Symbiosis between nanohaloarchaeon and haloarchaeon is based on utilization of different polysaccharides.

Nano-sized archaeota, with their small genomes and limited metabolic capabilities, are known to associate with other microbes, thereby compensating for their own auxotrophies. These diminutive and yet ubiquitous organisms thrive in hypersaline habitats that they share with haloarchaea. Here, we reveal the genetic and physiological nature of a nanohaloarchaeon-haloarchaeon association, with both microbes obtained from a solar saltern and reproducibly cultivated together in vitro. The nanohaloarchaeon Candidatus Nanohalobium constans LC1Nh is an aerotolerant, sugar-fermenting anaerobe, lacking key anabolic machinery and respiratory complexes. The nanohaloarchaeon cells are found physically connected to the chitinolytic haloarchaeon Halomicrobium sp. LC1Hm. Our experiments revealed that this haloarchaeon can hydrolyze chitin outside the cell (to produce the monosaccharide N-acetylglucosamine), using this beta-glucan to obtain carbon and energy for growth. However, LC1Hm could not metabolize either glycogen or starch (both alpha-glucans) or other polysaccharides tested. Remarkably, the nanohaloarchaeon's ability to hydrolyze glycogen and starch to glucose enabled growth of Halomicrobium sp. LC1Hm in the absence of a chitin. These findings indicated that the nanohaloarchaeon-haloarchaeon association is both mutualistic and symbiotic; in this case, each microbe relies on its partner's ability to degrade different polysaccharides. This suggests, in turn, that other nano-sized archaeota may also be beneficial for their hosts. Given that availability of carbon substrates can vary both spatially and temporarily, the susceptibility of Halomicrobium to colonization by Ca Nanohalobium can be interpreted as a strategy to maximize the long-term fitness of the host.

Functional microbiome deficits associated with ageing: Chronological age threshold.

Composition of the gut microbiota changes during ageing, but questions remain about whether age is also associated with deficits in microbiome function and whether these changes occur sharply or progressively. The ability to define these deficits in populations of different ages may help determine a chronological age threshold at which deficits occur and subsequently identify innovative dietary strategies for active and healthy ageing. Here, active gut microbiota and associated metabolic functions were evaluated using shotgun proteomics in three well-defined age groups consisting of 30 healthy volunteers, namely, ten infants, ten adults and ten elderly individuals. Samples from each volunteer at intervals of up to 6 months (n = 83 samples) were used for validation. Ageing gradually increases the diversity of gut bacteria that actively synthesize proteins, that is by 1.4-fold from infants to elderly individuals. An analysis of functional deficits consistently identifies a relationship between tryptophan and indole metabolism and ageing (p < 2.8e-8 ). Indeed, the synthesis of proteins involved in tryptophan and indole production and the faecal concentrations of these metabolites are directly correlated (r2  > .987) and progressively decrease with age (r2  > .948). An age threshold for a 50% decrease is observed ca. 11-31 years old, and a greater than 90% reduction is observed from the ages of 34-54 years. Based on recent investigations linking tryptophan with abundance of indole and other "healthy" longevity molecules and on the results from this small cohort study, dietary interventions aimed at manipulating tryptophan deficits since a relatively "young" age of 34 and, particularly, in the elderly are recommended.

Sulfur Respiration in a Group of Facultatively Anaerobic Natronoarchaea Ubiquitous in Hypersaline Soda Lakes.

