Search results for: nanopore sequencing
325 Application of ATP7B Gene Mutation Analysis in Prenatal Diagnosis of Wilson’s Disease
Authors: Huong M. T. Nguyen, Hoa A. P. Nguyen, Chi V. Phan, Mai P. T. Nguyen, Ngoc D. Ngo, Van T. Ta, Hai T. Le
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Wilson’s disease is an autosomal recessive disorder of copper metabolism, which is caused by mutation in copper- transporting P-type ATPase (ATP7B). The mechanism of this disease is a failure of hepatic excretion of copper to the bile, and it leads to copper deposits in the liver and other organs. Most clinical symptoms of Wilson’s disease can present as liver disease and/or neurologic disease. Objective: The goal of the study is prenatal diagnosis for pregnant women at high risk of Wilson’s disease in Northern Vietnam. Material and method: Three probands with clinically diagnosed liver disease were detected in the mutations of 21 exons and exon-intron boundaries of the ATP7B gene by direct Sanger-sequencing. Prenatal diagnoses were performed by amniotic fluid sampling from pregnant women in the 16th-18th weeks of pregnancy after the genotypes of parents with the probands were identified. Result: A total of three different mutations of the probands, including of S105*, P1052L, P1273G, were detected. Among three fetuses which underwent prenatal genetic testing, one fetus was homozygote; two fetuses were carriers. Conclusion: Genetic testing provided a useful method for prenatal diagnosis, and is a basis for genetic counseling.Keywords: ATP7B gene, genetic testing, prenatal diagnosis, pedigree, Wilson disease
Procedia PDF Downloads 455324 Utilization of Two Kind of Recycling Greywater in Irrigation of Syngonium SP. Plants Grown Under Different Water Regime
Authors: Sami Ali Metwally, Bedour Helmy Abou-Leila, Hussien I.Abdel-Shafy
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The work was carried out at the greenhouse of National Research Centre, Pot experiment was carried out during of 2020 and 2021 seasons aimed to study the effect of two types of water (two recycling gray water treatments((SMR (Sequencing Batch Reactor) and MBR(Membrane Biology Reactor) and three watering intervals 15, 20 and 25 days on Syangonium plants growth. Examination of data cleared that, (MBR) recorded increase in vegetative growth parameters, osmotic pressure, transpiration rate chlorophyll a,b,carotenoids and carbohydrate)in compared with SBR.As for water, intervalsthe highest values of most growth parameters were obtained from plants irrigated with after (20 days) compared with other treatments.15 days irrigation intervals recorded significantly increased in osmotic pressure, transpiration rate and photosynthetic pigments, while carbohydrate values recorded decreased. Interaction between water type and water intervals(SBR) recorded the highest values of most growth parameters by irrigation after 20 days. While the treatment (MBR)and irrigated after 25 days showed the highest values on leaf area and leaves fresh weight compared with other treatments.Keywords: grey water, water intervals, Syngonium plant, recycling water, vegetative growth
Procedia PDF Downloads 109323 Cholesterol-Lowering Effects of Lactobacillus plantarum Isolated from Northeastern Thai Fermented Vegetable Brassica juncea (L.)
Authors: T. Warinpramote, J. Sanguanjeen, P. Pholphakwaen, S. Kittisorayut, J. Kerdtubtim, S. Palachote, M. Taweechotipatr
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Cholesterol is a type of lipid molecule which is the significant risk factor for coronary heart disease. Currently, there are many cholesterol-lowering alternative treatments especially bile salt hydrolase positive lactobacilli. BSH can cleave the peptide linkage of bile salt, which results in removal of the amino acid group from the steroid core and increases production of the new bile acid by using more cholesterol. The purpose of this study was to isolate, and screen probiotic characteristics of lactobacilli from fermented Thai foods and further investigated for their comparative BSH activity. The result showed that 2 of 81 Lactobacillus strains, JPK2-2 and JPK3-2, isolated from Brassica juncea (L.) had significantly exhibited high BSH activity. In addition, these Lactobacillus strains showed their results that the ability to tolerate acid and bile salt. Furthermore, the using of 16S rDNA sequencing for definitive microbial identifications showed that these 2 strains belong to Lactobacillus plantarum. In the future, the L. plantarum with BSH activity strains JPK2-2 and JPK3-2 may be the candidate probiotics for application in functional foods and dairy products to improve in the patient with cardiovascular diseases.Keywords: Lactobacillus plantarum, probiotics, bile salt hydrolase, cholesterol-lowering, fermented Thai food
Procedia PDF Downloads 156322 Isolation, Identification and Crude Oil Biodegradation Potential of Providencia sp. BAZ 01
Authors: Aisami A., Z. A. Adamu, Lawan Bulama
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Due to growing issues of crude oil pollution in both marine and terrestrial environments, Billions to Trillions of US Dollars were spent over the years for the treatment of this spill. There is an urgent need for effective bioremediation strategies. This current study focuses on the isolation and characterization of a crude oil-degrading bacterium from hydrocarbon-contaminated soil samples. Soil samples were collected from an oil spill site and subjected to enrichment culture techniques in a mineral salt medium supplemented with crude oil as the singular carbon source. The isolates were screened for their crude oil-degrading capabilities using gravimetric analysis. The most efficient isolation was identified through 16S rRNA gene sequencing. Cultural and physical conditions such pH, temperature salinity and crude oil concentrations were optimized. The isolates showed significant crude oil degradation efficiency, reducing oil concentration (2.5%) by 75% within 15 days of incubation. The strain was identified as Providencia sp. through molecular characterization, the sequence was deposited at the NCBI Genbank with accession number MN880494. The bacterium exhibited optimal growth at 32.5°C, pH 7.0 to 7.5, and in the presence of 1.5% (w/v) NaCl. The isolated Providencia sp. shows encouraging potential for bioremediation of crude oil-contaminated environments. This study successfully isolated and characterized a crude oil-degrading Providencia sp., highlighting its potential in bioremediation.Keywords: crude oil degradation, providencia sp., bioremediation, hydrocarbon utilization, environmental pollution.
