Isolation, characterization and complete genome sequence of PhaxI: a phage of Escherichia coli O157 :H7

Bacteriophages are considered as promising biological agents for the control of infectious diseases. Sequencing of their genomes can ascertain the absence of antibiotic resistance, toxin or virulence genes. The anti-O157 :H7 coliphage, PhaxI, was isolated from a sewage sample in Iran. Morphological studies by transmission electron microscopy showed that it has an icosahedral capsid of 85–86 nm and a contractile tail of 115�15 nm. PhaxI contains dsDNA composed of 156 628 nt with a G+C content of 44.5 mol% that encodes 209 putative proteins. In MS analysis of phage particles, 92 structural proteins were identified. PhaxI lyses Escherichia coli O157 :H7 in Luria-Bertani medium and milk, has an eclipse period of 20 min and a latent period of 40 min, and has a burst size of about 420 particles per cell. PhaxI is a member of the genus ‘Viunalikevirus’ of the family Myoviridae and is specific for E. coli O157 : H7

Secretory expression and purification of a soluble NADH cytochrome b5 reductase enzyme from Mucorracemosus in Pichiapastoris based on codon usage adaptation

Abstract The genome of Mucor racemosus was analyzed to determine the relative levels of codon usage. The codon bias differred from that of Escherichia coli. The active, soluble isoform of NADH cytochrome b5 reductase containing 228 amino acids was successfully overexpressed and secreted using alpha factor in Pichia pastoris under the control of the alcohol oxidase promoter and finally purified. The culture medium and incubation time were optimized, and the maximum expression level observed was about 23 U/ml using X-33

Cloning, molecular characterization and expression of a cDNA encoding a functional NADH-cytochrome b5 reductase from Mucor racemosus PTCC 5305 in E. coli

The present work aims to study a new NADH-cytochrome b5 reductase (cb5r) from Mucor racemosus PTCC 5305. A cDNA coding for cb s r was isolated from a Mucor racemosus PTCC 5305 cDNA library. The nucleotide sequence of the cDNA including coding and sequences flanking regions was determined. The open reading frame starting from ATG and ending with TAG stop codon encoded 228 amino acids and displayed the closest similarity (73%) with Mortierella alpina cb s r. Lack of hydrophobic residues in the N-terminal sequence was apparent, suggesting that the enzyme is a soluble isoform. The coding sequence was then cloned in the pET16b transcription vector carrying an N-terminal-linked His-Tag® sequence and expressed in Escherichia coli BL21 (DE3). The enzyme was then homogeneously purified by a metal affinity column. The recombinant Mucor enzyme was shown to have its optimal activity at pH and temperature of about 7.5 and 40 °C, respectively. The apparent Km value was calculated to be 13 μM for ferricyanide. To our knowledge, this is the first report on cloning and expression of a native fungal soluble isoform of NADH-cytochrome b5 reductase in E. coli

Oral Immunotherapy Using Probiotic Ice Cream Containing Recombinant Food-Grade Lactococcus lactis Which Inhibited Allergic Responses in a BALB/c Mouse Model

This study was conducted to evaluate the effects of recombinant probiotic bacteria as a candidate for oral vaccine with the potential of treating allergy to Amaranthus retroflexus pollens. The main gene of this allergen, Ama r 2, was cloned into the food grade plasmid pNZ7025 and then was electrotransformed into the food grade Lactococcus lactis NZ1330. No expression was observed in the primary structure due to the distance between the ribosome binding site and the start codon. Therefore, the vector structure was corrected using the site-directed mutagenesis (SDM) technique. The cell extract of this strain was used for assessing the expression of the recombinant allergen in western blot analysis, and the existence of this protein with a molecular weight of 14.2 kDa was confirmed. To evaluate the efficacy of this strain in the treatment of allergies as an oral vaccine, probiotic ice cream was prepared. After the sensitization of mice, the treatment was performed by oral immunotherapy for 4 weeks, 4 to 5 times per week. 20 μl of functional ice cream with 1012 CFU/ml of r-L. lactis NZ1330 significantly reduced the serum IgE level. The levels of IFN-γ and TGF-β cytokines increased in the 20 μl ice cream treatment group as well as 40 μg/ml pure allergen compared with the PBS-treated group, and IL-4 cytokine levels decreased compared with the PBS-treated group. Overall, 20 μl ice cream with 1012 CFU/ml of the recombinant bacteria resulted in the best performance in terms of improving allergies to Th1 and Treg responses

Runx3 Expression Inhibits Proliferation and Distinctly Alters mRNA Expression of Bax in AGS and A549 Cancer Cells

Abstract Runx3, a member of Runt-related transcription factor (Runx) proteins with tumor suppressor effect, is a tissue-restricted and cancer related transcription factor that regulate cell proliferation and growth, as well as differentiation. In the present study, exogenous Run3 was transiently expressed in AGS (human gastric adenocarcinoma), with undetectable Runx3 protein and in A549 (human lung carcinoma) with low levels of endogenous Runx3 protein. The GFP tagged Runx3 was transfected into AGS and A549 cells using fugene6 and PolyFect and Runx3 expression was confirmed by fluorescent microscopy and RT-PCR. The effect of Runx3 transfection on cell proliferation was determined by MTT assay and the results were confirmed by the trypan blue dye exclusion method. The effect of Runx3 expression on mRNA expression of BCL2-associated X protein (Bax) was evaluated using RT-PCR. In AGS and A549 cells, Runx3 expression inhibited cell proliferation (p < 0.01). The growth inhibition was less in A549 cells. We show that Runx3 expression increases Bax mRNA expression in AGS cells when compared with control (p < 0.05), but no significant differences in mRNA expression was observed in both examined cells. Runx3 expression has antiproliferative effect in AGS cell perhaps via increase in expression of Bax. The effect of Runx3 on A549 cells' viability which has endogenous level of Runx3 is not related to Bax. These findings implicate a complex regulation by Runx3 in inhibition of cell proliferation utilizing Bax

