Development of a multiplex real-time PCR for contagious agalactia diagnosis in small ruminants

Contagious agalactia is an important disease worldwide that affects small ruminants. Clinical manifestations vary from mastitis, pneumonia, arthritis and keratoconjunctivitis to septicemia. Four mycoplasmal etiological agents have been identified: Mycoplasma (M.) agalactiae, M. mycoides subsp. capri, M. capricolum subsp. capricolum and M. putrefaciens. The current procedure for direct diagnosis, recommended by the World Organization for Animal Health, involves the isolation of one or several causative agents from clinical specimens and further time-consuming identification steps. The present study reports the development of a new multiplex real-time PCR (including an internal positive control) that detects all four pathogens simultaneously and distinguishes M. agalactiae from the others. First, intra- and inter-species polymorphisms of the two target house-keeping genes, namely polC and fusA, were analyzed to design primers and probes adapted to the diversity of currently circulating strains. The specificity and the sensitivity of the assay were then challenged and the limit of detection was found to be as low as 6 to 12 copies of the target genes. The assay requires further assessment on clinical specimens but its performances (notably low intra- and inter-assay variability) are already very promising for use in large-scale diagnosis and prophylactic surveys of contagious agalactia.     

Bilirubin oxidase from Magnaporthe oryzae: an attractive new enzyme for biotechnological applications

A novel bilirubin oxidase (BOD), from the rice blast fungus Magnaporthe oryzae, has been identified and isolated. The 64-kDa protein containing four coppers was successfully overexpressed in Pichia pastoris and purified to homogeneity in one step. Protein yield is more than 100?mg for 2?L culture, twice that of Myrothecium verrucaria. The k _cat/K _m ratio for conjugated bilirubin (1,513?mM^?1?s^?1) is higher than that obtained for the BOD from M. verrucaria expressed in native fungus (980?mM^?1?s^?1), with the lowest K _m measured for any BOD highly desirable for detection of bilirubin in medical samples. In addition, this protein exhibits a half-life for deactivation >300?min at 37?°C, high stability at pH?7, and high tolerance towards urea, making it an ideal candidate for the elaboration of biofuel cells, powering implantable medical devices. Finally, this new BOD is efficient in decolorizing textile dyes such as Remazol brilliant Blue R, making it useful for environmentally friendly industrial applications.     

Bilirubin oxidase from Bacillus pumilus: a promising enzyme for the elaboration of efficient cathodes in biofuel cells

A CotA multicopper oxidase (MCO) from Bacillus pumilus, previously identified as a laccase, has been studied and characterized as a new bacterial bilirubin oxidase (BOD). The 59 kDa protein containing four coppers, was successfully over-expressed in Escherichia coli and purified to homogeneity in one step. This 509 amino-acid enzyme, having 67% and 26% sequence identity with CotA from Bacillus subtilis and BOD from Myrothecium verrucaria, respectively, shows higher turnover activity towards bilirubin compared to other bacterial MCOs. The current density for O(2) reduction, when immobilized in a redox hydrogel, is only 12% smaller than the current obtained with Trachyderma tsunodae BOD. Under continuous electrocatalysis, an electrode modified with the new BOD is more stable, and has a higher tolerance towards NaCl, than a T. tsunodae BOD modified electrode. This makes BOD from B. pumilus an attractive new candidate for application in biofuel cells (BFCs) and biosensors.     

Roles of four Arabidopsis u-box e3 ubiquitin ligases in negative regulation of abscisic Acid-mediated drought stress responses

AtPUB18 and AtPUB19 are homologous U-box E3 ubiquitin ligases in Arabidopsis (Arabidopsis thaliana). AtPUB19 is a negative regulator of abscisic acid (ABA)-mediated drought responses, whereas the role of AtPUB18 in drought responses is unknown. Here, loss-of-function and overexpression tests identified AtPUB18 as a negative regulator in ABA-mediated stomatal closure and water stress responses. The atpub18-2atpub19-3 double mutant line displayed more sensitivity to ABA and enhanced drought tolerance than each single mutant plant; therefore, AtPUB18 and AtPUB19 are agonistic. Stomatal closure of the atpub18-2atpub19-3 mutant was hypersensitive to hydrogen peroxide (H(2)O(2)) but not to calcium, suggesting that AtPUB18 and AtPUB19 exert negative effects on the ABA signaling pathway downstream of H(2)O(2) and upstream of calcium. AtPUB22 and AtPUB23 are other U-box E3 negative regulators of drought responses. Although atpub22atpub23 was more tolerant to drought stress relative to wild-type plants, its ABA-mediated stomatal movements were highly similar to those of wild-type plants. The atpub18-2atpub19-3atpub22atpub23 quadruple mutant exhibited enhanced tolerance to drought stress as compared with each atpub18-2atpub19-3 and atpub22atpub23 double mutant progeny; however, its stomatal behavior was almost identical to the atpub18-2atpub19-3 double mutant in the presence of ABA, H(2)O(2), and calcium. Overexpression of AtPUB18 and AtPUB19 in atpub22atpub23 effectively hindered ABA-dependent stomatal closure, but overexpression of AtPUB22 and AtPUB23 in atpub18-2atpub19-3 did not inhibit ABA-enhanced stomatal closure, highlighting their ABA-independent roles. Overall, these results suggest that AtPUB18 has a linked function with AtPUB19, but is independent from AtPUB22 and AtPUB23, in negative regulation of ABA-mediated drought stress responses.     