Procedia PDF Downloads 44321 Characterization of Biosurfactant during Crude Oil Biodegradation Employing Pseudomonas sp. PG1: A Strain Isolated from Garage Soil
Authors: Kaustuvmani Patowary, Suresh Deka
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Oil pollution accidents, nowadays, have become a common phenomenon and have caused ecological and social disasters. Microorganisms with high oil-degrading performance are essential for bioremediation of petroleum hydrocarbon. In this investigation, an effective biosurfactant producer and hydrocarbon degrading bacterial strain, Pseudomonas sp.PG1 (identified by 16s rDNA sequencing) was isolated from hydrocarbon contaminated garage soil of Pathsala, Assam, India, using crude oil enrichment technique. The growth parameters such as pH and temperature were optimized for the strain and upto 81.8% degradation of total petroleum hydrocarbon (TPH) has been achieved after 5 weeks when grown in mineral salt media (MSM) containing 2% (w/v) crude oil as the carbon source. The biosurfactant production during the course of hydrocarbon degradation was monitored by surface tension measurement and emulsification activity. The produced biosurfactant had the ability to decrease the surface tension of MSM from 72 mN/m to 29.6 mN/m, with the critical micelle concentration (CMC)of 56 mg/L. The biosurfactant exhibited 100% emulsification activity on crude oil. FTIR spectroscopy and LCMS-MS analysis of the purified biosurfactant revealed that the biosurfactant is Rhamnolipidic in nature with several rhamnolipid congeners. Gas Chromatography-Mass spectroscopy (GC-MS) analysis clearly demonstrated that the strain PG1 efficiently degrade different hydrocarbon fractions of the crude oil. The study suggeststhat application of the biosurfactant producing strain PG1 as an appropriate candidate for bioremediation of crude oil contaminants.Keywords: petroleum hydrocarbon, hydrocarbon contamination, bioremediation, biosurfactant, rhamnolipid
Procedia PDF Downloads 355320 Maximizing Bidirectional Green Waves for Major Road Axes
Authors: Christian Liebchen
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Both from an environmental perspective and with respect to road traffic flow quality, planning so-called green waves along major road axes is a well-established target for traffic engineers. For one-way road axes (e.g. the Avenues in Manhattan), this is a trivial downstream task. For bidirectional arterials, the well-known necessary condition for establishing a green wave in both directions is that the driving times between two subsequent crossings must be an integer multiple of half of the cycle time of the signal programs at the nodes. In this paper, we propose an integer linear optimization model to establish fixed-time green waves in both directions that are as long and as wide as possible, even in the situation where the driving time condition is not fulfilled. In particular, we are considering an arterial along whose nodes separate left-turn signal groups are realized. In our computational results, we show that scheduling left-turn phases before or after the straight phases can reduce waiting times along the arterial. Moreover, we show that there is always a solution with green waves in both directions that are as long and as wide as possible, where absolute priority is put on just one direction. Compared to optimizing both directions together, establishing an ideal green wave into one direction can only provide suboptimal quality when considering prioritized parts of a green band (e.g., first few seconds).Keywords: traffic light coordination, synchronization, phase sequencing, green waves, integer programming
Procedia PDF Downloads 117319 Haplotypes of the Human Leukocyte Antigen-G Different HIV-1 Groups from the Netherlands
Authors: A. Alyami, S. Christmas, K. Neeltje, G. Pollakis, B. Paxton, Z. Al-Bayati
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The Human leukocyte antigen-G (HLA-G) molecule plays an important role in immunomodulation. To date, 16 untranslated regions (UTR) HLA-G haplotypes have been previously defined by sequenced SNPs in the coding region. From these, UTR-1, UTR-2, UTR-3, UTR-4, UTR-5, UTR-6 and UTR-7 are the most frequent 3’UTR haplotypes at the global level. UTR-1 is associated with higher levels of soluble HLA-G and HLA-G expression, whereas UTR-5 and UTR-7 are linked with low levels of soluble HLA-G and HLA-G expression. Human immunodeficiency virus type 1 (HIV-1) infection results in the progressive loss of immune function in infected individuals. The virus escape mechanism typically includes T lymphocytes and NK cell recognition and lyses by classical HLA-A and B down-regulation, which has been associated with non-classical HLA-G molecule up-regulation, respectively. We evaluated the haplotypes of the HLA-G 3′ untranslated region frequencies observed in three HIV-1 groups from the Netherlands and their susceptibility to develop infection. The three groups are made up of mainly men who have sex with men (MSM), injection drug users (IDU) and a high-risk-seronegative (HRSN) group. DNA samples were amplified with published primers prior sequencing. According to our results, the low expresser frequencies show higher in HRSN compared to other groups. This is indicating that 3’UTR polymorphisms may be identified as potential prognostic biomarkers to determine susceptibility to HIV.Keywords: Human leukocyte antigen-G (HLA-G) , men who have sex with men (MSM), injection drug users (IDU), high-risk-seronegative (HRSN) group, high-untranslated region (UTR)
Procedia PDF Downloads 154318 Elucidation of the Sequential Transcriptional Activity in Escherichia coli Using Time-Series RNA-Seq Data
Authors: Pui Shan Wong, Kosuke Tashiro, Satoru Kuhara, Sachiyo Aburatani
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Functional genomics and gene regulation inference has readily expanded our knowledge and understanding of gene interactions with regards to expression regulation. With the advancement of transcriptome sequencing in time-series comes the ability to study the sequential changes of the transcriptome. This method presented here works to augment existing regulation networks accumulated in literature with transcriptome data gathered from time-series experiments to construct a sequential representation of transcription factor activity. This method is applied on a time-series RNA-Seq data set from Escherichia coli as it transitions from growth to stationary phase over five hours. Investigations are conducted on the various metabolic activities in gene regulation processes by taking advantage of the correlation between regulatory gene pairs to examine their activity on a dynamic network. Especially, the changes in metabolic activity during phase transition are analyzed with focus on the pagP gene as well as other associated transcription factors. The visualization of the sequential transcriptional activity is used to describe the change in metabolic pathway activity originating from the pagP transcription factor, phoP. The results show a shift from amino acid and nucleic acid metabolism, to energy metabolism during the transition to stationary phase in E. coli.Keywords: Escherichia coli, gene regulation, network, time-series
