Quantitative, compositional, and immunohistochemical analyses of chondroitin sulfate, dermatan sulfate, and hyaluronan in internal organs of deer (Cervus nippon centralis and C. n. yesoensis) and cattle (Bos taurus).

Chondroitin sulfate (CS) + dermatan sulfate (DS) and hyaluronan (HA) concentrations and the sulfation patterns of CS-DS in the cartilaginous tissues and alimentary canals of Honshu Sika deer, Hokkaido Sika deer, and cattle were investigated in the present study. CS + DS concentrations were high in cartilaginous tissues, namely, the trachea and scapular cartilage region (5- 12 g*), and low in the alimentary canal (~0.3 g*). HA concentrations were low in cartilaginous tissues and the alimentary canal (~0.2 g*). All tissues mainly contained A-type [HexAGalNAc(4-sulfate)] and C-type [HexAGalNAc(6-sulfate)] CS + DS. The ratios of A-type/C-type CS + DS were 1.2- 3.1 and 0.9- 16.4 in cartilaginous tissues and the alimentary canal, respectively. CS + DS predominantly comprised ß-D-GlcA and α-L-IdoA in cartilaginous tissues and the alimentary canal, respectively. The alimentary canal characteristically contained up to 14 % highly sulfated E-type [HexAGalNAc(4,6-disulfate)] and D-type [HexA(2-sulfate)GalNAc(6-sulfate)] CS + DS. The specific distributions of CS and DS were immunohistochemically confirmed using CS + DS-specific antibodies. Although the omasum of cattle is more likely to have higher concentrations of CS + DS and HA, no significant species differences were observed in the concentrations or sulfation patterns of CS + DS among species for Honshu Sika deer, Hokkaido Sika deer, and cattle. (*per 100 g of defatted dry tissue).

Qualitative and quantitative analyses in sulfated glycosaminoglycans, chondroitin sulfate/dermatan sulfate, during 3 T3-L1 adipocytes differentiation.

Chondroitin sulfate/dermatan sulfate (CS/DS) is a member of glycosaminoglycans (GAGs) found in animal tissues. Major CS/DS subclasses, O, A, C, D, and E units, exist based on the sulfation pattern in d-glucuronic acid (GlcA) and N-acetyl-d-galactosamine repeating units. DS is formed when GlcA is epimerized into l-iduronic acid. Our study aimed to analyze the CS/DS profile in 3 T3-L1 cells before and after adipogenic induction. CS/DS contents, molecular weight (Mw), and sulfation pattern were analyzed by using high-performance liquid chromatography. CS/DS synthesis- and sulfotransferase-related genes were analyzed by reverse transcription real-time PCR. CS/DS amount was significantly decreased in the differentiated (DI) group compared to the non-differentiated (ND) group, along with a lower expression of CS biosynthesis-related genes, chondroitin sulfate N-acetylgalactosaminyltransferase 1 and 2, as well as chondroitin polymerizing factor. GAGs in the DI group also showed lower Mw than those of ND. Furthermore, the A unit was the major CS/DS in both groups, with a proportionally higher CS-A in the DI group. This was consistent with the expression of carbohydrate sulfotransferase 12 that encodes chondroitin 4-O-sulfotransferase, for CS-A formation. These qualitative and quantitative changes in CS/DS and CS/DS-synthases before and after adipocyte differentiation reveal valuable insights into adipocyte development.

Involvement of langerin in the protective function of a keratan sulfate-based disaccharide in an emphysema mouse model.

