Ulvans are not equal - Linkage and substitution patterns in ulvan polysaccharides differ with Ulva morphology.

Ulva are hardy green seaweeds that contain the sulfated polysaccharide ulvan and grow in two distinct morphologies: foliose and tubular. The authors hypothesise that ulvan from tubular species are more structurally complex than ulvans from foliose species. Herein, using standardised methods, the glycosyl linkage positions and sulfate ester substitutions of constituent monosaccharides of ulvan isolated from foliose (U. lacinulata and U. stenophylloides) and tubular (U. prolifera and U. ralfsii) species of Ulva were investigated. Comparison of native ulvans with 80 and 100 °C desulfated counterparts indicated that 4-linked rhamnose is predominantly 3-O-sulfated in all four ulvans. Ulvans from the foliose species predominantly contained →3,4)-Rhap-(1→, →4)-GlcAp-(1→ and →4)-IdoAp-(1→, collectively accounting for 67 to 81 mol% of the total linkages. In contrast, these same linkages in ulvans from the tubular species only collectively accounted for 29 to 36 mol%. Instead, ulvan from tubular species contained a combination of →2,3,4)-Rhap-(1→, terminal Rhap-(1→, →4)-GlcAp-(1→, →4)-Xylp-(1→, and/or →4)-Galp-(1→ in high proportions; some of the latter three residues were also likely O-2 sulfated. The results presented here suggest that ulvan from foliose species are predominantly unbranched polysaccharides composed of repeat disaccharides while ulvans from tubular species contain a greater diversity of branch and sulfate substitution locations.

Rational synthesis of a heparan sulfate saccharide that promotes the activity of BMP2.

Heparan sulfate (HS) is a glycosaminoglycan (GAG) found throughout nature and is involved in a wide range of functions including modulation of cell signalling via sequestration of growth factors. Current consensus is that the specificity of HS motifs for protein binding are individual for each protein. Given the structural complexity of HS the synthesis of libraries of these compounds to probe this is not trivial. Herein we present the synthesis of an HS decamer, the design of which was undertaken rationally from previously published data for HS binding to the growth factor BMP-2. The biological activity of this HS decamer was assessed in vitro, showing that it had the ability to both bind BMP-2 and increase its thermal stability as well as enhancing the bioactivity of BMP-2 in vitro in C2C12 cells. At the same time no undesired anticoagulant effect was observed. This decamer was then analysed in vivo in a rabbit model where higher bone formation, bone mineral density (BMD) and trabecular thickness were observed over an empty defect or collagen implant alone. This indicated that the HS decamer was effective in promoting bone regeneration in vivo.

Novel 4-O-ß-d-xylopyranosyl-3,6-anhydro-l-galactopyranosyl disaccharide units in a polysaccharide from the red alga Pyrophyllon subtumens.

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 ß-d-xylan-type polysaccharides found in many other red macroalgae. Instead, the polysaccharide backbone contained predominantly 4-linked ß-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)-ß-d-Xylp-(1 â†’ 4)-3,6-anhydro-l-Galp-(1 â†’ repeat unit in the polysaccharide.

The discovery of 12ß-methyl-17-epi-18-nor-bile acids as potent and selective TGR5 agonists.

Recent discoveries have demonstrated that the physiological function of bile acids extends to the regulation of diverse signaling processes through interactions with nuclear and G protein-coupled receptors, most notably the Farnesoid-X nuclear receptor (FXR) and the G protein-coupled bile acid receptor 1 (GPBAR1, also known as TGR5). Targeting such signaling pathways pharmacologically, i.e. with bile acid-derived therapeutics, presents great potential for the treatment of various metabolic, inflammatory immune, liver, and neurodegenerative diseases. Here we report the discovery of two potent and selective TGR5 agonists (NZP196 and 917). These compounds are the taurine conjugates of 6α-ethyl-substituted 12ß-methyl-18-nor-bile acids with the side chain being located on the α-face of the steroid scaffold. The compounds emerged from a screening effort of a diverse library of 12ß-methyl-18-nor-bile acids that were synthesized from 12ß-methyl-18-nor-chenodeoxycholic acid and its C17-epimer. Upon testing for FXR activity, both compounds were found to be inactive, thus revealing selectivity for TGR5.

Green Synthesis of Carbon Nanoparticles (CNPs) from Biomass for Biomedical Applications.