Procedia PDF Downloads 373317 The Isolation of Enterobacter Ludwigii Strain T976 from Nicotiana Tabacum L. Yunyan 97 and Its Application Study
Authors: Gao Qin, Hu Liwei, Dong Xiangzhou, Zhu Qifa, Cheng Tingming, Zhao Limei, Yang Mengmeng, Zhai Zhen, Dai Huaxin, Liang Taibo, Zhang Shixiang, Xue Chaoqun
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The functional strain T976 for starch degradation was isolated from Nicotiana tabacum L. Yunyan 97 tobacco leaves, the ratio of starch hydrolysis transparent circle diameter to colony diameter of the strain was 4.14, 16S rDNA sequencing identified these strains as Enterobacter ludwigii. Then Enterobacter ludwigii T976 was fermented and spaying Yunyan 97 plant in vigorous growing stage. The results of once spraying fermentation broth of Enterobacter ludwigii T976 showed that starch content of upper leaves decreased slightly, from 3.77% to 3.1%, the reducing sugar content increased from 4.39% to 5.53%, and the total sugar content increased from 5.82% to 7.39%. The chemical content was also checked after three time spraying. The starch content of middle leaves decreased from 5.63% to 3.74%, while the content of total sugar and reducing sugar decreased slightly. And the starch content of upper leaves decreased from 7.62% to 4.78%, the total sugar and reducing sugar decreased slightly, and starch content of middle leaf decreased from 6.27% to 3.62%, the total sugar and reducing sugar did not change much, and other chemical components were in a suitable range.Keywords: nicotiana tabacum, yunyan 97, leaf, starch, degradation, enterobacter ludwigii
Procedia PDF Downloads 57316 Production of Lignocellulosic Enzymes by Bacillus safensis LCX Using Agro-Food Wastes in Solid State Fermentation
Authors: Abeer A. Q. Ahmed, Tracey McKay
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The increasing demand for renewable fuels and chemicals is pressuring manufacturing industry toward finding more sustainable cost-effective resources. Lignocellulose, such as agro-food wastes, is a suitable equivalent to petroleum for fine chemicals and fuels production. The complex structure of lignocellulose, however, requires a variety of enzymes in order to degrade its components into their respective building blocks that can be used further for the production of various value added products. This study aimed to isolate bacterial strain with the ability to produce a variety of lignocellulosic enzymes. One bacterial isolate was identified by 16S rRNA gene sequencing and phylogenetic analysis as Bacillus safensis LCX found to have CMCase, xylanase, manganese peroxidase, lignin peroxidase, and laccase activities. The enzymes production was induced by growing Bacillus safensis LCX in solid state fermentation using wheat straw, wheat bran, and corn stover. The activities of enzymes were determined by specific colorimetric assays. This study presents Bacillus safensis LCX as a promising source for lignocellulosic enzymes. These findings can extend the knowledge on agro-food wastes valorization strategies toward a sustainable production of fuels and chemicals.Keywords: Bacillus safensis LCX, high valued chemicals, lignocellulosic enzymes, solid state fermentation
Procedia PDF Downloads 297315 Decision Tree Based Scheduling for Flexible Job Shops with Multiple Process Plans
Authors: H.-H. Doh, J.-M. Yu, Y.-J. Kwon, J.-H. Shin, H.-W. Kim, S.-H. Nam, D.-H. Lee
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This paper suggests a decision tree based approach for flexible job shop scheduling with multiple process plans, i. e. each job can be processed through alternative operations, each of which can be processed on alternative machines. The main decision variables are: (a) selecting operation/machine pair; and (b) sequencing the jobs assigned to each machine. As an extension of the priority scheduling approach that selects the best priority rule combination after many simulation runs, this study suggests a decision tree based approach in which a decision tree is used to select a priority rule combination adequate for a specific system state and hence the burdens required for developing simulation models and carrying out simulation runs can be eliminated. The decision tree based scheduling approach consists of construction and scheduling modules. In the construction module, a decision tree is constructed using a four-stage algorithm, and in the scheduling module, a priority rule combination is selected using the decision tree. To show the performance of the decision tree based approach suggested in this study, a case study was done on a flexible job shop with reconfigurable manufacturing cells and a conventional job shop, and the results are reported by comparing it with individual priority rule combinations for the objectives of minimizing total flow time and total tardiness.Keywords: flexible job shop scheduling, decision tree, priority rules, case study
Procedia PDF Downloads 358314 Rapid Detection and Differentiation of Camel Pox, Contagious Ecthyma and Papilloma Viruses in Clinical Samples of Camels Using a Multiplex PCR
Authors: A. I. Khalafalla, K. A. Al-Busada, I. M. El-Sabagh
