the TaqMan Real-time PCR assay. Results: Among these 31 medicals cannabis-induced ADRs patients, they were diagnosed with 22 (33.85%) tachycardia and 3 (4.62%) arrhythmia. There were 34 (52.31%) medical cannabis-tolerant controls who were included in this study.40 (61.53%) Thai patients were female, and 25 (38.46%) were male, with median age of 57 (range 27 – 87) years. In this study, we found none of the medical cannabis-induced ADRs carried CYP2C9*2 variant along with medical cannabis-tolerant control group. CYP2C9*3 variant (intermediate metabolizer, IM) was found just only one of thirty-one (3.23%) in the medical cannabis-induced ADRs and two of thirty-fourth (5.88%) in the tolerant controls. Conclusions: Thus, the distribution of CYP2C9 alleles offer a comprehensive view of pharmacogenomics marker in Thai population that could be used as a reference for worldwide to investigate the pharmacogenomics application.

Keywords: medical cannabis, adverse effect, CYP2C9, thai patients

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Authors: Rawan K. Al-Duaij, Salem A. Al-Salem

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Projects, the unique and temporary process of achieving a set of requirements have always been challenging; Planning the schedule and budget, managing the resources and risks are mostly driven by a similar past experience or the technical consultations of experts in the matter. With that complexity of Projects in Scope, Time, and execution environment, Adjustment Orders are tools to reflect changes to the original project parameters after Contract signature. Adjustment Orders are the official/legal amendments to the terms and conditions of a live Contract. Reasons for issuing Adjustment Orders arise from changes in Contract scope, technical requirement and specification resulting in scope addition, deletion, or alteration. It can be as well a combination of most of these parameters resulting in an increase or decrease in time and/or cost. Most business leaders (handling projects in the interest of the owner) refrain from using Adjustment Orders considering their main objectives of staying within budget and on schedule. Success in managing the changes results in uninterrupted execution and agreed project costs as well as schedule. Nevertheless, this is not always practically achievable. In this paper, a detailed study through utilizing Industrial Engineering & Systems Management tools such as Six Sigma, Data Analysis, and Quality Control were implemented on the organization’s five years records of the issued Adjustment Orders in order to investigate their prevalence, and time and cost impact. The analysis outcome revealed and helped to identify and categorize the predominant causations with the highest impacts, which were considered most in recommending the corrective measures to reach the objective of minimizing the Adjustment Orders impacts. Data analysis demonstrated no specific trend in the AO frequency in past five years; however, time impact is more than the cost impact. Although Adjustment Orders might never be avoidable; this analysis offers’ some insight to the procedural gaps, and where it is highly impacting the organization. Possible solutions are concluded such as improving project handling team’s coordination and communication, utilizing a blanket service contract, and modifying the projects gate system procedures to minimize the possibility of having similar struggles in future. Projects in the Oil and Gas sector are always evolving and demand a certain amount of flexibility to sustain the goals of the field. As it will be demonstrated, the uncertainty of project parameters, in adequate project definition, operational constraints and stringent procedures are main factors resulting in the need for Adjustment Orders and accordingly the recommendation will be to address that challenge.

Keywords: adjustment orders, data analysis, oil and gas sector, systems management

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Authors: Mohit Kumar, Beklashwar Salona, Shiv Murti, Mukesh Singh

