Plant Cell Wall Components Research Articles

Fungi isolated from Miscanthus and sugarcane: biomass conversion, fungal enzymes, and hydrolysis of plant cell wall polymers.

BackgroundBiofuel use is one of many means of addressing global change caused by anthropogenic release of fossil fuel carbon dioxide into Earth’s atmosphere. To make a meaningful reduction in fossil fuel use, bioethanol must be produced from the entire plant rather than only its starch or sugars. Enzymes produced by fungi constitute a significant percentage of the cost of bioethanol production from non-starch (i.e., lignocellulosic) components of energy crops and agricultural residues. We, and others, have reasoned that fungi that naturally deconstruct plant walls may provide the best enzymes for bioconversion of energy crops.ResultsPreviously, we have reported on the isolation of 106 fungi from decaying leaves of Miscanthus and sugarcane (Appl Environ Microbiol 77:5490–504, 2011). Here, we thoroughly analyze 30 of these fungi including those most often found on decaying leaves and stems of these plants, as well as four fungi chosen because they are well-studied for their plant cell wall deconstructing enzymes, for wood decay, or for genetic regulation of plant cell wall deconstruction. We extend our analysis to assess not only their ability over an 8-week period to bioconvert Miscanthus cell walls but also their ability to secrete total protein, to secrete enzymes with the activities of xylanases, exocellulases, endocellulases, and beta-glucosidases, and to remove specific parts of Miscanthus cell walls, that is, glucan, xylan, arabinan, and lignin.ConclusionThis study of fungi that bioconvert energy crops is significant because 30 fungi were studied, because the fungi were isolated from decaying energy grasses, because enzyme activity and removal of plant cell wall components were recorded in addition to biomass conversion, and because the study period was 2 months. Each of these factors make our study the most thorough to date, and we discovered fungi that are significantly superior on all counts to the most widely used, industrial bioconversion fungus, Trichoderma reesei. Many of the best fungi that we found are in taxonomic groups that have not been exploited for industrial bioconversion and the cultures are available from the Centraalbureau voor Schimmelcultures in Utrecht, Netherlands, for all to use.

Transcriptome Profiling ofhrf2Transgenic Rapeseed Revealed Genes Related to the Defense Responses and Signaling Pathways

ABSTRACTEctopic expression of the HarpinXooc–encoding genehrf2in rapeseed (Brassica napusL.) confers enhanced resistance toSclerotinia sclerotiorumand improved agronomic performance. To better understand the molecular mechanisms of harpin‐mediated plant responses, we developed a 3’–tag digital gene expression (DGE) method to compare the mRNA expression patterns of transgenic T‐4 rapeseed harboringhrf2and untransformed Yangyou 4 (Y4) at the global level. Solexa Illumina sequencing yielded 5,645,091 and 6,007,037 clean tags, corresponding to 14,237 and 14,773 genes for Y4 and T‐4, respectively. We identified 2114 differentially expressed genes (DEGs; log2 ratio ≥ 1, false discovery rate [FDR] ≤ 0.001) between transgenic T‐4 and untransformed Y4 that assigned to seven Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and 19 Gene Ontology categories. Numerous genes related to defense responses were differentially expressed in T‐4, representing categories such as hypersensitive response, defense proteins, plant cell wall components, and phenylpropanoid metabolism pathway. Furthermore, expression was largely modified in T‐4 in signaling pathways including G protein, Ca2+, ubiquitination, protein kinase, salicylic acid, jasmonic acid, abscisic acid, ethylene, and auxin hormones. We also identified numerous DEGs encoding transcription factors in T‐4. This analysis shows the breadth of molecular mechanisms of harpin‐mediated plant responses in genetically transformed plants. Furthermore, the DGE sequence resources identified in this study provide a platform for functional genomics studies of rapeseed.

Process–Structure–Function Relations of Pectin in Food

Pectin, a complex polysaccharide rich in galacturonic acid, has been identified as a critical structural component of plant cell walls. The functionality of this intricate macromolecule in fruit- and vegetable-based–derived products and ingredients is strongly determined by the nanostructure of its most abundant polymer, homogalacturonan. During food processing, pectic homogalacturonan is susceptible to various enzymatic as well as nonenzymatic conversion reactions modifying its structural and, hence, its functional properties. Consequently, a profound understanding of the various process–structure–function relations of pectin aids food scientists to tailor the functional properties of plant-based derived products and ingredients. This review describes the current knowledge on process–structure–function relations of pectin in foods with special focus on pectin's functionality with regard to textural attributes of solid plant-based foods and rheological properties of particulated fruit- and vegetable-derived products. In this context, both pectin research performed via traditional, ex situ physicochemical analyses of fractionated walls and isolated polymers and pectin investigation through in situ pectin localization are considered.

