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The invasive centric diatom Lindavia intermedia arrived in New Zealand in recent decades. It produces a pelagic mucilage, which forms an adhesive substance known as lake snow. While determining cell concentrations of L. intermedia is relatively straightforward, methods to estimate mucilage concentration in lake water have not yet been developed. Two methods for lake snow quantification are reported here: a dragged line, or “snow tow,” and in situ filtration. A chemical assay for hexosamine, as an estimate of chitin content, and an atomic absorption method, which measures silicon, were used to measure these components of lake snow. Samples from 4 lakes revealed a correlation between snow tow and filtration data; in principle, the snow tow method (used by municipal authorities) could be calibrated and made quantifiable using the filtration method. Expression of the chitin synthase gene in summer/autumn (but not in winter/spring) correlated with abundance of the chitin component of lake snow, aligning with anecdotal evidence for the timing of its development in lakes. By contrast, cell densities correlated poorly with lake snow abundance. Cell concentrations relative to chitin concentrations were also higher in winter/spring than in summer/autumn across lakes. Dry weight and silicate in lake snow relative to chitin varied by lake and season. The quantification of lake snow and its adhesiveness are important parameters for assessing its nuisance value and fouling characteristics and for developing measures to prevent and remediate its incursion in lakes.

Pectic polysaccharides from New Zealand (NZ) spinach (Tetragonia tetragonioides) and karaka berries (Corynocarpus laevigatus) were extracted and analyzed. NZ spinach polysaccharides comprised mostl...

International audience; Pectin is abundant in modern day diets, as it comprises the middle lamellae and one-third of the dry carbohydrate weight of fruit and vegetable cell walls. Currently there is no specialized model organism for studying pectin fermentation in the human colon, as our collective understanding is informed by versatile glycan-degrading bacteria rather than by specialist pectin degraders. Here we show that the genome of Monoglobus pectinilyticus possesses a highly specialized glycobiome for pectin degradation, unique amongst Firmicutes known to be in the human gut. Its genome encodes a simple set of metabolic pathways relevant to pectin sugar utilization, and its predicted glycobiome comprises an unusual distribution of carbohydrate-active enzymes (CAZymes) with numerous extracellular methyl/acetyl esterases and pectate lyases. We predict the M. pectinilyticus degradative process is facilitated by cell-surface S-layer homology (SLH) domain-containing proteins, which proteomics analysis shows are differentially expressed in response to pectin. Some of these abundant cell surface proteins of M. pectinilyticus share unique modular organizations rarely observed in human gut bacteria, featuring pectin-specific CAZyme domains and the cell wall-anchoring SLH motifs. We observed M. pectinilyticus degrades various pectins, RG-I, and galactan to produce polysaccharide degradation products (PDPs) which are presumably shared with other inhabitants of the human gut microbiome (HGM). This strain occupies a new ecological niche for a primary degrader specialized in foraging a habitually consumed plant glycan, thereby enriching our understanding of the diverse community profile of the HGM.

Marine pelagic mucilages (e.g., marine snow) have been reported to a greater extent than their lacustrine counterparts. A pelagic mucilage primarily comprised of chitin secretions from the invasive centric diatom, Lindavia intermedia, has been reported since the early 2000s, primarily from large, oligotrophic pre-alpine lakes of the South Island of New Zealand. To better understand the factors related to mucilage abundance, we monitored its abundance as well as factors potentially related to mucilage production over time in four mucilage-afflicted lakes. Temporal mucilage dynamics were episodic, with peaks in abundance occurring during any season, but most often during summer and autumn. Chitin was confirmed to be an important component of the mucilage, but the chitin content varied between 1 and 12% of the mucilage dry mass in the lakes. An RT-qPCR assay for chitin synthase gene overexpression in L. intermedia showed that overexpression occurred in summer and autumn, often when peaks in mucilage abundance also occurred. A correlation between mucilage and phytoplankton abundance was only observed in one of the lakes. Both dissolved reactive and total phosphorus concentrations were often below analytical detection limits in these lakes. Nitrate concentrations were also low and showed negative correlations with mucilage abundance. This suggests either that the secrection of chitinous mucilage by L. intermedia significantly depleted the available N in the water column or that mucilage facilitated N uptake by L. intermedia and/or other microorganisms associated with the mucilage. Pelagic mucilage in New Zealand lakes shares many characteristics of other conspicuous mucilage phenomena, including lake snow and marine snow. While our correlational analyses revealed some relationships and associations with mucilage abundance, the strengths of these were quite variable, indicating that as yet unstudied mucilage loss processes in these lakes (e.g., sedimentation, disintegration, decomposition, assimilation) likely also play important roles in regulating mucilage abundance.

