Search results for: genotyping by sequencing

Authors: M. Gogniashvili, I. Maisaia, A. Kotorashvili, N. Kotaria, T. Beridze

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Three types of plasmon (A, B and G) is typical for genus Triticum. In polyploid species - Triticum turgidum L. and Triticum aestivum L. plasmon B is detected. In the forthcoming paper, complete nucleotide sequence of chloroplast DNA of 11 representatives of Georgian wheat polyploid species, carrying plasmon B was determined. Sequencing of chloroplast DNA was performed on an Illumina MiSeq platform. Chloroplast DNA molecules were assembled using the SOAPdenovo computer program. All contigs were aligned to the reference chloroplast genome sequence using BLASTN. For detection of SNPs and Indels and phylogeny tree construction computer programs Mafft and Blast were used. Using Triticum aestivum L. subsp. macha (Dekapr. & Menabde) Mackey var. paleocolchicum Dekapr. et Menabde as a reference, 5 SNPs can be identified in chloroplast DNA of Georgian endemic polyploid wheat. The number of noncoding substitutions is 2, coding substitutions - 3. In comparison with reference DNA two - 38 bp and 56 bp inversions were observed in paleocolchicum subspecies. There were six 1 bp indels detected in Georgian polyploid wheats, all of them at microsatellite stretches. The phylogeny tree shows that subspecies macha, carthlicum and paleocolchicum occupy different positions. According to the simplified scheme based on SNP and indel data, the ancestral, female parent of the all studied polyploid wheat is unknown X predecesor, from which four lines were formed. 1 SNP and two inversions (38 bp and 56 bp) caused the formation of subsp. paleocolchicum. Three other lines are macha, durum and carthlicum lines. Macha line is further divided into two sublines (M_1 and M_4). Carthlicum line includes subsp.carthlicum and T.aestivum - C_1 - C_2 - A_1. One of the central question of wheat domestication is which people(s) participated in wheat domestication? It is proposed that the predecessors of Georgian peoples (Proto-Kartvelians) must be placed, on the evidence of archaic lexical and toponymic data, in the mountainous regions of the western and central part of the Little Caucasus (the Transcaucasian foothills) at least 4,000 years ago. One of the possibility to explain the ‘wheat puzzle’ is that Kartvelian speakers brought domesticated wheat species and subspecis from Fertile Crescent further north to South Caucasus.

Keywords: chloroplast DNA, sequencing, SNP, triticum

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Authors: Benjamin A. Ha, Marco Morselli, Xinhui Paige Zhang, Elizabeth A. C. Heath-Heckman, Jonathan B. Puritz, David K. Jacobs

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Next-generation sequencing has enhanced the ability to acquire massive amounts of sequence data to address classic population genetic questions for non-model organisms. Targeted approaches allow for cost effective or more precise analyses of relevant sequences; although, many such techniques require a known genome and it can be costly to purchase probes from a company. This is challenging for non-model organisms with no published genome and can be expensive for large population genetic studies. Expressed exome capture sequencing (EecSeq) synthesizes probes in the lab from expressed mRNA, which is used to capture and sequence the coding regions of genomic DNA from a pooled suite of samples. A normalization step produces probes to recover transcripts from a wide range of expression levels. This approach offers low cost recovery of a broad range of genes in the genome. This research project expands on EecSeq to investigate if mRNA from one taxon may be used to capture relevant sequences from a series of increasingly less closely related taxa. For this purpose, we propose to use the endangered Northern Tidewater goby, Eucyclogobius newberryi, a non-model organism that inhabits California coastal lagoons. mRNA will be extracted from E. newberryi to create probes and capture exomes from eight other taxa, including the more at-risk Southern Tidewater goby, E. kristinae, and more divergent species. Captured exomes will be sequenced, analyzed bioinformatically and phylogenetically, then compared to previously generated phylogenies across this group of gobies. This will provide an assessment of the utility of the technique in cross-species studies and for analyzing low genetic variation within species as is the case for E. kristinae. This method has potential applications to provide economical ways to expand population genetic and evolutionary biology studies for non-model organisms.

Keywords: coastal lagoons, endangered species, non-model organism, target capture method

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Authors: Corey F. Fitzgerald

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This study provides a comprehensive examination of the biomechanical sequencing inherent in pitching motions, coupled with an advanced methodology for interpreting gathered data to inform programming strategies. The analysis is conducted utilizing state-of-the-art biomechanical laboratory equipment capable of detecting subtle changes and deviations, facilitating highly informed decision-making processes. Through this presentation, the intricate dynamics of pitching sequences are meticulously discussed to highlight the complex movement patterns accessible and actionable for performance enhancement purposes in the weight room.

Keywords: sport science, applied biomechanics, strength and conditioning, applied research

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Authors: Pear-Rarin Leelakunakorn, Patompong Satapornpong

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Introduction: Dapsone is an anti-inflammatory and antibiotic drug that was widely used for the treatment of leprosy, acne fulminans, and dermatitis herpetiformis (DH). However, dapsone is the main cause that triggers severe cutaneous adverse reactions (SCARs), with a possibility of 0.4 to 3.6% of patients after initiating treatment. In fact, the mortality rate of dapsone-induced SCARs is approximately 9.9%. In previous studies, HLA-B*13:01 was strongly associated with dapsone-induced SCARs in Han Chinese, Thais, and Koreans. Nevertheless, the distribution of HLA-B*13:01 marker in each population might differ. Moreover, there were found that the association between HLA-C*03:04 and dapsone hypersensitivity syndrome in Han Chinese leprosy patients by OR = 9.00 and p-value = 2.23×10⁻¹⁹. Objective: The aim of this study was to investigate the distribution of HLA-C* 03:04 in Thailand's healthy population. Method: A total of 350 participants were HLA-C genotyping used sequence-specific oligonucleotides (PCR-SSOs). This study was approved by the Ethics Committee of Rangsit University Result : The most frequency of HLA -C alleles in Thais, consist of HLA -C* 01:02 (17.00 %), -C*08:01 (11.00%) , -C*07:02 (10.70%) , -C* 03:04 ( 9.10%) , -C* 03:02 (8.00%) , -C* 07:01 (6.30%), -C* 07:04 (4.60%), -C* 04:01 (4.40%) ,-C* 12:02 ( 4.30% ) ,and -C* 04:03(3.90%). Interestingly, HLA -C* 03:04 allele was similar to the distribution among Thais and other populations such as Eastern Europe (6.09%), Vietnam (7.42% ), East Croatia (2.25%), and Han Chinese (11.70%). Conclusion: Consequently, HLA-C*03:04 might serve as a pharmacogenetic marker for screening prior to initiation therapy with dapsone for prevention of dapsone-induced SCARs in Thai population.