A Molecular Description of Cellulose Biosynthesis

Cellulose is the most abundant biological macromolecule and is an extracellular linear polymer of glucose molecules. It is an essential component of plant cell walls but is also found in algae and bacteria. In bacteria, cellulose production frequently correlates with the formation of biofilms, a sessile, multi-cellular growth form. Cellulose synthesis and transport across the inner bacterial membrane is mediated by a complex of the multi-spanning catalytic BcsA subunit and the membrane-anchored periplasmic BcsB protein. We determined several crystal structures of the BcsA-B complex at intermediate states during cellulose synthesis and membrane translocation. The structures demonstrate how BcsA forms a cellulose-conducting channel and delineate conformational changes of the synthase underlying substrate binding, glycosyl transfer and polymer translocation. Furthermore, combining biochemical and structural data, we reveal the mechanism by which cyclic-di-GMP, a potent stimulator of bacterial biofilms and allosteric activator of bacterial cellulose synthase, regulates BcsA's activity.

Nano bio optically tunable composite nanocrystalline cellulose films

We utilize nanocellulose and semiconductor nanocrystals to reinforce and functionalize a biodegradable transparent film to create a transparent, strong and optically tunable plastic film.

Pectin Methylesterases: A Review

Pectin methylesterase (PME) is the first enzyme acting on pectin, a major component of plant cell wall. PME catalyzes reactions according to the double-displacement mechanism. In plants, PMEs can be classified on the basis of presence or absence of the PRO domain in pectin methylesterase into Type 1 and Type II. Type1 contains one to three PRO domains and two or three introns, type II PMEs are without PRO domain and with five or six introns. Once PMEs are secreted into the cell wall, mature PMEs exhibit three different modes of action: single chain mechanism, multiple chain mechanism and multiple attack mechanism. For bacterial and plant PMEs, single chain and multiple chain mechanism is used and for fungal PMEs only multiple chain mechanism has been proposed. PME activity is regulated by differential expression both spatially and temporally.

Pectin Nanostructure Visualization by Atomic Force Microscopy

Pectins, complex polysaccharides rich in galacturonic acid, are a major component of primary plant cell walls. These macromolecules regulate cell wall porosity and intercellular adhesion, being important in the control of cell expansion and differentiation through their effect on the rheological properties of the cell wall. In fruits, pectin disassembly during ripening is one the main event leading to textural changes and softening. Changes in pectic polymer size, composition and structure have been studied by conventional techniques, most of them relying on bulk analysis of a population of polysaccharides but studies of detailed structure of isolated polymer chains are scarce (Paniagua et al., 2014). Atomic force microscopy (AFM) is a versatile and powerful technique able to analyze force measurements, as well as to visualize roughness of biological samples at nanoscale (Morris et al., 2010). Using this technique, recent research has found a close relationship between pectin nanostructural complexity and texture and postharvest behavior in several fruits (Liu and Cheng, 2011; Cybulska et al., 2014; Posé et al., 2015). Here, we describe an AFM procedure to topographically visualize pectic polymers from fruit cell wall extracts that has successfully been used in the study of strawberry ripening (Posé et al., 2012; Posé et al., 2015). Thus, from AFM images the 3D structural analysis of isolated chains (length, height, and branch pattern) can be resolved at high magnification and with minimal sample preparation. A full description of AFM fundamentals and the different sampling modes are described in Morris et al. (2010).

