Baicalin-aluminum complex on the regulation of IPEC-1 infected with enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is the main bacterial cause of diarrhea in weaned piglets. Baicalin-aluminum (BA) complex is the main active ingredient of Scutellaria baicalensis Georgi extracted-aluminum complex, which has been used to treat diarrhea in weaning piglets, however the underlying mechanism remains unclear. To investigate the effects of the BA complex on the regulation of porcine intestinal epithelial (IPEC-1) cells infected with ETEC, IPEC-1 cells were incubated with an ETEC bacterial strain at a multiplicity of infection of 1 for 6 h and then treated with different concentrations of the BA complex for 6 h. ETEC infection increased the levels of cAMP and cGMP, upregulated CFTR (cystic fibrosis transmembrane conductance regulator) mRNA, and downregulated NHE4 mRNA in IPEC-1 cells. Treatment with the BA complex inhibited ETEC adhesion and the production of cAMP and cGMP, reduced CFTR mRNA expression, and increased NHE4 mRNA expression. Overall, the BA complex weakened the adhesion of ETEC to IPEC-1 cells, and inhibited cAMP/cGMP-CFTR signaling in IPEC-1 cells.

GPS2 promotes erythroid differentiation in K562 erythroleukemia cells primarily via NCOR1.

G protein pathway suppressor 2 (GPS2) has been shown to play a pivotal role in human and mouse definitive erythropoiesis in an EKLF-dependent manner. However, whether GPS2 affects human primitive erythropoiesis is still unknown. This study demonstrated that GPS2 positively regulates erythroid differentiation in K562 cells, which have a primitive erythroid phenotype. Overexpression of GPS2 promoted hemin-induced hemoglobin synthesis in K562 cells as assessed by the increased percentage of benzidine-positive cells and the deeper red coloration of the cell pellets. In contrast, knockdown of GPS2 inhibited hemin-induced erythroid differentiation of K562 cells. GPS2 overexpression also enhanced erythroid differentiation of K562 cells induced by cytosine arabinoside (Ara-C). GPS2 induced hemoglobin synthesis by increasing the expression of globin and ALAS2 genes, either under steady state or upon hemin treatment. Promotion of erythroid differentiation of K562 cells by GPS2 mainly relies on NCOR1, as knockdown of NCOR1 or lack of the NCOR1-binding domain of GPS2 potently diminished the promotive effect. Thus, our study revealed a previously unknown role of GPS2 in regulating human primitive erythropoiesis in K562 cells.

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.

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.

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.

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.

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.

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 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.

Oligosaccharides from fucosylated glycosaminoglycan prevent breast cancer metastasis in mice by inhibiting heparanase activity and angiogenesis.

The invasion and metastasis of tumor cells are the hallmarks of malignant diseases and the greatest obstacle to overcome. Heparanase-mediated degradation of heparan sulfate (HS) is the critical process for tumor angiogenesis and metastasis, therefore, heparanase become an attractive target for cancer research. Herein, we reported a native fucosylated glycosaminoglycan (nHG) extracted from sea cucumber Holothuria fuscopunctata and a depolymerized nHG (dHG) and its contained oligosaccharides (hs17, hs14, hs11, hs8 and hs5), acting as heparanase inhibitors. nHG and its derivatives have the ability to bind with heparanase directly, leading to significant inhibition of heparanase activity. Moreover, their apparent binding affinity to heparanase was comparable to their inhibitory effect, which was elevated along with the increase of chain length, similar to the effect of heparins. In addition, oligosaccharides inhibited the migration and invasion of 4T1 mammary carcinoma cells and human umbilical vein endothelial cells (HUVECs) and also suppressed tube formation in Matrigel matrix and angiogenesis in the chick chorioallantoic membrane (CAM) assay. In the metastatic mouse model, oligosaccharides exhibited practical antimetastatic effects on 4T1 mammary carcinoma cells. According to the reported anticoagulant activity and the low bleeding tendency of dHG and its oligosaccharides, the use of the oligosaccharides may lead to better effects on tumor patients with thrombosis tendency.

ABRO1 stabilizes the deubiquitinase BRCC3 through inhibiting its degradation mediated by the E3 ubiquitin ligase WWP2.

