Search results for: bacterial gene expression

Authors: Leo Nnamdi Ozurumba-Dwight

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Messenger ribooxynucleic acid (mRNA) molecules are compositional, protein-based. These proteins, encoding mRNA molecules (which collectively connote the transcriptome), when analyzed by RNA sequencing (RNAseq), unveils the nature of gene expression in the RNA. The obtained gene expression provides clues of cellular traits and their dynamics in presentations. These can be studied in relation to function and responses. RNAseq is a practical concept in Genomics as it enables detection and quantitative analysis of mRNA molecules. Single cell and spatial transcriptomics both present varying avenues for expositions in genomic characteristics of single cells and pooled cells in disease conditions such as cancer, auto-immune diseases, hematopoietic based diseases, among others, from investigated biological tissue samples. Single cell transcriptomics helps conduct a direct assessment of each building unit of tissues (the cell) during diagnosis and molecular gene expressional studies. A typical technique to achieve this is through the use of a single-cell RNA sequencer (scRNAseq), which helps in conducting high throughput genomic expressional studies. However, this technique generates expressional gene data for several cells which lack presentations on the cells’ positional coordinates within the tissue. As science is developmental, the use of complimentary pre-established tissue reference maps using molecular and bioinformatics techniques has innovatively sprung-forth and is now used to resolve this set back to produce both levels of data in one shot of scRNAseq analysis. This is an emerging conceptual approach in methodology for integrative and progressively dependable transcriptomics analysis. This can support in-situ fashioned analysis for better understanding of tissue functional organization, unveil new biomarkers for early-stage detection of diseases, biomarkers for therapeutic targets in drug development, and exposit nature of cell-to-cell interactions. Also, these are vital genomic signatures and characterizations of clinical applications. Over the past decades, RNAseq has generated a wide array of information that is igniting bespoke breakthroughs and innovations in Biomedicine. On the other side, spatial transcriptomics is tissue level based and utilized to study biological specimens having heterogeneous features. It exposits the gross identity of investigated mammalian tissues, which can then be used to study cell differentiation, track cell line trajectory patterns and behavior, and regulatory homeostasis in disease states. Also, it requires referenced positional analysis to make up of genomic signatures that will be sassed from the single cells in the tissue sample. Given these two presented approaches to RNA transcriptomics study in varying quantities of cell lines, with avenues for appropriate resolutions, both approaches have made the study of gene expression from mRNA molecules interesting, progressive, developmental, and helping to tackle health challenges head-on.

Keywords: transcriptomics, RNA sequencing, single cell, spatial, gene expression.

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Authors: Mudasir Ahmad Syed, Raashid Ahmd Wani, Mashooq Ahmad Dar, Uneeb Urwat, Riaz Ahmad Shah, Nazir Ahmad Ganai

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Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) is a primary avian pathogen responsible for severe intestinal pathology in younger chickens and economic losses. However, the Salmonella Typhimurium is also able to cause infection in humans, described by typhoid fever and acute gastro-intestinal disease. A study was conducted at days to investigate pathological, histopathological, haemato-biochemical, immunological and expression kinetics of NRAMP (natural resistance associated macrophage protein) gene family (NRAMP1 and NRAMP2) in broiler chickens following experimental infection of Salmonella Typhimurium at 0,1,3,5,7,9,11,13 and 15 days respectively. Infection was developed in birds through oral route at 2×108 CFU/ml. Clinical symptoms appeared 4 days post infection (dpi) and after one-week birds showed progressive weakness, anorexia, diarrhea and lowering of head. On postmortem examination, liver showed congestion, hemorrhage and necrotic foci on surface, while as spleen, lungs and intestines revealed congestion and hemorrhages. Histopathological alterations were principally observed in liver in second week post infection. Changes in liver comprised of congestion, areas of necrosis, reticular endothelial hyperplasia in association with mononuclear cell and heterophilic infiltration. Hematological studies confirm a significant decrease (P<0.05) in RBC count, Hb concentration and PCV. White blood cell count showed significant increase throughout the experimental study. An increase in heterophils was found up to 7dpi and a decreased pattern was observed afterwards. Initial lymphopenia followed by lymphocytosis was found in infected chicks. Biochemical studies showed a significant increase in glucose, AST and ALT concentration and a significant decrease (P<0.05) in total protein and albumin level in the infected group. Immunological studies showed higher titers of IgG in infected group as compared to control group. The real time gene expression of NRAMPI and NRAMP2 genes increased significantly (P<0.05) in infected group as compared to controls. The peak expression of NRAMP1 gene was seen in liver, spleen and caecum of infected birds at 3dpi, 5dpi and 7dpi respectively, while as peak expression of NRAMP2 gene in liver, spleen and caecum of infected chicken was seen at 9dpi, 5dpi and 9dpi respectively. This study has role in diagnostics and prognostics in the poultry industry for the detection of salmonella infections at early stages of poultry development.

Keywords: biochemistry, histopathology, NRAMP, poultry, real time expression, Salmonella Typhimurium

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Authors: Yuriy A. Knirel

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Enteric bacteria Escherichia coli is the predominant facultative anaerobe of the colonic flora, and some specific serotypes are associated with enteritis, hemorrhagic colitis, and hemolytic uremic syndrome. Shigella spp. are human pathogens that cause diarrhea and bacillary dysentery (shigellosis). They are in effect E. coli with a specific mode of pathogenicity. Strains of Salmonella enterica are responsible for a food-borne infection (salmonellosis), and specific serotypes cause typhoid fever and paratyphoid fever. All these bacteria are closely related in respect to structure and genetics of the lipopolysaccharide, including the O-polysaccharide part (O‑antigen). Being exposed to the bacterial cell surface, the O antigen is subject to intense selection by the host immune system and bacteriophages giving rise to diverse O‑antigen forms and providing the basis for typing of bacteria. The O-antigen forms of many bacteria are unique, but some are structurally and genetically related to others. The sequenced O-antigen gene clusters between conserved galF and gnd genes were analyzed taking into account the O-antigen structures established by us and others for all S. enterica and Shigella and most E. coli O-serogroups. Multiple genetic mechanisms of diversification of the O-antigen forms, such as lateral gene transfer and mutations, were elucidated and are summarized in the present paper. They include acquisition or inactivation of genes for sugar synthesis or transfer or recombination of O-antigen gene clusters or their parts. The data obtained contribute to our understanding of the origins of the O‑antigen diversity, shed light on molecular evolutionary relationships between the O-antigens of enteric bacteria, and open a way for studies of the role of gene polymorphism in pathogenicity.