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Pox and pox-like diseases of camels are a group of exanthematous skin conditions that have become increasingly important economically. They may be caused by three distinct viruses: camelpox virus (CMPV), camel contagious ecthyma virus (CCEV) and camel papillomavirus (CAPV). These diseases are difficult to differentiate based on clinical presentation in disease outbreaks. Molecular methods such as PCR targeting species-specific genes have been developed and used to identify CMPV and CCEV, but not simultaneously in a single tube. Recently, multiplex PCR has gained reputation as a convenient diagnostic method with cost- and time–saving benefits. In the present communication, we describe the development, optimization and validation a multiplex PCR assays able to detect simultaneously the genome of the three viruses in one single test allowing for rapid and efficient molecular diagnosis. The assay was developed based on the evaluation and combination of published and new primer sets, and was applied to the detection of 110 tissue samples. The method showed high sensitivity, and the specificity was confirmed by PCR-product sequencing. In conclusion, this rapid, sensitive and specific assay is considered a useful method for identifying three important viruses in specimens from camels and as part of a molecular diagnostic regime.Keywords: multiplex PCR, diagnosis, pox and pox-like diseases, camels
Procedia PDF Downloads 470313 Breast Cancer and BRCA Gene: A Study on Genetic and Environmental Interaction
Authors: Abhishikta Ghosh Roy
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Breast cancer is the most common malignancy among women globally, including India. Human breast cancer results from the genetic and environmental interaction. The present study attempts to understand the molecular heterogeneity of BRCA1 and BRCA2 genes, as well as to understand the association of various lifestyle and reproductive variables for the Breast Cancer risk. The study was conducted amongst 110 patients and 128 controls with total DNA sequencing of flanking and coding regions of BRCA1 BRCA2 genes that revealed ten Single Nucleotide Polymorphisms (SNPs) (6 novels). The controls selected for the study were age, sex and ethnic group matched. After written and informed consent biological samples were collected from the subjects. After detailed molecular analysis, significant (p < 0.005) molecular heterogeneity is revealed in terms of SNPs in BRCA1 (4 Exonic & 1 Intronic) and BRCA2 (2exonic and 3 Intronic) genes. The augmentation study investigated significant (p < 0.05) association with positive family history, early age at menarche, irregular menstrual periods, menopause, prolong contraceptive use, nulliparity, history of abortions, consumption of alcohol and smoking for breast cancer risk. To the best of authors knowledge, this study is the first of its kind, envisaged that the identification of the SNPs and modification of the lifestyle factors might aid to minimize the risk among the Bengalee Hindu females.Keywords: breast cancer, BRCA, lifestyle, India
Procedia PDF Downloads 114312 Association of AGT (M268T) Gene Polymorphism in Diabetes and Nephropathy in Pakistan
Authors: Syed M. Shahid, Rozeena Shaikh, Syeda N. Nawab, Abid Azhar
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Diabetes mellitus (DM) is a prevalent non-communicable disease worldwide. DM may lead to many vascular complications like hypertension, nephropathy, retinopathy, neuropathy and foot infections. Pathogenesis of diabetic nephropathy (DN) is implicated by the polymorphisms in genes encoding the specific components of renin angiotensin aldosterone system (RAAS) which include angiotensinogen (AGT), angiotensin-II receptor and angiotensin converting enzyme (ACE) genes. This study was designed to explore the possible association of AG (M268T) polymorphism in the patients of diabetes and nephropathy in Pakistan. Study subjects included 100 controls, 260 diabetic patients without renal insufficiency and 190 diabetic nephropathy patients with persistent albuminuria. Fasting blood samples were collected from all the subjects after getting institutional ethical approval and informed consent. The biochemical estimations, PCR amplification and direct sequencing for the specific region of AGT gene was carried out. A significantly high frequency of TT genotype and T allele of AGT (M268T) was observed in the patients of diabetes with nephropathy as compared to controls and diabetic patients without any known renal impairment. The TT genotype and T allele of AGT (M268T) polymorphism may be considered as a genetic risk factor for the development and progression of nephropathy in diabetes. Further cross sectional population studies would be of help to establish and confirm the observed possible association of AGT gene variations with development of nephropathy in diabetes.Keywords: RAAS, AGT (M268T), diabetes, nephropathy
Procedia PDF Downloads 528311 Classification of Multiple Cancer Types with Deep Convolutional Neural Network
Authors: Nan Deng, Zhenqiu Liu
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Thousands of patients with metastatic tumors were diagnosed with cancers of unknown primary sites each year. The inability to identify the primary cancer site may lead to inappropriate treatment and unexpected prognosis. Nowadays, a large amount of genomics and transcriptomics cancer data has been generated by next-generation sequencing (NGS) technologies, and The Cancer Genome Atlas (TCGA) database has accrued thousands of human cancer tumors and healthy controls, which provides an abundance of resource to differentiate cancer types. Meanwhile, deep convolutional neural networks (CNNs) have shown high accuracy on classification among a large number of image object categories. Here, we utilize 25 cancer primary tumors and 3 normal tissues from TCGA and convert their RNA-Seq gene expression profiling to color images; train, validate and test a CNN classifier directly from these images. The performance result shows that our CNN classifier can archive >80% test accuracy on most of the tumors and normal tissues. Since the gene expression pattern of distant metastases is similar to their primary tumors, the CNN classifier may provide a potential computational strategy on identifying the unknown primary origin of metastatic cancer in order to plan appropriate treatment for patients.Keywords: bioinformatics, cancer, convolutional neural network, deep leaning, gene expression pattern
Procedia PDF Downloads 301310 Cloning, Expression and Protein Purification of AV1 Gene of Okra Leaf Curl Virus Egyptian Isolate and Genetic Diversity between Whitefly and Different Plant Hosts
Authors: Dalia. G. Aseel
