Search results for: Ammonia monooxygenase α-subunit gene

Authors: L. L. Tundisi, A. Pessoa Jr., E. B. Tambourgi, E. Silveira, P. G. Mazzola

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L-asparaginase (L-ASNase) is the gold standard treatment for acute lymphoblastic leukemia that mainly affects pediatric patients; treatment increases survival from 20% to 90%. The characterization of other L-Asparaginases, apart from the most used from Escherichia coli and Erwinia chrysanthemi, has been reported, but the choice of the most appropriate is still under debate. This choice should be based on its pharmacokinetics, immune hypersensitivity, doses, prices, pharmacodynamics. The main factors influencing the antileukemic activity of ASNase are enzymatic activity, Km, glutaminase activity, clearance of the enzyme and development of resistance. However, most of the commercialized enzyme present an intrinsic glutaminase activity, which is responsible for some side effects. In this study, glutaminase free asparaginase produced from Aspergillus oryzae was precipitated in different percentages of ethanol (0–80%), until optimum ethanol concentration of 60% (w/w) was found. Following, precipitation of crude L-ASNase was performed in a single step, using 60% (w/w) ethanol, under constant agitation and temperature. It presented activity of 135.45 U/mg and after gel filtration chromatography with Sephadex G-the enzymatic activity was 322.02 U/mg. The apparent molecular mass of the purified L-ASNase fraction was estimated by 10% SDS-PAGE. Proteins were stained with Coomassie Brilliant Blue R-250. The molar mass range was from 10 kDa to 250 kDa. L-ASNase from Aspergillus oryzae was characterized aiming possible therapeutic use. Four different buffers (phosphate-citrate buffer pH 2.6 to 5.8; phosphate buffer pH 5.8 to 7.4; Tris - HCl pH 7.4 to 9.0; and carbonate buffer pH 9.8 to 10.6) were used to measure the optimum pH for L-ASNase activity. The optimum temperature for enzyme activity was measured at optimal pH conditions (Tris-HCl and phosphate buffer, pH 7.4) at different temperatures ranging from 5 to 55°C. All activities were calculated by quantifying the free ammonia, using the Nessler reagent. The kinetic parameters calculation, e.g. Michaelis-Menten constant (Km), maximum velocity (Vmax) and Hills coefficient (n), were performed by incubating the enzyme in different concentrations of the substrate at optimum conditions of pH and fitted on Hill’s equation. This glutaminase free asparaginase showed a low Km (3.39 mM and 3.81 mM) and enzymatic activity of 135.45 U/mg after precipitation with ethanol. After gel filtration chromatography it rose to 322.02 U/mg. Optimum activity was found between pH 5.8 - 9.0, best activity results with phosphate buffer pH 7.4 and Tris-HCl pH 7.4 and showed activity from 5°C to 55°C. These results indicate that L-ASNase from A. oryzae has the potential for human use.

Keywords: biopharmaceuticals, bioprocessing, bioproducts, biotechnology, enzyme activity, ethanol precipitation

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Authors: Ricardo Alberto Chiong Zevallos, Eduardo Moraes Rego Reis

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Pancreatic adenocarcinoma (PDAC) patients have a less than 10% 5-year survival rate. PDAC has no defined diagnostic and prognostic biomarkers. Gemcitabine is the first-line drug in PDAC and several other cancers. Long non-coding RNAs (lncRNAs) contribute to the tumorigenesis and are potential biomarkers for PDAC. Although lncRNAs aren’t translated into proteins, they have important functions. LncRNAs can decoy or recruit proteins from the epigenetic machinery, act as microRNA sponges, participate in protein translocation through different cellular compartments, and even promote chemoresistance. The chromatin remodeling enzyme EZH2 is a histone methyltransferase that catalyzes the methylation of histone 3 at lysine 27, silencing local expression. EZH2 is ambivalent, it can also activate gene expression independently of its histone methyltransferase activity. EZH2 is overexpressed in several cancers and interacts with lncRNAs, being recruited to a specific locus. EZH2 can be recruited to activate an oncogene or silence a tumor suppressor. The lncRNAs misregulation in cancer can result in the differential recruitment of EZH2 and in a distinct epigenetic landscape, promoting chemoresistance. The relevance of the EZH2-lncRNAs interaction to chemoresistant PDAC was assessed by Real Time quantitative PCR (RT-qPCR) and RNA Immunoprecipitation (RIP) experiments with naïve and gemcitabine-resistant PDAC cells. The expression of several lncRNAs and EZH2 gene targets was evaluated contrasting naïve and resistant cells. Selection of candidate genes was made by bioinformatic analysis and literature curation. Indeed, the resistant cell line showed higher expression of chemoresistant-associated lncRNAs and protein coding genes. RIP detected lncRNAs interacting with EZH2 with varying intensity levels in the cell lines. During RIP, the nuclear fraction of the cells was incubated with an antibody for EZH2 and with magnetic beads. The RNA precipitated with the beads-antibody-EZH2 complex was isolated and reverse transcribed. The presence of candidate lncRNAs was detected by RT-qPCR, and the enrichment was calculated relative to INPUT (total lysate control sample collected before RIP). The enrichment levels varied across the several lncRNAs and cell lines. The EZH2-lncRNA interaction might be responsible for the regulation of chemoresistance-associated genes in multiple cancers. The relevance of the lncRNA-EZH2 interaction to PDAC was assessed by siRNA knockdown of a lncRNA, followed by the analysis of the EZH2 target expression by RT-qPCR. The chromatin immunoprecipitation (ChIP) of EZH2 and H3K27me3 followed by RT-qPCR with primers for EZH2 targets also assess the specificity of the EZH2 recruitment by the lncRNA. This is the first report of the interaction of EZH2 and lncRNAs HOTTIP and PVT1 in chemoresistant PDAC. HOTTIP and PVT1 were described as promoting chemoresistance in several cancers, but the role of EZH2 is not clarified. For the first time, the lncRNA LINC01133 was detected in a chemoresistant cancer. The interaction of EZH2 with LINC02577, LINC00920, LINC00941, and LINC01559 have never been reported in any context. The novel lncRNAs-EZH2 interactions regulate chemoresistant-associated genes in PDAC and might be relevant to other cancers. Therapies targeting EZH2 alone weren’t successful, and a combinatorial approach also targeting the lncRNAs interacting with it might be key to overcome chemoresistance in several cancers.

