The Holothuria leucospilota genome elucidates sacrificial organ expulsion and bioadhesive trap enriched with amyloid-patterned proteins.

Some tropical sea cucumbers of the family Holothuriidae can efficiently repel or even fatally ensnare predators by sacrificially ejecting a bioadhesive matrix termed the Cuvierian organ (CO), so named by the French zoologist Georges Cuvier who first described it in 1831. Still, the precise mechanisms for how adhesiveness genetically arose in CO and how sea cucumbers perceive and transduce danger signals for CO expulsion during defense have remained unclear. Here, we report the first high-quality, chromosome-level genome assembly of Holothuria leucospilota, an ecologically significant sea cucumber with prototypical CO. The H. leucospilota genome reveals characteristic long-repeat signatures in CO-specific outer-layer proteins, analogous to fibrous proteins of disparate species origins, including spider spidroin and silkworm fibroin. Intriguingly, several CO-specific proteins occur with amyloid-like patterns featuring extensive intramolecular cross-ß structures readily stainable by amyloid indicator dyes. Distinct proteins within the CO connective tissue and outer surface cooperate to give the expelled matrix its apparent tenacity and adhesiveness, respectively. Genomic evidence offers further hints that H. leucospilota directly transduces predator-induced mechanical pressure onto the CO surface through mediation by transient receptor potential channels, which culminates in acetylcholine-triggered CO expulsion in part or in entirety. Evolutionarily, innovative events in two distinct regions of the H. leucospilota genome have apparently spurred CO's differentiation from the respiratory tree to a lethal defensive organ against predators.

A peek into the population genetics of white teatfish (Holothuria fuscogilva) of Kenya's south coast.

BACKGROUND: The white teatfish, Holothuria fuscogilva, is widely distributed in coastal areas, including waters around coral reefs and seagrasses in the Indo-Pacific. In Kenya, the species is distributed in shallow reefs with higher landings reported from the Vanga-Shimoni-Gazi seascape on the Kenyan south coast. Despite its high exploitation for export and its vulnerable and endangered statuses under IUCN and CITES respectively, Kenya's H. fuscogilva populations and how they may have been impacted by the fishing pressure have not been studied. METHODS: We estimated the genetic diversity and structure of H. fuscogilva population conveniently sampled from three sites in Kenyan south coast using the mitochondrial cytochrome oxidase subunit I (COI) gene sequences. We recorded 30 haplotypes with 43 polymorphic sites across the population. Furthermore, we estimated an overall high haplotype diversity and low nucleotide diversity of estimates of h = 0.970 ± 0.013 and π = 0.010 ± 0.001 respectively. CONCLUSIONS: These preliminary findings suggest several population outcomes, among them a fit population, which require confirming with more comprehensive study to inform strategies for the sustainable exploitation and management of the species.

Effects of hypo-osmotic stress on osmoregulation, antioxidant response, and energy metabolism in sea cucumber Holothuria moebii under desalination environment.

With global climate changing, hypo-salinity events are increasing in frequency and duration because of continuous rainfall and freshwater inflow, which causes reduced cytosolic osmolarity and cellular stress responses in aquatic animals. Sea cucumbers are considered stenohaline because they lack osmoregulatory organs and are vulnerable to salinity fluctuations. In this study, we performed multiple biochemical assays, de novo transcriptomics, and widely targeted metabolomics to comprehensively explore the osmoregulatory mechanisms and physiological responses of sea cucumber Holothuria moebii to hypo-osmotic stress, which is a representative specie that is frequently exposed to hypo-saline intertidal zones. Our results found that H. moebii contracted their ambulacral feet and oral tentacles, and the coelomic fluid ion concentrations were reduced to be consistent with the environment. The microvilli of intestines and respiratory trees underwent degeneration, and the cytoplasm exhibited swelling and vacuolation. Moreover, the Na+, K+, and Cl- concentrations and Na+/K+-ATPase activity were significantly reduced under hypo-osmotic stress. The decrease in protein kinase A activity and increase in 5'-AMP level indicated a significant inhibition of the cAMP signaling pathway to regulate ion concentrations. And small intracellular organic molecules (amino acids, nucleotides and their derivatives) also play crucial roles in osmoregulation through oxidative deamination of glutamate, nucleotide catabolism, and nucleic acid synthesis. Moreover, lysosomes and peroxisomes removed oxidative damage, whereas antioxidant metabolites, such as N-acetyl amino acids and glutathione, were increased to resist oxidative stress. With prolonged hypo-osmotic stress, glycerophospholipid metabolism was enhanced to maintain membrane stability. Furthermore, acyl-CoA-binding protein activity was significantly inhibited, and only a small amount of acylcarnitine was significantly accumulated, which indicated a disruption in energy metabolism. PPAR signaling pathway and choline content were up-regulated to promote fatty acid metabolism under hypo-osmotic stress. Overall, our results provide new insights into the osmoregulatory mechanisms and physiological responses of sea cucumbers to hypo-osmotic stress.

