Advantageous Direct Quantification of Viable Closely Related Probiotics in Petit-Suisse Cheeses under In Vitro Gastrointestinal Conditions by Propidium Monoazide - qPCR

Species-specific Quantitative Real Time PCR (qPCR) alone and combined with the use of propidium monoazide (PMA) were used along with the plate count method to evaluate the survival of the probiotic strains Lactobacillus acidophilus La-5 and Bifidobacterium animalis subsp. lactis Bb-12, and the bacteriocinogenic and potentially probiotic strain Lactobacillus sakei subsp. sakei 2a in synbiotic (F1) and probiotic (F2) petit-suisse cheeses exposed throughout shelf-life to in vitro simulated gastrointestinal tract conditions. The three strains studied showed a reduction in their viability after the 6 h assay. Bb-12 displayed the highest survival capacity, above 72.6 and 74.6% of the initial populations, respectively, by plate count and PMA-qPCR, maintaining population levels in the range or above 6 log CFU/g. The prebiotic mix of inulin and FOS did not offer any additional protection for the strains against the simulated gastrointestinal environment. The microorganisms'' populations were comparable among the three methods at the initial time of the assay, confirming the presence of mainly viable and culturable cells. However, with the intensification of the stress induced throughout the various stages of the in vitro test, the differences among the methods increased. The qPCR was not a reliable enumeration method for the quantification of intact bacterial populations, mixed with large numbers of injured and dead bacteria, as confirmed by the scanning electron microscopy results. Furthermore, bacteria plate counts were much lower (P<0.05) than with the PMA-qPCR method, suggesting the accumulation of stressed or dead microorganisms unable to form colonies. The use of PMA overcame the qPCR inability to differentiate between dead and alive cells. The combination of PMA and species-specific qPCR in this study allowed a quick and unequivocal way of enumeration of viable closely related species incorporated into probiotic and synbiotic petit-suisse cheeses and under stress conditions.     

Molecular cloning,expression, and hormonal regulation of the chicken microsomal triglyceride transfer protein

During an egg-laying cycle, oviparous animals transfer massive amounts of triglycerides, the major lipid component of very low density lipoprotein (VLDL), from the liver to the developing oocytes. A major stimulus for this process is the rise in estrogen associated with the onset of an egg-laying cycle. In mammals, the microsomal triglyceride transfer protein (MTP) is required for VLDL assembly and secretion. To enable studies to determine if MTP plays a role in basal and estrogen-stimulated VLDL assembly and secretion in an oviparous vertebrate, we have cloned and sequenced the chicken MTP cDNA. This cDNA encodes a protein of 893 amino acids with an N-terminal signal sequence. The primary sequence of chicken MTP is, on average, 65% identical to that of mammalian homologs, and 23% identical to the Drosophila melanogaster protein. We have obtained a clone of chicken embryo fibroblast cells that stably express the avian MTP cDNA and show that these cells display MTP activity as measured by the transfer of a fluorescently labeled neutral lipid. As in mammals, chicken MTP is localized to the endoplasmic reticulum as revealed by indirect immunofluorescence and by the fact that its N-linked oligosaccharide moiety remains sensitive to endoglycosidase H. Endogenous, enzymatically active MTP is also expressed in an estrogen receptor-expressing chicken hepatoma cell line that secretes apolipoprotein B-containing lipoproteins. In this cell line and in vivo, the expression and activity of MTP are not influenced by estrogen. Therefore, up-regulation of MTP in the liver is not required for the increased VLDL assembly during egg production in the chicken. This indicates that MTP is not rate-limiting, even for the massive estrogen-induced secretion of VLDL accompanying an egg-laying cycle.     

Extending a Model of Shift‐Work Tolerance

The present study contributes to theory and practice through the development of a model of shift‐work tolerance with the potential to indicate interventions that reduce nurses' intention toward turnover and increase job satisfaction in hospital‐based settings. Survey data from 1257 nurses were used to conduct structural equation modeling that examine the direct and indirect effects of supervisor and colleague support, team identity, team climate, and control over working environment on time‐based work/life conflict, psychological well‐being, physical symptoms, job satisfaction, and turnover intention. The analysis of the proposed model revealed a good fit The chi‐square difference test was non‐significant (χ²(26)=338.56), the fit indices were high (CFI=.923, NFI=.918, and NNFI=.868), the distribution of residuals was symmetric and approached zero, the average standardized residual was low (AASR=.04), and the standardized RMR was. 072. In terms of the predictor variable, the final model explained 48% of the variance in turnover intention. The data revealed considerable evidence of both direct effects on adjustment and complex indirect links between levels of adjustment and work‐related social support, team identity, team climate, and control. Nurses with high supervisor and coworker support experienced more positive team climates, identified more strongly with their team, and increased their perceptions of control over their work environment. This in turn lowered their appraisals of their time‐based work/life conflict, which consequently increased their psychological well‐being and job satisfaction and reduced their physical health symptoms and turnover intention. The type of shift schedule worked by the nurses influenced levels of turnover intention, control over work environment, time‐based work/life conflict, and physical symptoms.     