In this review, we summarize recent work on the "green synthesis" of carbon nanoparticles (CNPs) and their application with a focus on biomedical applications. Recent developments in the green synthesis of carbon nanoparticles, from renewable precursors and their application for environmental, energy-storage and medicinal applications are discussed. CNPs, especially carbon nanotubes (CNTs), carbon quantum dots (CQDs) and graphene, have demonstrated utility as high-density energy storage media, environmental remediation materials and in biomedical applications. Conventional fabrication of CNPs can entail the use of toxic catalysts; therefore, we discuss low-toxicity manufacturing as well as sustainable and environmentally friendly methodology with a focus on utilizing readily available biomass as the precursor for generating CNPs.

Variability in the composition of porcine mucosal heparan sulfates.

Commercial porcine intestinal mucosal heparan sulfate (HS) is a valuable material for research into its biological functions. As it is usually produced as a side-stream of pharmaceutical heparin manufacture, its chemical composition may vary from batch to batch. We analysed the composition and structure of nine batches of HS from the same manufacturer. Statistical analysis of the disaccharide compositions placed these batches in three categories: group A had high GlcNAc and GlcNS, and low GlcN typical of HS; group B had high GlcN and GlcNS, and low GlcNAc; group C had high di- and trisulfated, and low unsulfated and monosulfated disaccharide repeats. These batches could be placed in the same categories based on their 1H NMR spectra and molecular weights. Anticoagulant and growth factor binding activities of these HS batches did not fit within these same groups but were related to the proportions of more highly sulfated disaccharide repeats.

Structural characterization of ulvans extracted from blade (Ulva ohnoi) and filamentous (Ulva tepida and Ulva prolifera) species of cultivated Ulva.

Ulvans from Ulva ohnoi, Ulva tepida and Ulva prolifera were extracted under mild acidic conditions, isolated and their composition and structure determined. The ulvans contained mostly rhamnose (31.6-46.7 mol%) and glucuronic acid (26.6-37.5 mol%), with smaller amounts of xylose (3.4-10.4 mol%) and iduronic acid (3.1-7.6 mol%). In addition, the ulvan samples also contained galactose (4.4-26.0 mol%). Glycosyl linkage analysis showed that ulvan from U. ohnoi contained mostly →4)-GlcpA-(1→ and →3,4)-Rhap-(1→. Preparation of partially methylated alditol acetate standards of idose showed that U. ohnoi contained →4)-IdopA-(1→. In addition to these residues, glycosyl linkage analysis of U. tepida and U. prolifera showed the presence of →2,3,4)-Rhap-(1→, →4)-Xylp-(1→, →2,4)-GlcpA-(1→ and →3,4)-GlcpA-(1→. These two species also contained galactose linkages. These data, together with nuclear magnetic resonance (NMR) spectroscopy indicated that U. ohnoi comprised mostly of type A3S ulvanobiuronic acid repeats [→4)-ß-D-GlcpA-(1→4)-α-L-Rhap3S-(1→], together with smaller amounts of type B3S ulvanobiuronic acid repeats [→4)-α-L-IdopA-(1→4)-α-L-Rhap3S-(1→] and ulvanobiose (U3S [→4)-ß-D-Xylp-(1→4)-α-L-Rhap3S-(1→]). NMR spectra of U. tepida and U. prolifera showed resonances not detected in U. ohnoi, highlighting the complexity of the ulvans from these species. Regardless of the structural diversity of the ulvan samples there was very little antioxidant or inhibitory activity detected on enzymatic processes investigated.

Are all ulvans equal? A comparative assessment of the chemical and gelling properties of ulvan from blade and filamentous Ulva.

Green seaweeds of the genus Ulva are rich in the bioactive sulfated polysaccharide ulvan. Herein we characterise ulvan from Ulva species collected from the Bay of Plenty, Aotearoa New Zealand. Using standardised procedures, we quantified, characterised, and compared ulvans from blade (U. australis, U. rigida, U. sp. B, and Ulva sp.) and filamentous (U. flexuosa, U. compressa, U. prolifera, and U. ralfsii) Ulva species. There were distinct differences in composition and structure of ulvans between morphologies. Ulvan isolated from blade species had higher yields (14.0-19.3 %) and iduronic acid content (IdoA = 7-18 mol%), and lower molecular weight (Mw = 190-254 kDa) and storage moduli (G' = 0.1-6.6 Pa) than filamentous species (yield = 7.2-14.6 %; IdoA = 4-7 mol%; Mw = 260-406 kDa; G' = 22.7-74.2 Pa). These results highlight the variability of the physicochemical properties of ulvan from different Ulva sources, and identifies a morphology-based division within the genus Ulva.