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We report here a case of five-months old male baby, born as second child of non-consanguineous parents with no considerable history of genetic abnormality which was referred to our cytogenetic laboratory for chromosomal analysis. Physical dysmorphic facial features including mongoloid face, cleft palate, simian crease, and developmental delay were observed. We present this case with unique balanced autosomal translocation of t(3;10)(p21;p13). The risk of phenotypic abnormalities based on de novo balanced translocation was estimated to be 7%. The association of balanced chromosomal rearrangement with Down syndrome features such as multiple congenital anomalies, facial dysmorphism and congenital heart anomalies are very rare in a 5-months old male child. Trisomy-21 is not uncommon in chromosomal abnormality with the birth defect and balanced translocations are frequently observed in patients with secondary infertility or recurrent spontaneous abortion (RSA). Two ml heparinized peripheral blood cells cultured in RPMI-1640 for 72 hours supplemented with 20% fetal bovine serum, phytohemagglutinin (PHA), and antibiotics were used for chromosomal analysis. A total 30 metaphases images were captured using Olympus-BX51 microscope and analyzed using Bio-view karyotyping software through GTG-banding (G bands by trypsin and Giemsa) according to International System for Human Cytogenetic Nomenclature 2016. The results showed balanced translocation between short arm of chromosome # 3 and short arm of chromosome # 10. The karyotype of the child was found to be 46,XY,t(3;10)(p21; p13). Chromosomal abnormalities are one of the major causes of birth defect in new born babies. Also, balanced translocations are frequently observed in patients with secondary infertility or recurrent spontaneous abortion. The index case presented with dysmorphic facial features and had a balanced translocation 46,XY,t(3;10)(p21;p13). This translocation with break points at (p21; p13) has not been reported in the literature in a child with facial dysmorphism. To the best of our knowledge, this is the first report of novel balanced translocation t(3;10) with break points in a child with dysmorphic features. We found balanced chromosomal translocation instead of any trisomy or unbalanced aberrations along with some phenotypic abnormalities. Therefore, we suggest that such novel balanced translocation with abnormal phenotype should be reported in order to enable the pathologist, pediatrician, and gynecologist to have a better insight into the intricacies of chromosomal abnormalities and their associated phenotypic features. We hypothesized that dysmorphic features as seen in this case may be the result of change in the pattern of genes located at the breakpoint area in balanced translocations or may be due to deletion or mutation of genes located on the p-arm of chromosome # 3 and p-arm of chromosome # 10.

Keywords: balanced translocation, karyotyping, phenotypic abnormalities, facial dimorphisms

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Authors: Sadaf Moeez, Madiha Khalid, Zoya Khalid, Sania Shaheen, Sumbul Khalid

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Background: Inter-individual variation in response to metformin, which has been considered as a first line therapy for T2DM treatment is considerable. In the current study, it was aimed to investigate the impact of two genetic variants Leu125Phe (rs77474263) and Gly64Asp (rs77630697) in gene SLC47A1 on the clinical efficacy of metformin in T2DM Pakistani patients. Methods: The study included 800 T2DM patients (400 metformin responders and 400 metformin non-responders) along with 400 ethnically matched healthy individuals. The genotypes were determined by allele-specific polymerase chain reaction. In-silico analysis was done to confirm the effect of the two SNPs on the structure of genes. Association was statistically determined using SPSS software. Results: Minor allele frequency for rs77474263 and rs77630697 was 0.13 and 0.12. For SLC47A1 rs77474263 the homozygotes of one mutant allele ‘T’ (CT) of rs77474263 variant were fewer in metformin responders than metformin non-responders (29.2% vs. 35.5 %). Likewise, the efficacy was further reduced (7.2% vs. 4.0 %) in homozygotes of two copies of ‘T’ allele (TT). Remarkably, T2DM cases with two copies of allele ‘C’ (CC) had 2.11 times more probability to respond towards metformin monotherapy. For SLC47A1 rs77630697 the homozygotes of one mutant allele ‘A’ (GA) of rs77630697 variant were fewer in metformin responders than metformin non-responders (33.5% vs. 43.0 %). Likewise, the efficacy was further reduced (8.5% vs. 4.5%) in homozygotes of two copies of ‘A’ allele (AA). Remarkably, T2DM cases with two copies of allele ‘G’ (GG) had 2.41 times more probability to respond towards metformin monotherapy. In-silico analysis revealed that these two variants affect the structure and stability of their corresponding proteins. Conclusion: The present data suggest that SLC47A1 Leu125Phe (rs77474263) and Gly64Asp (rs77630697) polymorphisms were associated with the therapeutic response of metformin in T2DM patients of Pakistan.

