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Protein hydrolysates from sea cucumber ( Apostichopus japonicus ) gonads are rich in active materials with remarkable angiotensin-converting enzyme (ACE) inhibitory activity. Alcalase was used to hydrolyze sea cucumber gonads, and the hydrolysate was separated by the ultrafiltration membrane to produce a low-molecular-weight peptide component (less than 3 kDa) with good ACE inhibitory activity. The peptide component (less than 3 kDa) was isolated and purified using a combination method of ACE gel affinity chromatography and reverse high-performance liquid chromatography. The purified fractions were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the resulting products were filtered using structure-based virtual screening (SBVS) to obtain 20 peptides. Of those, three noncompetitive inhibitory peptides (DDQIHIF with an IC 50 value of 333.5 μmol·L -1 , HDWWKER with an IC 50 value of 583.6 μmol·L -1 , and THDWWKER with an IC 50 value of 1291.8 μmol·L -1 ) were further investigated based on their favorable pharmacochemical properties and ACE inhibitory activity. Molecular docking studies indicated that the three peptides were entirely enclosed within the ACE protein cavity, improving the overall stability of the complex through interaction forces with the ACE active site. The total free binding energies (ΔG total ) for DDQIHIF, HDWWKER, and THDWWKER were -21.9 Kcal·mol -1 , -71.6 Kcal·mol -1 , and -69.1 Kcal·mol -1 , respectively. Furthermore, a short-term assay of antihypertensive activity in spontaneously hypertensive rats (SHRs) revealed that HDWWKER could significantly decrease the systolic blood pressure (SBP) of SHRs after intravenous administration. The results showed that based on the better antihypertensive activity of the peptide in SHRs, the feasibility of targeted affinity purification and computer-aided drug discovery (CADD) for the efficient screening and preparation of ACE inhibitory peptide was verified, which provided a new idea of modern drug development method for clinical use.

The signal recognition particle (SRP) system delivers approximately 30% of the proteome to the endoplasmic reticulum (ER) membrane. SRP receptor alpha (SRα) binds to SRP for targeting nascent secreted proteins to the ER membrane in eukaryotic cells. In this study, the SRα homologous gene was identified in the sea cucumber, Apostichopus japonicus (AjSRα). AjSRα codes for 641 amino acids and has 54.94% identity with its mammalian homologs. Like mammalian SRα, it is expected to contain the SRP-alpha N domain, SRP54_N domain, and SRP54 domain. In addition, AjSRα is ubiquitously expressed in adult tissues and exhibits a sexually dimorphic expression pattern, with significantly higher expression in ovaries compared to testes. As a maternal factor, AjSRα can be continuously detected during embryonic development. Importantly, we first attempted to investigate its function by using lentiviral vectors for delivering SRα gene-specific shRNA, and we revealed that lentiviral vectors do not induce an upregulation of immune-related enzymes in sea cucumbers. However, compared to the dsRNA-based RNA interference (RNAi) method, lentivirus-mediated RNAi caused dynamic changes in gene expression at a later time. This study supplied the technical support for studying the functional mechanism of SRα in sea cucumbers., (© 2023. The Author(s).)

Low dissolved oxygen (LO) in seawater negatively affects aquatic animals and has received considerable attention. However, there is still much to learn about how echinoderms, which are keystone species in benthic ecosystems, respond to hypoxic stress. Here, we detected differentially expressed metabolites (DEMs) in sea cucumber (Apositchopus japonicus) between normoxic conditions (NC group) and hypoxic conditions (2 mg L -1 ) for 3 and 7 days (i.e., LO3 and LO7 groups). A total of 243, 298, and 178 DEMs were found in the NC versus LO3, NC vs. LO7, and LO3 vs. LO7 comparisons, respectively. Amino acids were the most abundant class of DEMs, and "biosynthesis of amino acids" was an enriched pathway in all three comparisons. Most of the enriched metabolite sets under hypoxic stress were related to metabolism. As the duration of the hypoxia treatment extended, the metabolism-related process maintained an upward trend, and signaling pathways maintained a downward trend. Thus, metabolism-related processes are affected in hypoxia-stressed sea cucumber, and amino acid metabolism is the most important process for adaption to hypoxic conditions, potentially function in osmotic regulation and energy regulation. Our results shed light on the adaptative strategies of sea cucumber to challenging environmental conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Atlantic sea cucumber is a benthic marine echinoderm found in Northwest Atlantic waters and is harvested mainly for its body wall. The body wall, along with internal organs and aquaphyrangeal bulb/flower, is a rich source of proteins, where the latter parts are often considered as processing discards. The objective of this research was to produce protein hydrolysates from sea cucumber tissues (body wall, flower, and internal organs) with bioactive properties associated with antioxidants, DNA and LDL cholesterol oxidation inhibition, and angiotensin-I-converting enzyme (ACE) inhibitory effects. The protein hydrolysates were prepared using food-grade commercial enzymes, namely Alcalase, Corolase, and Flavourzyme, individually and in combination, and found that the combination of enzymes exhibited stronger antioxidant potential than the individual enzymes, as well as their untreated counterparts. Similar trends were also observed for the DNA and LDL cholesterol oxidation inhibition and ACE-inhibitory properties of sea cucumber protein hydrolysates, mainly those that were prepared from the flower. Thus, the findings of this study revealed potential applications of sea cucumber-derived protein hydrolysates in functional foods, nutraceuticals, and dietary supplements, as well as natural therapeutics.

Background: Sea cucumber phospholipids, marine-derived lipids with high nutritional functions, have been proven to exhibit various biological activities. However, it is unclear how sea cucumber phospholipids regulate cholesterol (Chol) metabolism in atherosclerosis., Objectives: This study aimed to investigate the effects and mechanism of sea cucumber phospholipids on the metabolism of Chol and cholesterol esters (CE) in ApoE -/- mice, including plasmenyl phosphatidylethanolamine (PE-P) and plasmanyl phosphatidylcholine (PC-O)., Methods: Male ApoE -/- mice were fed with Chow diet, high-fat diet (HFD), and HFD supplemented with PC-O or PE-P, respectively. We integrated a targeted lipidomics strategy to classify and compare the cholesteryl esters according to their fatty acid types, and then analyzed the individual cholesteryl ester molecular species in the liver and serum of mice. Furthermore, the Chol metabolism-related genes and pathways were analyzed in high-fat-induced ApoE -/- mice., Results: Biochemical analysis showed that sea cucumber phospholipids significantly inhibit the generation of arterial plaque in ApoE -/- mice. Compared with the HFD group, PE-P significantly reduced the contents of SFA-CE and MUFA-CE in mice liver (P < 0.05), whereas PC-O particularly upregulated CE20:5 and CE22:6 in the serum of mice (P < 0.001). Furthermore, PC-O and PE-P inhibited the Chol synthesis pathway (Cyp7A1 and Cyp27A1), as well as promoted the catabolism of Chol by upregulating gene expressions of bile acid synthesis (Abcb11) and lysosomal activity (Lamp1), respectively., Conclusions: Sea cucumber phospholipids could ameliorate the atherosclerosis symptoms by regulating Chol metabolism. J Nutr 20xx;x:xx., (Copyright © 2023 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

