Self-tunable engineered yeast probiotics for the treatment of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a complex chronic inflammatory disorder of the gastrointestinal tract. Extracellular adenosine triphosphate (eATP) produced by the commensal microbiota and host cells activates purinergic signaling, promoting intestinal inflammation and pathology. Based on the role of eATP in intestinal inflammation, we developed yeast-based engineered probiotics that express a human P2Y2 purinergic receptor with up to a 1,000-fold increase in eATP sensitivity. We linked the activation of this engineered P2Y2 receptor to the secretion of the ATP-degrading enzyme apyrase, thus creating engineered yeast probiotics capable of sensing a pro-inflammatory molecule and generating a proportional self-regulated response aimed at its neutralization. These self-tunable yeast probiotics suppressed intestinal inflammation in mouse models of IBD, reducing intestinal fibrosis and dysbiosis with an efficacy similar to or higher than that of standard-of-care therapies usually associated with notable adverse events. By combining directed evolution and synthetic gene circuits, we developed a unique self-modulatory platform for the treatment of IBD and potentially other inflammation-driven pathologies.

Ghrelin effects on mitochondrial fitness modulates macrophage function.

Over the past years, systemic derived cues that regulate cellular metabolism have been implicated in the regulation of immune responses. Ghrelin is an orexigenic hormone produced by enteroendocrine cells in the gastric mucosa with known immunoregulatory roles. The mechanism behind the function of ghrelin in immune cells, such as macrophages, is still poorly understood. Here, we explored the hypothesis that ghrelin leads to alterations in macrophage metabolism thus modulating macrophage function. We demonstrated that ghrelin exerts an immunomodulatory effect over LPS-activated peritoneal macrophages, as evidenced by inhibition of TNF-α and IL-1ß secretion and increased IL-12 production. Concomitantly, ghrelin increased mitochondrial membrane potential and increased respiratory rate. In agreement, ghrelin prevented LPS-induced ultrastructural damage in the mitochondria. Ghrelin also blunted LPS-induced glycolysis. In LPS-activated macrophages, glucose deprivation did not affect ghrelin-induced IL-12 secretion, whereas the inhibition of pyruvate transport and mitochondria-derived ATP abolished ghrelin-induced IL-12 secretion, indicating a dependence on mitochondrial function. Ghrelin pre-treatment of metabolic activated macrophages inhibited the secretion of TNF-α and enhanced IL-12 levels. Moreover, ghrelin effects on IL-12, and not on TNF-α, are dependent on mitochondria elongation, since ghrelin did not enhance IL-12 secretion in metabolic activated mitofusin-2 deficient macrophages. Thus, ghrelin affects macrophage mitochondrial metabolism and the subsequent macrophage function.

Mitoimmunity-when mitochondria dictates macrophage function.

In the past decade, several reports have appointed the importance of mitochondria in the immune response. Our understanding of mitochondria evolved from a simple supplier of energy into a platform necessary for immunorregulation. Proinflammatory responses are associated with enhanced glycolytic activity and breakdown of the TCA cycle. Mitochondrial reactive species of oxygen (mROS) are key regulators of classically activated macrophages, with substantial impact in the anti-microbicidal activity and pro-inflammatory cytokine secretion of macrophages. The inflammasome activation in macrophages is dependent on mROS production and mitochondrial regulation and mitochondrial dynamics and functionality direct impact inflammatory responses. Alternative activated macrophage metabolism relies on fatty acid oxidation, and the mechanism responsible for this phenotype is not fully elucidated. Thus, cellular metabolism and mitochondria function is a key immunoregulatory feature of macrophage biology. In this review, we will provide insights into recently reported evidences of mitochondria-related metabolic nodes, which are important for macrophage physiology.

