Milk protein oxidation in healthy subjects:A preliminary study

The role of protein oxidation in the regulation of whole body protein metabolism is unknown. Previously, it was observed that vigorous exercise led to increased protein oxidation. To further characterise 13C-milk protein oxidation in healthy subjects, the oxidation of ingested 13C-protein after an overnight fast was measured using a non-invasive 13C-protein breath test. This approach enables the analysis of 13C-protein oxidation kinetics and the effect of interfering factors. It was found that the estimated maximal 13C-milk protein oxidation was 0.07 g min−1, corresponding to a theoretical maximal oxidation capacity of ≈1.4 g kg body weight−1 d−1. No indications were found for preferential oxidation of non-essential amino acids. Combined ingestion of 30 g 13C-whey protein with 30 g glucose resulted in a 19% decrease of 13C-whey protein oxidation. It was concluded that exogenous 13C-whey protein oxidation can be affected by other co-ingested nutrients like glucose

Effects of Argonaute on gene expression in Thermus thermophilus.

This is the final version of the article. Available from the publisher via the DOI in this record.BACKGROUND: Eukaryotic Argonaute proteins mediate RNA-guided RNA interference, allowing both regulation of host gene expression and defense against invading mobile genetic elements. Recently, it has become evident that prokaryotic Argonaute homologs mediate DNA-guided DNA interference, and play a role in host defense. Argonaute of the bacterium Thermus thermophilus (TtAgo) targets invading plasmid DNA during and after transformation. Using small interfering DNA guides, TtAgo can cleave single and double stranded DNAs. Although TtAgo additionally has been demonstrated to cleave RNA targets complementary to its DNA guide in vitro, RNA targeting by TtAgo has not been demonstrated in vivo. METHODS: To investigate if TtAgo also has the potential to control RNA levels, we analyzed RNA-seq data derived from cultures of four T. thermophilus strain HB27 variants: wild type, TtAgo knockout (Δago), and either strain transformed with a plasmid. Additionally we determined the effect of TtAgo on expression of plasmid-encoded RNA and plasmid DNA levels. RESULTS: In the absence of exogenous DNA (plasmid), TtAgo presence or absence had no effect on gene expression levels. When plasmid DNA is present, TtAgo reduces plasmid DNA levels 4-fold, and a corresponding reduction of plasmid gene transcript levels was observed. We therefore conclude that TtAgo interferes with plasmid DNA, but not with plasmid-encoded RNA. Interestingly, TtAgo presence stimulates expression of specific endogenous genes, but only when exogenous plasmid DNA was present. Specifically, the presence of TtAgo directly or indirectly stimulates expression of CRISPR loci and associated genes, some of which are involved in CRISPR adaptation. This suggests that TtAgo-mediated interference with plasmid DNA stimulates CRISPR adaptation.Funding: This study was financially supported by a TOP grant from the Netherlands Organisation for Scientific Research (NWO) to John van der Oost (NWO-TOP 854.10.003). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Time-dependent transcriptome profile of genes involved in triacylglycerol (TAG) and polyunsaturated fatty acid synthesis in Nannochloropsis gaditana during nitrogen starvation

In this research, the gene expression of genes involved in lipid metabolism of the eustigmatophyte alga Nannochloropsis gaditana was measured by transcriptomic data. This microalga can be used as a source of triacylglycerol (TAG) and the omega-3 fatty acid eicosapentaenoic acid (EPA). Insight in TAG and EPA production and regulation are needed to improve their productivity. Nitrogen starvation induces TAG accumulation in N. gaditana. Previous research showed that during nitrogen starvation, EPA was translocated from the polar lipids to TAG and de novo synthesized in N. gaditana. Therefore, the expression levels of genes involved in fatty acid translocation and de novo TAG synthesis were measured. Furthermore, the genes involved in de novo EPA synthesis such as elongases and desaturases were studied. The expression levels were measured during the first hours of nitrogen starvation and the subsequent period of 14 days. One phospholipid:diacylglycerol acyltransferase (PDAT) gene involved in translocation of fatty acids from membrane lipids to TAG was upregulated. In addition, several lipases were upregulated, suggesting that these enzymes might be responsible for the translocation of EPA to TAG. Most desaturases and elongases involved in de novo EPA synthesis were downregulated during nitrogen starvation, except for Δ9 desaturase which was upregulated. This upregulation correlates with the increase in oleic acid. Due to the presence of many hypothetical genes, improvement in annotation is needed to increase our understanding of these pathways and their regulation.</p