Sulfide is a common harmful substance in sediments, with an especially high risk for deposit feeder organisms. The sea cucumber Apostichopus japonicus is a typical benthic feeder, and its intestine is the first line of defense and serves as a crucial barrier function. In this study, histological, physiological, gut microbiota, and metabolomic analyses were performed to explore the toxic response in the intestine of juvenile A. japonicus exposed to 0, 0.8, and 1.6 mg/L sulfide stress for 96 h. The results revealed sulfide-induced intestinal inflammatory symptoms and oxidative stress. Moreover, gut bacterial composition was observed after sulfide exposure, with an increase in Proteobacteria and a decrease in Cyanobacteria and Planctomycetes. Specifically, sulfide increased a set of sulfide-removing bacteria and opportunistic pathogens while decreasing several putative beneficial substance-producing bacteria. The metabolomic analysis indicated that sulfide also disturbed metabolic homeostasis, especially lipid and energy metabolism, in intestine. Interestingly, several intestinal bacteria were further identified to be significantly correlated with metabolic changes; for example, the decreased abundance levels of Bacillus, Corynebacterium, and Psychromonas were positively correlated with important energy metabolites, including maleic acid, farnesyl pyrophosphate, thiamine, butynoic acid, and deoxycholic acid. Thus, our research provides new insights into the mechanisms associated with the intestinal metabolic and microbiota response involved in sulfide stress adaptation strategies of juvenile A. japonicus., Competing Interests: Declaration of Competing Interest All authors declare no conflict of interest. None of the materials presented in this manuscript has been previously published, nor are they under consideration for publication elsewhere., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Circular RNAs (circRNAs) are a kind of extensive and diverse covalently closed circular endogenous RNA, which exert crucial functions in immune regulation in mammals. However, the functions and mechanisms of circRNAs in invertebrates are largely unclarified. In our previous work, 261 differentially expressed circRNAs including circRNA432 (circ432) were identified from skin ulcer syndrome (SUS) diseased sea cucumber Apostichopus japonicus by RNA-seq. To better address the functional role of sea cucumber circRNAs, circ432 was first found to be significantly induced by Vibrio splendidus challenge and LPS exposure in this study. Knock-down circ432 could depress the V. splendidus-induced coelomocytes phagocytosis. Moreover, circ432 is validated to serve as the sponge of miR-2008, a differential expressed miRNA in SUS-diseased sea cucumbers, by Argonaute 2-RNA immunoprecipitation (AGO2-RIP) assay, luciferase reporter assay and RNA fluorescence in situ hybridization (FISH) in vitro. Engulfment and cell motility protein 1 (AjELMO1) is further demonstrated to be the target of miR-2008, and silencing AjELMO1 inhibits the V. splendidus-induced coelomocytes phagocytosis, and this phenomenon could be further suppressed by supplementing with miR-2008 mimics, suggesting that circ432 might regulate coelomocytes phagocytosis via miR-2008-AjELMO1 axis. We further confirm that the depressed coelomocytes' phagocytosis by circ432 silencing is consistent with the decreased abundance of AjELMO1, and could be recovered by miR-2008 inhibitors transfection. All our results provide the evidence that circ432 is involved in regulating pathogen-induced coelomocyte phagocytosis via sponge miR-2008 and promotes the abundance of AjELMO1. These findings will enrich the regulatory mechanism of phagocytosis in echinoderm and provide theoretical data for SUS disease prevention and control in sea cucumbers., (© 2023. The Author(s).)

The holothurian Eupentacta fraudatrix is capable of fully restoring its muscles after transverse dissection. Although the regeneration of these structures is well studied at the cellular level, the molecular basis of the process remains poorly understood. To identify genes that may be involved in the regulation of muscle regeneration, the transcriptome of the longitudinal muscle band of E. fraudatrix has been sequenced at different time periods post-injury. An analysis of the map of biological processes and pathways has shown that most genes associated with myogenesis decrease their expression during the regeneration. The only exception is the genes united by the GO term "heart valve development". This may indicate the antiquity of mechanisms of mesodermal structure transformation, which was co-opted into various morphogeneses in deuterostomes. Two groups of genes that play a key role in the regeneration have been analyzed: transcription factors and matrix metalloproteinases. A total of six transcription factor genes ( Ef-HOX5, Ef-ZEB2, Ef-RARB, Ef-RUNX1, Ef-SOX17, and Ef-ZNF318 ) and seven matrix metalloproteinase genes ( Ef-MMP11, Ef-MMP13, Ef-MMP13-1, Ef-MMP16-2, Ef-MMP16-3, Ef-MMP24, and Ef-MMP24-1 ) showing differential expression during myogenesis have been revealed. The identified genes are assumed to be involved in the muscle regeneration in holothurians.

The sulfated echinoside A (EA) and holothurin A (HA) are two prominent saponins in sea cucumber with high hemolytic activity but also superior lipid-lowering activity. Deglycosylated derivatives EA2 and HA2 exhibit low hemolysis compared to EA and HA, but their efficacies on lipid metabolism regulation remains unknown. In this study, fatty acids-treated HepG2 cells and orotic acid-treated rats were used to investigate the lipid-lowering effects of sea cucumber saponin derivatives. Both the saponin and derivatives could effectively alleviate lipid accumulation in HepG2 model, especially EA and EA2. Moreover, though the lipid-lowering effect of EA2 was not equal with EA at the same dosage of 0.05% in diet, 0.15% dosage of EA2 significantly reduced hepatic steatosis rate, liver TC and TG contents by 76%, 41.5%, and 63.7%, respectively, compared to control and reversed liver histopathological features to normal degree according to H&E stained sections. Possible mechanisms mainly included enhancement of fatty acids β-oxidation and cholesterol catabolism through bile acids synthesis and excretion, suppression of lipogenesis and cholesterol uptake. It revealed that the efficacy of EA2 on lipid metabolism regulation was dose-dependent, and 0.15% dosage of EA2 possessed better efficacy with lower toxicity compared to 0.05% dosage of EA.

Dietary supplementation of sea cucumber saponins and calorie restriction have been proved to be effective in alleviating obesity, but the differences of anti-obesity effects between sea cucumber saponins and energy restriction during weight loss and weight regain are still unknown. In the present study, high-fat-induced obesity mice were randomly divided into three groups, including a high-fat diet group (HF), an energy restriction by 40% group (HF-L), and a sea cucumber saponins group (HF-S), to compare the effects of dietary sea cucumber saponins and energy restriction on the weight, glucose, and lipid metabolism of obese mice during weight loss and weight regain. The results showed that dietary 0.06% sea cucumber saponins and limiting energy intake by 40% had the same weight loss effect. Interestingly, sea cucumber saponins could alleviate impaired glucose tolerance and insulin resistance caused by obesity. In addition, the inhibited SREBP-1c mediated lipogenesis might lead to the alleviation of weight regain after resuming the high-fat diet even when sea cucumber saponins were no longer supplemented. In contrast, limiting energy intake tended to promote lipid synthesis in the liver and white adipose tissue after restoring a high-fat diet, and inflammation was also induced. The findings indicated that sea cucumber saponins could replace calorie restriction to prevent obesity and might be used as a functional food or drug to resist obesity and related diseases caused by obesity.

The sea cucumber is prominent as a traditional remedy among Asians for wound healing due to its high capacity for regeneration after expulsion of its internal organs. A short peptide consisting of 45 amino acids from transcriptome data of Stichopus horrens (Sh-EGFl-1) shows a convincing capability to promote the growth of human melanoma cells. Molecular docking of Sh-EGFl-1 peptide with human epidermal growth factor receptor (hEGFR) exhibited a favorable intermolecular interaction, where most of the Sh-EGFl-1 residues interacted with calcium binding-like domains. A superimposed image of the docked structure against a human EGF-EGFR crystal model also gave an acceptable root mean square deviation (RMSD) value of less than 1.5 Å. Human cell growth was significantly improved by Sh-EGFl-1 peptide at a lower concentration in a cell proliferation assay. Gene expression profiling of the cells indicated that Sh-EGFl-1 has activates hEGFR through five epidermal growth factor signaling pathways; phosphoinositide 3-kinase (PI3K), mitogen-activated protein kinase (MAPK), phospholipase C gamma (PLC-gamma), Janus kinase-signal transducer and activator of transcription (JAK-STAT) and Ras homologous (Rho) pathways. All these pathways triggered cells' proliferation, differentiation, survival and re-organization of the actin cytoskeleton. Overall, this marine-derived, bioactive peptide has the capability to promote proliferation and could be further explored as a cell-growth-promoting agent for biomedical and bioprocessing applications.

