Bacterial and parasite co-infection in Mexican golden trout (Oncorhynchus chrysogaster) by Aeromonas bestiarum, Aeromonas sobria, Plesiomonas shigelloides and Ichthyobodo necator.

BACKGROUND: Bacterial infections are responsible of high economic losses in aquaculture. Mexican golden trout (Oncorhynchus chrysogaster) is a threatened native trout species that has been introduced in aquaculture both for species conservation and breeding for production and for which no studies of bacterial infections have been reported. CASE PRESENTATION: Fish from juvenile stages of Mexican golden trout showed an infectious outbreak in a farm in co-culture with rainbow trout (Oncorhynchus mykiss), showing external puntiform red lesions around the mouth and caudal pedunculus resembling furuncles by Aeromonas spp. and causing an accumulated mortality of 91%. Isolation and molecular identification of bacteria from lesions and internal organs showed the presence of Aeromonas bestiarum, Aeromonas sobria, Plesiomonas shigelloides and Ichthyobodo necator isolated from a single individual. All bacterial isolates were resistant to amoxicillin-clavulanic acid and cefazoline. P. shigelloides was resistant to third generation ß-lactamics. CONCLUSIONS: This is the first report of coinfection by Aeromonas bestiarum, Aeromonas sobria, Plesiomonas shigelloides and Ichthyobodo necator in an individual of Mexican golden trout in co-culture with rainbow trout. Resistance to ß-lactams suggests the acquisition of genetic determinants from water contamination by human- or livestock-associated activities.

The CSL proteins, versatile transcription factors and context dependent corepressors of the notch signaling pathway.

The Notch signaling pathway is a reiteratively used cell to cell communication pathway that triggers pleiotropic effects. The correct regulation of the pathway permits the efficient regulation of genes involved in cell fate decision throughout development. This activity relies notably on the CSL proteins, (an acronym for CBF-1/RBPJ-κ in Homo sapiens/Mus musculus respectively, Suppressor of Hairless in Drosophila melanogaster, Lag-1 in Caenorhabditis elegans) which is the unique transcription factor and DNA binding protein involved in this pathway. The CSL proteins have the capacity to recruit activation or repression complexes according to the cellular context. The aim of this review is to describe the different co-repressor proteins that interact directly with CSL proteins to form repression complexes thereby regulating the Notch signaling pathway in animal cells to give insights into the paralogous evolution of these co-repressors in higher eumetazoans and their subsequent effects at developmental processes.

Evaluation of DNA extraction methods of rumen microbial populations.

The dynamism of microbial populations in the rumen has been studied with molecular methods that analyze single nucleotide polymorphisms of ribosomal RNA gene fragments (rDNA). Therefore DNA of good quality is needed for this kind of analysis. In this work we report the evaluation of four DNA extraction protocols (mechanical lysis or chemical lysis with CTAB, ethylxanthogenate or DNAzol(®)) from ruminal fluid. The suitability of two of these protocols (mechanical lysis and DNAzol(®)) was tested on single-strand conformation polymorphism (SSCP) of rDNA of rumen microbial populations. DNAzol(®) was a simple method that rendered good integrity, yield and purity. With this method, subtle changes in protozoan populations were detected in young bulls fed with slightly different formulations of a supplement of multinutritional blocks of molasses and urea. Sequences related to Eudiplodinium maggi and a non-cultured Entodiniomorphid similar to Entodinium caudatum, were related to major fluctuating populations in an SSCP assay.

Cytocompatible antifungal acrylic resin containing silver nanoparticles for dentures.

BACKGROUND: Inhibition of Candida albicans on denture resins could play a significant role in preventing the development of denture stomatitis. The safety of a new dental material with antifungal properties was analyzed in this work. METHODS: Poly(methyl methacrylate) [PMMA] discs and PMMA-silver nanoparticle discs were formulated, with the commercial acrylic resin, Nature-Cryl™, used as a control. Silver nanoparticles were synthesized and characterized by ultraviolet-visible spectroscopy, dispersive Raman spectroscopy, and transmission electron microscopy. The antifungal effect was assessed using a luminescent microbial cell viability assay. Biocompatibility tests were carried out using NIH-3T3 mouse embryonic fibroblasts and a Jurkat human lymphocyte cell line. Cells were cultured for 24 or 72 hours in the presence or absence of the polymer formulations and analyzed using three different tests, ie, cellular viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and cell proliferation by enzyme-linked immunosorbent assay BrdU, and genomic DNA damage (Comet assay). Finally, the samples were evaluated mechanically, and the polymer-bearing silver nanoparticles were analyzed microscopically to evaluate dispersion of the nanoparticles. RESULTS: The results show that PMMA-silver nanoparticle discs significantly reduce adherence of C. albicans and do not affect metabolism or proliferation. They also appear not to cause genotoxic damage to cells. CONCLUSION: The present work has developed a new biocompatible antifungal PMMA denture base material.