Structural and Biochemical Basis for Mannan Utilization by Caldanaerobius polysaccharolyticus Strain ATCC BAA-17

Hemicelluloses, the polysaccharide component of plant cell walls, represent one of the most abundant biopolymers in nature. The most common hemicellulosic constituents of softwoods, such as conifers and cycads, are mannans consisting of a 1,4-linked β-mannopyranosyl main chain with branch decorations. Efforts toward the utilization of hemicellulose for bioconversion into cellulosic biofuels have resulted in the identification of several families of glycoside hydrolases that can degrade mannan. However, effective biofermentation of manno-oligosaccharides is limited by a lack of appropriate uptake route in ethanologenic organisms. Here, we used transcriptome sequencing to gain insights into mannan degradation by the thermophilic anaerobic bacterium Caldanaerobius polysaccharolyticus. The most highly up-regulated genes during mannan fermentation occur in a cluster containing several genes encoding enzymes for efficient mannan hydrolysis as well as a solute-binding protein (CpMnBP1) that exhibits specificity for short mannose polymers but exhibited the flexibility to accommodate branched polysaccharide decorations. Co-crystal structures of CpMnBP1 in complex with mannobiose (1.4-Å resolution) and mannotriose (2.2-Å resolution) revealed the molecular rationale for chain length and oligosaccharide specificity. Calorimetric analysis of several active site variants confirmed the roles of residues critical to the function of CpMnBP1. This work represents the first biochemical characterization of a mannose-specific solute-binding protein and provides a framework for engineering mannan utilization capabilities for microbial fermentation.

Uncovering the cultivable microbial diversity of costa rican beetles and its ability to break down plant cell wall components.

Coleopterans are the most diverse insect order described to date. These organisms have acquired an array of survival mechanisms through their evolution, including highly efficient digestive systems. Therefore, the coleopteran intestinal microbiota constitutes an important source of novel plant cell wall-degrading enzymes with potential biotechnological applications. We isolated and described the cultivable fungi, actinomycetes and aerobic eubacteria associated with the gut of larvae and adults from six different beetle families colonizing decomposing logs in protected Costa Rican ecosystems. We obtained 611 isolates and performed phylogenetic analyses using the ITS region (fungi) and 16S rDNA (bacteria). The majority of fungal isolates belonged to the order Hypocreales (26% of 169 total), while the majority of actinomycetes belonged to the genus Streptomyces (86% of 241 total). Finally, we isolated 201 bacteria spanning 19 different families belonging into four phyla: Firmicutes, α, β and γ-proteobacteria. Subsequently, we focused on microbes isolated from Passalid beetles to test their ability to degrade plant cell wall polymers. Highest scores in these assays were achieved by a fungal isolate (Anthostomella sp.), two Streptomyces and one Bacillus bacterial isolates. Our study demonstrates that Costa Rican beetles harbor several types of cultivable microbes, some of which may be involved in symbiotic relationships that enable the insect to digest complex polymers such as lignocellulose.

Identification of glycosyl hydrolases from a metagenomic library of microflora in sugarcane bagasse collection site and their cooperative action on cellulose degradation

Lignocellulose decomposition is a natural process involving the cooperative action of various glycosyl hydrolases (GH) on plant cell wall components. In this study, a metagenomic library was constructed to capture the genetic diversity of microbes inhabiting an industrial bagasse collection site. A variety of putative genes encoding GH families 2, 3, 5, 9, 11, and 16 were identified using activity-based screening, which showed low to moderate homology to various cellulases and hemicellulases. The recombinant GH9 endoglucanase (Cel9) and GH11 endo-xylanase (Xyn11) were thermophilic with optimal activity between 75°C and 80°C and the maximal activity at slightly acidic to neutral pH range. The enzymes exhibited cooperative activity with Trichoderma reesei cellulase on the degradation of lignocellulosic substrates. Mixture design showed positive interactions among the enzyme components. The optimal combination was determined to be 41.4% Celluclast, 18.0% Cel9, and 40.6% Xyn11 with the predicted relative reducing sugar of 658% when compared to Celluclastalone on hydrolysis of alkaline-pretreated bagasse. The work demonstrates the potential of lignocellulolytic enzymes from a novel uncultured microbial resource for enhancing efficiency of biomass-degrading enzyme systems for bio-industries.

