Susan M. Carnachan

Structural characterisation and rheological properties of a polysaccharide from sesame leaves (Sesamum radiatum Schumach. & Thonn.)

A polysaccharide from the leaves of Sesamum radiatum was extracted by maceration in deionized water followed by ethanol precipitation then chemically and physically characterised. Monosaccharide composition and linkages were determined by high performance anion exchange chromatography (HPAEC), gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy respectively. Sesamum gum was composed of glucuronic acid, mannose, galactose, and xylose with trace quantities of glucose, rhamnose and arabinose. Proton and (13)C NMR spectroscopy, and linkage analysis revealed a glucuronomannan based structure comprising a backbone of →4)-β-d-GlcpA-(1→2)-α-d-Manp-(1→ with side-chains of galactose and xylose. Hydrated sesamum gum displayed temperature independent viscoelastic properties with no thermal hysteresis. Intrinsic viscosity was determined to be 3.31 and 4.40dLg(-1) in 0.1M NaCl and deionised water respectively, while the critical concentration was determined to be 0.1% w/v. The characterisation performed in this study will help direct potential applications of this material in foods and pharmaceuticals.

Affinity Selection of FGF2‐Binding Heparan Sulfates for Ex Vivo Expansion of Human Mesenchymal Stem Cells

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.

Differential growth of bowel commensal Bacteroides species on plant xylans of differing structural complexity

Alterations to the composition of the bowel microbiota (dysbioses) are associated with particular diseases and conditions of humans. There is a need to discover new, indigestible polysaccharides which are selective growth substrates for commensal bowel bacteria. These substrates (prebiotics) could be added to food in intervention studies to correct bowel dysbiosis. A collection of commensal bacteria was screened for growth in culture using a highly-branched xylan produced by New Zealand flax. Two, Bacteroides ovatus ATCC 8483 and Bacteroides xylanisolvens DSM 18836 grew well on this substrate. The utilisation of the xylan was studied chromatographically and by constituent sugar analysis. The two closely related species utilised the xylan in different ways, and differently from their use of wheat arabinoxylan. The growth of Bacteroides species on other plant xylans having differing chemical structures was also investigated. Novel xylans expand the choice of potential prebiotics that could be used to correct bowel dysbioses.

Methylation analysis of polysaccharides: Technical Advice

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.

Structural and rheological studies of a polysaccharide mucilage from lacebark leaves (Hoheria populnea A. Cunn.)

A water-soluble mucilage extracted from the leaves of Hoheria populnea was chemically and physically studied. Monosaccharide composition and linkages were determined by high performance anion exchange chromatography (HPAEC), gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Lacebark mucilage was composed of rhamnose, galactose, galacturonic acid and glucuronic acid (2:1:2:1). Proton and 13C NMR spectroscopy, and linkage analysis, revealed a predominantly rhamnogalacturonan I-type (RG I-type) structure comprising of a backbone of →4]-α-D-GalpA-[1→2]-α-L-Rhap-[1→. Data indicated the mucilage likely comprises of a polymer containing several structurally discrete domains or possibly more than one discrete polymer. One domain contains a RG I-type backbone with branching at O-3 of GalpA residues to terminal β-D-GlcpA residues, another similarly contains a RG I-type backbone but is branched at O-4 of the Rhap residues to terminal GalpA residues or oligosaccharides containing α-linked 4-Galp and terminal GalpA residues. A possible third domain contains contiguous 2-Rhap residues, some branched at O-3. Hydrated mucilage exhibited pseudoplastic flow behaviour and viscoelastic properties of an entangled biopolymer network. These rheological behaviours were only slightly affected by pH and may prove advantageous in potential end-product applications including oral pharmaceuticals or as a food ingredient.

Poly Ethoxy Ethyl Glycinamide (PEE‐G) Dendrimers: Dendrimers Specifically Designed for Pharmaceutical Applications

Poly ethoxy ethyl glycinamide (PEE‐G) dendrimers have been specifically designed and synthesized with the aim of providing a readily available dendrimer scaffold that can be used to make products that can meet the stringent requirements of pharmaceutical applications. The synthesis has been refined to produce dendrimers that are of high HPLC purity. The suitability of PEE‐G dendrimers for their designed use has been verified by subsequent measurements to demonstrate that they are of high stability, high aqueous solubility, low cytotoxicity, low immunogenicity and with low in vivo toxicity in an escalating‐dose rat study. PEE‐G dendrimers therefore provide a useful scaffold for researchers wanting to develop dendrimer‐based drug candidates.

tuf Gene Sequence Variation in Bifidobacterium longum subsp. infantis Detected in the Fecal Microbiota of Chinese Infants

ABSTRACT Members of the bacterial genus Bifidobacterium generally dominate the fecal microbiota of infants. The species Bifidobacterium longum is prevalent, but the B. longum subsp. longum and B. longum subsp. infantis strains that are known to colonize the infant bowel are not usually differentiated in microbiota investigations. These subspecies differ in their capacities to metabolize human milk oligosaccharides (HMO) and may have different ecological and symbiotic roles in humans. Quantitative PCR provides a quick analytical method by which to accurately ascertain the abundances of target species in microbiotas and microcosms. However, amplification targets in DNA extracted from samples need to be dependably differential. We evaluated the tuf gene sequence as a molecular target for quantitative PCR measurements of the abundances of B. longum subsp. infantis and B. longum subsp. longum in fecal microbiotas. This approach resulted in the detection of a tuf gene variant (operational taxonomic unit 49 [OTU49]) in Chinese infants that has sequence similarities to both B. longum subsp. infantis and B. longum subsp. longum. We compared the genome sequence and growth and transcriptional characteristics of an OTU49 isolate cultured in HMO medium to those of other B. longum subsp. infantis cultures. We concluded from these studies that OTU49 belongs to B. longum subsp. infantis, that dependable quantitative PCR (qPCR) differentiation between the B. longum subspecies cannot be achieved by targeting tuf gene sequences, and that functional genes involved in carbohydrate metabolism might be better targets because they delineate ecological functions. IMPORTANCE High-throughput DNA sequencing methods and advanced bioinformatics analysis have revealed the composition and biochemical capacities of microbial communities (microbiota and microbiome), including those that inhabit the gut of human infants. However, the microbiology and function of natural ecosystems have received little attention in recent decades, so an appreciation of the dynamics of gut microbiota interactions is lacking. With respect to infants, rapid methodologies, such as quantitative PCR, are needed to determine the prevalences and proportions of different bifidobacterial species in observational and microcosm studies in order to obtain a better understanding of the dynamics of bifidobacterial nutrition and syntrophy, knowledge that might be used to manipulate the microbiota and perhaps ensure the better health of infants.