Sulfated Glycosaminoglycans Research Articles

The Crab Heparin-like Compound Exhibits a Strong Inhibitory Effect on Infections by Dengue Virus-2

Background: According to the World Health Organization (WHO), two-fifths of the world population is at risk of infection by DENV. There are no safe and effective vaccines established. Sulfated glycosaminoglycans such as heparin, used as anticoagulants, inhibit the initial step of dengue viral replication. Recently, an isolated heparin analogue Goniopsis cruentata (cCTH) has presented a low anticoagulant effect with reduced bleeding risk. Methods : The antiviral activity of cCTH and heparin compounds against DENV-2 in Vero cell culture was determined by quantitative RT-PCR (qRT-PCR) and titration. For this, four trials were carried out: treatment of the cells for 2 h before viral inoculation, concomitant viral inoculation treatment, treatment after viral inoculation and virucidal assay. Subsequently, the culture supernatants were collected for periods of 24, 48 and 72 h. Results: Our results demonstrated that cCTH and heparin showed antiviral activity against DENV-2. Conclusion : These data suggest that both compounds prevented viral replication in cultured Vero cells.

Macromolecular Interactions in Cartilage Extracellular Matrix Vary According to the Cartilage Type and Location.

To investigate GAG-ECM (glycosaminoglycan-extracellular matrix) interactions in different cartilage types. To achieve this, we first aimed to determine protocols for consistent calculation of GAG content between cartilage types. Auricular cartilage containing both collagen and elastin was used to determine the effect of lyophilization on GAG depletion activity. Bovine articular, auricular, meniscal, and nasal cartilage plugs were treated using different reagents to selectively remove GAGs. Sulfated glycosaminoglycan (sGAG) remaining in the sample after treatment were measured, and sGAG loss was compared between cartilage types. The results indicate that dry weight of cartilage should be measured prior to cartilage treatment in order to provide a more accurate reference for normalization. Articular, meniscal, and nasal cartilage lost significant amounts of sGAG for all reagents used. However, only hyaluronidase was able to remove significant amount of sGAG from auricular cartilage. Furthermore, hyaluronidase was able to remove over 99% of sGAG from all cartilage types except auricular cartilage where it only removed around 76% of sGAG. The results indicate GAG-specific ECM binding for different cartilage types and locations. In conclusion, lyophilization can be performed to determine native dry weight for normalization without affecting the degree of GAG treatment. To our knowledge, this is the first study to compare GAG-ECM interactions of different cartilage types using different GAG extraction methods. Degree of GAG depletion not only varied with cartilage type but also the same type from different anatomic locations. This suggests specific structure-function roles for GAG populations found in the tissues.

Transient anabolic effects of synovium in early post-traumatic osteoarthritis: a novel ex vivo joint tissue co-culture system for investigating synovium-chondrocyte interactions

Osteoarthritis (OA) is a serious joint disease with no disease-modifying medical treatment. To develop treatments targeting synovium, we must improve our understanding of the effects of OA-related changes in synovial physiology on joint tissue outcomes. The aim of this study was to investigate the effects of synovial pathology due to post-traumatic OA (PTOA) on articular chondrocyte physiology. We first developed and validated a novel joint tissue co-culture system to model the biological interactions between synovium and articular chondrocytes. Whole-joint synovial tissue from a surgical rat model of PTOA vs sham and surgical-naïve controls was placed into a co-culture system with adult primary articular chondrocytes (n=4-5). The effects of PTOA synovium on chondrocyte anabolic, inflammatory, and catabolic gene expression and sulfated glycosaminoglycan (sGAG) secretion and aggrecan synthesis were tested, and results from early and later stages of PTOA development were compared. Synovial injury by arthrotomy (sham surgery) alone decreased primary chondrocyte expression of genes including Col2a1 (0.36±0.15-fold) and Acan (0.41±0.28-fold). Early PTOA synovium rescued the suppression of Acan, induced increased sGAG secretion (3.94±0.44μg/mL vs surgery-naïve 2.41±0.55 and sham 2.92±0.73μg/mL controls), and upregulated Mmp3 (3.73±2.62-fold) and Prg4 (4.93±4.29-fold). These effects were lost with later stage PTOA synovium. Early PTOA synovium induces transient anabolic responses in articular chondrocytes rather than pro-inflammatory responses that would require inhibition. These results suggest that PTOA synovium plays at least a partially protective role and that loss of these protective effects may contribute to PTOA progression.

