Biological and molecular characterization of fEg-Eco19, a lytic bacteriophage active against an antibiotic-resistant clinical Escherichia coli isolate.

Characterization of bacteriophages facilitates better understanding of their biology, host specificity, genomic diversity, and adaptation to their bacterial hosts. This, in turn, is important for the exploitation of phages for therapeutic purposes, as the use of uncharacterized phages may lead to treatment failure. The present study describes the isolation and characterization of a bacteriophage effective against the important clinical pathogen Escherichia coli, which shows increasing accumulation of antibiotic resistance. Phage fEg-Eco19, which is specific for a clinical E. coli strain, was isolated from an Egyptian sewage sample. Phage fEg-Eco19 formed clear, sharp-edged, round plaques. Electron microscopy showed that the isolated phage is tailed and therefore belongs to the order Caudovirales, and morphologically, it resembles siphoviruses. The diameter of the icosahedral head of fEg-Eco19 is 68 ± 2 nm, and the non-contractile tail length and diameter are 118 ± 0.2 and 13 ± 0.6 nm, respectively. The host range of the phage was found to be narrow, as it infected only two out of 137 clinical E. coli strains tested. The phage genome is 45,805 bp in length with a GC content of 50.3% and contains 76 predicted genes. Comparison of predicted and experimental restriction digestion patterns allowed rough mapping of the physical ends of the phage genome, which was confirmed using the PhageTerm tool. Annotation of the predicted genes revealed gene products belonging to several functional groups, including regulatory proteins, DNA packaging and phage structural proteins, host lysis proteins, and proteins involved in DNA/RNA metabolism and replication.

In vivo toxicity and antitumor activity of newly green synthesized reduced graphene oxide/silver nanocomposites.

A novel biosynthesis of dual reduced graphene oxide/silver nanocomposites (rGO/AgNC) using the crude metabolite of Escherichia coli D8 (MF06257) strain and sunlight is introduced in this work. Physicochemical analysis of these rGO/AgNC revealed that they are sheet-like structures having spherically shaped silver nanoparticles (AgNPs) with an average particle size of 8 to 17 nm, and their absorption peak ranged from 350 to 450 nm. The biosynthesized rGO/AgNC were characterized by UV-vis and FT-IR spectra, X-ray diffraction, Zeta potential and transmission electron microscopy. After the injection of these nanocomposites to mice, their uptake by the kidney and liver has been proven by the ultrastructural observation and estimation of the hepatic and renal silver content. These nanocomposites caused a moderate toxicity for both organs. Changes in the liver and kidney functions and histopathological effects had been observed. The rGO/AgNC revealed a remarkable antitumor effect. They showed a dose-dependent cytotoxic effect on Ehrlich ascites carcinoma (EAC) cells in vitro. Treatment of mice bearing EAC tumors intraperitoneally with 10 mg/kg rGO/AgNC showed an antiproliferative effect on EAC cells, reduced ascites volume, and maintained mice survival. The results indicate that this green synergy of silver nanoparticles with reduced graphene oxide may have a promising potential in cancer therapy.

The nucleolar-related protein Dyskerin pseudouridine synthase 1 (DKC1) predicts poor prognosis in breast cancer.

BACKGROUND: Hypertrophy of the nucleolus is a distinctive cytological feature of malignant cells and corresponds to aggressive behaviour. This study aimed to identify the key gene associated with nucleolar prominence (NP) in breast cancer (BC) and determine its prognostic significance. METHODS: From The Cancer Genome Atlas (TCGA) cohort, digital whole slide images identified cancers having NP served as label and an information theory algorithm was applied to find which mRNA gene best explained NP. Dyskerin Pseudouridine Synthase 1 (DKC1) was identified. DKC1 expression was assessed using mRNA data of Molecular Taxonomy of Breast Cancer International Consortium (METABRIC, n = 1980) and TCGA (n = 855). DKC1 protein expression was assessed using immunohistochemistry in Nottingham BC cohort (n = 943). RESULTS: Nuclear and nucleolar expressions of DKC1 protein were significantly associated with higher tumour grade (p < 0.0001), high nucleolar score (p < 0.001) and poor Nottingham Prognostic Index (p < 0.0001). High DKC1 expression was associated with shorter BC-specific survival (BCSS). In multivariate analysis, DKC1 mRNA and protein expressions were independent risk factors for BCSS (p < 0.01). CONCLUSION: DKC1 expression is strongly correlated with NP and its overexpression in BC is associated with unfavourable clinicopathological characteristics and poor outcome. This has been a detailed example in the correlation of phenotype with genotype.