Comparative Analysis of Gut Bacterial Community Composition in Two Tropical Economic Sea Cucumbers under Different Seasons of Artificial Environment.

With the continuous rise of the sea cucumber aquaculture industry in China, the tropical sea cucumber aquaculture industry is also improving. However, research on the gut microorganisms of tropical sea cucumbers in captivity is scarce. In this study, high-throughput sequencing methods were used to analyze the gut microbial composition of Stichopus monotuberculatus and Holothuria scabra in the dry season and wet season of artificial environments. The results showed that 66 phyla were obtained in all samples, of which 59 phyla were obtained in the dry season, and 45 phyla were obtained in the wet season. The Tax4Fun analysis showed that certain gut bacterial communities affect the daily metabolism of two sea cucumber species and are involved in maintaining gut microecological balance in the gut of two sea cucumber species. In addition, compared with differences between species, PCoA and UPGMA clustering analysis showed the gut prokaryotes of the same sea cucumber species varied more in different seasons, indicating that the influence of environment was higher than the feeding choices of sea cucumbers under relatively closed conditions. These results revealed the gut bacterial community composition of S. monotuberculatus and H. scabra and the differences in gut bacterial structure between two sea cucumber species in different seasons were compared, which would provide the foundation for tropical sea cucumber aquaculture in the future.

Transcriptomic analysis reveals the early body wall regeneration mechanism of the sea cucumber Holothuria leucospilota after artificially induced transverse fission.

BACKGROUND: Sea cucumbers exhibit a remarkable ability to regenerate damaged or lost tissues and organs, making them an outstanding model system for investigating processes and mechanisms of regeneration. They can also reproduce asexually by transverse fission, whereby the anterior and posterior bodies can regenerate independently. Despite the recent focus on intestinal regeneration, the molecular mechanisms underlying body wall regeneration in sea cucumbers still remain unclear. RESULTS: In this study, transverse fission was induced in the tropical sea cucumber, Holothuria leucospilota, through constrainment using rubber bands. Histological examination revealed the degradation and loosening of collagen fibers on day-3, followed by increased density but disorganization of the connective tissue on day-7 of regeneration. An Illumina transcriptome analysis was performed on the H. leucospilota at 0-, 3- and 7-days after artificially induced fission. The differential expression genes were classified and enriched by GO terms and KEGG database, respectively. An upregulation of genes associated with extracellular matrix remodeling was observed, while a downregulation of pluripotency factors Myc, Klf2 and Oct1 was detected, although Sox2 showed an upregulation in expression. In addition, this study also identified progressively declining expression of transcription factors in the Wnt, Hippo, TGF-ß, and MAPK signaling pathways. Moreover, changes in genes related to development, stress response, apoptosis, and cytoskeleton formation were observed. The localization of the related genes was further confirmed through in situ hybridization. CONCLUSION: The early regeneration of H. leucospilota body wall is associated with the degradation and subsequent reconstruction of the extracellular matrix. Pluripotency factors participate in the regenerative process. Multiple transcription factors involved in regulating cell proliferation were found to be gradually downregulated, indicating reduced cell proliferation. Moreover, genes related to development, stress response, apoptosis, and cell cytoskeleton formation were also involved in this process. Overall, this study provides new insights into the mechanisms of whole-body regeneration and uncover potential cross-species regenerative-related genes.

The presence and distribution of gamma-aminobutyric acid and dopamine during the developmental stages of the sea cucumber, Holothuria scabra, with emphasis on settlement organs.