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Begomoviruses are economically important plant viruses that infect dicotyledonous plants and exclusively transmitted by the whitefly Bemisia tabaci. Here, replicative form was isolated from Okra, Cotton, Tomato plants and whitefly infected with Begomoviruses. Using coat protein specific primers (AV1), the viral infection was verified with amplicon at 450 bp. The sequence of OLCuV-AV1 gene was recorded and received an accession number (FJ441605) from Genebank. The phylogenetic tree of OLCuV was closely related to Okra leaf curl virus previously isolated from Cameroon and USA with nucleotide sequence identity of 92%. The protein purification was carried out using His-Tag methodology by using Affinity Chromatography. The purified protein was separated on SDS-PAGE analysis and an enriched expected size of band at 30 kDa was observed. Furthermore, RAPD and SDS-PAGE were used to detect genetic variability between different hosts of okra leaf curl virus (OLCuV), cotton leaf curl virus (CLCuV), tomato yellow leaf curl virus (TYLCuV) and the whitefly vector. Finally, the present study would help to understand the relationship between the whitefly and different economical crops in Egypt.Keywords: okra leaf curl virus, AV1 gene, sequencing, phylogenetic, cloning, purified protein, genetic diversity and viral proteins
Procedia PDF Downloads 150309 Phenotypic and Genotypic Diagnosis of Gaucher Disease in Algeria
Authors: S. Hallal, Z. Chami, A. Hadji-Lehtihet, S. Sokhal-Boudella, A. Berhoune, L. Yargui
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Gaucher disease is the most common lysosomal storage in our population, it is due to a deficiency of β –glucosidase acid. The enzyme deficiency causes a pathological accumulation of undegraded substrate in lysosomes. This metabolic overload is responsible for a multisystemic disease with hepatosplenomegaly, anemia, thrombocytopenia, and bone involvement. Neurological involvement is rare. The laboratory diagnosis of Gaucher disease consists of phenotypic diagnosis by determining the enzymatic activity of β - glucosidase by fluorimetric method, a study by genotypic diagnosis in the GBA gene, limiting the search recurrent mutations (N370S, L444P, 84 GG); PCR followed by an enzymatic digestion. Abnormal profiles were verified by sequencing. Monitoring of treated patients is provided by the determination of chitotriosidase. Our experience spaning a period of 6 years (2007-2014) has enabled us to diagnose 78 patients out of a total of 328 requests from the various departments of pediatrics, internal medicine, neurology. Genotypic diagnosis focused on the entire family of 9 children treated at pediatric CHU Mustapha, which help define the clinical form; or 5 of them had type III disease, carrying the L444P mutation in the homozygous state. Three others were composite (N370/L444P) (N370S/other unintended mutation in our study), and only in one family no recurrent mutation has been found. This molecular study permits screening of heterozygous essential for genetic counseling.Keywords: Gaucher disease, mutations, N370S, L444P
Procedia PDF Downloads 407308 Identification of Functional T Cell Receptors Reactive to Tumor Antigens from the T Cell Repertoire of Healthy Donors
Authors: Isaac Quiros-Fernandez, Angel Cid-Arregui
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Tumor-reactive T cell receptors (TCRs) are being subject of intense investigation since they offer great potential in adoptive cell therapies against cancer. However, the identification of tumor-specific TCRs has proven challenging, for instance, due to the limited expansion capacity of tumor-infiltrating T cells (TILs) and the extremely low frequencies of tumor-reactive T cells in the repertoire of patients and healthy donors. We have developed an approach for rapid identification and characterization of neoepitope-reactive TCRs from the T cell repertoire of healthy donors. CD8 T cells isolated from multiple donors are subjected to a first sorting step after staining with HLA multimers carrying the peptide of interest. The isolated cells are expanded for two weeks, after which a second sorting is performed using the same peptide-HLA multimers. The cells isolated in this way are then processed for single-cell sequencing of their TCR alpha and beta chains. Newly identified TCRs are cloned in appropriate expression vectors for functional analysis on Jurkat, NK92, and primary CD8 T cells and tumor cells expressing the appropriate antigen. We have identified TCRs specifically binding HLA-A2 presenting epitopes of tumor antigens, which are capable of inducing TCR-mediated cell activation and cytotoxicity in target cancer cell lines. This method allows the identification of tumor-reactive TCRs in about two to three weeks, starting from peripheral blood samples of readily available healthy donors.Keywords: cancer, TCR, tumor antigens, immunotherapy
Procedia PDF Downloads 70307 The Diversity of DRB1 Locus of Exon 2 of MHC Molecule of Sudanese Indigenous Desert Sheep
Authors: Muna A. Eissawi, Safaa Abed Elfataah, Haytham Hago, Fatima E Abukunna, Ibtisam Amin Goreish, Nahid Gornas
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The study examined and analyzed the genetic diversity of DRB1locus of exon 2 of major histocompatibility complex of Sudanese desert sheep using PCR-RFLP and DNA sequencing. Five hundred samples belonging to five ecotypes of Desert Sudanese sheep (Abrag (Ab), Ashgar (Ash), Hamari (H), Kabashi (K) and Watish (W) were included. Amplification of exon 2 of the DRB1 gene yielded (300bp) amplified product in different ecotypes. Nine different digestion patterns corresponding to Five distinct alleles were observed with Rsa1 digestion. Genotype (ag) was the most common among all ecotypes, with a percentage comprised (40.4 %). The Hardy-Weinberg equilibrium (HWE) test showed that the studied ecotypes have significantly deviated from the theoretical proportions of Rsa1 patterns; probability values of the Chi-square test for HWE for MHC-DRB1 gene in SDS were 0.00 in all ecotypes. The constructed phylogenetic tree revealed the relation of 22 Sudanese isolates with each other and showed the shared sequences with 47 published foreign sequences randomly selected from different geographic regions. The results of this study highlight the effect of heterozygosity of MHC genes of the Desert sheep of Sudan which may clarify some of genetic back ground of their disease resistance and adaptation to environment.Keywords: desert sheep, MHC, Ovar-DRB1, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP)
Procedia PDF Downloads 78306 Isolation and Screening of Fungal Strains for β-Galactosidase Production
Authors: Parmjit S. Panesar, Rupinder Kaur, Ram S. Singh
