Structural characterization of a distinct fucan sulfate from Pattalus mollis through an oligosaccharide mapping approach.

The elucidation of the precise structure of fucan sulfate is essential for understanding the structure-activity relationship and promoting potential biomedical applications. In this work, the structure of a distinct fucan sulfate fraction V (PmFS in Ref 15 and FSV in Ref 16 → PFV) from Pattalus mollis was investigated using an oligosaccharide mapping approach. Six size-homogeneous fractions were purified from the mild acid hydrolyzed PFV and identified as fucitols, disaccharides and trisaccharides by 1D/2D NMR and MS analysis. Significantly, the sulfation pattern, glycosidic linkages, and sequences of all the oligosaccharides were unambiguously identified. The common 2-desulfation of the reducing end residue of the oligosaccharides was observed. Overall, the backbone of PFV was composed of L-Fuc2S (major) and L-Fuc3S (minor) linked by α1,4 glycosidic bonds. Importantly, the branches contain both monosaccharide and disaccharide linked to the backbone by α1,3 glycosidic linkages. Thus, the tentative structure of natural PFV was shown to be {-(R-α1,3)-L-Fuc2S-α1,4-(L-Fuc2S/3S-α1,4)x-}n, where R is L-Fuc(2S)4S-α1,3/4-L-Fuc4S(0S)- or L-Fuc(2S)4S-. Our results provide insight into the heterogeneous structure of the fucan sulfate found in sea cucumbers. Additionally, PFV and its fractions showed strong anticoagulant and anti-iXase activities, which may be related to the distinct structure of PFV.

Structural Characterization and Anticoagulant Activities of a Keratan Sulfate-like Polysaccharide from the Sea Cucumber Holothuria fuscopunctata.

A sulfated polysaccharide (AG) was extracted and isolated from the sea cucumber H. fuscopunctata, consisting of GlcNAc, GalNAc, Gal, Fuc and lacking any uronic acid residues. Importantly, several chemical depolymerization methods were used to elucidate the structure of the AG through a bottom-up strategy. A highly sulfated galactose (oAG-1) and two disaccharides labeled with 2,5-anhydro-D-mannose (oAG-2, oAG-3) were obtained from the deaminative depolymerized product along with the structures of the disaccharide derivatives (oAG-4~oAG-6) identified from the free radical depolymerized product, suggesting that the repeating building blocks in a natural AG should comprise the disaccharide ß-D-GalS-1,4-D-GlcNAc6S. The possible disaccharide side chains (bAG-1) were obtained with mild acid hydrolysis. Thus, a natural AG may consist of a keratan sulfate-like (KS-like) glycosaminoglycan with diverse modifications, including the sulfation types of the Gal residue and the possible disaccharide branches α-D-GalNAc4S6S-1,2-α/ß-L-Fuc3S linked to the KS-like chain. Additionally, the anticoagulant activities of the AG and its depolymerized products (dAG1-9) were evaluated in vitro using normal human plasma. The AG could prolong activated partial thromboplastin time (APTT) in a dose-dependent manner, and the activity potency was positively related to the chain length. The AG and dAG1-dAG3 could prolong thrombin time (TT), while they had little effect on prothrombin time (PT). The results indicate that the AG could inhibit the intrinsic and common coagulation pathways.

Comparative Assessment of APTT Reagents for Evaluating Anticoagulant Sensitivity of Fucosylated Glycosaminoglycans (FGs) Derived from Sea Cucumbers.

