Clade 8 and Clade 6 Strains of Escherichia coli O157:H7 from Cattle in Argentina have Hypervirulent-Like Phenotypes

The hemolytic uremic syndrome (HUS) whose main causative agent is enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a disease that mainly affects children under 5 years of age. Argentina is the country with the highest incidence of HUS in the world. Cattle are a major reservoir and source of infection with E. coli O157:H7. To date, the epidemiological factors that contribute to its prevalence are poorly understood. Single nucleotide polymorphism (SNP) typing has helped to define nine E. coli O157:H7 clades and the clade 8 strains were associated with most of the cases of severe disease. In this study, eight randomly selected isolates of EHEC O157:H7 from cattle in Argentina were studied as well as two human isolates. Four of them were classified as clade 8 through the screening for 23 SNPs; the two human isolates grouped in this clade as well, while two strains were closely related to strains representing clade 6. To assess the pathogenicity of these strains, we assayed correlates of virulence. Shiga toxin production was determined by an ELISA kit. Four strains were high producers and one of these strains that belonged to a novel genotype showed high verocytotoxic activity in cultured cells. Also, these clade 8 and 6 strains showed high RBC lysis and adherence to epithelial cells. One of the clade 6 strains showed stronger inhibition of normal water absorption than E. coli O157:H7 EDL933 in human colonic explants. In addition, two of the strains showing high levels of Stx2 production and RBC lysis activity were associated with lethality and uremia in a mouse model. Consequently, circulation of such strains in cattle may partially contribute to the high incidence of HUS in Argentina.     

Biosynthesis of poly-β-hydroxybutyrate (PHB) with a high molecular mass by a mutant strain of Azotobacter vinelandii (OPN)

The aim of this study was to characterize the influence of the aeration conditions on the production of PHB and its molecular mass in a mutant strain of Azotobacter vinelandii (OPN), which carries a mutation on ptsN, the gene encoding enzyme IIA^Ntr, previously shown to increase the accumulation of PHB. Cultures of A. vinelandii wild-type strain OP and its mutant derivative strain OPN were grown in 500-mL flasks, containing 100 and 200 mL of PY sucrose medium. PHB production and its molecular mass were analyzed at the end of the culture. The molecular mass (MM) was significantly influenced by the aeration conditions and strain used. A polymer with a higher molecular weight was produced under low aeration conditions for both strains. A maximal molecular mass of 2,026 kDa (equivalent to 3,670 kDa measured by GPC) was obtained with strain OPN cultured under low-aeration conditions, reaching a value two-fold higher than that obtained from the parental strain OP (MM?=?1,013 kDa) grown under the same conditions. Aeration conditions and the ptsN mutation influence the molecular mass of the PHB produced by A. vinelandii affecting in turn its physico-chemical properties.     

The role of fructose-1, 6-diphosphate in cell migration and proliferation in an in vitro xenograft blood vessel model of vascular wound healing

Summary Both smooth muscle cells and endothelial cells play an important role in vascular wound healing. To elucidate the role of fructose-1, 6-diphosphate, cell proliferation and cell migration studies were performed with human endothelial cells and rat smooth muscle cells. To mimic blood vessels, endothelial and smooth muscle cells were used in 1:10, 1:5, and 1:1 concentrations, respectively, mimicking large-, mid-, and capillary-sized blood vessels. Cell migration was studied with fetal bovine serum-starved cells. For cell proliferation assay, cells were plated at 30–50% confluency and then starved. The cells were incubated for 48 h with fructose-1, 6-diphosphate at (per ml) 10 mg, 1 mg, 500 μg, 250 μg, 100 μg, and 10 μg, pulsed with tritiated-thymidine and incubated with 1 N NaOH for 30 min at room temperature, harvested, and counted. For migration assay, confluent cells were starved, wounded, and incubated for 24 h with same concentrations of fructose-1, 6-diphosphate as in proliferation assay. The cells were fixed and counted. Smooth muscle cell proliferation was inhibited by fructose-1, 6-diphosphate at 10 mg/ml. In the xenograft models of 1:10, 1:5, and 1:1 fructose-1, 6-diphosphate inhibited proliferation at 10 mg/ml. In migration studies 10 mg fructose-1, 6-diphosphate per ml was inhibitory to both cell types. In large-, mid-, and capillary-sized blood vessels, fructose-1, 6-diphosphate inhibited proliferation of both cell types at 10 mg/ml. At the individual cell level, fructose-1, 6-diphosphate is nonstimulatory to proliferation of endothelial cells while inhibiting migration, and it acts on smooth muscle cells by inhibiting both proliferation and migration.     