Whole-transcriptome analysis was used to investigate the molecular interplay between three bacterial species that are members of the human gut microbiota. Bacteroides ovatus, Subdoligranulum variabile, and Hungatella hathewayi formed associations in cocultures fed barley β-glucan, a constituent of dietary fiber. B. ovatus depolymerized β-glucan and released, but did not utilize, 3-O-β-cellobiosyl-d-glucose (DP3) and 3-O-β-cellotriosyl-d-glucose (DP4). These oligosaccharides provided growth substrates for S. variabile and H. hathewayi with a preference for DP4 in the case of the latter species. There was increased transcription of a B. ovatus mixed-linkage-β-glucan utilization locus, as well as carbohydrate transporters in S. variabile and H. hathewayi when in batch coculture. Increased transcription of the β-glucan utilization locus did not occur in continuous culture. Evidence for interactions relating to provision of cobalamin, alterations to signaling, and modulation of the “stringent response” (an adaptation to nutrient deprivation) were detected. Overall, we established a bacterial consortium based on barley β-glucan in vitro, which can be used to investigate aspects of the functional blueprint of the human gut microbiota. IMPORTANCE The microbial community, mostly composed of bacterial species, residing in the human gut degrades and ferments polysaccharides derived from plants (dietary fiber) that would not otherwise be digested. In this way, the collective metabolic actions of community members extract additional energy from the human diet. While the variety of bacteria present in the microbial community is well known, the formation of bacterial consortia, and the consequent interactions that result in the digestion of dietary polysaccharides, has not been studied extensively. The importance of our work was the establishment, under laboratory conditions, of a consortium of gut bacteria that formed around a dietary constituent commonly present in cereals. This enabled the metabolic interplay between the bacterial species to be studied. This kind of knowledge is required to construct an interactive, metabolic blueprint of the microbial community that inhabits the human gut.

A knowledge of the mobilities of the polysaccharides or parts of polysaccharides in a cell-wall preparation provides information about possible molecular interactions among the polysaccharides in the cell wall and the relative locations of polysaccharides within the cell wall. A number of solid-state

B. ovatus is a member of the human gut microbiota with a broad capability to degrade complex glycans. Here we show that B. ovatus degrades plant polysaccharides in a preferential order, and that glycan structural complexity plays a role in determining the prioritisation of polysaccharide usage.

Polysaccharides from feijoa fruit were extracted and analyzed; the composition of these polysaccharides conforms to those typically found in the primary cell walls of eudicotyledons. The two major polysaccharide extracts consisted of mainly pectic polysaccharides and hemicellulosic polysaccharides [xyloglucan (77%) and arabinoxylan (16%)]. A collection of commensal Bacteroides species was screened for growth in culture using these polysaccharide preparations and placed into five categories based on their preference for each substrate. Most of the species tested could utilize the pectic polysaccharides, but growth on the hemicellulose was more limited. Constituent sugar and glycosyl linkage analysis showed that species that grew on the hemicellulose fraction showed differences in their preference for the two polysaccharides in this preparation. Our data demonstrate that the members of the genus Bacteroides show differential hydrolysis of pectic polysaccharides, xyloglucan, and arabinoxylan, which might influence the structure and metabolic activities of the microbiota in the human gut.