Reverse methanogenesis and respiration in methanotrophic archaea

Anaerobic oxidation of methane (AOM) is catalyzed by anaerobic methane-oxidizing archaea (ANME) via a reverse and modified methanogenesis pathway. Methanogens can also reverse the methanogenesis pathway to oxidize methane, but only during net methane production (i.e., "trace methane oxidation"). In turn, ANME can produce methane, but only during net methane oxidation (i.e., enzymatic back flux). Net AOM is exergonic when coupled to an external electron acceptor such as sulfate (ANME-1, ANME-2abc, and ANME-3), nitrate (ANME-2d), or metal (oxides). In this review, the reversibility of the methanogenesis pathway and essential differences between ANME and methanogens are described by combining published information with domain based (meta)genome comparison of archaeal methanotrophs and selected archaea. These differences include abundances and special structure of methyl coenzyme M reductase and of multiheme cytochromes and the presence of menaquinones or methanophenazines. ANME-2a and ANME-2d can use electron acceptors other than sulfate or nitrate for AOM, respectively. Environmental studies suggest that ANME-2d are also involved in sulfate-dependent AOM. ANME-1 seem to use a different mechanism for disposal of electrons and possibly are less versatile in electron acceptors use than ANME-2. Future research will shed light on the molecular basis of reversal of the methanogenic pathway and electron transfer in different ANME types.The authors thank Stefanie Berger (RU,Nijmegen) for critical reading of the manuscript. This research is supported by the Soehngen Institute of Anaerobic Microbiology (SIAM) Gravitation Grant (024.002.002) of the Netherlands Ministry of Education, Culture and Science and the Netherlands Organisation for Scientific Research (NWO). Mike S. M. Jetten was further supported by ERC AG 339880 Eco-MoM and Alfons J. M. Stams was supported by ERC AG 323009 Novel Anaerobes.info:eu-repo/semantics/publishedVersio

Microbial systems for conversion of syngas to biobased products

Microbiology Centennial Symposium 2017 - Exploring Microbes for the Quality of Life (Book of Abstracts)Synthesis gas, a mixture of CO, H2, and CO2, can be created via gasification of any carbohydrate material. Fermentation of syngas by carboxydotrophic microbes allows for it to be converted into interesting bio-chemicals. Organisms involved in the fermentation of syngas use the CO or H2 in the gas as electron donor, fixating CO2 into the final end products. Currently acetate and ethanol are relatively well established products from syngas fermentation and there is interest to broaden the scope towards production of more complex products. However, genetic engineering of carboxydotrophic organisms and the knowledge of their metabolism is rather limited, making it difficult to create strains producing these products. A possible way to broaden the scope of products is via co-cultivation of microbes which can make use of each others products. We established a co-cultivation of Clostridium autoethanogenum, a well-known carboxydotrophic acetogen, together with Clostridium kluyveri, a well characterized organism employing the reverse -oxidation pathway. C. autoethanogenum uses the syngas to produce a mixture of acetate and ethanol. C. kluyveri subsequently uses these products to perform chain elongation. This results in a co-culture producing a mixture of C4 and C6 acids and alcohols using carbon monoxide as a sole substrate. This co-culture poses an interesting way for production of more complex and valuable products from syngas. Basic characterization of these co-cultures has been done and currently the research focus lies on how the species interact with each other and how environmental factors influence their production patterns and metabolism.info:eu-repo/semantics/publishedVersio

Short-term obeticholic acid treatment does not impact cholangiopathy in Cyp2c70-deficient mice with a human-like bile acid composition

Cyp2c70-/- mice with a human-like bile acid (BA) composition, lacking hydrophilic muricholic acids (MCAs), have been reported to display cholangiopathy and biliary fibrosis with female preponderance that can be reversed by ursodeoxycholic acid (UDCA). Obeticholic acid (OCA), a steroidal BA-like FXR agonist, has been shown to improve liver function in patients with primary biliary cholangitis and is approved as second-line treatment for patients with an inadequate response or intolerance to UDCA. Here, we investigated the impact of OCA on BA hydrophobicity and cholangiopathy in Cyp2c70-/- mice. Male and female wild-type (WT) and Cyp2c70-/- mice were fed a chow diet with or without 10 mg/kg/day OCA for 4 weeks. OCA accounted for 1-5% of biliary BAs, with larger enrichments in Cyp2c70-/- than in WT mice. In WT mice, OCA induced a more hydrophilic, MCA-rich BA pool. In Cyp2c70-/- mice, however, BA pool became more hydrophobic with a larger proportion of chenodeoxycholic acid, attributable to a reduction of BA 12α-hydroxylation. OCA treatment reduced fecal BA excretion, indicating repression of hepatic BA synthesis in both WT and Cyp2c70-/- mice. OCA did, however, not impact on markers of liver (dys)function in plasma nor did it ameliorate cholangiopathy and fibrosis in male or female Cyp2c70-/- mice. OCA treatment also did not affect the expression of genes involved in fibrosis, inflammation and cellular senescence. In conclusion, 4 weeks of OCA treatment oppositely modulates the hydrophobicity of the BA pool in WT and Cyp2c70-/- mice, but does not improve or worsen the characteristic sex-dependent liver pathology in Cyp2c70-/- mice

