Analysis Of Glycosaminoglycans Research Articles

Osteogenic differentiation enhances the MC3T3-E1 secretion of glycosaminoglycans with an affinity for basic fibroblast growth factor and bone morphogenetic protein-2.

IntroductionIt is generally recognized that a wide variety of morphogens and growth factors bind to the glycosaminoglycans (GAG) of proteoglycans (PG) to affect their bioavailability to ligands. Many growth factors involving in osteogenic differentiation require the GAG side chains to facilitate their interaction to the cell surface receptors and the biosynthesis of osteogenic proteins. The objective of this study is to investigate the secretion of GAG from MC3T3-E1 pre-osteoblasts of a murine bone calvaria during the osteogenic differentiation.MethodsWhen MC3T3-E1 cells were cultured in the differentiation medium (DM) containing bone morphogenetic protein (BMP)-2, the alkaline phosphatase activity, calcium content and the amount of basic fibroblast growth factor (bFGF)- or BMP-2-bound sulfated GAG were determined. Moreover, the disaccharide analysis of the GAG was performed.ResultsWhen MC3T3-E1 cells were cultured in the differentiation medium (DM) containing bone morphogenetic protein (BMP)-2, the alkaline phosphatase activity and calcium content were significantly enhanced compared with those of the BMP-2-free DM and normal medium with or without BMP-2. Significantly higher amount of GAG secreted was detected for cells cultured in the DM containing BMP-2, in contrast to other culture conditions. The GAG secreted had an affinity for BMP-2 and basic fibroblast growth factor (bFGF). The disaccharide analysis of GAG demonstrated that the percentage of ΔHexA α1,4GlcNSO3 and ΔHexA (2-OSO3) α1,4GlcNSO3 increased, but that of ΔHexA α1,4GlcNSO3(6-OSO3) decreased (ΔHexA: unsaturated uronic acid residue, GlcNSO3: N-sulfated glucosamine, ΔHexA (2-OSO3): unsaturated uronic acid 2-sulfate residue, GlcNSO3(6-OSO3): N-sulfated glucosamine 6-sulfated).ConclusionIt was found that the osteogenic differentiation allowed cells to enhance the secretion of GAG with an affinity for BMP-2 and bFGF.

Capillary Electrophoresis Separations of Glycans.

Capillary electrophoresis has emerged as a powerful approach for carbohydrate analyses since 2014. The method provides high resolution capable of separating carbohydrates by charge-to-size ratio. Principle applications are heavily focused on N-glycans, which are highly relevant to biological therapeutics and biomarker research. Advances in techniques used for N-glycan structural identification include migration time indexing and exoglycosidase and lectin profiling, as well as mass spectrometry. Capillary electrophoresis methods have been developed that are capable of separating glycans with the same monosaccharide sequence but different positional isomers, as well as determining whether monosaccharides composing a glycan are alpha or beta linked. Significant applications of capillary electrophoresis to the analyses of N-glycans in biomarker discovery and biological therapeutics are emphasized with a brief discussion included on carbohydrate analyses of glycosaminoglycans and mono-, di-, and oligosaccharides relevant to food and plant products. Innovative, emerging techniques in the field are highlighted and the future direction of the technology is projected based on the significant contributions of capillary electrophoresis to glycoscience from 2014 to the present as discussed in this review.

Complete solubilization of cartilage using the heat-stable protease thermolysin for comprehensive GAG analysis