Dietary fiber provides growth substrates for bacterial species that belong to the colonic microbiota of humans. The microbiota degrades and ferments substrates, producing characteristic short-chain fatty acid profiles. Dietary fiber contains plant cell wall-associated polysaccharides (hemicelluloses and pectins) that are chemically diverse in composition and structure. Thus, depending on plant sources, dietary fiber daily presents the microbiota with mixtures of plant polysaccharides of various types and complexity. We studied the extent and preferential order in which mixtures of plant polysaccharides (arabinoxylan, xyloglucan, β-glucan, and pectin) were utilized by a coculture of five bacterial species (Bacteroides ovatus, Bifidobacterium longum subspecies longum, Megasphaera elsdenii, Ruminococcus gnavus, and Veillonella parvula). These species are members of the human gut microbiota and have the biochemical capacity, collectively, to degrade and ferment the polysaccharides and produce short-chain fatty acids (SCFAs). B. ovatus utilized glycans in the order β-glucan, pectin, xyloglucan, and arabinoxylan, whereas B. longum subsp. longum utilization was in the order arabinoxylan, arabinan, pectin, and β-glucan. Propionate, as a proportion of total SCFAs, was augmented when polysaccharide mixtures contained galactan, resulting in greater succinate production by B. ovatus and conversion of succinate to propionate by V. parvula. Overall, we derived a synthetic ecological community that carries out SCFA production by the common pathways used by bacterial species for this purpose. Systems like this might be used to predict changes to the emergent properties of the gut ecosystem when diet is altered, with the aim of beneficially affecting human physiology. IMPORTANCE This study addresses the question as to how bacterial species, characteristic of the human gut microbiota, collectively utilize mixtures of plant polysaccharides such as are found in dietary fiber. Five bacterial species with the capacity to degrade polymers and/or produce acidic fermentation products detectable in human feces were used in the experiments. The bacteria showed preferential use of certain polysaccharides over others for growth, and this influenced their fermentation output qualitatively. These kinds of studies are essential in developing concepts of how the gut microbial community shares habitat resources, directly and indirectly, when presented with mixtures of polysaccharides that are found in human diets. The concepts are required in planning dietary interventions that might correct imbalances in the functioning of the human microbiota so as to support measures to reduce metabolic conditions such as obesity.

Pectic polysaccharides from New Zealand (NZ) spinach (

The depolymerisation of porcine mucosal heparan sulfate under the action of heparin lyases and analysis by size-exclusion chromatography (SEC) is described. Heparan sulfate treated to enzymic bond scission producing a Δ 4,5 double-bond and quantified by SEC with ultraviolet-visible (UV) spectroscopic detection (230 nm) indicated that the majority of the biopolymer (>85%) was reduced to disaccharides (degree of polymerisation (DP) = 2). However, analysis of the SEC eluant using refractive index (RI), which reflects the mass contribution of the oligosaccharides rather than the molar response of a UV chromophore, indicated that a considerable proportion of the digested HS, up to 43%, was present with DP >2. This was supported by a mass balance analysis. These results contradict the accepted literature where “complete digestion” is routinely reported. Herein we report on the composition and methodology utilised to ascertain the extent of depolymerization and disaccharide composition of this important biopolymer.

The future of human mesenchymal stem cells (hMSCs) as a successful cell therapy relies on bioprocessing strategies to improve the scalability of these cells without compromising their therapeutic ability. The culture-expansion of hMSCs can be enhanced by supplementation with growth factors, particularly fibroblast growth factor 2 (FGF2). The biological activity of FGF2 is controlled through interactions with heparan sulfate (HS) that facilitates ligand-receptor complex formation. We previously reported on an FGF2-interacting HS variant (termed HS2) isolated from embryonic tissue by anionic exchange chromatography that increased the proliferation and potency of hMSCs. Here, we detail the isolation of an FGF2 affinity-purified HS variant (HS8) using a scalable platform technology previously employed to generate HS variants with increased affinity for BMP-2 or VEGF165 . This process used a peptide sequence derived from the heparin-binding domain of FGF2 as a substrate to affinity-isolate HS8 from a commercially available source of porcine mucosal HS. Our data show that HS8 binds to FGF2 with higher affinity than to FGF1, FGF7, BMP2, PDGF-BB, or VEGF165 . Also, HS8 protects FGF2 from thermal destabilization and increases FGF signaling and hMSC proliferation through FGF receptor 1. Long-term supplementation of cultures with HS8 increased both hMSC numbers and their colony-forming efficiency without adversely affecting the expression of hMSC-related cell surface antigens. This strategy further exemplifies the utility of affinity-purifying HS variants against particular ligands important to the stem cell microenvironment and advocates for their addition as adjuvants for the culture-expansion of hMSCs destined for cellular therapy. J. Cell. Physiol. 232: 566-575, 2017. © 2016 Wiley Periodicals, Inc.