Apostichopus japonicus achieves intestinal regeneration in a short period after evisceration, and multiple genes are involved in this process. The transcriptome of A. japonicus was screened for regeneration-associated protein (Aj-Orpin), a gene that is specifically upregulated during intestinal regeneration. The expression and function of Aj-Orpin were identified and investigated in this study. The 5' and 3' RACE polymerase chain reaction (PCR) was used to clone the full-length cDNA of Aj-Orpin. The open reading frame codes for a 164 amino-acid protein with an EF-hand&#95;7 domain and overlapping signal peptides and transmembrane regions. Moreover, Aj-Orpin mRNA and protein expression during intestinal regeneration was investigated using real-time quantitative PCR and Western blot. The expression pattern of Aj-Orpin in the regenerating intestine was investigated using immunohistochemistry. The results showed that Aj-Orpin is an exocrine protein with two EF-hand-like calcium-binding domains. Expression levels were higher in the regenerating intestine than in the normal intestine, but protein expression changes lagged behind mRNA expression changes. Aj-Orpin was found to play a role in the formation of blastema and lumen. It was primarily expressed in the serosal layer and submucosa, suggesting that it might be involved in proliferation. These observations lay the foundation for understanding the role of Orpin-like in echinoderm intestinal regeneration.

Heavy-metal pollution has increasingly jeopardized the habitats of marine organisms including the sea cucumber, a seafloor scavenger vital to seawater bio-decontamination, ocean de-acidification and coral-reef protection. Normal physiology including immune functions of sea cucumbers is toxicologically modulated by marine metal pollutants such as cadmium (Cd). The processes underpinning Cd's toxic effects on immune systems in the sea cucumber, Holothuria leucospilota , are still poorly understood. To this end, we cloned and characterized a full-length caspase-9 ( Hl-CASP9 ) cDNA in the sea cucumber, Holothuria leucospilota . Hl-CASP9 mRNA levels evolved dynamically during embryonic development. Coelomocytes, a type of phagocytic immune effectors central to H. leucospilota immunity, were found to express Hl-CASP9 mRNA most abundantly. Hl-CASP9 protein structurally resembles caspases-2 and -9 in both invertebrate and vertebrate species, comprising a CARD domain and a CASc domain. Remarkably, Hl-CASP9 was transcriptionally sensitive to abiotic oxidative stress inducers including hydrogen peroxide (H 2 O 2 ), nitric oxide ( • NO) and cadmium (Cd), but insensitive to immunostimulants including lipopolysaccharide (LPS), and poly(I:C). Overexpression of Hl-CASP9 augmented mitochondria-dependent apoptosis in HEK293T cells, while knock-down of Hl-CASP9 blunted Cd-induced coelomocyte apoptosis in vivo . Overall, we illustrate that an evolutionarily ancient caspase-9-dependent pathway exists to sensitize coelomocytes to premature cell death precipitated by heavy metal pollutants, with important implications for negative modulation of organismal immune response in marine invertebrates., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Li, Chen, Wu, Li, Zhang, Jiang, Luo, Hu, Wong and Ren.)

Only some human organs, including the liver, are capable of very weak self-regeneration. Some marine echinoderms are very useful for studying the self-regeneration processes of organs and tissues. For example, sea cucumbers Eupentacta fraudatrix (holothurians) demonstrate complete restoration of all organs and the body within several weeks after their division into two parts. Therefore, these cucumbers are a prospective model for studying the general mechanisms of self-regeneration. However, there is no data available yet concerning biomolecules of holothurians, which can stimulate the processes of organ and whole-body regeneration. Investigation of these restoration mechanisms is very important for modern medicine and biology because it can help to understand which hormones, nucleic acids, proteins, enzymes, or complexes play an essential role in self-regeneration. It is possible that stable, polyfunctional, high-molecular-weight protein complexes play an essential role in these processes. It has recently been shown that sea cucumbers Eupentacta fraudatrix contain a very stable multiprotein complex of about 2000 kDa. The first analysis of possible enzymatic activities of a stable protein complex was carried out in this work, revealing that the complex possesses several protease and DNase activities. The complex metalloprotease is activated by several metal ions (Zn2+ > Mn2+ > Mg2+). The relative contribution of metalloproteases (~63.4%), serine-like protease (~30.5%), and thiol protease (~6.1%) to the total protease activity of the complex was estimated. Metal-independent proteases of the complex hydrolyze proteins at trypsin-specific sites (after Lys and Arg). The complex contains both metal-dependent and metal-independent DNases. Mg2+, Mn2+, and Co2+ ions were found to strongly increase the DNase activity of the complex.

DNAJ proteins function as co-chaperones of HSP70 and play key roles in cell physiology to promote protein folding and degradation, especially under environmental stress. Based on our previous study on HSP70, a systematic study of DNAJ was performed in sea cucumber Apostichopus japonicus using the transcriptomic and genomic data, identifying 43 AjDNAJ genes, including six AjDNAJA genes, eight AjDNAJB genes, and 29 AjDNAJC genes. Slight expansion and conserved genomic structure were observed using the phylogenetic and syntenic analysis. Differential period-specific and tissue-specific expression patterns of AjDNAJs were observed between adult and juvenile individuals during aestivation. Strong tissue-specific expression correlations between AjDNAJ and AjHSP70 genes were found, indicating that the involvements of AjHSP70IVAs in the aestivation of sea cucumbers were regulated by AjDNAJs. Several key genes with significant expression correlations, such as AjDNAJB4L and AjHSP70IVAs, were suggested to function together under heat stress. Together, these findings provide early insight into the involvement of AjDNAJs in the aestivation and their roles as co-chaperones of AjHSP70s., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Today, marine natural products are considered one of the main sources of compounds for drug development. Starfish and sea cucumbers are potential sources of natural products of pharmaceutical interest. Among their metabolites, polar steroids, triterpene glycosides, and polar lipids have attracted a great deal of attention; however, studying these compounds by conventional methods is challenging. The application of modern MS-based approaches can help to obtain valuable information about such compounds. This review provides an up-to-date overview of MS-based applications for starfish and sea cucumber bioactive compounds analysis. While describing most characteristic features of MS-based approaches in the context of starfish and sea cucumber metabolites, including sample preparation and MS analysis steps, the present paper mainly focuses on the application of MS-based metabolic profiling of polar steroid compounds, triterpene glycosides, and lipids. The application of MS in metabolomics studies is also outlined.

The protective effect of collagen peptide from Acaudina molpadioides (Amp) on acute kidney injury (AKI) in mice and its mechanism were explored. The results showed that Amp-fed could effectively improve the renal mass index and histopathological morphology. The levels of serum creatinine and urea nitrogen decreased significantly, while the antioxidant enzyme catalase (CAT) and superoxide dismutase (SOD) increased significantly in Amp-fed groups. Western blot results disclosed that Amp significantly upregulates the levels of heme oxygenase-1 (HO-1), Nrf2, p-PI3K, and p-AKT in the kidney. In addition, Amp could significantly downregulate the levels of nuclear factor NF-kappa-B (NF- κ B), tumor necrosis factor α (TNF- α ), inflammatory cytokines interleukin 6 (IL-6) and interleukin 1 β (IL-1 β ). These findings provide evidence that Amp plays a protective role in AKI via attenuation of oxidative stress and inflammation mediated by PI3K/AKT/Nrf2 and PI3K/AKT/NF- κ B pathways. This study laid a foundation for the application of Amp in the prevention of AKI., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2022 Wei Zhao et al.)