Optical Biomarkers for the Diagnosis of Osteoarthritis through Raman Spectroscopy: Radiological and Biochemical Validation Using Ex Vivo Human Cartilage Samples.

Osteoarthritis (OA) is the most common rheumatic disease, characterized by progressive articular cartilage degradation. Raman spectroscopy (RS) has been recently proposed as a label-free tool to detect molecular changes in musculoskeletal tissues. We used cartilage samples derived from human femoral heads to perform an ex vivo study of different Raman signals and ratios, related to major and minor molecular components of articular cartilage, hereby proposed as candidate optical biomarkers for OA. Validation was performed against the radiological Kellgren–Lawrence (K-L) grading system, as a gold standard, and cross-validated against sulfated glycosaminoglycans (sGAGs) and total collagens (Hyp) biochemical contents. Our results showed a significant decrease in sGAGs (SGAGs, A1063 cm−1/A1004 cm−1) and proteoglycans (PGs, A1375 cm−1/A1004 cm−1) and a significant increase in collagen disorganization (ColD/F, A1245 cm−1/A1270 cm−1), with OA severity. These were correlated with sGAGs or Hyp contents, respectively. Moreover, the SGAGs/HA ratio (A1063 cm−1/A960 cm−1), representing a functional matrix, rich in proteoglycans, to a mineralized matrix-hydroxyapatite (HA), was significantly lower in OA cartilage (K-L I vs. III–IV, p < 0.05), whilst the mineralized to collagenous matrix ratio (HA/Col, A960 cm−1/A920 cm−1) increased, being correlated with K-L. OA samples showed signs of tissue mineralization, supported by the presence of calcium crystals-related signals, such as phosphate, carbonate, and calcium pyrophosphate dihydrate (MGP, A960 cm−1/A1004 cm−1, MGC, A1070 cm−1/A1004 cm−1 and A1050 cm−1/A1004 cm−1). Finally, we observed an increase in lipids ratio (IL, A1450 cm−1/A1670 cm−1) with OA severity. As a conclusion, we have described the molecular fingerprint of hip cartilage, validating a panel of optical biomarkers and the potential of RS as a complementary diagnostic tool for OA.

Mechanobiological Interactions between Dynamic Compressive Loading and Viscoelasticity on Chondrocytes in Hydrazone Covalent Adaptable Networks for Cartilage Tissue Engineering.

Mechanobiological cues influence chondrocyte biosynthesis and are often used in tissue engineering applications to improve the repair of articular cartilage in load-bearing joints. In this work, the biophysical effects of an applied dynamic compression on chondrocytes encapsulated in viscoelastic hydrazone covalent adaptable networks (CANs) is explored. Here, hydrazone CANs exhibit viscoelastic loss tangents ranging from (9.03± 0.01) 10-4 to (1.67± 0.09) 10-3 based on the molar percentages of alkyl-hydrazone and benzyl-hydrazone crosslinks. Notably, viscoelastic alkyl-hydrazone crosslinks improve articular cartilage specific gene expression showing higher SOX9 expression in free swelling hydrogels and dynamic compression reduces hypertrophic chondrocyte markers (COL10A1, MMP13) in hydrazone CANs. Interestingly, dynamic compression also improves matrix biosynthesis in elastic benzyl-hydrazone controls but reduces biosynthesis in viscoelastic alkyl-hydrazone CANs. Additionally, intermediate levels of viscoelastic adaptability demonstrate the highest levels of matrix biosynthesis in hydrazone CANs, demonstrating on average 70 ± 4µg of sulfated glycosaminoglycans per day and 31 ± 3µg of collagen per day over one month in dynamic compression bioreactors. Collectively, the results herein demonstrate the role of matrix adaptability and viscoelasticity on chondrocytes in hydrazone CANs during dynamic compression, which may prove useful for tissue engineering applications in load-bearing joints.