Keywords: diabetes, T2DM, SLC47A1, Pakistan, polymorphism

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Authors: Lorico D. S. Lapitan Jr., Yihan Xu, Yuan Guo, Dejian Zhou

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The ability of ultrasensitive detection of specific genes and discrimination of single nucleotide polymorphisms is important for clinical diagnosis and biomedical research. Herein, we report the development of a new ultrasensitive approach for label-free DNA detection using magnetic nanoparticle (MNP) assisted rapid target capture/separation in combination with signal amplification using poly-enzyme tagged polymer nanobead. The sensor uses an MNP linked capture DNA and a biotin modified signal DNA to sandwich bind the target followed by ligation to provide high single-nucleotide polymorphism discrimination. Only the presence of a perfect match target DNA yields a covalent linkage between the capture and signal DNAs for subsequent conjugation of a neutravidin-modified horseradish peroxidase (HRP) enzyme through the strong biotin-nuetravidin interaction. This converts each captured DNA target into an HRP which can convert millions of copies of a non-fluorescent substrate (amplex red) to a highly fluorescent product (resorufin), for great signal amplification. The use of polymer nanobead each tagged with thousands of copies of HRPs as the signal amplifier greatly improves the signal amplification power, leading to greatly improved sensitivity. We show our biosensing approach can specifically detect an unlabeled DNA target down to 10 aM with a wide dynamic range of 5 orders of magnitude (from 0.001 fM to 100.0 fM). Furthermore, our approach has a high discrimination between a perfectly matched gene and its cancer-related single-base mismatch targets (SNPs): It can positively detect the perfect match DNA target even in the presence of 100 fold excess of co-existing SNPs. This sensing approach also works robustly in clinical relevant media (e.g. 10% human serum) and gives almost the same SNP discrimination ratio as that in clean buffers. Therefore, this ultrasensitive SNP biosensor appears to be well-suited for potential diagnostic applications of genetic diseases.

Keywords: DNA detection, polymer beads, signal amplification, single nucleotide polymorphisms

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Authors: Xianming Chen, Yu Lei, Meinan Wang

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The dikaryotic basidiomycete fungus, Puccinia striiformis, causes stripe rust, one of the most important diseases of wheat and barley worldwide. The pathogen is largely reproduced asexually, and asexual recombination has been hypothesized to be one of the mechanisms for the pathogen variations. To test the hypothesis and understand the genetic process of asexual recombination, somatic recombinant isolates were obtained under controlled conditions by inoculating susceptible host plants with a mixture of equal quantity of urediniospores of isolates with different virulence patterns and selecting through a series of inoculation on host plants with different genes for resistance to one of the parental isolates. The potential recombinant isolates were phenotypically characterized by virulence testing on the set of 18 wheat lines used to differentiate races of the wheat stripe rust pathogen, P. striiformis f. sp. tritici (Pst), for the combinations of Pst isolates; or on both sets of the wheat differentials and 12 barley differentials for identifying races of the barley stripe rust pathogen, P. striiformis f. sp. hordei (Psh) for combinations of a Pst isolate and a Psh isolate. The progeny and parental isolates were also genotypically characterized with 51 simple sequence repeat and 90 single-nucleotide polymorphism markers. From nine combinations of parental isolates, 68 potential recombinant isolates were obtained, of which 33 (48.5%) had similar virulence patterns to one of the parental isolates, and 35 (51.5%) had virulence patterns distinct from either of the parental isolates. Of the 35 isolates of distinct virulence patterns, 11 were identified as races that had been previously detected from natural collections and 24 were identified as new races. The molecular marker data confirmed 66 of the 68 isolates as recombinants. The percentages of parental marker alleles ranged from 0.9% to 98.9% and were significantly different from equal proportions in the recombinant isolates. Except for a couple of combinations, the greater or less contribution was not specific to any particular parental isolates as the same parental isolates contributed more to some of the progeny isolates but less to the other progeny isolates in the same combination. The unequal contributions by parental isolates appear to be a general role in somatic recombination for the stripe rust fungus, which may be used to distinguish asexual recombination from sexual recombination in studying the evolutionary mechanisms of the highly variable fungal pathogen.