Ferritin is considered to be an ubiquitous and conserved iron-binding protein that plays a crucial role in iron storage, detoxification, and immune response. Although ferritin is of critical importance for almost all kingdoms of life, there is a lack of knowledge about its role in the marine invertebrate sea cucumber (Apostichopus japonicus). In this study, we characterized the first crystal structure of A. japonicus ferritin (AjFER) at 2.75 Å resolution. The structure of AjFER shows a 4-3-2 symmetry cage-like hollow shell composed of 24 subunits, mostly similar to the structural characteristics of other known ferritin species, including the conserved ferroxidase center and 3-fold channel. The 3-fold channel consisting of three 3-fold negative amino acid rings suggests a potential pathway in which metal ions can be first captured by Asp120 from the outside environment, attracted by His116 and Cys128 when entering the channel, and then transferred by Glu138 from the 3-fold channel to the ferroxidase site. Overall, the presented crystal structure of AjFER may provide insights into the potential mechanism of the metal transport pathway for related marine invertebrate ferritins., (© 2022 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Tissue inhibitors of metalloproteinases (TIMPs) serve as matrix metalloproteinase (MMP) inhibitors in the pathogenesis of inflammatory diseases in vertebrates. We cloned and characterised the TIMP1 gene from Apostichopus japonicus using RACE approaches (designated as AjTIMP1). For Vibrio splendidus-challenged sea cucumbers, the peak expression of AjTIMP1 mRNAs in coelomocytes was detected at 24 h (23.44-fold) and remained at high levels (4.01-fold) until 72 h. Similarly, AjTIMP1 expression was upregulated in primary coelomocytes exposed to 10 μg mL -1 LPS. AjTIMP1 was expressed in all tissues, and the highest expression was observed in the body wall. Functional investigation revealed an imbalance in the ratio of AjMMP1/AjTIMP1 in the skin ulceration syndrome (SUS) diseased group; it was sharply up-regulated to 3.97:1 compared with the healthy group. Furthermore, when AjTIMP1 was knocked down using small interfering RNA (siRNA-KD) to 0.4-fold, AjMMP1 and AjMMP19 were upregulated to 1.99- and 1.85-fold, respectively. AjTIMP1 siRNA-KD can promote ROS production by 26.2%, whereas AjMMP1 siRNA-KD can eliminate the increase in ROS. In inflamed tissues, collagen I and III levels were decreased by 33.1% and 33.6%, respectively, in the AjTIMP1 siRNA group at 24 h AjTIMP1 was involved in the inflammatory response by mediating ROS formation and collagen degradation., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In recent years, sea cucumber has become a favorite healthcare food due to its characteristic prevention of cardiovascular diseases, suppression of tumors, as well as enhancement of immunity. In order to screen the anti-tumoral proteins or peptides from sea cucumber ( Apostichopus japonicus ), its cDNA library was analyzed, and a disintegrin-like and metalloproteinase with thrombospondin type 1 motif, member 13 (ADAMTS13)-like was found. ADAMTS13-like contains 10 thrombospondin 1 (TSP1) domains. Based on analysis of bioinformatics, the third TSP1 domain of this protein, which is further named Aj-Tspin, contains an arginine-glycine-aspartate (RGD) motif. Since our previous studies showed that the recombinant RGD-containing peptide from lampreys showed anti-tumoral activity, the third TSP1 domain of ADAMTS13-like was chosen to evaluate it's effect on tumor proliferation and metastasis, despite the fact it shares almost no homologue with disintegrins from other species. After artificial synthesis, its cDNA sequence, Aj-Tspin, which is composed of 56 amino acids, was subcloned into a pET23b vector and expressed as a recombinant Aj-Tspin (rAj-Tspin) in a soluble form with a molecular weight of 6.976 kDa. Through affinity chromatography, rAj-Tspin was purified as a single protein. Both anti-proliferation and immunofluorescence assays showed that rAj-Tspin suppressed the proliferation of Lewis lung carcinoma (LLC) cells through apoptosis. Adhesion assay also displayed that rAj-Tspin inhibited the adhesion of LLC cells to ECM proteins, including fibronectin, laminin, vitronectin and collagen. Lastly, rAj-Tspin also suppressed the migration and invasion of LLC cells across the filter in transwells. Thus, the above indicates that rAj-Tspin might act as a potential anti-tumoral drug in the future and could also provide information on the nutritional value of sea cucumber.

Regeneration is a key developmental process by which organisms recover vital tissue and organ components following injury or disease. A growing interest is focused on the elucidation and characterization of the molecular mechanisms involved in these regenerative processes. We have now analyzed the possible role of the Wnt/β-catenin pathway on the regeneration of the intestine in the sea cucumber Holothuria glaberrima. For this we have studied the expression in vivo of Wnt-associated genes and have implemented the use of Dicer-substrate interference RNA (DsiRNA) to knockdown the expression of β-catenin transcript on gut rudiment explants. Neither cell dedifferentiation nor apoptosis were affected by the reduction of β-catenin transcripts in the gut rudiment explants. Yet, the number of proliferating cells decreased significantly following the interference, suggesting that the Wnt/β-catenin signaling pathway plays a significant role in cell proliferation, but not in cell dedifferentiation nor apoptosis during the regeneration of the intestine. The development of the in vitro RNAi protocol is a significant step in analyzing specific gene functions involved in echinoderm regeneration., (Copyright © 2021. Published by Elsevier Inc.)

Echinoderms are marine deuterostomes with fascinating adaptation features such as aestivation and organ regeneration. However, post-transcriptional gene regulation by microRNAs (miRNAs) underlying these features are largely unexplored. Here, using homology-based and de novo approaches supported by expression data, we provided a comprehensive annotation of miRNA genes in the sea cucumber Apostichopus japonicus. By linkage and phylogenic analyses, we characterized miRNA genomic organization, evolutionary history and expression regulation. The results showed that sea cucumbers evolved a large number of new miRNAs, which tended to form polycistronic clusters via tandem duplication that had been especially active in the echinoderms. Most new miRNAs were weakly expressed, but miRNA clustering increased the expression level of clustered new miRNAs. The most abundantly expressed new miRNAs were organized in a single tandem cluster (cluster n2), which was activated during aestivation and intestine regeneration. Overall, our analyses suggest that clustering of miRNAs is important for their evolutionary origin, expression control, and functional cooperation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Genomic tRNA copy numbers determine cytoplasmic tRNA abundances, which in turn influence translation efficiency, but the underlying mechanism is not well understood. Using the sea cucumber Apostichopus japonicus as a model, we combined genomic sequence, transcriptome expression and ecological food resource data to study its codon usage adaptation. The results showed that, unlike intragenic non-coding RNAs, transfer RNAs (tRNAs) tended to be transcribed independently. This may be attributed to their specific Pol III promoters that lack transcriptional regulation, which may underlie the correlation between genomic copy number and cytoplasmic abundance of tRNAs. Moreover, codon usage optimization was mostly restrained by a gene's amino acid sequence, which might be a compromise between functionality and translation efficiency for stress responses were highly optimized for most echinoderms, while enzymes for saponin biosynthesis (LAS, CYPs and UGTs) were especially optimized in sea cucumbers, which might promote saponin synthesis as a defence strategy. The genomic tRNA content of A. japonicus was positively correlated with amino acid content in its natural food particles, which should promote its efficiency in protein synthesis. We propose that coevolution between genomic tRNA content and codon usage of sea cucumbers facilitates their saponin synthesis and survival using food resources with low nutrient content.

Certain sulfated glycans, including those from marine sources, can show potential effects against SARS-CoV-2. Here, a new fucosylated chondroitin sulfate (FucCS) from the sea cucumber Pentacta pygmaea (PpFucCS) (MW ∼10-60 kDa) was isolated and structurally characterized by NMR. PpFucCS is composed of {→3)-β-GalNAcX-(1→4)-β-GlcA-[(3→1)Y]-(1→}, where X = 4S (80%), 6S (10%) or nonsulfated (10%), Y = α-Fuc2,4S (40%), α-Fuc2,4S-(1→4)-α-Fuc (30%), or α-Fuc4S (30%), and S = SO 3 - . The anti-SARS-CoV-2 activity of PpFucCS and those of the FucCS and sulfated fucan isolated from Isostichopus badionotus (IbFucCS and IbSF) were compared with that of heparin. IC 50 values demonstrated the activity of the three holothurian sulfated glycans to be ∼12 times more efficient than heparin, with no cytotoxic effects. The dissociation constant (K D ) values obtained by surface plasmon resonance of the wildtype SARS-CoV-2 spike (S)-protein receptor-binding domain (RBD) and N501Y mutant RBD in interactions with the heparin-immobilized sensor chip were 94 and 1.8 × 10 3  nM, respectively. Competitive surface plasmon resonance inhibition analysis of PpFucCS, IbFucCS, and IbSF against heparin binding to wildtype S-protein showed IC 50 values (in the nanomolar range) 6, 25, and 6 times more efficient than heparin, respectively. Data from computational simulations suggest an influence of the sulfation patterns of the Fuc units on hydrogen bonding with GlcA and that conformational change of some of the oligosaccharide structures occurs upon S-protein RBD binding. Compared with heparin, negligible anticoagulant action was observed for IbSF. Our results suggest that IbSF may represent a promising molecule for future investigations against SARS-CoV-2., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Nine new mono-, di-, and trisulfated triterpene penta- and hexaosides, kurilosides A 3 ( 1 ), D 1 ( 2 ), G ( 3 ), H ( 4 ), I ( 5 ), I 1 ( 6 ), J ( 7 ), K ( 8 ), and K 1 ( 9 ) and two desulfated derivatives, DS-kuriloside L ( 10 ), having a trisaccharide branched chain, and DS-kuriloside M ( 11 ), having hexa- nor -lanostane aglycone with a 7(8)-double bond, have been isolated from the Far-Eastern deep-water sea cucumber Thyonidium (=Duasmodactyla) kurilensis (Levin) and their structures were elucidated based on 2D NMR spectroscopy and HR-ESI mass-spectrometry. Five earlier unknown carbohydrate chains and two aglycones (having a 16 β ,(20S)-dihydroxy-fragment and a 16 β -acetoxy,(20S)-hydroxy fragment) were found in these glycosides. All the glycosides 1 - 9 have a sulfate group at C-6 Glc, attached to C-4 Xyl1, while the positions of the other sulfate groups vary in different groups of kurilosides. The analysis of the structural features of the aglycones and the carbohydrate chains of all the glycosides of T. kurilensis showed their biogenetic relationships. Cytotoxic activities of the compounds 1 - 9 against mouse neuroblastoma Neuro 2a, normal epithelial JB-6 cells, and erythrocytes were studied. The highest cytotoxicity in the series was demonstrated by trisulfated hexaoside kuriloside H ( 4 ), having acetoxy-groups at C(16) and C(20), the latter one obviously compensated the absence of a side chain, essential for the membranolytic action of the glycosides. Kuriloside I 1 ( 6 ), differing from 4 in the lacking of a terminal glucose residue in the bottom semi-chain, was slightly less active. The compounds 1 - 3 , 5 , and 8 did not demonstrate cytotoxic activity due to the presence of hydroxyl groups in their aglycones.