Keywords: epigenetics, chemoresistance, long non-coding RNAs, pancreatic cancer, histone modification

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Authors: Adenike Adesanya, Nonthaphat Wong, Xiang-Yun Lan, Shea Ping Yip, Chien-Ling Huang

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Background: The most challenging mutation of the oncokinase BCR-ABL protein T315I, which is commonly known as the “gatekeeper” mutation and is notorious for its strong resistance to almost all tyrosine kinase inhibitors (TKIs), especially imatinib. Therefore, this study aims to identify T315I-dependent downstream microRNA (miRNA) pathways associated with drug resistance in chronic myeloid leukemia (CML) for prognostic and therapeutic purposes. Methods: T315I-carrying K562 cell clones (K562-T315I) were generated by the CRISPR-Cas9 system. Imatinib-treated K562-T315I cells were subjected to small RNA library preparation and next-generation sequencing. Putative lncRNA-miRNA-mRNA networks were analyzed with (i) DESeq2 to extract differentially expressed miRNAs, using Padj value of 0.05 as cut-off, (ii) STarMir to obtain potential miRNA response element (MRE) binding sites of selected miRNAs on lncRNA H19, (iii) miRDB, miRTarbase, and TargetScan to predict mRNA targets of selected miRNAs, (iv) IntaRNA to obtain putative interactions between H19 and the predicted mRNAs, (v) Cytoscape to visualize putative networks, and (vi) several pathway analysis platforms – Enrichr, PANTHER and ShinyGO for pathway enrichment analysis. Moreover, mitochondria isolation and transcript quantification were adopted to determine the new mechanism involved in T315I-mediated resistance of CML treatment. Results: Verification of the CRISPR-mediated mutagenesis with digital droplet PCR detected the mutation abundance of ≥80%. Further validation showed the viability of ≥90% by cell viability assay, and intense phosphorylated CRKL protein band being detected with no observable change for BCR-ABL and c-ABL protein expressions by Western blot. As reported by several investigations into hematological malignancies, we determined a 7-fold increase of H19 expression in K562-T315I cells. After imatinib treatment, a 9-fold increment was observed. DESeq2 revealed 171 miRNAs were differentially expressed K562-T315I, 112 out of these miRNAs were identified to have MRE binding regions on H19, and 26 out of the 112 miRNAs were significantly downregulated. Adopting the seed-sequence analysis of these identified miRNAs, we obtained 167 mRNAs. 6 hub miRNAs (hsa-let-7b-5p, hsa-let-7e-5p, hsa-miR-125a-5p, hsa-miR-129-5p, and hsa-miR-372-3p) and 25 predicted genes were identified after constructing hub miRNA-target gene network. These targets demonstrated putative interactions with H19 lncRNA and were mostly enriched in pathways related to cell proliferation, senescence, gene silencing, and pluripotency of stem cells. Further experimental findings have also shown the up-regulation of mitochondrial transcript and lncRNA MALAT1 contributing to the lncRNA-miRNA-mRNA networks induced by BCR-ABL T315I mutation. Conclusions: Our results have indicated that lncRNA-miRNA regulators play a crucial role not only in leukemogenesis but also in drug resistance, considering the significant dysregulation and interactions in the K562-T315I cell model generated by CRISPR-Cas9. In silico analysis has further shown that lncRNAs H19 and MALAT1 bear several complementary miRNA sites. This implies that they could serve as a sponge, hence sequestering the activity of the target miRNAs.

Keywords: chronic myeloid leukemia, imatinib resistance, lncRNA-miRNA-mRNA, T315I mutation

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Authors: Salma M. Wakil

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Advances in the field of genetics overwhelmed detecting large number of inherited disorders at the molecular level and directed to the development of innovative technologies. These innovations have led to gene sequencing, prenatal mutation detection, pre-implantation genetic diagnosis; population based carrier screening and genome wide analyses using microarrays. Microarrays are widely used in establishing clinical and diagnostic setup for genetic anomalies at a massive level, with the advent of cytoscan molecular karyotyping as a clinical utility card for detecting chromosomal aberrations with high coverage across the entire human genome. Unlike a regular karyotype that relies on the microscopic inspection of chromosomes, molecular karyotyping with cytoscan constructs virtual chromosomes based on the copy number analysis of DNA which improves its resolution by 100-fold. We have been investigating a large number of patients with Developmental Delay and Intellectual disability with this platform for establishing micro syndrome deletions and have detected number of novel CNV’s in the Arabian population with the clinical relevance.

Keywords: microarrays, molecular karyotyping, developmental delay, genetics

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Authors: Eugene Stepanov, Arkadi Ponossov

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Many mathematical models used in biological and other applications are ill-posed. The reason for that is the nature of differential equations, where the nonlinearities are assumed to be step functions, which is done to simplify the analysis. Prominent examples are switched systems arising from gene regulatory networks and neural field equations. This simplification leads, however, to theoretical and numerical complications. In the presentation, it is proposed to apply the theory of hybrid feedback controls to regularize the problem. Roughly speaking, one attaches a finite state control (‘automaton’), which follows the trajectories of the original system and governs its dynamics at the points of ill-posedness. The construction of the automaton is based on the classification of the attractors of the specially designed adjoint dynamical system. This ‘hybridization’ is shown to regularize the original switched system and gives rise to efficient hybrid numerical schemes. Several examples are provided in the presentation, which supports the suggested analysis. The method can be of interest in other applied fields, where differential equations contain step-like nonlinearities.

Keywords: hybrid feedback control, ill-posed problems, singular perturbation analysis, step-like nonlinearities

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Authors: Apichai Sawisit, Sirima Suvarnakuta Jantama, Sunthorn Kanchanatawee, Lonnie O. Ingram, Kaemwich Jantama

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Escherichia coli KJ122 was engineered to produce succinate from glucose using the wild type GalP for glucose uptake instead of the native phosphotransferase system (ptsI mutation). This strain ferments 10% (w/v) xylose poorly. Mutants were selected by serial transfers in AM1 mineral salts medium with 10% (w/v) xylose. Evolved mutants exhibited a similar improvement, co-fermentation of an equal mixture of xylose and glucose. One of these, AS1600a, produced 84.26±1.37 g/L succinate, equivalent to that produced by the parent (KJ122) strain from 10% glucose (85.46±1.78 g/L). AS1600a was sequenced and found to contain a mutation in galactose permease (GalP, G236D). Expressing the galP* mutation gene in KJ122ΔgalP resembled the xylose utilization phenotype of the mutant AS1600a. The strain AS1600a and KJ122ΔgalP (pLOI5746; galP*) also co-fermented a mixture of glucose, xylose, arabinose, and galactose in sugarcane bagasse hydrolysate for succinate production.

Keywords: xylose, furfural, succinate, sugarcane bagasse, E. coli

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Authors: Yu-Ju Hsieh, Lin-Hung Lin, Wen-Hui Chang

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This study was designed as an interventional research and intended to validate whether the introduction of drinking vinegar every day can reduce and even prevent urinary tract infection in Taiwan home stayed disabilities who using indwelling catheter. The data was collected from the subjects who have received home care case at northern Taiwan, according to the questionnaire and a medical records retroactive methodology, the subjects were informed and consent to drink 15ml of table vinegar in a daily diet, and through routine urine testing and culture study. Home care nurses would assist collecting urine at the point of before and after a meal from total 35 studied subjects per month, and total collected 4 times for testing. The results showed that when the average age of study subjects was 65.46 years and catheter indwelling time was 15 years, drinking table vinegar could inhibit the activity of E. coli O157: H7 and reduce its breeding. Before drinking table vinegar daily, the subjects’ urine pH value was 7.0-8.0, and the average was 7.5, and the urine PH value dropped to 6.5 after drinking table vinegar for a month. There were two purple urine cases whose urine were changed from purple to normal color after two weeks of drinking, and the protein and bacteria values of urine gradually improved. Urine smell unpleasant before attending to this study, and the symptom improved significantly only after 1 week, and the urine smell returned to normal ammonia and became clean after 1 month later. None of these subjects received treatment in a hospital due to urinary tract infection, and there were no signs of bleeding in all cases during this study. The subjects of this study are chronic patients with a long-term bedridden catheterization; drinking cranberry juice is an economic burden for them, and also highly prohibited for diabetes patients. By adapting to use cheaper table vinegar to acidified urine and improve its smell and ease Purple Urine Syndrome, to furthermore, proven urinary tract infection, it can also to reduce the financial burden on families, the cost of social resources and the rate of re-admission.

