Integrated microbiomics and metabolomics analysis reveals the influence of gut microbiota on the growth and metabolism of sea cucumber seedlings.

AIMS: This study explores the impact of gut microbiota on body metabolites and the growth rate of sea cucumber seedlings. METHODS AND RESULTS: A comprehensive analysis using metabolomics and microbiomics was conducted to ascertain the gut microbiota and body metabolites in sea cucumber seedlings exhibiting varying growth rates. Distinct changes in the intestinal flora were observed in correlation with different growth rates of sea cucumber seedlings. The microbial communities of faster-growing seedlings exhibited greater diversity and evenness of taxa. For example, the abundance of genera Rhodococcus, Woeseia, Lysobacter, Desulfuromonadia_Sva1033, and Flavobacteriaceae_NS5_marine_group was more than 24 times higher in the fast-growing group compared to the slow-growing group. Metabolomics analysis revealed an association between high growth rates of cucumber seedlings and discrepancies in metabolites, such as amino acids, lipids, and carbohydrates. Isorenieratene, possibly synthesized by Rhodococcus, was more than 2.5 times more abundant in the fast-growing group than the slow-growing group. Slow-growing seedlings showed considerable enrichment of environmental pollutants, such as antibiotics and drugs, while their colonies were devoid of bacteria capable of degrading such pollutants. In addition, significant differences were observed between groups in the biosynthesis of amino acids, metabolism of arginine and proline, biosynthesis of unsaturated fatty acids, and metabolism of linoleic acid. Moreover, significant correlations between the microbial genera and sea cucumber metabolites were identified through correlation analysis. CONCLUSIONS: Significant differences exist in the gut microbiota and metabolite composition among seedlings with varying growth rates. Microbes residing in the gut have the potential to influence the growth of seedlings through modulation of their metabolism.

Structure and Metabolically Oriented Efficacy of Fucoidan from Brown Alga Sargassum muticum in the Model of Colony Formation of Melanoma and Breast Cancer Cells.

This work reports the detailed structure of fucoidan from Sargassum miticum (2SmF2) and its ability to potentiate the inhibitory effect of glycolysis inhibitor 2-deoxy-d-glucose (2-DG). 2SmF2 was shown to be sulfated and acetylated galactofucan containing a main chain of alternating residues of 1,3- and 1,4-linked α-l-fucopyranose, fucose fragments with monotonous 1,3- and 1,4-type linkages (DP up to 3), α-d-Gal-(1→3)-α-L-Fuc disaccharides, and 1,3,4- and 1,2,4-linked fucose branching points. The sulfate groups were found at positions 2 and 4 of fucose and galactose residues. 2SmF2 (up to 800 µg/mL) and 2-DG (up to 8 mM) were not cytotoxic against MDA-MB-231 and SK-MEL-28 as determined by MTS assay. In the soft agar-based model of cancer cell colony formation, fucoidan exhibited weak inhibitory activity at the concentration of 400 µg/mL. However, in combination with low non-cytotoxic concentrations of 2-DG (0.5 or 2 mM), 2SmF2 could effectively inhibit the colony formation of SK-MEL-28 and MDA-MB-231 cells and decreased the number of colonies by more than 50% compared to control at the concentration of 200 µg/mL. Our findings reveal the metabolically oriented effect of fucoidan in combination with a glycolysis inhibitor that may be beneficial for a therapy for aggressive cancers.

Neiella holothuriorum sp. nov., isolated from the gut of a sea cucumber Apostichopus japonicus.