Holothurians, or sea cucumbers, belong to the phylum Echinodermata. They show good regenerative abilities. The present review provides an analysis of available data on the molecular aspects of regeneration mechanisms in holothurians. The genes and signaling pathways activated during the asexual reproduction and the formation of the anterior and posterior parts of the body, as well as the molecular mechanisms that provide regeneration of the nervous and digestive systems, are considered here. Damage causes a strong stress response, the signs of which are recorded even at late regeneration stages. In holothurian tissues, the concentrations of reactive oxygen species and antioxidant enzymes increase. Furthermore, the cellular and humoral components of the immune system are activated. Extracellular matrix remodeling and Wnt signaling play a major role in the regeneration in holothurians. All available morphological and molecular data show that the dedifferentiation of specialized cells in the remnant of the organ and the epithelial morphogenesis constitute the basis of regeneration in holothurians. However, depending on the type of damage, the mechanisms of regeneration may differ significantly in the spatial organization of regeneration process, the involvement of different cell types, and the depth of reprogramming of their genome (dedifferentiation or transdifferentiation).

Six new monosulfated triterpene tetra-, penta- and hexaosides, namely, the kurilosides A 1 ( 1 ), A 2 ( 2 ), C 1 ( 3 ), D ( 4 ), E ( 5 ) and F ( 6 ), as well as the known earlier kuriloside A ( 7 ), having unusual non-holostane aglycones without lactone, have been isolated from the sea cucumber Thyonidium (= Duasmodactyla) kurilensis (Levin) (Cucumariidae, Dendrochirotida) , collected in the Sea of Okhotsk near Onekotan Island from a depth of 100 m. Structures of the glycosides were established by 2D NMR spectroscopy and HR-ESI mass spectrometry. Kurilosides of the groups A and E contain carbohydrate moieties with a rare architecture (a pentasaccharide branched by C(4) Xyl1), differing from each other in the second monosaccharide residue (quinovose or glucose, correspondingly); kurilosides of the group C are characterized by a unique tetrasaccharide branched by a C(4) Xyl1 sugar chain; and kurilosides of the groups D and F are hexaosides differing from each other in the presence of an O -methyl group in the fourth (terminal) sugar unit. All these glycosides contain a sulfate group at C-6 of the glucose residue attached to C-4 Xyl1 and the non-holostane aglycones have a 9(11) double bond and lack γ-lactone. The cytotoxic activities of compounds 1 - 7 against mouse neuroblastoma Neuro 2a, normal epithelial JB-6 cells and erythrocytes were studied. Kuriloside A 1 ( 1 ) was the most active compound in the series, demonstrating strong cytotoxicity against the erythrocytes and JB-6 cells and a moderate effect against Neuro 2a cells.

Structural proteins play critical roles in the food quality, especially texture properties, of sea cucumbers and their products. Most of the previous studies on sea cucumbers focused on few individual proteins, which limited our understanding of how structural proteins influenced the quality of sea cucumbers. Inspired by the clarification of sea cucumber (Apostichopus japonicus) genome, we established an integrated data of structural proteins in the sea cucumber body wall. A portfolio of 2018 structural proteins was screened out from the sea cucumber annotated proteome by bioinformatics analysis. The portfolio was divided into three divisions, including extracellular matrix proteins, muscle proteins, and proteases, and further classified into 18 categories. The presence of 472 proteins in the sea cucumber body wall was confirmed by using a proteomics approach. Moreover, comparative proteomics analysis revealed the spatial distribution heterogeneity of structural proteins in the sea cucumber body wall at a molecular scale. This study suggested that future researches on sea cucumbers could be performed from an integrated perspective, which would reshape the component map of sea cucumber and provide novel insights into the understanding of how the food quality of sea cucumber was determined on a molecular level.

Sea cucumbers have been shown to have potential health benefits and are a rich source of several bioactive compounds, particularly triterpenoid saponins. However, most studies concentrate on the body wall, and little is known about the health effects of the coproducts. The objectives of this study were to determine the nutritional composition of a coproduct from the sea cucumber Cucumaria frondosa and the effects of the dietary consumption of this coproduct on cardiometabolic health in rats. Chemical, biochemical, and nutritional analyses were performed to characterize this coproduct. Forty (40) male Wistar rats were then equally divided into four groups and fed a purified control diet or a diet enriched with 0.5%, 1.5%, or 2.5% (by protein) of coproduct. After 28 days of feeding, the rats were sacrificed. Body and tissue weight, body composition, epididymal adipocyte diameter, plasma and hepatic lipids, glycemia, and insulinemia were measured at the end of the 28-day experiment. Analysis of the coproduct revealed high levels of protein, omega-3 fatty acids, minerals, and saponins. The 1.5% group had significantly smaller epididymal adipocytes vs. the control. We conclude that dietary administration of this sea cucumber coproduct at 1.5% doses decreases visceral adiposity, potentially decreasing the risk of cardiometabolic dysfunction. The coproduct's saponin content may contribute to the observed effects, but the impact of other components cannot be ruled out.

Collagen is the major fibrillar protein in most living organisms. Among the different types of collagen, type I collagen is the most abundant one in tissues of marine invertebrates. Due to the health-related risk factors and religious constraints, use of mammalian derived collagen has been limited. This triggers the search for alternative sources of collagen for both food and non-food applications. In this regard, numerous studies have been conducted on maximizing the utilization of seafood processing by-products and address the need for collagen. However, less attention has been given to marine invertebrates and their by-products. The present review has focused on identifying sea cucumber as a potential source of collagen and discusses the general scope of collagen extraction, isolation, characterization, and physicochemical properties along with opportunities and challenges for utilizing marine-derived collagen.

To promote the nutritional and pharmacological values of four sea cucumber species ( Holothuria poli , H. tubulosa , H. arguinensis , and H. sanctori ), harvested from the Algerian coast, we aimed to study their proximate composition, fatty acid profile and angiotensin-converting enzyme (ACE) inhibitory activity. Their phospholipids were also used to elaborate nanoliposomes and to encapsulate peptides obtained from the same source. After the physico-chemical characterization of nanoliposomes and peptides, in vitro analyses were realized. The four holothurian species showed a high amount of protein (49.26-69.34%), and an impressive lipid profile of 27 fatty acids, mainly composed of polar fatty acids (91.16-93.85%), with a high polyunsaturated fatty acids (PUFA) content (50.90-71.80%), particularly eicosapentaenoic acid (EPA) (5.07-8.76%) and docosahexaenoic acid (DHA) (4.86-7.25%). A high phospholipids amount was also found (55.20-69.85%), mainly composed of phosphatidylcholine (PC) (51.48-58.56%). Their peptide fractions exhibited a high ACE inhibitory activity (IC 50 0.30 to 0.51 mg/mL). The results also showed that the nanoliposomes do not induce cytotoxicity and cell death in human MSCs and no perturbation of proliferation for all the times and the tested concentrations, as well as the combined nanoliposomes and hydrolysates (HTS) at a concentration of 0.1 mg/mL. All four sea cucumbers show potential as a new source for omega-3, omega-6, and bioactive peptides., Competing Interests: The authors declare no conflict of interest.