Chronic obstructive pulmonary disease (COPD), which includes emphysema and chronic bronchitis, is now the third cause of death worldwide, and COVID-19 infection has been reported as an exacerbation factor of them. In this study, we report that the intratracheal administration of the keratan sulfate-based disaccharide L4 mitigates the symptoms of elastase-induced emphysema in a mouse model. To know the molecular mechanisms, we performed a functional analysis of a C-type lectin receptor, langerin, a molecule that binds L4. Using mouse BMDCs (bone marrow-derived dendritic cells) as langerin-expressing cells, we observed the downregulation of IL-6 and TNFa and the upregulation of IL-10 after incubation with L4. We also identified CapG (a macrophage-capping protein) as a possible molecule that binds langerin by immunoprecipitation combined with a mass spectrometry analysis. We identified a portion of the CapG that was localized in the nucleus and binds to the promoter region of IL-6 and the TNFa gene in BMDCs, suggesting that CapG suppresses the gene expression of IL-6 and TNFa as an inhibitory transcriptional factor. To examine the effects of L4 in vivo, we also generated langerin-knockout mice by means of genome editing technology. In an emphysema mouse model, the administration of L4 did not mitigate the symptoms of emphysema as well as the inflammatory state of the lung in the langerin-knockout mice. These data suggest that the anti-inflammatory effect of L4 through the langerin-CapG axis represents a potential therapeutic target for the treatment of emphysema and COPD.

Synthesis of biotinylated chondroitin sulfate DA and AD tetrasaccharides composed of hetero-type disaccharide units, and their interactions with the mAb MO-225.

Chondroitin sulfate (CS), a linear acidic polysaccharide, exhibits numerous biological activities that are dependent on sulfation patterns. CS oligosaccharides comprise repeating disaccharide units with different (hetero)-type sulfation patterns and are common in nature. We herein report the synthesis of the following biotinylated CS tetrasaccharides: CS-AD [ßGalNAc4S(1-4)ßGlcA(1-3)ßGalNAc6S(1-4)ßGlcA2S] and CS-DA [ßGalNAc6S(1-4)ßGlcA2S(1-3)ßGalNAc4S(1-4)ßGlcA], in a stereo-controlled manner. We also demonstrated that the CS-d-specific monoclonal antibody MO-225 bound more strongly to CS-DA than to CS-DD or -AD.

Altered sulfation status of FAM20C-dependent chondroitin sulfate is associated with osteosclerotic bone dysplasia.

Raine syndrome, a lethal osteosclerotic bone dysplasia in humans, is caused by loss-of-function mutations in FAM20C; however, Fam20c deficiency in mice does not recapitulate the human disorder, so the underlying pathoetiological mechanisms remain poorly understood. Here we show that FAM20C, in addition to the reported casein kinase activity, also fine-tunes the biosynthesis of chondroitin sulfate (CS) chains to impact bone homeostasis. Specifically, FAM20C with Raine-originated mutations loses the ability to interact with chondroitin 4-O-sulfotransferase-1, and is associated with reduced 4-sulfation/6-sulfation (4S/6S) ratio of CS chains and upregulated biomineralization in human osteosarcoma cells. By contrast, overexpressing chondroitin 6-O-sulfotransferase-1 reduces CS 4S/6S ratio, and induces osteoblast differentiation in vitro and higher bone mineral density in transgenic mice. Meanwhile, a potential xylose kinase activity of FAM20C does not impact CS 4S/6S ratio, and is not associated with Raine syndrome mutations. Our results thus implicate CS 4S/6S ratio imbalances caused by FAM20C mutations as a contributor of Raine syndrome etiology.

Synthesis of the matriglycan hexasaccharide, -3Xylα1-3GlcAß1-trimer and its interaction with laminin.

Matriglycan, a polysaccharide that is a pivotal part of the core M3 O-mannosyl glycan composed of the repeating disaccharide -3Xylα1-3GlcAß1-, interacts with laminin to stabilize muscle tissue. We herein report the synthesis of matriglycan-repeating hexasaccharides equipped with an alkyne linker to form glycoconjugates. The key step in the formation of an α-linked xylosyl glycoside was resolved by solvent-specific separation from an anomeric mixture. Successful glycan elongation was regio- and stereoselectively performed to obtain (-3Xylα1-3GlcAß1)3-O(C2H4O)3CH2CCH and the biotin conjugate. We also investigated interactions between matriglycan hexasaccharides and laminin-G-like domains 4 and 5 of laminin-α2 using saturation transfer difference-NMR. The dissociation constant obtained from bio-layer interferometry was estimated to be 7.5 × 10-8 M. These results indicate that a chemical approach may be applied to the reconstruction of muscle tissue.

Chemical and Chemo-Enzymatic Syntheses of Glycans Containing Ribitol Phosphate Scaffolding of Matriglycan.