Fucosylated glycosaminoglycans (FGs) derived from sea cucumbers exhibit potent intrinsic Xase (iXase) inhibition, anticoagulation, and antithrombosis. Plasma activated partial thromboplastin time (APTT), a widely used screening test worldwide, is crucial for evaluating anticoagulant efficacy. However, the applicability of these commercially available APTT reagents for assessing anticoagulation of FGs remains unreported. In this study, we investigated the disparity between ellagic acid and colloidal silica APTT reagents in evaluating anticoagulation of dHG-5 and dHLFG-4, two depolymerized FGs, and elucidated the underlying rationale. The results demonstrated that dHG-5 and dHLFG-4 exhibited heightened sensitivity to the ellagic acid APTT reagent both in vitro and in vivo, and did not significantly affect the activation of APTT reagents for plasma. In addition, both ellagic acid and colloidal silica APTT reagents inhibited the anti-iXase of dHG-5 and dHLFG-4, and the inhibition of the ellagic acid APTT reagent was less pronounced compared to the colloidal silica APTT reagent. These findings suggest that the reduced impact of the ellagic acid APTT reagent on the anti-iXase activity of dHG-5 and dHLFG-4 is responsible for the increased sensitivity in plasma APTT analysis. This study offers valuable insights into the characteristics of two APTT reagents applied for assessing the anticoagulant activity of FG-related compounds.

BRISC is required for optimal activation of NF-κB in Kupffer cells induced by LPS and contributes to acute liver injury.

BRISC (BRCC3 isopeptidase complex) is a deubiquitinating enzyme that has been linked with inflammatory processes, but its role in liver diseases and the underlying mechanism are unknown. Here, we investigated the pathophysiological role of BRISC in acute liver failure using a mice model induced by D-galactosamine (D-GalN) plus lipopolysaccharide (LPS). We found that the expression of BRISC components was dramatically increased in kupffer cells (KCs) upon LPS treatment in vitro or by the injection of LPS in D-GalN-sensitized mice. D-GalN plus LPS-induced liver damage and mortality in global BRISC-null mice were markedly attenuated, which was accompanied by impaired hepatocyte death and hepatic inflammation response. Constantly, treatment with thiolutin, a potent BRISC inhibitor, remarkably alleviated D-GalN/LPS-induced liver injury in mice. By using bone marrow-reconstituted chimeric mice and cell-specific BRISC-deficient mice, we demonstrated that KCs are the key effector cells responsible for protection against D-GalN/LPS-induced liver injury in BRISC-deficient mice. Mechanistically, we found that hepatic and circulating levels of TNF-α, IL-6, MCP-1, and IL-1ß, as well as TNF-α- and MCP-1-producing KCs, in BRISC-deleted mice were dramatically decreased as early as 1 h after D-GalN/LPS challenge, which occurred prior to the elevation of the liver injury markers. Moreover, LPS-induced proinflammatory cytokines production in KCs was significantly diminished by BRISC deficiency in vitro, which was accompanied by potently attenuated NF-κB activation. Restoration of NF-κB activation by two small molecular activators of NF-κB p65 effectively reversed the suppression of cytokines production in ABRO1-deficient KCs by LPS. In conclusion, BRISC is required for optimal activation of NF-κB-mediated proinflammatory cytokines production in LPS-treated KCs and contributes to acute liver injury. This study opens the possibility to develop new strategies for the inhibition of KCs-driven inflammation in liver diseases.

Branch distribution pattern and anticoagulant activity of a fucosylated chondroitin sulfate from Phyllophorella kohkutiensis.

Fucosylated chondroitin sulfate (FCS) extracted from Phyllophorella kohkutiensis (PkFCS) is composed of d-GalNAc, d-GlcA, l-Fuc and -SO42-. According to the defined structures revealed by NMR spectra of the branches released by mild acid hydrolysis and oligosaccharides generated by ß-eliminative depolymerization, the backbone of PkFCS is CS-E, and the branch types attached to C-3 of d-GlcA include l-Fuc2S4S, l-Fuc3S4S, l-Fuc4S, and the disaccharide α-d-GalNAc-1,2-α-l-Fuc3S4S with the ratio of 43:13:22:22. Notably, novel heptasaccharide and hendecasaccharide were identified that are branched with continuous distribution of the disaccharide. The structural sequences of the oligosaccharides indicate that three unique structural motifs are present in the entire PkFCS polymer, including a motif branched with randomly distributed different sulfated l-Fuc units, a motif containing regular l-Fuc2S4S branches and a motif enriched in α-d-GalNAc-1,2-α-l-Fuc3S4S. This is the first report about the distribution pattern of diverse branches in natural FCS. Natural PkFCS exhibited potent anticoagulant activity on APTT prolonging and anti-iXase activity. Regarding the structurally defined oligosaccharides with sulfated fucosyl side chains, octasaccharide (Pk4b) is the minimum fragment responsible for its anticoagulant activity correlated with anti-iXase. However, further glycosyl modification with a non-sulfated d-GalNAc at the C-2 position of l-Fuc3S4S could significantly decrease the anticoagulant and anti-iXase activity.