Experiments in sea cucumbers reveal how the physiological responses regulated by a neuropeptide called kisspeptin have evolved., Competing Interests: MA, PO No competing interests declared, (© 2020, Arnone and Oliveri.)

As a multifunctional protein, cyclophilin A (CypA) plays an important role in cell apoptosis. In our previous work, we found that CypA from Apostichopus japonicus (AjCypA), as a cofactor, could modulate nuclear translocation of NF-κB. However, the immune function of AjCypA is largely unknown. In the present study, we found that siRNA-mediated AjCypA knockdown in vivo significantly increased the coelomocyte apoptosis rate. In addition, the expression of B-cell lymphoma-2 (AjBcl-2, an anti-apoptosis gene) was synchronously downregulated. To better understand the connection between AjCypA and AjBcl-2 expression, we cloned the promoter of AjBcl-2 via genomic walking, which spanned 1870 bp and contained four potential binding sites of NF-κB. Dual-luciferase reporter assay revealed that the full-length sequence and all truncated fragments exhibited high transcriptional activity. Moreover, 1 μg/mL LPS exposure significantly increased the luciferase activity of P1 (-1870/+57) by 2.31-fold and 3.15-fold at 12 and 24 h, respectively. Furthermore, the four potential NF-κB binding sites and pCMV-Flag2C-AjNF-κB co-transfection assay demonstrated that NF-κB could regulate the expression of AjBcl-2 via the NF-κB binding sites of AjBcl-2 promoter. All results supported that AjCypA mediates coelomocyte apoptosis via NF-κB/AjBcl-2 signaling pathway in A. japonicus., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The holothurian Eupentacta fraudatrix is a unique organism for studying regeneration mechanisms. Moreover, E. fraudatrix can quickly restore parts of its body and entire organ systems, yet at the moment, there is no data on the participation of stem cells in the process. To the contrary, it has been repeatedly confirmed that this process is only due to the transformation of terminally differentiated cells. In this study, we examine changes in gene expression during gut regeneration of the holothurian E. fraudatrix. Transcriptomes of intestinal anlage of the three stages of regeneration, as well as the normal gut, were sequenced with an Illumina sequencer (San Diego, CA, USA). We identified 14,617 sea urchin protein homologs, of which 308 were transcription factors. After analysing the dynamics of gene expression during regeneration and the map of biological processes in which they participate, we identified 11 factors: Ef-EGR1, Ef-ELF, Ef-GATA3, Ef-ID2, Ef-KLF1/2/4, Ef-MSC, Ef-PCGF2, Ef-PRDM9, Ef-SNAI2, Ef-TBX20, and Ef-TCF24. With the exception of TCF24, they are all involved in the regeneration, development, epithelial-mesenchymal transition, and immune response in other animals. We suggest that these transcription factors may also be involved in the transdifferentiation of coelomic epithelial cells into enterocytes in holothurians.

Heat stress (HS) is an important factor for the survival of the marine organism Apostichopus japonicus . Lysine acetylation is a pivotal post-translational modification that modulates diverse physiological processes including heat shock response (HSR). In this study, 4028 lysine acetylation sites in 1439 proteins were identified in A. japonicus by acetylproteome sequencing. A total of 13 motifs were characterized around the acetylated lysine sites. Gene Ontology analysis showed that major acetylated protein groups were involved in "oxidation-reduction process", "ribosome", and "protein binding" terms. Compared to the control group, the acetylation quantitation of 25 and 41 lysine sites changed after 6 and 48 h HS. Notably, lysine acetyltransferase CREB-binding protein (CBP) was identified to have differential acetylation quantitation at multiple lysine sites under HS. Various chaperones, such as caseinolytic peptidase B protein homolog (CLBP), T-complex protein 1 (TCP1), and cyclophilin A (CYP1), showed differential acetylation quantitation after 48 h HS. Additionally, many translation-associated proteins, such as ribosomal proteins, translation initiation factor (IF), and elongation factors (EFs), had differential acetylation quantitation under HS. These proteins represented specific interaction networks. Collectively, our results offer novel insight into the complex HSR in A. japonicus and provide a resource for further mechanistic studies examining the regulation of protein function by lysine acetylation.

In this study, three synthetic zinc-chelating peptides (ZCPs) derived from sea cucumber hydrolysates with limited or none of the common metal-chelating amino-acid residues were analyzed by flame atomic absorption spectroscopy, circular dichroism spectroscopy, size exclusion chromatography, zeta-potential, Fourier transform infrared spectroscopy, Raman spectroscopy and nuclear magnetic resonance spectroscopy. The amount of zinc bound to the ZCPs reached maximum values with ZCP:zinc at 1:1, and it was not further increased by additional zinc presence. The secondary structures of ZCPs were slightly altered, whereas no formation of multimers was observed. Furthermore, zinc increased the zeta-potential value by neutralizing the negatively charged residues. Only free carboxyl in C-terminus of ZCPs was identified as the primary binding site of zinc. These results provide the theoretical foundation to understand the mechanism of zinc chelation by peptides., Competing Interests: Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: 13C NMR spectra of ZCPs and ZCP-zinc complexes from 160 to 180 ppm. Numbers represent designated carboxyl and acylamino, Figure S2: Full spectra of 13C NMR spectra of ZCPs and ZCP-zinc complexes, Figure 3: 1H NMR spectra of ZCPs and ZCP-zinc complexes (dissolved in DMSO-d6 or DMSO-d6/10%D2O). Numbers represent designated hydrogen.

The sea cucumber Apostichopus japonicus is a foodstuff with very high economic value in China, Japan and other countries in south-east Asia. It is at the heart of a multibillion-dollar industry and to meet demand for this product, aquaculture methods and facilities have been established. However, there are challenges associated with optimization of reproduction, feeding and growth in non-natural environments. Therefore, we need to learn more about the biology of A. japonicus, including processes such as aestivation, evisceration, regeneration and albinism. One of the major classes of molecules that regulate physiology and behaviour in animals are neuropeptides, and a few bioactive peptides have already been identified in A. japonicus. To facilitate more comprehensive investigations of neuropeptide function in A. japonicus, here we have analysed genomic and transcriptomic sequence data and proteomic data to identify neuropeptide precursors and neuropeptides in this species. We identified 44 transcripts encoding neuropeptide precursors or putative neuropeptide precursors, and in some instances neuropeptides derived from these precursors were confirmed by mass spectrometry. Furthermore, analysis of genomic sequence data enabled identification of the location of neuropeptide precursor genes on genomic scaffolds and linkage groups (chromosomes) and determination of gene structure. Many of the precursors identified contain homologs of neuropeptides that have been identified in other bilaterian animals. Precursors of neuropeptides that have thus far only been identified in echinoderms were identified, including L- and F-type SALMFamides, AN peptides and others. Precursors of several peptides that act as modulators of neuromuscular activity in A. japonicus were also identified. The discovery of a large repertoire of neuropeptide precursors and neuropeptides provides a basis for experimental studies that investigate the physiological roles of neuropeptide signaling systems in A. japonicus. Looking ahead, some of these neuropeptides may have effects that could be harnessed to enable improvements in the aquaculture of this economically important species.