Milk fermented by Propionibacterium freudenreichii induces apoptosis of HGT-1 human gastric cancer cells

Background

Gastric cancer is one of the most common cancers in the world. The “economically developed countries” life style, including diet, constitutes a risk factor favoring this cancer. Diet modulation may lower digestive cancer incidence. Among promising food components, dairy propionibacteria were shown to trigger apoptosis of human colon cancer cells, via the release of short-chain fatty acids acetate and propionate.

Methodology/Principal Findings

A fermented milk, exclusively fermented by P. freudenreichii, was recently designed. In this work, the pro-apoptotic potential of this new fermented milk was demonstrated on HGT-1 human gastric cancer cells. Fermented milk supernatant induced typical features of apoptosis including chromatin condensation, formation of apoptotic bodies, DNA laddering, cell cycle arrest and emergence of a subG1 population, phosphatidylserine exposure at the plasma membrane outer leaflet, reactive oxygen species accumulation, mitochondrial transmembrane potential disruption, caspase activation and cytochrome c release. Remarkably, this new fermented milk containing P. freudenreichii enhanced the cytotoxicity of camptothecin, a drug used in gastric cancer chemotherapy.

Conclusions/Significance

Such new probiotic fermented milk may thus be useful as part of a preventive diet designed to prevent gastric cancer and/or as a food supplement to potentiate cancer therapeutic treatments.     

Genomic characterization of the Taylorella genus

The Taylorella genus comprises two species: Taylorella equigenitalis, which causes contagious equine metritis, and Taylorella asinigenitalis, a closely-related species mainly found in donkeys. We herein report on the first genome sequence of T. asinigenitalis, analyzing and comparing it with the recently-sequenced T. equigenitalis genome. The T. asinigenitalis genome contains a single circular chromosome of 1,638,559 bp with a 38.3% GC content and 1,534 coding sequences (CDS). While 212 CDSs were T. asinigenitalis-specific, 1,322 had orthologs in T. equigenitalis. Two hundred and thirty-four T. equigenitalis CDSs had no orthologs in T. asinigenitalis. Analysis of the basic nutrition metabolism of both Taylorella species showed that malate, glutamate and alpha-ketoglutarate may be their main carbon and energy sources. For both species, we identified four different secretion systems and several proteins potentially involved in binding and colonization of host cells, suggesting a strong potential for interaction with their host. T. equigenitalis seems better-equipped than T. asinigenitalis in terms of virulence since we identified numerous proteins potentially involved in pathogenicity, including hemagluttinin-related proteins, a type IV secretion system, TonB-dependent lactoferrin and transferrin receptors, and YadA and Hep_Hag domains containing proteins. This is the first molecular characterization of Taylorella genus members, and the first molecular identification of factors potentially involved in T. asinigenitalis and T. equigenitalis pathogenicity and host colonization. This study facilitates a genetic understanding of growth phenotypes, animal host preference and pathogenic capacity, paving the way for future functional investigations into this largely unknown genus.     

In vivo evidence that TRAF4 is required for central nervous system myelin homeostasis

Tumor Necrosis Factor Receptor-Associated Factors (TRAFs) are major signal transducers for the TNF and interleukin-1/Toll-like receptor superfamilies. However, TRAF4 does not fit the paradigm of TRAF function in immune and inflammatory responses. Its physiological and molecular functions remain poorly understood. Behavorial analyses show that TRAF4-deficient mice (TRAF4-KO) exhibit altered locomotion coordination typical of ataxia. TRAF4-KO central nervous system (CNS) ultrastructure shows strong myelin perturbation including disorganized layers and disturbances in paranode organization. TRAF4 was previously reported to be expressed by CNS neurons. Using primary cell culture, we now show that TRAF4 is also expressed by oligodendrocytes, at all stages of their differentiation. Moreover, histology and electron microscopy show degeneration of a high number of Purkinje cells in TRAF4-KO mice, that was confirmed by increased expression of the Bax pro-apoptotic marker (immunofluorescence), TUNEL analysis, and caspase-3 activation and PARP1 cleavage (western blotting). Consistent with this phenotype, MAG and NogoA, two myelin-induced neurite outgrowth inhibitors, and their neuron partners, NgR and p75NTR were overexpressed (Q-RT-PCR and western blotting). The strong increased phosphorylation of Rock2, a RhoA downstream target, indicated that the NgR/p75NTR/RhoA signaling pathway, known to induce actin cytoskeleton rearrangement that favors axon regeneration inhibition and neuron apoptosis, is activated in the absence of TRAF4 (western blotting). Altogether, these results provide conclusive evidence for the pivotal contribution of TRAF4 to myelination and to cerebellar homeostasis, and link the loss of TRAF4 function to demyelinating or neurodegenerative diseases.     

The Revised Classification of Eukaryotes

This revision of the classification of eukaryotes, which updates that of Adl et al. [J. Eukaryot. Microbiol. 52 (2005) 399], retains an emphasis on the protists and incorporates changes since 2005 that have resolved nodes and branches in phylogenetic trees. Whereas the previous revision was successful in re‐introducing name stability to the classification, this revision provides a classification for lineages that were then still unresolved. The supergroups have withstood phylogenetic hypothesis testing with some modifications, but despite some progress, problematic nodes at the base of the eukaryotic tree still remain to be statistically resolved. Looking forward, subsequent transformations to our understanding of the diversity of life will be from the discovery of novel lineages in previously under‐sampled areas and from environmental genomic information.