Ribitol phosphate modifications to the core M3 O-mannosyl glycan are important for the functional maturation of α-dystroglycan. Three sequentially extended partial structures of the core M3 O-mannosyl glycan including a tandem ribitol phosphate were regio- and stereo-selectively synthesized: Rbo5P-3GalNAcß, Rbo5P-1Rbo5P-3GalNAcß, and Xylß1-4Rbo5P-1Rbo5P-3GalNAcß (Rbo5P, d-ribitol-5-phosphate; GalNAc, N-acetyl-d-galactosamine; Xyl, d-xylose). Rbo5P-3GalNAcß with p-nitrophenyl at the aglycon part served as a substrate for ribitol phosphate transferase (FKRP, fukutin-related protein), and its product was glycosylated by the actions of a series of glycosyltransferases, namely, ribitol xylosyltransferase 1 (RXYLT1), ß1,4-glucuronyltransferase 1 (B4GAT1), and like-acetyl-glucosaminyltransferase (LARGE). Rbo5P-3GalNAcß equipped with an alkyne-type aglycon was also active for FKRP. The molecular information obtained on FKRP suggests that Rbo5P-3GalNAcß derivatives are the minimal units required as the acceptor glycan for Rbo5P transfer and may serve as a precursor for the elongation of the core M3 O-mannosyl glycan.

Chondroitin Sulfates Control Invasiveness of the Basal-Like Breast Cancer Cell Line MDA-MB-231 Through ROR1.

Extracellular and cell surface chondroitin sulfates (CSs) regulate cancer cell properties, including proliferation and invasion. Thus, it is necessary to understand the mechanisms underlying their roles in cancer. Although we have shown that CS has an inherent ability to enhance the invasive activity of the human triple-negative breast cancer cell line MDA-MB-231, its molecular mechanism remains elusive. Here, we focused on receptor tyrosine kinase-like orphan receptor 1 (ROR1) and dickkopf WNT signaling pathway inhibitor 1 (DKK1). MDA-MB-231 cells express high levels of ROR1; their invasive potential depends on ROR1 signaling. Although accumulating evidence has demonstrated that ROR1 is associated with aggressive breast-cancer phenotypes, the whole picture of its biological function remains poorly understood. In this study, we examined whether CS controls ROR1 function. Surface plasmon resonance analysis indicated that CSs were bound to ROR1 in the presence of WNT5A. The invasive activity of MDA-MB-231 cells enhanced by CSs was completely suppressed by ROR1 knockdown. In addition, knockdown of the CS biosynthetic enzymes CHST11 and CHST15 inhibited invasive activity, even in the presence of ROR1. These results suggest that CS is required to induce an ROR1-dependent, aggressive MDA-MB-231 phenotype. ROR1 signaling in MDA-MB-231 cells activated c-Jun N-terminal kinase (JNK), leading to increased invasive potential; moreover, exogenous CSs activated JNK. MDA-MB-231 cells express DKK1, a tumor suppressor factor that binds to CS, at high levels. Knockdown of DKK1 enhanced CS-stimulated tumor invasion activity of MDA-MB-231 cells, suggesting that DKK1 sequesters CS to block ROR1/JNK signaling. These results showed that CSs promotes cancer aggressiveness through the ROR1-JNK axis in MDA-MB-231 cells.

Compositional analysis of chondroitin/dermatan sulfate in rhesus monkeys (Macaca mulatta).

Glycosaminoglycans (GAGs) are found in various tissues and are involved in many physiological functions. Since the rhesus monkey (Macaca mulatta) is the most widely used nonhuman primate in biomedical research, an understanding of the compositions of GAGs in their tissues is important. The aim of this study was to determine the content and sulfation pattern of disaccharides contained in several tissues of the rhesus monkey. The chondroitin sulfate (CS)/dermatan sulfate (DS) hybrid chain was extracted from several tissues of female and male rhesus monkeys. Compositional analysis was performed after digestion with chondroitinases ABC and ACI to reveal the sulfation pattern of the CS/DS hybrid chain. This study revealed that the major CS/DS disaccharide units present in the tissues were A and C types. The E and iE types were specifically distributed not only in the tracheal tissue but also in gastrointestinal tissues.