Herpes simplex virus type 1 (HSV1) induces the formation of the characteristic abnormal molecules of Alzheimer's disease (AD) brains, beta-amyloid, and abnormally phosphorylated, AD-like tau (P-tau). Formation of these molecules is inhibited by treatment with the antiviral agent acyclovir (ACV), which prevents viral DNA replication. A totally different mechanism of antiviral action against herpes simplex viruses is shown by sulfated fucans. The antiviral activity of sulfated fucans from five brown algae (Scytothamnus australis, Marginariella boryana, Papenfussiella lutea, Splachnidium rugosum and Undaria pinnatifida) was investigated in relation to the HSV1-induced formation of beta-amyloid, and AD-like tau. Antiviral activity was also related to specific structural features of these polysaccharides. Four sulfated fucan extracts each prevented the accumulation of HSV1-induced beta-amyloid and AD-like tau in HSV1-infected Vero cells. The structures of these extracts had some similarities but also key differences, indicating that a number of structural features can cause antiviral activity. The most active sulfated fucan combined with acyclovir was particularly effective, so may be particularly suitable for further experimental testing in order to develop treatment protocols for AD patients, with the aim of slowing or stopping disease progression.

A novel anaerobic pectinolytic bacterium (strain 14T) was isolated from human faeces. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 14T belonged to the family Ruminococcaceae , but was located separately from known clostridial clusters within the taxon. The closest cultured relative of strain 14T was Acetivibrio cellulolyticus (89.7 % sequence similarity). Strain 14T shared ~99 % sequence similarity with cloned 16S rRNA gene sequences from uncultured bacteria derived from the human gut. Cells were Gram-stain-positive, non-motile cocci approximately 0.6 µm in diameter. Strain 14T fermented pectins from citrus peel, apple, and kiwifruit as well as carbohydrates that are constituents of pectins and hemicellulose, such as galacturonic acid, xylose, and arabinose. TEM images of strain 14T, cultured in association with plant tissues, suggested extracellular fibrolytic activity associated with the bacterial cells, forming zones of degradation in the pectin-rich regions of middle lamella. Phylogenetic and phenotypic analysis supported the differentiation of strain 14T as a novel genus in the family Ruminococcaceae . The name Monoglobus pectinilyticus gen. nov., sp. nov. is proposed; the type strain is 14T (JCM 31914T=DSM 104782T).

Water-soluble, non-starch polysaccharides from plants are used commercially in a wide range of food and non-food applications. The increasing range of applications for natural polysaccharides means that there is growing demand for plant-derived polysaccharides with different functionalities. The geographical isolation of New Zealand and its unique flora presents opportunities to discover new polysaccharides with novel properties for a range of applications. This review brings together data published since the year 2000 on the composition and structure of exudate gums, mucilages, and storage polysaccharides extracted from New Zealand endemic land plants. The structures and properties of these polysaccharides are compared with the structures of similar polysaccharides from other plants. The current commercial use of these polysaccharides is reviewed and their potential for further exploitation discussed.

Bone morphogenetic protein (BMP)-2 is a potent bone healing compound produced at sites of bone trauma. Here we present a therapeutic strategy to harness the activity of endogenously produced BMP-2 by delivery of an affinity-matched heparan sulfate (HS) glycos aminoglycan biomaterial that increases the bioavailability, bioactivity and half-life of this growth factor. We have developed a robust, cost effective, peptide-based affinity platform to isolate a unique BMP-2 binding HS variant from commercially available preparations of HS, so removing the manufacturing bottleneck for their translation into the clinic. This affinity-matched HS enhanced BMP-2-induced osteogenesis through improved BMP-2 kinetics and receptor modulation, prolonged pSMAD signaling and reduced interactions with its antagonist noggin. When co-delivered with a collagen implant, the HS was as potent as exogenous BMP-2 for the healing of critical-sized bone defects in rabbits. This affinity platform can be readily tuned to isolate HS variants targeted ata range of clinically-relevant growth and adhesive factors.

