Changes in dietary fiber intake in mice reveal associations between colonic mucin O-glycosylation and specific gut bacteria

The colonic mucus layer, comprised of highly O-glycosylated mucins, is vital to mediating host-gut microbiota interactions, yet the impact of dietary changes on colonic mucin O-glycosylation and its associations with the gut microbiota remains unexplored. Here, we used an array of omics techniques including glycomics to examine the effect of dietary fiber consumption on the gut microbiota, colonic mucin O-glycosylation and host physiology of high-fat diet-fed C57BL/6J mice. The high-fat diet group had significantly impaired glucose tolerance and altered liver proteome, gut microbiota composition, and short-chain fatty acid production compared to normal chow diet group. While dietary fiber inclusion did not reverse all high fat-induced modifications, it resulted in specific changes, including an increase in the relative abundance of bacterial families with known fiber digesters and a higher propionate concentration. Conversely, colonic mucin O-glycosylation remained similar between the normal chow and high-fat diet groups, while dietary fiber intervention resulted in major alterations in O-glycosylation. Correlation network analysis revealed previously undescribed associations between specific bacteria and mucin glycan structures. For example, the relative abundance of the bacterium Parabacteroides distasonis positively correlated with glycan structures containing one terminal fucose and correlated negatively with glycans containing two terminal fucose residues or with both an N-acetylneuraminic acid and a sulfate residue. This is the first comprehensive report of the impact of dietary fiber on the colonic mucin O-glycosylation and associations of these mucosal glycans with specific gut bacteria

Taloudellisten kannusteiden käyttö vähähiilisen rakentamisen ohjauksessa : TALO-hankkeen loppuraportti

Rakennetun ympäristön osuus Suomen energiankulutuksesta ja kasvihuonekaasupäästöistä on tällä hetkellä huomattavan suuri. Yleisen arvion mukaan rakennuksissa käytetään lähes 40 prosenttia kokonaisenergian kulutuksesta, ja rakentaminen, rakennusten lämmitys ja sähkönkäyttö aiheuttavat yli 30 prosenttia kasvihuonekaasupäästöistä Suomessa. Rakennusmateriaalien osuus rakennuksen elinkaaren aikaisesta hiilijalanjäljestä arvioitiin aiemmin vähäiseksi käytön aikaisiin päästöihin verrattuna. Rakennusmateriaalien tuotannosta aiheutuneiden päästöjen vaikutus on kuitenkin sitä merkittävämpi, mitä energiatehokkaampia rakennukset ovat ja mitä enemmän rakennuksissa hyödynnetään ympäristön uusiutuvia energialähteitä. Myös energiantuotannon päästöjen vähentyessä rakennusmateriaalien päästöjen vaikutus korostuu. Ympäristöministeriön tavoitteena on, että rakennuksen elinkaaren aikaisia kasvihuonekaasupäästöjä ohjataan lainsäädännöllä vuoteen 2025 mennessä. Tässä selvityksessä arvioidaan neljän taloudellisen ohjauskeinon vaikutuksia vähähiilisen rakentamisen edistämisessä. Ohjauskeinoja arvioidaan mahdollisen raja-arvo-ohjauksen rinnalla vähähiilisen uudisrakentamisen edistämisessä ja itsenäisinä keinoina vähähiilisen korjausrakentamisen edistämisessä. Rakennustyypeistä tässä selvityksessä arvioidut taloudelliset ohjauskeinot kohdistuisivat asuinkerrostaloihin. Uudisrakentamisen arvioidut ohjauskeinot ovat: (1) valtion avustus, (2) kiinteistöverosta vapauttaminen, ja (3) lisärakennusoikeuden myöntäminen. Tuen saamisen kriteerinä on erittäin vähähiilinen rakentaminen. Erittäin vähähiilisen asuinkerrostalon kriteeri määritettiin tässä tarkastelussa elinkaarisen hiilijalanjälkilaskennan perusteella. Korjausrakentamisen arvioitu ohjauskeino on avustus asuinkerrostalojen vähähiilisiin korjauksiin. Avustuksen kriteerinä olisi käytönaikaisen energiankulutuksen päästöjen voimakas lasku. Selvityksen tulosten perusteella valtion tuki erittäin vähähiiliselle uudisrakentamiselle ja asuinkerrosten vähähiilisten korjausten avustukset saisivat aikaan vähähiilisiä asuinkerrostaloja ja korjausrakentamisessa kohtuuhintaisia päästövähennyksiä. Sen sijaan kiinteistöverosta vapauttaminen viideksi vuodeksi ei ole riittävä kannuste rakentaa erittäin vähähiilisiä asuinkerrostaloja. Lisärakennusoikeuden myöntäminen puolestaan on tulosten mukaan riittävä kannuste rakentajalle, mutta sen käyttöönoton laajuus perustuisi kuntien halukkuuteen ottaa ohjauskeino käyttöön

