FAM20B-catalyzed glycosaminoglycans control murine tooth number by restricting FGFR2b signaling.

BACKGROUND: The formation of supernumerary teeth is an excellent model for studying the molecular mechanisms that control stem/progenitor cell homeostasis needed to generate a renewable source of replacement cells and tissues. Although multiple growth factors and transcriptional factors have been associated with supernumerary tooth formation, the regulatory inputs of extracellular matrix in this regenerative process remains poorly understood. RESULTS: In this study, we present evidence that disrupting glycosaminoglycans (GAGs) in the dental epithelium of mice by inactivating FAM20B, a xylose kinase essential for GAG assembly, leads to supernumerary tooth formation in a pattern reminiscent of replacement teeth. The dental epithelial GAGs confine murine tooth number by restricting the homeostasis of Sox2(+) dental epithelial stem/progenitor cells in a non-autonomous manner. FAM20B-catalyzed GAGs regulate the cell fate of dental lamina by restricting FGFR2b signaling at the initial stage of tooth development to maintain a subtle balance between the renewal and differentiation of Sox2(+) cells. At the later cap stage, WNT signaling functions as a relay cue to facilitate the supernumerary tooth formation. CONCLUSIONS: The novel mechanism we have characterized through which GAGs control the tooth number in mice may also be more broadly relevant for potentiating signaling interactions in other tissues during development and tissue homeostasis.

Can natural fibers be a silver bullet? Antibacterial cellulose fibers through the covalent bonding of silver nanoparticles to electrospun fibers.

Natural cotton was dissolved in a room-temperature ionic liquid 1-ethyl-3-methyl acetate and wet-jet electrospun to obtain nanoscale cotton fibers with a substantially reduced diameter-and therefore an increased surface area-relative to natural cotton fibers. The resulting nano-cotton fibers were esterified with trityl-3-mercaptopropionic acid, which after selective de-tritylation afforded nano-cotton fibers containing reactive thiol functionality. Silver nanoparticles that were covalently attached to these sulfhydryl groups were assembled next. The microstructure of the resulting nanocomposite was characterized, and the antibacterial activity of the resulting nano-cotton Ag-nanoparticle composite was also studied. This nanocomposite showed significant activity against both Gram-negative and Gram-positive bacteria.

Ganoderenic acid D-loaded functionalized graphene oxide-based carrier for active targeting therapy of cervical carcinoma.

Ganoderenic acid D (GAD), extracted from the Chinese herb Ganoderma lucidum, was loaded onto a graphene oxide-polyethylene glycol-anti-epidermal growth factor receptor (GO-PEG-EGFR) carrier to develop a targeting antitumor nanocomposite (GO-PEG@GAD). The carrier was fabricated from PEG and anti-EGFR aptamer modified GO. Targeting was mediated by the grafted anti-EGFR aptamer, which targets the membrane of HeLa cells. Physicochemical properties were characterized by transmission electron microscopy, dynamic light scattering, X-ray powder diffraction, and Fourier transform infrared spectroscopy. High loading content (77.3 % ± 1.08 %) and encapsulation efficiency (89.1 % ± 2.11 %) were achieved. Drug release continued for approximately 100 h. The targeting effect both in vitro and in vivo was confirmed by confocal laser scanning microscopy (CLSM) and imaging analysis system. The mass of the subcutaneous implanted tumor was significantly decreased by 27.27 ± 1.23 % after treatment with GO-PEG@GAD compared with the negative control group. Moreover, the in vivo anti-cervical carcinoma activity of this medicine was due to activation of the intrinsic mitochondrial pathway.

Adsorption of doxorubicin on poly(methyl methacrylate)-chitosan-heparin-coated activated carbon beads.

