A Proteomic Analysis of Fibre Degradation and Assimilation by Butyrivibrio Proteoclasticus

<p>Butyrivibrio proteoclasticus B316T is a Gram-positive, lignocellulose degrading bacterium that is prevalent in the rumen of animals grazing pasture, and is one of only a few rumen microbes known to degrade and utilise xylan in vitro. Xylan is a hemicellulose that comprises up to 45% of the polysaccharide component of ruminant forages. Often as little as 30% of the total energy content of forages is utilised by the ruminant due to poor hemicellulose degradation by the fibrolytic rumen microbes. An opportunity exists to improve forage degradation in the rumen, which is predicted to improve the productivity of forage fed ruminants. A clearer understanding of the strategies employed by fibrolytic rumen microbes to degrade and utilise lignocellulose is important in realising this goal. Almost 10% of the B. proteoclasticus genome encodes proteins involved in polysaccharide metabolism and transport, which includes 134 fibrolytic enzymes that are active upon plant fibre. Many of these are clustered into one of 36 polysaccharide utilisation loci that also contain transmembrane transporters, transcriptional regulators, environmental sensors and genes involved in further polysaccharide metabolism. Gel-based and gel-free proteomic analyses of the cytosolic, cell-associated, and secreted fractions of cells grown on xylan were used to identify proteins involved in the degradation, assimilation, and metabolism of hemicellulose. A set of 416 non-redundant proteins were identified, which included 12 extracellular and 24 cytosolic polysaccharidases, and 59 proteins involved in the uptake and further metabolism of polysaccharide degradation products, many of which were substrate-binding protein components of ATP-driven transporter systems. In cells grown on xylan, several of these proteins displayed significant protein abundance changes relative to cells grown on the monomeric sugar xylose, in a pattern that reflected the growth substrates used. A model of xylan degradation by B. proteoclasticus based on these results hypothesises that B. proteoclasticus attacks the xylan backbone and main substituent groups of hemicellulose in the extracellular space, assimilates the xylooligosaccharides and performs the final stages of degradation within the cell. These results provide insight into a xylan degrading enzyme system that has evolved to efficiently degrade and utilise hemicellulose, extend our understanding of the enzymes that are likely to play important roles in hemicellulose degradation, and support the notion that Butyrivibrio species are important contributors to rumen fibre degradation.</p>

A Proteomic Analysis of Fibre Degradation and Assimilation by Butyrivibrio Proteoclasticus

<p>Butyrivibrio proteoclasticus B316T is a Gram-positive, lignocellulose degrading bacterium that is prevalent in the rumen of animals grazing pasture, and is one of only a few rumen microbes known to degrade and utilise xylan in vitro. Xylan is a hemicellulose that comprises up to 45% of the polysaccharide component of ruminant forages. Often as little as 30% of the total energy content of forages is utilised by the ruminant due to poor hemicellulose degradation by the fibrolytic rumen microbes. An opportunity exists to improve forage degradation in the rumen, which is predicted to improve the productivity of forage fed ruminants. A clearer understanding of the strategies employed by fibrolytic rumen microbes to degrade and utilise lignocellulose is important in realising this goal. Almost 10% of the B. proteoclasticus genome encodes proteins involved in polysaccharide metabolism and transport, which includes 134 fibrolytic enzymes that are active upon plant fibre. Many of these are clustered into one of 36 polysaccharide utilisation loci that also contain transmembrane transporters, transcriptional regulators, environmental sensors and genes involved in further polysaccharide metabolism. Gel-based and gel-free proteomic analyses of the cytosolic, cell-associated, and secreted fractions of cells grown on xylan were used to identify proteins involved in the degradation, assimilation, and metabolism of hemicellulose. A set of 416 non-redundant proteins were identified, which included 12 extracellular and 24 cytosolic polysaccharidases, and 59 proteins involved in the uptake and further metabolism of polysaccharide degradation products, many of which were substrate-binding protein components of ATP-driven transporter systems. In cells grown on xylan, several of these proteins displayed significant protein abundance changes relative to cells grown on the monomeric sugar xylose, in a pattern that reflected the growth substrates used. A model of xylan degradation by B. proteoclasticus based on these results hypothesises that B. proteoclasticus attacks the xylan backbone and main substituent groups of hemicellulose in the extracellular space, assimilates the xylooligosaccharides and performs the final stages of degradation within the cell. These results provide insight into a xylan degrading enzyme system that has evolved to efficiently degrade and utilise hemicellulose, extend our understanding of the enzymes that are likely to play important roles in hemicellulose degradation, and support the notion that Butyrivibrio species are important contributors to rumen fibre degradation.</p>

Reduced B12 uptake and increased gastrointestinal formate are associated with archaeome-mediated breath methane emission in humans