Fiber Supplements Derived From Sugarcane Stem, Wheat Dextrin and Psyllium Husk Have Different In Vitro Effects on the Human Gut Microbiota

There is growing public interest in the use of fiber supplements as a way of increasing dietary fiber intake and potentially improving the gut microbiota composition and digestive health. However, currently there is limited research into the effects of commercially available fiber supplements on the gut microbiota. Here we used an in vitro human digestive and gut microbiota model system to investigate the effect of three commercial fiber products; NutriKane™, Benefiber® and Psyllium husk (Macro) on the adult gut microbiota. The 16S rRNA gene amplicon sequencing results showed dramatic fiber-dependent changes in the gut microbiota structure and composition. Specific bacterial OTUs within the families Bacteroidaceae, Porphyromonadaceae, Ruminococcaceae, Lachnospiraceae, and Bifidobacteriaceae showed an increase in the relative abundances in the presence of one or more fiber product(s), while Enterobacteriaceae and Pseudomonadaceae showed a reduction in the relative abundances upon addition of all fiber treatments compared to the no added fiber control. Fiber-specific increases in SCFA concentrations showed correlation with the relative abundance of potential SCFA-producing gut bacteria. The chemical composition, antioxidant potential and polyphenolic content profiles of each fiber product were determined and found to be highly variable. Observed product-specific variations could be linked to differences in the chemical composition of the fiber products. The general nature of the fiber-dependent impact was relatively consistent across the individuals, which may demonstrate the potential of the products to alter the gut microbiota in a similar, and predictable direction, despite variability in the starting composition of the individual gut microbiota

Trichoderma reesei proteasome and genome-wide effects of the expression of mutant cellobiohydrolase I

Thesis (PhD) -- Macquarie University, Faculty of Science, Dept. of Chemistry and Biomolecular Sciences, 2009.Bibliography: leaves 178-214.Trichoderma reesei has a naturally high capacity for protein secretion and is currently employed for industrial production of a range of enzymes and recombinant gene products for a variety of biotechnological applications. A major limitation for the use of T. reesei as a universal production host is that industrial-scale production of heterologous proteins often results in lower yields than those achieved from native proteins. One reason for the low secretion yields of heterologous proteins is their improper folding and consequent elimination from the cell by the protein quality control mechanisms mediated by the unfolded protein response and the ER-associated degradation. Proteasome plays an important role in protein quality control by degradation the misfolded or aberrant proteins. In the current study three different mutant versions of the main secreted protein, cellobiohydrolase I (CBHI) tagged with the fluorescent protein Venus, were produced in T. reesei and their effects on physiology and gene expression were explored. The transcriptional response of the fungal hyphae was determined by CustomArrayTM 12K slides at three different time points. Potential interaction between the mutant CBHIs and the fungal proteasome was studied by fluorescence and the immunoelectron microscopy. -- A new rapid purification method for the fungal proteasome was developed during this study followed by separation of the proteasome subunit proteins by 2DE. Several proteasome interacting proteins (PIPs) were also identified. The purified 26S proteasome was visualised by transmission electron microscopy. The three mutant CBHI strains differed in terms of protein production and CBHI enzyme activity, although there were similarities between them showing a 'pulsing'-phenomenon both in protein secretion and transcription of the CBHI mRNA. Interestingly only one of the mutant CBHI strains could secrete the Venus-tagged fusion protein into the culture medium. -- The genome wide transcriptional study showed that two mutations in the cbh1 core gene did not cause UPR or ERAD activation, even though physiological signs of the stress were evident. Four and five mutations in the cbh1 core gene lead to expression changes in genes related to UPR and ERAD pathways and the physiological indications of stress were also seen under the light microscope. A new finding was up-regulation of a group of genes involved in 'ribosome structure and synthesis' in all mutant CBHI strains. In previous studies, secretion stress has been applied to fungal hyphae by drugs such as dithiothreitol (DTT) or tunicamycin, which seem to result in a different feedback to the protein translation machinery. -- Fluorescence and immunoelectron microscopy studies supported the microarray results indicating that four mutations in the cbh1 core gene lead to the interaction of the mutant CBHI with the 20S proteasome and at least partial retention of the mutant CBHI protein in the fungal hyphae.Mode of access: World Wide Web.240 leaves ill. (some col.