Characterization of p-hydroxycinnamate catabolism in a soil Actinobacterium.

p-Hydroxycinnamates, such as ferulate and p-coumarate, are components of plant cell walls and have a number of commercial applications. Rhodococcus jostii RHA1 (RHA1) catabolizes ferulate via vanillate and the β-ketoadipate pathway. Here, we used transcriptomics to identify genes in RHA1 that are upregulated during growth on ferulate versus benzoate. The upregulated genes included three transcriptional units predicted to encode the uptake and β-oxidative deacetylation of p-hydroxycinnamates: couHTL, couNOM, and couR. Neither ΔcouL mutants nor ΔcouO mutants grew on p-hydroxycinnamates, but they did grow on vanillate. Among several p-hydroxycinnamates, CouL catalyzed the thioesterification of p-coumarate and caffeate most efficiently (k(cat)/K(m) = ∼ 400 mM(-1) s(-1)). p-Coumarate was also RHA1's preferred growth substrate, suggesting that CouL is a determinant of the pathway's specificity. CouL did not catalyze the activation of sinapate, in similarity to two p-coumaric acid:coenzyme A (CoA) ligases from plants, and contains the same bulged loop that helps determine substrate specificity in the plant homologues. The couO mutant accumulated 4-hydroxy-3-methoxyphenyl-β-ketopropionate in the culture supernatant when incubated with ferulate, supporting β-oxidative deacetylation. This phenotype was not complemented with a D257N variant of CouO, consistent with the predicted role of Asp257 as a metal ligand in this amidohydrolase superfamily member. These data suggest that CouO functionally replaces the β-ketothiolase and acyl-CoA thioesterase that occur in canonical β-oxidative pathways. Finally, the transcriptomics data suggest the involvement of two distinct formaldehyde detoxification pathways in vanillate catabolism and identify a eugenol catabolic pathway. The results of this study augment our understanding of the bacterial catabolism of aromatics from renewable feedstocks.

Extensive sampling of basidiomycete genomes demonstrates inadequacy of the white-rot/brown-rot paradigm for wood decay fungi.

Basidiomycota (basidiomycetes) make up 32% of the described fungi and include most wood-decaying species, as well as pathogens and mutualistic symbionts. Wood-decaying basidiomycetes have typically been classified as either white rot or brown rot, based on the ability (in white rot only) to degrade lignin along with cellulose and hemicellulose. Prior genomic comparisons suggested that the two decay modes can be distinguished based on the presence or absence of ligninolytic class II peroxidases (PODs), as well as the abundance of enzymes acting directly on crystalline cellulose (reduced in brown rot). To assess the generality of the white-rot/brown-rot classification paradigm, we compared the genomes of 33 basidiomycetes, including four newly sequenced wood decayers, and performed phylogenetically informed principal-components analysis (PCA) of a broad range of gene families encoding plant biomass-degrading enzymes. The newly sequenced Botryobasidium botryosum and Jaapia argillacea genomes lack PODs but possess diverse enzymes acting on crystalline cellulose, and they group close to the model white-rot species Phanerochaete chrysosporium in the PCA. Furthermore, laboratory assays showed that both B. botryosum and J. argillacea can degrade all polymeric components of woody plant cell walls, a characteristic of white rot. We also found expansions in reducing polyketide synthase genes specific to the brown-rot fungi. Our results suggest a continuum rather than a dichotomy between the white-rot and brown-rot modes of wood decay. A more nuanced categorization of rot types is needed, based on an improved understanding of the genomics and biochemistry of wood decay.

A comprehensive study on relating cell-wall components of lignocellulosic biomass to oxygenated species formed during pyrolysis

This study was conducted to fundamentally understand the relationship between biomass composition and key pyrolysis oxygenates that affect the quality of bio-oil. Unlike many studies where surrogates are used to probe the effect of biomass characteristics on pyrolysis; in this investigation, we used well-characterized biomass feedstocks with different natural varying levels of plant cell-wall components. With this approach, possible interaction effects of the biomass components which are not revealed in model studies are captured. Selected oxygenates from the pyrolysis of debarked sugar maple, poplar, willow, switchgrass, and hot-water extracted sugar maple were correlated with their composition (namely, glucan, xylan, arabinan, mannan, acetyl, lignin, extractives and ash). The pyrolysis was conducted at 550°C using a bench scale pyroprobe-GC/MS and Pearson correlation coefficients were used to statistically analyze the data. Quantitatively, relationships between the biomass components and oxygenated species such as acetic acid, levoglucosan, hydroxyacetone, methyl pyruvate, furfural, 2-furanone, 5-hydroxymethyl furfural, 4-allyl-2,6-dimethoxyphenol, coniferyl alcohol, and 2-methoxy-4-vinylphenol were established. Statistically significant correlation between the acetyl content and acetic acid yield indicate that deacetylation of the lignocellulosic cell wall could help in reducing the acidity of bio-oils. Likewise, other strong correlations suggest that carbonyls could be reduced by lowering the hemicellulose fraction. The data also indicated possible interaction between lignin and polysaccharides on the formation of specific oxygenates. For example, it was found that feedstocks with high lignin content tend to produce high yields of hydroxyacetaldehyde. Similarly, feedstocks with high glucan fraction yield more phenolic aldehydes and ketones. Overall, it can be inferred from the results that both genomic science and pretreatment could be used as tools to modify the heterogeneity of lignocellulosics to reduce the complexity of biomass pyrolysis and improve prospects for producing better quality bio-oils.