Keywords: enteric bacteria, O-antigen gene cluster, polysaccharide biosynthesis, polysaccharide structure

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Authors: Mahbuba Rahman, Sabri Boughorbel, Scott Presnell, Charlie Quinn, Darawan Rinchai, Damien Chaussabel, Nico Marr

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Collections of large-scale datasets made available in public repositories can be used to identify and fill gaps in biomedical knowledge. But first, these data need to be made readily accessible to researchers for analysis and interpretation. Here a collection of transcriptome datasets was made available to investigate the functional programming of human hematopoietic cells in early life. Thirty two datasets were retrieved from the NCBI Gene Expression Omnibus (GEO) and loaded in a custom, interactive web application called the Gene Expression browser (GXB), designed for visualization and query of integrated large-scale data. Multiple sample groupings and gene rank lists were created based on the study design and variables in each dataset. Web links to customized graphical views can be generated by users and subsequently be used to graphically present data in manuscripts for publication. The GXB tool also enables browsing of a single gene across datasets, which can provide information on the role of a given molecule across biological systems. The dataset collection is available online. As a proof-of-principle, one of the datasets (GSE25087) was re-analyzed to identify genes that are differentially expressed by regulatory T cells in early life. Re-analysis of this dataset and a cross-study comparison using multiple other datasets in the above mentioned collection revealed that PMCH, a gene encoding a precursor of melanin-concentrating hormone (MCH), a cyclic neuropeptide, is highly expressed in a variety of other hematopoietic cell types, including neonatal erythroid cells as well as plasmacytoid dendritic cells upon viral infection. Our findings suggest an as yet unrecognized role of MCH in immune regulation, thereby highlighting the unique potential of the curated dataset collection and systems biology approach to generate new hypotheses which can be tested in future mechanistic studies.

Keywords: early-life, GEO datasets, PMCH, interactive query, systems biology

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Authors: Tania Tzong, Chao-Yi Teng, Tzong-Yuan Wu

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Currently, Chikungunya virus (CHIKV) transmitted to humans by Aedes mosquitoes has distributed from Africa to Southeast Asia, South America, and South Europe. However, little is known about the antigenic targets for immunity, and there are no licensed vaccines or specific antiviral treatments for the disease caused by CHIKV. Baculovirus has been recognized as a novel vaccine vector with attractive characteristic features of an optional vaccine delivery vehicle. This approach provides the safety and efficacy of CHIKV vaccine. In this study, bi-cistronic recombinant baculoviruses vAc-CMV-CHIKV26S-Rhir-EGFP and vAc-CMV-pH-CHIKV26S-Lir-EGFP were produced. Both recombinant baculovirus can express EGFP reporter gene in insect cells to facilitate the recombinant virus isolation and purification. Examination of vAc-CMV-CHIKV26S-Rhir-EGFP and vAc-CMV-pH-CHIKV26S-Lir-EGFP showed that this recombinant baculovirus could induce syncytium formation in insect cells. Unexpectedly, the immunofluorescence assay revealed the expression of E1 and E2 of CHIKV structural proteins in insect cells infected by vAc-CMV-CHIKV26S-Rhir-EGFP. This result may imply that the CMV promoter can induce the transcription of CHIKV26S in insect cells. There are also E1 and E2 expression in mammalian cells transduced by vAc-CMV-CHIKV26S-Rhir-EGFP and vAc-CMV-pH-CHIKV26S-Lir-EGFP. The expression of E1 and E2 proteins of insect and mammalian cells was validated again by Western blot analysis. The vector construction with dual tandem promoters, which is polyhedrin and CMV promoter, has higher expression of the E1 and E2 of CHIKV structural proteins than the vector construction with CMV promoter only. Most of the E1 and E2 proteins expressed in mammalian cells were glycosylated. In the future, the expression of structural proteins of CHIKV in mammalian cells is expected can form virus-like particle, so it could be used as a vaccine for chikungunya virus.

Keywords: chikungunya virus, virus-like particle, vaccines, baculovirus expression vector system

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Authors: Arfan Ali, Idrees Ahmad Nasir

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The critical evaluation of tolerance in tomato plants against the induced saline soil were assessed by transcript analysis of genes coding for products potentially involved in stress tolerance. A reverse transcriptase PCR experiment was performed with Hsp90-1, MT2, and GR1like protein genes using RNA isolated from different tissues of tomato plants. Four strains of Bacillus magisterium were inoculated with 100 Mm & 200 Mm concentrations of salt. Eleven treatments each ten replica pots were installed in green house experiment and the parameters taken into account were morphological (length, weight, number of leaves, leaf surface area), chemical (anthocyanin, chlorophyll-a, chlorophyll-b, carotenoids) and biological (gene expression). Results bare a response i.e. highest response of MT2 like gene was at 24 hpi and the highest levels of GR1 like protein transcript accumulation were detected at 36 hpi. The chemical and morphological parameters at diverse salt concentrations bequeath superlative response amongst strains which candidly flank on Zm7 and Zm4. Therefore, Bacillus magisterium Zm7 strains and somehow Zm4 strain can be used in saline condition to make plants tolerant. The overall performance of strains Zm7, Zm6, and Zm4 was found better for all studied traits under salt stress conditions. Significant correlations among traits root length, shoot length, number of leaves, leaf surface area, carotenoids, anthocyanin, chlorophyll-a and chlorophyll-b were found and suggested that the salt tolerance in tomato may be improved through the use of PGPR strains.

Keywords: Bacillus magisterium, gene expression glutathione reductase, metallothionein, PGPR, Rhizobacteria, saline

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Authors: Naoki Yamamoto, Hidenobu Ozaki, Taiichiro Ookawa, Youming Liu, Kazunori Okada, Aiping Zheng

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Rice sheath blight is one of the destructive fungal diseases in rice. We have thought that rice sheath blight resistance is a polygenic trait. Host-pathogen interactions and secondary metabolites such as lignin and phytoalexins are likely to be involved in defense against R. solani. However, to our knowledge, it is still unknown how sheath blight resistance can be enhanced in rice breeding. To seek for an alternative genetic factor that contribute to sheath blight resistance, we mined relevant allelic variations from rice core collections created in Japan. Based on disease lesion length on detached leaf sheath, we selected 30 varieties of the top tail-end and the bottom tail-end, respectively, from the core collections to perform genome-wide association mapping. Re-sequencing reads for these varieties were used for calling single nucleotide polymorphisms among the 60 varieties to create a SNP panel, which contained 1,137,131 homozygous variant sites after filitering. Association mapping highlighted a locus on the long arm of chromosome 11, which is co-localized with three sheath blight QTLs, qShB11-2-TX, qShB11, and qSBR-11-2. Based on the localization of the trait-associated alleles, we identified an ankyryn repeat-containing protein gene (ANK-M) as an uncharacterized candidate factor for rice sheath blight resistance. Allelic distributions for ANK-M in the whole rice population supported the reliability of trait-allele associations. Gene expression characteristics were checked to evaluiate the functionality of ANK-M. Since an ANK-M homolog (OsPIANK1) in rice seems a basal defense regulator against rice blast and bacterial leaf blight, ANK-M may also play a role in the rice immune system.