The ubiquity of strictly anaerobic sulfur-respiring haloarchaea in hypersaline systems with circumneutral pH has shaken a traditional concept of this group as predominantly aerobic heterotrophs. Here, we demonstrated that this functional group of haloarchaea also has its representatives in hypersaline alkaline lakes. Sediments from various hypersaline soda lakes showed high activity of sulfur reduction only partially inhibited by antibiotics. Eight pure cultures of sulfur-reducing natronoarchaea were isolated from such sediments using formate and butyrate as electron donors and sulfur as an electron acceptor. Unlike strict anaerobic haloarchaea, these novel sulfur-reducing natronoarchaea are facultative anaerobes, whose metabolic capabilities were inferred from cultivation experiments and genomic/proteomic reconstruction. While sharing many physiological traits with strict anaerobic haloarchaea, following metabolic distinctions make these new organisms be successful in both anoxic and aerobic habitats: the recruiting of heme-copper quinol oxidases as terminal electron sink in aerobic respiratory chain and the utilization of formate, hydrogen or short-chain fatty acids as electron donors during anaerobic growth with elemental sulfur. Obtained results significantly advance the emerging concept of halo(natrono)archaea as important players in the anaerobic sulfur and carbon cycling in various salt-saturated habitats.

Young and Especially Senescent Endothelial Microvesicles Produce NADPH: The Fuel for Their Antioxidant Machinery.

In a previous study, we demonstrated that endothelial microvesicles (eMVs) have a well-developed enzymatic team involved in reactive oxygen species detoxification. In the present paper, we demonstrate that eMVs can synthesize the reducing power (NAD(P)H) that nourishes this enzymatic team, especially those eMVs derived from senescent human umbilical vein endothelial cells. Moreover, we have demonstrated that the molecules that nourish the enzymatic machinery involved in NAD(P)H synthesis are blood plasma metabolites: lactate, pyruvate, glucose, glycerol, and branched-chain amino acids. Drastic biochemical changes are observed in senescent eMVs to optimize the synthesis of reducing power. Mitochondrial activity is diminished and the glycolytic pathway is modified to increase the activity of the pentose phosphate pathway. Different dehydrogenases involved in NADPH synthesis are also increased. Functional experiments have demonstrated that eMVs can synthesize NADPH. In addition, the existence of NADPH in eMVs was confirmed by mass spectrometry. Multiphoton confocal microscopy images corroborate the synthesis of reducing power in eMVs. In conclusion, our present and previous results demonstrate that eMVs can act as autonomous reactive oxygen species scavengers: they use blood metabolites to synthesize the NADPH that fuels their antioxidant machinery. Moreover, senescent eMVs have a stronger reactive oxygen species scavenging capacity than young eMVs.

Proteomic Analysis of Methanonatronarchaeum thermophilum AMET1, a Representative of a Putative New Class of Euryarchaeota, "Methanonatronarchaeia".

The recently discovered Methanonatronarchaeia are extremely halophilic and moderately thermophilic methyl-reducing methanogens representing a novel class-level lineage in the phylum Euryarchaeota related to the class Halobacteria. Here we present a detailed analysis of 1D-nano liquid chromatography-electrospray ionization tandem mass spectrometry data obtained for "Methanonatronarchaeum thermophilum" AMET1 grown in different physiological conditions, including variation of the growth temperature and substrates. Analysis of these data allows us to refine the current understanding of the key biosynthetic pathways of this triple extremophilic methanogenic euryarchaeon and identify proteins that are likely to be involved in its response to growth condition changes.

The Antioxidant Machinery of Young and Senescent Human Umbilical Vein Endothelial Cells and Their Microvesicles.

We examine the antioxidant role of young and senescent human umbilical vein endothelial cells (HUVECs) and their microvesicles (MVs). Proteomic and Western blot studies have shown young HUVECs to have a complete and well-developed antioxidant system. Their MVs also contain antioxidant molecules, though of a smaller and more specific range, specialized in the degradation of hydrogen peroxide and the superoxide anion via the thioredoxin-peroxiredoxin system. Senescence was shown to be associated with a large increase in the size of the antioxidant machinery in both HUVECs and their MVs. These responses might help HUVECs and their MVs deal with the more oxidising conditions found in older cells. Functional analysis confirmed the antioxidant machinery of the MVs to be active and to increase in size with senescence. No glutathione or nonpeptide antioxidant (ascorbic acid and vitamin E) activity was detected in the MVs. Endothelial cells and MVs seem to adapt to higher ROS concentrations in senescence by increasing their antioxidant machinery, although this is not enough to recover completely from the senescence-induced ROS increase. Moreover, MVs could be involved in the regulation of the blood plasma redox status by functioning as ROS scavengers.