Temperature affects the hypoxia tolerance of neotropical Cichlid Geophagus brasiliensis

Oxygen and temperature are the most limiting factors in aquatic environments. Several species are exposed to variations of these factors in water because of physical, chemical and biological processes. The objective of this study was to evaluate the metabolic profile and the tolerance to the hypoxia of Geophagus brasiliensis exposed to changes in temperature and oxygen availability. The fish were exposed to 20 and 90% of oxygen saturation combined with different temperatures (20°, 24° and 28° C) for 8 h. Hepatic and muscular glycogen, as well as the activities of lactate dehydrogenase (LDH), malate dehydrogenase (MDH), citrate synthase (CS) and their ratios were evaluated. Both glycogen and MDH activity showed a significant difference in the liver. While CS showed increased activity only in the heart. The increase in LDH activity in the white muscle shows the importance of the anaerobic pathway as energy source in this tissue. The MDH / LDH ratio increased in all tissues, while CS / LDH increased in the liver and decreased in the heart. Based on the results of the present study it may be concluded that this species used the anaerobic metabolism as the main strategy for hypoxia tolerance.(AU)

O oxigênio e a temperatura são os fatores mais limitantes em ambientes aquáticos. Várias espécies são expostas a variações destes fatores na água como resultado de processos físicos, químicos e biológicos. O estudo objetivou avaliar o perfil metabólico e a tolerância à hipóxia de Geophagus brasiliensis expostos a alterações na temperatura e disponibilidade de oxigênio. Os peixes foram expostos a 20% e 90% de saturação de oxigênio combinadas com diferentes temperaturas (20 ° C, 24 ° C e 28 ° C) durante 8h. Foram avaliados o glicogénio hepático e muscular, assim como as atividades das enzimas lactato desidrogenase (LDH), malato desidrogenase (MDH), citrato sintase (CS) e suas razões. Tanto o glicogênio quanto a atividade da MDH apresentaram diferença significativa no fígado. Enquanto a CS apresentou aumento de sua atividade apenas no coração. O aumento da atividade LDH no músculo branco mostra a importância da via anaeróbia como fonte de energia neste tecido. A razão MDH/LDH aumentou em todos os tecidos, enquanto CS/LDH apresentou aumento no fígado e diminuição no coração. Os resultados obtidos permitem concluir que esta espécie utilizou o metabolismo anaeróbio como principal estratégia de tolerância à hipóxia.(AU)

Standardization of a Continuous Assay for Glycosidases and Its Use for Screening Insect Gut Samples at Individual and Populational Levels.

Glycoside Hydrolases (GHs) are enzymes able to recognize and cleave glycosidic bonds. Insect GHs play decisive roles in digestion, in plant-herbivore, and host-pathogen interactions. GH activity is normally measured by the detection of a release from the substrate of products as sugars units, colored, or fluorescent groups. In most cases, the conditions for product release and detection differ, resulting in discontinuous assays. The current protocols result in using large amounts of reaction mixtures for the obtainment of time points in each experimental replica. These procedures restrain the analysis of biological materials with limited amounts of protein and, in the case of studies regarding small insects, implies in the pooling of samples from several individuals. In this respect, most studies do not assess the variability of GH activities across the population of individuals from the same species. The aim of this work is to approach this technical problem and have a deeper understanding of the variation of GH activities in insect populations, using as models the disease vectors Rhodnius prolixus (Hemiptera: Triatominae) and Lutzomyia longipalpis (Diptera: Phlebotominae). Here we standardized continuous assays using 4-methylumbelliferyl derived substrates for the detection of α-Glucosidase, ß-Glucosidase, α-Mannosidase, N-acetyl-hexosaminidase, ß-Galactosidase, and α-Fucosidase in the midgut of R. prolixus and L. longipalpis with results similar to the traditional discontinuous protocol. The continuous assays allowed us to measure GH activities using minimal sample amounts with a higher number of measurements, resulting in data that are more reliable and less time and reagent consumption. The continuous assay also allows the high-throughput screening of GH activities in small insect samples, which would be not applicable to the previous discontinuous protocol. We applied continuous GH measurements to 90 individual samples of R. prolixus anterior midgut homogenates using a high-throughput protocol. α-Glucosidase and α-Mannosidase activities showed the normal distribution in the population. ß-Glucosidase, ß-Galactosidase, N-acetyl-hexosaminidase, and α-Fucosidase activities showed non-normal distributions. These results indicate that GHs fluorescent-based high-throughput assays apply to insect samples and that the frequency distribution of digestive activities should be considered in data analysis, especially if a small number of samples is used.