The study of fibroblast growth factor-2, sulfated glycosaminoglycans and oxyproline with mitral valve prolapse in combination with type 1 diabetes mellitus

The aim. The aim of the study was to conduct a comparative analysis of fibroblast growth factor-2, the total content and fractions of sulfated glycosoaminoglycans as well as free and peptide-bounding oxyproline as the markers of connective tissue metabolism in young patients with mitral valve prolapse, type 1 diabetes mellitus and the combination of both indications.&#x0D; Materials and methods. 93 patients between 19 and 33 years old with either mitral valve prolapse, or type 1 diabetes or the combination of both indications were examined. Group 1 was represented by 36 patients with the monomorbid type 1 diabetes mellitus. Group 2 consisted of 33 patients with type 1 diabetes mellitus and mitral valve prolapse. The comparison group included 24 people with diagnosed mitral valve prolapse. The concentration of fibroblast growth factor-2 in blood plasma was determined by the enzyme immunoassay using a Quantikine reagent kit (Human FGF basic Immunoassay), manufactured by R&amp;D Systems, Inc. (USA) and expressed in pg/ml. The total content and fractions of glycosaminoglycans in blood serum were determined by the method of N. G. Stern et al. and expressed in units of optical density. The level of free and peptide-bounding oxyproline in blood serum were determined by the method of P. N. Sharaev and expressed in µmol/L.&#x0D; Results. Fibroblast growth factor-2 was higher in group 2, where it was 23.7±0.25 pg/ml compared to the control group – 14.20±0.22 pg/ml (p &lt;0.01). There was also a significant difference in the levels of fibroblast growth factor-2 between groups 1 where it equaled 15.33±0.24 pg/ml and 2 – 23.71±0.25 pg/ml (p&lt;0.01). The total content of glycosaminoglycans in comparison with the control group, where it was equal to 9.7±0.62 odu, was higher in all groups of patients: in group 1 it was 12.07±1.04 odu, in the comparison group it was 11.75±0.83 odu, in the group with the combined pathology it was 13.32±1.59 odu (p&lt;0.05). The values of glycosaminoglycans II fraction, were higher in group 2 – 4.96±0.59 odu. The level of peptide-bound oxyproline, just as the level of free oxyproline, significantly increased in patients with comorbid pathology in group 2, compared to the control group: 16.06±1.54 µmol/l versus 8.7±0.81 µmol/l (p&lt;0.01) respectively.&#x0D; Conclusions. The values of fibroblast growth factor-2, glycosaminoglycans II fraction, free and peptide-bound oxyproline were significantly higher in patients with diabetes mellitus and mitral valve prolapse in comparison with the control group. Fibroblast growth factor-2 was higher in patients with comorbid pathology compared to the monomorbid diabetes patients.

Biochemical and Morphological Abnormalities of Subchondral Bone and Their Association with Cartilage Degeneration in Spontaneous Osteoarthritis.

This study aims to investigate how biochemical composition in subchondral bone (SB) relates to the sulfated glycosaminoglycan (sGAG) content of articular cartilage (AC) in the knee joint of guinea pigs from the early to moderate osteoarthritis (OA). Male Dunkin Hartley strain guinea pigs were grouped according to age (1, 3, 6, and 9months, with 10 guinea pigs in each group). The biochemical properties of the AC and SB in the tibial plateau of the guinea pigs were determined through histology and Raman spectroscopy, respectively. Furthermore, the microstructures of the SB were investigated using micro-computed tomography (micro-CT) and histology. Increased thickness and bone mineral density (BMD) and decreased porosity were observed in the subchondral plate (SP) with the progression of spontaneous OA, accompanied by a decreasing trend in sGAG integrated optical density (IOD) of AC. Compared with the changes in the microstructure of subchondral bone, the content of sGAG was more correlated to the changes in the mineral/matrix ratio of subchondral bone. The mineralization of the matrix was significantly correlated to the content of sGAG compared with crystallinity/maturity and Type B carbonate substitution. PO43- ν1/Amide III was more correlated to the content of sGAG than PO43- ν1/Amide I, PO43- ν1/CH2 wag during the progression of spontaneous osteoarthritis. This study demonstrated that the mineralization of subchondral bone plays a crucial role in the pathogenesis of OA. Future studies may access to the mineralization of subchondral bone in addition to its microstructure in the study for pathogenesis and early diagnosis of osteoarthritis.