Increased Abundance of Proteins Involved in Resistance to Oxidative and Nitrosative Stress at the Last Stages of Growth and Development of Leishmania amazonensis Promastigotes Revealed by Proteome Analysis.

Leishmania amazonensis is one of the major etiological agents of the neglected, stigmatizing disease termed american cutaneous leishmaniasis (ACL). ACL is a zoonosis and rodents are the main reservoirs. Most cases of ACL are reported in Brazil, Bolivia, Colombia and Peru. The biological cycle of the parasite is digenetic because sand fly vectors transmit the motile promastigote stage to the mammalian host dermis during blood meal intakes. The amastigote stage survives within phagocytes of the mammalian host. The purpose of this study is detection and identification of changes in protein abundance by 2DE/MALDI-TOF/TOF at the main growth phases of L. amazonensis promastigotes in axenic culture and the differentiation process that takes place simultaneously. The average number of proteins detected per gel is 202 and the non-redundant cumulative number is 339. Of those, 63 are differentially abundant throughout growth and simultaneous differentiation of L. amazonensis promastigotes. The main finding is that certain proteins involved in resistance to nitrosative and oxidative stress are more abundant at the last stages of growth and differentiation of cultured L. amazonensis promastigotes. These proteins are the arginase, a light variant of the tryparedoxin peroxidase, the iron superoxide dismutase, the regulatory subunit of the protein kinase A and a light HSP70 variant. These data taken together with the decrease of the stress-inducible protein 1 levels are additional evidence supporting the previously described pre-adaptative hypothesis, which consists of preparation in advance towards the amastigote stage.

Differential protein abundance in promastigotes of nitric oxide-sensitive and resistant Leishmania chagasi strains.

PURPOSE: Leishmania chagasi is the causative agent of zoonotic visceral leishmaniasis in Brazil. Domestic and stray dogs are the main reservoirs. The life cycle of the parasite involves two stages. Promastigotes are extracellular and develop within the sand fly gut. Amastigotes survive inside the harsh environment of the phagolysosome of mammalian host phagocytes, which display the nitric oxide defense mechanism. Surprisingly, we were able to isolate promastigotes that are also resistant to NO. This finding may be explained by the preadaptative hypothesis. An insight into the proteome of NO-sensitive and resistant promastigotes is presented herein. EXPERIMENTAL DESIGN: Total protein extracts were prepared from promastigote cultures of an NO-sensitive and a resistant strain at early-logarithmic, mid-logarithmic and stationary phase. A population enriched in metacyclic promastigotes was also isolated by Percoll gradient centrifugation. In vitro infectivity of both strains was compared. Differential protein abundance was analyzed by 2DE-MALDI-TOF/TOF. The most striking results were tested at the mRNA level by qRT-PCR. Three biological replicates were performed in all cases. RESULTS: NO-resistant L. chagasi promastigotes are more infective than NO-sensitive ones. Among the differentially abundant spots, 40 proteins could be successfully identified in the sensitive strain and 38 in resistant promastigotes. CONCLUSIONS AND CLINICAL RELEVANCE: The increase of G6PD and the decrease of ARG and GPX transcripts and proteins contribute to NO resistance in L. chagasi promastigotes. These proteins may be studied as potential drug targets and/or vaccine candidates in the future.

Improved sake metabolic profile during fermentation due to increased mitochondrial pyruvate dissimilation.