Synthesis, characterization and antiproliferative studies of the enantiomers of cis-[(1,2-camphordiamine)dichloro]platinum(II) complexes

The platinum(II) complex cis-[(1S,2R,3S)-1,7,7-trimethylbicyclo[2.2.1]heptane-2,3-diamine]dichloroplatinum(II) (1) and its enantiomer (2) have been synthesized and physically and spectroscopically characterized. To obtain the enantiopure complexes the chiral pool approach was applied. The synthetic pathway has four steps, starting from (+/-)-diphenylethylenediamine (DPEDA) (3) and the natural products (1S)-camphorquinone or (1R)-camphorquinone to obtain enantiomers 1 and 2, respectively. The interaction of the Pt(II) complexes with DNA was studied by several techniques: circular dichroism, electrophoresis on agarose gel and atomic force microscopy (AFM). These studies showed differences in the degree of interaction between both enantiomers and DNA (calf thymus DNA and plasmid pBR322 DNA). The cytotoxicity of enantiomers 1 and 2 against the HL-60 cell line was studied by in vitro tests of antiproliferative activity, incubating during both 24 h and 72 h. An important difference of activity was found between both enantiomers regarding the IC50 data at 24 h of incubation. Thus, complex 1 showed to be much more active than its enantiomer 2.     

Evaluating the influence of off-shore cage aquaculture on the benthic ecosystem in Alghero Bay (Sardinia,Italy) using AMBI and M-AMBI

The impact of an off-shore fish farm in Alghero Bay (northwest Sardinia, Italy) on the benthic ecosystem was investigated in 2007 and 2008. In addition to studying the chemical and physical characteristics of the area (i.e., currents and sediment analyses), some biological analyses were also performed. The AZTI's Marine Biotic Index (AMBI) and the multivariate AMBI (M-AMBI) were calculated, which are being used in assessing the ecological status of benthic communities within the European Water Framework Directive (WFD). Clear impact gradients were detected according to both methods; they are related to farm production, prevailing currents, and characteristics of the area (i.e., water depth and distance to the cages). The site affected most was detected within 84 m from the cages; the area that no longer showed effects was over 907 m from the cages. The gradient is shown by decreasing AMBI values and percentage of opportunistic species and increasing richness, diversity, and the presence of sensitive species. This study highlights the importance of setting reference conditions for different areas when calculating M-AMBI. These reference conditions correspond to those in undisturbed sites in the opposite direction of the prevailing currents within the area.     

Influence of organic co-solvents on the activity and substrate specificity of feruloyl esterases

Organic co-solvents can expand the use of enzymes in lignocellulose deconstruction through making substrates more soluble and thus more accessible. In choosing the most adequate co-solvent for feruloyl esterases, hydrolysis of methyl p-hydroxycinnamates by three pure enzymes (and a multi-enzyme preparation) was evaluated. Low concentrations of dimethylsulfoxide (DMSO) enhanced hydrolysis by two of the enzymes while at levels >20%, activity was reduced. DMSO also enhanced acetyl esterase-type activity of the enzymes. The co-solvent effect was different for each enzyme-substrate couple, indicating that other factors are also involved. Kinetic studies with a Talaromyces stipitatus feruloyl esterase showed low concentrations of dimethylsulfoxide enhanced the hydrolytic rate while K_m also increased. Moreover, long-term incubation (96 h) of an Aspergillus niger feruloyl esterase in dimethylsulfoxide:water provided to the enzyme the ability to hydrolyze methyl p-coumarate, suggesting an active-site re-arrangement. Dimethylsulfoxide (10-30%) is proposed as an adequate co-solvent for feruloyl esterase treatment of water-insoluble substrates.     

Microbial metabolism of chlorosalicylates: effect of prolonged subcultivation on constructed strains

The hybrid strain Pseudomonas sp. WR4016 was subcultivated with increasing concentrations of 5-chlorosalicylate (510 mM) as sole carbon source over a period of 9 months. At intervals of approximately 3 months derivative strains WR4017, WR4018 and WR4019 were isolated which exhibited higher growth rates and increased substrate tolerance. Comparative analysis of the turnover rates of the key enzymes in chlorosalicylate degradation showed that the adaptation process did not result from structural modifications of these proteins. Instead, balanced over-production of the salicylate hydroxylase and catechol 1,2-dioxygenase prevented the accumulation of toxic chlorocatechols and accounted for the reduction of the doubling times with 4- or 5-chlorosalicylate. A comparative analysis of a genetically engineered chlorosalicylate degrader PL300-1 showed similar regulatory patterns as the most advanced isolate WR4019 from the adaptation series.