The phosphoinositide-3-kinase-Akt signaling pathway is important for Staphylococcus aureus internalization by endothelial cells.

Internalization of Staphylococcus aureus in bovine endothelial cells (BEC) is increased by tumor necrosis factor alpha stimulation and NF-κB activation. Because the phosphoinositide-3-kinase (PI3K)-Akt signaling pathway also modulates NF-κB activity, we considered whether the internalization of S. aureus by BEC is associated with the activity of PI3K and Akt. We found a time- and multiplicity of infection-dependent phosphorylation of Akt on Ser473 in BEC infected with S. aureus. This phosphorylation was inhibited by LY294002 (LY), indicating the participation of PI3K. Inhibition of either PI3K with LY or wortmannin, or Akt with SH-5, strongly reduced the internalization of S. aureus. Transfection of BEC with a dominant-negative form of the Akt gene significantly decreased S. aureus internalization, whereas transfection with the constitutively active mutant increased the number of internalized bacterium. Inhibition of PDK1 activity with OSU-03012 did not affect the level of S. aureus internalization, demonstrating that phosphorylation of Akt on Thr308 is not important for this process. Compared to the untreated control, the adherence of S. aureus to the surface of BEC was unaltered when cells were transfected or incubated with the pharmacological inhibitors. Furthermore, Akt activation by internalized S. aureus triggered a time-dependent phosphorylation of glycogen synthase kinase-3α (GSK-3α) on Ser21 and GSK-3ß on Ser9 that was partially inhibited with SH-5. Finally, treatment of BEC with LY prior to S. aureus infection inhibited the NF-κB p65 subunit phosphorylation on Ser536, indicating the involvement of PI3K. These results suggest that PI3K-Akt activity is important for the internalization of S. aureus and phosphorylation of GSK-3α, GSK-3ß, and NF-κB.

Gluconacetobacter diazotrophicus levansucrase is involved in tolerance to NaCl, sucrose and desiccation, and in biofilm formation.

Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium and endophyte of sugarcane, which expresses levansucrase, a fructosyltransferase exoenzyme with sucrose hydrolytic and levan biosynthetic activities. As a result of their physical properties, the levan can provide protection against stress caused by abiotic or biotic factors and participate in the formation of biofilms. In this study, we investigated the construction and function of a levansucrase-defective mutant of G. diazotrophicus. The lsdA mutant showed a decreased tolerance (65.5%) to 50-150 mM NaCl and a decrease of 89% in 876 mM (30%) sucrose, a reduction (99%) in tolerance to desiccation after 18 h, and a decrease (36.9-58.5%) in the ability to form cell aggregates on abiotic surfaces. Complementation of the mutant with the complete lsdA gene leads to a recovery of the ability to grow on sucrose-containing medium and to form slimy colonies, the ability to form the cell aggregates on abiotic surfaces and the tolerance to NaCl. This report demonstrates the importance of levansucrase in environmental adaptation of G. diazotrophicus under high osmotic stress and in biofilm formation.