Keywords: HLA-C*03:04, SCARs, thai population, allele frequency

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Authors: Feng Zhang

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Kashin-Beck disease (KBD) is a chronic osteochondropathy. The mechanism of hand growth and development failure of KBD remains elusive now. In this study, we conducted a two-stage genome-wide association study (GWAS) of palmar length-width ratio (LWR) of KBD, totally involving 493 Chinese Han KBD patients. Affymetrix Genome Wide Human SNP Array 6.0 was applied for SNP genotyping. Association analysis was conducted by PLINK software. Imputation analysis was performed by IMPUTE against the reference panel of the 1000 genome project. In the GWAS, the most significant association was observed between palmar LWR and rs2071358 of COL2A1 gene (P value = 4.68×10-8). Imputation analysis identified 3 SNPs surrounding rs2071358 with significant or suggestive association signals. Replication study observed additional significant association signals at both rs2071358 (P value = 0.017) and rs4760608 (P value = 0.002) of COL2A1 gene after Bonferroni correction. Our results suggest that COL2A1 gene was a novel susceptibility gene involved in the growth and development failure of hand of KBD.

Keywords: Kashin-Beck disease, genome-wide association study, COL2A1, hand

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Authors: Muhammad Fiaz, Muhammad Saqlain, Bernard M. Y. Cheung, S. M. Saqlan Naqvi, Ghazala Kaukab Raja

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Background: Association of C allele of rs662799 SNP of APOA5 gene with metabolic syndrome (MetS) has been reported in different populations around the world. A case control study was conducted to explore the relationship of rs662799 variants (T/C) with the MetS and the associated risk phenotypes in a population of Pakistani origin. Methods: MetS was defined according to the IDF criteria. Blood samples were collected from the Pakistan Institute of Medical Sciences, Islamabad, Pakistan for biochemical profiling and DNA extraction. Genotyping of rs662799 was performed using mass ARRAY, iPEX Gold technology. A total of 712 unrelated case and control subjects were genotyped. Data were analyzed using Plink software and SPSS 16.0. Results: The risk allele C of rs662799 showed highly significant association with MetS (OR=1.5, Ρ=0.002). Among risk phenotypes, dyslipidemia, and obesity showed strong association with SNP (OR=1.49, p=0.03; OR =1.46, p=0.01) respectively in models adjusted for age and gender. Conclusion: The rs662799C allele is a significant risk marker for MetS in the local Pakistani population studied. The effect of the SNP is more on dyslipidemia than the other components of the MetS.

Keywords: metabolic syndrome, APOA5, rs662799, dyslipidemia, obesity

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Authors: Shameel Pervez, Saad Aziz Durrani, Ibatsam Khokhar

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Pectinase is an enzyme that breaks down pectin, a compound responsible for structural integrity of the plant. Pectin is difficult to break down mechanically and the cost is very high, that is why many industries including food industries use pectinase enzyme produced by microbes for pectin breakdown. Apple (Malus domestica) is an important fruit in terms of market value. Every year, millions of apples are wasted due to post-harvest rot caused by fungi. Fungi are natural decomposers of our ecosystem and are infamous for post-harvest rot of apple fruit but at the same time they are prized for their high production of valuable extracellular enzymes such as pectinase. In this study, fungi belonging to different genus were isolated from rotten apples. Rotten samples of apple were picked from different markets of Lahore. After surface sterilization, the rotten parts were cut into small pieces and placed onto MEA media plates for three days. Afterwards, distinct colonies were picked and purified by sub-culturing. The isolates were identified to genus level through the study of basic colony morphology and microscopic features. The isolates were then subjected to screening for pectinase activity on MS media to compare pectinase production and were then subsequently tested for pathogenic activity through wound suspension method to evaluate the pathogenic activity of isolates in comparison with their pectinolytic activity. A total of twelve fungal strains were isolates from rotten apples. They were belonging to genus Penicillium, Alternaria, Paecilomyces and Rhizopus. Upon screening for pectinolytic activity, isolates Pen 1, Pen 4, and Rz showed high pectinolytic activity and were further subjected to DNA isolation and partial sequencing for species identification. The results of partial sequencing were combined with in-depth study of morphological features revealing Pen 1 as Penicillium janthinellum, Pen 4 as Penicillium griseofulvum, and Rz as Rhizopus microsporus. Pathogenic activity of all twelve isolates was evaluated. Penicillium spp. were highly pathogenic and destructive and same was the case with Paecilomyces sp. and Rhizopus sp. However, Alternaria spp. were found to be more consistent in their pathogenic activity, on all types of apples.

Keywords: apple, pectinase, fungal pathogens, penicillium, rhizopus

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

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

Keywords: Acanthamoeba, DNA methylation, RNA sequencing, virulence

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Authors: Naso Isaiah Thanavisuth, Patompong Satapornpong

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Introduction: Covid-19 has been a global pandemic for some time now, causing severe symptoms to patients that received the virus. However, there has been no report on this gene in the Thai population. Objective: Our aim in this study is to explore and compare the frequency of HLA-C allele that is associated with severe COVID-19 symptoms in Thais and other populations. Method: 200 general Thai population were enrolled in this study. The genotyping of HLA -C alleles were determined by the polymerase chain reaction with sequence-specific oligonucleotide probes (PCR-SSOP) and Luminex®IS 100 system (Luminex Corporation, Austin, Texas, USA). Results: We found that the frequency of alleles HLA-C* 01:02 (16.00%), HLA-C* 08:01(10.50%), HLA-C* 03:04 (10.25%),HLA-C* 07:02 (10.00%), HLA-C* 03:02 (9.25%), HLA-C* 07:01 (6.75%), HLA-C* 04:01 (5.00%), HLA-C* 06:02 (4.00%), HLA-C* 04:03 (4.00%), and HLA-C* 07:04 (3.75%) were more common in the Thai population. HLA-C* 01:02 (16.00%) allele was the highest frequency in the North, Center, and North East groups in Thailand, but there was the South region that was not significantly different when compared with the other groups of the region. Additionally, HLA-C∗14:02 allele was similarly distributed in Thais (3.00%), African Americans (1.98%), Caucasians (2.08%), Hispanics (1.71%), North American Natives (1.34%) and Asians (5.01%) by p-value = 0.6506, 0.6506, 0.6506, 0.6135 and 0.7182, respectively. Conclusion: Genetic variation database is important to identify HLA can be a risk factor for severe COVID-19 in many populations. In this study, we will support the research of the HLA markers for screening severe COVID-19 in many populations.

