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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. [ABSTRACT FROM AUTHOR]

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. • Bacteroides ovatus has a broad capability to degrade complex glycans. • B. ovatus degrades plant glycans in a preferential order. • Glycan structural complexity plays a role in determining prioritisation of glycan usage. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

• Garlic polysaccharides were predominantly pectins. • Pectins extracted from skin biomass were mainly homogalacturonan. • Pectins from leaf biomass comprised both HG and RG-I. Polysaccharides from garlic waste leaf and skin biomass have been isolated using a sequential extraction protocol and characterised using constituent sugar composition and linkage analysis, spectroscopy, chromatography and dilute solution viscometry. The results revealed that the isolated polysaccharides were predominantly pectins. The predominant monosaccharide in all samples was galacturonic acid (>61 %), followed by galactose and rhamnose. The pectins extracted from skin biomass were mainly homogalacturonan (83–91 %), whereas those extracted from leaf biomass comprised both homogalacturonan (62–65 %) and rhamnogalacturonan-I (35–38 %). The degree of methyl esterification of uronic acids in all samples was 44–56 %. The peak molecular weight of the main polysaccharide population in each sample was ∼ 350 x103 g/mol, with leaf extracts and the skin acidic extract containing a second, lower molecular weight peak. Overall, waste garlic biomass is a potential resource for commercial pectin extraction for use in food or pharmaceutical industries. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Thalli of the endemic epiphytic New Zealand red seaweed Pyrophyllon subtumens are known to contain a high level of xylose and a notable amount of arabinose but the extracted polysaccharide has not been characterised. The linkage/substitution of individual sugars within the water-soluble polysaccharide extract and various derivatives were determined by chemical and spectroscopic methods. No 3-linked sugars nor any d -galactose were found, which excluded agar-, carrageenan- or mixed 3-linked/4-linked β- d -xylan-type polysaccharides found in many other red macroalgae. Instead, the polysaccharide backbone contained predominantly 4-linked β- d -xylopyranosyl, 4-linked 3,6-anhydro- l -galactopyranosyl and 4-linked l -galactopyranosyl units. Some of each type of sugar were sulfated at various positions. Some xylosyl units were substituted at the 2- or 3-position with l -arabinosyl units. The polysaccharide is complex and likely contains a range of structures. However, partial sequencing was successfully used to recover and identify a novel disaccharide 4- O - d -xylopyranosyl-3,6-anhdydro- l -galactopyranose, which indicates a unique →4)-β- d -Xyl p -(1 → 4)-3,6-anhydro- l -Gal p -(1 → repeat unit in the polysaccharide. [Display omitted] [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]