Verotoxin-producing Escherichia coli in Spain: prevalence,serotypes, and virulence genes of O157:H7 and non-O157 VTEC in ruminants,raw beef products,and humans

In Spain, as in many other countries, verotoxin-producing Escherichia coli (VTEC) strains have been frequently isolated from cattle, sheep, and foods. VTEC strains have caused seven outbreaks in Spain (six caused by E. coli O157:H7 and one by E. coli O111:H- [nonmotile]) in recent years. An analysis of the serotypes indicated serological diversity. Among the strains isolated from humans, serotypes O26:H11, O111:H-, and O157:H7 were found to be more prevalent. The most frequently detected serotypes in cattle were O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and OUT (O untypeable):H19. Different VTEC serotypes (e.g., O5:H-, O6:H10, O91:H-, O117:H-, O128:H-, O128:H2, O146:H8, O146:H21, O156:H-, and OUT:H21) were found more frequently in sheep. These observations suggest a host serotype specificity for some VTEC. Numerous bovine and ovine VTEC serotypes detected in Spain were associated with human illnesses, confirming that ruminants are important reservoirs of pathogenic VTEC. VTEC can produce one or two toxins (VT1 and VT2) that cause human illnesses. These toxins are different proteins encoded by different genes. Another virulence factor expressed by VTEC is the protein intimin that is responsible for intimate attachment of VTEC and effacing lesions in the intestinal mucosa. This virulence factor is encoded by the chromosomal gene eae. The eae gene was found at a much less frequency in bovine (17%) and ovine (5%) than in human (45%) non-O157 VTEC strains. This may support the evidence that the eae gene contributes significantly to the virulence of human VTEC strains and that many animal non-O157 VTEC strains are less pathogenic to humans.     

Optimization study of doxorubicin liposomal preparations coated with laminin fragments

Immunoliposomes, coated with two peptide sequences and loaded with doxorubicin, were prepared. The influence of different parameters in the sequential steps of liposomal preparations was studied as, for instance, lipid composition, size reduction methods, elimination of non-entrapped drug, and peptide coating sequence. Results were evaluated, such as entrapment efficiency, phospholipid/drug and phospholipid/peptide relationship, and size of final preparations. Effective size reduction was only achieved through probe sonication and the presence of peptides on the surface of liposomes, which does not modify, significantly, the final phospholipid/drug relationship, related to the initial values; however, they promoted a slight increase in the size of final preparations. Dialysis was the most suitable method to wash liposomes from reactants, drug and peptides, as well as being the cleanest process to avoid microbial contamination without significant dilution. Peptide coating yields were similar for liposomal compositions presenting free carboxyl groups on the surface. As determined by other authors, the presence of polyethylene glycol monomethoxy chains on the surface reduces the reactivity of NPGE carboxylic groups.     

Stability of human mesenchymal stem cells during in vitro culture: considerations for cell therapy

Ex vivo expansion and manipulation of human mesenchymal stem cells are important approaches to immunoregulatory and regenerative cell therapies. Although these cells show great potential for use, issues relating to their overall nature emerge as problems in the field. The need for extensive cell quantity amplification in vitro to obtain sufficient cell numbers for use, poses a risk of accumulating genetic and epigenetic abnormalities that could lead to sporadic malignant cell transformation. In this study, we have examined human mesenchymal stem cells derived from bone marrow, over extended culture time, using cytogenetic analyses, mixed lymphocyte reactions, proteomics and gene expression assays to determine whether the cultures would retain their potential for use in subsequent passages. Results indicate that in vitro cultures of these cells demonstrated chromosome variability after passage 4, but their immunomodulatory functions and differentiation capacity were maintained. At the molecular level, changes were observed from passage 5 on, indicating initiation of differentiation. Together, these results lead to the hypothesis that human mesenchymal stem cells cultures can be used successfully in cell therapy up to passage 4. However, use of cells from higher passages would have to be analysed case by case.     

Environmental DNA fragment conferring early and increased sporulation and antibiotic production in Streptomyces species

Here we describe the rep gene, isolated from an environmental DNA library, which when transformed into Streptomyces species resulted in increased production of secondary metabolites and accelerated sporulation. We show that Streptomyces lividans strains bearing rep are particularly useful as expression hosts for heterologous antibiotic production.     