Articular cartilage comprises collagens, proteoglycans, and glycosaminoglycans (GAGs) together with water, in hyaline matrixes. Articular cartilage is resistant to proteolytic solubilization for comprehensive GAG analyses partly because of assemblies of collagen fibers with thermolabile hydrogen bonds. In this study, we used the heat-stable protease thermolysin to digest collagen in solid articular cartilage at 70 °C and compared the efficiencies of collagen digestion and GAG extraction to those with collagenase digestion at 50 °C. Overnight digestion with thermolysin completely solubilized cartilage, whereas collagenase with >10-times higher proteolytic activity digested <20% of collagen. Following thermolysin treatments, almost all GAGs were extracted from the cartilage, whereas only 56% of GAGs were extracted after collagenase digestion. Disaccharide analyses of extracted GAG chains revealed >98% extraction efficiencies of several GAG classes from thermolysin-treated cartilage, compared with <60% extraction efficiencies using collagenase, depending on GAG classes. These results indicate that thermolysin allows complete GAG extraction from solid articular cartilage and that complete solubilization is required for accurate and reproducible analyses of cartilage GAGs. Hence, thermolysin offers a tool for complete solubilization of cartilage prior to comprehensive GAGomic analysis, and is likely applicable to other collagen-rich tissues such as ligaments, skin, and blood vessels.

Fast, sensitive method for trisaccharide biomarker detection in mucopolysaccharidosis type 1

Certain recessively inherited diseases result from an enzyme deficiency within lysosomes. In mucopolysaccharidoses (MPS), a defect in glycosaminoglycan (GAG) degradation leads to GAG accumulation followed by progressive organ and multiple system dysfunctions. Current methods of GAG analysis used to diagnose and monitor the diseases lack sensitivity and throughput. Here we report a LC-MS method with accurate metabolite mass analysis for identifying and quantifying biomarkers for MPS type I without the need for extensive sample preparation. The method revealed 225 LC-MS features that were >1000-fold enriched in urine, plasma and tissue extracts from untreated MPS I mice compared to MPS I mice treated with iduronidase to correct the disorder. Levels of several trisaccharides were elevated >10000-fold. To validate the clinical relevance of our method, we confirmed the presence of these biomarkers in urine, plasma and cerebrospinal fluid from MPS I patients and assessed changes in their levels after treatment.

Sensitive method for glycosaminoglycan analysis of tissue sections

A simple, isocratic HPLC method based on HILIC-WAX separation, has been developed for analyzing sulfated disaccharides of glycosaminoglycans (GAGs). To our best knowledge, this is the first successful attempt using this special phase in nano-HPLC–MS analysis. Mass spectrometry was based on negative ionization, improving both sensitivity and specificity. Detection limit for most sulfated disaccharides were approximately 1 fmol; quantitation limits 10 fmol. The method was applied for glycosaminoglycan profiling of tissue samples, using surface digestion protocols. This novel combination provides sufficient sensitivity for GAG disaccharide analysis, which was first performed using prostate cancer tissue microarrays. Preliminary results show that GAG analysis may be useful for identifying cancer related changes in small amounts of tissue samples (ca. 10 μg).

Platelet rich concentrate enhances mesenchymal stem cells capacity to repair focal cartilage injury in rabbits

BackgroundIt has been previously suggested that the use of regenerative promoters, which include bone marrow-derived mesenchymal stem cells (MSCs) or natural growth factors supplement such as platelet-rich concentrate (PRC) could promote cartilage regeneration. However, the notion that the concurrent use of both promoters may provide a synergistic effect that improves the repair outcome of focal cartilage injury has not been previously demonstrated. This study was thus conducted to determine whether the concomitant use of PRC could further enhance the reparative potential of MSCs encapsulated in alginate transplanted into focal cartilage injury in rabbits. MethodsArtifically created full thickness cartilage defects were made on the weight-bearing region of medial femoral condyles in bilateral knees of New Zealand White rabbits (N = 30). After one month, the right knee was treated with either i) PRC (n = 10), ii) MSCs (n = 10), or, iii) a combination of PRC and MSCs (PRC + MSC) (n = 10), all encapsulated in alginate. The left knee remained untreated (control). Rabbits were sacrificed at 3 and 6 months after treatment. Cartilage tissue regeneration was accessed using ICRS morphologic scoring, histologic grading by O'Driscoll scoring, immunohistochemical staining and quantitative analysis of glycosaminoglycans (GAG) per total protein content. ResultsAt 3 months, transplantation using PRC alone was equally effective as MSCs in inducing the repair of cartilage defects. However, PRC + MSC resulted in significantly higher ICRS and O’Driscoll scores (p < 0.05) as compared to other groups. The regenerated tissues from the PRC + MSC group also had stronger staining for Safranin-O and collagen type II. By 6 months, in addition to superior ICRS and O’Driscoll scores as well as stronger staining, glycosaminoglycan per total protein content was also significantly higher (p < 0.05) in the PRC + MSC group (3.4 ± 0.3 μg/mg) as compared to the MSC (2.6 ± 0.2 μg/mg) or PRC (2.1 ± 0.2 μg/mg) groups. ConclusionPRC enhances the reparative effects of MSC in treating focal articular cartilage injuries.