Keywords: HLA-C * 14:02, COVID-19, allele frequency, Thailand

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Authors: Yelin Zhao, Xinxiu Li, Martin Smelik, Oleg Sysoev, Firoj Mahmud, Dina Mansour Aly, Mikael Benson

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Background: Early diagnosis of pancreatic cancer is clinically challenging due to vague, or no symptoms, and lack of biomarkers. Polygenic risk score (PRS) scores may provide a valuable tool to assess increased or decreased risk of PC. This study aimed to develop such PRS by filtering genetic variants identified by GWAS using transcriptional programs identified by single-cell RNA sequencing (scRNA-seq). Methods: ScRNA-seq data from 24 pancreatic ductal adenocarcinoma (PDAC) tumor samples and 11 normal pancreases were analyzed to identify differentially expressed genes (DEGs) in in tumor and microenvironment cell types compared to healthy tissues. Pathway analysis showed that the DEGs were enriched for hundreds of significant pathways. These were clustered into 40 “programs” based on gene similarity, using the Jaccard index. Published genetic variants associated with PDAC were mapped to each program to generate program PRSs (pPRSs). These pPRSs, along with five previously published PRSs (PGS000083, PGS000725, PGS000663, PGS000159, and PGS002264), were evaluated in a European-origin population from the UK Biobank, consisting of 1,310 PDAC participants and 407,473 non-pancreatic cancer participants. Stepwise Cox regression analysis was performed to determine associations between pPRSs with the development of PC, with adjustments of sex and principal components of genetic ancestry. Results: The PDAC genetic variants were mapped to 23 programs and were used to generate pPRSs for these programs. Four distinct pPRSs (P1, P6, P11, and P16) and two published PRSs (PGS000663 and PGS002264) were significantly associated with an increased risk of developing PC. Among these, P6 exhibited the greatest hazard ratio (adjusted HR[95% CI] = 1.67[1.14-2.45], p = 0.008). In contrast, P10 and P4 were associated with lower risk of developing PC (adjusted HR[95% CI] = 0.58[0.42-0.81], p = 0.001, and adjusted HR[95% CI] = 0.75[0.59-0.96], p = 0.019). By comparison, two of the five published PRS exhibited an association with PDAC onset with HR (PGS000663: adjusted HR[95% CI] = 1.24[1.14-1.35], p < 0.001 and PGS002264: adjusted HR[95% CI] = 1.14[1.07-1.22], p < 0.001). Conclusion: Compared to published PRSs, scRNA-seq-based pPRSs may be used not only to assess increased but also decreased risk of PDAC.

Keywords: cox regression, pancreatic cancer, polygenic risk score, scRNA-seq, UK biobank

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Authors: Zi-Han Li, Zi-Jun Sun, Dong-Yuan Chang, Li Zhu, Min Chen, Ming-Hui Zhao

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Aims: A genome-wide association study (GWAS) reported that patients with the rs55703767 minor allele in collagen type IV α3 chain encoding gene COL4A3 showed protection against diabetic kidney disease (DKD) in type 1 diabetes mellitus (T1DM). However, the role of rs55703767 in type 2 DKD has not been elucidated. The aim of the current study was to investigate the association between COL4A3 variant rs55703767 and DKD risk in Chinese patients with type 2 diabetes mellitus (T2DM). Methods: This nested case-control study was performed on 1311 patients who had T2DM for at least 10 years, including 580 with DKD and 731 without DKD. We detected the genotypes of all patients by TaqMan SNP Genotyping Assay and analyzed the association between COL4A3 variant rs55703767 and DKD risk. Results: Genetic analysis revealed that there was no significant difference between T2DM patients with DKD and those without DKD regarding allele or genotype frequencies of rs55703767, and the effect of this variant was not hyperglycemia specific. Conclusion: Our findings suggested that there was no detectable association between the COL4A3 variant rs55703767 and the susceptibility to DKD in the Chinese T2DM population.

Keywords: collagen type IV α3 chain, gene polymorphism, type 2 diabetes, diabetic kidney disease

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Authors: Ethel E. Phiri, Lionel Hartzenberg, Percy Chimwamuromba, Emmanuel Nepolo, Jens Kossmann, James R. Lloyd

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Orphan crops are underresearched and underutilized food plant species that have not been categorized as major food crops, but have the potential to be economically and agronomically significant. They have been documented to have the ability to tolerate extreme environmental conditions. However, limited research has been conducted to uncover their potential as food crop species. The New Partnership for Africa’s Development (NEPAD) has classified Marama bean, Tylosema esculentum, as an orphan crop. The plant is one of the 101 African orphan crops that must have their genomes sequenced, assembled, and annotated in the foreseeable future. Marama bean is a perennial leguminous plant that primarily grows in poor, arid soils in southern Africa. The plants produce large tubers that can weigh as much as 200kg. While the foliage provides fodder, the tuber is carbohydrate rich and is a staple food source for rural communities in Namibia. Also, the edible seeds are protein- and oil-rich. Marama Bean plants respond rapidly to increased temperatures and severe water scarcity without extreme consequences. Advances in molecular biology and biotechnology have made it possible to effectively transfer technologies between model- and major crops to orphan crops. In this research, the aim was to assemble the first transcriptomic analysis of Marama Bean RNA-sequence data. Many model plant species have had their genomes sequenced and their transcriptomes assembled. Therefore the availability of transcriptome data for a non-model crop plant species will allow for gene identification and comparisons between various species. The data has been sequenced using the Ilumina Hiseq 2500 sequencing platform. Data analysis is underway. In essence, this research will eventually evaluate the potential use of Marama Bean as a crop species to improve its value in agronomy. data for a non-model crop plant species will allow for gene identification and comparisons between various species. The data has been sequenced using the Ilumina Hiseq 2500 sequencing platform. Data analysis is underway. In essence, this researc will eventually evaluate the potential use of Marama bean as a crop species to improve its value in agronomy.

Keywords: 101 African orphan crops, RNA-Seq, Tylosema esculentum, underutilised crop plants

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Authors: Tamar Lin, Nufar Buchshtab, Yifat Elharar, Julian Nicenboim, Rotem Edgar, Iddo Weiner, Lior Zelcbuch, Ariel Cohen, Sharon Kredo-Russo, Inbar Gahali-Sass, Naomi Zak, Sailaja Puttagunta, Merav Bassan