Keywords: table vinegar, urinary tract infection, disability patients, long-term indwelling catheter

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Authors: Ellana Boada, Eric Santos-Clotas, Alba Cabrera-Codony, Maria Martin, Lluis Baneras, Frederic Gich

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Volatile methylsiloxanes (VMS) are a group of manmade silicone compounds widely used in household and industrial applications that end up on the biogas produced through the anaerobic digestion of organic matter in landfills and wastewater treatment plants. The presence of VMS during the biogas energy conversion can cause damage on the engines, reducing the efficiency of this renewable energy source. Non regenerative adsorption onto activated carbon is the most widely used technology to remove siloxanes from biogas, while new trends point out that biotechnology offers a low-cost and environmentally friendly alternative to conventional technologies. The first objective of this research was to enrich, isolate and identify bacterial species able to grow using siloxane molecules as a sole carbon source: anoxic wastewater sludge was used as initial inoculum in liquid anoxic enrichments, adding D4 (as representative siloxane compound) previously adsorbed on activated carbon. After several months of acclimatization, liquid enrichments were plated onto solid media containing D4 and thirty-four bacterial isolates were obtained. 16S rRNA gene sequencing allowed the identification of strains belonging to the following species: Ciceribacter lividus, Alicycliphilus denitrificans, Pseudomonas aeruginosa and Pseudomonas citronellolis which are described to be capable to degrade toxic volatile organic compounds. Kinetic assays with 8 representative strains revealed higher cell growth in the presence of D4 compared to the control. Our second objective was to characterize the community composition and diversity of the microbial community present in the enrichments and to elucidate whether the isolated strains were representative members of the community or not. DNA samples were extracted, the 16S rRNA gene was amplified (515F & 806R primer pair), and the microbiome analyzed from sequences obtained with a MiSeq PE250 platform. Results showed that the retrieved isolates only represented a minor fraction of the microorganisms present in the enrichment samples, which were represented by Alpha, Beta, and Gamma proteobacteria as dominant groups in the category class thus suggesting that other microbial species and/or consortia may be important for D4 biodegradation. These results highlight the need of additional protocols for the isolation of relevant D4 degraders. Currently, we are developing molecular tools targeting key genes involved in siloxane biodegradation to identify and quantify the capacity of the isolates to metabolize D4 in batch cultures supplied with a synthetic gas stream of air containing 60 mg m⁻³ of D4 together with other volatile organic compounds found in the biogas mixture (i.e. toluene, hexane and limonene). The isolates were used as inoculum in a biotrickling filter containing lava rocks and activated carbon to assess their capacity for siloxane removal. Preliminary results of biotrickling filter performance showed 35% of siloxane biodegradation in a contact time of 14 minutes, denoting that biological siloxane removal is a promising technology for biogas upgrading.

Keywords: bacterial cultivation, biogas upgrading, microbiome, siloxanes

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Authors: Alexis John de Britto

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Studies were performed to select the superior genotypes based on intra-specific variations, caused by phytogeographical, climatic and edaphic parameters of three anti cancer drug yielding mangrove plants such as Acanthus ilicifolius L., Calophyllum inophyllum L. and Excoecaria agallocha L. using ISSR (Inter Simple Sequence Repeats) markers and phytochemical analysis such as preliminary phytochemical tests, TLC, HPTLC, HPLC and antioxidant tests. The plants were collected from five different geographical locations of the East Coast of south India. Genetic heterozygosity, Nei’s gene diversity, Shannon’s information index and Percentage of polymorphism between the populations were calculated using POPGENE software. Cluster analysis was performed using UPGMA algorithm. AMOVA and correlations between genetic diversity and soil factors were analyzed. Combining the molecular and phytochemical variations superior genotypes were selected. Conservation constraints and methods of efficient exploitation of the species are discussed.

Keywords: anti-cancer drug yielding plants, DNA fingerprinting, phytochemical analysis, selection of superior genotypes

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Authors: Henrik L. Funke, Lars Ulke-Winter, Sandra Gelbrich, Lothar Kroll

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This work reports about an approach for an automatic adaptation of concrete formulations based on genetic algorithms (GA) to optimize a wide range of different fit-functions. In order to achieve the goal, a method was developed which provides a numerical description of a fibre reinforced concrete (FRC) mixture regarding the production technology and the property spectrum of the concrete. In a first step, the FRC mixture with seven fixed components was characterized by varying amounts of the components. For that purpose, ten concrete mixtures were prepared and tested. The testing procedure comprised flow spread, compressive and bending tensile strength. The analysis and approximation of the determined data was carried out by GAs. The aim was to obtain a closed mathematical expression which best describes the given seven-point cloud of FRC by applying a Gene Expression Programming with Free Coefficients (GEP-FC) strategy. The seven-parametric FRC-mixtures model which is generated according to this method correlated well with the measured data. The developed procedure can be used for concrete mixtures finding closed mathematical expressions, which are based on the measured data.

Keywords: concrete design, fibre reinforced concrete, genetic algorithms, GEP-FC

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Authors: Ivana Cabarkapa, Zorica Tomicic, Olivera Duragic