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Enzymes are the biocatalysts which catalyze the biochemical processes and thus have a wide variety of applications in the industrial sector. β-Galactosidase (E.C. 3.2.1.23) also known as lactase, is one of the prime enzymes, which has significant potential in the dairy and food processing industries. It has the capability to catalyze both the hydrolytic reaction for the production of lactose hydrolyzed milk and transgalactosylation reaction for the synthesis of prebiotics such as lactulose and galactooligosaccharides. These prebiotics have various nutritional and technological benefits. Although, the enzyme is naturally present in almonds, peaches, apricots and other variety of fruits and animals, the extraction of enzyme from these sources increases the cost of enzyme. Therefore, focus has been shifted towards the production of low cost enzyme from the microorganisms such as bacteria, yeast and fungi. As compared to yeast and bacteria, fungal β-galactosidase is generally preferred as being extracellular and thermostable in nature. Keeping the above in view, the present study was carried out for the isolation of the β-galactosidase producing fungal strain from the food as well as the agricultural wastes. A total of more than 100 fungal cultures were examined for their potential in enzyme production. All the fungal strains were screened using X-gal and IPTG as inducers in the modified Czapek Dox Agar medium. Among the various isolated fungal strains, the strain exhibiting the highest enzyme activity was chosen for further phenotypic and genotypic characterization. The strain was identified as Rhizomucor pusillus on the basis of 5.8s RNA gene sequencing data.Keywords: beta-galactosidase, enzyme, fungal, isolation
Procedia PDF Downloads 253305 Genomic Imprinting as a Possible Epigenetic Cause of Esophageal Atresia
Authors: M. Błoch, P. Karpiński, P. Gasperowicz, R. Płoski, A. Lebioda, P. Skiba, A. Rozensztrauch, D. Patkowski, R. Śmigiel
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Introduction: The cause of the isolated form of esophageal atresia has been yet unknown. Objectives: The primary objective of this study was to indicate epigenetic factors which may play an important role in the etiopathogenesis of esophageal atresia. Methods: We recruited a group of 6 pairs of twins, among whom one of the twins developed EA. The selection of such a group for testing allows for excluding external factors (e.g., infections, drugs, toxins) as the cause of the birth defect. The analyzes were performed with the use of genetic material isolated from the whole blood and esophagus tissue of a patient with EA. The reduced representation bisulphite sequencing (RRBS) technique was used to study the change in the genomic imprinting -a change in the expression of genes, which may be the epigenetic cause of EA. Results: In the course of the analyzes, significant hypomethylation and hypermethylation regions were identified. 65 genes with probably increased expression and 65 with decreased expression were selected. These genes have not been marked in literature as possibly pathogenic in esophageal atresia. However, their participation in the pathogenesis of esophageal atresia cannot be clearly excluded. Conclusion: We suggest a role of hypomethylation or hypermethylation of selected genes as one of the possible epigenetic factors in EA pathogenesis. The use of the RRBS technique in the search for the cause of EA is pioneer research; therefore, it seems necessary to extend the research group to new patients with EA. Acknowledgment: The work was supported by the National Science Centre, Poland, under research project 2016/21/N/NZ5/01927.Keywords: esophageal atresia, epigenetics, embryonic development, surgery, genes expression, twins
Procedia PDF Downloads 75304 Secondary Metabolites Identified from a Pseudoalteromonas rubra Bacterial Strain Isolated from a Fijian Marine Alga
Authors: James Sinclair, Katy Soapi, Brad Carte
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The marine environment has continuously demonstrated to be a rich source of secondary metabolites and bioactive compounds that can address the many pharmaceutical problems facing mankind. The emergence of multidrug resistant pathogens has caused scientists to explore contemporary ways of combating these super bugs. A red-pigmented bacterial strain isolated from a marine alga collected in Fiji was identified to be Pseudoalteromonas rubra from 16s rRNA sequencing. This bacterial strain was cultured using a yeast-peptone media and incubated for five days. The ethyl acetate extract of this bacterium was subjected to chromatographic separation techniques such as vacuum liquid chromatography, flash chromatography, size exclusion chromatography and high-pressure liquid chromatography to yield the pure compound and a number of semi-pure fractions. The crude extract and subsequent purified fractions were analyzed by ultraviolet/visible spectroscopy and mass spectroscopy and was found to contain the compounds ivermectin, stenothricin, cyclo-L-pro-L-val, prodigiosin, mycophenolic acid, phenazine-1-carboxylic acid, eplerenone, staurosporine and pseudoalteromone A. The structure of the pure compound, pseudoalteromone A, was elucidated using NMR 1H, 13C, 1H-1H COSY, HSQC and HMBC spectroscopic data.Keywords: Pseudoalteromonas rubra, Pseudoalteromone A, secondary metabolites, structure elucidation
Procedia PDF Downloads 215303 Bacteriological Characterization of Drinking Water Distribution Network Biofilms by Gene Sequencing Using Different Pipe Materials
Authors: M. Zafar, S. Rasheed, Imran Hashmi
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Very little is concerned about the bacterial contamination in drinking water biofilm which provide a potential source for bacteria to grow and increase rapidly. So as to understand the microbial density in DWDs, a three-month study was carried out. The aim of this study was to examine biofilm in three different pipe materials including PVC, PPR and GI. A set of all these pipe materials was installed in DWDs at nine different locations and assessed on monthly basis. Drinking water quality was evaluated by different parameters and characterization of biofilm. Among various parameters are Temperature, pH, turbidity, TDS, electrical conductivity, BOD, COD, total phosphates, total nitrates, total organic carbon (TOC) free chlorine and total chlorine, coliforms and spread plate counts (SPC) according to standard methods. Predominant species were Bacillus thuringiensis, Pseudomonas fluorescens , Staphylococcus haemolyticus, Bacillus safensis and significant increase in bacterial population was observed in PVC pipes while least in cement pipes. The quantity of DWDs bacteria was directly depended on biofilm bacteria and its increase was correlated with growth and detachment of bacteria from biofilms. Pipe material also affected the microbial community in drinking water distribution network biofilm while Similarity in bacterial species was observed between systems due to same disinfectant dose, time period and plumbing pipes.Keywords: biofilm, DWDs, pipe material, bacterial population