In the sea cucumber, Holothuria scabra, the competent larvae require main settlement organs (SOs), including the ciliary bands (CiBs), tentacles (Ts), podia (PDs), and cues from neurotransmitters, including gamma-aminobutyric acid (GABA) and dopamine (DA), for successful settlement. In the present study, we investigated the spatial distribution of GABA and DA in the developmental stages of H. scabra, with special emphasis on SOs by detecting immunoreactivity (-ir) against these two neurotransmitters. Strong GABA-ir and DA-ir cells and fibers were specifically detected in several SO structures, including CiBs, CiB cells (CiBCs), and long cilia (LCi), of H. scabra larvae. Additionally, we found intense GABA-ir and DA-ir cells in the epithelial lining of bud-papillae (BP) and mesothelium (Me) in the stem (S) region of Ts in larvae and juveniles. Intense GABA-ir and DA-ir were observed in the epineural nerve plexus (ENP) and hyponeural nerve plexus (HNP) of Ts in H. scabra pentactula and juvenile stages. Staining for these two neurotransmitters was particularly intense in the PDs and their nerve fibers. We also found significant changes in the numbers of GABA-ir and DA-ir-positive cells and intensities in the CiBs, Ts, and PDs during the developmental stages. Taken together, we are the first to report on the existence and distribution of GABAergic and dopaminergic systems in structures associated with the settlement. Our findings provide new and important insights into the possible functions of these two neurotransmitters in regulating the settlement of this sea cucumber species.

Isolation and characterization of 21 microsatellite loci for the sea cucumber Holothuria (Halodeima) atra (Echinodermata, Holothuroidea) reveal low asexual propagation through time in Reunion Island (southwestern Indian Ocean).

BACKGROUND: Holothuria (Halodeima) atra Jaeger, 1833 is a tropical sea cucumber usually harvested for the "bêche-de-mer" trade market. It has been reported to reproduce both sexually, through gamete spawning, or asexually, through fission. To date, no study has ever investigated clonality, nor genetic connectivity, among its populations, using microsatellite markers. METHODS AND RESULTS: We isolated the first 21 microsatellite loci specific for H. atra, which were then used to investigate clonal diversity, genetic structure and diversity among 44 H. atra individuals sampled in Reunion Island (southwestern Indian Ocean), over two seasons. All 21 loci were polymorphic, with number of alleles per locus ranging from 2 to 10. No repetitive multi-locus genotype (MLG) and few clonal lineages (MLL) were found. Observed heterozygosities per locus and season ranged from 0.000 to 0.909, while expected heterozygosities ranged from 0.290 to 0.882. Four loci were at Hardy-Weinberg equilibrium for both seasons, all others presenting a deficit of heterozygotes in one or both seasons. Meanwhile, no genetic differentiation was detected between seasons, according to assignment tests and global FST. CONCLUSIONS: These results suggest low asexual propagation in this population. These loci represent useful tools to better understand reproductive strategies and population connectivity of H. atra, and thus provide relevant knowledge for efficient management.

Uptake and depuration of three common antibiotics in benthic organisms: Sea cucumber (Holothuria tubulosa), snakelocks anemone (Anemonia sulcata) and beadlet anemone (Actinia equina).

Antibiotics are widely used drugs in human and veterinary medicine, which has attracted great attention in relation to the development of bacterial resistance, currently a problem of great concern for governments and states, as it is related to the resurgence of infectious diseases already eradicated. Understanding the bioaccumulation of antibiotics in aquatic organisms is an important key to understanding their risk assessment. The present study was designed to study the bioaccumulation of target antibiotics in relevant organisms inhabiting benthic marine environments. The uptake and elimination of ciprofloxacin (CIP), sulfamethoxazole (SMX) and trimethoprim (TMP) were investigated in sea cucumbers (Holothuria tubulosa), snakelock anemone (Anemonia sulcata) and beadlet anemone (Actinia equina) under controlled laboratory conditions. The results show that antibiotics have a particular trend over time during all periods of absorption and depuration. The tissue distribution of antibiotics in sea cucumber is strongly influenced by the structure of the compounds, while CIP is concentrated in the body wall; TMP is concentrated in the digestive tract. Two different approaches were used to estimate bioconcentration factors (BCFs) in different animal models, based on toxicokinetic data and measured steady-state concentrations. The BCF ranges were 456-2731 L/kg, 6-511 L/kg and 9-100 L/kg for TMP, CIP and SMX, respectively. The estimated BCF values obtained classify TMP as cumulative in A. equina and H. tubulosa, underlining the potential bioconcentration in these marine organisms. A correlation was observed between the BCFs of the target antibiotics and the octanol-water distribution coefficient (Dow) (r2 > 0.7). The animal-specific BCF followed the order of beadlet anemone > sea cucumber > snakelock anemone.

Exploring in vitro effect of silver nanoparticles and Holothuria parva extracts on kinetics and stability of α-amylase.