A Gram-stain negative, aerobic, rod-shaped bacterium, designated 126T, was isolated from the intestinal content of a sea cucumber, Apostichopus japonicus, in China. Strain 126T was found to grow optimally at 25-28 °C and pH 7.5-8.0 in marine 2216 E medium, with tolerance of 1-7% (w/v) NaCl. Strain 126T is motile by means of one to several polar flagella. The dominant fatty acids of strain 126T were identified as C16:1 ω7c/C16:1 ω6c (29.5%), C18:1 ω7c/C18:1 ω6c (19.8%) and C16:0 (16.7%). The respiratory quinone was found to be Q-8. The polar lipid profile was found to be mainly composed of phosphatidylglycerol and phosphatidylethanolamine. The total length of the draft genome is approximately 4.2 × 106 bp, encoding 3655 genes and 3576 coding sequences. The G + C content of the genomic DNA is 48.0%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain 126T belongs to the genus Neiella and is closely related to Neiella marina J221T (96.5%). Genomic comparisons of 126T to N. marina J221T revealed that they had similar genome size, G + C content and complement of clusters of orthologous groups. However, average nucleotide identity and digital DNA-DNA hybridization values between strains126T and N. marina J221T was 75.5% and 19.7%, which could distinguish the strains. On the basis of these phenotypic and genotypic data, strain 126T is concluded to represent a novel species, for which the name Neiella holothuriorum sp. nov. is proposed. The type strain is 126T (= GDMCC 1.2530T = KCTC 82829T).

Characterization and Caco-2 Cell Transport Assay of Chito-Oligosaccharides Nano-Liposomes Based on Layer-by-Layer Coated.

Chito-oligosaccharides (COSs) were encapsulated by the film-ultrasonic method into three nano-liposomes, which were uncoated liposomes (COSs-Lip), chitosan-coated liposomes (CH-COSs-Lip), and sodium alginate (SA)/chitosan (CH)-coated liposomes (SA/CH-COSs-Lip). The physicochemical and structural properties, as well as the stability and digestive characteristics, of all three nano-liposomes were assessed in the current study. Thereafter, the characteristics of intestinal absorption and transport of nano-liposomes were investigated by the Caco-2 cell monolayer. All nano-liposomes showed a smaller-sized distribution with a higher encapsulation efficiency. The ζ-potential, Z-average diameter (Dz), and polydispersity index (PDI) demonstrated that the stability of the SA/CH-COSs-Lip had much better stability than COSs-Lip and CH-COSs-Lip. In addition, the transport of the nano-liposomes via the Caco-2 cell monolayer indicated a higher transmembrane transport capacity. In summary, the chitosan and sodium alginate could serve as potential delivery systems for COSs to fortify functional foods and medicines.

Bioactive isopimarane diterpenes from the fungus, Epicoccum sp. HS-1, associated with Apostichopus japonicus.

One new isopimarane diterpene (1), together with two known compounds, 11-deoxydiaporthein A (2) and iso-pimara-8(14),15-diene (3) were isolated from the culture of Epicoccum sp., which was associated with Apostichopus japonicus. Their structures were determined by the analysis of 1D and 2D NMR, as well as mass spectroscopic data. The absolute configuration of Compound 1 was deduced by a single-crystal X-ray diffraction experiment using CuKα radiation. In the bioactivity assay, both Compounds 1 and 2 exhibited α-glucosidase inhibitory activity with IC50 values of 4.6 ± 0.1 and 11.9 ± 0.4 µM, respectively. This was the first report on isopimarane diterpenes with α-glucosidase inhibitory activity.

Extraction, preliminary characterization and evaluation of in vitro antitumor and antioxidant activities of polysaccharides from Mentha piperita.

This study describes the extraction, preliminary characterization and evaluation of the in vitro antitumor and antioxidant activities of polysaccharides extracted from Mentha piperita (MPP). The optimal parameters for the extraction of MPP were obtained by Box-Behnken experimental design and response surface methodology (RSM) at the ratio of water to raw material of 20, extraction time of 1.5 h and extraction temperature at 80 °C. Chemical composition analysis showed that MPP was mainly composed of glucuronic acid, galacturonic acid, glucose, galactose and arabinose, and the molecular weight of its two major fractions were estimated to be about 2.843 and 1.139 kDa, respectively. In vitro bioactivity experiments showed that MPP not only inhibited the growth of A549 cells but possessed potent inhibitory action against DNA topoisomerase I (topo I), and an appreciative antioxidant action as well. These results indicate that MPP may be useful for developing safe natural health products.

