Development and use of a new medium to detect yeasts of the genera Dekkera/Brettanomyces

Aims: The objectives of this work were to develop a selective and/or differential medium able to effciently recover Dekkera/Brettanomyces sp. from wine-related environments and to determine the relationship between these yeasts and the 4-ethylphenol content in a wide range of wines. Methods and Results: The selectivity of the developed medium was provided by the addition of ethanol, as single carbon source, and cycloheximide. The inclusion of bromocresol green evidenced acid-producing strains. The inclusion of p-coumaric acid, substrate for the production of 4-ethylphenol, enabled the differentiation by smell of Dekkera/Brettanomyces sp. from all other yeast species growing in the medium. The medium was used either by plating after membrane filtration or by the Most Probable Number (MPN) technique. In 29 white and 88 red randomly collected wines, these yeasts were found only in red wines at levels up to 2500 MPN ml±1, but constituted less than 1% of the total microbial flora. In red wines, 84% showed detectable amounts of 4-ethylphenol up to 4430 lg l±1 while 28% of the white wines showed detectable levels up to 403 lg l±1. Conclusions: The use of the medium proposed in this work evidenced the presence of low relative populations of Dekkera/Brettanomyces sp. even in wines contaminated by fast-growing yeasts and moulds. Significance and Impact of the Study: Further ecological studies on Dekkera/Brettanomyces sp. should take into account the use of highly specific culture media in order to establish their true occurrence in natur

Factors affecting the production of 4-ethylphenol by the yeast Dekkera bruxellensis in enological conditions

"Food Microbiology" article in PressThe conversion of p-coumaric acid into 4-ethylphenol was studied in Dekkera bruxellensis ISA 1791 under defined conditions in synthetic media. The production of 4-ethylphenol occurred roughly between mid-exponential growth phase and the beginning of the stationary phase. This behaviour was observed when glucose was the only energy and carbon source, the conversion rate being close to 90%. Ethanol, as the single energy source, yielded conversion rates close to 80% while in the presence of trehalose and acetic acid conversion rates lower than 10% were obtained. The production of 4-ethylphenol was not observed when the cells were maintained in buffer solution without carbon and energy sources. The precursor of 4-ethylphenol, p-coumaric acid, was not utilized as energy and carbon source. Furthermore, it was shown that 4-vinylphenol may be used as a precursor of 4-ethylphenol in the absence of pcoumaric acid. Growth and 4-ethylphenol production were inhibited by increasing concentrations of ethanol, being fully prevented at 13% (v/v) ethanol. The cultivation of strain ISA 1791 in mixed culture with Saccharomyces cerevisiae, in synthetic medium, showed that the cell numbers of D. bruxellensis increased from 104 cfu/ml to 5 109 cfu/ml. Laboratory microvinifications of white and red juices inoculated with as low as 10 cfu/ml of D. bruxellensis and 107 cells/ml of S. cerevisiae showed growth of D. bruxellensis to levels of about 5 108 cfu/ml. In addition, 4-ethylphenol production by D. bruxellensis was observed only after complete fermentation of the grape juice

Canine parvovirus : a predicting canine model for sepsis

Research Areas: Veterinary SciencesBackground: Sepsis is a severe condition associated with high prevalence and mortality rates. Parvovirus enteritis is a predisposing factor for sepsis, as it promotes intestinal bacterial translocation and severe immunosuppression. This makes dogs infected by parvovirus a suitable study population as far as sepsis is concerned. The main objective of the present study was to evaluate the differences between two sets of SIRS (Systemic Inflammatory Response Syndrome) criteria in outcome prediction: SIRS 1991 and SIRS 2001. The possibility of stratifying and classifying septic dogs was assessed using a proposed animal adapted PIRO (Predisposition, Infection, Response and Organ dysfunction) scoring system. Results: The 72 dogs enrolled in this study were scored for each of the PIRO elements, except for Infection, as all were considered to have the same infection score, and subjected to two sets of SIRS criteria, in order to measure their correlation with the outcome. Concerning SIRS criteria, it was found that the proposed alterations on SIRS 2001 (capillary refill time or mucous membrane colour alteration) were significantly associated with the outcome (OR = 4.09, p < 0.05), contrasting with the 1991 SIRS criteria (p = 0.352) that did not correlate with the outcome. No significant statistical association was found between Predisposition (p = 1), Response (p = 0.1135), Organ dysfunction (p = 0.1135), total PIRO score (p = 0.093) and outcome. To explore the possibility of using the SIRS criteria as a fast decision-making tool, a Fast-andFrugal tree (FFT) was created with a sensitivity of 92% and a specificity of 29%. Conclusion: These results suggest that increasing the SIRS criteria specificity may improve their prognostic value and their clinical usefulness. In order to improve the proposed PIRO scoring system outcome prediction ability, more specific criteria should be added, mainly inflammatory and organ dysfunction biomarkers.info:eu-repo/semantics/publishedVersio

Purinergic mechanism in the immune system: A signal of danger for dendritic cells

There is increasing appreciation that injured or stressed cells release molecules endowed with the ability to modulate dendritic cell maturation. The role of these molecules is thought to be that of alerting the body of an impending danger, and initiate and shape the subsequent immune response. Nucleotides are perfectly suited for this task as they are easily released upon damage of the cell membrane, rapidly diffuse in the extracellular environment and ligate specific plasma membrane receptors expressed by dendritic cells and other mononuclear phagocytes. A better knowledge of the modulation of dendritic cell responses by extracellular nucleotides may provide novel routes to enhance the immune response and increase the efficacy of vaccination