The molecular weight of ulvan affects the in vitro inflammatory response of a murine macrophage.

Ulvan, a sulfated polysaccharide extracted from the green seaweed genus Ulva, has bioactive properties including an immunomodulating capacity. The immunomodulatory capacity of ulvan from Ulva ohnoi, however, has not been assessed in detail. We depolymerised purified ulvan from U. ohnoi to obtain a range of molecular weight fractions (Mw 7, 9, 13, 21, 209 kDa), which were characterised by constituent sugar analysis, SEC-MALLS, and NMR. Ulvan fractions contained 48.8-54.7 mol% rhamnose, 32.5-35.9 mol% glucuronic acid, 4.5-7.3 mol% iduronic acid, and 3.3-5.6 mol% xylose. 1H and 13C NMR was consistent with hydrolysis occurring at the anomeric centre without further modification to the oligosaccharide structure. The in vitro immunomodulatory effect of ulvan fractions was quantified by measuring levels of inflammatory-mediating signalling molecules released from LPS-stimulated RAW264.7 murine macrophages. All ulvan fractions showed no toxicity on RAW264.7 cells at concentrations below 100 µg mL-1 over 48 h. Secreted interleukin-10 and prostaglandin E2 demonstrated an anti-inflammatory effect by higher molecular weight ulvan fractions at 100 µg mL-1. To a lesser extent, these fractions also enhanced the LPS-induced inflammation through minor increases of IL-1ß and IL-6. This study confirms that ulvan from U. ohnoi has a mild in vitro immunomodulatory effect.

Reisolation and NMR characterization of the satratoxins G and H.

The exquisitely cytotoxic macrolides, satratoxins G and H, have been reisolated from a solvent extract of a rice culture inoculated with Stachybotrys chartarum to be used as high-purity reference compounds for analytical analyses. Extensive chromatographic separation realized the compounds that were fully recharacterized in two solvents by 1D- and 2D-NMR spectroscopy, revealing some discrepancies in the nuclear magnetic resonance (NMR) data as compared with the previously reported values found in the literature. Detailed spectra are provided in order to aid future identification and dereplication.

Therapeutic Potential of Neurotrophins for Repair After Brain Injury: A Helping Hand From Biomaterials.

Stroke remains the leading cause of long-term disability with limited options available to aid in recovery. Significant effort has been made to try and minimize neuronal damage following stroke with use of neuroprotective agents, however, these treatments have yet to show clinical efficacy. Regenerative interventions have since become of huge interest as they provide the potential to restore damaged neural tissue without being limited by a narrow therapeutic window. Neurotrophins, such as brain-derived neurotrophic factor (BDNF), and their high affinity receptors are actively produced throughout the brain and are involved in regulating neuronal activity and normal day-to-day function. Furthermore, neurotrophins are known to play a significant role in both protection and recovery of function following neurodegenerative diseases such as stroke and traumatic brain injury (TBI). Unfortunately, exogenous administration of these neurotrophins is limited by a lack of blood-brain-barrier (BBB) permeability, poor half-life, and rapid degradation. Therefore, we have focused this review on approaches that provide a direct and sustained neurotrophic support using pharmacological therapies and mimetics, physical activity, and potential drug delivery systems, including discussion around advantages and limitations for use of each of these systems. Finally, we discuss future directions of biomaterial drug-delivery systems, including the incorporation of heparan sulfate (HS) in conjunction with neurotrophin-based interventions.

Polysaccharides from New Zealand Native Plants: A Review of Their Structure, Properties, and Potential Applications.

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.

Utilization of Complex Pectic Polysaccharides from New Zealand Plants (Tetragonia tetragonioides and Corynocarpus laevigatus) by Gut Bacteroides Species.