Extracorporeal filter cartridges, filled with an activated carbon bead (ACB) adsorbent, have been used for removal of overdosed cancer drugs from the blood. Coatings on adsorbent matrices, poly(methyl methacrylate) (PMMA)/activated carbon bead and PMMA/chitosan/heparin/ACB composites, were tested to improve their biocompatibility and blood compatibility. PMMA coating on ACBs was accomplished in a straightforward manner using a PMMA solution in ethyl acetate. A one-step hybrid coating of ACBs with PMMA-anticoagulant heparin required the use of acetone and water co-solvents. Multilayer coatings with three components, PMMA, chitosan, and heparin, involved three steps: PMMA was first coated on ACBs; chitosan was then coated on the PMMA-coated surface; and finally, heparin was covalently attached to the chitosan coating. Surface morphologies were studied by scanning electron microscopy. X-ray photoelectron spectroscopy confirmed the -SO(3)(-) group. Adsorption, of a chemotherapy drug (doxorubicin) from both water and phosphate-buffered saline, by the coated ACBs was examined. The adsorption isotherm curves were fitted using the Freundlich model. The current adsorption system might find potential applications in the removal of high-dose regional chemotherapy drugs while maintaining high efficiency, biocompatibility, and blood compatibility.

Chemoenzymatic synthesis of sulfur-linked sugar polymers as heparanase inhibitors.

Complex carbohydrates (glycans) are major players in all organisms due to their structural, energy, and communication roles. This last essential role involves interacting and/or signaling through a plethora of glycan-binding proteins. The design and synthesis of glycans as potential drug candidates that selectively alter or perturb metabolic processes is challenging. Here we describe the first reported sulfur-linked polysaccharides with potentially altered conformational state(s) that are recalcitrant to digestion by heparanase, an enzyme important in human health and disease. An artificial sugar donor with a sulfhydryl functionality is synthesized and enzymatically incorporated into polysaccharide chains utilizing heparosan synthase. Used alone, this donor adds a single thio-sugar onto the termini of nascent chains. Surprisingly, in chain co-polymerization reactions with a second donor, this thiol-terminated heparosan also serves as an acceptor to form an unnatural thio-glycosidic bond ('S-link') between sugar residues in place of a natural 'O-linked' bond. S-linked heparan sulfate analogs are not cleaved by human heparanase. Furthermore, the analogs act as competitive inhibitors with > ~200-fold higher potency than expected; as a rationale, molecular dynamic simulations suggest that the S-link polymer conformations mimic aspects of the transition state. Our analogs form the basis for future cancer therapeutics and modulators of protein/sugar interactions.

Digestibility of squash polysaccharide under simulated salivary, gastric and intestinal conditions and its impact on short-chain fatty acid production in type-2 diabetic rats.

In vitro digestive conditions were simulated to investigate the digestibility of polysaccharides prepared from squash (SPS). A small amount of free glucose monosaccharide was released after salivary and intestinal digestion due to the breakdown of α-(1 → 4)-glucose linkages and may form SPS or a starch impurity. At the same time, there was no obvious change in molecular weight distribution and reducing sugar content throughout this digestion period, demonstrating that the main structure of SPS was relatively stable under the simulated digestive conditions. Thus, most SPS can be transported intact to the large intestine. In addition, SPS alleviated type 2 diabetes (T2D) in rats. Moreover, the content of short-chain fatty acids (SCFAs) in the colon significantly increased after treatment with SPS. The present research provides insight into the non-digestibility of SPS, and suggests its utility to alleviate T2D by promoting the production of SCFA in the colon.

Fucosylated Chondroitin Sulfate 9-18 Oligomers Exhibit Molecular Size-Independent Antithrombotic Activity while Circulating in the Blood.