Kaempferia parviflora Extract Alleviated Rat Arthritis, Exerted Chondroprotective Properties In Vitro, and Reduced Expression of Genes Associated with Inflammatory Arthritis.

Kaempferia parviflora Wall. ex Baker (KP) has been reported to attenuate cartilage destruction in rat model of osteoarthritis. Previously, we demonstrated that KP rhizome extract and its active components effectively suppressed mechanisms associated with RA in SW982 cells. Here, we further evaluated the anti-arthritis potential of KP extract by using multi-level models, including a complete Freund’s adjuvant-induced arthritis and a cartilage explant culture model, and to investigate the effects of KP extract and its major components on related gene expressions and underlying mechanisms within cells. In arthritis rats, the KP extract reduced arthritis indexes, with no significant changes in biological parameters. In the cartilage explant model, the KP extract exerted chondroprotective potential by suppressing sulfated glycosaminoglycans release while preserving high accumulation of proteoglycans. In human chondrocyte cell line, a mixture of the major components equal to their amounts in KP extract showed strong suppression the expression of genes-associated inflammatory joint disease similar to that of the extract. Additionally, KP extract significantly suppressed NF-κB and MAPK signaling pathways. The suppressing expression of necroptosis genes and promoted anti-apoptosis were also found. Collectively, these results provided supportive evidence of the anti-arthritis properties of KP extract, which are associated with its three major components.

A biphasic response of polymerized Type 1 collagen architectures to dermatan sulfate.

Collagen I, the most abundant extracellular matrix (ECM) protein in vertebrate tissues provides mechanical durability to tissue microenvironments and regulates cell function. Its fibrillogenesis in biological milieu is predominantly regulated by dermatan sulfate proteoglycans, proteins conjugated with iduronic acid-containing dermatan sulfate (DS) glycosaminoglycans (GAG). Although DS is known to regulate tissue function through its modulation of Coll I architecture, a precise understanding of the latter remains elusive. We investigated this problem by visualizing the fibrillar pattern of fixed Coll I gels polymerized in the presence of varying concentrations of DS using second harmonic generation microscopy. Measuring mean second harmonic generation signal (which estimates the ordering of the fibrils), and surface occupancy (which estimates the space occupied by fibrils) supported by confocal reflectance microscopy, our observations indicated that the effect on fibril pattern of DS is contextual upon the latter's concentrations: Lower levels of DS resulted in sparse disorganized fibrils; higher levels restore organization, with fibrils occupying greater space. An appropriate change in elasticity as a result of DS levels was also observed through atomic force microscopy. Examination of dye-based GAG staining and scanning electron microscopy suggested distinct constitutions of Coll I gels when polymerized with higher and lower levels of DS. We observed that adhesion of the invasive ovarian cancer cells SKOV3 decreased for lower DS levels but was partially restored at higher DS levels. Our study shows how the Coll I gel pattern-tuning of DS is of relevance for understanding its biomaterial applications and possibly, pathophysiological functions.

Synthetic heparan sulfate standards and machine learning facilitate the development of solid-state nanopore analysis

The application of solid-state (SS) nanopore devices to single-molecule nucleic acid sequencing has been challenging. Thus, the early successes in applying SS nanopore devices to the more difficult class of biopolymer, glycosaminoglycans (GAGs), have been surprising, motivating us to examine the potential use of an SS nanopore to analyze synthetic heparan sulfate GAG chains of controlled composition and sequence prepared through a promising, recently developed chemoenzymatic route. A minimal representation of the nanopore data, using only signal magnitude and duration, revealed, by eye and image recognition algorithms, clear differences between the signals generated by four synthetic GAGs. By subsequent machine learning, it was possible to determine disaccharide and even monosaccharide composition of these four synthetic GAGs using as few as 500 events, corresponding to a zeptomole of sample. These data suggest that ultrasensitive GAG analysis may be possible using SS nanopore detection and well-characterized molecular training sets.

Glycosaminoglycans: Carriers and Targets for Tailored Anti-Cancer Therapy.