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Background: Atopic dermatitis (AD) is a chronic, relapsing inflammatory skin disorder that is characterized by dry skin and flares of eczematous lesions and intense pruritus. Multiple lines of evidence suggest that AD is associated with increased colonization by Staphylococcus aureus, which contributes to disease pathogenesis through the release of virulence factors that affect both keratinocytes and immune cells, leading to disruption of the skin barrier and immune cell dysfunction. The aim of the current study is to develop a bacteriophage-based product that specifically targets S. aureus. Methods: For the discovery of phage, environmental samples were screened on 118 S. aureus strains isolated from skin samples, followed by multiple enrichment steps. Natural phages were isolated, subjected to Next-generation Sequencing (NGS), and analyzed using proprietary bioinformatics tools for undesirable genes (toxins, antibiotic resistance genes, lysogeny potential), taxonomic classification, and purity. Phage host range was determined by an efficiency of plating (EOP) value above 0.1 and the ability of the cocktail to completely lyse liquid bacterial culture under different growth conditions (e.g., temperature, bacterial stage). Results: Sequencing analysis demonstrated that the 118 S. aureus clinical strains were distributed across the phylogenetic tree of all available Refseq S. aureus (~10,750 strains). Screening environmental samples on the S. aureus isolates resulted in the isolation of 50 lytic phages from different genera, including Silviavirus, Kayvirus, Podoviridae, and a novel unidentified phage. NGS sequencing confirmed the absence of toxic elements in the phages’ genomes. The host range of the individual phages, as measured by the efficiency of plating (EOP), ranged between 41% (48/118) to 79% (93/118). Host range studies in liquid culture revealed that a subset of the phages can infect a broad range of S. aureus strains in different metabolic states, including stationary state. Combining the single-phage EOP results of selected phages resulted in a broad host range cocktail which infected 92% (109/118) of the strains. When tested in vitro in a liquid infection assay, clearance was achieved in 87% (103/118) of the strains, with no evidence of phage resistance throughout the study (24 hours). A S. aureus host was identified that can be used for the production of all the phages in the cocktail at high titers suitable for large-scale manufacturing. This host was validated for the absence of contaminating prophages using advanced NGS methods combined with multiple production cycles. The phages are produced under optimized scale-up conditions and are being used for the development of a topical formulation (BX005) that may be administered to subjects with atopic dermatitis. Conclusions: A cocktail of natural phages targeting S. aureus was effective in reducing bacterial burden across multiple assays. Phage products may offer safe and effective steroid-sparing options for atopic dermatitis.

Keywords: atopic dermatitis, bacteriophage cocktail, host range, Staphylococcus aureus

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Authors: Ireneusz Majsterek, Karolina Przybylowska, Lukasz Dziki, Adam Dziki, Jacek Kabzinski

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Colorectal cancer (CRC) is one of the deadliest cancers. Every year we see an increase in the number of cases, and in spite of intensive research etiology of the disease remains unknown. For many years, researchers are seeking to associate genetic factors with an increased risk of CRC, so far it has proved to be a compelling link between the MMR system of DNA repair and hereditary nonpolyposis colorectal cancers (HNPCC). Currently, research is focused on finding the relationship between the remaining DNA repair systems and an increased risk of developing colorectal cancer. The aim of the study was to determine the relationship between gene polymorphisms Ser835Ser of XPF gene and Gly23Ala of XPA gene–elements of NER DNA repair system, and modulation of the risk of colorectal cancer in the Polish population. Determination of the molecular basis of carcinogenesis process and predicting increased risk will allow qualifying patients to increased risk group and including them in preventive program. We used blood collected from 110 patients diagnosed with colorectal cancer. The control group consisted of equal number of healthy people. Genotyping was performed by TaqMan method. The obtained results indicate that the genotype 23Gly/Ala of XPA gene is associated with an increased risk of colorectal cancer, while 23Ala/Ala as well as TCT allele of Ser835Ser of XPF gene may reduce the risk of CRC.

Keywords: NER, colorectal cancer, XPA, XPF, polymorphisms

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Authors: Artem Kim, Clara Savary, Christele Dubourg, Wilfrid Carre, Houda Hamdi-Roze, Valerie Dupé, Sylvie Odent, Marie De Tayrac, Veronique David

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Holoprosencephaly (HPE) is a rare congenital brain malformation resulting from the incomplete separation of the two cerebral hemispheres. It is characterized by a wide phenotypic spectrum and a high degree of locus heterogeneity. Genetic defects in 16 genes have already been implicated in HPE, but account for only 30% of cases, suggesting that a large part of genetic factors remains to be discovered. HPE has been recently redefined as a complex multigenic disorder, requiring the joint effect of multiple mutational events in genes belonging to one or several developmental pathways. The onset of HPE may result from accumulation of the effects of multiple rare variants in functionally-related genes, each conferring a moderate increase in the risk of HPE onset. In order to decipher the genetic basis of HPE, unconventional patterns of inheritance involving multiple genetic factors need to be considered. The primary objective of this study was to uncover possible disease causing combinations of multiple rare variants underlying HPE by performing trio-based Whole Exome Sequencing (WES) of familial cases where no molecular diagnosis could be established. 39 families were selected with no fully-penetrant causal mutation in known HPE gene, no chromosomic aberrations/copy number variants and without any implication of environmental factors. As the main challenge was to identify disease-related variants among a large number of nonpathogenic polymorphisms detected by WES classical scheme, a novel variant prioritization approach was established. It combined WES filtering with complementary gene-level approaches: transcriptome-driven (RNA-Seq data) and clinically-driven (public clinical data) strategies. Briefly, a filtering approach was performed to select variants compatible with disease segregation, population frequency and pathogenicity prediction to identify an exhaustive list of rare deleterious variants. The exome search space was then reduced by restricting the analysis to candidate genes identified by either transcriptome-driven strategy (genes sharing highly similar expression patterns with known HPE genes during cerebral development) or clinically-driven strategy (genes associated to phenotypes of interest overlapping with HPE). Deeper analyses of candidate variants were then performed on a family-by-family basis. These included the exploration of clinical information, expression studies, variant characteristics, recurrence of mutated genes and available biological knowledge. A novel bioinformatics pipeline was designed. Applied to the 39 families, this final integrated workflow identified an average of 11 candidate variants per family. Most of candidate variants were inherited from asymptomatic parents suggesting a multigenic inheritance pattern requiring the association of multiple mutational events. The manual analysis highlighted 5 new strong HPE candidate genes showing recurrences in distinct families. Functional validations of these genes are foreseen.

Keywords: complex genetic disorder, holoprosencephaly, multiple rare variants, whole exome sequencing

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Authors: Iryna P. Dzieciuch, Michael D. Putman

