Ethidium and propidium monoazide: comparison of potential toxicity on Vibrio sp. viability.

Vibrio sp., ubiquitous in the aquatic ecosystem, are bacteria of interest because of their involvement in human health, causing gastroenteritis after ingestion of seafood, as well as their role in vibriosis leading to severe losses in aquaculture production. Their ability to enter a viable but non-culturable (VBNC) state under stressful environmental conditions may lead to underestimation of the Vibrio population by traditional microbiological enumeration methods. As a result, using molecular methods in combination with EMA or PMA allows the detection of viable (VBNC and culturable viable) cells. In this study, the impact of the EMA and PMA was tested at different concentrations on the viability of several Vibrio species. We compared the toxicity of these two DNA-binding dyes to determine the best pretreatment to use with qPCR to discriminate between viable and dead Vibrio cells. Our results showed that EMA displayed lethal effects for each strain of V. cholerae and V. vulnificus tested. In contrast, the concentrations of PMA tested had no toxic effect on the viability of Vibrio cells studied. These results may help to achieve optimal PMA-qPCR methods to detect viable Vibrio sp. cells in food and environmental samples.

A pan-European ring trial to validate an International Standard for detection of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus in seafoods.

Globally, vibrios represent an important and well-established group of bacterial foodborne pathogens. The European Commission (EC) mandated the Comite de European Normalisation (CEN) to undertake work to provide validation data for 15 methods in microbiology to support EC legislation. As part of this mandated work programme, merging of ISO/TS 21872-1:2007, which specifies a horizontal method for the detection of V. parahaemolyticus and V. cholerae, and ISO/TS 21872-2:2007, a similar horizontal method for the detection of potentially pathogenic vibrios other than V. cholerae and V. parahaemolyticus was proposed. Both parts of ISO/TS 21872 utilized classical culture-based isolation techniques coupled with biochemical confirmation steps. The work also considered simplification of the biochemical confirmation steps. In addition, because of advances in molecular based methods for identification of human pathogenic Vibrio spp. classical and real-time PCR options were also included within the scope of the validation. These considerations formed the basis of a multi-laboratory validation study with the aim of improving the precision of this ISO technical specification and providing a single ISO standard method to enable detection of these important foodborne Vibrio spp.. To achieve this aim, an international validation study involving 13 laboratories from 9 countries in Europe was conducted in 2013. The results of this validation have enabled integration of the two existing technical specifications targeting the detection of the major foodborne Vibrio spp., simplification of the suite of recommended biochemical identification tests and the introduction of molecular procedures that provide both species level identification and discrimination of putatively pathogenic strains of V. parahaemolyticus by the determination of the presence of theromostable direct and direct related haemolysins. The method performance characteristics generated in this have been included in revised international standard, ISO 21872:2017, published in July 2017.

Evaluation of most-probable-number-PCR method with internal amplification control for the counting of total and pathogenic Vibrio parahaemolyticus in frozen shrimps.

The most-probable-number (MPN) method is often time-consuming for the isolation, detection, and quantification of Vibrio parahaemolyticus from natural sources. MPN counting of V. parahaemolyticus bacteria usually involves the isolation of typical V. parahaemolyticus colonies on selective medium, with subsequent confirmation by biochemical identification. In this study, we evaluated the use of a PCR on MPN enrichment cultures (MPN-PCR) for the direct detection of total and pathogenic V. parahaemolyticus cells in frozen shrimp. This reaction targeted the R72H, tdh, and trh sequences. An internal amplification control was added to the samples before R72H amplification. There was an excellent correlation between the results of the two methods for artificially inoculated and natural shrimp samples. Of 36 natural samples, 28 tested positive for the presence of V. parahaemolyticus, with an MPN value of 2 × 10(-1) to 9.2 × 10(1) per g. No pathogenic V. parahaemolyticus cells were detected. The test had a detection limit of one V. parahaemolyticus organism per g and was completed within two working days. These results support the use of the combination of PCR with MPN for the detection of total or potentially pathogenic V. parahaemolyticus cells in frozen shrimp.

Total and pathogenic Vibrio parahaemolyticus in shrimp: fast and reliable quantification by real-time PCR.

Vibrio parahaemolyticus is found in aquatic environments and is the leading cause of gastroenteritis due to seafood consumption worldwide. We evaluated a quantitative real-time PCR (Q-PCR) assay with hydrolysis probes, to determine whether this method could be used for the efficient counting of total, tdh and trh-positive V. parahaemolyticus in shrimps. We assessed the specificity of this assay, using 62 strains from 12 non target bacterial species of the Vibrio, Photobacterium, Shewanella and Aeromonas genera. Only V. parahaemolyticus with the appropriate target gene generated a fluorescent signal. We assessed the robustness of this assay, by analyzing spiked shrimps by Q-PCR and traditional culture methods, using most probable number (MPN)-PCR. After a 6h enrichment period, the assay successfully detected total and pathogenic V. parahaemolyticus in shrimps samples spiked with less than 5 V. parahaemolyticuscells/g of shrimp. The Q-PCR results obtained were compared with those obtained by most probable number (MPN)-PCR format. An excellent correlation between the two methods was observed in all cases (R² > 0.9742). The application of this Q-PCR assay to 85 natural shrimp samples also resulted in the successful quantification of V. parahaemolyticus in this matrix, and the counts obtained were correlated with those obtained by (MPN)-PCR (P=0.2598). Thus, this rapid and sensitive Q-PCR can be used to quantify V. parahaemolyticus in natural shrimp samples. This assay could be proposed, in response to the demands of the European Commission, as a tool for testing for the presence of vibrios in crustaceans, making it possible to legislate in this domain.