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Antimicrobial resistance represents one of the major challenges facing humanity in the last decades. Increasing antibiotic-resistant pathogens indicates the need for the development of alternative antibacterial drugs and new treatment strategies. One of the innovative emerging treatments in overcoming multidrug-resistant pathogens certainly represents the inhibition anti-quorum sensing system. For most of the food-borne pathogens, the expression of the virulence depends on their capability communication with other members of the population by means of quorum sensing (QS). QS represents a specific way of bacterial intercellular communication, which enabled owing to their ability to detect and to respond to cell population density by gene regulation. QS mechanisms are responsible for controls the pathogenesis, virulence luminescence, motility, sporulation and biofilm formation of many organisms by regulating gene expression. Therefore, research in this field is being an attractive target for the development of new natural antibacterial agents. Anti-QS compounds are known to have the ability to prohibit bacterial pathogenicity. Considering the importance of quorum sensing during bacterial pathogenesis, this research has been focused on evaluation anti - QS properties of four essential oils (EOs) Origanum heracleoticum, Origanum vulgare, Thymus vulgare, and Thymus serpyllum, using biomonitor strain of Chromobacterium violaceum CV026. Tests conducted on Luria Bertani agar supplemented with N hexanol DL homoserine lacton (HHL) 10µl/50ml of agar. The anti-QS potential of the EOs was assayed in a range of concentrations of 200 – 0.39 µl/ml using the disc diffusion method. EOs of Th. vulgaris and T. serpyllum were exhibited anti-QS activity indicated by a non- pigmented ring with a dilution-dependent manner. The lowest dilution of EOs T. vulgaris and T. serpyllum in which they exhibited visually detectable inhibition of violacein synthesis was 6.25 µl/ml for both tested EOs. EOs of O. heracleoticum and O. vulgare were displayed different active principles, i.e., antimicrobial activity indicated by the inner clear ring and anti-QS activity indicated by the outer non-pigmented ring, in a concentration-dependent manner. The lowest dilution of EOs of O. heracleoticum and O. vulgare in which exhibited visually detectable inhibition of violacein synthesis was 1.56 and 3.25 µl/ml, respectively. Considering that, the main constituents of the tested EOs represented by monoterpenes (carvacrol, thymol, γ-terpinene, and p-cymene), anti - QS properties of tested EOs can be mainly attributed to their activity. In particular, from the scientific literature, carvacrol and thymol show a sub-inhibitory effect against foodborne pathogens. Previous studies indicated that sub-lethal concentrations of carvacrol reduced the mobility of bacteria due to the ability of interference using QS mechanism between the bacterial cells, and thereby reducing the ability of biofilm formation The precise mechanism by which carvacrol inhibits biofilm formation is still not fully understood. Our results indicated that EOs displayed different active principles, i.e., antimicrobial activity indicated by the inner clear ring and anti-QS activity indicated by an outer non- pigmented ring with visually detectable inhibition of violacein. Preliminary results suggest that EOs represent a promising alternative for effective control of the emergence and spread of resistant pathogens.

Keywords: anti-quorum sensing activity, Chromobacterium violaceum, essential oils, violacein

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Authors: Mohammad Asadpour, Gholamreza Razmi, Gholamreza Mohammadi, Abolghasem Naghibi

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Cryptosporidium Spp., protozoan parasites of the phylum Apicomplexa, have a wide spectrum of hosts including humans, domestic animals and wild mammals, birds, reptiles, amphibians and fish. Dairy cattle have been identified in numerous reports as a major source of environmental contamination with this pathogen. In this study, a Polymerase Chain Reaction (PCR), Restriction Fragment Length Polymorphism (RFLP) analysis of the Small-Subunit (SSU) rRNA gene was used to detect and identify Cryptosporidium Spp. in 300 fecal specimens from 1 to 30 days pre-wean calves in 10 farms in Mashhad, Iran. Eighty five (28.3%) and forty five (15%) of the specimens were positive for Cryptosporidium by microscopic and PCR examination respectively. Restriction digestion of the PCR products by VSPI and Ssp1 restriction enzymes and analysis of sequence data revealed the presence of C. parvum, bovine genotype in all isolates. Our findings suggest that cattle can be a source of Cryptosporidial infections for humans and animals in Mashhad area. This is the first published description of Cryptosporidium sub genotyping in Mashhad.

Keywords: cryptosporidium, genotype, dairy calves, 18S rRNA, Mashhad

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Authors: Asiye Ciftci, Zeki Kaya

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Populus nigra is one of the most economically and ecologically important forest trees in Turkey, well known for its rapid growth, good ability to vegetative propagation and the extreme uses of its wood. Due to overexploitation, loss of natural distribution area and extreme hybridization and introgression, Populus nigra is one of the most threatened tree species in Turkey and Europe. Using 20 nuclear microsatellite loci, the genetic structure of European black poplar populations along the two largest rivers of Turkey was analyzed. All tested loci were highly polymorphic, displaying 5 to 15 alleles per locus. Observed heterozygosity (overall Ho = 0.79) has been higher than the expected (overall He = 0.58) in each population. Low level of genetic differentiation among populations (FST= 0,03) and excess of heterozygotes for each river were found. Human-mediated dispersal, phenotypic selection, high level of gene flow and extensive circulations of clonal materials may cause those situations. The genetic data obtained from this study could provide the basis for efficient in situ and ex-situ conservation and restoration of species natural populations in its natural habitat as well as having sustainable breeding and poplar plantations in the future.

Keywords: populus, clonal, loci, ex situ

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Authors: Austin Belfiori, Kevin Rinek, Zach Barcroft, Jennifer Berglind

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Diet influences the composition of the gut microbiota and host's health. Disruption of the balance among the microbiota, epithelial cells, and resident immune cells in the intestine is involved in the pathogenesis of inflammatory bowel disease (IBD). To study the role of gut microbiota in intestinal inflammation, the microbiome of control mice (C57BL6) given a gluten-containing standard diet versus C57BL6 mice given the gluten-free (GF) feed (n=10 in each group) was examined. All mice received the 3% DSS for 5 days. Throughout the study, feces were collected and processed for DNA extraction and MiSeq Illumina sequencing of V4 region of bacterial 16S rRNA gene. Alpha and beta diversities and compositional differences at phylum and genus levels were determined in intestinal microbiota. The mice receiving the GF diet showed a significantly increased abundance of Firmicutes and a decrease of Bacteroides and Lactobacillus at phylum level. Therefore, the gluten free diet led to reductions in beneficial gut bacteria populations. These findings indicate a role of wheat gluten in dysbiosis of the intestinal microbiota.

Keywords: gluten, colitis, microbiota, DSS, dextran sulphate sodium

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Authors: Ghulam Abbas Shah, Maqsood Sadiq, Ahsan Yasin

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Massive global plastic production, combined with poor degradation and recycling, leads to significant environmental pollution from microplastics, whose effects on plants in the soil remain understudied. Besides, effective mitigation strategies and their impact on ammonia (NH₃) emissions under varying fertilizer management practices remains sketchy. Therefore, the objectives of the study were (i) to determine the impact of organic fertilizers on the toxicity of microplastics in sorghum and physicochemical characteristics of microplastics-contaminated soil and (ii) to assess the impacts of these fertilizers on NH₃ emissions from this soil. A field experiment was conducted using sorghum as a test crop. Treatments were: (i) Control (C), (ii) Microplastics (MP), (iii) Inorganic fertilizer (IF), (iv) MPIF, (v) Farmyard manure (FM), (vi) MPFM, (vii) Biochar (BC), and (viii) MPBC, arranged in a randomized complete block design (RCBD) with three replicates. Microplastics of polyvinyl chloride (PVC) were applied at a rate of 1.5 tons ha-¹, and all fertilizers were applied at the recommended dose of 90 kg N ha-¹. Soil sampling was done before sowing and after harvesting the sorghum, with samples analyzed for chemical properties and microbial biomass. Crop growth and yield attributes were measured. In a parallel pot experiment, NH₃ emissions were measured using passive flux samplers over 72 hours following the application of treatments similar to those used in the field experiment. Application of MPFM, MPBC and MPIF reduced soil mineral nitrogen by 8, 20 and 38% compared to their sole treatments, respectively. Microbial biomass carbon (MBC) was reduced by 19, 25 and 59% in MPIF, MPBC and MPFM as compared to their sole application, respectively. Similarly, the respective reduction in microbial biomass nitrogen (MBN) was 10, 27 and 66%. The toxicity of microplastics was mitigated by MPFM and MPBC, each with only a 5% reduction in grain yield of sorghum relative to their sole treatments. The differences in nitrogen uptake between BC vs. MPBC, FM vs. MPFM, and IF vs. MPIF were 8, 10, and 12 kg N ha-¹, respectively, indicating that organic fertilizers mitigate microplastic toxicity in the soil. NH₃ emission was reduced by 5, 11 and 20% after application of MPFM, MPBC and MPIF than their sole treatments, respectively. The study concludes that organic fertilizers such as FM and BC can effectively mitigate the toxicity of microplastics in soil, leading to improved crop growth and yield.