Glycosyl linkage (methylation) analysis is used widely for the structural determination of oligo- and poly-saccharides. The procedure involves derivatisation of the individual component sugars of a polysaccharide to partially methylated alditol acetates which are analysed and quantified by gas chromatography-mass spectrometry. The linkage positions for each component sugar can be determined by correctly identifying the partially methylated alditol acetates. Although the methods are well established, there are many technical aspects to this procedure and both careful attention to detail and considerable experience are required to achieve a successful methylation analysis and to correctly interpret the data generated. The aim of this article is to provide the technical details and critical procedural steps necessary for a successful methylation analysis and to assist researchers (a) with interpreting data correctly and (b) in providing the comprehensive data required for reviewers to fully assess the work.

The future of human mesenchymal stem cells (hMSCs) as a successful cell therapy relies on bioprocessing strategies to improve the scalability of these cells without compromising their therapeutic ability. The culture-expansion of hMSCs can be enhanced by supplementation with growth factors, particularly fibroblast growth factor 2 (FGF2). The biological activity of FGF2 is controlled through interactions with heparan sulfate (HS) that facilitates ligand-receptor complex formation. We previously reported on an FGF2-interacting HS variant (termed HS2) isolated from embryonic tissue by anionic exchange chromatography that increased the proliferation and potency of hMSCs. Here, we detail the isolation of an FGF2 affinity-purified HS variant (HS8) using a scalable platform technology previously employed to generate HS variants with increased affinity for BMP-2 or VEGF

Intestinal pathogens use the host's excessive inflammatory cytokine response, designed to eliminate dangerous bacteria, to disrupt epithelial gut wall integrity and promote their tissue invasion. We sought to develop a non-antibiotic-based approach to prevent this injury. Molecular docking studies suggested that glycosylated dendrimers block the TLR4-MD-2-LPS complex, and a 13.6 kDa polyamidoamine (PAMAM) dendrimer glucosamine (DG) reduced the induction of human monocyte interleukin (IL)-6 by Gram-negative bacteria. In a rabbit model of shigellosis, PAMAM-DG prevented epithelial gut wall damage and intestinal villous destruction, reduced local IL-6 and IL-8 expression, and minimized bacterial invasion. Computational modelling studies identified a 3.3 kDa polypropyletherimine (PETIM)-DG as the smallest likely bioactive molecule. In human monocytes, high purity PETIM-DG potently inhibited Shigella Lipid A-induced IL-6 expression. In rabbits, PETIM-DG prevented Shigella-induced epithelial gut wall damage, reduced local IL-6 and IL-8 expression, and minimized bacterial invasion. There was no change in β-defensin, IL-10, interferon-β, transforming growth factor-β, CD3 or FoxP3 expression. Small and orally delivered DG could be useful for preventing gut wall tissue damage in a wide spectrum of infectious diarrhoeal diseases. –>See accompanying article http://dx.doi.org/10.1002/emmm.201201668

The therapeutic use of VEGF165 to stimulate blood vessel formation for the treatment of peripheral arterial disease or cardiovascular-related disease has met with limited success. Here we describe an affinity-isolated heparan sulfate glycotherapeutic (HS7+ve) that binds to, and enhances the bioactivity of, VEGF165. Application of HS7+ve complexed with VEGF165 results in enhanced VEGF165–VEGFR2 interaction, prolonged downstream pErk1/2 signalling, and increased cell proliferation and tube formation in HUVECs, compared with VEGF165 alone. The pro-angiogenic potential of HS7+ve was further assessed in vivo using the chick embryo chorioallantoic membrane (CAM) assay. Exogenous dosing with HS7+ve alone significantly enhanced the formation of new blood vessels with potencies comparable to VEGF165. These results demonstrate the potential for vascular therapy of glycotherapeutic agents targeted at augmenting the bioactivity of VEGF165. ASTAR (Agency for Sci., Tech. and Research, S’pore)