Isolation and Characterization of a Chinese Hamster Ovary Heparan Sulfate Cell Mutant Defective in Both Met Receptor Binding and Hepatocyte Growth Factor NK1/Met Signaling

Background/Aims: The up-regulation of hepatocyte growth factor/receptor, HGF/Met, signal transduction is observed in most of human cancers. Specific heparan sulfate structures enhance the HGF/Met signaling at both cell and animal-based model systems. Biochemical studies indicate that heparan sulfate interacts with HGF and a natural occurring splicing variant NK1 of HGF with similar affinity. However, it is currently unknown if cell surface heparan sulfate binds to Met at physiological conditions and if specific cell surface heparan sulfate structures are required for effective HGF/Met or NK1/Met signaling. Methods: An established flow sorting strategy was used to isolate a soluble Met recombinant protein-binding positive or negative CHO cell clones different only in specific heparan sulfate structures. The cell surface bindings were imaged by confocal microscopy and flow cytometry analysis. Glucosamine vs. galactosamine contents from media-, cell surface-, and cell association glycosaminoglycans were quantified by HPLC. ³⁵S-sulfate labeled glycosaminoglycans were characterized by anion exchange and size-exclusion HPLC. Heparan sulfate disaccharide compositions were determined by HPLC-MS analysis. Western blot analyses of MAPK-p42/44 were used to monitor HGF- and NK1-facillated Met signaling. Results: CHO-Positive but not CHO-Negative cell surface heparan sulfate bound to Met recombinant protein and HGF/NK1 further promoted the binding. Overall glycosaminoglycan analysis results indicated that the CHO-Negative cells had reduced amount of heparan sulfate, shorter chain length, and less 6-O-sulfated disaccharides compared to that of CHO-Positive cells. Moreover, CHO-Negative cells were defective in NK1/Met but not HGF/Met signaling. Conclusions: This study demonstrated that soluble Met recombinant protein bound to cell surface HS at physiological conditions and a Met /HGF or NK1/HS ternary signaling complex might be involved in Met signaling. Shorter HS chains and reduced 6-O-sulfation might be responsible for reduced Met binding and the diminished NK1-initiated signaling in the CHO-Negative cells. The unique CHO-Positive and CHO-Negative cell clones established in current study should be effective tools for studying the role of specific glycosaminoglycan structures in regulating Met signaling. Such knowledge should be useful in developing glycosaminoglycan-based compounds that target HGF/Met signaling.

Comprehensive analysis of glycosaminoglycans from the edible shellfish

We have previously reported that the keratan sulfate (KS) disaccharide was branched to the C-3 position of glucuronate in chondroitin sulfate (CS)-E derived from the Mactra chinensis. We carried out the comprehensive disaccharide analysis of GAGs from 10 shellfish, Ruditapes philippinarum, Scapharca broughtonii, Mizuhopecten yessoensis, Turbo cornutus, Crassostrea nippona, Corbicula japonica, Mytilus galloprovincialis, Neptunea intersculpta, Pseudocardium sachalinense and Crassostrea gigas, to better understand the glycan structures in marine organisms. The contents of CS, heparan sulfate and hyaluronic acid and their compositions depend on the species of shellfish. Interestingly, a peak corresponding to a pentasaccharide containing KS disaccharide was observed when GAGs from T. cornutus was treated with chondroitinase (Chase) ACII but not Chase ABC. In addition, unidentified peaks were also observed when CS derived from R. philippinarum, S. broughtonii were treated with Chase ACII. These results suggest the presence of additional unidentified structure of CS in these shellfish.