A genomic view on syntrophic versus non-syntrophic lifestyle of anaerobic fatty acid-degrading bacteria

In sulfate-reducing and methanogenic environments complex biopolymers are degraded by fermentative micro-organisms that produce hydrogen, carbon dioxide and short chain fatty acids. Degradation of short chain fatty acids can be coupled to methanogenesis or to sulfate reduction. We applied a genomic approach to understand why some bacteria are able to grow in syntrophy with methanogens and others are not. Bacterial strains were selected based on genome availability and upon their ability to grow on short chain fatty acids alone or in syntrophic association with methanogens. Systematic functional domain profiling allowed us to shed light on this fundamental and ecologically important question. Extra-cytoplasmic formate dehydrogenases, including their maturation protein are a typical difference between syntrophic and non-syntrophic butyrate and propionate degraders. Furthermore, two domains with a currently unknown function seem to be associated with the ability of syntrophic growth. One is putatively involved in capsule or biofilm production and a second in cell division, shape-determination or sporulation. Some sulfate reducing bacteria have never been tested for syntrophic growth, but as all crucial domains were found in their genomes, it is possible that these are able to grow in syntrophic association with methanogens. In addition, profiling domains involved in electron transfer mechanisms revealed the important role of the Rnfcomplex and the formate transporter in syntrophy, and indicates that DUF224 may have a role in electron transfer in bacteria that show syntrophic growth

Comparative genomics highlights symbiotic capacities and high metabolic flexibility of the marine genus Pseudovibrio

Pseudovibrio is a marine bacterial genus members of which are predominantly isolated from sessile marine animals, and particularly sponges. It has been hypothesised that Pseudovibrio spp. form mutualistic relationships with their hosts. Here, we studied Pseudovibrio phylogeny and genetic adaptations that may play a role in host colonization by comparative genomics of 31 Pseudovibrio strains, including 25 sponge isolates. All genomes were highly similar in terms of encoded core metabolic pathways, albeit with substantial differences in overall gene content. Based on gene composition, Pseudovibrio spp. clustered by geographic region, indicating geographic speciation. Furthermore, the fact that isolates from the Mediterranean Sea clustered by sponge species suggested host-specific adaptation or colonization. Genome analyses suggest that Pseudovibrio hongkongensis UST20140214-015BT is only distantly related to other Pseudovibrio spp., thereby challenging its status as typical Pseudovibrio member. All Pseudovibrio genomes were found to encode numerous proteins with SEL1 and tetratricopeptide repeats, which have been suggested to play a role in host colonization. For evasion of the host immune system, Pseudovibrio spp. may depend on type III, IV and VI secretion systems that can inject effector molecules into eukaryotic cells. Furthermore, Pseudovibrio genomes carry on average seven secondary metabolite biosynthesis clusters, reinforcing the role of Pseudovibrio spp. as potential producers of novel bioactive compounds. Tropodithietic acid, bacteriocin and terpene biosynthesis clusters were highly conserved within the genus, suggesting an essential role in survival e.g. through growth inhibition of bacterial competitors. Taken together, these results support the hypothesis that Pseudovibrio spp. have mutualistic relations with sponges

Role of bile acid receptor FXR in development and function of brown adipose tissue

Bile acids act as signalling molecules that contribute to maintenance of energy homeostasis in mice and humans. Activation of G-protein-coupled bile acid receptor TGR5 induces energy expenditure in brown adipose tissue (BAT). However, a role for the nuclear bile acid receptor Farnesoid X receptor (FXR) in BAT has remained ambiguous. We aimed to study the potential role of FXR in BAT development and functioning. Here we demonstrate low yet detectable expression of the α1/2 isoforms of FXR in murine BAT that markedly decreases upon cold exposure. Moderate adipose tissue-specific FXR overexpression in mice induces pronounced BAT whitening, presenting with large intracellular lipid droplets and extracellular collagen deposition. Expression of thermogenic marker genes including the target of Tgr5, Dio2, was significantly lower in BAT of chow-fed aP2-hFXR mice compared to wild-type controls. Transcriptomic analysis revealed marked up-regulation of extracellular matrix formation and down-regulation of mitochondrial functions in BAT from aP2-hFXR mice. In addition, markers of cell type lineages deriving from the dermomyotome, such as myocytes, as well as markers of cellular senescence were strongly induced. The response to cold and β3-adrenergic receptor agonism was blunted in these mice, yet resolved BAT whitening. Newborn cholestatic Cyp2c70-/- mice with a human-like bile acid profile also showed distinct BAT whitening and upregulation of myocyte-specific genes, while thermogenic markers were down-regulated. Ucp1 expression inversely correlated with plasma bile acid levels. Therefore, bile acid signalling via FXR has a role in BAT function already early in tissue development. Functionally, FXR activation appears to oppose TGR5-mediated thermogenesis