Keywords: microplastics, soil characteristics, crop n uptake, biochar, NH₃ emissions

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Authors: Delly C. Lestari, Linosefa, Ardiana Kusumaningrum, Andi Yasmon, Anis Karuniawati

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The objective of this study is to determine the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) harboring mecA genes from screening isolates among intensive care unit (ICU) patients. All MRSA screening isolates from ICU’s patients of Cipto Mangunkusumo Hospital during 2011 and 2014 were included in this study. Identification and susceptibility test was performed using Vitek2 system (Biomereux®). PCR was conducted to characterize the SCCmec of S. aureus harboring the mecA gene on each isolate. Patient’s history of illness was traced through medical record. 24 isolates from 327 screening isolates were MRSA positive (7.3%). From PCR, we found 17 (70.8%) isolates carrying SCCmec type I, 3 (12.5%) isolates carrying SCCmec type III, and 2 (8.3%) isolates carrying SCCmec type IV. In conclusion, SCCmec type I is the most prevalent MRSA colonization among ICU patients in Cipto Mangunkusumo Hospital.

Keywords: MRSA, mecA genes, ICU, colonization

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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: Apotiola Z. O., Anyakorah C. I., Kuforiji O. O.

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Sensory and microbial properties of fresh and canned Calocybe indica (milky mushroom) were evaluated. The mushroom was grown under a controlled environment with hardwood (Cola nitida) and rice bran substrate (4:1) canned in a brine solution of salt and citric acid. Analysis was carried out using standard methods. The overall acceptability ranged between 5.62 and 6.50, with sample S30 adjudged the best. In all, significant differences p<0.01 exist in the panelist judgment. Thus, the incorporation of salt and citric acid at 3.5g and 1.5g, respectively, improved sensory attributes such as texture, aroma, color, and overall acceptability. There was no coliform and fungi growth on the samples throughout the storage period. The bacterial count, on the other hand, was observed only in the fifth and sixth week of the storage period which varied between 0.2 to 0.9 x 103 cfu/g. The highest value was observed in sample S20 of the sixth week of storage, while the lowest value was recorded in sample S30 of the sixth week of storage. Based on 16S rRNA gene sequencing, bacterial species were taxonomically confirmed as Bacillus thuringiensis. The percentile compositions and Sequence ID of the bacterial species in the mushroom was 90%.

Keywords: bacterial count, microbial property, sensory, sawdust, texture

Procedia PDF Downloads 53

Authors: Gizachew Mulugeta Manahelohe, Khidmet Safarovich Shikhaliev

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There has been a significant surge in interest in the synthesis of heterocyclic compounds that contain hydroquinoline fragments. This surge can be attributed to the broad range of pharmaceutical and industrial applications that these compounds possess. The present study provides a comprehensive account of the synthesis of both linear and fused heterocyclic systems that incorporate hydroquinoline fragments. Furthermore, the pharmacological activity spectra of the synthesized compounds were assessed using the in silico method, employing the prediction of activity spectra of substances (PASS) program. Hydroquinoline nitriles 7 and 8 were prepared through the reaction of the corresponding hydroquinolinecarbaldehyde using a hydroxylammonium chloride/pyridine/toluene system and iodine in aqueous ammonia under ambient conditions, respectively. 2-Phenyl-1,3-oxazol-5(4H)-ones 9a,b and 10a,b were synthesized via the condensation of compounds 5a,b and 6a,b with hippuric acid in acetic acid in 30–60% yield. When activated, 7-methylazolopyrimidines 11a and b were reacted with N-alkyl-2,2,4-trimethyl-1,2,3,4-tetrahydroquinoline-6-carbaldehydes 6a and b, and triazolo/pyrazolo[1,5-a]pyrimidin-6-yl carboxylic acids 12a and b were obtained in 60–70% yield. The condensation of 7-hydroxy-1,2,3,4-tetramethyl-1,2-dihydroquinoline 3 h with dimethylacetylenedicarboxylate (DMAD) and ethyl acetoacetate afforded cyclic products 16 and 17, respectively. The condensation reaction of 6-formyl-7-hydroxy-1,2,2,4-tetramethyl-1,2-dihydroquinoline 5e with methylene-active compounds such as ethyl cyanoacetate/dimethyl-3-oxopentanedioate/ethyl acetoacetate/diethylmalonate/Meldrum’s acid afforded 3-substituted coumarins containing dihydroquinolines 19 and 21. Pentacyclic coumarin 22 was obtained via the random condensation of malononitrile with 5e in the presence of a catalytic amount of piperidine in ethanol. The biological activities of the synthesized compounds were assessed using the PASS program. Based on the prognosis, compounds 13a, b, and 14 exhibited a high likelihood of being active as inhibitors of gluconate 2-dehydrogenase, as well as possessing antiallergic, antiasthmatic, and antiarthritic properties, with a probability value (Pa) ranging from 0.849 to 0.870. Furthermore, it was discovered that hydroquinoline carbonitriles 7 and 8 tended to act as effective progesterone antagonists and displayed antiallergic, antiasthmatic, and antiarthritic effects (Pa = 0.276–0.827). Among the hydroquinolines containing coumarin moieties, compounds 17, 19a, and 19c were predicted to be potent progesterone antagonists, with Pa values of 0.710, 0.630, and 0.615, respectively.

Keywords: heterocyclic compound, hydroquinoline, Vilsmeier–Haack formulation, quinolone

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Authors: Manal Farea, Adam Husein, Ahmad S. Halim, Zurairah Berahim, Nurul A. Abdullah, Khairani I. Mokhtar, Kasmawati Mokhtar

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Endodontic furcal perforation remains both an endodontic and a periodontal problem. Regeneration of cementum is very essential for the perforation repair. The aim of this study was to investigate the role of Hertwig's epithelial root sheath (HERS) cells on the cementogenic differentiation of stem cells derived from human exfoliated deciduous teeth (SHED) in the presence of chitosan scaffold-TGFβ1. HERS cells were isolated and characterized then co-cultured with SHED with/without chitosan scaffold-TGFβ1. SHED proliferation was assessed by PrestoBlue. Alkaline phosphatase activity, mineralization behaviour and gene/protein expression of cemento/osteoblast phenotype of SHED were evaluated. Results of the present study showed that HERS cells in association with chitosan-TGFβ1 enhanced proliferation and cemento/osteogenic differentiation of SHED. Our novel co-culture system confirmed the potential effect of HERS cells to stimulate the differentiation of SHED along the cementoblastic lineage which was triggered in the presence of chitosan-TGFβ1. This approach possesses a novel therapeutic strategy for future endodontic perforation and periodontitis.