Marine macroalgae (seaweed) are an excellent source of novel bioactive metabolites. The biorefinery concept applied to seaweed facilitates the extraction of many chemical constituents from the same biomass ensuring that the resource is used fully, generating few residues through a succession of extraction steps. In the present study, the biomass of the carragenophyte Solieria filiformis (Rhodophyta, Gigartinales) cultured in an integrated multi-trophic aquaculture (IMTA) system was evaluated to obtain valuable products by a biorefinery approach. Enzymatic-assisted extraction (EAE) and microwave-assisted extraction (MAE) were the eco-friendly technologies used to ensure an environmentally friendly valorization of the biomass. Three valuable products were successfully recovered: a water-soluble extract rich in proteins and sulfated polysaccharides suitable as a food supplement; a lipid fraction rich in polyunsaturated fatty acids (PUFAs) with potential to be used in the nutraceutical industry; and a pure ι-carrageenan with a powerful antiviral activity against Herpes simplex virus (EC 50 = 6.3 µg mL -1 ) comparable to the commercial antiviral acyclovir (EC 50 = 3.2⁻5.4 µg mL -1 ).

Sea cucumbers produce saponins as a chemical defense mechanism, however their cells can tolerate the cytotoxic nature of these chemicals. To elucidate the molecular mechanisms behind this tolerance a suite of complementary biophysical tools was used, firstly using liposomes for in vitro techniques then using in silico approaches for a molecular-level insight. The holothuroid saponin Frondoside A, caused significantly less permeabilization in liposomes containing a Δ 7 holothuroid sterol than those containing cholesterol and resulted in endothermic interactions versus exothermic interactions with cholesterol containing liposomes. Lipid phases simulations revealed that Frondoside A has an agglomerating effect on cholesterol domains, however, induced small irregular Δ 7 sterol clusters. Our results suggest that the structural peculiarities of holothuroid sterols provide sea cucumbers with a mechanism to mitigate the sterol-agglomerating effect of saponins, and therefore to protect their cells from the cytotoxicity of the saponins they produce.

Impaired wound healing is a major clinical problem in patients with diabetes and is the leading cause of lower limb amputation. This study is aimed to observe the effects of small molecule oligopeptides isolated from sea cucumber (SCCOPs) on the wound healing process in diabetic mice. Ninety db/db male mice were divided into five groups, including the model control group, whey protein group (0.50 g/kg) and three SCCOPs dose groups (0.25 g/kg, 0.50 g/kg and 1.00 g/kg). Additionally, 18 db/m male mice were used as normal control group. After full-thickness incisions on the dorsum, mice in SCCOPs-treated groups were intragastrically administered SCCOPs, while others were administered vehicle or whey protein. Mice were sacrificed on days 4, 7 and 14. The wound healing condition, inflammatory response, angiogenesis, collagen deposition, oxidative stress and nutritional status were evaluated. A pathological report showed increased vascularisation, collagen deposition and epithelialisation in SCCOPs-treated groups. SCCOPs-treated mice showed decreased C-reactive protein (CRP), interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α, chemokine (C-C motif) ligand 2 (CCL2) and reactive oxygen species (ROS) contents, and increased IL-10, stromal cell-derived factor-1 alpha (SDF-1α), nitric oxide (NO), albumin (ALB), prealbumin (PA) and transferrin (TRF) levels and vascular endothelial growth factor (VEGF) expression. All parameters were significant ( p < 0.05) in comparison to model control group. These results suggest that treatment with SCCOPs can promote significant wound healing in diabetic mice., Competing Interests: The authors declare no conflict of interest.

The sea cucumber, an important economic species, has encountered high mortality since 2013 in northern China because of seasonal environmental stress such as hypoxia, high temperature, and low salinity. MicroRNAs (miRNAs) are important in regulating gene expression in marine organisms in response to environmental change. In this study, high-throughput sequencing was used to investigate alterations in miRNA expression in the sea cucumber under different levels of dissolved oxygen (DO). Nine small RNA libraries were constructed from the sea cucumber respiratory trees. A total of 26 differentially expressed miRNAs, including 12 upregulated and 14 downregulated miRNAs, were observed in severe hypoxia (DO 2 mg/L) compared with mild hypoxia (DO 4 mg/L) and normoxic conditions (DO 8 mg/L). Twelve differentially expressed miRNAs were clustered in severe hypoxia. In addition, real-time PCR revealed that 14 randomly selected differentially expressed miRNAs showed significantly increased expressions in severe hypoxia and the expressions of nine miRNAs, including key miRNAs such as Aja-miR-1, Aja-miR-2008, and Aja-miR-184, were consistent with the sequencing results. Moreover, gene ontology and pathway analyses of putative target genes suggest that these miRNAs are important in redox, transport, transcription, and hydrolysis under hypoxia stress. Notably, novel-miR-1, novel-miR-2, and novel-miR-3 were specifically clustered and upregulated in severe hypoxia, which may provide new insights into novel "hypoxamiR" identification. These results will provide a basis for future studies of miRNA regulation and molecular adaptive mechanisms in sea cucumbers under hypoxia stress., (Copyright © 2017 Huo et al.)

Sea cucumbers belonging to echinoderm are traditionally used as tonic food in China and other Asian countries. They produce abundant biologically active triterpene glycosides. More than 300 triterpene glycosides have been isolated and characterized from various species of sea cucumbers, which are classified as holostane and nonholostane depending on the presence or absence of a specific structural unit γ(18,20)-lactone in the aglycone. Triterpene glycosides contain a carbohydrate chain up to six monosaccharide units mainly consisting of d-xylose, 3-O-methy-d-xylose, d-glucose, 3-O-methyl-d-glucose, and d-quinovose. Cytotoxicity is the common biological property of triterpene glycosides isolated from sea cucumbers. Besides cytotoxicity, triterpene glycosides also exhibit antifungal, antiviral and hemolytic activities. This review updates and summarizes our understanding on diverse chemical structures of triterpene glycosides from various species of sea cucumbers and their important biological activities. Mechanisms of action and structural-activity relationships (SARs) of sea cucumber glycosides are also discussed briefly., Competing Interests: The authors declare no conflict of interest.

The Far Eastern sea cucumber Eupentacta fraudatrix is an inhabitant of shallow waters of the south part of the Sea of Japan. This animal is an interesting and rich source of triterpene glycosides with unique chemical structures and various biological activities. The objective of this study was to investigate composition and distribution in various body components of triterpene glycosides of the sea cucumber E. fraudatrix . We applied LC-ESI MS (liquid chromatography-electrospray mass spectrometry) of whole body extract and extracts of various body components for metabolic profiling and structure elucidation of triterpene glycosides from the E. fraudatrix . Totally, 54 compounds, including 26 sulfated, 18 non-sulfated and 10 disulfated glycosides were detected and described. Triterpene glycosides from the body walls, gonads, aquapharyngeal bulbs, guts and respiratory trees were extracted separately and the distributions of the detected compounds in various body components were analyzed. Series of new glycosides with unusual structural features were described in E. fraudatrix , which allow clarifying the biosynthesis of these compounds. Comparison of the triterpene glycosides contents from the five different body components revealed that the profiles of triterpene glycosides were qualitatively similar, and only some quantitative variabilities for minor compounds were observed.

The sea cucumber (Apostichopus japonicus) is an economically important aquaculture species in China. However, the serious individual growth variation often caused financial losses to farmers and the genetic mechanisms are poorly understood. In the present study, the extensively analysis at the transcriptome level for individual growth variation in sea cucumber was carried out. A total of 118946 unigenes were assembled from 255861 transcripts, with N50 of 1700. Of all unigenes, about 23% were identified with at least one significant match to known databases. In all four pair of comparison, 1840 genes were found to be expressed differently. Global hypometabolism was found to be occurred in the slow growing population, based on which the hypothesis was raised that growth retardation in individual growth variation of sea cucumber is one type of dormancy which is used to be against to adverse circumstances. Besides, the pathways such as ECM-receptor interaction and focal adhesion were enriched in the maintenance of cell and tissue structure and communication. Further, 76645 SSRs, 765242 SNPs and 146886 ins-dels were detected in the current study providing an extensive set of data for future studies of genetic mapping and selective breeding. In summary, these results will provides deep insight into the molecular basis of individual growth variation in marine invertebrates, and be valuable for understanding the physiological differences of growth process.