The influence of hyperglycemia on the remodeling of urethral connective tissue in pregnant rats

ObjectiveTo analyze the distribution and quantification of the key structural extracellular matrix components of the urethral tissue in a rat model of hyperglycemia and pregnancy. Study designA total of 120 female Wistar rats were distributed into the following four experimental groups: virgin, pregnant, hyperglycemic and hyperglycemic + pregnant groups. The urethra was harvested for histochemical, morphometric, immunohistochemical, Western blot and glycosaminoglycan analyses. All protocols were approved by the Institutional Animal Care and Use Committee of Botucatu Medical School (process number 828–2010). ResultsThe hyperglycemic + pregnant group showed significantly increased stiffness in urethral tissue. The total striated muscle was decreased, with increased deposition of collagen fibers around the muscle fibers and a change in the organization of the collagen fibrils. An increase in the relative collagen type I/III ratio and a decrease in total glycosaminoglycans were also observed. ConclusionsThis study provides the first line of experimental evidence supporting a metabolic relationship between hyperglycemia and urethral remodeling of connective tissue in pregnant rats. The different organization of the collagen fibrils and the profile of glycosaminoglycans found in urethral samples suggest that the pathology of the urethral fibromuscular system could be related to hyperglycemia-induced pelvic floor dysfunction in women, which has direct clinical implications with the possibility to develop new multidisciplinary treatments for improving the health care of these women.

Glycosaminoglycan Analysis by Cryogenic Messenger-Tagging IR Spectroscopy Combined with IMS-MS.

We combine ion mobility spectrometry with cryogenic, messenger-tagging, infrared spectroscopy and mass spectrometry to identify different isomeric disaccharides of chondroitin sulfate (CS) and heparan sulfate (HS), which are representatives of two major subclasses of glycosaminoglycans. Our analysis shows that while CS and HS disaccharide isomers have similar drift times, they can be uniquely distinguished by their vibrational spectrum between ∼3200 and 3700 cm-1 due to their different OH hydrogen-bonding patterns. We suggest that this combination of techniques is well suited to identify and characterize glycan isomers directly, which presents tremendous challenges for existing methods.

Chondrocytes from congenital microtia possess an inferior capacity for in vivo cartilage regeneration to healthy ear chondrocytes.

The remnant auricular cartilage from microtia has become a valuable cell source for ear regeneration. It is important to clarify the issue of whether the genetically defective microtia chondrocytes could engineer cartilage tissue comparable to healthy ear chondrocytes. In the current study, the histology and cell yield of native microtia and normal ear cartilage were investigated, and the biological characteristics of derived chondrocytes examined, including proliferation, chondrogenic phenotype and cell migration. Furthermore, the in vivo cartilage-forming capacity of passaged microtia and normal auricular chondrocytes were systematically compared by seeding them onto polyglycolic acid/polylactic acid scaffold to generate tissue engineered cartilage in nude mice. Through histological examinations and quantitative analysis of glycosaminoglycan, Young's modulus, and the expression of cartilage-related genes, it was found that microtia chondrocytes had a slower dedifferentiation rate with the decreased expression of stemness-related genes, and weaker migration ability than normal ear chondrocytes, and the microtia chondrocytes-engineered cartilage was biochemically and biomechanically inferior to that constructed using normal ear chondrocytes. This study provides valuable information for the clinical application of the chondrocytes derived from congenital microtia to engineer cartilage. Copyright © 2016 John Wiley & Sons, Ltd.

Single Stage Tandem Mass Spectrometry Assignment of the C-5 Uronic Acid Stereochemistry in Heparan Sulfate Tetrasaccharides using Electron Detachment Dissociation.