Interaction of Escherichia coli heat-labile enterotoxin B-pentamer with exopolysaccharides from Leuconostoc mesenteroides P35: Insights from surface plasmon resonance and molecular docking studies

International audienceIn this study, the interaction of exopolysaccharides from Leuconostoc mesenteroides P35 (EPS-LM) with Escherichia coli heat-labile enterotoxin B-pentamer (LTB) was investigated at different concentrations and temperatures by using surface plasmon resonance (SPR) and molecular docking approaches. FT-IR spectral analysis together with HPTLC analysis revealing that glucose is the only constitutive monosaccharide of EPS-LM suggests that its structure is composed of dextran with α-D (1 → 6) glycosidic linkages. SPR analysis revealed the high affinity of EPS-LM for immobilized LTB toxin (KA=(2.05 ± 0.04) × 106 mol.L−1 at 37°C). The binding process was spontaneous (ΔG0), and entropy-driven (ΔS>0) with an increase of KA with temperature. This suggests that EPS-LM - LTB interaction is dominated by hydrophobic forces. The binding affinity of EPS-LM to LTB had negligible dependence on enthalpy (ΔH = 0.084 kJ mol−1). Further, molecular docking results suggested the presence of some binding sites of EPS-LM on the LTB through hydrophobic forces (Lys, Asp, Arg, Glu) and also hydrogen bonding (Glu) in the hydrophobic core of LTB. Besides autodock studies, Schiffer-Edmundson helical wheel diagrams of LTB in α-helix domain suggested that LTB hydrophobic core is a highly effective region, which was able to form favorable non-polar interactions of the protein's binding surface (with amino acids residues such as Tyr, Leu, Ile) with EPS-LM. This study provided thus further insights into the interactions between EPS-LM and LTB, suggesting that EPS produced by some LAB, such as EPS produced by Ln. mesenteroides P35 strain are good candidates to inhibit E. coli toxin activity

Effect of the interaction of nisin Z with various polysaccharides on its antibacterial activity

International audienceThe effect of various polysaccharides commonly used as thickening or gelling agents in foods on the antibacterial activity of nisin Z was investigated. Addition of 1 g.L−1 sodium alginate in tryptic soy broth resulted in a four-fold increase of minimal inhibitory (MIC) and bactericidal (MBC) concentrations of nisin Z against Listeria innocua ATCC 33090 and Kocuria rhizophila ATCC 9341, while the addition of citrus pectin or various exopolysaccharides (Leuconostoc mesenteroides P35 exopolysaccharides, low (9–11 kDa) or medium (60–76 kDa) molecular weight dextran from Ln. mesenteroides and 2 kefirans (extracted from kefir grains provided by Crokfun or Kefiralia)) did not increase MIC and MBC of nisin Z against these bacteria. Zeta potential determination at pH 7 of the nisin Z, bacteria and polysaccharides allowed us to observe that only nisin Z had a slightly positive zeta potential, while the bacteria, sodium alginate and highly methoxylated citrus pectin zeta potentials were lower than −20 mV and zeta potentials of all exopolysaccharides were between 0 and - 10 mV. It was thus proposed that anionic sodium alginate, which is the only polysaccharide having a lower zeta potential than both bacteria, forms complexes stabilized by electrostatic interactions with oppositely charged nisin Z, thereby limiting the quantity of “free” nisin Z interacting with nisin-susceptible bacteria. This hypothesis was substantiated by the observation of nisin Z - sodium alginate aggregation at pH 7 and the estimation of the apparent affinity of sodium alginate for “free” nisin Z and immobilized nisin Z by partition experiments and by surface plasmon resonance analysis, respectively. This study thus provides useful information for food formulation regarding the effect of different polysaccharides on nisin Z antibacterial activity and some understanding of how interactions between nisin Z, polysaccharides, and nisin Z - sensitive bacteria are explanatory

Assessment of Cytokine Expression Profile in Acute Myeloid Leukemia Patients Before and After Chemotherapy

OBJECTIVE: One of the major goals of cancer treatment is the monitoring of chemotherapeutic protocols. Quantitative and comparative cytokine expression profiling could be reliable to be used for biomarkers in deadly and fast-growing cancers such as acute myeloid leukemia (AML). The present study aims to assess and further validate cytokines with probable effects on proliferation and maturation of blood cells in AML. METHODS: Gene expression levels of IL-1β, IL-10, IL-8, TNF-α, and IFN-γ were analyzed before and after chemotherapy and after granulocyte colony-stimulating factor (G-CSF) therapy in 46 AML patients by an in-house quantitative comparative RT-PCR method. RESULTS: Our findings indicated that although the gene expression level of TNF-α was almost constant in all 3 samples, IL-1β, IL-8, and IL-10 expression levels showed a decrease after chemotherapy and an increase after G-CSF therapy. On the other hand, the expression level of IFN-γ had a different pattern with an increase after chemotherapy and a decrease after G-CSF therapy. CONCLUSION: Taken together, the results of this study are in support of the idea that the analyzed cytokines could be useful biomarkers for AML treatment monitoring. However, further molecular epidemiological investigations are suggested to elaborate more cancer monitoring biomarkers