Lysine 117 Residue Is Essential for the Function of the Hepatocyte Nuclear Factor 1α.

Hepatocyte nuclear factor 1α (HNF1α) plays essential roles in controlling development and metabolism; its mutations are clearly linked to the occurrence of maturity-onset diabetes of the young (MODY3) in humans. Lysine 117 (K117) to glutamic acid (E117) mutation in the HNF1α gene has been clinically associated with MODY3, but no functional data on this variant are available. Here, we addressed the role of lysine 117 in HNF1α function using a knock-in animal model and site-directed mutagenesis. HNF1α K117E homozygous mice exhibited dwarfism, hepatic dysfunction, renal Fanconi syndrome, and progressive wasting syndrome. These phenotypes were very similar to those of mice with complete HNF1α deficiency, suggesting that K117 is critical to HNF1α functions. K117E homozygotes developed diabetes in the early postnatal period. The relative deficiency of serum insulin levels and the normal response to insulin treatment in homozygous mice were markedly similar to those in the MODY3 disorder in humans. Moreover, K117E heterozygous mutant causes age-dependent glucose intolerance, which is similar to the pathogenesis of MODY3 as well. K117 mutants significantly reduced the overall transactivation and DNA binding capacity of HNF1α by disrupting dimerization. Collectively, our findings reveal a previously unappreciated role of POU domain of HNF1α in homodimerization and provide important clues for identifying the molecular basis of HNF1α-related diseases such as MODY3. ARTICLE HIGHLIGHTS: HNF1α K117E homozygous mice exhibited dwarfism, hepatic dysfunction, renal Fanconi syndrome, and progressive wasting syndrome. K117E homozygotes developed diabetes in the early postnatal period. K117E heterozygous mutant causes age-dependent glucose intolerance, which is similar to the pathogenesis of maturity-onset diabetes of the young. K117 mutants significantly reduced the overall transactivation and DNA binding capacity of HNF1α by disrupting dimerization.

VPS37C facilitates erythroid differentiation by promoting EKLF stability.

The process of erythroid differentiation is orchestrated at the molecular level by a complex network of transcription factors. Erythroid Krüppel-like factor (EKLF/KLF1) is a master erythroid gene regulator that directly regulates most aspects of terminal erythroid differentiation. However, the underlying regulatory mechanisms of EKLF protein stability are still largely unknown. In this study, we identified Vacuolar protein sorting 37 C (VPS37C), a core subunit of the Endosomal sorting complex required for transport-I (ESCRT-I) complex, as an essential regulator of EKLF stability. Our study showed that VPS37C interacts with EKLF and prevents K48-linked polyubiquitination of EKLF and proteasome-mediated EKLF degradation, thus enhancing EKLF protein stability and transcriptional activity. VPS37C overexpression in murine erythroleukemia (MEL) cells promotes hexamethylene bisacetamide (HMBA)-induced erythroid differentiation manifested by up-regulating erythroid-specific EKLF target genes and increasing benzidine-positive cells. In contrast, VPS37C knockdown inhibits HMBA-induced MEL cell erythroid differentiation. Particularly, the restoration of EKLF expression in VPS37C-knockdown MEL cells reverses erythroid-specific gene expression and hemoglobin production. Collectively, our study demonstrated VPS37C is a novel regulator of EKLF ubiquitination and degradation, which plays a positive role in erythroid differentiation of MEL cells by enhancing EKLF protein stability.