Extracellular NAD and ATP: Partners in immune cell modulation

Extracellular NAD and ATP exert multiple, partially overlapping effects on immune cells. Catabolism of both nucleotides by extracellular enzymes keeps extracellular concentrations low under steady-state conditions and generates metabolites that are themselves signal transducers. ATP and its metabolites signal through purinergic P2 and P1 receptors, whereas extracellular NAD exerts its effects by serving as a substrate for ADP-ribosyltransferases (ARTs) and NAD glycohydrolases/ADPR cyclases like CD38 and CD157. Both nucleotides activate the P2X7 purinoceptor, although by different mechanisms and with different characteristics. While ATP activates P2X7 directly as a soluble ligand, activation via NAD occurs by ART-dependent ADP-ribosylation of cell surface proteins, providing an immobilised ligand. P2X7 activation by either route leads to phosphatidylserine exposure, shedding of CD62L, and ultimately to cell death. Activation by ATP requires high micromolar concentrations of nucleotide and is readily reversible, whereas NAD-dependent stimulation begins at low micromolar concentrations and is more stable. Under conditions of cell stress or inflammation, ATP and NAD are released into the extracellular space from intracellular stores by lytic and non-lytic mechanisms, and may serve as ‘danger signals–to alert the immune response to tissue damage. Since ART expression is limited to naïve/resting T cells, P2X7-mediated NAD-induced cell death (NICD) specifically targets this cell population. In inflamed tissue, NICD may inhibit bystander activation of unprimed T cells, reducing the risk of autoimmunity. In draining lymph nodes, NICD may eliminate regulatory T cells or provide space for the preferential expansion of primed cells, and thus help to augment an immune response

Shaping immune responses through the activation of dendritic cells–P2 receptors

Dendritic cells (DCs) activate and shape the adaptive immune response by capturing antigens, migrating to peripheral lymphoid organs where naïve T cells reside, expressing high levels of MHC and costimulatory molecules and secreting cytokines and chemokines. DCs are endowed with a high degree of functional plasticity and their functions are tightly regulated. Besides initiating adaptive immune responses, DCs play a key role in maintaining peripheral tolerance toward self-antigens. On the basis of the information gathered from the tissue where they reside, DCs adjust their functional activity to ensure that protective immunity is favoured while unwanted or exaggerated immune responses are prevented. A wide variety of signals from neighbouring cells affecting DC functional activity have been described. Here we will discuss the complex role of extracellular nucleotides in the regulation of DC function and the role of P2 receptors as possible tools to manipulate immune responses

Activation of the P2X7 ion channel by soluble and covalently bound ligands

The homotrimeric P2X7 purinergic receptor has sparked interest because of its capacity to sense adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD) released from cells and to induce calcium signaling and cell death. Here, we examine the response of arginine mutants of P2X7 to soluble and covalently bound ligands. High concentrations of ecto-ATP gate P2X7 by acting as a soluble ligand and low concentrations of ecto-NAD gate P2X7 following ADP-ribosylation at R125 catalyzed by toxin-related ecto-ADP-ribosyltransferase ART2.2. R125 lies on a prominent cysteine-rich finger at the interface of adjacent receptor subunits, and ADP-ribosylation at this site likely places the common adenine nucleotide moiety into the ligand-binding pocket of P2X7

The role of P2 receptors in controlling infections by intracellular pathogens

A growing number of studies have demonstrated the importance of ATPe-signalling via P2 receptors as an important component of the inflammatory response to infection. More recent studies have shown that ATPe can also have a direct effect on infection by intracellular pathogens, by modulating membrane trafficking in cells that contain vacuoles that harbour intracellular pathogens, such as mycobacteria and chlamydiae. A conserved mechanism appears to be involved in controlling infection by both of these pathogens, as a role for phospholipase D in inducing fusion between lysosomes and the vacuoles has been demonstrated. Other P2-dependent mechanisms are most likely operative in the cases of pathogens, such as Leishmania, which survive in an acidic phagolysosomal-like compartment. ATPe may function as a ‘danger signal–that alerts the immune system to the presence of intracellular pathogens that damage the host cell, while different intracellular pathogens have evolved enzymes or other mechanisms to inhibit ATPe-mediated signalling, which should, thus, be viewed as virulence factors for these pathogens

P2X7 receptor: Death or life?

The P2X7 plasma membrane receptor is an intriguing molecule that is endowed with the ability to kill cells, as well as to activate many responses and even stimulate proliferation. Here, the authors give an overview on the multiplicity and complexity of P2X7-mediated responses, discussing recent information on this receptor. Particular attention has been paid to early and late signs of apoptosis and necrosis linked to activation of the receptor and to the emerging field of P2X7 function in carcinogenesis

Probenecid Blocks Human P2X7 Receptor-Induced Dye Uptake via a Pannexin-1 Independent Mechanism

P2X7 is a ligand-gated ion channel which is activated by ATP and displays secondary permeability characteristics. The mechanism of development of the secondary permeability pathway is currently unclear, although a role for the hemichannel protein pannexin-1 has been suggested. In this study we investigated the role of pannexin-1 in P2X7-induced dye uptake and ATP-induced IL-1β secretion from human monocytes. We found no pharmacological evidence for involvement of pannexin-1 in P2X7-mediated dye uptake in transfected HEK-293 cells with no inhibition seen for carbenoxolone and the pannexin-1 mimetic inhibitory peptide, 10Panx1. However, we found that probenecid inhibited P2X7-induced cationic and anionic dye uptake in stably transfected human P2X7 HEK-293 cells. An IC50 value of 203 μM was calculated for blockade of ATP-induced responses at human P2X7. Probenecid also reduced dye uptake and IL-1β secretion from human CD14+ monocytes whereas carbenoxolone and 10Panx1 showed no inhibitory effect. Patch clamp and calcium indicator experiments revealed that probenecid directly blocks the human P2X7 receptor