Connecting lignin-degradation pathway with pre-treatment inhibitor sensitivity of Cupriavidus necator.

To produce lignocellulosic biofuels economically, the complete release of monomers from the plant cell wall components, cellulose, hemicellulose, and lignin, through pre-treatment and hydrolysis (both enzymatic and chemical), and the efficient utilization of these monomers as carbon sources, is crucial. In addition, the identification and development of robust microbial biofuel production strains that can tolerate the toxic compounds generated during pre-treatment and hydrolysis is also essential. In this work, Cupriavidus necator was selected due to its capabilities for utilizing lignin monomers and producing polyhydroxylbutyrate (PHB), a bioplastic as well as an advanced biofuel intermediate. We characterized the growth kinetics of C. necator in pre-treated corn stover slurry as well as individually in the pre-sence of 11 potentially toxic compounds in the saccharified slurry. We found that C. necator was sensitive to the saccharified slurry produced from dilute acid pre-treated corn stover. Five out of 11 compounds within the slurry were characterized as toxic to C. necator, namely ammonium acetate, furfural, hydroxymethylfurfural (HMF), benzoic acid, and p-coumaric acid. Aldehydes (e.g., furfural and HMF) were more toxic than the acetate and the lignin degradation products benzoic acid and p-coumaric acid; furfural was identified as the most toxic compound. Although toxic to C. necator at high concentration, ammonium acetate, benzoic acid, and p-coumaric acid could be utilized by C. necator with a stimulating effect on C. necator growth. Consequently, the lignin degradation pathway of C. necator was reconstructed based on genomic information and literature. The efficient conversion of intermediate catechol to downstream products of cis,cis-muconate or 2-hydroxymuconate-6-semialdehyde may help improve the robustness of C. necator to benzoic acid and p-coumaric acid as well as improve PHB productivity.

Heterologous expression of a Penicillium purpurogenum pectin lyase in Pichia pastoris and its characterization

Lignocellulose is the major component of plant cell walls and it represents a great source of renewable organic matter. One of lignocellulose constituents is pectin. Pectin is composed of two basic structures: a ‘smooth’ region and a ‘hairy’ region. The ‘smooth’ region (homogalacturonan) is a linear polymer of galacturonic acid residues with α-(1→4) linkages, substituted by methyl and acetyl residues. The ‘hairy’ region is more complex, containing xylogalacturonan and rhamnogalacturonans I and II. Among the enzymes which degrade pectin (pectinases) is pectin lyase (E.C. 4.2.2.10). This enzyme acts on highly esterified homogalacturonan, catalysing the cleavage of α-(1→4) glycosidic bonds between methoxylated residues of galacturonic acid by means of β-elimination, with the formation of 4,5-unsaturated products. In this work, the gene and cDNA of a pectin lyase from Penicillium purpurogenum have been sequenced, and the cDNA has been expressed in Pichia pastoris. The gene is 1334 pb long, has three introns and codes for a protein of 376 amino acid residues. The recombinant enzyme was purified to homogeneity and characterized. Pectin lyase has a molecular mass of 45 kDa as determined by SDS-PAGE. It is active on highly esterified pectin, and decreases 40 % the viscosity of pectin with a degree of esterification ≥85 %. The enzyme showed no activity on polygalacturonic acid and pectin from citrus fruit 8 % esterified. The optimum pH and temperature for the recombinant enzyme are 6.0 and 50 °C, respectively, and it is stable up to 50 °C when exposed for 3 h. A purified pectin lyase may be useful in biotechnological applications such as the food industry where the liberation of toxic methanol in pectin degradation should be avoided.