The tumor microenvironment (TME) is composed of cancerous, non-cancerous, stromal, and immune cells that are surrounded by the components of the extracellular matrix (ECM). Glycosaminoglycans (GAGs), natural biomacromolecules, essential ECM, and cell membrane components are extensively altered in cancer tissues. During disease progression, the GAG fine structure changes in a manner associated with disease evolution. Thus, changes in the GAG sulfation pattern are immediately correlated to malignant transformation. Their molecular weight, distribution, composition, and fine modifications, including sulfation, exhibit distinct alterations during cancer development. GAGs and GAG-based molecules, due to their unique properties, are suggested as promising effectors for anticancer therapy. Considering their participation in tumorigenesis, their utilization in drug development has been the focus of both industry and academic research efforts. These efforts have been developing in two main directions; (i) utilizing GAGs as targets of therapeutic strategies and (ii) employing GAGs specificity and excellent physicochemical properties for targeted delivery of cancer therapeutics. This review will comprehensively discuss recent developments and the broad potential of GAG utilization for cancer therapy.

Cell-to-cell transmission of p53 aggregates: a novel player in oncology?

ABSTRACT The mutants of the tumor suppressor protein p53 form protein aggregates. It has been proposed that these aggregates propagate like prions, albeit the detailed mechanism of the propagation is unclear. Our recent study revealed that sulfated glycosaminoglycans, especially highly sulfated domains of heparan sulfate (heparan sulfate S-domains), participate in cancer pathology by mediating transcellular propagation of p53 aggregates.

Complete biosynthesis of a sulfated chondroitin in Escherichia coli

Sulfated glycosaminoglycans (GAGs) are a class of important biologics that are currently manufactured by extraction from animal tissues. Although such methods are unsustainable and prone to contamination, animal-free production methods have not emerged as competitive alternatives due to complexities in scale-up, requirement for multiple stages and cost of co-factors and purification. Here, we demonstrate the development of single microbial cell factories capable of complete, one-step biosynthesis of chondroitin sulfate (CS), a type of GAG. We engineer E. coli to produce all three required components for CS production–chondroitin, sulfate donor and sulfotransferase. In this way, we achieve intracellular CS production of ~27 μg/g dry-cell-weight with about 96% of the disaccharides sulfated. We further explore four different factors that can affect the sulfation levels of this microbial product. Overall, this is a demonstration of simple, one-step microbial production of a sulfated GAG and marks an important step in the animal-free production of these molecules.

Morphological aspects of orbital defect reconstruction in rats with elastin-based biomaterial

The objective of the study was to identify morphological aspects of replacement of xenogeneic decellularized elastin matrix (ХDEM) transplanted into a bone defect of the upper orbital wall in rats. Materials and methods. The experiment was performed on 60 Wistar rats with artificially created 7×4 mm defect in the upper edge of their orbit. In the experimental group (n=30), DХEM was placed in the defect area. Its size matched the size of the defect, and it was attached with a suture material (50 μm silk). Soft tissues were sutured layer by layer in the control group (n=30). Tissue excision was performed after 1, 3 and 12 months. Histological, immunohistochemical and electron microscopic methods were employed. Results. We were gradually replacing DХEM with bone tissue against the background of a pronounced reaction of CD68+/MMP-9+ macrophages, which implied its resorption and lysis. Osteogenesis occurred via intramembranous ossification and endochondral ossification, which was preceded by centripetal migration of endothelial kidneys with subsequent differentiation into capillaries and overgrowth of loose fibrous connective tissue containing progenitor cells. The microenvironment, represented by reticulin fibers, TGF-β1, and sulfated glycosaminoglycans, could contribute to the differentiation of progenitor cells in the osteogenic direction and to osteogenesis per se. In the control group, the defect remained open throughout the experiment. Conclusion. Decellularized biomaterial, based on elastin matrix, has osteoconductive and osteoinductive properties and can serve an adequate biomimetic for reconstruction of the bone defects.

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining.

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

Human Osteochondral Explants: Reliable Biomimetic Models to Investigate Disease Mechanisms and Develop Personalized Treatments for Osteoarthritis.