Re-expansion pulmonary edema after thoracentesis for iatrogenic pneumothorax and severe subcutaneous emphysema.

Subclavian central venous catheterization can cause severe complications, including tension pneumothorax, subcutaneous emphysema, and pneumomediastinum. Re-expansion pulmonary edema after thoracentesis is a life-threatening complication.

Reconsideration of the Semaphorin-3A Binding Motif Found in Chondroitin Sulfate Using Galnac4s-6st-Knockout Mice.

The chondroitin sulfate (CS)-rich dense extracellular matrix surrounding neuron cell bodies and proximal dendrites in a mesh-like structure is called a perineuronal net (PNN). CS chains in PNNs control neuronal plasticity by binding to PNN effectors, semaphorin-3A (Sema3A) and orthodenticle homeobox 2. Sema3A recognizes CS-containing type-E disaccharide units (sulfated at O-4 and O-6 of N-acetylgalactosamine). Type-E disaccharide units are synthesized by N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST). In this study, we demonstrated that Sema3A accumulates in the PNNs surrounding parvalbumin cells, even in mice deficient in GalNAc4S-6ST. In addition, there were no differences in the number and structure of PNNs visualized by Cat316 antibody and Wisteria floribunda lectin, which recognize CS chains, between wild type and GalNAc4S-6ST knockout mice. Therefore, we re-examined the Sema3A binding motif found in CS chains using chemically synthesized CS tetrasaccharides. As a result, we found that non-sulfated GalNAc residues at the non-reducing termini of CS chains are required for the binding of Sema3A.

Stereo- and Regioselective Synthesis of O-Mannosyl Glycan Containing Matriglycan and a Part of Tandem Ribitol Phosphate.

We herein successfully synthesized two pivotal structures of O-mannosyl glycan: (1) the matriglycan-repeating tetrasaccharide Xylα1-3GlcAß1-3Xylα1-3GlcAß and (2) the link between matriglycan and a part of tandem ribitol phosphate, Xylα1-3GlcAß1-4Xylß1-4Rbo, in a regio- and stereocontrolled manner. The disaccharide unit with the α-linkage of xylose was obtained by adopting the conformational fixation of the xylopyranoside ring and a specific solvation system of diastereoselective solubility.

Accurate and quick predictor of necrotizing soft tissue infection: Usefulness of the LRINEC score and NSTI assessment score.

OBJECTIVE: The Laboratory Risk Indicator for Necrotizing Fasciitis (LRINEC) score is a diagnostic tool for necrotizing soft tissue infection (NSTI), which is validated and is considered to have high diagnostic value. However, some experts criticize LRINEC score for consisting of laboratory test results only. METHODS: In this single-center retrospective study, we created a new scoring system (NSTI assessment score; NAS), which also incorporated vital signs as another diagnostic tool for NSTI using cases from our hospital and also evaluated diagnostic accuracy of LRINEC score. We identified NSTI predictors by comparing 24 NSTI patients and 80 non NSTI patients using uni- and multivariate logistic regression analysis, and created NAS based on odds ratio of variables which are statistically significant in the multivariate model. RESULTS: We identified mean arterial pressure, C-reactive protein, hemoglobin, serum creatinine, and glucose as a predictor for NSTI. The maximum value of NAS was 11 points with the cut-off value of 6. Sensitivity, specificity, positive predictive value, and negative predictive value of the NAS for diagnosis of NSTI were 87.5%, 91.3%, 75.0%, and 96.1%, respectively. Area under the receiver operating characteristic curve was 0.926 (0.851-1.00) for the NAS and 0.903 (0.833-0.973) for the LRINEC score, and they were not statistically different (p = 0.167). CONCLUSION: The NAS has high diagnostic accuracy in predicting NSTI, and is comparable with the LRINEC score. The NAS needs to be validated in other cohorts in the future.

A folded-blister pill pack causes ileal perforation.

Ileal perforation by an accidental ingestion of a blister pill pack is rare. A detailed history of the present illness is the initial key to proceed appropriate investigations. Computed tomography is the gold standard for diagnosis of the disease.

Key diagnostic characteristics of fever of unknown origin in Japanese patients: a prospective multicentre study.