Effect of mineral oil on Potato virus Y acquisition by Rhopalosiphum padi

Seed potato crops are currently sprayed weekly with mineral oil to prevent transmission of the Potato virus Y (PVY; Potyviridae: Potyvirus), one of the most prevalent and important non‐persistent viruses affecting potato production. In spite of its wide usage as inhibitor of virus transmission, the mode of action for mineral oil is poorly known. The objective of this study was to quantify the effect of dosage and time from application of mineral oil on the inhibition of PVY acquisition. The bird cherry‐oat aphid, Rhopalosiphum padi (L.) (Hemiptera: Aphididae), known as vector of PVY, was used in all the experiments. The results indicated that mineral oil efficiently decreased PVY acquisition by 75 and 70% 1 day after application of 5 and 10 l ha^?1, respectively. The inhibition effect decreased with time from application; mineral oil inhibits acquisition for less than 4 days at 5 l ha^?1 and between 8 and 12 days at 10 l ha^?1. As mineral oil was detected in the body of fewer aphids when they fed on plants 1 day after oil application, a change in the aphid probing behaviour on mineral oil‐treated plants was deduced. These results support the hypothesis that mineral oil physically inhibits the binding of the virus at the tip of the stylets.     

Common gene variants in RAD51, XRCC2 and XPD are not associated with clinical outcome in soft-tissue sarcoma patients

BackgroundDNA repair mechanisms play a major role in cancer risk and progression. Germline variants in DNA repair genes may result in altered gene function and/or activity, thereby causing inter-individual differences in a patient's tumor recurrence capacity. In genes of the DNA repair pathway the gene variants RAD51 rs1801320 G > C, XRCC2 rs3218536 G > A and XPD rs13181 A > C have been previously related to genetic predisposition and prognosis of various cancer entities. In this study we investigated the association between these polymorphisms and time to recurrence (TTR) and overall survival (OS) in soft-tissue sarcoma (STS) patients after curative surgery.MethodsTwo hundred sixty STS patients were included in this retrospective study. Germline DNA was genotyped by 5′-exonuclease (TaqMan) technology. Kaplan Meier curves and multivariate Cox proportional models were calculated for TTR and OS.ResultsA statistically significant association was observed between tumor grade and adjuvant radiotherapy and TTR and between tumor grade and OS. No association was found between RAD51 rs1801320 G > C, XRCC2 rs3218536 G > A and XPD rs13181 A > C and TTR and OS in univariate and multivariate analysis.ConclusionOur results underline a prognostic effect of tumor grade and adjuvant radiotherapy in STS patients but indicate no association between RAD51 rs1801320 G > C, XRCC2 rs3218536 G > A and XPD rs13181 A > C and clinical outcome in STS patients after curative surgery.     

Novel extracellular and nuclear caspase-1 and inflammasomes propagate inflammation and regulate gene expression: a comprehensive database mining study

Background

Caspase-1 is present in the cytosol as an inactive zymogen and requires the protein complexes named “inflammasomes” for proteolytic activation. However, it remains unclear whether the proteolytic activity of caspase-1 is confined only to the cytosol where inflammasomes are assembled to convert inactive pro-caspase-1 to active caspase-1.

Methods

We conducted meticulous data analysis method?s on proteomic, protein interaction, protein intracellular localization, and gene expressions of 114 experimentally identified caspase-1 substrates and 38 caspase-1 interaction proteins in normal physiological conditions and in various pathologies.

Results

We made the following important findings: (1) Caspase-1 substrates and interaction proteins are localized in various intracellular organelles including nucleus and secreted extracellularly; (2) Caspase-1 may get activated in situ in the nucleus in response to intra-nuclear danger signals; (3) Caspase-1 cleaves its substrates in exocytotic secretory pathways including exosomes to propagate inflammation to neighboring and remote cells; (4) Most of caspase-1 substrates are upregulated in coronary artery disease regardless of their subcellular localization but the majority of metabolic diseases cause no significant expression changes in caspase-1 nuclear substrates; and (5) In coronary artery disease, majority of upregulated caspase-1 extracellular substrate-related pathways are involved in induction of inflammation; and in contrast, upregulated caspase-1 nuclear substrate-related pathways are more involved in regulating cell death and chromatin regulation.

Conclusions

Our identification of novel caspase-1 trafficking sites, nuclear and extracellular inflammasomes, and extracellular caspase-1-based inflammation propagation model provides a list of targets for the future development of new therapeutics to treat cardiovascular diseases, inflammatory diseases, and inflammatory cancers.

     

Rhipsalis (Cactaceae): loss and gain of floral rewards is mirrored in range sizes and distribution patterns of species

Autogamous species are usually distinguishable from xenogamous relatives by smaller flowers, fewer or even no floral rewards and lower pollen–ovule (P/O) ratios. Many Rhipsalis spp. are small flowered, selfing and include the most widespread species in Cactaceae. However, Rhipsalis also includes a large number of narrowly endemic species and is most diverse in the Atlantic rainforests of Brazil. To investigate the evolution of floral function and the correlation between floral function and range size, we analysed display size, floral reward and P/O ratios of Rhipsalis and its closest relatives, reconstructed ancestral traits and related these patterns to the distributions and range sizes of the species. Display size and sugar amount are reduced in subgenera Goniorhipsalis and Rhipsalis and secondarily increased in Phyllarthrorhipsalis, whereas the P/O ratio is decreased in subgenera Rhipsalis and Phyllarthrorhipsalis. We interpret this pattern as a switch from a predominantly xenogamous to an autogamous reproductive system, followed by a return to a predominantly xenogamous system. None of the floral parameters shows significant correlations with range size, except for display size. Nevertheless, those species with the smallest flowers, lowest sugar amounts per flower and lowest P/O ratios occur either outside southeastern Brazil and/or have comparatively large distribution ranges. Almost all Rhipsalis spp. occurring outside the Atlantic rainforests are restricted to the clade formed by subgenera Rhipsalis and Phyllarthrorhipsalis. Thus, we believe that the evolution of an autogamous reproduction system enabled this lineage of Rhipsalis to diversify and spread in the Atlantic rainforests, in the rest of the Neotropics and even spread to the Old World, where it is the only member of the family.