Procedia PDF Downloads 347302 The Occurrence of Sporeformers in Processed Milk from Household Refrigerators and The Effect of Heat Treatment on Bacillus Spores Activation
Authors: Sarisha Devnath, Oluwatosin A. Ijabadeniyi
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In recent years milk contamination has become a major problem in households; due to the likely occurrence of bacteria, even after the milk has been processed. One such genus of bacteria causing unwanted growth is Bacillus. This research project looks at the presence of spore formers in processed milk from household refrigerators and the effect of pasteurization and high temperature on Bacillus spores activation. 24 samples each of UHT milk and pasteurised milk from 24 households were sampled for the presence of spore formers. While anaerobic spore formers were not found in any of the samples, the average aerobic spore formers in UHT milk and pasteurized milk however were 5.77 cfu/ml and 5.88 cfu/ml respectively. After sequencing, it was detected that the mixed culture contained Bacillus cereus, for both pasteurised and UHT milk samples. For the activation study, raw milk samples were collected and subjected to four different temperatures; 65˚C, 72˚C, 80˚C, 100˚C respectively. Samples were stored for 7 days at 5˚C and 10˚C and analysed daily. The average aerobic spore formers in raw milk for samples stored at 5˚C range between 4.67-6.00 cfu/ml while it ranges between 4.84-6.00 cfu/ml at 10˚C, signifying that the high temperatures could have resulted in germination of dominant spores. Statistical analysis conducted on these results indicated a significant difference between the numbers of colonies present at the different treatment temperatures the bacterium was exposed to. This work showed that household milk may constitute public health risk furthermore; pasteurization and higher temperatures may not be effective to remove aerobic spore formers because of Bacillus spores activation.Keywords: sporeformers, bacillus, spores, activation, milk
Procedia PDF Downloads 440301 Multi-Omics Investigation of Ferroptosis-Related Gene Expression in Ovarian Aging and the Impact of Nutritional Intervention
Authors: Chia-Jung Li, Kuan-Hao Tsui
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As women age, the quality of their oocytes deteriorates irreversibly, leading to reduced fertility. To better understand the role of Ferroptosis-related genes in ovarian aging, we employed a multi-omics analysis approach, including spatial transcriptomics, single-cell RNA sequencing, human ovarian pathology, and clinical biopsies. Our study identified excess lipid peroxide accumulation in aging germ cells, metal ion accumulation via oxidative reduction, and the interaction between ferroptosis and cellular energy metabolism. We used multi-histological prediction of ferroptosis key genes to evaluate 75 patients with ovarian aging insufficiency and then analyzed changes in hub genes after supplementing with DHEA, Ubiquinol CoQ10, and Cleo-20 T3 for two months. Our results demonstrated a significant increase in TFRC, GPX4, NCOA4, and SLC3A2, which were consistent with our multi-component prediction. We theorized that these supplements increase the mitochondrial tricarboxylic acid cycle (TCA) or electron transport chain (ETC), thereby increasing antioxidant enzyme GPX4 levels and reducing lipid peroxide accumulation and ferroptosis. Overall, our findings suggest that supplementation intervention significantly improves IVF outcomes in senescent cells by enhancing metal ion and energy metabolism and enhancing oocyte quality in aging women.Keywords: multi-omics, nutrients, ferroptosis, ovarian aging
Procedia PDF Downloads 104300 Poultry as a Carrier of Chlamydia gallinacea
Authors: Monika Szymańska-Czerwińsk, Kinga Zaręba-Marchewka, Krzysztof Niemczuk
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Chlamydiaceae are Gram-negative bacteria distributed worldwide in animals and humans. One of them is Chlamydia gallinacea recently discovered. Available data show that C. gallinacea is dominant chlamydial agent found in poultry in European and Asian countries. The aim of the studies was screening of poultry flocks in order to evaluate frequency of C. gallinacea shedding and genetic diversity. Sampling was conducted in different regions of Poland in 2019-2020. Overall, 1466 cloacal/oral swabs were collected in duplicate from 146 apparently healthy poultry flocks including chickens, turkeys, ducks, geese and quails. Dry swabs were used for DNA extraction. DNA extracts were screened using a Chlamydiaceae 23S rRNA real-time PCR assay. To identify Chlamydia species, specific real-time PCR assays were performed. Furthermore, selected samples were used for sequencing based on ompA gene fragments and variable domains (VD1-2, VD3-4). In total, 10.3% of the tested flocks were Chlamydiaceae-positive (15/146 farms). The presence of Chlamydiaceae was confirmed mainly in chickens (13/92 farms) but also in turkey (1/19 farms) and goose (1/26 farms) flocks. Eleven flocks were identified as C. gallinacea-positive while four flocks remained unclassified. Phylogenetic analysis revealed at least 16 genetic variants of C. gallinacea. Research showed that Chlamydiaceae occur in a poultry flock in Poland. The strains of C. gallinacea as dominant species show genetic variability.Keywords: C. gallinacea, emerging agent, poultry, real-time PCR
Procedia PDF Downloads 105299 Characterization of Solanum tuberosum Ammonium Transporter Gene Using Bioinformatics Approach
Authors: Adewole Tomiwa Adetunji, Francis Bayo Lewu, Richard Mundembe