The analysis of heparan sulfate (HS) glycosaminoglycans presents many challenges, due to the high degree of structural heterogeneity arising from their non-template biosynthesis. Complete structural elucidation of glycosaminoglycans necessitates the unambiguous assignments of sulfo modifications and the C-5 uronic acid stereochemistry. Efforts to develop tandem mass spectrometric-based methods for the structural analysis of glycosaminoglycans have focused on the assignment of sulfo positions. The present work focuses on the assignment of the C-5 stereochemistry of the uronic acid that lies closest to the reducing end. Prior work with electron-based tandem mass spectrometry methods, specifically electron detachment dissociation (EDD), have shown great promise in providing stereo-specific product ions, such as the B3´ -CO2, which has been found to distinguish glucuronic acid (GlcA) from iduronic acid (IdoA) in some HS tetrasaccharides. The previously observed diagnostic ions are generally not observed with 2-O-sulfo uronic acids or for more highly sulfated heparan sulfate tetrasaccharides. A recent study using electron detachment dissociation and principal component analysis revealed a series of ions that correlate with GlcA versus IdoA for a set of 2-O-sulfo HS tetrasaccharide standards. The present work comprehensively investigates the efficacy of these ions for assigning the C-5 stereochemistry of the reducing end uronic acid in 33 HS tetrasaccharides. A diagnostic ratio can be computed from the sum of the ions that correlate to GlcA to those that correlate to IdoA. Graphical Abstract ᅟ.

Piperine ameliorates collagenase-induced Achilles tendon injury in the rat

ABSTRACTTendinopathy is a common clinical pathology found in athletes and workers with mixed treatment results. Piperine, a major alkaloid found in the black and long pepper, has been demonstrated to have variety of pharmacological properties such as analgesic and anti-inflammatory effects. The present study was designed to investigate the effects of piperine on collagenase-induced Achilles tendon injury. Rats were intratendineously injected with collagenase in the right Achilles tendon, followed by intragastrical administration of piperine (100 mg/kg). Morphological structure and biochemical analysis of glycosaminoglycans, hydroxyproline, collagen III, and the activity of matrix metallopeptidases in the tendon tissues were performed. Our results showed that collagenase injection resulted in clear degenerative changes in the tendon. Administration of piperine improved the morphological structure of tendon, increased glycosaminoglycans and hydroxyproline levels, and inhibited the expression and activities of MMP-2 and MMP-9. Furthermore, piperine inhibited the activation of ERK and p38 signaling pathways in injured tendon. These results indicate a beneficial role of piperine against collagenase-induced tendon injury.

Isolation and Analysis of Proteoglycans and Glycosaminoglycans from Archaeological Bones and Teeth.

We have developed methods for isolating proteoglycans and glycosaminoglycans from archaeological bones and teeth. These methods have been previously published (Coulson- Thomas et al., 2015 ) and are described here in more detail. In the case of glycosaminoglycans, the method was a previously described method ( Nader et al., 1999 ) which we optimized for archeological samples.

Implementation of infrared and Raman modalities for glycosaminoglycan characterization in complex systems

Glycosaminoglycans (GAGs) are natural, linear and negatively charged heteropolysaccharides which are incident in every mammalian tissue. They consist of repeating disaccharide units, which are composed of either sulfated or non-sulfated monosaccharides. Depending on tissue types, GAGs exhibit structural heterogeneity such as the position and degree of sulfation or within their disaccharide units composition being heparin, heparan sulfate, chondroitine sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid. They are covalently linked to a core protein (proteoglycans) or as free chains (hyaluronan). GAGs affect cell properties and functions either by direct interaction with cell receptors or by sequestration of growth factors. These evidences of divert biological roles of GAGs make their characterization at cell and tissue levels of importance. Thus, non-invasive techniques are interesting to investigate, to qualitatively and quantitatively characterize GAGs in vitro in order to use them as diagnostic biomarkers and/or as therapeutic targets in several human diseases including cancer. Infrared and Raman microspectroscopies and imaging are sensitive enough to differentiate and classify GAG types and subtypes in spite of their close molecular structures. Spectroscopic markers characteristic of reference GAG molecules were identified. Beyond these investigations of the standard GAG spectral signature, infrared and Raman spectral signatures of GAG were searched in complex biological systems like cells. The aim of the present review is to describe the implementation of these complementary vibrational spectroscopy techniques, and to discuss their potentials, advantages and disadvantages for GAG analysis. In addition, this review presents new data as we show for the first time GAG infrared and Raman spectral signatures from conditioned media and live cells, respectively.