Although the decrease in pyruvate secretion by brewer's yeasts during fermentation has long been desired in the alcohol beverage industry, rather little is known about the regulation of pyruvate accumulation. In former studies, we developed a pyruvate under-secreting sake yeast by isolating a strain (TCR7) tolerant to ethyl α-transcyanocinnamate, an inhibitor of pyruvate transport into mitochondria. To obtain insights into pyruvate metabolism, in this study, we investigated the mitochondrial activity of TCR7 by oxigraphy and (13) C-metabolic flux analysis during aerobic growth. While mitochondrial pyruvate oxidation was higher, glycerol production was decreased in TCR7 compared with the reference. These results indicate that mitochondrial activity is elevated in the TCR7 strain with the consequence of decreased pyruvate accumulation. Surprisingly, mitochondrial activity is much higher in the sake yeast compared with CEN.PK 113-7D, the reference strain in metabolic engineering. When shifted from aerobic to anaerobic conditions, sake yeast retains a branched mitochondrial structure for a longer time than laboratory strains. The regulation of mitochondrial activity can become a completely novel approach to manipulate the metabolic profile during fermentation of brewer's yeasts.

Comprehensive analysis of gluten in processed foods using a new extraction method and a competitive ELISA based on the R5 antibody.

The only treatment for coeliac disease is to follow a strict, life-long gluten-free diet. It is therefore essential to use a highly sensitive, specific technique for gluten analysis in foods. Nowadays, the usual method for determining gluten content in gluten-free foods, internationally accepted by the Codex Alimentarius Commission, is the R5 antibody-based sandwich ELISA, combined with the cocktail-extraction solution. This technique requires at least two epitopes in the protein, but in hydrolysed foods, proteins are fragmented during food processing and converted into peptides in which only one toxic epitope may appear. Consequently, it was necessary to develop a new competitive immunoassay that, together with a reliable, compatible extraction solution, would provide a complete gluten analysis in any kind of food. We analysed commercial foods and home-made maize breads spiked with a known amount of gliadins using the sandwich R5 ELISA and the new competitive R5 ELISA that has been developed. These foods had previously been extracted with 60% ethanol/water, the cocktail solution or the new extracting solution called UPEX (universal prolamin and glutelin extractant solution). The complementary SDS-PAGE and western blot techniques were also used to confirm the gluten content. The limits of detection and quantification of the competitive R5 ELISA were 0.36 and 1.22 ng/ml of gliadins, respectively. The intra- and inter-assay precisions based on two samples were, respectively, 7.3% and 5.4% for the first sample and 9.9% and 6.3% for the second. This new assay was a better technique than the sandwich R5 ELISA for detecting gliadins quantitatively in hydrolysed foods. Regarding the extraction procedure, we did not find any significant interference from components of the UPEX solution at the concentration used. In addition, the UPEX solution extraction was compatible with the R5 western blot and mass spectrometry techniques. The competitive R5 ELISA we developed, combined with the UPEX solution described here, is a very useful tool for detecting and quantifying gluten in any kind of food samples, including heat-treated and/or hydrolysed ones.

Measurement of wheat gluten and barley hordeins in contaminated oats from Europe, the United States and Canada by Sandwich R5 ELISA.

OBJECTIVES: We have investigated the extent of contamination with wheat, barley, rye or a mixture of these cereals in a large number of grains and commercial oats. We have also attempted to identify the type of cereal contaminant. METHODS: Sandwich R5 ELISA (using either gliadins or hordeins as standards), western blot, matrix-assisted laser desorption/ionization time-of-flight mass spectrometric and quantitative real-time PCR (Q-PCR) techniques have been used to analyze a total of 134 oats, comprising grains and commercial oat products collected from Europe, the United States and Canada. RESULTS: Twenty-five of the 134 pure, uncontaminated oat varieties were found to have undetectable levels of gluten, whereas most of the 109 grains and commercial oat products were mainly contaminated with mixtures of wheat, barley and rye, barley being the predominant contaminant. The percentages of these cereals in the oat samples have been calculated by specific wheat, barley and rye Q-PCR systems. The oat samples were grouped according to the avenin spectra determined by the mass spectrometric technique. The data confirmed that contaminated oat foods, based on the same variety, could have different levels of wheat, barley and rye contamination. CONCLUSION: This study has verified that contamination with wheat gliadins or barley hordeins in oat samples can be measured by the Sandwich R5 ELISA, using either gliadins or hordeins as standards, and also the importance of using confirmatory techniques (such as western blot, Q-PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) to confirm that most oats are contaminated with mixtures of wheat, barley and rye.