Pimarane diterpenes from the fungus Epicoccum sp. HS-1 associated with Apostichopus japonicus.

Three new pimarane diterpenes (1, 2 and 3) as well as a known compound 4, were isolated from the marine-derived fungus HS-1 from Apostichopus japonicus. Their structures and relative stereochemistry of 1-3 were elucidated using a combination of NMR spectroscopy and CD. In the primary bioassay, compounds 1, 2 and 4 inhibited the growth of KB and KBv200 with IC(50) of 3.51, 2.34 µg/mL, 20.74, 14.47 µg/mL, and 3.86, 6.52 µg/mL, respectively.

Structural characterization of a P-selectin and EGFR dual-targeting fucoidan from Sargassum fusiforme.

In this study, we obtained fucoidans SFP, SHP, STP, and FVP from Sargassum fusiforme, Sargassum horneri, Sargassumthunbergii, and Fucus vesiculosus, respectively. Chitosan/fucoidan nanoparticles (Cs/F NPs) were prepared using the fucoidans mentioned above. SFP NPs and SHP NPs showed strong binding abilities to P-selectin and epithelial growth factor receptor (EGFR). Given the yields from the alga, SFP was first selected to explore the structural characteristics of the P-selectin and EGFR dual-targeting fucoidan. SFP had an estimated molecular weight of 739 kDa and was mainly composed of galactose (26.57%, mol%) and fucose (66.81%), with minor amounts of mannose (2.54%), glucosamine (0.42%), and glucose (3.66%). Galactose and fucose accounted for thevast majority. Further investigation, including methylation analysis, one- and two-dimensional nuclear magnetic resonance, and mass spectroscopy, was performed to reveal the fine structure of SFP. The results indicated that SFP mainly consisted of â†’ 3)-α-l-Fucp-(1→, →4)-α-l-Fucp-(1→, →3,4)-α-l-Fucp-(1→, →3)-ß-d-Galp-(1→, and minor â†’ 6)-ß-d-Galp-(1→, partially sulfated at the C-4 of â†’ 3)-α-l-Fucp-(1→, C-3 of â†’ 4)-α-l-Fucp-(1→, C-3 of â†’ 6)-ß-d-Galp-(1→, and C-6 of â†’ 3)-ß-d-Galp-(1 â†’ . Sulfated fuco- and galactofuco-segments formed the branches.

[The study of the secondary metabolites from fungus Paecilomyces sp].

OBJECTIVE: To get active secondary metabolites from the fungus Paecilomyces sp.. METHODS: The strain Paecilomyces sp. was further grown in solid-substrate fermentation cultures, the metabolites were got by application of different separation techniques, such as silica gel, Sephadex LH-20 column chromatography, and reversed-phase high performance liquid chromatography. Their structures were identified by comprehensive spectroscopic methods. RESULTS: Four compounds were isolated and identified as Cerebroside C (1), Cerebroside D (2), 2-Hydroxybenzyl alcohol (3), 2-(4-Hydroxyphenyl) ethanol. CONCLUSION: Four compounds are isolated from Paecilomyces sp. for the first time.

Hypolipidemic effect of soluble dietary fibers prepared from Asparagus officinalis and their effects on the modulation of intestinal microbiota.

The soluble dietary fiber from Asparagus officinalis (ASDF) was successively prepared using enzymolysis combined with spray-drying technology. High-performance liquid chromatography analysis showed that ASDF contained two polysaccharide fractions with the average molecular weight of 2.77 × 105 and 6.44 × 103 Da, and was composed of mannose, rhamnose, galacturonic acid, glucose, galactose, and arabinose with a molecular ratio of 19.93:1.02:1.94:32.17:1.00:1.91, respectively. ASDF showed potential in vitro antioxidant activities. The oral administration of ASDF significantly reduced the levels of total cholesterol, triglyceride, and low-density lipoprotein cholesterol in HD-induced mice serum. Furthermore, 16S rRNA gene sequencing analysis showed that ASDF significantly affected the composition of intestinal microbiota, especially reducing the Firmicutes/Bacteroidotetes ratio and the relative abundances of Desulfobacterota, Proteobacteria, Actinobacteriota and increasing that of Muribaculaceae, Bacteroides, and Alloprevotella. These results demonstrated that the intake of ASDF could regulate intestinal microbiota and serum lipid levels in hyperlipidemic conditions.