OBJECTIVE: To identify the key diagnostic features and causes of fever of unknown origin (FUO) in Japanese patients. DESIGN: Multicentre prospective study. SETTING: Sixteen hospitals affiliated with the Japanese Society of Hospital General Medicine, covering the East and West regions of Japan. PARTICIPANTS: Patient aged ≥20 years diagnosed with classic FUO (axillary temperature≥38.0°C at least twice within a 3-week period, cause unknown after three outpatient visits or 3 days of hospitalisation). A total of 141 cases met the criteria and were recruited from January 2016 to December 2017. INTERVENTION: Japanese standard diagnostic examinations. OUTCOME MEASURES: Data collected include usual biochemical blood tests, inflammatory markers (erythrocyte sedimentation rate (ESR), C reactive (CRP) protein level, procalcitonin level), imaging results, autopsy findings (if performed) and final diagnosis. RESULTS: The most frequent age group was 65-79 years old (mean: 58.6±9.1 years). The most frequent cause of FUO was non-infectious inflammatory disease. After a 6-month follow-up period, 21.3% of cases remained undiagnosed. The types of diseases causing FUO were significantly correlated with age and prognosis. Between patients with and without a final diagnosis, there was no difference in CRP level between patients with and without a final diagnosis (p=0.121). A significant difference in diagnosis of a causative disease was found between patients who did or did not receive an ESR test (p=0.041). Of the 35 patients with an abnormal ESR value, 28 (80%) had causative disease identified. CONCLUSIONS: Age may be a key factor in the differential diagnosis of FUO; the ESR test may be of value in the FUO evaluation process. These results may provide clinicians with insight into the management of FUO to allow adequate treatment according to the cause of the disease.

Cardiac involvement in heavy and light chain amyloidosis: A case report and literature review.

INTRODUCTION: Heavy and light chain amyloidosis is an extremely rare condition. There are few reports referring to the clinical impact of cardiac involvement in heavy and light chain amyloidosis, and the significance of myocardial impairment has not yet been completely explained. PATIENT CONCERNS: A 66-year-old Japanese man was admitted to our hospital presenting with nephrotic syndrome and congestive heart failure. DIAGNOSIS: Kidney and endoscopic gastric mucosal biopsy demonstrated congophilic hyalinization in most of the glomeruli and surrounding vessel walls, which were highly positive for immunoglobulin A and lambda. Finally, the patient was diagnosed as an atypical multiple myeloma with systemic heavy and light chain amyloidosis. INTERVENTIONS: The patient was referred to hematology for further treatment and was moved to another hospital for the administration of chemotherapy using melphalan and dexamethasone. OUTCOMES: The patient was still alive after 15-month follow-up from the initial diagnosis. CONCLUSION: Initial screening and follow-up for cardiac involvement are important for heavy and light chain amyloidosis. Further investigation for the prognosis of heavy and light chain amyloidosis is required to improve the strategies of diagnosis and treatment options for patients with this disease.

Chondroitin sulfate-D promotes neurite outgrowth by acting as an extracellular ligand for neuronal integrin αVß3.

BACKGROUND: Chondroitin sulfate (CS) chains are prominent extra/pericellular matrix components in the central nervous system (CNS) and can exert positive or negative regulatory effects on neurite outgrowth, depending on the CS structure and the amount. Despite the remarkable abilities of highly sulfated forms of CS chains to enhance neurite outgrowth, the neuronal recognition systems for such promotional CS chains, including CS-D polysaccharide, remain to be fully elucidated. METHODS: We explored the molecular basis of the CS-D-mediated neurite extension using primary hippocampal neurons cultured on substrate precoated with CS-D polysaccharides, and evaluated functional involvement of a distinct integrin heterodimer as a novel neuronal CS receptor for CS-D. RESULTS: We identified an extracellular matrix receptor, integrin αVß3, as a functional receptor for CS-D. CS-D, but not CS-C (a precursor form of CS-D) showed significant binding affinity toward recombinant integrin αVß3 heterodimer and activated intracellular signaling(s) involving focal adhesion kinase (FAK) and Src/Fyn kinase. Functional blockade of the respective players for integrin signaling abrogated the promotional effects of CS-D. We also found the existence of CS-D-induced integrin activation system in neuronal stem/progenitor cell population. CONCLUSIONS: The neuronal cell surface integrin αVß3 can function as a CS receptor for a highly sulfated CS subtype, CS-D. GENERAL SIGNIFICANCE: Our findings are the first to demonstrate that CS-dependent neurite outgrowth promotion is exerted via direct activation of specific integrin heterodimers on neuronal cell surfaces, providing new insights into understanding the CS-sensing machineries that regulate CNS development and regeneration.