Glycosaminoglycans detection methods: Applications of mass spectrometry

Glycosaminoglycans (GAGs) are long blocks of negatively charged polysaccharides. They are one of the major components of the extracellular matrix and play multiple roles in different tissues and organs. The accumulation of undegraded GAGs causes mucopolysaccharidoses (MPS). GAGs are associated with other pathological conditions such as osteoarthritis, inflammation, diabetes mellitus, spinal cord injury, and cancer. The need for further understanding of GAG functions and mechanisms of action boosted the development of qualitative and quantitative (alcian blue, toluidine blue, paper and thin layer chromatography, gas chromatography, high pressure liquid chromatography, capillary electrophoresis, 1,9-dimethylmethylene blue, enzyme linked-immunosorbent assay, mass spectrometry) techniques. The availability of quantitative techniques has facilitated translational research on GAGs into the medical field for: 1) diagnosis, monitoring, and screening for MPS; 2) analysis of GAG synthetic and degradation pathways; and 3) determination of physiological and pathological roles of GAGs. This review provides a history of development of GAG assays and insights about the use of tandem mass spectrometry and its applications for GAG analysis.

Glycosaminoglycan levels and structure in a mucopolysaccharidosis IIIA mice and the effect of a highly secreted sulfamidase engineered to cross the blood-brain barrier.

Mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo A) is a neurodegenerative lysosomal storage disorder caused by the deficiency of sulphamidase enzyme (SGSH) leading to accumulation of heparan sulfate (HS). We quantitatively and structurally characterize primary stored HS and other glycosaminoglycans (GAGs) possibly accumulated through a secondary storage in brain, liver, kidney and lung of MPS IIIA mouse model. This analysis was also performed in MPS IIIA mice upon the intravenous treatment with an engineered human sulphamidase (chimeric hSGSH) capable to increase its secretion from the liver and to cross the blood-brain barrier. MPS IIIA animals showed a huge accumulation of HS, from ~15 up to ~24-times higher than wild type and also of hyaluronic acid (HA) (from 2.5 up to ~5.0-times more) and chondroitin sulfate (CS)/dermatan sulfate (DS) (from ~2 up to ~5-times more) in all studied organs. We also observed a significant increase in the overall HS charge density and in particular of 2-O-sulfation in MPS IIIA mice organs. 8months after a systemic treatment with an engineered SGSH, the enzyme was highly efficient in the reduction of all accumulated GAGs in liver, brain and lung up to values of wild type mice. On the contrary, even if reduced, GAGs levels still remained significantly elevated in kidney. Overall data obtained by this detailed analysis of GAGs in the different organs of affected and treated animals with chimeric hSGSH may have implications for the evaluation of an effective therapeutic option of MPS IIIA and for the reduction of related neuropathology.

UPLC-MS/MS detection of disaccharides derived from glycosaminoglycans as biomarkers of mucopolysaccharidoses