La lipofección incrementa la eficiencia de mutagénesis dirigida en células troncoembrionarias de ratón E14 TG2a / Lipofection improves gene targeting efficiency in E14 TG2a mouse embryonic stem cells

Electroporation has been the method of election for transfection of murine embryonic stem cells for over 15 years; however, it is a time consuming protocol because it requires large amounts of DNA and cells, as well as expensive and delicate equipment. Lipofection is a transfection method that requires lower amounts of cells and DNA than electroporation, and has proven to be efficient in a large number of cell lines. It has been shown that after lipofection, mouse embryonic stem cells remain pluripotent, capable of forming germ line chimeras and can be transfected with greater efficiency than with electroporation; however, gene targeting of mouse embryonic stem cells by lipofection has not been reported. The objective of this work was to find out if lipofection can be used as efficiently as electroporation for regular gene targeting protocols. This context compares gene targeting efficiency between these techniques in mouse embryonic stem cells E14TG2a, using a gene replacement type vector. No differences were found in gene targeting efficiency between groups; however, lipofection was three times more efficient than electroporation in transfection efficiency, which makes lipofection a less expensive alternative method to produce gene targeting in mouse embryonic stem cells.

Durante los últimos 15 años se ha demostrado que la electroporación representa el método ideal para la transfección de células troncoembrionarias de ratón; sin embargo, demanda grandes cantidades de ADN y células, así como equipo caro y delicado, ello hace que este proceso sea costoso y laborioso. La lipofección es un método de transfección que requiere menos de células y ADN que la electroporación; asimismo, ha probado ser eficiente en gran número de líneas celulares. Se ha demostrado que después de lipofectar células troncoembrionarias de ratón, éstas mantienen su pluripotencia y son capaces de formar quimeras de línea germinal y se transfectan con mayor eficiencia que con electroporación, pero no se ha notificado la mutagénesis dirigida mediante la lipofección de células troncoembrionarias de ratón. El objetivo del presente trabajo fue saber si la lipofección puede ser utilizada con la misma o mayor eficiencia que la electroporación para los protocolos regulares de mutagénesis dirigida; en este contexto, se compara la eficiencia en mutagénesis dirigida entre estas técnicas en células troncoembrionarias de ratón E14TG2a, utilizando un vector de reemplazo. Entre las células transfectadas no se hallan diferencias en la eficiencia en mutagénesis dirigida entre grupos; sin embargo, los resultados que aquí se ofrecen muestran que la lipofección es tres veces más eficiente en la transfección, lo cual indica que la lipofección es un método alternativo menos costoso para obtener mutagénesis dirigida en células troncoembrionarias de ratón.

TNF-alpha reduces the level of Staphylococcus epidermidis internalization by bovine endothelial cells.

Staphylococcus epidermidis is an environmental opportunistic pathogen associated with bovine intramammary infections. In bacterial infections, the endothelial tissue plays an important role during inflammation and it is the target of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha). Therefore, this work was designed to explore the effect of TNF-alpha on the interaction of S. epidermidis with bovine endothelial cells (BEC). We show that cell signaling activated by TNF-alpha caused a marked reduction in the number of intracellular S. epidermidis, suggesting that molecules participating in this pathway were involved in the internalization of this bacterium. We also found that S. epidermidis internalization was not associated with basal levels of nuclear factor kappa B (NF-kappaB) activity because the intracellular number of bacteria recovered after treating BEC with the NF-kappaB inhibitors, SN50 or BAY 11-7083, was similar to that of the untreated control. Interestingly, inhibition of the basal activity of JNK with SP600125 and p38 with SB203580 caused a decrease in the number of intracellular S. epidermidis. These results suggest that activation of the signaling pathway initiated by TNF-alpha could play an important role in the phagocytosis of this bacterium. However, the basal activity of NF-kappaB was shown not to be important for the internalization process of S. epidermidis.

Structural analysis of point mutations in the Hairless gene and their association with the activity of the Hairless protein.

The Notch signaling pathway (NSP) is an important intercellular communication mechanism that regulates embryo development and adult physiological functions. The Hairless (H) protein is a powerful antagonist of the NSP by its interaction with the Suppressor of Hairless (Su[H]) protein, recruiting the corepressors Gro and CtBP. In the present work, we examined the role of several important amino acids in different H protein domains analyzing four mutant lines of Drosophila melanogaster. The mutant alleles were evaluated by single-strand conformational polymorphism (SSCP) analysis and we located mutated regions at different positions along the sequence of the Hairless gene.

The capacity of bovine endothelial cells to eliminate intracellular Staphylococcus aureus and Staphylococcus epidermidis is increased by the proinflammatory cytokines TNF-alpha and IL-1beta.