A fucoidan from Sargassum fusiforme with novel structure and its regulatory effects on intestinal microbiota in high-fat diet-fed mice.

A fucoidan SFP, having novel structure, was extracted from Sargassum fusiforme. It had a molecular weight of 703 kDa and was composed of fucose and galactose with the ratio of 73.16:26.84 (mol%). Structural analyses showed that it mainly consisted of 1,3-, 1,4-, 1,3,4-linked-α-l-Fucp and 1,3-, 1,6-linked-ß-d-Galp, with partial sulfation at C-4, C-3 of fucose units and C-6, C-3 of galactose units. The branches consisted of sulfated fucosyl and galactofucosyl oligosaccharides. The regulatory effects of SFP on the intestinal microbiota in high-fat diet-fed mice were investigated. The high-dosage SFP exhibited good hypolipidemic effects, especially in regulating the high-densitylipoproteincholesterol, non-esterified fatty acid levels and lipase activity. It also significantly decreased the ratio of phyla Firmicutes/Bacteroidetes (P < 0.05). Besides, SFP had certain effects on the richness and diversity of intestinal microbiota. Therefore, SFP exhibited novel structure and certain beneficial effects on the disorder of intestinal microbiota in high-fat diet-fed mice.

Expression, purification, and characterization of thermolabile hemolysin (TLH) from Vibrio alginolyticus.

Hemolysin is a putative pathogenicity factor in many bacterial pathogens. In this study, a DNA fragment containing the open reading frame (1254 bp) of the thermolabile hemolysin gene (tlh) from Vibrio alginolyticus V05 was amplified and cloned into the expression plasmid pET-24d(+). The deduced amino acid sequence of the thermolabile hemolysin (TLH) shared 94 and 83% identity with the lecithin-dependent hemolysin (LDH)/TLH of V. parahaemolyticus and V. harveyi thermolabile hemolysin (VHH), respectively. The sequence analysis also indicated that it contained a GDSL lipase domain like VHH. The recombinant protein with a predicted molecular mass of 47.2 kDa was expressed in the Escherichia coli strain BL21 (DE3) as a His-tag fused protein. TLH purified by the nickel-nitrilotriacetic acid (Ni-NTA) His-Bind Resin method showed phospholipase activity on an egg yolk emulsion plate and hemolytic activity against flounder erythrocytes with a specific activity of 18 hemolytic units microg(-1). The addition of divalent cations at different concentrations decreased hemolytic activity of the purified TLH, but monavalent cations did not affect hemolytic activity. The hemolytic activity of TLH was also markedly inhibited by protein modification reagents, i.e. beta-mercaptoethanol, phenylmethylsulfonyl fluoride, and 5,5'-dithio-bis(2-nitrobenzoic acid). Moreover, TLH was toxic to zebrafish when injected intraperitoneally, with a median lethal dose (LD50) of 0.8 microg protein g(-1) fish. This work shows that TLH could potentially be developed as a vaccine and used as a diagnostic tool for vibriosis.

[The secondary metabolites of the HS-3 Alternaria sp. fungus associated with holothurians].

OBJECTIVE: The metabolites of HS-3 associated with holothurians were studied, which was identified by molecular biology as Alternaria sp.. METHODS: The holothurians were gathered from the Sea of Zhifu Islet, Shandong Province. HS-3 Alternaria sp. was culternitived in potato medium, and four compound was got by TLC, chromatography and HPLC, and 1-hydroxyl-3-methylanthracene-9,10-dione (1), chrysophanol (2), sterigmatocystin (3) and cerebroside (4) were elucated by modern spectrum. CONCLUSION: All of this provides scientific data for further study of holothurians, and the four coumpouns are isolated from the microbe associated with holothurians for the first time.