Mucopolysaccharidoses (MPSs) are a group of disorders resulting from primary defects in lysosomal enzymes involved in the degradation of glycosaminoglycans (GAGs). Depending on the specific enzyme defect, the catabolism of one or more GAGs is blocked leading to accumulation in tissues and biological fluids. GAG measurements are important for high-risk screening, diagnosis, monitoring treatment efficacy, and patient follow up. The dimethylmethylene blue (DMB) spectrophotometric method commonly used in most biochemical genetics laboratories relies on a non-specific total GAG analysis which has led to false positive results, and even false negative results (mainly for MPS III and IV patients). The main objective of our project was to devise and validate a reliable tandem mass spectrometry multiplex analysis for the urine quantitation of four GAGs (dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and chondroitin sulfate (CS)) for an eventual technological transfer to the clinic. The developed methodology is rapid (7 min) and our results showed good intraday and interday precision (RSDs ≤ 8.7%) and accuracy (Biases range: −12.0%–18.4%). Linearity was good (r2 > 0.995) for DS, HS, CS, and KS calibration curves. In comparison with the DMB spectrophotometric method, this multiplex tandem mass spectrometry method allows GAG fractionation, thus a differentiation of MPS types, except for MPS I and II which are characterized by the same GAG profile. The devised method is a useful and reliable tool for diagnosis of MPS patients, as well as their monitoring and follow up, as shown by longitudinal studies.

Systematic protein interactome analysis of glycosaminoglycans revealed YcbS as a novel bacterial virulence factor.

Microbial pathogens have evolved several strategies for interacting with host cell components, such as glycosaminoglycans (GAGs). Some microbial proteins involved in host–GAG binding have been described; however, a systematic study on microbial proteome–mammalian GAG interactions has not been conducted. Here, we used Escherichia coli proteome chips to probe four typical mammalian GAGs, heparin, heparan sulphate (HS), chondroitin sulphate B (CSB), and chondroitin sulphate C (CSC), and identified 185 heparin-, 62 HS-, 98 CSB-, and 101 CSC-interacting proteins. Bioinformatics analyses revealed the unique functions of heparin- and HS-specific interacting proteins in glycine, serine, and threonine metabolism. Among all the GAG-interacting proteins, three were outer membrane proteins (MbhA, YcbS, and YmgH). Invasion assays confirmed that mutant E. coli lacking ycbS could not invade the epithelial cells. Introducing plasmid carrying ycbS complemented the invading defects at ycbS lacking E. coli mutant, that can be further improved by overexpressing ycbS. Preblocking epithelial cells with YcbS reduced the percentage of E. coli invasions. Moreover, we observed that whole components of the ycb operon were crucial for invasion. The displacement assay revealed that YcbS binds to the laminin-binding site of heparin and might affect the host extracellular matrix structure by displacing heparin from laminin.

Abnormal osteogenic and chondrogenic differentiation of human mesenchymal stem cells from patients with adolescent idiopathic scoliosis in response to melatonin.

Abnormalities of membranous and endochondral ossification in patients with adolescent idiopathic scoliosis (AIS) remain incompletely understood. To investigate abnormalities in the melatonin signaling pathway and cellular response to melatonin in AIS, a case-control study of osteogenic and chondrogenic differentiation was performed using human mesenchymal stem cells (hMSCs). AIS was diagnosed by physical and radiographic examination. hMSCs were isolated from the bone marrow of patients with AIS and control subjects (n=12 each), and purified by density gradient centrifugation. The expression levels of melatonin receptors (MTs) 1 and 2 were detected by western blotting. Osteogenic and chondrogenic differentiation was induced by culturing hMSCs in osteogenic and chondrogenic media containing vehicle or 50 nM melatonin. Alkaline phosphatase (ALP) activity assays, quantitative glycosaminoglycan (GAG) analysis, and reverse transcription-quantitative polymerase chain reaction analysis were performed. Compared with controls, MT2 demonstrated low expression in the AIS group. Melatonin increased ALP activity, GAG synthesis and upregulated the expression of genes involved in osteogenic and chondrogenic differentiation including, ALP, osteopontin, osteocalcin, runt-related transcription factor 2, collagen type II, collagen type X, aggrecan and sex-determining region Y-box 9 in the normal control hMSCs, but did not affect the AIS groups. Thus, AIS hMSCs exhibit abnormal cellular responses to melatonin during osteogenic and chondrogenic differentiation, which may be associated with abnormal membranous and endochondral ossification, and skeletal growth. These results indicate a potential modulating role of melatonin via the MT2 receptor on abnormal osteogenic and chondrogenic differentiaation in patients with AIS.