The fungal community and its interaction with the concentration of short-chain fatty acids in the faeces of Chenghua,Yorkshire and Tibetan pigs

Despite their important roles in host nutrition and metabolism, and potential to cause disease, our knowledge of the fungal community in the mammalian gut is quite limited. To date, diversity and composition of fungi in swine gut still remains unknown. Therefore, the first internal transcribed spacer of fungi in faecal samples from three breeds of pigs (10 pigs for each breed) was sequenced based on an Illumina HiSeq 2500 platform, and the relationship between the fungal community and the concentrations of main short-chain fatty acids (SCFAs) was also analysed. Results indicated that Chenghua (local, higher body fat rate), Yorkshire (foreign, higher lean meat and growth rate) and Tibetan (plateau, stronger disease resistance) pigs harboured distinct fungal community. The Basidiomycota and Ascomycota presented as the two predominant phyla, with Loreleia, Russula and Candida as the top three genera in all samples. Network analysis revealed a total of 35 correlations among different fungal genera, with 27 (77.14%) positive and 8 (22.86%) negative pairwise interactions. Canonical correspondence analysis suggested that fungi in the faeces of pigs were more correlated to the concentration of acetate and butyrate rather than propionate. Spearman’s correlation further showed that Tomentella was positively correlated to both acetate and butyrate, and Loreleia was positively correlated to propionate (P < 0.05), while Nephroma and Taiwanofungus were negatively correlated to acetate and propionate (P < 0.05). These findings expanded our knowledge on the intestinal fungi in pigs with different genotypes and phenotypes, indicating that fungi may play an indispensable role during the metabolism of host and the maintenance of intestinal health. The cross-feeding between fungi and other microorganisms may be crucial during the digestion of dietary carbohydrates and the associated physiological processes, which is worthy to be further studied.     

Improved lineshape and sensitivity in the HNCO-family of triple resonance experiments

A simple scheme is presented for the suppression of dispersive contributions to cross peaks in HNCO-type spectra where the ¹⁵N chemical shift is recorded in a constant-time manner immediately prior to the transfer from ¹⁵ N to ¹HN at the end of the sequence. These dispersive contributions arise when the delay for refocusing the ¹⁵N-¹³CO one-bond coupling is set to less than 0.5/¹J_N,CO and when ² J_HN,CO 0. Improvements in sensitivity in ¹HN detected experiments recorded on ¹⁵ N,¹³C-labeled samples can be realized by application of ¹³CO/¹³ decoupling during acquisition. Sensitivity gains on the order of 15% and 5% have been obtained for an SH3 domain (62 residues) and maltose binding protein (370 residues), respectively.     

A 4D TROSY-based pulse scheme for correlating 1HNi,15Ni,13Cαi,13C′i−1 chemical shifts in high molecular weight, 15N,13C, 2H labeled proteins

A 4D TROSY-based triple resonance experiment, 4D-HNCO_i–1CA_i, is presented which correlates intra-residue ¹HN, ¹⁵N, ¹³ C chemical shifts with the carbonyl (¹³C) shift of the preceding residue. The experiment is best used in concert with recently described 4D TROSY-HNCOCA and -HNCACO experiments [Yang, D. and Kay, L.E. (1999) J. Am. Chem. Soc., 121, 2571–2575]. In cases where degeneracy of (¹HN,¹⁵N) spin pairs precludes assignment using the HNCOCA and HNCACO, the HNCO_i–1CA_i often allows resolution of the ambiguity by linking the ¹³C and ¹³C spins surrounding the (¹HN,¹⁵N) pair. The experiment is demonstrated on a sample of ¹⁵N, ¹³C, ² H labeled maltose binding protein in complex with -cyclodextrin that tumbles with a correlation time of 46 ns.     

TROSY-based HNCO pulse sequences for the measurement of 1HN-15N, 15N-13CO, 1HN-13CO, 13CO-13Cα and 1HN-13Cα dipolar couplings in 15N, 13C, 2H-labeled proteins

HNCO-based 3D pulse schemes are presented for measuring ¹HN-¹⁵N,¹⁵N-¹³CO, ¹HN-¹³CO,¹³CO-¹³C and ¹HN-¹³C dipolar couplings in ¹⁵N,¹³C,²-labeled proteins. The experiments are based on recently developed TROSY methodology for improving spectral resolution and sensitivity. Data sets recorded on a complex of Val, Leu, Ile (1 only) methyl protonated ¹⁵N,¹³C,²H-labeled maltose binding protein and -cyclodextrin as well as ¹⁵N,¹³C,²H-labeled human carbonic anhydrase II demonstrate that precise dipolar couplings can be obtained on proteins in the 30–40 kDa molecular weight range. These couplings will serve as powerful restraints for obtaining global folds of highly deuterated proteins.     