Diabetes is a chronic metabolic disorder characterized by elevated blood glucose levels. Major limitations of synthetic drugs, including high cost, efficacy, and adverse side effects, have prompted researchers to seek more effective and low-cost alternative therapies with fewer adverse effects. Marine life forms are considered the most important sources of biologically active natural products due to their secondary metabolites. In this research, sea cucumber Holothuria parva was collected from coastal areas of Bandar Lengeh, Hormozgan, Iran, and was then subjected to extraction. The results showed that compounds extracted from Holothuria parva had a stimulatory effect on enzyme activity, and in the presence of these compounds, the Vmax value of the enzyme was increased about two times, while the Km value was reduced. The phosphate buffer form of extracts had the greatest impact on enzyme activity. Upon an increase in the concentration of silver nanoparticles (AgNPs), the α-amylase activity was inhibited in parallel. Silver nanoparticles exhibited the highest enzyme inhibition with an IC50 of 0.86 mg/ml. Silver nanoparticles showed anti-α-amylase activity and had the ability to decrease intestinal glucose uptake in diabetic individuals when prescribed as a novel supplementary medicine.

A Distinct Saponin Profile Drives an Olfactory-Mediated Aggregation in the Aquacultivated Sea Cucumber Holothuria scabra.

Intraspecific chemical communication between echinoderms has often been limited to prespawning aggregation. However, sea cucumber farmers have long observed year-round adult aggregation as a potential source of disease propagation and the suboptimal use of available sea pen acreage and food resources. In this study, through spatial distribution statistics, we demonstrated the significant aggregation of the aquacultivated sea cucumber Holothuria scabra both as adults in large sea-based pens and as juveniles in laboratory-based aquaria, proving that aggregation in these animals is not only observed during spawning. The role of chemical communication in aggregation was investigated using olfactory experimental assays. Our study established that the sediment that H. scabra feeds on as well as the water preconditioned by conspecifics induced positive chemotaxis in juvenile individuals. More specifically, through comparative mass spectrometry, a distinct triterpenoid saponin profile/mixture was identified to be a pheromone allowing sea cucumber intraspecific recognition and aggregation. This "attractive" profile was characterized as containing disaccharide saponins. This "attractive" aggregation-inducing saponin profile was, however, not conserved in starved individuals that were no longer attractive to other conspecifics. In summary, this study sheds new light on the pheromones in echinoderms. It highlights the complexity of the chemical signals detected by sea cucumbers and suggests a role of saponins well beyond that of a simple toxin.

Proliferative Effect of Aqueous Extract of Sea Cucumber (Holothuria parva) Body Wall on Human Umbilical Cord Mesenchymal Stromal/Stem Cells.

Sea cucumber extracts and their bioactive compounds have the potential for stem cell proliferation induction and for their beneficial therapeutic properties. In this study, human umbilical cord mesenchymal stromal/stem cells (hUC-MSCs) were exposed to an aqueous extract of Holothuria parva body walls. Proliferative molecules were detected using gas chromatography-mass spectrometry (GC-MS) analysis in an aqueous extract of H. parva. The aqueous extract concentrations of 5, 10, 20, 40, and 80 µg/mL and 10 and 20 ng/mL of human epidermal growth factor (EGF) as positive controls were treated on hUC-MSCs. MTT, cell count, viability, and cell cycle assays were performed. Using Western blot analysis, the effects of extracts of H. parva and EGF on cell proliferation markers were detected. Computational modeling was done to detect effective proliferative compounds in the aqueous extract of H. parva. A MTT assay showed that the 10, 20, and 40 µg/mL aqueous extract of H. parva had a proliferative effect on hUC-MSCs. The cell count, which was treated with a 20 µg/mL concentration, increased faster and higher than the control group (p < 0.05). This concentration of the extract did not have a significant effect on hUC-MSCs' viability. The cell cycle assay of hUC-MSCs showed that the percentage of cells in the G2 stage of the extract was biologically higher than the control group. Expression of cyclin D1, cyclin D3, cyclin E, HIF-1α, and TERT was increased compared with the control group. Moreover, expression of p21 and PCNA decreased after treating hUC-MSCs with the extract. However, CDC-2/cdk-1 and ERK1/2 had almost the same expression as the control group. The expression of CDK-4 and CDK-6 decreased after treatment. Between the detected compounds, 1-methyl-4-(1-methyl phenyl)-benzene showed better affinity to CDK-4 and p21 than tetradecanoic acid. The H. parva aqueous extract showed proliferative potential on hUC-MSCs.

