Purification and Structural Analyses of Sulfated Polysaccharides from Low-Value Sea Cucumber Stichopus naso and Anticoagulant Activities of Its Oligosaccharides.

Three polysaccharides (SnNG, SnFS and SnFG) were purified from the body wall of Stichopus naso. The physicochemical properties, including monosaccharide composition, molecular weight, sulfate content, and optical rotation, were analyzed, confirming that SnFS and SnFG are sulfated polysaccharides commonly found in sea cucumbers. The highly regular structure {3)-L-Fuc2S-(α1,}n of SnFS was determined via a detailed NMR analysis of its oxidative degradation product. By employing ß-elimination depolymerization of SnFG, tri-, penta-, octa-, hendeca-, tetradeca-, and heptadeca-saccharides were obtained from the low-molecular-weight product. Their well-defined structures confirmed that SnFG possessed the backbone of {D-GalNAc4S6S-ß(1,4)-D-GlcA}, and each GlcA residue was branched with Fuc2S4S. SnFS and SnFG are both structurally the simplest version of natural fucan sulfate and fucosylated glycosaminoglycan, facilitating the application of low-value sea cucumbers S. naso. Bioactivity assays showed that SnFG and its derived oligosaccharides exhibited potent anticoagulation and intrinsic factor Xase (iXase) inhibition. Moreover, a comparative analysis with the series of oligosaccharides solely branched with Fuc3S4S showed that in oligosaccharides with lower degrees of polymerization, such as octasaccharides, Fuc2S4S led to a greater increase in APTT prolongation and iXase inhibition. As the degree of polymerization increases, the influence from the sulfation pattern diminishes, until it is overshadowed by the effects of molecular weight.

Paenibacillus sedimenti sp. nov., isolated from freshwater wetland sediment.

A Gram-stain-positive, motile, rod-shaped, facultatively anaerobic bacterium, designated strain WST5T, isolated from sediment was characterized using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain WST5T was most closely related to Paenibacillus aestuarii CJ25T (96.8 % similarity). The genome size of the WST5T was 6.5 Mb, contained 4500 predicted protein-coding genes, and had a DNA G+C content of 46.6%. The values of whole-genome average nucleotide identity analysis and digital DNA-DNA hybridization between strain WST5T and its closely related type strains were less than 76 and 25.6 %, respectively. The predominant cellular fatty acids (>10 %) were anteiso-C15 : 0 and C16 : 1 ω5c and the main menaquinone was MK-7. The major polar lipids were identified as diphospholidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and two unknown aminophospholipids. Based on the results of phenotypic, genotypic, chemotaxonomic and phylogenetic analyses, strain WST5T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus sedimentum sp. nov. is proposed. The type strain is WST5T (=NBRC 115194 T=CGMCC 1.18706T).

Pseudomonas pratensis sp. nov., Isolated from Grassland Soil from Inner Mongolia, China.

A novel bacterial strain, designated MHJ-10JT, was isolated from a soil sample obtained from a grassland in Inner Mongolia, China. MHJ-10JT strain could grow at 4-37 °C (optimum: 30 °C) and pH 4-9 (optimum: pH 6), as well as in the presence of 0-6% NaCl (optimum: 1%). Cells of strain MHJ-10JT are Gram-negative, rod-shaped, and motile. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain MHJ-10JT was most closely related to Pseudomonas lutea OK2T (98.5% 16S rRNA gene sequence similarity). The values of the average nucleotide identities (ANI) and digital DNA-DNA hybridization (dDDH) between strain MHJ-10JT and its related species were all below 80.5% and 24.4%, respectively, which are significantly lower than the thresholds of 95% for ANI and 70% for DDH for species delineation. The genomic G + C content of the MHJ-10JT strain is 64.8 mol%. Based on the phenotypic, genotypic, chemotaxonomic, and phylogenetic analyses, strain MHJ-10JT can be assigned to the genus Pseudomonas. In this study, we propose that strain MHJ-10JT be classified as a novel species belonging to the genus Pseudomonas with the species name Pseudomonas pratensis sp. nov. The type strain of the proposed novel species is MHJ-10JT (= KCTC 82206T = CGMCC 17322T).

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.

miR-4486 enhances cisplatin sensitivity of gastric cancer cells by restraining the JAK3/STAT3 signalling pathway.

Along with the occurrence of cisplatin resistance, treatment on gastric cancer (GC) becomes difficult. Therefore, researches on new therapeutic methods to revert cisplatin resistance are becoming increasingly urgent. qRT-PCR was used to quantify the expression of miR-4486, JAK3 in SGC-7901 or SGC-7901/DDP cell lines. WB was utilized to analyze the expression of JAK3, STAT3 and p-STAT3 in SGC-7901/DDP cell lines. CCK-8 assay was used to determine the IC50 of cisplatin on both cell lines and cell viability of SGC-7901/DDP cell lines. The target relationship between miR-4486 and JAK3 was determined by luciferase assay. MiR-4486 expression on apoptosis of SGC-7901/DDP cell lines was determined by flow cytometry. qRT-PCR testified that miR-4486 decreased in SGC-7901/DDP cells, and the expression of miR-4486 mimic increased significantly compared with miR-4486 NC. By CCK-8 assay, the IC50 of cisplatin on both cell lines were 9 µg/mL and 81.3 µg/mL, and overexpression of miR-4486 decreased the viability of SGC-7901/DDP cells. Compared with DDP group, the expression of miR-4486 accelerated SGC-7901/DDP cells apoptosis. Dual-luciferase assay suggested that JAK3 was the target gene of miR-4486. qRT-PCR and WB proved that miR-4486/JAK3 axis inhibit the activation of JAK3/STAT3 pathway, and JAK3 overexpression can partly reverse this. As shown by CCK-8 and flow cytometry, miR-4486 overexpression decreased viability and stimulated apoptosis of SGC-7901/DDP cells. However, JAK3 overexpression can also partly revert this. miR-4486 overexpression could decrease viability and improve apoptosis of SGC-7901/DDP cells to revert its cisplatin-resistance, and the mechanism may be related to JAK3/STAT3 signalling pathway.