Modification of a virulence-associated phenotype after growth of Listeria monocytogenes on food.

AIM: To assess the effect of different foods, which have been implicated or not in cases of listeriosis, on the in vitro virulence-associated phenotype level of different Listeria monocytogenes strains. METHODS AND RESULTS: The virulence-associated phenotype level of L. monocytogenes was studied with the in vitro cell test based on a plaque-forming assay with a human adenocarcinoma cell line (HT-29) monolayer. Three strains of L. monocytogenes were grown in preparations (homogenate, 1-mum filtrate or 0.2-mum filtrate) of different food extracts ['rillettes' (potted minced pork), milk, raw salmon and cold-smoked salmon] or in a control medium, brain heart infusion (BHI). The bacterial suspensions grown in food extracts or in BHI at 37 degrees C were diluted with their growth medium (food extract or BHI) or with minimum essential medium before seeding on confluent HT-29 cell monolayers. Filtration of food extracts had no significant effect on the plaque numbers formed by the bacteria. A significant decrease in the plaque numbers was noted for the three strains when they grew in the rillettes extracts, compared with the other food extracts and BHI. The levels of in vitro virulence-associated phenotype of the strains after growth in the rillettes extract were similar to or lower than that of the hypovirulent internal reference strain L. monocytogenes 442. After growth in milk and cold-smoked salmon, the impact on virulence-associated phenotype depended on the strain. In contrast, plaque-forming assay indicated increased virulence-associated phenotype when the strains were switched from a nutrient-rich medium (food extract or BHI) to a minimum essential medium. CONCLUSIONS: In vitro virulence-associated phenotype level of the studied strains grown in BHI or cold-smoked salmon was the same as the control virulent strain EGD. In contrast, the nutrients present in rillettes may therefore substantially reduce the number of plaques but not the growth of L. monocytogenes. The utilization of minimum essential medium as diluent attenuates changes the effect of the food extract on virulence-associated phenotype in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: In the experimental design of this study, we showed that the nature of the food could affect the in vitro virulence-associated phenotype level of L. monocytogenes.

Influence of prolactin on the differentiation of mouse B-lymphoid precursors.

Development and activation of immune cells are submitted to hormonal influences, as illustrated by the roles of corticosteroids in thymus, pregnancy-related estrogens in B-cell development, or prolactin (PRL) on T-cell generation and function. We have analyzed the putative role of PRL in B lymphopoiesis and differentiation. We chose as an experimental model the interleukin (IL)-3 dependent BaF-3 pro-B cell line, which was transfected with the rat long form of the PRL receptor (PRL-R) and transferred from IL-3- to PRL-enriched media. When stimulated with PRL, the PRL-R transfectants underwent some changes characteristic of B-cell differentiation: (a) IL-2R alpha chain became positively controlled by PRL; (b) antiapoptotic Bcl-2 protein was induced by PRL in a dose-dependent manner; and (c) transcription of the pre-B cell receptor encoding the lambda5 gene was strongly up-regulated. We attempted to evaluate the differentiation-promoting activity of PRL in more physiological conditions, and the presence of PRL-R in bone marrow B-cell precursors was revealed. Furthermore, PRL promoted significant expansions of defined B-lineage cell populations in short-term bone marrow cell cultures. These findings suggest that PRL, in collaboration with other cytokines and hormonal influences, modulates B-cell development.

Differential involvement of the transcription factor Blimp-1 in T cell-independent and -dependent B cell differentiation to plasma cells.