Pectin Metabolism and Assembly in the Cell Wall of the Charophyte Green Alga Penium margaritaceum

The pectin polymer homogalacturonan (HG) is a major component of land plant cell walls and is especially abundant in the middle lamella. Current models suggest that HG is deposited into the wall as a highly methylesterified polymer, demethylesterified by pectin methylesterase enzymes and cross-linked by calcium ions to form a gel. However, this idea is based largely on indirect evidence and in vitro studies. We took advantage of the wall architecture of the unicellular alga Penium margaritaceum, which forms an elaborate calcium cross-linked HG-rich lattice on its cell surface, to test this model and other aspects of pectin dynamics. Studies of live cells and microscopic imaging of wall domains confirmed that the degree of methylesterification and sufficient levels of calcium are critical for lattice formation in vivo. Pectinase treatments of live cells and immunological studies suggested the presence of another class of pectin polymer, rhamnogalacturonan I, and indicated its colocalization and structural association with HG. Carbohydrate microarray analysis of the walls of P. margaritaceum, Physcomitrella patens, and Arabidopsis (Arabidopsis thaliana) further suggested the conservation of pectin organization and interpolymer associations in the walls of green plants. The individual constituent HG polymers also have a similar size and branched structure to those of embryophytes. The HG-rich lattice of P. margaritaceum, a member of the charophyte green algae, the immediate ancestors of land plants, was shown to be important for cell adhesion. Therefore, the calcium-HG gel at the cell surface may represent an early evolutionary innovation that paved the way for an adhesive middle lamella in multicellular land plants.

Structural characterization of arabinoxylans from two African plant species Eragrostis nindensis and Eragrostis tef using various mass spectrometric methods

The arabinoxylans are one of the main components of plant cell walls and are known to play major roles in plant tissues properties depending in particular on their structural features. It has been recently shown that one of the strategies developed by resurrection plants to overcome dehydration is based on cell wall composition. For this purpose, the structural characterization of arabinoxylans from desiccation-tolerant grass Eragrostis nindensis (E. nindensis) was compared with its close relative, the desiccation-sensitive Eragrostis tef (E. tef) in order to further understand mechansism of desiccation tolerance in resurrection plants. Ion mobility spectrometry coupled to mass spectrometry (IM-MS) in combination with the conventional mass spectrometric approaches, including matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), electrospray ionization multistage tandem mass spectrometry (ESI-MS(n)) and gas chromatography/mass spectrometry (GC/MS), were used to characterize arabinoxylan fragments obtained after endo-xylanase digestion of leave extracts from E. nindensis and E. tef. Whole fingerprinting by MALDI-MS analysis showed the presence of various arabinoxylan fragments within leaves of E. nindensis and E. tef. The monosaccharide composition and some linkage information were determined by GC/MS experiments. Information regarding the branching and sequence details was obtained by ESI-MS(n) experiments after sample permethylation. The presence of structural isomeric ions with different collision cross sections was evidenced by IM-MS which could be differentiated using ESI-MS(n). We have shown that an orthogonal approach, and especially IM-MS associated to ESI-MS(n) (n = 2 to 4) and GC/MS allowed characterization of arabinoxylan fragments of E. nindensis and E. tef and revealed the presence of isomeric structures. The same arabinoxylan structures were identified for both species but in different relative abundance. Moreover, this work illustrated that IM-MS can efficiently separate isomeric structures and advantageously complements the conventional mass spectrometric methodologies used for arabinoxylan structural characterization.

Bacteria, phages and pigs: the effects of in-feed antibiotics on the microbiome at different gut locations

Disturbance of the beneficial gut microbial community is a potential collateral effect of antibiotics, which have many uses in animal agriculture (disease treatment or prevention and feed efficiency improvement). Understanding antibiotic effects on bacterial communities at different intestinal locations is essential to realize the full benefits and consequences of in-feed antibiotics. In this study, we defined the lumenal and mucosal bacterial communities from the small intestine (ileum) and large intestine (cecum and colon) plus feces, and characterized the effects of in-feed antibiotics (chlortetracycline, sulfamethazine and penicillin (ASP250)) on these communities. 16S rRNA gene sequence and metagenomic analyses of bacterial membership and functions revealed dramatic differences between small and large intestinal locations, including enrichment of Firmicutes and phage-encoding genes in the ileum. The large intestinal microbiota encoded numerous genes to degrade plant cell wall components, and these genes were lacking in the ileum. The mucosa-associated ileal microbiota harbored greater bacterial diversity than the lumen but similar membership to the mucosa of the large intestine, suggesting that most gut microbes can associate with the mucosa and might serve as an inoculum for the lumen. The collateral effects on the microbiota of antibiotic-fed animals caused divergence from that of control animals, with notable changes being increases in Escherichia coli populations in the ileum, Lachnobacterium spp. in all gut locations, and resistance genes to antibiotics not administered. Characterizing the differential metabolic capacities and response to perturbation at distinct intestinal locations will inform strategies to improve gut health and food safety.