Fucosylated chondroitin sulfate (FCS) oligosaccharides extracted from sea cucumber and depolymerized exhibit potent anticoagulant activity. Knowledge of the antithrombotic activity of different size oligosaccharides and their fucose (Fuc) branch sulfation pattern should promote their development for clinical applications. We prepared highly purified FCS trisaccharide repeating units from hexasaccharide (6-mer) to octadecasaccharide (18-mer), including those with 2,4-disulfated and 3,4-disulfated Fuc branches. All 10 oligosaccharides were identified by their nuclear magnetic resonance structures and ESI-FTMS spectroscopy. In vitro anticoagulant activities and surface plasmon resonance binding tests indicated those of larger molecular sizes and 2,4-disulfated Fuc branches showed stronger anticoagulant effects with respect to anti-FXase activity, as well as stronger binding to FIXa among various clotting proteins. However, both types of FCS 9-mer to 18-mer exhibited molecular size-independent potent antithrombotic activity in vivo at the same dose. In addition, both types of the FCS 6-mer exhibited favorable antithrombotic activity in vivo, although they showed weak anticoagulant activity in vitro. Combining absorption and metabolism studies, we conclude that FCS 9-18 oligomers could remain in the circulation to interact with various clotting proteins to prevent thrombus formation, and appreciable quantities of these oligomers could be excreted through the kidneys. All FCS 9-18 oligomers also resulted in no bleeding, hypotension, or platelet aggregation risk during blood circulation. Thus, FCS 9-18 oligomers with 2,4-disulfated or 3,4-disulfated Fuc branches exhibit potent and safe antithrombotic activity needed for clinical applications.

Structural characterization of a clinically described heparin-like substance in plasma causing bleeding.

Heparin-like substances (HLS) have been described in various clinical situations, including in settings of liver disease associated with infection, transplant, and metastasis. HLS are generally attributed to circulating glycosaminoglycans. Initial results for this patient showed coagulopathy due to liver disease without HLS. Two weeks after liver transplantation, a 10 year-old female with liver failure patient began to bleed from catheter insertion sites, mouth, and nares and HLS was suspected. The patient subsequently died and these clinical samples resulted in the isolation of a single heparan sulfate (HS) present at high concentrations in the plasma. Analysis of this HS showed it had an intermediate between heparin and HS with low antithrombin-mediated anticoagulant activity. We speculate that this 10-year old patient might have a platelet function defect influenced by this unusual HS. Endothelial defects not measurable by our methods might have also contributed to the observed bleeding complications.

IO4- -stimulated crosslinking of catechol-conjugated hydroxyethyl chitosan as a tissue adhesive.

Catechol-functionalized polymers are of particular interest because of their strong water-resistant adhesive properties. Hydroxymethyl chitosan (HECTS) has been used as an implantable biomaterial having good water solubility, biodegradability and biocompatibility. Here, hydrocaffeic acid (HCA) grafted HECTS (HCA-g-HECTS) was prepared through carbodiimide coupling and the tethered catechol underwent periodate ( IO4- )-stimulated mono and double cross-linking with genipin. The gelation time of these cross-linked HCA-g-HECTS hydrogels decreased with increasing molar ratio of cross-linker to grafted catechol group, increasing temperature, or the addition of genipin. Under the same molar ratio of cross-linker to catechol, IO4- -induced cross-linked HCA-g-HECTS hydrogels exhibited much stronger storage modulus and temperature stability than hydrogels made by Fe3+ -triggered cross-linking. The IO4- -stimulated HCA-g-HECTS hydrogels were biocompatible on a cellular level when the molar ratio of IO4- to catechol group was less than 0.5:1. The hydrogels prepared with a 0.125:1 molar ratio of IO4- to catechol group exhibited high adhesion strength of 73.56 kPa against wet rat skin, and a higher adhesion strength than sutures in a rat wound closure model. This biocompatible IO4- -stimulated HCA-g-HECTS hydrogel may represent a promising new tissue adhesive. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 582-593, 2019.

Amphiphilic bromelain-synthesized oligo-phenylalanine grafted with methoxypolyethylene glycol possessing stabilizing thermo-responsive emulsion properties.