Oligomer-guided recognition of two fucan sulfate from Bohadschia argus and inhibition of P-selectin binding to its ligand.

Fucan sulfate (FS) from sea cucumber shows intriguing structure and extensive activities. Here, three homogeneous FS (BaFSI - III) were obtained from Bohadschia argus, followed with physicochemical properties analyses including monosaccharide composition, molecular weight, and sulfate content. BaFSI was proposed to carry a unique distribution pattern of sulfate groups as a novel sequence composed of domain A and domain B that formed by different FucS residues, markedly differing from FS reported before, according to the analyses of 12 oligosaccharides and a representative residual saccharide chain. BaFSII possessed a highly regular structure {4-L-Fuc3S-α1,}n according to its peroxide depolymerized product. BaFSIII was confirmed as a FS mixture bearing similar structural characteristics with BaFSI and BaFSII by means of mild acid hydrolysis and oligosaccharide analysis. Bioactivity assays showed that BaFSI and BaFSII could potently inhibit P-selectin binding to PSGL-1 and HL-60 cells. Structure-activity relationship analysis showed that molecular weight and sulfation pattern were the essential factors for the potent inhibition. Meanwhile, an acid hydrolysate of BaFSII with a molecular weight about 15 kDa exhibited a comparable inhibition with the native BaFSII. Given the potent activity and highly regular structure of BaFSII, it shows great potential for development as a P-selectin inhibitor.

Analysis of the Risk Factors for Negative Emotions in Patients with Esophageal Cancer During the Peri-Radiotherapy Period and Their Effects on Malnutrition.

Background: Esophageal cancer has a high incidence in China. Many patients also have a heavy psychological burden due to clinical features such as wasting and choking on food. This study analyzed the risk factors of negative emotions in esophageal cancer patients during the peri-radiotherapy period and its effects on malnutrition. Methods: We retrospectively analyzed 339 patients with esophageal cancer during the peri-radiotherapy who received treatment at our hospital from April 2017 to April 2020, and followed up for 3 years. t test and Chi-square test were used to analyze the relationship between patients' negative emotions and clinical data. Binary logistics regression was performed to analyze the independent risk factors for the occurrence of negative mood and malnutrition in the patients. Kaplan-Meier survival curves were used to analyze survival rates. Results: Our results showed that 18.3% of patients undergoing radiotherapy for esophageal cancer had negative emotions, and 41.9% suffered from malnutrition. The results of the binary logistic regression analysis showed that monthly household income (OR = 0.470, P = 0.022), the TNM stage (OR = 2.030, P = 0.044), concomitant gastrointestinal symptoms (OR = 2.071, P = 0.024), sleep status (OR = 2.540, P = 0.003), swallowing disorders (OR = 1.919, P = 0.048), and post-radiotherapy complications were independent risk factors for the development of negative emotions in patients. Negative emotions (OR = 2.547, P = 0.038) were also a risk factor for malnutrition in patients with esophageal cancer. Conclusion: Many patients with esophageal cancer suffer from anxiety and depression in the peri-radiotherapy period, which might lead to complications such as malnutrition or aggravate and affect the prognosis of patients. Therefore, psychological care should be provided based on conventional care to effectively relieve their psychological pressure, and improve their prognosis and quality of life.

Branched Chondroitin Sulfate Oligosaccharides Derived from the Sea Cucumber Acaudina molpadioides Stimulate Neurite Outgrowth.