Keywords: molecular markers, Puccinia striiformis, somatic recombination, stripe rust

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Authors: Soodabeh Shahid Sales, Azam Rastgar Moghadam, Mehrane Mehramiz, Malihe Entezari, Kazem Anvari, Mohammad Sadegh Khorrami, Saeideh Ahmadi Simab, Ali Moradi, Seyed Mahdi Hassanian, Majid Ghayour-Mobarhan, Gordon A. Ferns, Amir Avan

Abstract:

Background Esophageal cancer has been reported as the eighth most common cancer universal and the seventh cause of cancer-related death in men .recent studies have revealed that cytochrome P450, family 1, subfamily B, polypeptide 1, which plays a role in metabolizing xenobiotics, is associated with different cancers. Therefore in the present study, we investigated the impact of CYP1B1-rs1056836 on esophagus squamous cell carcinoma (ESCC) patients. Method: 317 subjects, with and without ESCC were recruited. DNA was extracted and genotyped via Real-time PCR-Based Taq Man. Kaplan Meier curves were utilized to assess overall and progression-free survival. To evaluate the relationship between patients clinicopathological data, genotypic frequencies, disease prognosis, and patients survival, Pearson chi-square and t-test were used. Logistic regression was utilized to assess the association between the risk of ESCC and genotypes. Results: the genotypic frequency for GG, GC, and CC are respectively 58.6% , 29.8%, 11.5% in the healthy group and 51.8%, 36.14% and 12% in ESCC group. With respect to the recessive genetic inheritance model, an association between the GG genotype and stage of ESCC were found. Also, statistically significant results were not found for this variation and risk of ESCC. Patients with GG genotype had a decreased risk of nodal metastasis in comparison with patients with CC/CG genotype, although this link was not statistically significant. Conclusion: Our findings illustrated the correlation of CYP1B1-rs1056836 as a potential biomarker for ESCC patients, supporting further studies in larger populations in different ethnic groups. Moreover, further investigations are warranted to evaluate the association of emerging marker with dietary intake and lifestyle.

Keywords: Cytochrome P450, esophagus squamous cell carcinoma, dietary intake, lifestyle

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Authors: E. Syngelaki, C. C. F. Schinkel, S. Klatt, E. Hörandl

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Environmental influence can alter the conditions for plant development and can trigger changes in epigenetic variation. Thus, the exposure to abiotic environmental stress can lead to different DNA methylation profiles and may have evolutionary consequences for adaptation. Epigenetic control mechanisms may further influence mode of reproduction. The alpine species R. kuepferi has diploid and tetraploid cytotypes, that are mostly sexual and facultative apomicts, respectively. Hence, it is a suitable model system for studying the correlations of mode of reproduction, ploidy, and environmental stress. Diploid and tetraploid individuals were placed in two climate chambers and treated with low (+7°C day/+2°C night, -1°C cold shocks for three nights per week) and warm (control) temperatures (+15°C day/+10°C night). Subsequently, methylation sensitive-Amplified Fragment-Length Polymorphism (AFPL) markers were used to screen genome-wide methylation alterations triggered by stress treatments. The dataset was analyzed for four groups regarding treatment (cold/warm) and ploidy level (diploid/tetraploid), and also separately for full methylated, hemi-methylated and unmethylated sites. Patterns of epigenetic variation suggested that diploids differed significantly in their profiles from tetraploids independent from treatment, while treatments did not differ significantly within cytotypes. Furthermore, diploids are more differentiated than the tetraploids in overall methylation profiles of both treatments. This observation is in accordance with the increased frequency of apomictic seed formation in diploids and maintenance of facultative apomixis in tetraploids during the experiment. Global analysis of molecular variance showed higher epigenetic variation within groups than among them, while locus-by-locus analysis of molecular variance showed a high number (54.7%) of significantly differentiated un-methylated loci. To summarise, epigenetic variation seems to depend on ploidy level, and in diploids may be correlated to changes in mode of reproduction. However, further studies are needed to elucidate the mechanism and possible functional significance of these correlations.