Keywords: cementogenesis, co-culture system, HERS, SHED

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Authors: Sireetorn Kuharat, Anwar Beg

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In this study, multi-mode heat transfer characteristics of spacecraft solar collectors are investigated computationally. Two-dimensional steady-state incompressible laminar Newtonian viscous convection-radiative heat transfer in a rectangular solar collector geometry. The ANSYS FLUENT finite volume code (version 17.2) is employed to simulate the thermo-fluid characteristics. Several radiative transfer models are employed which are available in the ANSYS workbench, including the classical Rosseland flux model and the more elegant P1 flux model. Mesh-independence tests are conducted. Validation of the simulations is conducted with a computational Harlow-Welch MAC (Marker and Cell) finite difference method and excellent correlation. The influence of aspect ratio, Prandtl number (Pr), Rayleigh number (Ra) and radiative flux model on temperature, isotherms, velocity, the pressure is evaluated and visualized in color plots. Additionally, the local convective heat flux is computed and solutions are compared with the MAC solver for various buoyancy effects (e.g. Ra = 10,000,000) achieving excellent agreement. The P1 model is shown to better predict the actual influence of solar radiative flux on thermal fluid behavior compared with the limited Rosseland model. With increasing Rayleigh numbers the hot zone emanating from the base of the collector is found to penetrate deeper into the collector and rises symmetrically dividing into two vortex regions with very high buoyancy effect (Ra >100,000). With increasing Prandtl number (three gas cases are examined respectively hydrogen gas mixture, air and ammonia gas) there is also a progressive incursion of the hot zone at the solar collector base higher into the solar collector space and simultaneously a greater asymmetric behavior of the dual isothermal zones. With increasing aspect ratio (wider base relative to the height of the solar collector geometry) there is a greater thermal convection pattern around the whole geometry, higher temperatures and the elimination of the cold upper zone associated with lower aspect ratio.

Keywords: thermal convection, radiative heat transfer, solar collector, Rayleigh number

Procedia PDF Downloads 113

Authors: Doaa Hashad, Amany Elbanna, Abeer Ibrahim, Gihan Khedr

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Background: H19 is one of the long non coding RNAs (LncRNA) that is related to the progression of many diseases including cancers. This work was carried out to study the level of the long non-coding RNA; H119, in plasma of patients with gastric cancer (GC) and to assess its significance in their clinical management. Methods: A total of sixty-two participants were enrolled in the present study. The first group included thirty-two GC patients, while the second group was formed of thirty age and sex matched healthy volunteers serving as a control group. Plasma samples were used to assess H19 gene expression using real time quantitative PCR technique. Results: H19 expression was up-regulated in GC patients with positive correlation to TNM cancer stages. Conclusions: Up-regulation of H19 is closely associated with gastric cancer and correlates well with tumor staging. Convenient, efficient quantification of H19 in plasma using real time PCR technique implements its role as a potential noninvasive prognostic biomarker in gastric cancer, that predicts patient’s outcome and most importantly as a novel target in gastric cancer treatment with better performance achieved on using both CEA and H19 simultaneously.

Keywords: biomarker, gastric, cancer, LncRNA

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Authors: Jesús M. Martín, Francisco Corona, Dolores Hidalgo

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Landfills present long-term threats to soil, air, groundwater and surface water due to the formation of greenhouse gases (methane gas and carbon dioxide) and leachate from decomposing garbage. The composition of leachate differs from site to site and also within the landfill. The leachates alter with time (from weeks to years) since the landfilled waste is biologically highly active and their composition varies. Mainly, the composition of the leachate depends on factors such as characteristics of the waste, the moisture content, climatic conditions, degree of compaction and the age of the landfill. Therefore, the leachate composition cannot be generalized and the traditional treatment models should be adapted in each case. Although leachate composition is highly variable, what different leachates have in common is hazardous constituents and their potential eco-toxicological effects on human health and on terrestrial ecosystems. Since leachate has distinct compositions, each landfill or dumping site would represent a different type of risk on its environment. Nevertheless, leachates consist always of high organic concentration, conductivity, heavy metals and ammonia nitrogen. Leachate could affect the current and future quality of water bodies due to uncontrolled infiltrations. Therefore, control and treatment of leachate is one of the biggest issues in urban solid waste treatment plants and landfills design and management. This work presents a treatment model that will be carried out "in-situ" using a cost-effective novel technology that combines solar evaporation/condensation plus forward osmosis. The plant is powered by renewable energies (solar energy, biomass and residual heat), which will minimize the carbon footprint of the process. The final effluent quality is very high, allowing reuse (preferred) or discharge into watercourses. In the particular case of this work, the final effluents will be reused for cleaning and gardening purposes. A minority semi-solid residual stream is also generated in the process. Due to its special composition (rich in metals and inorganic elements), this stream will be valorized in ceramic industries to improve the final products characteristics.

Keywords: forward osmosis, landfills, leachate valorization, solar evaporation

Procedia PDF Downloads 198

Authors: M. N. Hanina, M. Hairul Shahril, I. Ismatul Nurul Asyikin, A. K. Abdul Jalil, M. R. Salina, M. R. Maryam, M. Rosfarizan

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The extracellular proteins secreted by bacteria may be increased in stressful surroundings, such as in the presence of antibiotics. It appears that many antibiotics, when used at low concentrations, have in common the ability to activate or repress gene transcription, which is distinct from their inhibitory effect. There have been comparatively few studies on the potential of antibiotics as a specific chemical signal that can trigger a variety of biological functions. Therefore, this study was carried out to determine the effect of Streptomycin Sulfate in regulating extracellular proteins secreted by Bacillus subtilis ATCC21332. Results of Microdilution assay showed that the Minimum Inhibition Concentration (MIC) of Streptomycin Sulfate on B. subtilis ATCC21332 was 2.5 mg/ml. The bacteria cells were then exposed to Streptomycin Sulfate at concentration of 0.01 MIC before being further incubated for 48h to 72 h. The extracellular proteins secreted were then isolated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins profile revealed that three additional bands with approximate sizes of 30 kDa, 22 kDa and 23 kDa were appeared for the treated bacteria with Streptomycin Sulfate. Thus, B. subtilis ATCC21332 in stressful condition with the presence of Streptomycin Sulfate at low concentration could induce the extracellular proteins secretion.