Fucosylated chondroitin sulfate (FCS) from the sea cucumber Acaudina molpadioides (FCSAm) is the first one that was reported to be branched by disaccharide GalNAc-(α1,2)-Fuc3S4S (15%) and sulfated Fuc (85%). Here, four size-homogenous fractions, and seven oligosaccharides, were separated from its ß-eliminative depolymerized products. Detailed NMR spectroscopic and MS analyses revealed the oligomers as hexa-, hepta-, octa-, and nonasaccharide, which further confirmed the precise structure of native FCSAm: it was composed of the CS-E-like backbone with a full content of sulfation at O-4 and O-6 of GalNAc in the disaccharide repeating unit, and the branches consisting of sulfated fucose (Fuc4S and Fuc2S4S) and heterodisaccharide [GalNAc-(α1,2)-Fuc3S4S]. Pharmacologically, FCSAm and its depolymerized derivatives, including fractions and oligosaccharides, showed potent neurite outgrowth-promoting activity in a chain length-dependent manner. A comparison of analyses among oligosaccharides revealed that the sulfate pattern of the Fuc branches, instead of the heterodisaccharide, could affect the promotion intensity. Fuc2S4S and the saccharide length endowed the neurite outgrowth stimulation activity most.

Mild acid hydrolysis on Fucan sulfate from Stichopus herrmanni: Structures, depolymerization mechanism and anticoagulant activity.

Fucan sulfate (FS) from sea cucumbers possesses linear sequences with repeating units that differ principally in the pattern of sulfation and position of glycosidic linkage. FS from Stichopus herrmanni (ShFS) was preliminarily identified as the relatively simple structure {-3)-L-Fuc2S-(α1-}n. Herein, mild acid hydrolysis was employed on ShFS to yield 21 oligosaccharides. Analyses on their structures complemented the features of ShFS to refine its spectral signal assignments. Combining with the methylation analysis, unit L-Fuc2S4S was determined as the micro-component in its intact structure. The irregularity came from minor sulfation on O-4. Temperature and acid concentration were the critical parameters to the depolymerization and formation of oligosaccharides. Meanwhile, a three-step cleavage of the hydrolysis mechanism involving the partial O-2 de-sulfation and preferential cleavage between Fuc0S and Fuc2S4S was proposed. Bioactivity assays revealed that the Mw 15-16 kDa conferred the potent anticoagulation via inhibiting the thrombin activity mediated by heparin cofactor II.

Re-understanding of structure and anticoagulation: Fucosylated chondroitin sulfate from sea cucumber Ludwigothurea grisea.

Fucosylated chondroitin sulfate (FCS) from sea cucumber Ludwigothurea grisea (FCSLg) is the first one that reported to bear the di-fucosyl branches. Here we deciphered it by analyzing the physicochemical properties and its derivatives. Oligosaccharides prepared by selective cleavage of glycosidic linkages presented the mono-fucose and heterodisaccharide branches in FCSLg. The disaccharide branch was determined as d-GalNAcR1-(α1,2)-l-FucR2 rather than the di-fucosyl branch, where R1 was 4-mono-O- or 4,6-di-O-sulfation, and R2 was 3-mono-O- or 3,4-di-O-sulfation, respectively. The diversity of sulfation patterns in branches complicated the structure. These results give us a new understanding of FCSLg and provided a reliable method to decipher the FCS with complex branches. Bioanalysis of chemically modified derivatives showed that modulating the molecular mass could enhance the Xase target selectivity. Side chains conferred the Xase complex inhibition by binding to FIXa with a high affinity. Whether monosaccharide and disaccharide branches have differential effects needs to be further explored.

ExpressVis: a biologist-oriented interactive web server for exploring multi-omics data.