Fucosylated chondroitin sulfate (FCS) from sea cucumbers is composed of a chondroitin sulfate (CS) central core and branches of sulfated fucose. The structure of this complex glycosaminoglycan is usually investigated via nuclear magnetic resonance (NMR) analyses of the intact molecule, ergo through a top-down approach, which often yield spectra with intricate sets of signals. Here we employed a bottom-up approach to analyze the FCSs from the sea cucumbers Isostichopus badionotus and Ludwigothurea grisea from their basic constituents, viz. CS cores and sulfated fucose branches, obtained via systematic fragmentation through mild acid hydrolysis. Oligosaccharides derived from the central CS core were analyzed via NMR spectroscopy and the disaccharides produced using chondroitin sulfate lyase via SAX-HPLC. The CS cores from the two species were similar, showing only slight differences in the proportions of 4- or 6-monosulfated and 4,6-disulfated β-d-GalNAc. Sulfated fucose units released from the FCSs were analyzed via NMR and ESI-HRMS spectroscopies. The fucose units from each species presented extensive qualitative differences, but quantitative assessments of these units were hindered, mostly because of their extensive desulfation during the hydrolysis. The bottom-up analysis performed here has proved useful to explore the structure of FCS through a sum-of-the-parts approach in a qualitative manner. We further demonstrate that under specific acidification conditions particular fucose branches can be removed preferentially from FCS. Preparation of derivatives enriched with particular fucose branches could be useful for studies on "structure vs. biological function" of FCS., (© The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

This study evaluated the effect of pH on the activity of antioxidant and immune enzymes in the sea cucumber Isostichopus badionotus exposed to different temperatures. The organisms (530 ±110 g) were exposed to 16, 20, 24, 28, 30, 34 and 36°C for 6 h to evaluate thermal limits at two water pH values (treatment = 7.70; control = 8.17). For the thermal tolerance experiment, the organisms were exposed to sublethal temperature of 34°C for 3, 6, 12, 24, and 48 h. I. badionotus showed signs of thermal stress by synthesizing heat shock protein 70 (hsp70) at the cold (16°C) and warm thermal limits (34°C). The glutathione peroxidase (GPx) showed a negative correlation with superoxide dismutase (SOD) activity in modulating the effect of oxidative stress at different temperature levels. Specifically, GPx activity was maximal at the extremes of the cold and warm temperatures (16, 20, and 36°C) tested, while contrarily, the SOD activity increased significantly in the narrow range of temperature between 28 and 30°C, as a part of a reaction to offset oxidative damage. The effect of pH on the expression of hsp70 was not significant, whereas the antioxidant enzymes activity was stimulated at pH 7.70. Mucosal immunity, evidenced by the activation of the phenoloxidase (PO) system, increased above the basal level at pH 7.70 and at 28, 30, and 34°C. Independent of pH, the temperature of 34°C was identified as the 12 h-sublethal upper limit for I. badionotus.

The sea cucumber, Apostichopus japonicus, is a good model for studying environmentally-induced aestivation by a marine invertebrate. One of the central requirements of aestivation is the repression of energy-expensive cellular processes such as cell cycle progression. The present study identified the gene structure of the cell cycle regulator, cyclin B, and detected the expression levels of this gene over three stages of the annual aestivation-arousal cycle. Furthermore, the DNA methylation characteristics of cyclin B were analyzed in non-aestivation and deep-aestivation stages of sea cucumbers. We found that the cyclin B promoter contains a CpG island, three CCAAT-boxes and three cell cycle gene homology regions (CHRs). Application of qRT-PCR analysis showed significant downregulation of cyclin B transcript levels during deep-aestivation in comparison with non-aestivation in both intestine and longitudinal muscle, and these returned to basal levels after arousal from aestivation. Methylation analysis of the cyclin B core promoter revealed that its methylation level showed significant differences between non-aestivation and deep-aestivation stages (p<0.05) and interestingly, a positive correlation between Cyclin B transcripts expression and methylation levels of the core promoter was also observed. Our findings suggest that cell cycle progression may be reversibly arrested during aestivation as indicated by the changes in cyclin B expression levels and we propose that DNA methylation is one of the regulatory mechanisms involved in cyclin B transcriptional variation., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Context: Sea cucumbers have been consumed as tonic, food, and nutrition supplements for many years., Objective: The objective of this study is to investigate the antiobesity and lipid-lowering effects of sea cucumber extracts in in vitro and in vivo models and elucidate the mechanism of action of the extracts on obesity and dyslipidemia., Materials and Methods: The 60% ethanol extracts from the body walls of 10 different sea cucumbers were investigated for the inhibition of pancreatic lipase (PL) activity in vitro. The optimal active extract (SC-3) was further chemically analyzed by LC-MS and UV. And 0.1% and 0.2% of SC-3 was mixed with a high-fat diet to treat C57/BL6 mice for 6 weeks or 2 weeks as preventive and therapeutic study. The body weight, serum, and liver lipid profile in the mice were investigated., Results: The crude extract of Pearsonothuria graeffei Semper (Holothuriidae) inhibited the PL activity by 36.44% of control at 0.5 μg/mL. SC-3 and echinoside A inhibited PL with an IC50 value at 2.86 μg/mL and 0.76 μM. 0.1% of SC-3 reduced the body weight (23.0 ± 0.62 versus 26.3 ± 0.76 g), the serum TC (2.46 ± 0.04 versus 2.83 ± 0.12 mmol/L), TG (0.19 ± 0.08 versus 0.40 ± 0.03 mmo/L), and LDL-c (0.48 ± 0.02 versus 0.51 ± 0.02 mmol/L), and liver TC (1.19 ± 0.17 versus 1.85 ± 0.13 mmol/mg) and TG (6.18 ± 0.92 versus 10.87 ± 0.97 mmol/mg) contents of the obese C57BL/six mice on a high-fat diet., Discussion and Conclusion: Sea cucumber may be used for developing antiobesity and antihyperlipidemia drugs.

The present study is focused on the intestinal absorption of sea cucumber saponins. We determined the pharmacokinetic characteristics and bioavailability of Echinoside A and Holotoxin A₁; the findings indicated that the bioavailability of Holotoxin A₁ was lower than Echinoside A. We inferred that the differences in chemical structure between compounds was a factor that explained their different characteristics of transport across the intestine. In order to confirm the absorption characteristics of Echinoside A and Holotoxin A₁, we examined their transport across Caco-2 cell monolayer and effective permeability by single-pass intestinal perfusion. The results of Caco-2 cell model indicate that Echinoside A is transported by passive diffusion, and not influenced by the exocytosis of P-glycoprotein (P-gp, expressed in the apical side of Caco-2 monolayers as the classic inhibitor). The intestinal perfusion also demonstrated well the absorption of Echinoside A and poor absorption of Holotoxin A₁, which matched up with the result of the Caco-2 cell model. The results demonstrated our conjecture and provides fundamental information on the relationship between the chemical structure of these sea cucumber saponins and their absorption characteristics, and we believe that our findings build a foundation for the further metabolism study of sea cucumber saponins and contribute to the further clinical research of saponins.

In this study, three typical members representative of different arginine metabolic pathways were firstly identified from Apostichopus japonicus, including nitric oxide synthase (NOS), arginase, and agmatinase. Spatial expression analysis revealed that the AjNOS transcript presented negative expression patterns relative to those of Ajarginase or Ajagmatinase in most detected tissues. Furthermore, Vibrio splendidus-challenged coelomocytes and intestine, and LPS-exposed primary coelomocytes could significantly induce AjNOS expression, followed by obviously inhibited Arginase and AjAgmatinase transcripts at the most detected time points. Silencing the three members with two specific siRNAs in vivo and in vitro collectively indicated that AjNOS not only compete with Ajarginase but also with Ajagmatinase in arginine metabolism. Interestingly, Ajarginase and Ajagmatinase displayed cooperative expression profiles in arginine utilization. More importantly, live pathogens of V. splendidus and Vibrio parahaemolyticus co-incubated with primary cells also induced NO production and suppressed arginase activity in a time-dependent at an appropriate multiplicity of infection (MOI) of 10, without non-pathogen Escherichia coli. When increasing the pathogen dose (MOI = 100), arginase activity was significantly elevated, and NO production was depressed, with a larger magnitude in V. splendidus co-incubation. The present study expands our understanding of the connection between arginine's metabolic and immune responses in non-model invertebrates.