Keywords: apomixis, cold stress, DNA methylation, Ranunculus kuepferi

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Authors: Muhammad Ashfaq, Muhammad Saleem Haider, Muhammad Ali, Muhammad Sajjad, Amna Ali, Urooj Mubashar

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Phenotypic diversity was confirmed by measuring different morphological traits i.e. seed traits (seed length, seed width, seed thickness, seed length-width ratio, 1000 grain weight) and root-shoot traits (shoot length, root length, shoot fresh weight, root fresh weight, root-shoot ratio, root numbers and root thickness). Variance and association study of desirable traits determine the genotypic differences among the rice germplasm. All the traits showed significant differences among the genotypes. The traits were studied in Randomized complete block design (RCBD) at different water levels. Some traits showed positive correlation with each other and beneficial for increasing the yield and production of the crop. Seed thickness has positive correlation with seed length and seed width (r= 0.104**, r=0.246**). On the other hand, various root shoot traits showed positive highly significant association at different water levels i.e. root length, fresh root weight, root thickness, shoot thickness and root numbers. Our main focus to study the performance/correlation of root shoots traits under stress condition. Fresh root weight, shoot thickness and root numbers showed positive significant association with shoot length, root length, fresh root and shoot weight (r=0.2530**, r=0.2891**, r=0.4626**, r=0.4515**, r=0.5781**, r=0.7164**, r=0.0603**, r= 0.5570**, r=0.5824**). Long root length genotypes favors and suitable for drought stress conditions and screening of diverse genotypes for the further development of new plant material that performing well under different environmental conditions. After screening genetic diversity of potential rice, lines were studied to check the polymorphism by using some SSR markers. DNA was extracted, and PCR analyses were done to study PIC values and allelic diversity of the genotypes. The main objective of this study is to screen out the genotypes on the basis of various genotypic and phenotypic traits.

Keywords: rice, morphological traits, association, germplasm, genetic diversity, water levels, variation

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Authors: Karolina Rutkowska, Hubert Pausch, Jolanta Oprzadek, Krzysztof Flisikowski

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The placenta is one of the most important organs that plays a crucial role in the fetal growth and development. Placenta dysfunction is one of the primary cause of the intrauterine growth restriction (IUGR). Cattle have the cotyledonary placenta which consists of two anatomical parts: fetal and maternal. In the case of cattle during the first months of pregnancy, it is very easy to separate maternal caruncle from fetal cotyledon tissue, easier in fact than removing an ordinary glove from one's hand. Which in fact make easier to conduct tissue-specific molecular studies. Typically, animal models for the study of IUGR are created using surgical methods and malnutrition of the pregnant mother or in the case of mice by genetic modifications. However, proposed cattle model with MIMT1Del/WT deletion is unique because it was created without any surgical methods what significantly distinguish it from the other animal models. The primary objective of the study was to identify differential expression of selected miRNAs in the placenta from normal and intrauterine growth restricted fetuses. There was examined the expression of miRNA in the fetal and maternal part of the placenta from 24 fetuses (12 samples from the fetal part of the placenta and 12 samples from maternal part of the placenta). In the study, there was done miRNAs sequencing in the placenta of MIMT1Del/WT fetuses and MIMT1WT/WT fetuses. Then, there were selected miRNAs that are involved in fetal growth and development. Analysis of miRNAs expression was conducted on ABI7500 machine. miRNAs expression was analyzed by reverse-transcription polymerase chain reaction (RT-PCR). As the reference gene was used SNORD47. The results were expressed as 2ΔΔCt: ΔΔCt = (Ctij − CtSNORD47j) − (Cti1 − CtSNORD471). Where Ctij and CtSNORD47j are the Ct values for gene i and for SNORD47 in a sample (named j); Cti1 and CtSNORD471 are the Ct values in sample 1. Differences between groups were evaluated with analysis of variance by using One-Way ANOVA. Bonferroni’s tests were used for interpretation of the data. All normalised miRNA expression values are expressed on a value of natural logarithm. The data were expressed as least squares mean with standard errors. Significance was declared when P < 0.05. The study shows that miRNAs expression depends on the part of the placenta where they origin (fetal or maternal) and on the genotype of the animal. miRNAs offer a particularly new approach to study IUGR. Corresponding tissue samples were collected according to the standard veterinary protocols according to the European Union Normative for Care and Use of Experimental Animals. All animal experiments were approved by the Animal Ethics Committee of the State Provincial Office of Southern Finland (ESAVI-2010-08583/YM-23).

Keywords: placenta, intrauterine growth restriction, miRNA, cattle

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