E-NTPDase and E-ADA activities are altered in lymphocytes of patients with indeterminate form of Chagas' disease

Trypanosoma cruzi infection triggers a chronic inflammatory process in human host and purinergic system ecto-enzymes play an important role in modulating the inflammatory and immune responses. In this study, it was investigated ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase; EC 3.6.1.5; CD39) and ecto-adenosine deaminase (E-ADA; EC 3.5.4.4) activities in lymphocytes from patients with indeterminate form of Chagas' disease (IFCD). Twenty-five IFCD patients and 25 healthy subjects (control group) were selected. The peripheral lymphocytes were isolated and E-NTPDase and E-ADA activities were determined. Adenine nucleotides and adenosine levels were determined in serum by HPLC and the E-NTPDase1 expression in lymphocytes by Western blot analysis. E-NTPDase (ATP and ADP as substrates) and E-ADA (adenosine as substrate) activities were decreased in lymphocytes from IFCD patients (P<0.05 and P<0.01, respectively), while the E-NTPDase1 expression presented no changes in these patients. Serum ATP levels showed to be decreased (P<0.05) and both AMP (P<0.01) and adenosine (P<0.001) levels were increased in the IFCD group. The enzymatic alterations observed are in agreement with the immune response against T. cruzi infection in IFCD patients, since the decreased extracellular ATP and the increased adenosine levels trigger a Th2 anti-inflammatory response, which it is associated to adaptation of host to parasite, preventing clinical progress of disease.     

β-catenin confers resistance to PI3K and AKT inhibitors and subverts FOXO3a to promote metastasis in colon cancer

The Wnt–β-catenin and PI3K-AKT-FOXO3a pathways have a central role in cancer. AKT phosporylates FOXO3a, relocating it from the cell nucleus to the cytoplasm, an effect that is reversed by PI3K and AKT inhibitors. Simultaneous hyperactivation of the Wnt–β-catenin pathway and inhibition of PI3K-AKT signaling promote nuclear accumulation of β-catenin and FOXO3a, respectively, promoting cell scattering and metastasis by regulating a defined set of target genes. Indeed, the anti-tumoral AKT inhibitor API-2 promotes nuclear FOXO3a accumulation and metastasis of cells with high nuclear β-catenin content. Nuclear β-catenin confers resistance to the FOXO3a-mediated apoptosis induced by PI3K and AKT inhibitors in patient-derived primary cultures and in corresponding xenograft tumors in mice. This resistance is reversed by XAV-939, an inhibitor of Wnt–β-catenin signaling. In the presence of high nuclear β-catenin content, activation of FOXO3a by PI3K or AKT inhibitors makes it behave as a metastasis inductor rather than a proapoptotic tumor suppressor. We show that it is possible to evaluate the β-catenin status of patients' carcinomas and the response of patient-derived cells to target-directed drugs that accumulate FOXO3a in the nucleus before deciding on a course of treatment. We propose that this evaluation could be essential to the provision of a safer and more effective personalized treatment.     

Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting

Functional analysis of genome sequences requires methods for cloning DNA of interest. However, existing methods, such as library cloning and screening, are too demanding or inefficient for high-throughput application to the wealth of genomic data being delivered by massively parallel sequencing. Here we describe direct DNA cloning based on the discovery that the full-length Rac prophage protein RecE and its partner RecT mediate highly efficient linear-linear homologous recombination mechanistically distinct from conventional recombineering mediated by Redαβ from lambda phage or truncated versions of RecET. We directly cloned all ten megasynthetase gene clusters (each 10–52 kb in length) from Photorhabdus luminescens into expression vectors and expressed two of them in a heterologous host to identify the metabolites luminmycin A and luminmide A/B. We also directly cloned cDNAs and exactly defined segments from bacterial artificial chromosomes. Direct cloning with full-length RecE expands the DNA engineering toolbox and will facilitate bioprospecting for natural products.     

Ensayo de formación y cuantificación de biopelículas mixtas de Candida albicans y Staphylococcus aureus

This study quantifies the production of single and mixed biofilms of Candida albicans and Staphylococcus aureus to determine if such mixed biofilms have synergistic effects. Assays were performed using polystyrene microtitre plates of 96 wells, metabolic activity was measured by the enzymatic reduction of a tetrazolium salt (XTT) and colorimetric changes were measured at 490 nm. Confocal scanning laser microscopy was used to visualise the biofilms of each microorganism and its growth kinetics. The highest levels of biofilm formation were observed in mixed biofilms, followed by those of Candida albicans only, with the lowest levels of biofilm formation being detected for Staphylococcus aureus; all together these results suggest a synergistic relationship between the tested microorganisms.     

The -2518 A/G polymorphism in the monocyte chemoattractant protein 1 gene is associated with the risk of developing systemic lupus erythematosus in Argentinean patients: a multicenter study

Systemic lupus erythematosus (SLE) is a systemic, autoimmune disorder. Monocyte chemoattractant protein 1 (MCP-1), a chemokine involved in the recruitment and migration of monocytes/macrophages, has been shown to be increased in the plasma of SLE patients. The aim of our study was to evaluate the possible association of the polymorphism -2518 of the MCP-1 gene with the risk of developing SLE, manifesting lupus nephritis (LN) and with other clinical features of SLE in an Argentinean population. A group of 171 SLE patients and 120 control subjects were examined. Genotypic and allelic frequencies of the MCP-1 -2518 A/G polymorphism showed significant differences between the SLE and the control groups (p=0.001 and p=0.01, respectively). However, the polymorphism showed no association with LN or with the other clinical variables studied. Our results suggest that the presence of the MCP-1 -2518 A/G polymorphism might be a risk factor for developing SLE in genetically predisposed individuals, but it does not seem to have a role in the evolution of the disease in the Argentinean population.     

Production of a monoclonal antibody by ascites, hollow fiber system, and transgenic plants for vaccine production using CB.Hep-1 mAb as a study case

Monoclonal antibody (mAb) production methods (ascites, in vitro technologies, transgenic animals, and dicot or monocot transgenic plants; moss, algae) have been improved since they were first developed in 1975. In this study, we illustrate a summary of a study case in which mice, a hollow fiber system, and tobacco transgenic plants were assessed for the production of mAb for vaccine manufacturing and vaccine production.