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Humidity is known to degrade Navy ship electronic equipment, especially in hot moist environments. If left untreated, it can cause significant and permanent damage. Even rigorous inspection and frequent clean-up would not prevent further equipment contamination and degradation because of the constant presence of favorable growth conditions for many microorganisms. Generally, relative humidity levels of less than 60% will inhibit corrosion in electronic equipment, but because NAVY electronics often operate in hot and humid environments, prevention via dehumidification is not always possible. Currently, there is no defined research that fully describes key mechanisms which cause electronics and its coating degradation. The corrosive action of most bacteria is mainly developed through (i) mycelium adherence to the metal plates, (ii) facilitation the formation of pitting areas, (iii) production of organic acids such as citric, iso-citric, cis-aconitic, alpha-ketoglutaric, which are corrosive to electronic equipment and its components. Our approach studies corrosive action in electronic equipment: circuit-board, wires and connections that are exposed in the humid environment that gets worse during condensation. In our new approach the technical task is built on work with the bacterial communities in public areas, bacterial genetics, bioinformatics, biostatistics and Scanning Electron Microscopy (SEM) of corroded circuit boards. Based on these methods, we collect and examine environmental samples from biofilms of the corroded and non-corroded sites, where bacterial contamination of electronic equipment, such as machine racks and shore boats, is an ongoing concern. Sample collection and sample analysis is focused on addressing the key questions identified above through the following tasks: laboratory sample processing and evaluation under scanning electron microscopy, initial sequencing and data evaluation; bioinformatics and data analysis. Preliminary results from scanning electron microscopy (SEM) have revealed that metal particulates and alloys in corroded samples consists mostly of Tin ( < 40%), Silicon ( < 4%), Sulfur ( < 1%), Aluminum ( < 2%), Magnesium ( < 2%), Copper ( < 1%), Bromine ( < 2%), Barium ( <1%) and Iron ( < 2%) elements. We have also performed X 12000 magnification of the same sites and that proved existence of undisrupted biofilm organelles and crystal structures. Non-corrosion sites have revealed high presence of copper ( < 47%); other metals remain at the comparable level as on the samples with corrosion. We have performed X 1000 magnification on the non-corroded at the sites and have documented formation of copper crystals. The next step of this study, is to perform metagenomics sequencing at all sites and to compare bacterial composition present in the environment. While copper is nontoxic to the living organisms, the process of bacterial adhesion creates acidic environment by releasing citric, iso-citric, cis-aconitic, alpha-ketoglutaric acidics, which in turn release copper ions Cu++, which that are highly toxic to the bacteria and higher order living organisms. This phenomenon, might explain natural “antibiotic” properties that are lacking in elements such as tin. To prove or deny this hypothesis we will use next - generation sequencing (NGS) methods to investigate types and growth cycles of bacteria that from bacterial biofilm the on corrosive and non-corrosive samples.

Keywords: bacteria, biofilm, circuit board, copper, corrosion, electronic equipment, organic acids, tin

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Authors: Shakir H. Mohammed Al-Alwany, Saad Hasan M. Ali, Ibrahim Mohammed S. Shnawa

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The study was aimed to define the percentage of detection of high-oncogenic risk types of HPV and their genotyping in archival tissue specimens that ranged from apparently healthy tissue to invasive breast cancer by using one of the recent versions of In Situ Hybridization(ISH) 0.2. To find out rational significance of such genotypes as well as over expressed products of mutants P53 and RB genes on the severity of underlying breast cancers. The DNA of HPV was detected in 46.5 % of tissues from breast cancers while HPV DNA in the tissues from benign breast tumours was detected in 12.5%. No HPV positive–ISH reaction was detected in healthy breast tissues of the control group. HPV DNA of genotypes (16, 18, 31 and 33) was detected in malignant group in frequency of 25.6%, 27.1%, 30.2% and 12.4%, respectively. Over expression of p53 was detected by IHC in 51.2% breast cancer cases and in 50% benign breast tumour group, while none of control group showed P53- over expression. Retinoblastoma protein was detected by IHC test in 49.7% of malignant breast tumours, 54.2% of benign breast tumours but no signal was reported in the tissues of control group. The significance prevalence of expression of mutated p53 & Rb genes as well as detection of high-oncogenic HPV genotypes in patients with breast cancer supports the hypothesis of an etiologic role for the virus in breast cancer development.

Keywords: human papilloma virus, P53, RB, breast cancer

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Authors: Hanbyul Kim, Hye-Jin Kin, Taehun Park, Woo Jun Sul, Susun An

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Skin is the largest organ of the human body and can provide the diverse habitat for various microorganisms. The ecology of the skin surface selects distinctive sets of microorganisms and is influenced by both endogenous intrinsic factors and exogenous environmental factors. The diversity of the bacterial community in the skin also depends on multiple host factors: gender, age, health status, location. Among them, age-related changes in skin structure and function are attributable to combinations of endogenous intrinsic factors and exogenous environmental factors. Skin aging is characterized by a decrease in sweat, sebum and the immune functions thus resulting in significant alterations in skin surface physiology including pH, lipid composition, and sebum secretion. The present study gives a comprehensive clue on the variation of skin microbiota and the correlations between ages by analyzing and comparing the metagenome of skin microbiome using Next Generation Sequencing method. Skin bacterial diversity and composition were characterized and compared between two different age groups: younger (20 – 30y) and older (60 - 70y) Xian, Chinese women. A total of 73 healthy women meet two conditions: (I) living in Xian, China; (II) maintaining healthy skin status during the period of this study. Based on Ribosomal Database Project (RDP) database, skin samples of 73 participants were enclosed with ten most abundant genera: Chryseobacterium, Propionibacterium, Enhydrobacter, Staphylococcus and so on. Although these genera are the most predominant genus overall, each genus showed different proportion in each group. The most dominant genus, Chryseobacterium was more present relatively in Young group than in an old group. Similarly, Propionibacterium and Enhydrobacter occupied a higher proportion of skin bacterial composition of the young group. Staphylococcus, in contrast, inhabited more in the old group. The beta diversity that represents the ratio between regional and local species diversity showed significantly different between two age groups. Likewise, The Principal Coordinate Analysis (PCoA) values representing each phylogenetic distance in the two-dimensional framework using the OTU (Operational taxonomic unit) values of the samples also showed differences between the two groups. Thus, our data suggested that the composition and diversification of skin microbiomes in adult women were largely affected by chronological and physiological skin aging.

Keywords: next generation sequencing, age, Xian, skin microbiome

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Authors: L. Salomon, P. Sklenar

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The Andes hold the highest plant species diversity in the world. How this occurred is one of the most intriguing questions in studies addressing the origin and patterning of plant diversity worldwide. Recently, the explosive adaptive radiations found in high Andean groups have been pointed as triggers to this spectacular diversity. The Andes is the species-richest area for the biggest genus from the Asteraceae family: Senecio. There, the genus presents an incredible diversity of species, striking growth form variation, and large niche span. Even when some studies tried to disentangle the evolutionary story for some Andean species in Senecio, they obtained partially resolved and low supported phylogenies, as expected for recently radiated groups. The high-throughput sequencing (HTS) approaches have proved to be a powerful tool answering phylogenetic questions in those groups whose evolutionary stories are recent and traditional techniques like Sanger sequencing are not informative enough. Although these tools have been used to understand the evolution of an increasing number of Andean groups, nowadays, their scope has not been applied for Senecio. This project aims to contribute to a better knowledge of the mechanisms shaping the hyper diversity of Senecio in the Andean region, using HTS focusing on Senecio ser. Culcitium (Asteraceae), recently recircumscribed. Firstly, reconstructing a highly resolved and supported phylogeny, and after assessing the role of allopatric differentiation, hybridization, and genome duplication in the diversification of the group. Using the Hyb-Seq approach, combining target enrichment using Asteraceae COS loci baits and genome skimming, more than 100 new accessions were generated. HybPhyloMaker and HybPiper pipelines were used for the phylogenetic analyses, and another pipeline in development (Paralogue Wizard) was used to deal with paralogues. RAxML was used to generate gene trees and Astral for species tree reconstruction. Phyparts were used to explore as first step of gene tree discordance along the clades. Fully resolved with moderated supported trees were obtained, showing Senecio ser. Culcitium as monophyletic. Within the group, some species formed well-supported clades with morphologically related species, while some species would not have exclusive ancestry, in concordance with previous studies using amplified fragment length polymorphism (AFLP) showing geographical differentiation. Discordance between gene trees was detected. Paralogues were detected for many loci, indicating possible genome duplications; ploidy level estimation using flow cytometry will be carried out during the next months in order to identify the role of this process in the diversification of the group. Likewise, TreeSetViz package for Mesquite, hierarchical likelihood ratio congruence test using Concaterpillar, and Procrustean Approach to Cophylogeny (PACo), will be used to evaluate the congruence among different inheritance patterns. In order to evaluate the influence of hybridization and Incomplete Lineage Sorting (ILS) in each resultant clade from the phylogeny, Joly et al.'s 2009 method in a coalescent scenario and Paterson’s D-statistic will be performed. Even when the main discordance sources between gene trees were not explored in detail yet, the data show that at least to some degree, processes such as genome duplication, hybridization, and/or ILS could be involved in the evolution of the group.