Background Methane is an end product of microbial fermentation in the human gastrointestinal tract. This gas is solely produced by an archaeal subpopulation of the human microbiome. Increased methane production has been associated with abdominal pain, bloating, constipation, IBD, CRC or other conditions. Twenty percent of the (healthy) Western populations innately exhale substantially higher amounts (>5 ppm) of this gas. The underlying principle for differential methane emission and its effect on human health is not sufficiently understood. Results We assessed the breath methane content, the gastrointestinal microbiome, its function and metabolome, and dietary intake of one-hundred healthy young adults (female: n = 52, male: n = 48; mean age =24.1). On the basis of the amount of methane emitted, participants were grouped into high methane emitters (CH4 breath content 5–75 ppm) and low emitters (CH4 < 5 ppm). The microbiomes of high methane emitters were characterized by a 1000-fold increase in Methanobrevibacter smithii. This archaeon co-occurred with a bacterial community specialized on dietary fibre degradation, which included members of Ruminococcaceae and Christensenellaceae. As confirmed by metagenomics and metabolomics, the biology of high methane producers was further characterized by increased formate and acetate levels in the gut. These metabolites were strongly correlated with dietary habits, such as vitamin, fat and fibre intake, and microbiome function, altogether driving archaeal methanogenesis. Conclusions This study enlightens the complex, multi-level interplay of host diet, genetics and microbiome composition/function leading to two fundamentally different gastrointestinal phenotypes and identifies novel points of therapeutic action in methane-associated disorders.

A New Method of Rice Moisture Content Determination Using Voxel Weighting-Based from Radio Tomography Images

This manuscript presents a new method to monitor and localize the moisture distribution in a rice silo based on tomography images. Because the rice grain is naturally hygroscopic, the stored grains’ quality depends on their level of moisture content. Higher moisture content leads to fibre degradation, making the grains too frail and possibly milled. If the moisture is too low, the grains become brittle and are susceptible to higher breakage. At present, the single-point measurement method is unreliable because the moisture build-up inside the silo might be distributed unevenly. In addition, this method mostly applies gravimetric analysis, which is destructive. Thus, we proposed a radio tomographic imaging (RTI) system to address these problems. Four simulated phantom profiles at different percentages of moisture content were reconstructed using Newton’s One-Step Error Reconstruction and Tikhonov Regularization algorithms. This simulation study utilized the relationship between the maximum voxel weighting of the reconstructed RTI image and the percentage of moisture content. The outcomes demonstrated promising results, in which the weighting voxel linearly increased with the percentage of moisture content, with a correlation coefficient higher than 0.95 was obtained. Therefore, the results support the possibility of using the RTI approach for monitoring and localizing the moisture distribution inside the rice silo.

The effects of compound treatment of Aspergillus oryzae and fibrolytic enzyme on in vitro degradation, gas production and fermentative profile of maize silage and sugarcane silage

The present study was conducted to evaluate the effect of a live culture of Aspergillus oryzae (A; CCT4359) and fibrolytic enzyme (E; Fibrozyme Alltech Inc.) on fibre digestibility by a gas production bioassay and in vitro degradation of maize silage and sugarcane silage. A completely randomized design trial was performed to evaluate: A doses (0, 20, 60 and 100 mg/l), E doses (0, 160, 320 and 480 mg/l) and roughage source (R; maize and sugarcane silage) in a 4 × 4 × 2 factorial arrangement. The inclusion of increasing doses of A and E increased dry matter and neutral detergent fibre in vitro digestibility linearly, but for E this effect occurred only in maize silage. There was a linear increase in the potential for gas production at the highest dose of A only in sugarcane silage, with no effect on lag time (L). Increasing doses of E increased the volume of gases produced linearly, and a trend of linear reduction of L, regardless of the roughage. There was a linear reduction in ammonia-nitrogen concentration in response to increasing doses of A and E, and an increase in acetic acid concentration at the highest dose of A, regardless of roughage. The additives had no synergistic effect on gas production and digestibility, but were efficient in altering the fermentative pattern, demonstrating the potential to increase fibre degradation.

Plant cell wall chemistry: implications for ruminant utilisation

Ruminants have adapted to cope with bulky, fibrous forage diets by accommodating a large, diverse microbial population in the reticulo-rumen. Ruminants are dependent on forages as their main sources of energy and other nutrients. Forages are comprised of a complex matrix of cellulose, hemicellulose, protein, minerals and phenolic compounds (including lignin and tannins) with various linkages; many of which are poorly defined. The composition and characteristics of polysaccharides vary greatly among forages and plant cell walls. Plant cell walls are linked and packed together in tight configurations to resist degradation, and hence their nutritional value to animals varies considerably, depending on composition, structure and degradability. An understanding of the inter-relationship between the chemical composition and the degradation of plant cell walls by rumen microorganisms is of major economic importance to ruminant production. Increasing the efficiency of fibre degradation in the rumen has been the subject of extensive research for many decades. This review summarises current knowledge of forage chemistry in order to develop strategies to increase efficiency of forage utilisation by ruminants.