BRCA1/BRCA2-containing complex subunit 3 (BRCC3) is a lysine 63-specific deubiquitinase involved in multiple biological processes, such as DNA repair and immune responses. However, the regulation mechanism for BRCC3 protein stability is still unknown. Here, we demonstrate that BRCC3 is mainly degraded through the ubiquitin-proteasome pathway. The HECT-type E3 ubiquitin ligase WWP2 modulates BRCC3 ubiquitination and degradation. ABRO1, a subunit of the BRCC36 isopeptidase complex (BRISC), competes with WWP2 to bind to BRCC3, thereby preventing WWP2-mediated BRCC3 ubiquitination and enhancing BRCC3 stability. Functionally, we show that lentivirus-mediated overexpression of WWP2 in murine macrophages inhibits NLRP3 inflammasome activation by decreasing BRCC3 protein level. This study provides the first insights into the regulation of BRCC3 stability and expands our knowledge about the physiological function of WWP2.

NLRP3 is dispensable for d-galactosamine/lipopolysaccharide-induced acute liver failure.

The nucleotide-binding domain and leucine-rich repeat-containing family pyrin domain containing 3 (NLRP3) inflammasome is involved in various acute and chronic liver diseases, however, it is not clear whether NLRP3 contributes to d-Galactosamine (D-GalN) plus lipopolysaccharide (LPS)-induced acute liver failure (ALF). This study aims to investigate the role of NLRP3 inflammasome in D-GalN/LPS-induced fatal hepatitis. We found that Nlrp3-/- and WT mice showed similar mortality against a lethal dose of D-GalN/LPS treatment. Serum ALT and AST levels, as well as liver necrosis area and hepatocyte apoptosis, were not significantly different between Nlrp3-/- and WT mice at 6 h after D-GalN/LPS injection. Moreover, the numbers of intrahepatic F4/80+ cells and Ly6G+ cells were comparable in two genotype mice following D-GalN/LPS treatment. Besides, Nlrp3-/- mice had reduced IL-1ß levels but similar TNF-α, IL-6, and MCP-1 levels compared with WT mice upon D-GalN/LPS administration. Our findings revealed that NLRP3 ablation does not protect mice from D-GalN/LPS-induced fatal hepatitis and has a marginal effect on intrahepatic inflammatory response upon D-GalN/LPS treatment. This suggests that NLRP3 inflammasome does not appear to be a major contributor to D-GalN/LPS-induced ALF.

The effects of baicalin on piglets challenged with Glaesserella parasuis.

Glaesserella parasuis (G. parasuis) causes porcine vascular inflammation and damage. Baicalin is reported to have antioxidant and anti-inflammatory functions. However, whether baicalin protects piglets against G. parasuis challenge and the potential protective mechanism have not been investigated. Therefore, in this study, we comprehensively examined the protective efficacy of baicalin in piglets challenged with G. parasuis and the possible protective mechanism. Our results show that baicalin attenuated the release of the inflammation-related cytokines interleukin (IL) 1ß, IL6, IL8, IL10, and tumour necrosis factor α (TNF-α) and reduced high mobility group box 1 (HMGB1) production and cell apoptosis in piglets infected with G. parasuis. Baicalin also inhibited the activation of the mitogen-activated protein kinase (MAPK) signalling pathway and protected piglets against G. parasuis challenge. Taken together, our data suggest that baicalin could protect piglets from G. parasuis by reducing HMGB1 release, attenuating cell apoptosis, and inhibiting MAPK signalling activation, thereby alleviating the inflammatory response induced by the bacteria. Our results suggest that baicalin has utility as a novel therapeutic drug to control G. parasuis infection.

A new fucosylated glycosaminoglycan containing disaccharide branches from Acaudina molpadioides: Unusual structure and anti-intrinsic tenase activity.