Keywords: adaptive radiations, Andes, genome duplication, hybridization, Senecio

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Authors: Javed Mohammed

Abstract:

Right now, bio-medical infrastructure lags well behind the curve. Our healthcare system is dispersed and disjointed; medical records are a bit of a mess; and we do not yet have the capacity to store and process the crazy amounts of data coming our way from widespread whole-genome sequencing. And then there are privacy issues. Despite these infrastructure challenges, some researchers are plunging into bio medical Big Data now, in hopes of extracting new and actionable knowledge. They are doing delving into molecular-level data to discover bio markers that help classify patients based on their response to existing treatments; and pushing their results out to physicians in novel and creative ways. Computer scientists and bio medical researchers are able to transform data into models and simulations that will enable scientists for the first time to gain a profound under-standing of the deepest biological functions. Solving biological problems may require High-Performance Computing HPC due either to the massive parallel computation required to solve a particular problem or to algorithmic complexity that may range from difficult to intractable. Many problems involve seemingly well-behaved polynomial time algorithms (such as all-to-all comparisons) but have massive computational requirements due to the large data sets that must be analyzed. High-throughput techniques for DNA sequencing and analysis of gene expression have led to exponential growth in the amount of publicly available genomic data. With the increased availability of genomic data traditional database approaches are no longer sufficient for rapidly performing life science queries involving the fusion of data types. Computing systems are now so powerful it is possible for researchers to consider modeling the folding of a protein or even the simulation of an entire human body. This research paper emphasizes the computational biology's growing need for high-performance computing and Big Data. It illustrates this article’s indispensability in meeting the scientific and engineering challenges of the twenty-first century, and how Protein Folding (the structure and function of proteins) and Phylogeny Reconstruction (evolutionary history of a group of genes) can use HPC that provides sufficient capability for evaluating or solving more limited but meaningful instances. This article also indicates solutions to optimization problems, and benefits Big Data and Computational Biology. The article illustrates the Current State-of-the-Art and Future-Generation Biology of HPC Computing with Big Data.

Keywords: high performance, big data, parallel computation, molecular data, computational biology

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Authors: Shagufta Jabeen, Faez J. Firdaus Abdullah, Zunita Zakaria, Nurulfiza M. Isa, Yung C. Tan, Wai Y. Yee, Abdul R. Omar

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Pasteurella multocida (PM) is an important veterinary opportunistic pathogen particularly associated with septicemic pasteurellosis, pneumonic pasteurellosis and hemorrhagic septicemia in cattle and buffaloes. P. multocida serotype A has been reported to cause fatal pneumonia and septicemia. Pasteurella multocida subspecies multocida of serotype A Malaysian isolate PMTB2.1 was first isolated from buffaloes died of septicemia. In this study, the genome of P. multocida strain PMTB2.1 was sequenced using third-generation sequencing technology, PacBio RS2 system and analyzed bioinformatically via de novo analysis followed by in-depth analysis based on comparative genomics. Bioinformatics analysis based on de novo assembly of PacBio raw reads generated 3 contigs followed by gap filling of aligned contigs with PCR sequencing, generated a single contiguous circular chromosome with a genomic size of 2,315,138 bp and a GC content of approximately 40.32% (Accession number CP007205). The PMTB2.1 genome comprised of 2,176 protein-coding sequences, 6 rRNA operons and 56 tRNA and 4 ncRNAs sequences. The comparative genome sequence analysis of PMTB2.1 with nine complete genomes which include Actinobacillus pleuropneumoniae, Haemophilus parasuis, Escherichia coli and five P. multocida complete genome sequences including, PM70, PM36950, PMHN06, PM3480, PMHB01 and PMTB2.1 was carried out based on OrthoMCL analysis and Venn diagram. The analysis showed that 282 CDs (13%) are unique to PMTB2.1and 1,125 CDs with orthologs in all. This reflects overall close relationship of these bacteria and supports the classification in the Gamma subdivision of the Proteobacteria. In addition, genomic distance analysis among all nine genomes indicated that PMTB2.1 is closely related with other five Pasteurella species with genomic distance less than 0.13. Synteny analysis shows subtle differences in genetic structures among different P.multocida indicating the dynamics of frequent gene transfer events among different P. multocida strains. However, PM3480 and PM70 exhibited exceptionally large structural variation since they were swine and chicken isolates. Furthermore, genomic structure of PMTB2.1 is more resembling that of PM36950 with a genomic size difference of approximately 34,380 kb (smaller than PM36950) and strain-specific Integrative and Conjugative Elements (ICE) which was found only in PM36950 is absent in PMTB2.1. Meanwhile, two intact prophages sequences of approximately 62 kb were found to be present only in PMTB2.1. One of phage is similar to transposable phage SfMu. The phylogenomic tree was constructed and rooted with E. coli, A. pleuropneumoniae and H. parasuis based on OrthoMCL analysis. The genomes of P. multocida strain PMTB2.1 were clustered with bovine isolates of P. multocida strain PM36950 and PMHB01 and were separated from avian isolate PM70 and swine isolates PM3480 and PMHN06 and are distant from Actinobacillus and Haemophilus. Previous studies based on Single Nucleotide Polymorphism (SNPs) and Multilocus Sequence Typing (MLST) unable to show a clear phylogenetic relatedness between Pasteurella multocida and the different host. In conclusion, this study has provided insight on the genomic structure of PMTB2.1 in terms of potential genes that can function as virulence factors for future study in elucidating the mechanisms behind the ability of the bacteria in causing diseases in susceptible animals.

