Avaliação da interação entre leveduras isoladas de kombucha e bactérias probióticas / Evaluation of the interaction between isolated kombucha yeasts and probiotic bacteria

Um dos maiores desafios no desenvolvimento de produtos probióticos é entender como os microrganismos interagem entre si e com o hospedeiro. Quando falamos em alimentos fermentados tradicionais, este obstáculo aumenta porque a matriz alimentar já possui um microbioma intrínseco. No entanto, também é conhecido que muitos microrganismos podem interagir e cooperar para sobreviver quando condições de estresse são encontradas. Assim, o objetivo deste trabalho foi isolar leveduras de quatro diferentes kombuchas em distintos momentos fermentativos e verificar a influência que leveduras isoladas de kombucha têm na manutenção da viabilidade da bactéria probiótica Bifidobacterium animalis subsp. lactis HN019 em condições de aerobiose. Meyerozyma guilliermondii, Candida albicans, Rhodotorula mucilaginosa e Pichia membranifaciens foram leveduras encontradas nas kombuchas, das quais as duas últimas favoreceram a manutenção da alta viabilidade de HN019 em cocultura por 14 dias. Observou-se a viabilidade da bactéria acima de 9 log ao longo de todo o experimento, o que não foi observado em monocultura. Ademais, utilizou-se de análise de autoagregação, hidrofobicidade, atividade enzimática de proteases e fosfolipases das leveuras para analisar seu potencial patogênico. Observou-se que R. mucilaginosa demonstrou características semelhantes à Saccharomyces cerevisiae subsp. boulardii, e sua interação benéfica com HN019 reforça a possibilidade de que esta levedura seja uma chave para a inserção da bactéria em uma kombucha probiótica. Análises metabólicas foram realizadas e encontrou-se uma vasta diversidade de dipeptídeos, principalmente os compostos de prolina, durante a cocultura da bactéria com as leveduras. Tais dipeptídeos apresentam importantes mecanismos de ação no controle biológico e quorum sensing de bactérias e leveduras, e supostamente regulam a manutenção das relações mutualísticas entre ambos microrganismo

One of the biggest challenges in the development of probiotic products is to understand how microorganisms interact with each other and with the host. When we talk about traditional fermented foods, this obstacle increases because the food matrix already has an intrinsic microbiome. However, it is also known that many microorganisms can interact and cooperate to survive when stressful situations are encountered. Thus, the objective of this work was to isolate yeasts from four different kombuchas at different fermentation times and to verify the influence that yeasts isolated from kombucha have on maintaining the viability of the probiotic bacterium Bifidobacterium animalis subsp. lactis HN019 under aerobic conditions. Meyerozyma guilliermondii, Candida albicans, Rhodotorula mucilaginosa and Pichia membranifaciens were yeasts found in kombuchas, of which the last two favored the maintenance of HN019 high viability in co-culture for 14 days. Bacteria viability above 9 log was observed throughout the experiment, which was not observed in monoculture. In addition, analysis of autoaggregation, hydrophobicity, enzyme activity of proteases and phospholipases of yeasts was used to analyze their pathogenic potential. It was observed that R. mucilaginosa demonstrated characteristics similar to Saccharomyces cerevisiae subsp. boulardii, and its beneficial interaction with HN019 reinforces the possibility that this yeast is a key to the insertion of the bacterium in a probiotic kombucha. Metabolic analysis were performed and a wide diversity of dipeptides, mainly proline-based, was found during the co-culture of the bacteria with the yeasts. Such dipeptides have important mechanisms of action in the biological control and quorum sensing of bacteria and yeast, and supposedly regulate the maintenance of mutualistic relationships between both microorganism

Smac mimetic compounds potentiate interleukin-1beta-mediated cell death.

Smac mimetic compounds (SMCs) potentiate TNFα-mediated cancer cell death by targeting the inhibitor of apoptosis (IAP) proteins. In addition to TNFα, the tumor microenvironment is exposed to a number of pro-inflammatory cytokines, including IL-1ß. Here, we investigated the potential impact of IL-1ß on SMC-mediated death of cancer cells. Synergy was seen in a subset of a diverse panel of 21 cancer cell lines to the combination of SMC and IL-1ß treatment, which required IL-1ß-induced activation of the NF-κB pathway. Elevated NF-κB activity resulted in the production of TNFα, which led to apoptosis dependent on caspase-8 and RIP1. In addition, concurrent silencing of cIAP1, cIAP2, and X-linked IAP by siRNA was most effective for triggering IL-1ß-mediated cell death. Importantly, SMC-resistant cells that produced TNFα in response to IL-1ß treatment were converted to an SMC-sensitive phenotype by c-FLIP knockdown. Reciprocally, ectopic expression of c-FLIP blocked cell death caused by combined SMC and IL-1ß treatment in sensitive cancer cells. Together, our study indicates that a positive feed-forward loop by pro-inflammatory cytokines can be exploited by SMCs to induce apoptosis in cancer cells.