Keywords: allele mining, GWAS, QTL, rice sheath blight

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Authors: Rachel Matar, Maxime Merheb

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Chemical drugs have been used for many centuries as the only way to cure diseases until the novel gene therapy has been created in 1960. Gene therapy is based on the insertion, correction, or inactivation of genes to treat people with genetic illness (1). Gene therapy has made wonders in Parkison’s, Alzheimer and multiple sclerosis. In addition to great promises in the healing of deadly diseases like many types of cancer and autoimmune diseases (2). This method implies the use of recombinant DNA technology with the help of different viral and non-viral vectors (3). It is nowadays used in somatic cells as well as embryos and gametes. Beside all the benefits of gene therapy, this technique is deemed by some opponents as an ethically unacceptable treatment as it implies playing with the genes of living organisms.

Keywords: gene therapy, genetic disease, cancer, multiple sclerosis

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Authors: Daina Jonkus, Liga Paura, Tatjana Sjakste, Kristina Dokane

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The aim of this study was to analyse PRKAG3 and RYR1 gene and genotypes frequencies in Latvian White pigs’ breed. Genotypes of RYR1 gene two loci (rs196953058 and rs323041392) in 89 exon and PRKAG3 gene two loci (rs196958025 and rs344045190) in gene promoter were detected in 103 individuals of Latvian white pigs’ breed. Analysis of RYR1 gene loci rs196953058 shows all individuals are homozygous by T allele and all animals are with genotypes TT, its mean - in 2769 position is Phenylalanine. Analysis of RYR1 gene loci rs323041392 shows all individuals are homozygous by G allele and all animals are with genotypes GG, its mean - in 4119 positions is Asparagine. In loci rs196953058 and rs323041392, there were no gene polymorphisms. All analysed individuals by two loci rs196953058-rs323041392 have TT-GG genotypes or Phe-Asp amino acids. In PRKAG3 gene loci rs196958025 and rs344045190 there was gene polymorphisms. In both loci frequencies for A allele was higher: 84.6% for rs196958025 and 73.0% for rs344045190. Analysis of PRKAG3 gene loci rs196958025 shows 74% of individuals are homozygous by An allele and animals are with genotypes AA. Only 4% of individuals are homozygous by G allele and animals are with genotypes GG, which is associated with pale meat colour and higher drip loss. Analysis of PRKAG3 gene loci rs344045190 shows 46% of individuals are homozygous with genotypes AA and 54% of individuals are heterozygous with genotypes AG. There are no individuals with GG genotypes. According to the results, in Latvian white pigs population there are no rs344435545 (RYR1 gene) CT heterozygous or TT recessive homozygous genotypes, which is related to the meat quality and pigs’ stress syndrome; and there are 4% rs196958025 (PRKAG3 gene) GG recessive homozygote genotypes, which is related to the meat quality. Acknowledgment: the investigation is supported by VPP 2014-2017 AgroBioRes Project No. 3 LIVESTOCK.

Keywords: genotype frequencies, pig, PRKAG3, RYR1

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Authors: Michelle C. S. Azevedo, Priscila M. Colavite, Carolina F. Francisconi, Ana P. Trombone, Gustavo P. Garlet

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VIP (vasoactive intestinal peptide) know as a potential protective factor in the view of its marked immunosuppressive properties. In this work, we investigated a possible association of VIP with the clinical status of experimental periapical granulomas and the association with expression markers in the lesions potentially associated with periapical lesions pathogenesis. C57BL/6WT mice were treated or not with recombinant VIP. Animals with active/progressive (N=40), inactive/stable (N=70) periapical granulomas and controls (N=50) were anesthetized and the right mandibular first molar was surgically opened, allowing exposure of dental pulp. Endodontic pathogenic bacterial strains were inoculated: Porphyromonas gingivalis, Prevotella nigrescens, Actinomyces viscosus, and Fusobacterium nucleatum subsp. polymorphum. The cavity was not sealed after bacterial inoculation. During lesion development, animals were treated or not with recombinant VIP 3 days post infection. Animals were killed after 3, 7, 14, and 21 days of infection and the jaws were dissected. The extraction of total RNA from periodontal tissues was performed and the integrity of samples was checked. qPCR reaction using TaqMan chemistry with inventoried primers were performed in ViiA7 equipment. The results, depicted as the relative levels of gene expression, were calculated in reference to GAPDH and β-actin expression. Periodontal tissues from upper molars were vested and incubated supplemented RPMI, followed by processing with 0.05% DNase. Cell viability and couting were determined by Neubauer chamber analysis. For flow cytometry analysis, after cell counting the cells were stained with the optimal dilution of each antibody; (PE)-conjugated and (FITC)-conjugated antibodies against CD4, CD25, FOXP3, IL-4, IL-17 and IFN-γ antibodies, as well their respective isotype controls. Cells were analyzed by FACScan and CellQuest software. Results are presented as the number of cells in the periodontal tissues or the number of positive cells for each marker in the CD4+FOXp3+, CD4+IL-4+, CD4+IFNg+ and CD4+IL-17+ subpopulations. The levels mRNA were measured by qPCR. The VIP expression was predominated in inactive lesions, as well part of the clusters of cytokine/Th markers identified as protective factors and a negative correlation between VIP expression and lesion evolution was observed. A quantitative analysis of IL1β, IL17, TNF, IFN, MMP2, RANKL, OPG, IL10, TGFβ, CTLA4, COL5A1, CTGF, CXCL11, FGF7, ITGA4, ITGA5, SERP1 and VTN expression was measured in experimental periapical lesions treated with VIP 7 and 14 days after lesion induction and healthy animals. After 7 days, all targets presented a significate increase in comparison to untreated animals. About migration kinetics, profile of chemokine receptors expression of TCD4+ subsets and phenotypic analysis of Tregs, Th1, Th2 and Th17 cells during the course of experimental periodontal disease evaluated by flow cytometry and depicted as the number of positive cells for each marker. CD4+IFNg+ and CD4+FOXp3+ cells migration were significate increased 7 days post VIP treatment. CD4+IL17+ cells migration were significate increased 7 and 14 days post VIP treatment, CD4+IL4+ cells migration were significate increased 14 and 21 days post VIP treatment compared to the control group. In conclusion, our experimental data support VIP involvement in determining the inactivity of periapical lesions. Financial support: FAPESP #2015/25618-2.