In the era of life-omics, huge amounts of multi-omics data have been generated and widely used in biomedical research. It is challenging for biologists with limited programming skills to obtain biological insights from multi-omics data. Thus, a biologist-oriented platform containing visualization functions is needed to make complex omics data digestible. Here, we propose an easy-to-use, interactive web server named ExpressVis. In ExpressVis, users can prepare datasets; perform differential expression analysis, clustering analysis, and survival analysis; and integrate expression data with protein-protein interaction networks and pathway maps. These analyses are organized into six modules. Users can use each module independently or use several modules interactively. ExpressVis displays analysis results in interactive figures and tables, and provides comprehensive interactive operations in each figure and table, between figures or tables in each module, and among different modules. It is freely accessible at https://omicsmining.ncpsb.org.cn/ExpressVis and does not require login. To test the performance of ExpressVis for multi-omics studies of clinical cohorts, we re-analyzed a published hepatocellular carcinoma dataset and reproduced their main findings, suggesting that ExpressVis is convenient enough to analyze multi-omics data. Based on its complete analysis processes and unique interactive operations, ExpressVis provides an easy-to-use solution for exploring multi-omics data.

HPS protects the liver against steatosis, cell death, inflammation, and fibrosis in mice with steatohepatitis.

Hepassocin (HPS) is a hepatokine associated with metabolic regulation and development of non-alcoholic steatohepatitis (NASH). However, previous reports on HPS are controversial and its true function is not yet understood. Here, we demonstrated that hepatic HPS expression levels were upregulated in short-term feeding and downregulated in long-term feeding in high-fat diet (HFD)- and methionine- and choline-deficient (MCD) diet-fed mice, as well as in genetically obese (ob/ob) mice. HFD- and MCD-induced hepatic steatosis, inflammation, apoptosis, and fibrosis were more pronounced in HPS knockout mice than in the wild-type mice. Moreover, HPS depletion aggravated HFD-induced insulin resistance. By contrast, HPS administration improved MCD- or HFD-induced liver phenotypes and insulin resistance in HPS knockout and wild-type mice. Mechanistic studies revealed that MCD-induced hepatic oxidative stress was significantly increased by HPS deficiency and could be attenuated by HPS administration. Furthermore, palmitic acid-induced lipid accumulation and oxidative stress were exclusively enhanced in HPS knockout hepatocytes and diminished by HPS cotreatment. These data suggest that HPS ameliorates NASH in mice, at least in part, by inhibiting the oxidative stress. HPS expression levels are downregulated in human fatty liver tissues, suggesting that it may play an important protective role in NASH. Collectively, our findings provide clear genetic evidence that HPS has beneficial effects on the development of steatohepatitis in mice and suggest that upregulating HPS signaling may represent an effective treatment strategy for NASH.

Protein phosphatase 2A catalytic subunit ß suppresses PMA/ionomycin-induced T-cell activation by negatively regulating PI3K/Akt signaling.

The precise regulation of the T-cell activation process is critical for overall immune homeostasis. Although protein phosphatase 2A (PP2A) is required for T-cell development and function, the role of PPP2CB, which is the catalytic subunit ß isoform of PP2A, remains unknown. In the present study, using a T cell-specific knockout mouse of PPP2CB (PPP2CBfl/fl Lck-Cre+ ), we demonstrated that PPP2CB was dispensable for T-cell development in the thymus and peripheral lymphoid organs. Furthermore, PPP2CB deletion did not affect T-cell receptor (TCR)-induced T-cell activation or cytokine-induced T-cell responses; however, it specifically enhanced phorbol myristate acetate (PMA) plus ionomycin-induced T-cell activation with increased cellular proliferation, elevated CD69 and CD25 expression, and enhanced cytokine production (inteferon-γ, interleukin-2 and tumor necrosis factor). Mechanistic analyses suggested that the PPP2CB deletion enhanced activation of the phosphoinositide 3-kinase/Akt signaling pathway and Ca2+ flux following stimulation with PMA plus ionomycin. Moreover, the specific PI3K inhibitor rescued the augmented cell activation in PPP2CB-deficient T cells. Using mass spectrometry-based phospho-peptide analysis, we identified potential substrates of PPP2CB during PMA plus ionomycin-induced T-cell activation. Collectively, our study provides evidence of the specific role of PPP2CB in controlling PMA plus ionomycin-induced T-cell activation.