The neuroactive peptide N-acetylaspartylglutamate is not an agonist at the metabotropic glutamate receptor subtype 3 of metabotropic glutamate receptor.

The peptide N-acetylaspartylglutamate (NAAG) is present in high concentrations in the mammalian central nervous system. Various mechanisms have been proposed for its action, including selective activation of the metabotropic glutamate receptor (mGluR) subtype 3, its action at the N-methyl-D-aspartate receptor, or the production of glutamate by its hydrolysis catalyzed by an extracellular protease. To re-examine its agonist activity at mGluR3, we coexpressed human or rat mGluR3 with G protein inward rectifying channels in Xenopus laevis oocytes. High-performance liquid chromatography analysis of commercial sources of NAAG showed 0.38 to 0.48% glutamate contamination. Although both human and rat mGluR3 were highly sensitive to glutamate, with EC(50) values of 58 and 28 nM, respectively, purified NAAG (100 microM) had little activity (7.7% of full activation by glutamate). Only in the millimolar range did it show significant activity, possibly due to residual traces of glutamate remaining in the purified NAAG preparations. In contrast, the unpurified NAAG sample did produce a full agonist response with mGluR3 coexpressed with G alpha(15), with an EC(50) of 120 microM, as measured by a calcium release assay. This response can be explained by the 0.38 to 0.48% glutamate contamination. Our results suggest that NAAG may not have a direct agonist activity at the mGluR3 receptor. Thus, several in vivo and in vitro published results that did not address the issue of glutamate contamination of NAAG preparations may need to be re-evaluated.

[The original nootropic and neuroprotective drug noopept potentiates the anticonvulsant activity of valproate in mice].

The influence of the original dipeptide drug noopept, known to possess nootrope, neuroprotector, and anxiolytic properties, on the anticonvulsant activity of the antiepileptic drug valproate has been studied on the model of corazole-induced convulsions in mice. Neither a single administration of noopept (0.5 mg/kg, i.p.) nor its repeated introduction in 10 or 35 days enhanced the convulsant effect of corazole, which is evidence that noopept alone does not possess anticonvulsant properties. Prolonged (five weeks) preliminary administration of noopept enhanced the anticonvulsant activity of valproate. This result justifies the joint chronic administration of noopept in combination with valproate in order to potentiate the anticonvulsant effect of the latter drug. In addition, the administration of noopept favorably influences the cognitive functions and suppresses the development of neurodegenerative processes.

NAAG peptidase inhibitor increases dialysate NAAG and reduces glutamate, aspartate and GABA levels in the dorsal hippocampus following fluid percussion injury in the rat.

Traumatic brain injury (TBI) produces a rapid and excessive elevation in extracellular glutamate that induces excitotoxic brain cell death. The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) is reported to suppress neurotransmitter release through selective activation of presynaptic group II metabotropic glutamate receptors. Therefore, strategies to elevate levels of NAAG following brain injury could reduce excessive glutamate release associated with TBI. We hypothesized that the NAAG peptidase inhibitor, ZJ-43 would elevate extracellular NAAG levels and reduce extracellular levels of amino acid neurotransmitters following TBI by a group II metabotropic glutamate receptor (mGluR)-mediated mechanism. Dialysate levels of NAAG, glutamate, aspartate and GABA from the dorsal hippocampus were elevated after TBI as measured by in vivo microdialysis. Dialysate levels of NAAG were higher and remained elevated in the ZJ-43 treated group (50 mg/kg, i.p.) compared with control. ZJ-43 treatment also reduced the rise of dialysate glutamate, aspartate, and GABA levels. Co-administration of the group II mGluR antagonist, LY341495 (1 mg/kg, i.p.) partially blocked the effects of ZJ-43 on dialysate glutamate and GABA, suggesting that NAAG effects are mediated through mGluR activation. The results are consistent with the hypothesis that inhibition of NAAG peptidase may reduce excitotoxic events associated with TBI.