A fucosylated glycosaminoglycan (AmFG) was extracted from the sea cucumber Acaudina molpadioides. And a series of oligosaccharides were purified from the size-homogeneous fractions, which were prepared from the ß-eliminative depolymerized AmFG. According to "bottom-up" strategy, the precise structure of AmFG was elucidated by analyzing the structures of these purified oligosaccharides, combining with NMR analysis of its free-radical depolymerized product. It contained a CS-E-like backbone, and each GlcUA was branched with a mono- or di-sulfated fucose (Fuc) at O-3. Intriguingly, besides two types of monosaccharide branches, Fuc2S4S (60 %) and Fuc4S (25 %), that were common in FG, AmFG also contained an unusual disaccharide branch GalNAc-α1,2-Fuc3S4S (15 %); this is the first report of such a structure in a glycosaminoglycan. Biological assays indicated that native AmFG and its oligosaccharides had potent anticoagulant and intrinsic tenase (iXase) inhibitory activities in a chain length-dependent manner. For these oligosaccharides, octasaccharide was the minimum structural fragment for potent anti-iXase activity, and the disaccharide branch might enhance this activity.

GPS2 promotes erythroid differentiation by control of the stability of EKLF protein.

Erythropoiesis is a complex multistage process that involves differentiation of early erythroid progenitors to enucleated mature red blood cells, in which lineage-specific transcription factors play essential roles. Erythroid Krüppel-like factor (EKLF/KLF1) is a pleiotropic erythroid transcription factor that is required for the proper maturation of the erythroid cells, whose expression and activation are tightly controlled in a temporal and differentiation stage-specific manner. Here, we uncover a novel role of G-protein pathway suppressor 2 (GPS2), a subunit of the nuclear receptor corepressor/silencing mediator of retinoic acid and thyroid hormone receptor corepressor complex, in erythrocyte differentiation. Our study demonstrates that knockdown of GPS2 significantly suppresses erythroid differentiation of human CD34+ cells cultured in vitro and xenotransplanted in nonobese diabetic/severe combined immunodeficiency/interleukin-2 receptor γ-chain null mice. Moreover, global deletion of GPS2 in mice causes impaired erythropoiesis in the fetal liver and leads to severe anemia. Flow cytometric analysis and Wright-Giemsa staining show a defective differentiation at late stages of erythropoiesis in Gps2-/- embryos. Mechanistically, GPS2 interacts with EKLF and prevents proteasome-mediated degradation of EKLF, thereby increasing EKLF stability and transcriptional activity. Moreover, we identify the amino acids 191-230 region in EKLF protein, responsible for GPS2 binding, that is highly conserved in mammals and essential for EKLF protein stability. Collectively, our study uncovers a previously unknown role of GPS2 as a posttranslational regulator that enhances the stability of EKLF protein and thereby promotes erythroid differentiation.

EDAG mediates Hsp70 nuclear localization in erythroblasts and rescues dyserythropoiesis in myelodysplastic syndrome.

During human erythroid maturation, Hsp70 translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. Failure of Hsp70 to localize to the nucleus was found in Myelodysplastic syndrome (MDS) erythroblasts and can induce dyserythropoiesis, with arrest of maturation and death of erythroblasts. However, the mechanism of the nuclear trafficking of Hsp70 in erythroblasts remains unknown. Here, we found the hematopoietic transcriptional regulator, EDAG, to be a novel binding partner of Hsp70 that forms a protein complex with Hsp70 and GATA-1 during human normal erythroid differentiation. EDAG overexpression blocked the cytoplasmic translocation of Hsp70 induced by EPO deprivation, inhibited GATA-1 degradation, thereby promoting erythroid maturation in an Hsp70-dependent manner. Furthermore, in myelodysplastic syndrome (MDS) patients with dyserythropoiesis, EDAG is dramatically down-regulated, and forced expression of EDAG has been found to restore the localization of Hsp70 in the nucleus and elevate the protein level of GATA-1 to a significant extent. In addition, EDAG rescued the dyserythropoiesis of MDS patients by increasing erythroid differentiation and decreasing cell apoptosis. This study demonstrates the molecular mechanism of Hsp70 nuclear sustaining during erythroid maturation and establishes that EDAG might be a suitable therapeutic target for dyserythropoiesis in MDS patients.

Effects of Native Fucosylated Glycosaminoglycan, Its Depolymerized Derivatives on Intrinsic Factor Xase, Coagulation, Thrombosis, and Hemorrhagic Risk.