Methods for isolation and cultivation of filamentous fungi

Filamentous fungi are important organisms for basic discovery, industry, and human health. Their natural growth environments are extremely variable, a fact reflected by the numerous methods developed for their isolation and cultivation. Fungal culture in the laboratory is usually carried out on agar plates, shake flasks, and bench top fermenters starting with an inoculum that typically features fungal spores. Here we discuss the most popular methods for the isolation and cultivation of filamentous fungi for various purposes with the emphasis on enzyme production and molecular microbiology

Secretion of Proteases by an Opportunistic Fungal Pathogen <i>Scedosporium aurantiacum</i>

<div><p><i>Scedosporium aurantiacum</i> is an opportunistic filamentous fungus increasingly isolated from the sputum of cystic fibrosis patients, and is especially prevalent in Australia. At the moment, very little is known about the infection mechanism of this fungus. Secreted proteases have been shown to contribute to fungal virulence in several studies with other fungi. Here we have compared the profiles of proteases secreted by a clinical isolate <i>Scedosporium aurantiacum</i> (WM 06.482) and an environmental strain (WM 10.136) grown on a synthetic cystic fibrosis sputum medium supplemented with casein or mucin. Protease activity was assessed using class-specific substrates and inhibitors. Subtilisin-like and trypsin-like serine protease activity was detected in all cultures. The greatest difference in the secretion of proteases between the two strains occurred in mucin-supplemented medium, where the activities of the elastase-like, trypsin-like and aspartic proteases were, overall, 2.5–75 fold higher in the clinical strain compared to the environmental strain. Proteases secreted by the two strains in the mucin-supplemented medium were further analyzed by mass spectrometry. Six homologs of fungal proteases were identified from the clinical strain and five from the environmental strain. Of these, three were common for both strains including a subtilisin peptidase, a putative leucine aminopeptidase and a PA-SaNapH-like protease. Trypsin-like protease was identified by mass spectrometry only in the clinical isolate even though trypsin-like activity was present in all cultures. In contrast, high elastase-like activity was measured in the culture supernatant of the clinical strain but could not be identified by mass spectrometry searching against other fungi in the NCBI database. Future availability of an annotated genome will help finalise identification of the <i>S</i>. <i>aurantiacum</i> proteases.</p></div

Classes and activities of specific secreted proteases in three media.

<p>Activities of specific proteases were expressed as the amount of ρ-NA or MCA released in one minute per mg of dry biomass. Substrates and test conditions are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0169403#pone.0169403.t001" target="_blank">Table 1</a>. Data represent mean ± SD (3 biological repeats). SCFM: Synthetic CF sputum medium; SCFM+C (casein added); SCFM+M (mucin added). C: Clinical strain, E: environmental strain.</p

Inhibition of protease activity in day-4 culture supernatant of <i>S</i>. <i>aurantiacum</i> in three media.

<p>Percentage changes in the protease activity in the supernatant with an inhibitor relative to the non-inhibited control supernatant, set as 100% for each culture (dotted line). Data represent mean ± SD (3 biological repeats). A. Clinical strain; B. Environmental strain. SCFM: Synthetic CF sputum medium; SCFM+C (casein added); SCFM+M (mucin added).</p