Along humoral immune responses, different stimuli drive the differentiation of B lymphocytes to Ig-secreting plasma cells in discrete microenvironments. The Blimp-1 transcription factor is up-regulated early during the transition of mature B cells to IgM-secreting plasma cells. In the present study, we have examined the requirement of Blimp-1 in plasma cell formation after both T cell-independent (LPS) and -dependent (CD40 + IL-4, Th cell lines) stimulation of spleen B cells. B lymphocyte-induced maturation protein (Blimp-1) was expressed early after in vitro LPS stimulation, mainly in a population of IgM+Syndecan+CD43+ preplasma cells. In contrast, the BSAP transcription factor expressed in mature B cells was down-regulated during the differentiation to plasma cells. Treatment of these cultures with Blimp-1-specific antisense phosphorothioate oligonucleotides suppressed both Blimp-1 protein levels and the emergence of IgM+Syndecan+ cells and plasma cells. However, T-B cell cocultures of spleen B cells from C3H/HeJ (H-2k) mice and syngeneic autoreactive SR.10 Th2 cells submitted to the anti-Blimp-1 therapy did not show any significant reduction in IgM- and IgG1-secreting plasma cell formation. Spleen B cells treated with anti-CD40 mAb + IL-4 differentiated to IgG1-secreting cells without significant transcription of the Blimp-1 gene; anti-Blimp-1 treatment subsequently did not have any effect in the later cultures. Altogether, these results suggest that Blimp-1 transcription factor specifically promotes T cell-independent B cell differentiation to plasma cells, probably at preplasma cell stages. In contrast, T cell-dependent plasma cell formation likely evolves through Blimp-1-independent pathways.

Developmental hematopoiesis.

Hematopoiesis is a developmental process that evolves throughout the lifespan of an individual. Most work in the field has focused on events occurring in the adult bone marrow (BM). In the embryo, blood and endothelial cell generation begins very early after gastrulation, in defined intraembryonic mesodermic sites. Recent multidisciplinary studies, taking advantage of classic embryological and gene targeting technology in various species, have provided a new image of embryofetal lymphohemopoiesis, which includes the suggestion of developmental compartmentalization or waves. The first hematopoietic stem cells (HSC) migrate further and home in an ordered sequence of supporting microenvironments depending on scarcely known molecular requirements. These early hematopoietic progenitors show important differences in their cell biology and differentiation potentialities with respect to those present in adult stages; this fact, together with specific microenvironmental influences, define a process that diverges significantly from that occurring in the BM. Here, we update the latest developments in the field, the new understanding of lymphohemopoiesis in prenatal life, and the novel questions that this emerging paradigm is producing.

Identification and characterization of a new oncogene derived from the regulatory subunit of phosphoinositide 3-kinase.

p85/p110 phosphoinositide 3-kinase (PI3K) is a heterodimer composed of a p85-regulatory and a p110-catalytic subunit, which is involved in a variety of cellular responses including cytoskeletal organization, cell survival and proliferation. We describe here the cloning and characterization of p65-PI3K, a mutant of the regulatory subunit of PI3K, which includes the initial 571 residues of the wild type p85alpha-protein linked to a region conserved in the eph tyrosine kinase receptor family. We demonstrate that this mutation, obtained from a transformed cell, unlike previously engineered mutations of the regulatory subunit, induces the constitutive activation of PI3K and contributes to cellular transformation. This report links the PI3K enzyme to mammalian tumor development for the first time.

Antigenic phenotype and gene expression pattern of lymphohemopoietic progenitors during early mouse ontogeny.

Hemopoiesis, initiated in the early embryo yolk sac (YS) (7.5-8 days postcoitum (pc) in mouse), takes place thereafter in sites successively seeded by extrinsic hemopoietic stem cells (HSC). Since the existence of intraembryonic HSC has been proven experimentally in some vertebrates, it is also likely that not all HSC originate in the YS in mammals, as previously thought. Candidate intraembryonic sites that may be active in producing HSC before liver colonization are the para-aortic splanchnopleura (P-Sp) and the aorta-gonads-mesonephros region (AGM). Here we explore these sites directly for the presence of cells with hemopoietic-specific surface molecules and gene activities. The Ags c-kit, AA4.1, Mac-1, and Sca-1 begin to be expressed on some P-Sp and AGM cells, making it possible to distinguish subpopulations that evolve according to reproducible developmental patterns. On the basis of RAG-1 gene transcription, the first lymphoid precursors in the mouse embryo appear to be present 9.5 to 10 days pc in P-Sp/AGM and YS. Starting B-cell lymphopoiesis (9-12 days pc) is characterized by nonexpression of the surrogate light chain lambda 5-encoding gene and biased usage of IgH DJ4 rearrangements. In the 12.5- to 13.5-day-pc fetal liver (FL), a switch occurs, characterized by the random use of all IgH DJ and the detection of lambda 5 gene transcripts.

Synthesis and antitumour activity of fluorinated 1-aza and 1,8-diazaanthraquinones.

A series of 3-fluoro-1-aza- and 1,8-diazaanthraquinones, structurally related to the antitumour antibiotic diazaquinomycin A, have been prepared by Diels-Alder reactions of 2-fluoro-2-propenal N, N-dimethylhydrazone and the corresponding quinones. These compounds showed potent in vitro activity against different tumour cell lines. They also showed some selectivity towards rapid-growth tumours when compared to other non-fluorinated analogues. In contrast with diazaquinomycin A, the azaanthraquinones studied here have not shown significant activity as thymidylate synthase inhibitors. Some compounds showed a high activity as inhibitors of protein, DNA and RNA biosynthesis.