Staphylococcus aureus is a pathogenic bacterium causing clinical and subclinical bovine mastitis. Infections of the udder by S. aureus are frequently associated with the presence of Staphylococcus epidermidis, an opportunistic pathogen. We reported previously that the capacity of bovine endothelial cells (BEC) to endocytize S. aureus is associated with the activation of NF-kappaB and modulated by the proinflammatory cytokines TNF-alpha and IL-1beta. In this work, we explore the ability of BEC to eliminate intracellular S. aureus and S. epidermidis and their response to these cytokines. Time-kinetics survival experiments indicated that BEC eliminate intracellular S. epidermidis more efficiently. Replication of S. aureus, but not S. epidermidis, inside BEC was evident by an increase in intracellular bacteria recovered at 2 h postinfection. Afterwards, the intracellular number of staphylococci decreased gradually, reaching the lowest value at 24 h. Treatment of BEC with TNF-alpha or IL-1beta potentiated the capacity of BEC to eliminate both Staphylococcus species at the times tested. These results indicate that activation of the intrinsic antistaphylococcal response in BEC, enhanced by TNF-alpha and IL-1beta, is effective to eliminate S. aureus and S. epidermidis and suggest that endothelial cells may play a prominent role in the defense against infections caused by these bacteria.

Innate immune response of bovine mammary gland to pathogenic bacteria responsible for mastitis.

Mastitis (mammary gland inflammation) is one of the most important bovine diseases causing economic losses to dairy producers. Mammary gland inflammation is a consequence of the activity of a number of cell and soluble factors that function together to eliminate invading microorganisms. The factors involved in this inflammatory response differ depending on the infectious agent. This review analyzes the factors involved in the immunologic mechanisms against the main pathogenic bacteria causing mastitis, and emphasizes the innate immune response of the mammary gland. Knowledge, at the molecular level, of the mammary gland immune response during infection by pathogenic bacteria is fundamental to the design of effective therapies to control and eradicate bovine mastitis.

Regulatory role of the 3' untranslated region (3'UTR) of rat 5' deiodinase (D1). effects on messenger RNA translation and stability.

The previous findings that both a long and a short type 1 deiodinase (D1) mRNA are present in different tissues and that the D1 gene contains two potential polyA signals suggest that the two mRNAs result from differential polyA signal usage. In this study, we examined the properties of the two D1 mRNAs generated in HEK 293 cells by the alternative use of each of the poly A signals in order to ascertain the potential regulatory role of the 3'UTR of this gene. Our results showed that the long mRNA is less stable, but that it is translated more efficiently than the short mRNA. The net result of these differences is a higher D1 activity with the long message. These data suggest that the D1 3'UTR may play an important role in regulating the stability and translational efficiency of the D1 mRNA, both of which could be physiologically relevant when the demand for D1 activity is high.

Immortalization of bovine umbilical vein endothelial cells: a model for the study of vascular endothelium.

Endothelial cells perform a large array of physiological functions that are influenced by their cellular heterogeneity in the different vascular beds. Vein endothelial cells isolated from the umbilical cords are commonly used to study vascular endothelium. Primary cultures of these cells, however, have low proliferative capacity and a limited life span. We have immortalized bovine umbilical vein endothelial cells (BUVEC) by transfection with an expression vector containing the human papillomavirus type 16 E6E7 oncogenes. Expression of E6E7 extended the life span of BUVEC from 40 to more than 1-20 cell replication cycles with no signs of senescence. Four immortalized clones were isolated and found to maintain endothelial cell properties, such as the uptake of acetylated low density lipoprotein, the expression of the von Willebrand protein, the binding of endothelial cell-specific lectins and proliferative responses to the specific endothelial cell mitogen, vascular endothelial growth factor. Moreover, clone BVE-E6E7-1, like its wild-type counterparts, expressed prolactin mRNA and decreased its proliferation in response to the anti-angiogenic 16-kDa fragment of prolactin. This clone showed little signs of genetic instability as revealed by centrosome and chromosome number analysis. Thus, immortalized E6E7 BUVEC cell lines retain endothelial cell characteristics and could facilitate studies to investigate the action of regulatory factors of vascular endothelium. Moreover, being the first non-human umbilical vein endothelial cell lines, their use should provide insights into the mechanisms governing species-related heterogeneity of endothelial cells.