Keywords: chronic inflammation, cytokines, osteolytic lesions, VIP (Vasoactive Intestinal Peptide)

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Authors: Denis Mustafov, Emmanouil Karteris, Maria Braoudaki

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Glioblastoma multiform (GBM) is a heterogenous primary brain tumour that kills most affected patients. To the authors best knowledge, despite all research efforts there is no early diagnostic biomarker for GBM. MicroRNAs (miRNAs) are short non-coding RNA molecules which are deregulated in many cancers. The aim of this research was to determine miRNAs with a diagnostic impact and to potentially identify promising therapeutic targets for glioblastoma multiform. In silico analysis was performed to identify deregulated miRNAs with diagnostic relevance for glioblastoma. The expression profiles of the chosen miRNAs were then validated in vitro in the human glioblastoma cell lines A172 and U-87MG. Briefly, RNA extraction was carried out using the Trizol method, whilst miRNA extraction was performed using the mirVANA miRNA isolation kit. Quantitative Real-Time Polymerase Chain Reaction was performed to verify their expression. The presence of five target proteins within the A172 cell line was evaluated by Western blotting. The expression of the CORO1C protein within 32 GBM cases was examined via immunohistochemistry. The miRNAs identified in silico included miR-21-5p, miR-34a and miR-128a. These miRNAs were shown to target deregulated GBM genes, such as CDK6, E2F3, BMI1, JAG1, and CORO1C. miR-34a and miR-128a showed low expression profiles in comparison to a control miR-RNU-44 in both GBM cell lines suggesting tumour suppressor properties. Opposing, miR-21-5p demonstrated greater expression indicating that it could potentially function as an oncomiR. Western blotting revealed expression of all five proteins within the A172 cell line. In silico analysis also suggested that CORO1C is a target of miR-128a and miR-34a. Immunohistochemistry demonstrated that 75% of the GBM cases showed moderate to high expression of CORO1C protein. Greater understanding of the deregulated expression of miR-128a and the upregulation of CORO1C in GBM could potentially lead to the identification of a promising diagnostic biomarker signature for glioblastomas.

Keywords: non-coding RNAs, gene expression, brain tumours, immunohistochemistry

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Authors: D. Hammad, D. P. Tonge

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Rheumatoid arthritis (RA) is a disabling and common autoimmune disease during which the body's immune system attacks healthy tissues. This results in complicated and long-lasting actions being carried out by the immune system, which typically only occurs when the immune system encounters a foreign object. In the case of RA, the disease affects millions of people and causes joint inflammation, ultimately leading to the destruction of cartilage and bone. Interestingly, the disease mechanism still remains unclear. It is likely that RA occurs as a result of a complex interplay of genetic and environmental factors including an imbalance in the microorganism population inside our body. The human microbiome or microbiota is an extensive community of microorganisms in and on the bodies of animals, which comprises bacteria, fungi, viruses, and protozoa. Recently, the development of molecular techniques to characterize entire bacterial communities has renewed interest in the involvement of the microbiome in the development and progression of RA. We believe that an imbalance in some of the specific bacterial species in the gut, mouth and other sites may lead to atopobiosis; the translocation of these organisms into the blood, and that this may lead to changes in immune system status. The aim of this study was, therefore, to characterize the microbiome of RA serum samples in comparison to healthy control subjects using 16S rRNA gene amplification and sequencing. Serum samples were obtained from healthy control volunteers and from patients with RA both prior to, and following treatment. The bacterial community present in each sample was identified utilizing V4 region 16S rRNA amplification and sequencing. Bacterial identification, to the lowest taxonomic rank, was performed using a range of bioinformatics tools. Significantly, the proportions of the Lachnospiraceae, Ruminococcaceae, and Halmonadaceae families were significantly increased in the serum of RA patients compared with healthy control serum. Furthermore, the abundance of Bacteroides and Lachnospiraceae nk4a136_group, Lachnospiraceae_UGC-001, RuminococcaceaeUCG-014, Rumnococcus-1, and Shewanella was also raised in the serum of RA patients relative to healthy control serum. These data support the notion of a blood microbiome and reveal RA-associated changes that may have significant implications for biomarker development and may present much-needed opportunities for novel therapeutic development.

Keywords: blood microbiome, gut and oral bacteria, Rheumatoid arthritis, 16S rRNA gene sequencing

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Authors: Meenakshi Srivastava, A. K. Mishra

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This study characterizes the metabolic versatility of denitrifying bacterial communities residing in the paddy soil using the GC-MS based Phospholipid Fatty Acid (PLFA) analyses simultaneously with nosZ gene based PCR-DGGE (Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis) and real time Q-PCR analysis. We have analyzed the abundance of nitrous oxide reductase (nosZ) genes, which was subsequently related to soil PLFA profile and DGGE based denitrifier community structure. Soil denitrifying bacterial community comprised majority or dominance of Ochrobactrum sp. following Cupriavidus and uncultured bacteria strains in paddy soil of selected sites. Initially, we have analyzed the abundance of the nitrous oxide reductase gene (nosZ), which was found to be related with PLFA based lipid profile. Chandauli of Eastern UP, India represented greater amount of lipid content (C18-C20) and denitrifier’s diversity. This study suggests the positive co-relation between soil PLFA profiles, DGGE, and Q-PCR data. Thus, a close networking among metabolic abilities and taxonomic composition of soil microbial communities existed, and subsequently, such work at greater extent could be helpful in managing nutrient dynamics as well as microbial dynamics of paddy soil ecosystem.