Scleraxis expressing scleral cells respond to inflammatory stimulation

The sclera is an ocular tissue rich of collagenous extracellular matrix, which is built up and maintained by relatively few, still poorly characterized fibroblast-like cells. The aims of this study are to add to the characterization of scleral fibroblasts and to examine the reaction of these fibroblasts to inflammatory stimulation in an ex vivo organotypic model. Scleras of scleraxis-GFP (SCX-GFP) mice were analyzed using immunohistochemistry and qRT-PCR for the expression of the tendon cell associated marker genes scleraxis (SCX), mohawk and tenomodulin. In organotypic tissue culture, explanted scleras of adult scleraxis GFP reporter mice were exposed to 10 ng/ml recombinant interleukin 1-ß (IL1-ß) and IL1-ß in combination with dexamethasone. The tissue was then analyzed by immunofluorescence staining of the inflammation- and fibrosis-associated proteins IL6, COX-2, iNOS, connective tissue growth factor, MMP2, MMP3, and MMP13 as well as for collagen fibre degradation using a Collagen Hybridizing Peptide (CHP) binding assay. The mouse sclera displayed a strong expression of scleraxis promoter-driven GFP, indicating a tendon cell-like phenotype, as well as expression of scleraxis, tenomodulin and mohawk mRNA. Upon IL1-ß stimulation, SCX-GFP+ cells significantly upregulated the expression of all proteins analysed. Moreover, IL1-ß stimulation resulted in significant collagen degradation. Adding the corticosteroid dexamethasone significantly reduced the response to IL1-ß stimulation. Collagen degradation was significantly enhanced in the IL1-ß group. Dexamethasone demonstrated a significant rescue effect. This work provides insights into the characteristics of scleral cells and establishes an ex vivo model of scleral inflammation.

A toolbox for the comprehensive analysis of small volume human intestinal samples that can be used with gastrointestinal sampling capsules

Detailed knowledge on the fate of dietary components inside the human intestinal tract is lacking. Access to this inner world of digestion is now possible through novel human gastrointestinal sampling capsules. Due to the novelty of such devices, no methodology has been published to stabilise and analyse the resulting samples. A complicating factor is that excretion of such capsules in faeces may take days, while degradation of the dietary components continues. Therefore a stabilising reagent should be pre-loaded in the capsule to ensure the measurement of a representative sample. Considering the small volume of recovered samples, analytical methods must be optimized to collect as many data as possible from little material. We present a complete workflow for stabilising and analysing the fermentation status of dietary fibres in such samples, including microbiota, fibre degradation, and short chain fatty acids. The final quenching reagent was designed based on safety and effectiveness to inhibit fructo- and galacto-oligosaccharides degradation and short chain fatty acids production by human ileostomy microbiota, and subsequently validated in faecal samples. The final composition of the stock quenching reagent is 175 mM Tris, 525 mM NaCl, 35 mM EDTA, 12% SDS, and 8 M urea at pH 8.5.

Reduced B12 Uptake and Increased Gastrointestinal Formate Drive Archaeome-Mediated Breath Methane Emission in Humans

Background Methane is an end product of microbial fermentation in the human gastrointestinal tract. This gas is solely produced by an archaeal subpopulation of the human microbiome. Increased methane production has been associated with abdominal pain, bloating, constipation, IBD, CRC or other conditions. Twenty percent of the (healthy) Western populations innately exhale substantially higher amounts (>5 ppm) of this gas. The underlying principle for differential methane emission and its effect on human health was still not sufficiently understood. Results We assessed the breath methane content, gastrointestinal microbiome, metagenome, metabolome, and eating behavior of one-hundred healthy young adults (female: n = 52, male: n = 48; mean age =24.1). On the basis of the amount of methane emitted, participants were grouped into high methane emitters (CH4 breath content 5-75 ppm) and low emitters (CH4 < 5 ppm). The microbiomes of high methane emitters were characterized by a 1000-fold increase in Methanobrevibacter smithii. This archaeon co-occurred with a bacterial community specialized on dietary fibre degradation, which included members of Ruminococcaceae and Christensenellaceae. As confirmed by metagenomics and metabolomics, the biology of high methane producers was further characterized by increased formate and acetate levels in the gut. These metabolites were strongly correlated with dietary habits, such as vitamin, fat and fibre intake, host genetics, and microbiome function, altogether driving archaeal methanogenesis. Conclusions This study enlightens the complex, multi-level interplay of host diet, genetics and microbiome composition/function leading to two fundamentally different gastrointestinal phenotypes and identifies novel points of therapeutic action in methane-associated disorders.

Nutritional qualities of corn cob and waste paper incubated with edible mushroom

Studies were conducted to evaluate corn cob and waste paper cultured with edible mushroom (Pleurotus sajor) as a means of improving their nutritive value for livestock animals. Waste paper and corn cob were 14.2 and 47.7% DM respectively. Crude fibre degradation was 25.0% for corn cob and 7.21% for waste paper which was intermediate to 34.0 and 10.30% of the untreated samples. Digestibility of the fungus treated paper and corn cob were assessed using 24 Wistar Albino rats. Dry matter digestibility of the paper and the corn cob were improved by incubating with the mushroom with an increase of 30 and 40% waste paper and corn cob respectively. The crude protein digestibility and crude fibre digestibility were improved by incubation with mushroom compared with untreated samples. It was concluded that such biodegradation and the concurrent 30.2 and 27.8 percentage unit increases in crude protein digestibility of corn cob and waste paper may in fact improve the nutritive value for livestock with no detriment to the health (organ and blood) of the animals.