IntroductionLikely due to ignored heterogeneity in disease pathophysiology, osteoarthritis (OA) has become the most common disabling joint disease, without effective disease-modifying treatment causing a large social and economic burden. In this study we set out to explore responses of aged human osteochondral explants upon different OA-related perturbing triggers (inflammation, hypertrophy and mechanical stress) for future tailored biomimetic human models.MethodsHuman osteochondral explants were treated with IL-1β (10 ng/ml) or triiodothyronine (T3; 10 nM) or received 65% strains of mechanical stress (65% MS). Changes in chondrocyte signalling were determined by expression levels of nine genes involved in catabolism, anabolism and hypertrophy. Breakdown of cartilage was measured by sulphated glycosaminoglycans (sGAGs) release, scoring histological changes (Mankin score) and mechanical properties of cartilage.ResultsAll three perturbations (IL-1β, T3 and 65% MS) resulted in upregulation of the catabolic genes MMP13 and EPAS1. IL-1β abolished COL2A1 and ACAN gene expression and increased cartilage degeneration, reflected by increased Mankin scores and sGAGs released. Treatment with T3 resulted in a high and significant upregulation of the hypertrophic markers COL1A1, COL10A1 and ALPL. However, 65% MS increased sGAG release and detrimentally altered mechanical properties of cartilage.ConclusionWe present consistent and specific output on three different triggers of OA. Perturbation with the pro-inflammatory IL-1β mainly induced catabolic chondrocyte signalling and cartilage breakdown, while T3 initiated expression of hypertrophic and mineralization markers. Mechanical stress at a strain of 65% induced catabolic chondrocyte signalling and changed cartilage matrix integrity. The major strength of our ex vivo models was that they considered aged, preserved, human cartilage of a heterogeneous OA patient population. As a result, the explants may reflect a reliable biomimetic model prone to OA onset allowing for development of different treatment modalities.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40744-021-00287-y.

Iron triggers the early stages of cartilage degeneration in vitro: The role of articular chondrocytes

ObjectiveArthropathy is a major clinical problem in patients with hemochromatosis, the most common genetic disorder of iron overload. The pathological features of hemochromatosis arthropathy (HA) are heterogeneous and its specific nature remains unknown. One important drawback is the lack of proper in vitro models. The aim of the present study was to set up a model to investigate the biological response of cartilage to iron exposure. DesignBovine articular cartilage explants were incubated with ferric citrate for up to 9 days. We evaluated chondrocyte viability, iron deposition, and biomarkers of cartilage degradation in the conditioned medium. ResultsIron accumulated within chondrocytes, which was associated with programmed cell death through chondroptosis. Iron treatment increased the release of sulfated glycosaminoglycans (sGAG), a component of the extracellular matrix, into the medium (p=0.0189). This was dependent on the presence of viable chondrocytes and was associated with increased activity of matrix-degrading metalloproteinases (MMP) (pro/active MMP-9, p=0.0317; pro MMP-2, p=0.0092; active MMP-2, p=0.0288). Co-treatment with the broad MMP/aggrecanase inhibitor prinomastat reduced iron-mediated sGAG release (0.02 ​μM, p=0.0425; 2 ​μM, p=0.0014), confirming that iron induces sGAG release via the activation of catabolic enzymes. Notably, iron-treated cartilage continued to release an increased amount of sGAG into the medium for 6 days after termination of the ferric citrate treatment (p=0.0259). ConclusionsIron triggers the early stages of cartilage degeneration. Removal of iron exposure does not prevent further damage to the cartilage, thus providing a possible explanation why HA is not prevented after iron depletion by phlebotomy treatment.

Gelatin–Poly (γ-Glutamic Acid) Hydrogel as a Potential Adhesive for Repair of Intervertebral Disc Annulus Fibrosus