Keywords: comparative genomics, DNA sequencing, phage, phylogenomics

Procedia PDF Downloads 188

Authors: Ghazi Chabchoub, Mouna Feki, Mohamed Abid, Hammadi Ayadi

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Autoimmune thyroid diseases (AITDs), including Graves’ disease (GD) and Hashimoto’s thyroiditis (HT), are inherited as complex traits. Genetic factors associated with AITDs have been tentatively identified by candidate gene and genome scanning approaches. We analysed three intragenic microsatellite markers in the thyroid hormone receptor associated protein 2 gene (THRAP2), mapped near D12S79 marker, which have a potential role in immune function and inflammation [THRAP2-1(TG)n, THRAP2-2 (AC)n and THRAP2-3 (AC)n]. Our study population concerned 12 patients affected with AITDs belonging to a multiplex Tunisian family with high prevalence of AITDs. Fluorescent genotyping was carried out on ABI 3100 sequencers (Applied Biosystems USA) with the use of GENESCAN for semi-automated fragment sizing and GENOTYPER peak-calling software. Statistical analysis was performed using the non parametric Lod score (NPL) by Merlin software. Merlin outputs non-parametric NPLall (Z) and LOD scores and their corresponding asymptotic P values. The analysis for three intragenic markers in the THRAP2 gene revealed strong evidence for linkage (NPL=3.68, P=0.00012). Our results suggested the possible role of THRAP2 gene in AITDs susceptibility in this family.

Keywords: autoimmunity, autoimmune disease, genetic, linkage analysis

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

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Dilated cardiomyopathy (DCM) is a severe cardiovascular disorder characterized by progressive systolic dysfunction due to cardiac chamber dilatation and inefficient myocardial contractility often leading to chronic heart failure. Recently, a genome-wide association studies (GWASs) on DCM indicate that the ZBTB17 gene rs10927875 single nucleotide polymorphism is associated with DCM. The aim of the study was to identify the distribution of ZBTB17 gene rs10927875 polymorphism in 50 Slovak patients with DCM and 80 healthy control subjects using the Custom Taqman®SNP Genotyping assays. Risk factors detected at baseline in each group included age, sex, body mass index, smoking status, diabetes and blood pressure. The mean age of patients with DCM was 52.9±6.3 years; the mean age of individuals in control group was 50.3±8.9 years. The distribution of investigated genotypes of rs10927875 polymorphism within ZBTB17 gene in the cohort of Slovak patients with DCM was as follows: CC (38.8%), CT (55.1%), TT (6.1%), in controls: CC (43.8%), CT (51.2%), TT (5.0%). The risk allele T was more common among the patients with dilated cardiomyopathy than in normal controls (33.7% versus 30.6%). The differences in genotype or allele frequencies of ZBTB17 gene rs10927875 polymorphism were not statistically significant (p=0.6908; p=0.6098). The results of this study suggest that ZBTB17 gene rs10927875 polymorphism may be a risk factor for susceptibility to DCM in Slovak patients with DCM. Studies of numerous files and additional functional investigations are needed to fully understand the roles of genetic associations.

Keywords: ZBTB17 gene, rs10927875 polymorphism, dilated cardiomyopathy, cardiovascular disorder

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Authors: H. Imad, Y. Cheah, O. Aamera

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The aims of this research are to study the mitochondrial non-coding region by using the Sanger sequencing technique and establish the degree of variation characteristics of a fragment. FTA® Technology (FTA™ paper DNA extraction) utilized to extract DNA. A portion of a non-coding region encompassing positions 37 to 340 amplified in accordance with the Anderson reference sequence. PCR products purified by EZ-10 spin column then sequenced and detected by using the ABI 3730xL DNA Analyzer. New polymorphic positions 57, 63, and 101 are described may in future be suitable sources for identification purpose. The data obtained can be used to identify variable nucleotide positions characterized by frequent occurrence most promising for identification variants.

Keywords: encompassing nucleotide positions 37 to 340, HVII, Iraq, mitochondrial DNA, polymorphism, frequency

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Authors: N. C. van der Merwe, J. Oosthuizen, M. F. Makhetha, J. Adams, B. K. Dajee, S-R. Schneider

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Women representing high-risk breast cancer families, who tested negative for pathogenic mutations in BRCA1 and BRCA2, are four times more likely to develop breast cancer compared to women in the general population. Sequencing of genes involved in genomic stability and DNA repair led to the identification of novel contributors to familial breast cancer risk. These include BLM and PALB2. Bloom's syndrome is a rare homozygous autosomal recessive chromosomal instability disorder with a high incidence of various types of neoplasia and is associated with breast cancer when in a heterozygous state. PALB2, on the other hand, binds to BRCA2 and together, they partake actively in DNA damage repair. Archived DNA samples of 66 BRCA1/2 negative high-risk breast cancer patients were retrospectively selected based on the presence of an extensive family history of the disease ( > 3 affecteds per family). All coding regions and splice-site boundaries of both genes were screened using High-Resolution Melting Analysis. Samples exhibiting variation were bi-directionally automated Sanger sequenced. The clinical significance of each variant was assessed using various in silico and splice site prediction algorithms. Comprehensive screening identified a total of 11 BLM and 26 PALB2 variants. The variants detected ranged from global to rare and included three novel mutations. Three BLM and two PALB2 likely pathogenic mutations were identified that could account for the disease in these extensive breast cancer families in the absence of BRCA mutations (BLM c.11T > A, p.V4D; BLM c.2603C > T, p.P868L; BLM c.3961G > A, p.V1321I; PALB2 c.421C > T, p.Gln141Ter; PALB2 c.508A > T, p.Arg170Ter). Conclusion: The study confirmed the contribution of pathogenic mutations in BLM and PALB2 to the familial breast cancer burden in South Africa. It explained the presence of the disease in 7.5% of the BRCA1/2 negative families with an extensive family history of breast cancer. Segregation analysis will be performed to confirm the clinical impact of these mutations for each of these families. These results justify the inclusion of both these genes in a comprehensive breast and ovarian next generation sequencing cancer panel and should be screened simultaneously with BRCA1 and BRCA2 as it might explain a significant percentage of familial breast and ovarian cancer in South Africa.