Pectic polysaccharides from New Zealand (NZ) spinach (Tetragonia tetragonioides) and karaka berries (Corynocarpus laevigatus) were extracted and analyzed. NZ spinach polysaccharides comprised mostly homogalacturonan (64.4%) and rhamnogalacturonan I (5.8%), with side chains of arabinan (8.1%), galactan (2.2%), and type II arabinogalactan (7.1%); karaka berry polysaccharides comprised homogalacturonan (21.8%) and rhamnogalacturonan I (10.0%), with greater proportions of side chains (arabinan, 15.6%; galactan, 23.8%; and type II arabinogalactan, 19.3%). Screening of gut commensal Bacteroides showed that six were able to grow on the NZ spinach extract, while five were able to grow on the karaka berry extract. Analysis of the polysaccharides remaining after fermentation, by size-exclusion chromatography and constituent sugar analysis, showed that the Bacteroides species that grew on these two substrates showed preferences for the different pectic polysaccharide types. Our data suggest that, to completely degrade and utilize the complex pectin structures found in plants, members of Bacteroides and other bowel bacteria work as metabolic consortia.

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.

Retention of the Structure and Function of Heparan Sulfate Biomaterials After Gamma Irradiation.

Heparan sulfate (HS) is a highly heterogeneous polysaccharide implicated in many important biological processes. Our previous work has demonstrated that a particular affinity-selected HS (referred to henceforth as "HS3") is capable of enhancing the osteogenic effects of bone morphogenetic protein 2 (BMP2). Here, we gamma-irradiated HS with 26 kGy of ionizing radiation to determine how this affected the structure, composition, and function. Initial structural studies were performed on a commercial preparation of HS as a proof-of-concept. Gamma irradiation of this HS preparation did not significantly alter its structure or composition compared to nonirradiated material, as demonstrated by proton nuclear magnetic resonance spectroscopy, molecular weight analysis using size exclusion chromatography, and disaccharide compositional analysis. When HS3 was gamma irradiated, no significant effect on binding affinity toward BMP2 was observed, based on competitive surface plasmon resonance and differential scanning fluorimetry assays. Furthermore, irradiation did not significantly affect HS3's ability to synergistically enhance the osteogenic effects of BMP2 in vitro; as measured by the relative abundance of osteogenic transcripts in transdifferentiating C2C12 murine myoblasts. Additionally, no significant differences were observed in the levels of alkaline phosphatase (ALP) or calcium deposition in C2C12s treated with BMP2, together with the irradiated, or nonirradiated HS3. Irradiation of HS3 incorporated into collagen type I sponges did not affect its ability to enhance BMP2-mediated ALP expression in C2C12 cells. Our data confirm that gamma irradiation is a cost-effective and viable solution for the sterilization of HS species that allows the retention of its structure and biological function. The work suggests an effective way to incorporate clinically compatible HS species into orthotic implants, scaffolds, and other medical devices for use in the treatment of a range of diseases and disorders.

Isolation and characterization of roridin E.

The commonly occurring, high-cytotoxicity macrolide roridin E has been re-isolated from Stachybotrys chartarum and characterized by 1-D and 2-D NMR spectroscopy. Assignment of the spectral data for roridin E revealed differences from the accepted literature, and spectra are reported herein to aid in future identification. For the first time confirmation of structure was provided by a crystallographic solution for roridin E. Copyright © 2016 John Wiley & Sons, Ltd.

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. © 2016 Wiley Periodicals, Inc.

Heparan Sulfate Identification and Characterisation: Method I.Heparan Sulfate Identification by NMR Analysis.

Heparin and heparan sulfate (HS) may be purified from complex biological matrices and are often isolated in sub-milligram quantities but not unequivocally identified by spectroscopic means. This protocol details a methodology to rapidly assess the gross compositional features and approximate purity of HS by 1H nuclear magnetic resonance. A complimentary method for identification and characterisation of heparan sulfate can be found at Carnachan and Hinkley (2017).

Heparan Sulfate Identification and Characterisation: Method II. Enzymatic Depolymerisation and SAX-HPLC Analysis to Determine Disaccharide Composition.

Heparan sulfate (HS) is purified from complex matrices and often not fully characterised to validate its assignment. The characterisation of heparins and heparan sulfates through enzymatic depolymerisation and subsequent strong anion-exchange high performance liquid chromatography (SAX-HPLC) analysis and quantitation of the resulting disaccharides is a critical tool for assessing the structural composition of this class of compound. This protocol details a methodology to reproducibly determine the disaccharide composition of heparan sulfate by enzymatic depolymerisation and SAX-HPLC analysis. A complementary method for identification and characterisation of heparan sulfate can be found at Carnachan and Hinkley (2017).

Determining the extent of heparan sulfate depolymerisation following heparin lyase treatment.

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 Δ4,5 double-bond and quantified by SEC with ultraviolet-visible (UV) spectroscopic detection (230nm) 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.