Keywords: denaturing gradient gel electrophoresis, DGGE, nitrifying and denitrifying bacteria, PLFA, Q-PCR

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Authors: Dalal Alghawas, Jetty Lee, Kaisa Poutanen, Hani El-Nezami

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Oat β-glucan a water soluble non starch linear polysaccharide has been approved as a cholesterol lowering agent by various food safety administrations and is commonly used to reduce the risk of heart disease. The molecular weight of oat β-glucan can vary depending on the extraction and fractionation methods. It is not clear whether the molecular weight has a significant impact at reducing the acceleration of atherosclerosis. The aim of this study was to investigate three different oat β-glucan fractionations on the development of atherosclerosis in vivo. With special focus on plaque stability and the intestinal barrier function. To test this, ApoE-/- female mice were fed a high fat diet supplemented with oat bran, high molecular weight (HMW) oat β-glucan fractionate and low molecular weight (LMW) oat β-glucan fractionate for 16 weeks. Atherosclerosis risk markers were measured in the plasma, heart and aortic tree. Plaque size was measured in the aortic root and aortic tree. ICAM-1, VCAM-1, E-Selectin, P-Selectin, protein levels were assessed from the aortic tree to determine plaque stability at 16 weeks. The expression of p22phox at the aortic root was evaluated to study the NADPH oxidase complex involved in nitric oxide bioavailability and vascular elasticity. The tight junction proteins E-cadherin and beta-catenin from western blot analyses were analysed as an intestinal barrier function test. Plasma LPS, intestinal D-lactate levels and hepatic FMO gene expression were carried out to confirm whether the compromised intestinal barrier lead to endotoxemia. The oat bran and HMW oat β-glucan diet groups were more effective than the LMW β-glucan diet group at reducing the plaque size and showed marked improvements in plaque stability. The intestinal barrier was compromised for all the experimental groups however the endotoxemia levels were higher in the LMW β-glucan diet group. The oat bran and HMW oat β-glucan diet groups were more effective at attenuating the development of atherosclerosis. Reasons for this could be due to the LMW oat β-glucan diet group’s low viscosity in the gut and the inability to block the reabsorption of cholesterol. Furthermore the low viscosity may allow more bacterial endotoxin translocation through the impaired intestinal barrier. In future food technologists should carefully consider how to incorporate LMW oat β-glucan as a health promoting food.

Keywords: Atherosclerosis, beta glucan, endotoxemia, intestinal barrier function

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Authors: A. Elragig, S. Townley, H. Dreiwi

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In this paper we introduce a bacteria-leukocyte model with bacteria chemotaxsis. We assume that bacteria develop a tactic defense mechanism as a response to Leukocyte phagocytosis. We explore the effect of this tactic motion on Turing space in two parameter spaces. A fine tuning of bacterial chemotaxis shows a significant effect on developing a non-uniform steady state.

Keywords: chemotaxis-diffusion driven instability, bacterial chemotaxis, mathematical biology, ecology

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Authors: Hamid Mustafa, Adeela Ajmal, Kim EuiSoo, Noor-ul-Ain

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World wild, buffalo production is considered as most important component of food industry. Efficient buffalo production is related with reproductive performance of this species. Lack of knowledge of reproductive efficiency and its related genes in buffalo species is a major constraint for sustainable buffalo production. In this study, we performed some bioinformatics analysis on Follicle Stimulating Hormone Receptor (FSHR) gene and explored the possible relationship of this gene among different buffalo breeds and with other farm animals. We also found the evolution pattern for this gene among these species. We investigate CDS lengths, Stop codon variation, homology search, signal peptide, isoelectic point, tertiary structure, motifs and phylogenetic tree. The results of this study indicate 4 different motif in this gene, which are Activin-recp, GS motif, STYKc Protein kinase and transmembrane. The results also indicate that this gene has very close relationship with cattle, bison, sheep and goat. Multiple alignment (MA) showed high conservation of motif which indicates constancy of this gene during evolution. The results of this study can be used and applied for better understanding of this gene for better characterization of Follicle Stimulating Hormone Receptor (FSHR) gene structure in different farm animals, which would be helpful for efficient breeding plans for animal’s production.

Keywords: buffalo, FSHR gene, bioinformatics, production

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Authors: Baharuddin Patandjengi, A. Pabborong, T. Kuswinanti

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Bacterial ring rot caused by a gram-positive Coryneform bacterium Corynebacterium michiganensis subsp. sepedonicus is an important disease on potato crops in the world. The disease still belongs to an A1 quarantine pathogen in Indonesia, although it was found in West Java since 2013. The objective of this study was to know the presence of bacterial ring rot in four potato district areas in South Sulawesi. Infected samples were collected from potato fields and storage warehouses in Enrekang, Gowa, Jeneponto and Bantaeng districts. Potato tuber samples were cut and observed their vasiculer vessels and the bacterial ooze was used for isolation on Nutrient Agar and Nutrient Broth–Yeast extract medium. Bacterial isolates were then morphologically and physiologically characterized. A patogenicity test on eggplant and molecular characterization using PCR with specific primer for Cms (50F and Cms 50 R) was revealed for further identification. The results showed that Cms has become widespread in four districts of South Sulawesi. The bacterial ringrot disease incidence in these districts was reached above 30 %. All of 14 bacterial isolates that identified before using standard methods of EPPO, showed DNA band in size of 224 bp in PCR test, which indicated positively belong to C. michiganensis subsp. sepedonicus.

Keywords: bacterial ring rot, clavibacter michiganensis pv. sepedonicus, PCR, potato

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Authors: Sevil Yildiz

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Audio visual communication is a type of collective expression. Collective expression activity informs the masses, gives direction to opinions and establishes public opinion. Due to these characteristics, audio visual communication must be subjected to special restrictions. This has been stipulated in both the Constitution and the European Human Rights Agreement. This paper aims to review freedom of expression and its restriction in audio visual media. For this purpose, the authorisation of the Radio and Television Supreme Council to impose sanctions as an independent administrative authority empowered to regulate the field of audio visual communication has been reviewed with regard to freedom of expression and its limits.

Keywords: audio visual media, freedom of expression, its limits, radio and television supreme council

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Authors: Shahram Nanekarania, Majid Goodarzia

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Calpastatin and callipyge have been known as one of the candidate genes in meat quality and quantity. Calpastatin gene has been located to chromosome 5 of sheep and callipyge gene has been localized in the telomeric region on ovine chromosome 18. The objective of this study was identification of calpastatin and callipyge genes polymorphism and analysis of genotype structure in population of Lori sheep kept in Iran. Blood samples were taken from 120 Lori sheep breed and genomic DNA was extracted by salting out method. Polymorphism was identified using the PCR-RFLP technique. The PCR products were digested with MspI and FaqI restriction enzymes for calpastatin gene and callipyge gene, respectively. In this population, three patterns were observed and AA, AB, BB genotype have been identified with the 0.32, 0.63, 0.05 frequencies for calpastatin gene. The results obtained for the callipyge gene revealed that only the wild-type allele A was observed, indicating that only genotype AA was present in the population under consideration.