Identification and Functional Analysis of Temperate Siphoviridae Bacteriophages of Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic pathogen that presents a serious clinical challenge due to its increasing resistance to all available antibiotics. Phage therapy has been introduced recently to treat antibiotic-incurable A. baumannii infections. In search for new A. baumannii specific bacteriophages, 20 clinical A. baumannii strains were used in two pools in an attempt to enrich phages from sewage. The enrichment resulted in induction of resident prophage(s) and three temperate bacteriophages, named vB_AbaS_fEg-Aba01, vB_AbaS_fLi-Aba02 and vB_AbaS_fLi-Aba03, all able to infect only one strain (#6597) of the 20 clinical strains, were isolated. Morphological characteristics obtained by transmission electron microscopy together with the genomic information revealed that the phages belong to the family Siphoviridae. The ca. 35 kb genomic sequences of the phages were >99% identical to each other. The linear ds DNA genomes of the phages contained 10 nt cohesive end termini, 52-54 predicted genes, an attP site and one tRNA gene each. A database search revealed an >99% identical prophage in the genome of A.baumannii strain AbPK1 (acc. no. CP024576.1). Over 99% identical prophages were also identified from two of the original 20 clinical strains (#5707 and #5920) and both were shown to be spontaneously inducible, thus very likely being the origins of the isolated phages. The phage vB_AbaS_fEg-Aba01 was also able to lysogenize the susceptible strain #6597 demonstrating that it was fully functional. The phages showed a very narrow host range infecting only two A.baumannii strains. In conclusion, we have isolated and characterized three novel temperate Siphoviridae phages that infect A.baumannii.

Heterologous expression, purification, immobilization and characterization of recombinant α-amylase AmyLa from Laceyella sp. DS3.

AmyLa α-amylase gene from Laceyella sp. DS3 was heterologously expressed in E. coli BL21. E. coli BL21 maximally expressed AmyLa after 4 h of adding 0.02 mM IPTG at 37 °C. The recombinant AmyLa α-amylase was purified 2.19-fold through gel filtration and ion exchange chromatography. We immobilized the purified recombinant AmyLa α-amylase on four carriers; chitosan had the best efficiency. The recombinant free and the immobilized AmyLa α-amylase showed optimum activity in the pH ranges of 6.0-7.0 and 4.0-7.0, respectively and possessed an optimum temperature of 55 °C. The free enzyme had activation energy, Km, and Vmax of 291.5 kJ, 1.5 mg/ml, and 6.06 mg/min, respectively. The immobilized enzyme had activation energy, Km, and Vmax of 309.74 kJ, 6.67 mg/ml, and 50 mg/min, respectively. The immobilized enzyme was calcium-independent and insensitive (relative to the free enzyme) to metals. It could also be reused for seven cycles.

Allyl rhodanine azo dye derivatives: Potential antimicrobials target d-alanyl carrier protein ligase and nucleoside diphosphate kinase.

3-Allyl-5-(4-arylazo)-2-thioxothiazolidine-4-one (HLn ) ligands (where n = 1 to 3) were hypothesized to have antimicrobial activities mediated through inhibition of new antimicrobial targets. The ligands (HLn ) were synthesized and characterized by infrared (IR) and 1 H nuclear magnetic resonance (1 H NMR) spectra. The ligands (HLn ) were in silico screened to their potential inhibition to models of d-alanyl carrier protein ligase (DltA) (from Bacillus cereus, PDB code 3FCE) and nucleoside diphosphate kinase (NDK) (from Staphylococcus aureus; PDB code 3Q8U). HL3 ligand has the best energy and mode of binding to both NDK and DltA, even though its binding to DltA was stronger than that to NDK. In antimicrobial activity of HL3 ligand, morphological and cytological changes in HL3 -treated bacteria agreed with the in silico results. The HL3 ligand showed significant antimicrobial activity against B. cereus, S. aureus, and Fusarium oxysporium. The HL3 -treated bacterial cells appeared malformed and incompletely separated. Its cell walls appeared electron-lucent and ruptured. They contained more mesosomes than normal cells. It was found that the HL3 ligand represented as a bactericide against B. cereus and S. aureusby blocking target DltA, and may target NDK.

Occurrence of cyanobacteria and microcystin toxins in raw and treated waters of the Nile River, Egypt: implication for water treatment and human health.