Keywords: Bacillus subtilis ATCC21332, streptomycin sulfate, extracellular proteins, antibiotics

Procedia PDF Downloads 277

Authors: Khiem M. Nguyen, Ming C. Yang

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Chloroplast pigments are extremely important during photosynthesis since they play essential roles in light absorption and energy transfer. Therefore, understanding the efficiency of chlorophyll (Chl) biosynthesis could facilitate enhancement in photo-assimilates accumulation, and ultimately, in crop yield. The Chl-deficient mutants have been used extensively to study the Chl biosynthetic pathways and the biogenesis of the photosynthetic apparatus. Rice (Oryza sativa L.) is one of the most leading food crops, serving as staple food for many parts of the world. To author’s best knowledge, Chl b–lacking rice has been found; however the molecular mechanism of Chl biosynthesis still remains unclear compared to wild-type rice. In this study, the ultrastructure analysis, photosynthetic properties, and transcriptome profile of wild-type rice (Norin No.8, N8) and its Chl b-lacking mutant (Chlorina 1, C1) were examined. The finding concluded that total Chl content and Chl b content in the C1 leaves were strongly reduced compared to N8 leaves, suggesting that reduction in the total Chl content contributes to leaf color variation at the physiological level. Plastid ultrastructure of C1 possessed abnormal thylakoid membranes with loss of starch granule, large number of vesicles, and numerous plastoglobuli. The C1 rice also exhibited thinner stacked grana, which was caused by a reduction in the number of thylakoid membranes per granum. Thus, the different Chl a/b ratio of C1 may reflect the abnormal plastid development and function. Transcriptional analysis identified 23 differentially expressed genes (DEGs) and 671 transcription factors (TFs) that were involved in Chl metabolism, chloroplast development, cell division, and photosynthesis. The transcriptome profile and DEGs revealed that the gene encoding PsbR (PSII core protein) was down-regulated, therefore suggesting that the lower in light-harvesting complex proteins are responsible for the lower photosynthetic capacity in C1. In addition, expression level of cell division protein (FtsZ) genes were significantly reduced in C1, causing chloroplast division defect. A total of 19 DEGs were identified based on KEGG pathway assignment involving Chl biosynthesis pathway. Among these DEGs, the GluTR gene was down-regulated, whereas the UROD, CPOX, and MgCH genes were up-regulated. Observation through qPCR suggested that later stages of Chl biosynthesis were enhanced in C1, whereas the early stages were inhibited. Plastid structure analysis together with transcriptomic analysis suggested that the Chl a/b ratio was amplified both by the reduction in Chl contents accumulation, owning to abnormal chloroplast development, and by the enhanced conversion of Chl b to Chl a. Moreover, the results indicated the same Chl-cycle pattern in the wild-type and C1 rice, indicating another Chl b degradation pathway. Furthermore, the results demonstrated that normal grana stacking, along with the absence of Chl b and greatly reduced levels of Chl a in C1, provide evidence to support the conclusion that other factors along with LHCII proteins are involved in grana stacking. The findings of this study provide insight into the molecular mechanisms that underlie different Chl a/b ratios in rice.

Keywords: Chl-deficient mutant, grana stacked, photosynthesis, RNA-Seq, transcriptomic analysis

Procedia PDF Downloads 112

Authors: N. A. Kazemi Sefat, M. M. Mohammadi, J. Hadjati, S. Talebi, M. Ajami, H. Daneshvar

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Colorectal cancer metastases result in a significant number of cancer related deaths. Histone deacetylase (HDAC) inhibitors induce growth arrest and apoptosis in a variety of human cancer cells. Sodium butyrate (SB) is a short chain fatty acid, belongs to HDAC inhibitors which is released in the colonic lumen as a consequence of fiber fermentation. In this study, we are about to assess the effect of sodium butyrate on HCT116 human colorectal carcinoma cell line. The viability of cells was measured by microscopic morphologic study and MTT assay. After 48 hours, treatments more than 10 mM lead to cell injury in HCT116 by increasing cell granulation and decreasing cell adhesion (p>0.05). After 72 hours, treatments at 10 mM and more lead to significant cell injury (p<0.05). Our results may suggest that the gene expression which is contributed in cell proliferation and apoptosis has been changed under pressure of HDAC inhibition.

Keywords: colorectal cancer, sodium butyrate, cytotoxicity, MTT

Procedia PDF Downloads 357

Authors: Alexander A. Stakheev, Larisa V. Samokhvalova, Sergey K. Zavriev

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Fusarium fungi are among the most devastating plant pathogens distributed all over the world. Significant reduction of grain yield and quality caused by Fusarium leads to multi-billion dollar annual losses to the world agricultural production. These organisms can also cause infections in immunocompromised persons and produce the wide range of mycotoxins, such as trichothecenes, fumonisins, and zearalenone, which are hazardous to human and animal health. Identification of Fusarium fungi based on the morphology of spores and spore-forming structures, colony color and appearance on specific culture media is often very complicated due to the high similarity of these features for closely related species. Modern Fusarium taxonomy increasingly uses data of crossing experiments (biological species concept) and genetic polymorphism analysis (phylogenetic species concept). A number of novel Fusarium sibling species has been established using DNA barcoding techniques. Species recognition is best made with the combined phylogeny of intron-rich protein coding genes and ribosomal DNA sequences. However, the internal transcribed spacer of (ITS), which is considered to be universal DNA barcode for Fungi, is not suitable for genus Fusarium, because of its insufficient variability between closely related species and the presence of non-orthologous copies in the genome. Nowadays, the translation elongation factor 1 alpha (TEF1α) gene is the “gold standard” of Fusarium taxonomy, but the search for novel informative markers is still needed. In this study, we used two novel DNA markers, frataxin (FXN) and heat shock protein 90 (HSP90) to discover phylogenetic relationships between Fusarium species. Multilocus phylogenetic analysis based on partial sequences of TEF1α, FXN, HSP90, as well as intergenic spacer of ribosomal DNA (IGS), beta-tubulin (β-TUB) and phosphate permease (PHO) genes has been conducted for 120 isolates of 19 Fusarium species from different climatic zones of Russia and neighboring countries using maximum likelihood (ML) and maximum parsimony (MP) algorithms. Our analyses revealed that FXN and HSP90 genes could be considered as informative phylogenetic markers, suitable for evolutionary and taxonomic studies of Fusarium genus. It has been shown that PHO gene possesses more variable (22 %) and parsimony informative (19 %) characters than other markers, including TEF1α (12 % and 9 %, correspondingly) when used for elucidating phylogenetic relationships between F. avenaceum and its closest relatives – F. tricinctum, F. acuminatum, F. torulosum. Application of novel DNA barcodes confirmed the fact that F. arthrosporioides do not represent a separate species but only a subspecies of F. avenaceum. Phylogeny based on partial PHO and FXN sequences revealed the presence of separate cluster of four F. avenaceum strains which were closer to F. torulosum than to major F. avenaceum clade. The strain F-846 from Moldova, morphologically identified as F. poae, formed a separate lineage in all the constructed dendrograms, and could potentially be considered as a separate species, but more information is needed to confirm this conclusion. Variable sites in PHO sequences were used for the first-time development of specific qPCR-based diagnostic assays for F. acuminatum and F. torulosum. This work was supported by Russian Foundation for Basic Research (grant № 15-29-02527).