The glycosidic bond cleavage and desulfation investigation of fucosylated glycosaminoglycan during mild acid hydrolysis through structural analysis of the resulting oligosaccharides.

Mild acid hydrolysis is a common method to study the chemical structure of fucosylated glycosaminoglycan (FG). It was generally considered that the fucose branches α-L-FucS-(1, of FG could be hydrolyzed selectively in mild acid. This report focused on the selectivity of glycosidic bond cleavage and extensive desulfation characteristics of the backbone during mild acid hydrolysis. The hydrolyzed product of native SvFG (dfSvFG) was prepared by mild acid hydrolysis in 0.1 M H2SO4 at 100 °C for 2 h. A series of oligosaccharides were purified by GPC and SAX-HPLC from dfSvFG, then they were analyzed by HPGPC, 1D/2D NMR and ESI-Q-TOF-MS. The precise structure of these oligosaccharides was elucidated to be trisaccharides, tetrasaccharides and pentasaccharides, indicating SvFG branches hydrolyzed basically and its' backbone composed of repeating ß-D-GlcA-(1,3)-D-GalNAc and ß-D-GalNAc-(1,4)-D-GlcA unit. The prevalent presence of the GlcA residues at the non-reducing terminal of these oligosaccharides, suggesting the glycosidic bond of ß-D-GalNAc-(1,4)-D-GlcA was more susceptible to acid than that of ß-D-GlcA-(1,3)-D-GalNAc during mild acid hydrolysis. Moreover, the sulfate ester groups in GalNAc4S6S unit could also be hydrolyzed by acid, and it at position C-4 was more susceptible to hydrolysis than that at C-6. This extensive degradation and desulfation of the backbone should be taken into consideration when mild acid hydrolysis was used in elucidating the exact structure or structure-activity relationship of native FG.

Hemgn Protects Hematopoietic Stem and Progenitor Cells Against Transplantation Stress Through Negatively Regulating IFN-γ Signaling.

Hematopoietic stem and progenitor cells (HSPCs) possess the remarkable ability to regenerate the whole blood system in response to ablated stress demands. Delineating the mechanisms that maintain HSPCs during regenerative stresses is increasingly important. Here, it is shown that Hemgn is significantly induced by hematopoietic stresses including irradiation and bone marrow transplantation (BMT). Hemgn deficiency does not disturb steady-state hematopoiesis in young mice. Hemgn-/- HSPCs display defective engraftment activity during BMT with reduced homing and survival and increased apoptosis. Transcriptome profiling analysis reveals that upregulated genes in transplanted Hemgn-/- HSPCs are enriched for gene sets related to interferon gamma (IFN-γ) signaling. Hemgn-/- HSPCs show enhanced responses to IFN-γ treatment and increased aging over time. Blocking IFN-γ signaling in irradiated recipients either pharmacologically or genetically rescues Hemgn-/- HSPCs engraftment defect. Mechanistical studies reveal that Hemgn deficiency sustain nuclear Stat1 tyrosine phosphorylation via suppressing T-cell protein tyrosine phosphatase TC45 activity. Spermidine, a selective activator of TC45, rescues exacerbated phenotype of HSPCs in IFN-γ-treated Hemgn-/- mice. Collectively, these results identify that Hemgn is a critical regulator for successful engraftment and reconstitution of HSPCs in mice through negatively regulating IFN-γ signaling. Targeted Hemgn may be used to improve conditioning regimens and engraftment during HSPCs transplantation.

Structural characterization of oligosaccharides from free radical depolymerized fucosylated glycosaminoglycan and suggested mechanism of depolymerization.