β-D-glucan inhibits endocrine-resistant breast cancer cell proliferation and alters gene expression

Endocrine therapies have been successfully used for breast cancer patients with estrogen receptor α (ERα) positive tumors, but ∼40% of patients relapse due to endocrine resistance. β-glucans are components of plant cell walls that have immunomodulatory and anticancer activity. The objective of this study was to examine the activity of β-D-glucan, purified from barley, in endocrine-sensitive MCF-7 versus endocrine-resistant LCC9 and LY2 breast cancer cells. β-D-glucan dissolved in DMSO but not water inhibited MCF-7 cell proliferation in a concentration-dependent manner as measured by BrdU incorporation with an IC50 of ∼164±12 μg/ml. β-D-glucan dissolved in DMSO inhibited tamoxifen/endocrine-resistant LCC9 and LY2 cell proliferation with IC50 values of 4.6±0.3 and 24.2±1.4 μg/ml, respectively. MCF-10A normal breast epithelial cells showed a higher IC50 ∼464 μg/ml and the proliferation of MDA-MB-231 triple negative breast cancer cells was not inhibited by β-D-glucan. Concentration-dependent increases in the BAX/BCL2 ratio and cell death with β-D-glucan were observed in MCF-7 and LCC9 cells. PCR array analysis revealed changes in gene expression in response to 24-h treatment with 10 or 50 μg/ml β-D-glucan that were different between MCF-7 and LCC9 cells as well as differences in basal gene expression between the two cell lines. Select results were confirmed by quantitative real-time PCR demonstrating that β-D-glucan increased RASSF1 expression in MCF-7 cells and IGFBP3, CTNNB1 and ERβ transcript expression in LCC9 cells. Our data indicate that β-D-glucan regulates breast cancer-relevant gene expression and may be useful for inhibiting endocrine-resistant breast cancer cell proliferation.

Measurement of rumen dry matter and neutral detergent fiber degradability of feeds by Fourier-transform infrared spectroscopy

This study explored the potential of partial least squares (PLS) and Fourier-transform infrared spectroscopy (FTIR) to predict rumen dry matter (DM) and neutral detergent fiber (NDF) degradation parameters of a wide range of feeds for ruminants, as an alternative to the in situ method. In total, 663 samples comprising 80 different feed types were analyzed. In situ DM and NDF degradabilities were determined as follows: effective degradability (ED), rumen soluble fraction (A), degradable but not soluble fraction (B), rate of degradation of the B fraction (C), and indigestible NDF (iNDF). Infrared spectra of dry samples were collected by attenuated total reflectance from 600 to 4000cm−1. Feeds were randomly classified into 2 subsets of samples with representation of all feed types; one subset was used to develop regression models using partial least squares, and the second subset was used to conduct an external validation of the models. This study indicated that universal models containing all feed types and specific models containing concentrate feeds could provide only a relatively poor estimation of in situ DM degradation parameters because of compositional heterogeneity. More research, such as a particle size distribution analysis, is required to determine whether this lack of accuracy was due to limitations of the FTIR approach, or simply due to methodological error associated with the in situ method. This latter hypothesis may explain the low accuracy observed in the prediction of degradation rates if there was physical leakage of fine particles from the mesh bags used during in situ studies. In contrast, much better predictions were obtained when models were developed for forage feeds alone. Models for forages led to accurate predictions of DMA, DMB, NDFED, and NDF concentration (R2=0.91, 0.89, 0.85, and 0.79, standard error = 4.34, 5.97, 4.59, and 4.41% of DM, respectively), and could be used for screening of DMED, NDFC, and iNDF. These models relied on certain regions of the FTIR spectrum (900–1150 and 1500–1700cm−1), which are mainly compatible with absorption of plant cell wall components, such as cellulose, pectin, lignin, cutin, and suberin, but also with nonstructural carbohydrates and certain active compounds. In conclusion, FTIR spectroscopy could be considered a low-cost alternative to in situ measurements in feed evaluation.