Monitoring of cyanobacteria and their associated toxins has intensified in raw water sources of drinking water treatment plants (WTPs) in most countries of the world. However, it is not explored yet for Egyptian WTPs. Therefore, this study was undertaken to investigate the occurrence of cyanobacteria and their microcystin (MC) toxins in the Nile River source water of Damietta WTP during warm months (April-September 2013) and to evaluate the removal efficiency of both cyanobacterial cells and MCs by conventional methods used in this plant as a representative of Egyptian drinking WTPs. The results showed that the source water at the intake of Damietta WTP contained dense cyanobacterial population (1.1-6.6 × 107 cells L(-1)) dominated by Microcystis aeruginosa. This bloom was found to produce MC-RR and MC-LR. Both cyanobacterial cell density and intracellular MCs in the intake source water increased with the increase in temperature and nutrients during the study period, with maximum values obtained in August. During treatment processes, cyanobacterial cells were incompletely removed by coagulation/flocculation/sedimentation (C/F/S; 91-96.8%) or sand filtration (93.3-98.9%). Coagulation/flocculation induced the release of MCs into the ambient water, and the toxins were not completely removed or degraded during further treatment stages (filtration and chlorination). MCs in outflow tank water were detected in high concentrations (1.1-3.6 µg L - 1), exceeding WHO provisional guideline value of 1 µg L - 1 for MC-LR in drinking water. Based on this study, regular monitoring of cyanobacteria and their cyanotoxins in the intake source water and at different stages at all WTPs is necessary to provide safe drinking water to consumers or to prevent exposure of consumers to hazardous cyanobacterial metabolites.

Purification, sequencing, and biochemical characterization of a novel calcium-independent α-amylase AmyTVE from Thermoactinomyces vulgaris.

α-Amylase from Thermoactinomyces vulgaris was highly purified 48.9-fold by ammonium sulfate precipitation, gel filtration on Sephadex G-50 column, and ion exchange chromatography column of DEAE-cellulose. The molecular weight of the enzyme was estimated to be 135 and 145 kDa by SDS-PAGE. Its high molecular weight is due to high glycosylation. The purified amylase exhibited maximal activity at pH 6.0 to 7.0 and was stable in the range of pH 4.0 to 9.0. The optimum temperature for its activity was 50 °C. The enzyme half-life time was 120 min at 50 °C, suggesting intermediate temperature stable α-amylase. The enzyme was sensitive to different metal ions, including NaCl, CoCl(2), and CaCl(2), and to different concentrations of EDTA. The enzyme activity was inhibited in the presence of 1 mM CaCl(2), suggesting that it is a calcium-independent α-amylase. The TLC showed that the amylase hydrolyzed starch to produce large maltooligosaccharides as the main products. A 1.1-kb DNA fragment of the putative α-amylase gene (amy TVE) from T. vulgaris was amplified by using two specific newly designed primers. Sequencing analysis showed 56.2 % similarity to other Thermoactinomyces α-amylases with two conserved active sites confirming its function.

Immunochemical identification and partial characterization of a native hepatitis C viral non-structural 4 antigen in sera of HCV infected patients.

BACKGROUND: The identification of native HCV antigens may prove very useful in the diagnosis and early treatment of HCV infection. Here, we aimed to identify and partially characterize a native HCV-NS4 antigen. METHODS: The western blot, ELISA and immunohistochemical staining were used to identify the native antigen in sera and liver biopsies of HCV serotype 4 infected patients. RESULTS: The native NS4 antigen was identified in serum at 27-kDa molecular weight, in addition to a lower approximately 21-kDa antigen. The purified HCV antigen showed a polypeptide band at 27-kDa when analyzed by silver stained SDS-PAGE and a single peak at 7.6 min by capillary zone electrophoresis. The immunostaining pattern of hepatocytes was cytoplasmic with mainly coarse granular and diffuse pattern based on specific rabbit antisera to the native HCV antigen. A highly significant correlation (r=0.797, p<0.0001) was shown between serum concentrations of the HCV-NS4 antigen and HCV-RNA. Also, antigen detection rates were increased (p<0.05) with the progression of liver disease. CONCLUSION: A native HCV-NS4 antigen was identified and partially characterized as 27-kDa protein and the NS4 antigenemia based ELISA test can serve as a useful addition to HCV diagnostic methods, especially under field condition.

Production and immobilization of alpha-amylase from Bacillus subtilis.

Alpha-amylase production by Bacillus subtilis was studied under different cultivation conditions. The maximum alpha-amylase production occurred after an incubation period of 48 h, temperature 40 degrees C and pH 7.5. Among the defined carbohydrates, starch (1%) was the best carbon source. The organism grew better and produced high levels of alpha-amylase using peptone as nitrogen source. The produced alpha-amylase was immobilized on various carriers by different methods and the properties of the enzyme were compared before and after immobilization. The optimum pH of the immobilized enzyme was changed to acidic range. The optimum reaction temperature of immobilized enzyme was shifted slightly to 70-80 degrees C. Both of Km values and Vmax and thermal stability of immobilized enzyme were found to be higher than that of free one. Among the tested metals CaCl2 exerted a stimulating effect on the activity of alpha-amylase.