Methanotrophs Contribute to Nitrogen Fixation in Emergent Macrophytes.

Root-associated aerobic methanotroph plays an important role in reducing methane emissions from wetlands. In this study, we examined the activity of methane-dependent nitrogen fixation and active nitrogen-fixing bacterial communities on the roots of Typha angustifolia and Scirpus triqueter using a 15N-N2 feeding experiment and a cDNA-based clone library sequence of the nifH gene, respectively. A 15N-N2 feeding experiment showed that the N2 fixation rate of S. triqueter (1.74 µmol h-1 g-1 dry weight) was significantly higther than that of T. angustifolia (0.48 µmol h-1 g-1 dry weight). The presence of CH4 significantly increased the incorporation of 15N-labeled N2 into the roots of both plants, and the rate of CH4-dependent N2 fixation of S. triqueter (5.6 µmol h-1 g-1 dry weight) was fivefold higher than that of T. angustifolia (0.94 µmol h-1 g-1 dry weight). The active root-associated diazotrophic communities differed between the plant species. Diazotrophic Methylosinus of the Methylocystaceae was dominant in S. triqueter, while Rhizobium of the Rhizobiaceae was dominant in T. angustifolia. However, there were no significant differences in the copy numbers of nifH between plant species. These results suggest that N2 fixation was enhanced by the oxidation of CH4 in the roots of macrophytes grown in natural wetlands and that root-associated Methylocystacea, including Methylosinus, contribute to CH4 oxidation-dependent N2 fixation.

miR-4486 reverses cisplatin-resistance of colon cancer cells via targeting ATG7 to inhibiting autophagy.

Cisplatin (DDP) resistance is one of the main causes of treatment failure in patients with colon cancer (CC). Autophagy is a key mechanism of resistance to chemotherapy. Since autophagy-related 7 (ATG7) has been reported to be involved in the regulation of autophagy and DDP resistance for lung and esophageal cancer, the present study aimed to explore the functions of microRNA (miR)-4486 in the autophagy-mediated DDP resistance of CC. The expression level of miR-4486 in HCT116, DDP-resistant HCT116 cells (HCT116/DDP), SW480 and DDP-resistant SW480 cells (SW480/DDP) was quantified by reverse transcription-quantitative PCR. Western blotting was utilized to analyze the expression of ATG7, autophagy-related proteins Beclin 1 and LC3-I/II, as well as apoptosis-related proteins Bcl-2, Bax and cleaved-caspase 3 in HCT116/DDP and SW480/DDP cells. The half maximal inhibitory concentration of DDP on all cell lines and the cell viability of HCT116/DDP and SW480/DDP cells were measured using Cell Counting Kit 8 assay. Luciferase assay was used to examine the potential targets of miR-4486 and ATG7. The effects of upregulating mimic miR-4486 expression on the apoptosis and autophagy of HCT116/DDP and SW480/DDP cells were determined by flow cytometry and electron microscopy, respectively. It was found that miR-4486 expression was significantly decreased in HCT116/DDP and SW480/DDP cells compared with that in HCT116 and SW480 cells. Overexpression of miR-4486 could increase the sensitivity of HCT116/DDP and SW480/DDP cells to DDP by reducing cell viability, promoting apoptosis and inhibiting autophagy through downregulating Beclin 1 expression and the LC3-II/LC3-I ratio. Additionally, ATG7 was identified to be a target gene of miR-4486, where ATG7 overexpression could partially reverse the effects of miR-4486 on cell viability and apoptosis by promoting the formation of autophagosomes. In conclusion, the present results demonstrated that miR-4486 could reverse DDP resistance in HCT116/DDP and SW480/DDP cells by targeting ATG7 to inhibit autophagy.

Characteristics of the tree shrew gut virome.

The tree shrew (Tupaia belangeri) has been proposed as an alternative laboratory animal to primates in biomedical research in recent years. However, characteristics of the tree shrew gut virome remain unclear. In this study, the metagenomic analysis method was used to identify the features of gut virome from fecal samples of this animal. Results showed that 5.80% of sequence reads in the libraries exhibited significant similarity to sequences deposited in the viral reference database (NCBI non-redundant nucleotide databases, viral protein databases and ACLAME database), and these reads were further classified into three major orders: Caudovirales (58.0%), Picornavirales (16.0%), and Herpesvirales (6.0%). Siphoviridae (46.0%), Myoviridae (45.0%), and Podoviridae (8.0%) comprised most Caudovirales. Picornaviridae (99.9%) and Herpesviridae (99.0%) were the primary families of Picornavirales and Herpesvirales, respectively. According to the host types and nucleic acid classifications, all of the related viruses in this study were divided into bacterial phage (61.83%), animal-specific virus (34.50%), plant-specific virus (0.09%), insect-specific virus (0.08%) and other viruses (3.50%). The dsDNA virus accounted for 51.13% of the total, followed by ssRNA (33.51%) and ssDNA virus (15.36%). This study provides an initial understanding of the community structure of the gut virome of tree shrew and a baseline for future tree shrew virus investigation.