Free radical depolymerization is a common method in structural analysis of polysaccharides, the major challenge is the analysis of the cleavage site and characterization of newly formed ends in this reaction. Here, a fucosylated glycosaminoglycan from H. fuscopunctata (HfFG) was depolymerized by H2O2 and a series of oligosaccharides were purified and their structures were elucidated. For non-reducing ends of the trisaccharides were intact GalNAc4S6S, the cleavage site should mainly be the ß(1,3) linkages between GlcA and GalNAc in the backbone of FG. Meanwhile, the reducing ends of the disaccharides and trisaccharides were almost dicarboxylic acid derivatives of GlcA, possibly arising from oxidative breaking of the CC bond of GlcA at the reducing ends. In addition, glycosidic linkages in D-GalNAc-ß(1,4)-D-GlcA and L-FucS-α(1,3)-D-GlcA located at the reducing end could be cleaved, and the released GalNAc4S6S were oxidized to N-acetylgalactosaminic acid.

Structural characterization and anticoagulant analysis of the novel branched fucosylated glycosaminoglycan from sea cucumber Holothuria nobilis.

Glycosaminoglycan HnFG was extracted from sea cucumber Holothuria nobilis. Its chemical structure was characterized by analyzing the physicochemical properties, oligosaccharides from its mild acid hydrolysates and depolymerized products. The disaccharide d-GalNAc4S6S-α1,2-l-Fuc3S-ol found in its mild acid hydrolysates provided a clue for the presence of a unique disaccharide-branch in HnFG. Furthermore, it was confirmed by a series of oligosaccharides from the low-molecular weight HnFG prepared by ß-eliminative depolymerization. Combining with the analysis of its peroxide depolymerized products, the precise structure of HnFG was determined: A chondroitin sulfate E (CS-E)-like backbone branched with sulfated monofucoses (~67%) and disaccharides d-GalNAcS-α1,2-l-Fuc3S (~33%) at O-3 position of each GlcUA. This is the first report on the novel branches in glycosaminoglycan. Biologically, the native and depolymerized HnFG showed potent activities in prolonging the activated partial thrombin time (APTT) and inhibiting intrinsic coagulation Xase (iXase), whereas the oligosaccharides (degree of polymerization ≤6) had no obvious effects.

Five distinct fucan sulfates from sea cucumber Pattalus mollis: Purification, structural characterization and anticoagulant activities.

Fucan sulfates from echinoderm possess characteristic structures and various biological activities. Herein, comprehensive methods including enzymolysis, ion-exchange chromatography and size exclusion chromatography lead to the purification of five fucan sulfates (FSI, FSII, FSIII, FSIV, FSV) from the sea cucumber Pattalus mollis. Chemical composition analysis showed that they were all composed of l-fucose. Their sulfate content was determined by a conductimetric method. The molecular weight (Mw) of FSI, FSII, FSIII, FSIV and FSV were measured as 238.3 kDa, 81.0 kDa, 82.0 kDa, 23.2 kDa and 6.12 kDa, respectively. Detailed NMR spectroscopic analysis revealed that the structural sequence of FSI and FSII was →3)-l-FucS-α(1→, where FucS were Fuc2S4S (10%), Fuc2S (44%), Fuc0S (10%), Fuc4S (36%), that of FSIII was →4)-l-Fuc2S-(α1 â†’ 4)-l-Fuc2S-(α1 â†’ 4)-l-Fuc0S/3S-(α1→, where Fuc0S and Fuc3S were in equal molar, and that FSIV was →4)-l-Fuc2S3S-(α1 â†’ 4)-l-Fuc2S3S-(α1 â†’ 4)-l-Fuc2S-(α1→4)-l-Fuc2S-(α1 â†’ 4)-l-Fuc2S-(α1 â†’ 4)-l-Fuc2S-(α1 â†’ . This is the first report that such a diversity of fucan sulfates were obtained from the same sea cucumber species. Biological activity showed that FSI, FSII, FSIII and FSIV exhibited potent anticoagulant by prolonging the APTT. Among them, FSII, FSIII and FSIV showed the similar potency, while FSI owned the strongest. Structure-activity relationships analysis showed that molecular weight and sulfation degree should be the crucial factors for the activity.