Keywords: polymorphism, calpastatin, callipyge, PCR-RFLP, Lori sheep

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Authors: Hye Kyung Kim, Myung-Gyou Kim, Kang-Hyun Leem

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Postmenopausal osteoporosis is characterized by low bone density which leads to increased bone fragility and greater susceptibility to fracture. Current treatments for osteoporosis are dominated by drugs that inhibit bone resorption although they also suppress bone formation that may contribute to pathogenesis of osteonecrosis. To restore the extensive bone loss, there is a great need for anabolic treatments that induce osteoblasts to build new bone. Pre-osteoblastic cells produce proteins of the extra-cellular matrix, including type I collagen at first, and then to successively produce alkaline phosphatase (ALP) and osteocalcin during differentiation to osteoblasts. Finally, osteoblasts deposit calcium. Present study investigated the effects of egg yolk peptide (EYP) on osteogenic activities and bone matrix gene expressions in human osteoblastic MG-63 cells. The effects of EYP on cell proliferation, alkaline phosphatase (ALP) activity, collagen synthesis, and mineralization were measured. The expression of osteogenic genes including COL1A1 (collagen, type I, alpha 1), ALP, BGLAP (osteocalcin), and SPP1 (secreted phosphoprotein 1, osteopontin) were measured by quantitative realtime PCR. EYP dose-dependently increased MG-63 cell proliferation, ALP activity, collagen synthesis, and calcium deposition. Furthermore, COL1A1, ALP, and SPP1 gene expressions were increased by EYP treatment. Present study suggested that EYP treatment enhanced osteogenic activities and increased bone matrix osteogenicgenes. These results could provide a mechanistic explanation for the bone-strengthening effects of EYP.

Keywords: egg yolk peptide, osteoblastic MG-63 cells, alkaline phosphatase, collagen synthesis, osteogenic genes, COL1A1, osteocalcin, osteopontin

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Authors: Ricardo de Oliveira Orsi, Aline Astolfi, Daniel Diego Mendes, Isabella Cristina de Castro Lippi, Jaine da Luz Scheffer, Yan Souza Lima, Juliana Lunardi, Giovanna do Padro Ribeiro, Samir Moura Kadri

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NECTAR Brazilian group (Center of Education, Science, and Technology in Rational Beekeeping) conducted studies on the pesticides honey bees effects using the transcriptome sequencing (RNA-Seq) analyzes for gene expression studies. In this way, we analyzed the effects of Pyraclostrobin and Fipronil on the honey bees with 21 old-days (forager) in laboratory conditions. For this, frames containing sealed brood were removed from the beehives and maintenance on the stove (32°C and 75% humidity) until the bees were born. So, newly emerged workers were marked on the pronotum with a non-toxic pen and reintroduced into their original hives. After 21 days, 120 marked bees were collected with an entomological forces and immediately stored in Petri dishes, perforated to ensure ventilation, and kept fasted for 3 hours. These honeybees were exposed to food contaminated or not with the sublethal dose of Pyraclostrobin (850 ppb/bee) or Fipronil (2.5 ppb/bee). After four hours of exposure, 15 bees from each treatment were referred to transcriptome analysis. Total RNA analysis was extracted from the brain pools (03 brains per pool) using the TRIzol® reagent protocol according to the manufacturer's instructions. cDNA libraries were constructed, and the FASTQC program was used to check adapter content and assess the quality of raw reads. Differential expression analysis was performed with the DESeq2 package. Genes that had an adjusted value of less than 0.05 were considered to be significantly up-regulated. Regarding the Pyraclostrobin, alterations were observed in the pattern of 17 gene related to of antioxidant system, cellular respiration, glucose metabolism, and regulation of juvenile hormone and the hormone insulin. Glyphosate altered the 10 gene related to the digestive system, exoskeleton composition, vitamin E transport, and antioxidant system. The results indicate that the necessity of studies using the sublethal doses to evaluate the pesticides uses and risks on crops and its effects on the honey bees.

Keywords: beekeeping, honey bees, pesticides, transcriptome

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Authors: Zelikha Labbani

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The in vitro isolated microspore culture is the most powerful androgenic pathway to produce doubled haploid plants in the short time. To deviate a microspore toward embryogenesis, a number of factors, different for each species, must concur at the same time and place. Once induced, the microspore undergoes numerous changes at different levels, from overall morphology to gene expression. Induction of microspore embryogenesis not only implies the expression of an embryogenic program, but also a stress-related cellular response and a repression of the gametophytic program to revert the microspore to a totipotent status. As haploid single cells, microspore became a strategy to achieve various objectives particularly in genetic engineering. In this communication we would show the most recent advances in the producing haploid embryos via in vitro isolated microspore culture.

Keywords: in vitro isolated microspore culture, success, haploid cells, bioinformatics, biomedicine

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Authors: Iman Farasat, Howard M. Salis

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The CRISPR/Cas9 system has revolutionized genome engineering by enabling site-directed and high-throughput genome editing, genome insertion, and gene knockdowns in several species, including bacteria, yeast, flies, worms, and human cell lines. This technology has the potential to enable human gene therapy to treat genetic diseases and cancer at the molecular level; however, the current CRISPR/Cas9 system suffers from seemingly sporadic off-target genome mutagenesis that prevents its use in gene therapy. A comprehensive mechanistic model that explains how the CRISPR/Cas9 functions would enable the rational design of the guide-RNAs responsible for target site selection while minimizing unexpected genome mutagenesis. Here, we present the first quantitative model of the CRISPR/Cas9 genome mutagenesis system that predicts how guide-RNA sequences (crRNAs) control target site selection and cleavage activity. We used statistical thermodynamics and law of mass action to develop a five-step biophysical model of cas9 cleavage, and examined it in vivo and in vitro. To predict a crRNA's binding specificities and cleavage rates, we then compiled a nearest neighbor (NN) energy model that accounts for all possible base pairings and mismatches between the crRNA and the possible genomic DNA sites. These calculations correctly predicted crRNA specificity across 5518 sites. Our analysis reveals that cas9 activity and specificity are anti-correlated, and, the trade-off between them is the determining factor in performing an RNA-mediated cleavage with minimal off-targets. To find an optimal solution, we first created a scheme of safe-design criteria for Cas9 target selection by systematic analysis of available high throughput measurements. We then used our biophysical model to determine the optimal Cas9 expression levels and timing that maximizes on-target cleavage and minimizes off-target activity. We successfully applied this approach in bacterial and mammalian cell lines to reduce off-target activity to near background mutagenesis level while maintaining high on-target cleavage rate.