Glycan sulfation patterns define autophagy flux at axon tip via PTPRσ-cortactin axis.

Chondroitin sulfate (CS) and heparan sulfate (HS) are glycosaminoglycans that both bind the receptor-type protein tyrosine phosphatase PTPRσ, affecting axonal regeneration. CS inhibits axonal growth, while HS promotes it. Here, we have prepared a library of HS octasaccharides and, together with synthetic CS oligomers, we found that PTPRσ preferentially interacts with CS-E-a rare sulfation pattern in natural CS-and most HS oligomers bearing sulfate and sulfamate groups. Consequently, short and long stretches of natural CS and HS, respectively, bind to PTPRσ. CS activates PTPRσ, which dephosphorylates cortactin-herein identified as a new PTPRσ substrate-and disrupts autophagy flux at the autophagosome-lysosome fusion step. Such disruption is required and sufficient for dystrophic endball formation and inhibition of axonal regeneration. Therefore, sulfation patterns determine the length of the glycosaminoglycan segment that bind to PTPRσ and define the fate of axonal regeneration through a mechanism involving PTPRσ, cortactin and autophagy.

Compositional analysis of the glycosaminoglycan family in velvet antlers of Sika deer (Cervus nippon) at different growing stages.

Glycosaminoglycans (GAG) from the velvet antlers of Sika deer (Cervus nippon) at the different growing stages (Fukurozuno, Anshi, and Santajo) of bred and wild deer were isolated and their concentrations and sulfation patterns were analyzed. GAG were digested with chondroitinase ABC, ACI, heparinase-I and -III, and keratanase-II into the corresponding repeating disaccharides of chondroitin sulfate (CS), dermatan sulfate (DS), hyaluronan, heparan sulfate (HS), and keratan sulfate. Cartilaginous tissues contained CS-DS at high concentrations with an almost equal ratio of 4- and 6-sulfates, while 4-sulfate-type CS-DS predominantly occupied ossified tissues, but at low concentrations. High O- and N-sulfation degrees of HS correspond to high ossification. Dynamic quantitative changes in CS-DS and compositional changes in CS-DS and HS were closely associated with the mineralization of deer antlers.

Can the Wish to Receive Intensive Treatment in Elderly Patients with Respiratory Tract Infection Be Predicted?

Objective Almost no Japanese elderly patients have an advance directive (AD). Our aim was to determine whether or not the wish to receive intensive care in elderly patients with respiratory tract infection could be predicted from the prehospital data. Methods In this retrospective study, we reviewed patients ≥65 years of age with respiratory tract infection who had been transferred to our hospital by ambulance between September 2014 and August 2016. The patients were divided into two groups according to whether or not they wished to receive intensive treatment. We placed patients without a wish to receive intensive treatments (WITs) in Group A and patients with a WITs in Group B. We then analyzed parameters that could be determined in the prehospital phase and compared the findings between the groups. Results Thirty-seven patients were in Group A, and 67 patients were in Group B. None of the patients in this study had an AD. There were significant differences in the age, rate of residence in an extended care facility, frequency of inability to care for oneself fully, frequency of dementia, number of prescribed drugs, and Glasgow coma scale (GCS) on a univariate analysis. A logistic regression analysis showed that the inability to care for oneself fully [odds ratio (OR): 4.521, 95% confidence interval (CI): 2.024-10.096, p<0.001] and a low GCS (OR 0.885, 95%CI 0.838-0.935, p<0.001) were related to a WITs. Conclusion Elderly patients who are unable to care for themselves and who have a low GCS in the prehospital stage are likely not to want intensive treatment.