A native fucosylated glycosaminoglycan from sea cucumber Holothuria fuscopunctata (nHG), mainly branched with Fuc3S4S, exhibited potent anticoagulant activity by intrinsic tenase iXase (FIXa-FVIIIa complex) and antithrombin-dependent factor IIa (FIIa) inhibition, but also had the effects of FXII activation and platelet aggregation. For screening a selective iXase inhibitor, depolymerized nHG (dHG-1 ∼ -6) and a pure octasaccharide (oHG-8) were prepared. Like nHG, dHG-1 ∼ -6 and oHG-8 could potently inhibit iXase, and competitive binding assay indicated that dHG-5 and oHG-8 could bind to FIXa. Nevertheless, dHG-5 and oHG-8 had no effects on FXII and platelet activation. nHG, dHG-5, and oHG-8 could significantly prolong the activated partial thromboplastin time of human, rat, and rabbit plasma. In the rat deep venous thrombosis model, dHG-5 and oHG-8 showed potent antithrombotic effects in a dose-dependent manner, while the thrombus inhibition rate of nHG at high dose was markedly reduced. Additionally, dHG-5 and oHG-8 did not increase bleeding at the doses up to 10-fold of the effectively antithrombotic doses compared with nHG and low molecular weight heparin in the mice tail-cut model. Considering that dHG-5 possesses strong anti-iXase and antithrombotic activities, and its preparation process is simpler and its yield is higher compared with oHG-8, it might be a promising antithrombotic candidate.

NLRC4 Mutation in flagellin-derived peptide CBLB502 ligand-binding domain reduces the inflammatory response but not radioprotective activity.

Bacterial flagellin is a pathogen-associated molecular pattern recognized by surface-localized Toll-like receptor 5 (TLR5) and cytosolic NOD-like receptor protein 4 (NLRC4). CBLB502, derived from Salmonella flagellin, exhibits high radioprotective efficacy in mice and primates by regulating TLR5 and the nuclear factor kappa B (NF-κB) signaling pathway. In this study, we examined the effects of CBLB502 and mutations in its NLRC4- and TLR5-binding domains on radioprotective efficacy and the immune inflammatory response. The results showed that CBLB502 mutation with I213A in the TLR5-binding domain significantly reduced NF-κB activity and radioprotective activity, whereas CBLB502 mutation with L292A in NLRC4-binding domain did not. Additionally, CBLB502 with both mutations greatly reduced NF-κB activity and eliminated radioprotection in mice. In contrast, NLRC4-binding domain mutation reduced the secretion of inflammatory interleukin-1ß and interleukin-18. CBLB502 exerts its radioprotective effects through both the TLR5 and NLRC4 pathways. Additionally, deletion in the NLRC4-binding domain did not reduce radioprotective activity but reduced the inflammatory response.

Precise structures and anti-intrinsic tenase complex activity of three fucosylated glycosaminoglycans and their fragments.

Fucosylated glycosaminoglycan (FG), a glycosaminoglycan derivative containing distinct sulfated fucose (FucS) branches, displays potent anticoagulant activity by inhibiting the intrinsic tenase complex (iXase). Herein, AmFG, SvFG and HaFG from three species of sea cucumbers were isolated and depolymerized by ß-eliminative cleavage. Three series of fragments, A1-A4, S1-S4 and H1-H4, were purified from the depolymerized FGs. Based on structural analysis of these fragments, three FGs were deduced as -{→4)-[L-FucS-α(1→3)]-D-GlcA-ß(1→3)-D-GalNAc4S6S-ß(1}n-. The structures differed in sulfation types of FucS, namely, most of FucS in AmFG was Fuc3S4S, but the FucS in SvFG was Fuc2S4S, while the FucS in HaFG was Fuc3S4S, Fuc2S4S and Fuc4S. However, all FucS branches attached to C-3 of GlcA as monosaccharides. Anticoagulant and anti-iXase assays showed the octasaccharide is the minimum fragment for potent anticoagulant activity via anti-iXase irrespective of FucS types. Among FG fragments with same degree of polymerization, oligosaccharides containing Fuc2S4S had more potent anti-iXase activity.