Study Design. An in vitro experimental study testing a Gelatin–poly (γ-glutamic acid) hydrogel for disc repair. Objective. To evaluate the cytocompatibility and degradability of the above mentioned hydrogel for intervertebral disc annular fibrosis (AF) repair. Summary of Background Data. No repair strategies for correcting annular defects in lumbar discectomy have been clinically well recognized. Exogenous supplementation of regenerative materials to fill defects is a minimally invasive way to restore compromised mechanical properties. The injected materials, most commonly gelatin-based materials with cross-linking agents, serve as sealants and as a scaffold for incorporating biomaterials for augmentation. However, cytotoxicity of hydrogel crosslinking agents is of concern in developing viable materials. Methods. This in vitro experimental study evaluated a newly developed gelatin-based hydrogel for intervertebral disc AF repair. Mechanical strength was augmented by γ-PGA, and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC) was used for material crosslinking. Isolated bovine tail intervertebral discs (IVDs) were used to test the hydrogel, and hydrogel surface monolayer AF cell culture was used to investigate efficacy in hydrogel constructs of different EDC concentrations. Cell metabolic activity was evaluated with Alamar blue assay, cell viability assay with live/dead stain, and sulfated glycosaminoglycan (GAG) and double strain DNA were quantified to evaluate proliferation of implanted cells and synthesis of extracellular matrix (ECM) proteins. Results. EDC concentrations from 10 to 40 mM resulted in significant decreases in AF cell proliferation without obvious influence on cell viability. Higher EDC concentrations resulted in decreased percentage of Alamar blue reduction and GAG and DNA concentration, but did not affect GAG/DNA and live–dead ratios. Degradation tests revealed that higher EDC concentrations decreased the hydrogel degradation rate. Conclusion. The developed gelatin–poly (γ-PGA) hydrogel with 20 mM EDC concentration provides an effective gap-filling biomaterial with good cytocompatibility, suggesting substantial promise for use as a sealant for small AF defects. Level of Evidence: N/A

CA10 regulates neurexin heparan sulfate addition via a direct binding in the secretory pathway

Neurexins are presynaptic adhesion molecules that shape the molecular composition of synapses. Diversification of neurexins in numerous isoforms is believed to confer synapse-specific properties by engaging with distinct ligands. For example, a subset of neurexin molecules carry a heparan sulfate (HS) glycosaminoglycan that controls ligand binding, but how this post-translational modification is controlled is not known. Here, we observe that CA10, a ligand to neurexin in the secretory pathway, regulates neurexin-HS formation. CA10 is exclusively found on non-HS neurexin and CA10 expressed in neurons is sufficient to suppress HS addition and attenuate ligand binding and synapse formation induced by ligands known to recruit HS. This effect is mediated by a direct interaction in the secretory pathway that blocks the primary step of HS biosynthesis: xylosylation of the serine residue. NMR reveals that CA10 engages residues on either side of the serine that can be HS-modified, suggesting that CA10 sterically blocks xylosyltransferase access in Golgi. These results suggest a mechanism for the regulation of HS on neurexins and exemplify a new mechanism to regulate site-specific glycosylations.

N-Glycans and sulfated glycosaminoglycans contribute to the action of diverse Tc toxins on mammalian cells

Tc toxin is an exotoxin composed of three subunits named TcA, TcB and TcC. Structural analysis revealed that TcA can form homopentamer that mediates the cellular recognition and delivery processes, thus contributing to the host tropism of Tc toxin. N-glycans and heparan sulfates have been shown to act as receptors for several Tc toxins. Here, we performed two independent genome-wide CRISPR-Cas9 screens, and have validated glycans and sulfated glycosaminoglycans (sGAGs) as Tc toxin receptors also for previously uncharacterized Tc toxins. We found that TcdA1 form Photorhabdus luminescens W14 (TcdA1W14) can recognize N-glycans via the RBD-D domain, corroborating previous findings. Knockout of N-glycan processing enzymes specifically blocks the intoxication of TcdA1W14-assembled Tc toxin. On the other hand, our results showed that sGAG biosynthesis pathway is involved in the cell surface binding of TcdA2TT01 (TcdA2 from P. luminescens TT01). Competition assays and biolayer interferometry demonstrated that the sulfation group in sGAGs is required for the binding of TcdA2TT01. Finally, based on the conserved domains of representative TcA proteins, we have identified 1,189 putative TcAs from 1,039 bacterial genomes. These TcAs are categorized into five subfamilies. Each subfamily shows a good correlation with both genetic organization of the TcA protein(s) and taxonomic origin of the genomes, suggesting these subfamilies may utilize different mechanisms for cellular recognition. Taken together, our results support the previously described two different binding modalities of Tc toxins, leading to unique host targeting properties. We also present the bioinformatics data and receptor screening strategies for TcA proteins, provide new insights into understanding host specificity and biomedical applications of Tc toxins.