Hot water pretreatment-induced significant metabolite changes in the sea cucumber Apostichopus japonicus.

Hot water pretreatment of sea cucumbers potentially changes nutritional benefits. This study aimed to quantify hot water pretreatment-induced changes in metabolite profiles of sea cucumber body walls. ICP-OES, GC-MS, and LC-MS analyses of untreated- (UT-BW), hot water-treated body walls (HW-BW) of Apostichopus japonicus, and the hot water extract (HW-E) determined significant losses of minerals (25-50% w/w), protein (~11% w/w), carbohydrate (33% w/w), saponins (~41% w/w), and spermidine (100%), a potential antipsychotic from hot water-treated samples. Multivariate comparisons of HW-BW with UT-BW and HW-BW with HW-E showed increases in amino acids and fatty acids, suggesting hot water-induced degradation or transformation or easier extraction of protein, lipid or other components. Presence of 80 to 88.5% of compounds in the HW-E and lower DHA, EPA and glycerophospholipids levels in HW-BW suggested extraction of these metabolites. These data indicate that novel processing technologies are required to preserve the full nutritional benefits of sea cucumbers.

Molecular cloning, characterization, and expression analysis of the CXCR4 gene from Turbot: Scophthalmus maximus.

Chemokine receptor 4 (CXCR4) belongs to the large superfamily of G protein-coupled receptors. The EST sequence of CXCR4 from turbot (Scophthalmus maximus L.) was obtained from a subtractive cDNA library. In the present study, the full-length cDNA sequence of turbot CXCR4 was obtained, and sequence analysis indicated that its primary structure was highly similar to CXCR4 from other vertebrates. Quantitative real-time PCR demonstrated that the highest expression level of turbot CXCR4 was in the spleen following injection with physiological saline (PS). After turbot were challenged with Vibrio harveyi, the lowest expression level of CXCR4 was detected at 8 hours in the spleen and 12 hours in the head kidney, and then increased gradually to 36 hours. These findings suggested that CXCR4 may play a significant role in the immune response of turbot.

Molecular cloning, characterization and expression analysis of cathepsin D gene from turbot Scophthalmus maximus.

Cathepsin D is a lysosomal endoproteolytic aspartic proteinase which also has been found in endosomes of macrophage. It is thought to play key roles in the developmental and physiological process of animals. The EST sequence of turbot (Scophthalmus maximus L.) cathepsin D was obtained from a subtractive cDNA library. In the present study, 5'-RACE and 3'-RACE were carried out to obtain the complete cDNA sequence of turbot cathepsin D, which contained a 91 bp 5'-UTR, a 1191 bp open reading frame encoding 396 amino acids, and a 329 bp 3'-UTR. The deduced amino acid sequence of the cathepsin D consisted of a signal peptide of 18 aa, a leader peptide extending 43 aa, and a mature peptide of 335 aa. BLAST analysis revealed that turbot cathepsin D shared high similarity with other known cathepsin D, and it showed significant homology with that of Barramundi (Lates calcarifer B., 89% aa similarity). Quantitative real-time PCR (q PCR) demonstrated that the highest expression level of the turbot cathepsin D was in liver. After turbot were challenged with Vibrio harveyi, the lowest expression levels of cathepsin D in liver, spleen and head kidney were detected at 8 h. This result was different from the expression of MHCII of which the expression lever was increased upon challenge. The expression levels of cathepsin D in liver and head kidney increased gradually after 8 h and exceeded the background level after 24 h. In spleen, the expression level was reinforced after 8 h and kept at level that was higher than the original level after 12 h. The results suggested that cathepsin D might process antigens for presentation to the immune system and have synergetic effect with apoptosis pathway until 12 h after injection.

Genetic diversity between two Vibrio anguillarum strains exhibiting different virulence by suppression subtractive hybridization.