NMR solution structure and dynamics of an exchangeable apolipoprotein,Locusta migratoria apolipophorin III

We report here the NMR structure and backbone dynamics of an exchangeable apolipoprotein, apoLp-III, from the insect Locusta migratoria. The NMR structure adopts an up-and-down elongated five-helix bundle, which is similar to the x-ray crystal structure of this protein. A short helix, helix 4', is observed that is perpendicular to the bundle and fully solvent-exposed. NMR experimental parameters confirm the existence of this short helix, which is proposed to serve as a recognition helix for apoLp-III binding to lipoprotein surfaces. The L. migratoria apoLp-III helix bundle displays several characteristic structural features that regulate the reversible lipoprotein binding activity of apoLp-III. The buried hydrophilic residues and exposed hydrophobic residues readily adjust the marginal stability of apoLp-III, facilitating the helix bundle opening. Specifically, upon lipoprotein binding the locations and orientations of the buried hydrophilic residues modulate the apoLp-III helix bundle to adopt a possible opening at the hinge that is opposite the recognition short helix, helix 4'. The backbone dynamics provide additional support to the recognition role of helix 4' and this preferred conformational adaptation of apoLp-III upon lipid binding. In this case, the lipid-bound open conformation contains two lobes linked by hinge loops. One lobe contains helices 2 and 3, and the other lobe contains helices 1, 4, and 5. This preferred bundle opening is different from the original proposal on the basis of the x-ray crystal structure of this protein (Breiter, D. R., Kanost, M. R., Benning, M. M., Wesenberg, G., Law, J. H., Wells, M. A., Rayment, I., and Holden, H. M. (1991) Biochemistry 30, 603-608), but it efficiently uses helix 4' as the recognition short helix. The buried interhelical H-bonds are found to be mainly located between the two lobes, potentially providing a specific driving force for the helix bundle recovery of apoLp-III from the lipid-bound open conformation. Finally, we compare the NMR structures of Manduca sexta apoLp-III and L. migratoria apoLp-III and present a united scheme for the structural basis of the reversible lipoprotein binding activity of apoLp-III.     

Proteomic Analysis of Fetal Ovary Reveals That Ovarian Developmental Potential Is Greater in Meishan Pigs than in Yorkshire Pigs

Time-dependent expression of functional proteins in fetal ovaries is important to understand the developmental process of the ovary. This study was carried out to enhance our understanding of the developmental process of porcine fetal ovaries and to better address the differences in fetal ovary development of local and foreign pigs. The objective of the present study is to test the expression of key proteins that regulate the growth and development of fetal ovaries in Meishan and Yorkshire porcine breeds by using proteomics technology. Six Meishan and 6 Yorkshire pregnant gilts were used in this experiment. Fetal ovaries were obtained from Yorkshire and Meishan gilts on days 55 and 90 of the gestation period. Using 2D-DIGE (two dimensional-difference in gel electrophoresis) analysis, the results showed that there are about 1551 and 1400 proteins in gilt fetal ovaries on days 55 and 90, respectively of the gestation. Using MALDI TOF-TOF MS analysis, 27 differentially expressed proteins were identified in the fetal ovaries of the 2 breeds on day 55 of gestation, and a total of 18 proteins were identified on day 90 of gestation. These differentially expressed proteins were involved in the regulation of biological processes (cell death, stress response, cytoskeletal proteins) and molecular functions (enzyme regulator activity). We also found that alpha-1-antitrypsin, actin, vimentin, and PP2A proteins promote the formation of primordial follicles in the ovaries of Yorkshire pigs on day 55 of gestation while low expression heat shock proteins and high expression alpha-fetoproteins (AFP) may promote Meishan fetal ovarian follicular development on day 90 of gestation. These findings provide a deeper understanding of how reduced expression of heat shock proteins and increased expression of AFP can significantly reduce the risk of reproductive disease in obese Meishan sows. Our study also shows how these proteins can increase the ovulation rate and may be responsible for the low reproductive efficiency reported in other obese breeds. The ovarian developmental potential was found to be greater in Meishan pigs than in Yorkshire pigs.     

Nutrition and health relevant regulation of intestinal sulfur amino acid metabolism

Sulfur amino acids (SAA), particularly methionine and cysteine, are critical for the gut to maintain its functions including the digestion, absorption and metabolism of nutrients, the immune surveillance of the intestinal epithelial layer and regulation of the mucosal response to foreign antigens. However, the metabolism of SAA in the gut, specifically the transmethylation of methionine, will result in a net release of homocysteine, which is shown to be associated with cardiovascular disease and stroke. Furthermore, the extensive catabolism of dietary methionine by the intestine or by luminal microbes may result in a decrease in nutritional efficiency. Therefore, the regulation of SAA metabolism in the gut is not only nutritionally relevant, but also relevant to the overall health and well-being. The superiority of dl-2-hydroxy-4-methylthiobutyrate to dl-methionine in decreasing homocysteine production, alleviating stress responses, and reducing the first-pass intestinal metabolism of dietary methionine may provide a promising implication for nutritional strategies to manipulate SAA metabolism and thus to improve the nutrition and health status of animals and perhaps humans.