Structural basis for the oligomerization-mediated regulation of NLRP3 inflammasome activation.

SignificanceThe nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) is a pattern recognition receptor that forms an inflammasome. The cryo-electron microscopy structure of the dodecameric form of full-length NLRP3 bound to the clinically relevant NLRP3-specific inhibitor MCC950 has established the structural basis for the oligomerization-mediated regulation of NLRP3 inflammasome activation and the mechanism of action of the NLRP3 specific inhibitor. The inactive NLRP3 oligomer represents the NLRP3 resting state, capable of binding to membranes and is likely disrupted for its activation. Visualization of the inhibitor binding mode will enable optimization of the activity of NLRP3 inflammasome inhibitor drugs.

Effects of feed intake on the diversity and population density of homoacetogens in the large intestine of pigs.

OBJECTIVE: Homoacetogens play important roles in the production of acetate in the large intestine of monogastric mammals. However, their diversity in the porcine large intestine is still unknown. Marker gene analysis was performed to assess the effects of energy level on the diversity and population densities of homoacetogens in porcine feces. METHODS: Crossbred pigs were fed high or low energy-level diets. The high-intake (HI) diet was sufficient to allow a daily gain of 1.2 kg. The low-intake (LI) diet provided 0.6 times the amount of energy as the HI diet. Genetic diversity was analyzed using formyltetrahydrofolate synthetase (FTHFS) gene (fhs) clone libraries derived from fecal DNA samples. fhs DNA copy numbers were quantified using real-time PCR. RESULTS: A wide variety of fhs sequences was recovered from animals in both treatments. No differences in fhs clone libraries between the HI and LI groups were found. During the experimental period, no significant differences in the proportion of fhs copy numbers were observed between the two treatment groups. CONCLUSION: This is the first reported molecular diversity analysis using specific homoacetogen marker genes from the large intestines of pigs. There was no observable effect of feed intake on acetogen diversity.

Diversity and population density of methanogens in the large intestine of pigs fed diets of different energy levels.

Marker gene analysis was performed to assess the effect of energy level on the diversity and population density of methanogens in pig fecal material. Crossbred pigs were fed high or low energy level diets, a high-energy (HE) diet that satisfied daily gain at 1.2 kg, and a low-energy (LE) diet with amount of 0.6 times of the HE diet. Growth performance and short-chain fatty acid in feces were examined. Diversity of methanogen was analyzed by the α-subunit of methyl coenzyme-M reductase gene (mcrA) clone library from fecal DNA. The DNA copy numbers of mcrA were quantified by real-time PCR. There was no difference in the concentration and composition of short-chain fatty acid between treatments. Differences in the mcrA clone library were observed between HE and LE treatments (p < 0.05). Ninety-five percent of cloned sequence affiliated genus Methanobrevibacter in the feces of the pig regardless of treatments. During the experimental period, no significant difference in the proportion of copy numbers of mcrA against that of 16S rRNA gene of total bacteria was observed between treatments. In conclusion, feeding energy level affected composition of methanogens in the large intestine of the pig, while population density of methanogen was not affected.

Changes in faecal bacteria during fattening in finishing swine.

Body fat accumulation in mice and human is linked to the percentage of Firmicutes and Bacteroidetes, two bacterial phyla dominant in the large intestine. However, little is known about the relationship between the composition of the gut microbiota and fattening in pig. This study aimed to investigate the abundance of Firmicutes, Bacteroidetes, and Bacteroides, which is the major genus within Bacteroidetes, in porcine faeces during fattening. Ten 4-month-old crossbred pigs were given free access to commercial feed for fattening and water for 14 weeks. Daily feed intake and body weight were measured every 2 weeks. Faecal samples were collected at 0, 4, 8, and 14 weeks, and plasma samples were collected every 2 weeks. Daily feed intake increased until 8 weeks, and then decreased. Body weight increased with fattening during the experimental period. Feed efficiency showed high values at 0-4 and 6-8 weeks. The level of Firmicutes increased (P < 0.05), whereas those of Bacteroides and Bacteroidetes decreased (P < 0.05) with fattening. The total short chain fatty acid content in the faeces increased (P < 0.05) with fattening until 8 weeks and then decreased (P < 0.05) at 14 weeks. There were no significant relationships between the level of Firmicutes and feed intake or plasma leptin concentration. The levels of Bacteroidetes and Bacteroides correlated with feed intake, body weight, and plasma leptin or plasma urea nitrogen (PUN) concentration. Our results suggested that the level of Firmicutes increased and those of Bacteroidetes and Bacteroides decreased with increase in feed intake and body weight, similar to previous results obtained for mice and human. However, energy extraction from feed was not influenced by compositional alteration of gut flora, because daily gain and feed efficiency did not show high values towards the end of the fattening period. Manipulating the gut microbiota might help improve fattening performance, although further studies are necessary to understand the relationships between the composition of gut microbiota and energy absorption.

Crystal structure of NOD2 and its implications in human disease.

Nucleotide-binding oligomerization domain-containing protein 2 (NOD2), a member of the NOD-like receptors family, are crucial for innate immune responses. Mutations of NOD2 have been associated with chronic inflammatory disorders such as Crohn's disease (CD), Blau syndrome (BS) and early-onset sarcoidosis (EOS), but little is known about its signalling mechanism and the role it plays in these diseases. Here, we report the crystal structure of rabbit NOD2 in an ADP-bound state. The structure reveals an inactive closed conformation in which the subdomains in the NOD domain are closely packed by ADP-mediated and inter-domain interactions. Mapping of the BS- or EOS-associated gain-of-function mutations reveals that most of these mutations are located in the NOD subdomain interfaces, and are likely to disrupt the inner domain interactions, facilitating a conformational change to the active form. Conversely, mutations associated with CD are distributed throughout the protein, some of which may affect oligomer formation and ligand binding.