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Plants require nitrogen (N) to support desired production levels. There is a need for better understanding of N transport mechanism in order to improve N assimilation by plant root. Nitrogen is available to plants in the form of nitrate or ammonium, which are transported into the cell with the aid of various transport proteins. Ammonium transporters (AMTs) play a role in the uptake of ammonium, the form in which N is preferentially absorbed by plants. Solanum tuberosum AMT1 (StAMT1) was amplified, sequenced and characterized using molecular biology and bioinformatics methods. Nucleotide database sequences were used to design 976 base pairs AMT1-specific primers which include forward primer 5’- GCCATCGCCGCCGCCGG-3’ and reverse primer 5’-GGGTCAGATCCATACCCGC-3’. These primers were used to amplify the Solanum tuberosum AMT1 internal regions. Nucleotide sequencing, alignment and phylogenetic analysis assigned StAMT1 to the AMT1 family due to the clade and high similarity it shared with other plant AMT1 genes. The deduced amino acid sequences showed that StAMT1 is 92%, 83% and 76% similar to Solanum lycopersicum LeAMT1.1, Lotus japonicus LjAMT1.1, and Solanum lycopersicum LeAMT1.2 respectively. StAMT1 fragments were shown to correspond to the 5th-10th trans-membrane domains. Residue StAMT1 D15 is predicted to be essential for ammonium transport, while mutations of StAMT1 S76A may further enhance ammonium transport.Keywords: ammonium transporter, bioinformatics, nitrogen, primers, Solanum tuberosum
Procedia PDF Downloads 229298 Impact of a Locally-Prepared Fermented Alcoholic Beverage from Jaggery on the Gut Bacterial Profile of the Tea-Tribal Populations of Assam, India
Authors: Rupamoni Thakur, Madhusmita Dehingia, Narayan C. Talukdar, Mojibur R. Khan
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The human gut is an extremely active fermentation site and is inhabited by diverse bacterial species. Consumption of alcoholic beverages has been shown to substantially modulate the human gut bacterial profile (GBP) of an individual. Assam, a major north-eastern state of India, is home to a number of tribal populations of which the tea-tribes form a major community. These tea-tribal communities are known to prepare and consume a locally-prepared alcoholic beverage from fermented jaggery, whose chemical composition is unknown. In this study, we demonstrate the effect of daily intake of the locally-prepared alcoholic beverage on the GBP of the tea-tribal communities and correlate it with the changes in the biochemical biomarkers of the population. The fecal bacterial diversity of 40 drinkers and 35 non-drinking healthy individuals were analyzed by polymerase chain reaction (PCR)–denaturing gradient gel electrophoresis (DGGE). The results suggested that the GBP was significantly modulated in the fermented-beverage consuming subjects. Significant difference was also observed in the serum biochemical parameters such as triglyceride, total cholesterol and the liver marker enzymes (ASAT/ALAT and GGT). Further studies to identify the GBP of drinkers vs non-drinkers through Next-generation Sequencing (NGS) analysis and to correlate the changes with the biochemical biomarkers of the population is underway.Keywords: alcoholic beverage, gut bacterial profile, PCR-DGGE analysis, tea-tribes of India
Procedia PDF Downloads 330297 Identification of Two Novel Carbapenemase Gene Variants from a Carbapenem-Resistant Aeromonas Veronii Environmental Isolate
Authors: Rafael Estrada, Cristian Ruiz Rueda
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Carbapenems are last-resort antibiotics used in clinical settings to treat antibiotic-resistant bacterial infections. Thus, the emergence and spread of resistance to carbapenems is a major public health concern. Here, we have studied a carbapenem-resistant Aeromonas veronii strain previously isolated from a water sample from Sam Simeon Creek (Hearst San Simeon State Park, CA). Analysis of this isolate using disk-diffusion, CarbaNP, eCIM and mCIM assays revealed that it was resistant to amoxicillin-clavulanic acid and all carbapenems tested and that this isolate produced a potentially novel carbapenemase of the Metallo-β-lactamase family. Whole genome sequencing analysis revealed that this A. veronii isolate carries a novel variant of the blacₚₕₐ class β-carbapenemase gene that was closely related to the blacₚₕₐ₇ gene of Aeromonas jandaei. This isolate also carried a novel variant of the blaₒₓₐ class D carbapenemase gene that was most closely related to the blaₒₓₐ-₉₁₂ gene found in other Aeromonas veronii isolates. Finally, we also identified a novel class C β-lactamase gene moderately related to the blaFₒₓ-₁₇ gene of Providencia stuartii and other blaFₒₓ variants identified in Klebsiella pneumoniae, Escherichia coli and other Enterobacteriaceae. Overall, our findings reveal that environmental isolates are an important reservoir of multiple carbapenemases and other β-lactamases of clinical significance.Keywords: β-lactamases, carbapenem, antibiotic-resistant, aeromonas veronii
Procedia PDF Downloads 93296 Distribution and Taxonomy of Marine Fungi in Nha Trang Bay and Van Phong Bay, Vietnam
Authors: Thu Thuy Pham, Thi Chau Loan Tran, Van Duy Nguyen
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Marine fungi play an important role in the marine ecosystems. Marine fungi also supply biomass and metabolic products of industrial value. Currently, the biodiversity of marine fungi along the coastal areas of Vietnam has not yet been studied fully. The objective of this study is to assess the spatial and temporal diversity of planktonic fungi from the coastal waters of Nha Trang Bay and Van Phong Bay in Central Vietnam using culture-dependent and independent approach. Using culture-dependent approach, filamentous fungi and yeasts were isolated on selective media and then classified by phenotype and genotype based on the sequencing of ITS (internal transcribed spacers) regions of rDNA with two primer pairs (ITS1F_KYO2 and ITS4; NS1 and NS8). Using culture-independent approach, environmental DNA samples were isolated and amplified using fungal-specific ITS primer pairs. A total of over 160 strains were isolated from 10 seawater sampling stations at 50 cm depth. They were classified into diverse genera and species of both yeast and mold. At least 5 strains could be potentially novel species. Our results also revealed that planktonic fungi were molecularly diverse with hundreds of phylotypes recovered across these two bays. The results of the study provide data about the distribution and taxonomy of mycoplankton in this area, thereby allowing assessment of their positive role in the biogeochemical cycle of coastal ecosystems and the development of new bioactive compounds for industrial applications.Keywords: biodiversity, ITS, marine fungi, Nha Trang Bay, Van Phong Bay
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