Selective expression of presenilin 1 in neural progenitor cells rescues the cerebral hemorrhages and cortical lamination defects in presenilin 1-null mutant mice

Mice with a null mutation of the presenilin 1 gene (Psen1(-/-)) die during late intrauterine life or shortly after birth and exhibit multiple CNS and non-CNS abnormalities, including cerebral hemorrhages and altered cortical development. The cellular and molecular basis for the developmental effects of Psen1 remain incompletely understood. Psen1 is expressed in neural progenitors in developing brain, as well as in postmitotic neurons. We crossed transgenic mice with either neuron-specific or neural progenitor-specific expression of Psen1 onto the Psen1(-/-) background. We show that neither neuron-specific nor neural progenitor-specific expression of Psen1 can rescue the embryonic lethality of the Psen1(-/-) embryo. Indeed neuron-specific expression rescued none of the abnormalities in Psen1(-/-) mice. However, Psen1 expression in neural progenitors rescued the cortical lamination defects, as well as the cerebral hemorrhages, and restored a normal vascular pattern in Psen1(-/-) embryos. Collectively, these studies demonstrate that Psen1 expression in neural progenitor cells is crucial for cortical development and reveal a novel role for neuroectodermal expression of Psen1 in development of the brain vasculature.     

What do mitochondrial diseases teach us about normal mitochondrial functions...that we already knew: threshold expression of mitochondrial defects

This paper shows how metabolic control analysis (MCA) can help to explain two important features of mitochondrial diseases: (i) the existence of a threshold in the expression of the complex deficiencies on the respiratory flux or on ATP synthesis, i.e. the fact that it is necessary to have a large complex deficiency in order to observe a substantial decrease in these fluxes; (ii) the tissue specificity, i.e. the fact that all tissues are not affected, even if the complex deficiency is present in all of them. We also show the limits of MCA, particularly when considering the in vivo situation. However, MCA offers a new way to consider mitochondrial diseases. The fact that fluxes only slightly change, when a complex is affected, is done at the expense of great changes in intermediate metabolite concentrations; intermediate metabolites situated upstream from the deficient complex are more reduced, leading to a greater generation of free radicals. This could bring an explanation for the diseases observed in conditions where the mitochondrial rate of ATP synthesis is only slightly affected.     

Comparison of Protein-Extraction Methods for Gills of the Shore Crab,Carcinus maenas (L.), and Application to 2DE

As it is well-established that protein extraction constitutes a crucial step for two-dimensional electrophoresis (2DE), this work was done as a prerequisite to further the study of alterations in the proteome in gills of the shore crab Carcinus maenas under contrasted environmental conditions. Because of the presence of a chitin layer, shore crab gills have an unusual structure. Consequently, they are considered as a hard tissue and represent a challenge for optimal protein extraction. In this study, we compared three published extraction procedures for subsequent applications to 2DE: the first one uses homogenization process, the second one included an additional TCA-acetone precipitation step, and finally, the third one associated grinding in liquid nitrogen (N₂) and TCA-acetone precipitation. Extracted proteins were then resolved using 1DE and 2DE. Although interesting patterns were obtained using 1DE with the three methods, only the one involving grinding in liquid N₂ and TCA-acetone precipitation led to proper resolution after 2DE, showing a good level of reproducibility at technical (85%) and biological (84%) levels. This last method is therefore proposed for analysis of gill proteomes in the shore crab.     

The larger attachment glycoprotein of respiratory syncytial virus produced in primary human bronchial epithelial cultures reduces infectivity for cell lines

Respiratory syncytial virus (RSV) infects the upper and lower respiratory tracts and can cause lower respiratory tract infections in children and elders. RSV has traditionally been isolated, grown, studied and quantified in immortalized cell lines, most frequently HEp-2 cells. However, in vivo RSV infection is modeled more accurately in primary well differentiated human bronchial epithelial (HBE) cultures where RSV targets the ciliated cells and where the putative RSV receptor differs from the receptor on HEp-2 cells. The RSV attachment (G) glycoprotein in virions produced by HEp-2 cells is a highly glycosylated 95 kDa protein with a 32 kDa peptide core. However, virions produced in HBE cultures, RSV (HBE), contain an even larger, 170 kDa, G protein (LgG). Here we show that LgG is found in virions from both subgroups A and B lab-adapted and clinical isolates. Unexpectedly, RSV (HBE) virions were approximately 100-fold more infectious for HBE cultures than for HEp-2 cells. Surprisingly, the cause of this differential infectivity, was reduced infectivity of RSV (HBE) on HEp-2 cells rather than enhanced infectivity on HBE cultures. The lower infectivity of RSV(HBE) for HEp-2 cells is caused by the reduced ability of LgG to interact with heparan sulfate proteoglycans (HSPG), the RSV receptor on HEp-2 cells. The discovery of different infectivity corresponding with the larger form of the RSV attachment protein when produced by HBE cultures highlights the importance of studying a virus produced by its native host cell and the potential impact on quantifying virus infectivity on cell lines where the virus entry mechanisms differ from their natural target cell.