OBJECTIVE AND METHODS: Vibrio anguillarum, a halophilic Gram-negative bacterium, is the causative agent of vibriosis in fish. V. anguillarum strain VIB72 was defined as having high virulence whereas strain CW1 was defined as having low virulence on the basis of their different LD50 values to zebra fish. Suppression subtractive hybridization (SSH) was used to identify genetic differences between these two strains. RESULTS: After screening, 59 subtracted library clones were isolated which were specific for strain VIB72, and the DNA sequences of these clones were determined. Seventeen fragments showed high homology to the genes of known functions in other bacteria. This includes soluble lytic murein transglycosylase, mobilization protein (MobA, MobC), transposase (IS66), resistance-related protein (metallo-beta-lactamase and acetyltransferase family), toxin protein (DT-201 and alveicin A immunity protein), ATP-dependent endonuclease of OLD family like protein, SocE and GTP-binding protein HflX (high frequency of lysogenization). These fragments may represent parts of putative pathogenicity islands (PAIs) in V. anguillarum. The remaining fragments showed no significant homology to any known genes. CONCLUSION: The results indicated that SSH was successful in identifying genetic differences and putative virulence genes among different strains of V. anguillarum.

Three new oleanane-type triterpenoid saponins from the seeds of Celosia cristata L.

Phytochemical investigation of the 1-butanol soluble fraction of 60% ethanol extract of the seeds of Celosia cristata L. led to the identification of three new oleanane-type triterpenoid saponins. Using 1D and 2D NMR experiment methods, ESI-MS analysis and acid hydrolysis, their structures were identified as 3-O-[ß-D-xylopyranosyl-(1 â†’ 3)-ß-D-glucuronopyranosyl]-2ß-hydroxy-oleanolic acid-28-O-ß-D-glucopyranoside (1), 3-O-[ß-D-xylopyranosyl-(1 â†’ 3)-ß-D-glucuronopyranosyl]-2ß, 23-dihydroxy-oleanolic acid-28-O-ß-D-glucopyranoside (2) and 3-O-[ß-D-glucopyranosyl-(1 â†’ 4)-ß-D-glucopyranosyl]-2-hydroxyl-medicagenic acid-28-O-ß-D-glucopyranosyide (3), respectively.

Isolation and Characterization of Antiangiogenesis Compounds from the Fungus Aspergillus terreus Associated with Apostichopus japonicus Using Zebrafish Assay.

Three compouds, (+)-butyrolactone IV (1), butyrolactone I (2) and terrelactone A (3) were isolated from the fungus Aspergillus terreus associated with Apostichopusjaponicus from the Yellow Sea in China; their structures were elucidated by spectral methods. Compounds I and 2 were shown to have moderate antiangiogenesis activity when tested using-the zebrafish assay. This is the first report of butyrolactones with antiangiogenesis activity.

Cellulase-assisted extraction, characterization, and bioactivity of polysaccharides from Polygonatum odoratum.

Cellulase-assisted extraction of polysaccharides from Polygonatum odoratum (CPP) was optimized by response surface methodology (RSM) and the extracted CPP's preliminary chemical characteristics, as well as antioxidant and immunomodulatory activities were also investigated. The optimal extraction parameters comprised an extraction temperature of 58.21 °C, an extraction time of 3.18 h, pH value of 5.8, and cellulase amount of 6.0%. Under these conditions, the relative yield was 15.76%, higher than the yield achieved with hot water extracted polysaccharide (HPP). Chemical composition analysis demonstrated that CPP and HPP consisted of mannose, glucosamine, rhamnose, glucose, galactose, and arabinose with a molecular ratio of 7.80:1.08:1.63:65.93:3.58:1.00 and 11.22:0.23:0.23:17.59:2.73:9.10, respectively. The molecular weight distribution of CPP was lower and more homogeneous compared with HPP. CPP exhibited stronger antioxidant activities than HPP, including DPPH radical scavenging activity and reducing power. Both CPP and HPP could significantly promote the proliferation and neutral red phagocytosis of RAW 264.7 macrophage cells in vitro. These results indicate that the cellulase-assisted extraction method influenced the physicochemical characteristics, and consequently, the functional activities of polysaccharides, suggesting the cellulose-assisted method may be a viable option for extraction polysaccharides from P. odoratum.