A thermo-responsive amphiphile was developed from oligo-phenylalanine [oligo(Phe)]. The hydrophobic moiety of the amphiphile, oligo(Phe) was synthesized via reverse hydrolysis catalyzed by bromelain in dimethyl sulfoxide and dioxane solutions. The production of oligo(Phe) increased by 80.7% by screening suitable reaction conditions. The average degree of polymerization of oligo(Phe) was determined to be four by 1H NMR. By grafting with aldehyde-ended methoxypolyethylene glycol (mPEG), oligo(Phe) was converted to amphiphilic oligo(Phe)-mPEG. The surface tension of oligo(Phe)-mPEG solution increased with decreasing chain length of the mPEG moiety. Cytotoxicity studies showed oligo(Phe)-mPEGs are biocompatible. On varying temperature, a reversible phase transition of oligo(Phe)-mPEG solutions could be observed. N-octane-in-water emulsions and 0.5% beta-carotene containing squalene-in-water emulsions stabilized by oligo(Phe)-mPEGs occurred at 25 °C but de-emulsification took place at >40 °C. Emulsification could be restored once the separated mixture cooled and re-homogenized. The emulsification/de-emulsification cycling could be repeated many times. The time required for de-emulsification decreased with elevated temperature but increased with a reduced concentration of oligo(Phe)-mPEGs and a reduction in the chain length of the mPEG moiety.

A comparative secretome analysis of industrial Aspergillus oryzae and its spontaneous mutant ZJGS-LZ-21.

Aspergillus oryzae koji plays a crucial role in fermented food products due to the hydrolytic activities of secreted enzymes. In the present study, we performed a comparative secretome analysis of the industrial strain of Aspergillus oryzae 3.042 and its spontaneous mutantZJGS-LZ-21. One hundred and fifty two (152) differential protein spots were excised (p<0.05), and 25 proteins were identified. Of the identified proteins, 91.3% belonged to hydrolytic enzymes acting on carbohydrates or proteins. Consistent with their enzyme activities, the expression of 14 proteins involved in the degradation of cellulose, hemicellulose, starch and proteins, increased in the ZJGS-LZ-21isolate. In particular, increased levels of acid protease (Pep) may favor the degradation of soy proteins in acidic environments and promote the cleavage of allergenic soybean proteins in fermentation, resulting in improvements of product safety and quality. The ZJGS-LZ-21 isolate showed higher protein secretion and increased hydrolytic activities than did strain 3.042, indicating its promising application in soybean paste fermentation.

Analysis of the biochemical mechanisms for the endocrine actions of fibroblast growth factor-23.

Fibroblast growth factor (FGF)-23 has emerged as an endocrine regulator of phosphate and of vitamin D metabolism. It is produced in bone and, unlike other FGFs, circulates in the bloodstream to ultimately regulate phosphate handling and vitamin D production in the kidney. Presently, it is unknown which of the seven principal FGF receptors (FGFRs) transmits FGF23 biological activity. Furthermore, the molecular basis for the endocrine mode of FGF23 action is unclear. Herein, we performed surface plasmon resonance and mitogenesis experiments to comprehensively characterize receptor binding specificity. Our data demonstrate that FGF23 binds and activates the c splice isoforms of FGFR1-3, as well as FGFR4, but not the b splice isoforms of FGFR1-3. Interestingly, highly sulfated and longer glycosaminoglycan (GAG) species were capable of promoting FGF23 mitogenic activity. We also show that FGF23 induces tyrosine phosphorylation and inhibits sodium-phosphate cotransporter Npt2a mRNA expression using opossum kidney cells, a model kidney proximal tubule cell line. Removal of cell surface GAGs abolishes the effects of FGF23, and exogenous highly sulfated GAG is capable of restoring FGF23 activity, suggesting that proximal tubule cells naturally express GAGs that are permissive for FGF23 action. We propose that FGF23 signals through multiple FGFRs and that the unique endocrine actions of FGF23 involve escape from FGF23-producing cells and circulation to the kidney, where highly sulfated GAGs most likely act as cofactors for FGF23 activity. Our biochemical findings provide important insights into the molecular mechanisms by which dysregulated FGF23 signaling leads to disorders of hyper- and hypophosphatemia.