Keywords: DNA barcode, fusarium, identification, phylogenetics, taxonomy

Procedia PDF Downloads 319

Authors: Seyedeh Nasim Mirbahari, Taha Azad, Mehdi Totonchi

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Oncolytic viruses, which only replicate in tumor cells, are being extensively studied for their use in cancer therapy. A particular virus known as the vaccinia virus, a member of the poxvirus family, has demonstrated oncolytic abilities glioma. Treating Glioma with traditional methods such as chemotherapy and radiotherapy is quite challenging. Even though oncolytic viruses have shown immense potential in cancer treatment, their effectiveness in glioblastoma treatment is still low. Therefore, there is a need to improve and optimize immunotherapies for better results. In this study, we have designed oncoVV-TT, which can more effectively target tumor cells while minimizing replication in normal cells by replacing the thymidine kinase gene with a luc-p2a-GFP gene expression cassette. Human glioblastoma cell line U251 MG, rat glioblastoma cell line C6, and non-tumor cell line HFF were plated at 105 cells in a 12-well plates in 2 mL of DMEM-F2 medium with 10% FBS added to each well. Then incubated at 37°C. After 16 hours, the cells were treated with oncoVV-TT at an MOI of 0.01, 0.1 and left in the incubator for a further 24, 48, 72 and 96 hours. Viral replication assay, fluorescence imaging and viability tests, including trypan blue and crystal violet, were conducted to evaluate the cytotoxic effect of oncoVV-TT. The finding shows that oncoVV-TT had significantly higher cytotoxic activity and proliferation rates in tumor cells in a dose and time-dependent manner, with the strongest effect observed in U251 MG. To conclude, oncoVV-TT has the potential to be a promising oncolytic virus for cancer treatment, with a more cytotoxic effect in human glioblastoma cells versus rat glioma cells. To assess the effectiveness of vaccinia virus-mediated viral therapy, we have tested U251mg and C6 tumor cell lines taken from human and rat gliomas, respectively. The study evaluated oncoVV-TT's ability to replicate and lyse cells and analyzed the survival rates of the tested cell lines when treated with different doses of oncoVV-TT. Additionally, we compared the sensitivity of human and mouse glioma cell lines to the oncolytic vaccinia virus. All experiments regarding viruses were conducted under biosafety level 2. We engineered a Vaccinia-based oncolytic virus called oncoVV-TT to replicate specifically in tumor cells. To propagate the oncoVV-TT virus, HeLa cells (5 × 104/well) were plated in 24-well plates and incubated overnight to attach to the bottom of the wells. Subsequently, 10 MOI virus was added. After 48 h, cells were harvested by scraping, and viruses were collected by 3 sequential freezing and thawing cycles followed by removal of cell debris by centrifugation (1500 rpm, 5 min). The supernatant was stored at −80 ◦C for the following experiments. To measure the replication of the virus in Hela, cells (5 × 104/well) were plated in 24-well plates and incubated overnight to attach to the bottom of the wells. Subsequently, 5 MOI virus or equal dilution of PBS was added. At the treatment time of 0 h, 24 h, 48 h, 72 h and 96 h, the viral titers were determined under the fluorescence microscope (BZ-X700; Keyence, Osaka, Japan). Fluorescence intensity was quantified using the imagej software according to the manufacturer’s protocol. For the isolation of single-virus clones, HeLa cells seeded in six-well plates (5×105 cells/well). After 24 h (100% confluent), the cells were infected with a 10-fold dilution series of TianTan green fluorescent protein (GFP)virus and incubated for 4 h. To examine the cytotoxic effect of oncoVV-TT virus ofn U251mg and C6 cell, trypan blue and crystal violet assay was used.

Keywords: oncolytic virus, immune therapy, glioma, vaccinia virus

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Authors: Mohamed A. Dkhil, Denis Delic, Saleh Al-Quraishy

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Coccidiosis causes considerable economic loss in the poultry industry. The current study aimed to investigate the response of goblet cells as well as the induced tissue damage during Eimeria papilata infection. Mice were infected with sporulated E. papillata oocyts. On day 5 post-infection, the fecal output was determined. Also, the jejunum was prepared for the histological, histochemical and molecular studies. Our results revealed that the intestinal coccidian infection with E. papillata induced a marked goblet cell hypoplasia and depleted mucus secretion. Also, the infection was able to alter the jejuna architecture and increased the apoptotic cells inside the villi. In addition, the real time PCR results indicated that, the inflammatory cytokines TNF-α, iNOS, IFN-y and IL-1β were significantly up-regulated. In contrast, the mRNA expression patterns of IL-6 in response to E. papillata infection did not differ significantly between control and infected mice. Moreover, the mRNA expression of TLR4 was significantly up-regulated, whereas the expression of MUC2 is significantly down-regulated upon infection. Further studies are required to understand the regulatory mechanisms of goblet cells related genes.

Keywords: goblet cells, Eimeria papillata, mice, jejunum

Procedia PDF Downloads 269

Authors: Palash Soni, Vivek Kumar Gaba, Shubhankar Bhowmick, Bidyut Mazumdar

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Refrigeration technology is a fast developing field at the present era since it has very wide application in both domestic and industrial areas. It started from the usage of simple ice coolers to store food stuffs to the present sophisticated cold storages along with other air conditioning system. A variety of techniques are used to bring down the temperature below the ambient. Adsorption refrigeration technology is a novel, advanced and promising technique developed in the past few decades. It gained attention due to its attractive property of exploiting unlimited natural sources like solar energy, geothermal energy or even waste heat recovery from plants or from the exhaust of locomotives to fulfill its energy need. This will reduce the exploitation of non-renewable resources and hence reduce pollution too. This work is aimed to develop a model for a solar adsorption refrigeration system and to simulate the same for different operating conditions. In this system, the mechanical compressor is replaced by a thermal compressor. The thermal compressor uses renewable energy such as solar energy and geothermal energy which makes it useful for those areas where electricity is not available. Refrigerants normally in use like chlorofluorocarbon/perfluorocarbon have harmful effects like ozone depletion and greenhouse warming. It is another advantage of adsorption systems that it can replace these refrigerants with less harmful natural refrigerants like water, methanol, ammonia, etc. Thus the double benefit of reduction in energy consumption and pollution can be achieved. A thermodynamic model was developed for the proposed adsorber, and a universal MATLAB code was used to simulate the model. Simulations were carried out for a different operating condition for the silicagel-methanol working pair. Various graphs are plotted between regeneration temperature, adsorption capacities, the coefficient of performance, desorption rate, specific cooling power, adsorption/desorption times and mass. The results proved that adsorption system could be installed successfully for refrigeration purpose as it has saving in terms of power and reduction in carbon emission even though the efficiency is comparatively less as compared to conventional systems. The model was tested for its compliance in a cold storage refrigeration with a cooling load of 12 TR.

Keywords: adsorption, refrigeration, renewable energy, silicagel-methanol

Procedia PDF Downloads 201