Keywords: biophysical model, CRISPR, Cas9, genome editing

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Authors: Ali Bayram, Burak Uz, Remzi Yiğiter

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The impairment of lipid metabolism in the central nervous system has been suggested as a critical factor of Alzheimer’s disease (AD) pathogenesis. Homo sapiens acyl-coenyme A synthetase short-chain family member 2 (ACSS2) gene encodes the enzyme acetyl-Coenzyme A synthetase (AMP forming; AceCS) providing acetyl-coenzyme A (Ac-CoA) for various physiological processes, such as cholesterol and fatty acid synthesis, as well as the citric acid cycle. We investigated ACSS2, transcript variant 1 (ACSS2*1), mRNA levels in the peripheral blood mononuclear cells (PBMC) of patients with AD and compared them with the controls. The study group comprised 50 patients with the diagnosis of AD who have applied to Gaziantep University Faculty of Medicine, and Department of Neurology. 49 healthy individuals without any neurodegenerative disease are included as controls. ACSS2 mRNA expression in PBMC of AD/control patients was 0.495 (95% confidence interval: 0.410-0.598), p= .000000001902). Further studies are needed to better clarify this association.

Keywords: Alzheimer’s disease, ACSS2 Genes, mRNA expression, RT-PCR

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Authors: Philip Semaha, Zhongfang Lei, Ziwen Zhao, Sen Liu, Zhenya Zhang, Kazuya Shimizu

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The increasing generation of saline wastewater through various industrial activities is becoming a global concern for activated sludge (AS) based biological treatment which is widely applied in wastewater treatment plants (WWTPs). As for the AS process, an increase in wastewater salinity has negative impact on its overall performance. The advent of conventional aerobic granular sludge (AGS) or bacterial AGS biotechnology has gained much attention because of its superior performance. The development of algal-bacterial AGS could enhance better nutrients removal, potentially reduce aeration cost through symbiotic algae-bacterial activity, and thus, can also reduce overall treatment cost. Nonetheless, the potential of salt stress to decrease biomass growth, microbial activity and nutrient removal exist. Up to the present, little information is available on saline wastewater treatment by algal-bacterial AGS. To the authors’ best knowledge, a comparison of the two AGS systems has not been done to evaluate nutrients removal capacity in the context of salinity increase. This study sought to figure out the impact of salinity on the algal-bacterial AGS system in comparison to bacterial AGS one, contributing to the application of AGS technology in the real world of saline wastewater treatment. In this study, the salt concentrations tested were 0 g/L, 1 g/L, 5 g/L, 10 g/L and 15 g/L of NaCl with 24-hr artificial illuminance of approximately 97.2 µmol m¯²s¯¹, and mature bacterial and algal-bacterial AGS were used for the operation of two identical sequencing batch reactors (SBRs) with a working volume of 0.9 L each, respectively. The results showed that salinity increase caused no apparent change in the color of bacterial AGS; while for algal-bacterial AGS, its color was progressively changed from green to dark green. A consequent increase in granule diameter and fluffiness was observed in the bacterial AGS reactor with the increase of salinity in comparison to a decrease in algal-bacterial AGS diameter. However, nitrite accumulation peaked from 1.0 mg/L and 0.4 mg/L at 1 g/L NaCl in the bacterial and algal-bacterial AGS systems, respectively to 9.8 mg/L in both systems when NaCl concentration varied from 5 g/L to 15 g/L. Almost no ammonia nitrogen was detected in the effluent except at 10 g/L NaCl concentration, where it averaged 4.2 mg/L and 2.4 mg/L, respectively, in the bacterial and algal-bacterial AGS systems. Nutrients removal in the algal-bacterial system was relatively higher than the bacterial AGS in terms of nitrogen and phosphorus removals. Nonetheless, the nutrient removal rate was almost 50% or lower. Results show that algal-bacterial AGS is more adaptable to salinity increase and could be more suitable for saline wastewater treatment. Optimization of operation conditions for algal-bacterial AGS system would be important to ensure its stably high efficiency in practice.

Keywords: algal-bacterial aerobic granular sludge, bacterial aerobic granular sludge, Nutrients removal, saline wastewater, sequencing batch reactor

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Authors: Vandana Singh, Subash Singh

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Introduction: Oral squamous cell carcinoma is the most common malignant tumor of the oral cavity. Normally the death of cell and the growth are active processes and depend not only on external factors but also on the expression of genes like Bcl-2, which activate and inhibit apoptosis. The term Bcl-2 is an acronym for B-cell lymphoma/ leukemia -2 genes. Objectives: An attempt was made to evaluate Bcl-2 oncoprotein expression in patients with oral precancer and cancer and to assess possible correlation between Bcl-2 oncoprotein expression and clinicopathological features of oral precancer and cancer. Material and Methods: This is a selective prospective clinical and immunohistochemical study. Clinicopathological examination is correlated with immunohistochemical findings. The immunolocalization of Bcl-2 protein is performed using the labeled streptavidin biotin (LSAB) method. To visualize the reaction, 3, 3-diaminobenzidine (DAB) is used. Results: Bcl-2 expression was positive in 11 [36.66 %, low Bcl-2 expression 3 (10.00 %), moderate Bcl-2 expression 7 (23.33 %), and high Bcl-2 expression 1 (3.33 %)] oral cancer cases and in 14 [87.50 %, low expression 8 (50 %), moderate expression 6 (37.50 %)] precancer cases. Conclusion: On the basis of the results of our study we conclude that positive Bcl-2 expression may be an indicator of poor prognosis in oral cancer and precancer. Relevance: It has been reported that there is deregulation of Bcl-2 expression during progression from oral epithelial dysplasia to squamous cell carcinoma. It can be used for revealing progression of epithelial dysplasia to malignancy and as a prognostic marker in oral precancer and cancer.