Keywords: Bloom Syndrome, familial breast cancer, PALB2, South Africa

Procedia PDF Downloads 236

Authors: Moussa I. Mohamed, Turki, K. A. Al-Faraj, Abdullah A. Al-Arfaj, Ashgan M. Hessain

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The objective of the present study is to detect the incidences of Salmonella enterica from different poultry farms located in Egypt on molecular basis. During the summer of 2012, a total of 1800 cloacal swabs were collected from poultry farms located I Cairo, Egypt to be subjected for isolation of Salmonella enteric. Moreover, a total of 300 samples of poultry and poultry products were collected from different retail establishment markets in Cairo, Egypt including, 150 local whole frozen chickens, 50 imported whole frozen chickens, 100 local chicken cut samples. The highest rate of isolation 8% was obtained from imported frozen chickens and local chicken cuts, followed by local frozen chickens 6.66% and finally rectal swabs from apparently health chickens 6.4 %. Salmonella Typhimurium and Salmonella Enteritidis were most frequent among the total Salmonella isolates. Multiplex-PCR for the rapid detection of Salmonella Typhimurium and Salmonella Enteritidis from field samples especially after pre-enrichment on Rappaport-Vassiliadis (RV) selective broth (PCR-RV), revealed the same positive samples. Therefore PCR-RV technique is rabid, time saving and applicable to detect Salmonella serovars directly from chicken samples. Moreover, detecting Salmonella Typhimurium and Salmonella Enteritidis by this assay was carried out within 2 days opposed to 5–6 d by the bacteriological and serological methods.

Keywords: Salmonella enterica, Salmonella typhimurium, Salmonella enteritidis enrichment, multiplex-PCR

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Authors: Naso Isaiah Thanavisuth

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Medical cannabis can be used for treatment including pain, multiple sclerosis, Parkinson's disease, and cancer. 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 and CYP2C9*3 on exon 7 to decrease enzyme activity. Notwithstanding, there is no data describing whether the variant of CYP2C9 genes are apharmacogenetics marker for the prediction of THC-induced AEs in Thai patients. We want to investigate the association between CYP2C9 gene and THC-induced AEs in Thai patients. We enrolled 39 Thai patients with medical cannabis treatment who were classified by clinical data. The CYP2C9*2 and *3 genotyping were conducted using the TaqMan real time PCR assay. All Thai patients who received the medical cannabis consist of twenty-four (61.54%) patients 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. Although, our results indicate 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 the prevention of medical cannabis-induced AEs.

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

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Authors: Rachel Danielson, Brendan Bohannan, S.M. Tsai, Kyle Meyer, Jorge L.M. Rodrigues

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The Amazon Rainforest is a global diversity hotspot and crucial carbon sink, but approximately 20% of its total extent has been deforested- primarily for the establishment of cattle pasture. Understanding the impact of this large-scale disturbance on soil microbial community composition and activity is crucial in understanding potentially consequential shifts in nutrient or greenhouse gas cycling, as well as adding to the body of knowledge concerning how these complex communities respond to human disturbance. In this study, surface soils (0-10cm) were collected from three forests and three 45-year-old pastures in Rondonia, Brazil (the Amazon state with the greatest rate of forest destruction) in order to determine the impact of forest conversion on microbial communities involved in nitrogen fixation. Soil chemical and physical parameters were paired with measurements of microbial activity and genetic profiles to determine how community composition and process rates relate to environmental conditions. Measuring both the natural abundance of 15N in total soil N, as well as incorporation of enriched 15N2 under incubation has revealed that conversion of primary forest to cattle pasture results in a significant increase in the rate of nitrogen fixation by free-living diazotrophs. Quantification of nifH gene copy numbers (an essential subunit encoding the nitrogenase enzyme) correspondingly reveals a significant increase of genes in pasture compared to forest soils. Additionally, genetic sequencing of both nifH genes and transcripts shows a significant increase in the diversity of the present and metabolically active diazotrophs within the soil community. Levels of both organic and inorganic nitrogen tend to be lower in pastures compared to forests, with ammonium rather than nitrate as the dominant inorganic form. However, no significant or consistent differences in total, extractable, permanganate-oxidizable, or loss-on-ignition carbon are present between the two land-use types. Forest conversion is associated with a 0.5- 1.0 unit pH increase, but concentrations of many biologically relevant nutrients such as phosphorus do not increase consistently. Increases in free-living diazotrophic community abundance and activity appear to be related to shifts in carbon to nitrogen pool ratios. Furthermore, there may be an important impact of transient, low molecular weight plant-root-derived organic carbon on free-living diazotroph communities not captured in this study. Preliminary analysis of nitrogenase gene variant composition using NovoSeq metagenomic sequencing indicates that conversion of forest to pasture may significantly enrich vanadium-based nitrogenases. This indication is complemented by a significant decrease in available soil molybdenum. Very little is known about the ecology of diazotrophs utilizing vanadium-based nitrogenases, so further analysis may reveal important environmental conditions favoring their abundance and diversity in soil systems. Taken together, the results of this study indicate a significant change in nitrogen cycling and diazotroph community composition with the conversion of the Amazon Rainforest. This may have important implications for the sustainability of cattle pastures once established since nitrogen is a crucial nutrient for forage grass productivity.

Keywords: free-living diazotrophs, land use change, metagenomic sequencing, nitrogen fixation

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Authors: Busra Mutlu Ipek, Merve Mutlu, Ahmet Mutlu

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Novel coronavirus COVID-19, which has recently influenced the world, poses a great threat to humanity. In order to overcome this challenging situation, scientists are working on developing effective vaccine against coronavirus. Many experts and researchers have also produced articles and done studies on this highly important subject. In this direction, this special topic was chosen for article to make a contribution to this area. The purpose of this article is to perform RNA sequence analysis of selected virus forms in the Coronaviridae family and predict/classify SARS-CoV-2 (COVID-19) from other selected complete genomes in coronaviridae family using proposed Artificial Neural Network(ANN) algorithm.

Keywords: Coronaviridae family, COVID-19, RNA sequencing, ANN, neural network

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Authors: Rasheed F. G., Hassan H. A., Shehu F. A., Mukhtar M. M., Muhammad Y. Y., Ibrahim S. S., Shehu D., Abdulsalam K., N. Abdullahi

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A cross sectional randomized, descriptive cross sectional study was conducted on the frequency of GSTT1 null alleles in patients diagnosed with type-2-diabetes mellitus (T2DM). A total of 40 patients with T2DM and 10 non-diabetic controls were included in the study. GSTT1 null-alleles genotyping was carried out using multiplex PCR amplification to amplify GSTT1 gene (460bp) while using β-globulin (250bp) as an internal control. The results showed that 55% of T2DM patients had BMI within reference limits, 13% are overweight. Additionally, patients with T2DM were found to have significantly higher (p<0.05) serum levels of glucose, total cholesterol, triglyceride and low density lipoprotein. Furthermore, the presence of null genotype of GSTT1 (deletion in GSTT1) was observed in 28% of diabetic patients. Subjects with GSTT1 deletion have significantly higher (p<0.05) levels of serum glucose, low-density lipoprotein and total cholesterol when compared with individuals without deletion (diabetic and non-diabetic). This results suggests that the deletion of GSTT1 gene might serve as a predisposing factor in the development of T2DM and dyslipideamia

Keywords: diabetes, glutathione-S-transferase, lipid profile, PCR, polymorphism.

Procedia PDF Downloads 96