Keywords: BcL-2, immunohistochemistry, oral cancer, oral precancer

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Authors: Tahira Hussain, Ilhom Rahamkulov, Muhammad Aasim, Ugur Pirlak, Emre Aksoy, Mehmet Emin Caliskan, Allah Bakhsh

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RNA interference (RNAi) has proved its usefulness in functional genomic research on insects recently and is considered potential strategy in crop improvement for the control of insect pests. The different insect pests incur significant losses to potato yield worldwide, Colorado Potato Beetle (CPB) being most notorious one. The present study focuses to knock down highly specific 20-hydroxyecdysone hormone-receptor complex interaction by using RNAi approach to silence Ecdysone receptor (EcR) gene of CPB in transgenic potato plants expressing dsRNA of EcR gene. The partial cDNA of Ecdysone receptor gene of CPB was amplified using specific primers in sense and anti-sense orientation and cloned in pRNAi-GG vector flanked by an intronic sequence (pdk). Leaf and internodal explants of Lady Olympia, Agria and Granola cultivars of potato were infected with Agrobacterium strain LBA4404 harboring plasmid pRNAi-CPB, pRNAi-GFP (used as control). Neomycin phosphotransferase (nptII) gene was used as a plant selectable marker at a concentration of 100 mg L⁻¹. The primary transformants obtained have shown proper integration of T-DNA in plant genome by standard molecular analysis like polymerase chain reaction (PCR), real-time PCR, Sothern blot. The transgenic plants developed out of these cultivars are being evaluated for their efficacy against larvae as well adults of CPB. The transgenic lines are expected to inhibit expression of EcR protein gene, hindering their molting process, hence leading to increased potato yield.

Keywords: plant mediated RNAi, molecular strategy, ecdysone receptor, insect metamorphosis

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Authors: Yan Li, Qingxiang Meng, Bo Zhou, Zhenming Zhou

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The objective of this study was to compare the effects of ensiled mulberry leaves (EML), and sun-dried mulberry fruit pomace (SMFP) on fecal bacterial communities in Simmental crossbred finishing steers fed the following 3 diets: a standard TMR diet, standard diet containing EML and standard diet containing SMFP, and the diets had similar protein and energy levels. Bacterial communities in the fecal content were analyzed using Illumina Miseq sequencing of the V4 region of the 16S rRNA gene amplification. Quantitative real-time PCR was used to detect the selected bacterial species in the feces. Most of the sequences were assigned to phyla Firmicutes (56.67%) and Bacteroidetes(35.90%), followed by Proteobacteria(1.86%), Verrucomicrobia(1.80%) and Tenericutes(1.37%). And the predominant genera included the 5-7N15 (5.91%), CF231 (2.49%), Oscillospira (2.33%), Paludibacter (1.23%) and Akkermansia(1.11%). As for the treatments, no significant differences were observed in Firmicutes (p = 0.28), Bacteroidetes (p = 0.63), Proteobacteria (p = 0.46), Verrucomicrobia (p = 0.17) and Tenericutes (p = 0.75). On the genus level, classified genera with high abundance (more than 0.1%) mainly came from two phyla: Bacteroidetes and Firmicutes. Also no differences were observed in most genera level, 5-7N15 (p = 0.21), CF231 (p = 0.62), Oscillospira (p = 0.9), Paludibacter (p = 0.33) and Akkermansia (p = 0.37), except that rc4-4 were lower in the CON and SMFP groups compared to the EML animals (p = 0.02). Additionally, there were no differences in richness estimate and diversity indices (p > 0.16), and treatments had no significant effect on most selected bacterial species in the fecal (p > 0.06), except that Ruminococcus albus were higher in the EML group (p < 0.01) and Streptococcus bovis were lower in the CON group (p < 0.01). In conclusion, diets supplemented with EML and SMFP have little influence on fecal bacterial community composition in finishing steers.

Keywords: fecal bacteria community composition, sequencing, ensiled mulberry leaves (EML), sun-dried mulberry fruit pomace (SMFP)

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Authors: Roxane A. Legaie, Kjiana E. Schwab, Caroline E. Gargett

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Studies looking at the changes in gene expression from RNAseq data often make use of linear models. It is also common practice to focus on a subset of data for a comparison of interest, leaving aside the samples not involved in this particular comparison. This work shows the importance of including all observations in the modeling process to better estimate variance parameters, even when the samples included are not directly used in the comparison under test. The human endometrium is a dynamic tissue, which undergoes cycles of growth and regression with each menstrual cycle. The mesenchymal stem cells (MSCs) present in the endometrium are likely responsible for this remarkable regenerative capacity. However recent studies suggest that MSCs also plays a role in the pathogenesis of endometriosis, one of the most common medical conditions affecting the lower abdomen in women in which the endometrial tissue grows outside the womb. In this study we compared gene expression profiles between MSCs and non-stem cell counterparts (‘non-MSC’) obtained from women with (‘E’) or without (‘noE’) endometriosis from RNAseq. Raw read counts were used for differential expression analysis using a linear model with the limma-voom R package, including either all samples in the study or only the samples belonging to the subset of interest (e.g. for the comparison ‘E vs noE in MSC cells’, including only MSC samples from E and noE patients but not the non-MSC ones). Using the full dataset we identified about 100 differentially expressed (DE) genes between E and noE samples in MSC samples (adj.p-val < 0.05 and |logFC|>1) while only 9 DE genes were identified when using only the subset of data (MSC samples only). Important genes known to be involved in endometriosis such as KLF9 and RND3 were missed in the latter case. When looking at the MSC vs non-MSC cells comparison, the linear model including all samples identified 260 genes for noE samples (including the stem cell marker SUSD2) while the subset analysis did not identify any DE genes. When looking at E samples, 12 genes were identified with the first approach and only 1 with the subset approach. Although the stem cell marker RGS5 was found in both cases, the subset test missed important genes involved in stem cell differentiation such as NOTCH3 and other potentially related genes to be used for further investigation and pathway analysis.

Keywords: differential expression, endometriosis, linear model, RNAseq

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Authors: R. Majidzadeh Heravi, M. Sankian, H. Kermanshahi, M. R. Nassiri, A. Heravi Moussavi, S. A. Lari, A. R. Varasteh

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The use of probiotics engineered to express specific enzymes has been the subject of considerable attention in poultry industry because of increased nutrient availability and reduced cost of enzyme supplementation. Phytase enzyme is commonly added to poultry feed to improve digestibility and availability of phosphorus from plant sources. To construct a probiotic with potential of phytate degradation, phytase gene (appA) from E. coli was cloned and transformed into two probiotic bacteria Lactobacillus salivarius and Lactococcus lactis. L. salivarous showed plasmid instability, unable to express the gene. The expression of appA gene in L. lactis was analyzed by detecting specific RNA and zymography assay. Phytase enzyme was isolated from cellular extracts of recombinant L. lactis, showing a 46 kDa band upon the SDS-PAGE analysis. Zymogram also confirmed the phytase activity of the 46 kDa band corresponding to the enzyme. An enzyme activity of 4.9U/ml was obtained in cell extracts of L. lactis. The growth of native and recombinant L. lactis was similar in the presence of two concentrations of ox bile.

Keywords: Lactobacillus salivarus, Lactococcuslactis, recombinant, phytase, poultry

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