Anti-Parkinson Effects of Holothuria leucospilota-Derived Palmitic Acid in Caenorhabditis elegans Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease which is still incurable. Sea cucumber-derived compounds have been reported to be promising candidate drugs for treating age-related neurological disorders. The present study evaluated the beneficial effects of the Holothuria leucospilota (H. leucospilota)-derived compound 3 isolated from ethyl acetate fraction (HLEA-P3) using Caenorhabditis elegans PD models. HLEA-P3 (1 to 50 µg/mL) restored the viability of dopaminergic neurons. Surprisingly, 5 and 25 µg/mL HLEA-P3 improved dopamine-dependent behaviors, reduced oxidative stress and prolonged lifespan of PD worms induced by neurotoxin 6-hydroxydopamine (6-OHDA). Additionally, HLEA-P3 (5 to 50 µg/mL) decreased α-synuclein aggregation. Particularly, 5 and 25 µg/mL HLEA-P3 improved locomotion, reduced lipid accumulation and extended lifespan of transgenic C. elegans strain NL5901. Gene expression analysis revealed that treatment with 5 and 25 µg/mL HLEA-P3 could upregulate the genes encoding antioxidant enzymes (gst-4, gst-10 and gcs-1) and autophagic mediators (bec-1 and atg-7) and downregulate the fatty acid desaturase gene (fat-5). These findings explained the molecular mechanism of HLEA-P3-mediated protection against PD-like pathologies. The chemical characterization elucidated that HLEA-P3 is palmitic acid. Taken together, these findings revealed the anti-Parkinson effects of H. leucospilota-derived palmitic acid in 6-OHDA induced- and α-synuclein-based models of PD which might be useful in nutritional therapy for treating PD.

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

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

The potential adverse effects of acrylamide on the oxidative stress response, fatty acids profile, and histopathological aspect of the Mediterranean Holothuria forskali respiratory tree.

Acrylamide (ACR), organic compound, has been widely used owing to its broad spectrum of chemical and industrial applications. This study aims at evaluating the response of the antioxidant defense system, fatty acid composition and histopathological aspect in the respiratory trees of Holothuria forskali against ACR exposure under laboratory conditions. Holothuries were exposed to 5, 10, and 20 mg L-1 ACR concentrations for 5 days. A significant increase in n-6 polyunsaturated fatty acids levels especially the arachidonic acid (ARA, C20:4n-6) and its precursor linoleic acid (LA, C18:2n-6) in ACR-treated organisms. Regarding the n-3 levels, eicosapentaenoic acid (EPA, C20:5n-3) levels were increased in treated groups despite an acute decrease in docosahexaenoic acid (DHA, C22:6n-3) levels was observed. Our results showed a significant increase in hydrogen peroxide, malondialdehyde, protein carbonyl, and metallothionein levels along with an alteration of the antioxidants status in all treated sea cucumbers. The exposure to ACR prompted the inhibition of Acetylcholinesterase activity in a concentration-dependent manner. The histopathological aspect was marked especially with the infiltration of coelomic cells which confirms our biochemical findings. Our study provided novel insights to create a link between redox status and fatty acid composition disruptions to better understand ACR-triggered toxicity.

Isolation of Extracellular Vesicles of Holothuria (Sea Cucumber Eupentacta fraudatrix).

Extracellular vesicles (EVs), carriers of molecular signals, are considered a critical link in maintaining homeostasis in mammals. Currently, there is growing interest in studying the role of EVs, including exosomes (subpopulation of EVs), in animals of other evolutionary levels, including marine invertebrates. We have studied the possibility of obtaining appropriate preparations of EVs from whole-body extract of holothuria Eupentacta fraudatrix using a standard combination of centrifugation and ultracentrifugation. However, the preparations were heavily polluted, which did not allow us to conclude that they contained vesicles. Subsequent purification by FLX gel filtration significantly reduced the pollution but did not increase vesicle concentration to a necessary level. To detect EVs presence in the body of holothurians, we used transmission electron microscopy of ultrathin sections. Late endosomes, producing the exosomes, were found in the cells of the coelom epithelium covering the gonad, digestive tube and respiratory tree, as well as in the parenchyma cells of these organs. The study of purified homogenates of these organs revealed vesicles (30-100 nm) morphologically corresponding to exosomes. Thus, we can say for sure that holothurian cells produce EVs including exosomes, which can be isolated from homogenates of visceral organs.

Polysaccharides from Holothuria leucospilota Relieve Loperamide-Induced Constipation Symptoms in Mice.

A vital bioactive component of marine resources is Holothuria leucospilota polysaccharides (HLP). This study examined whether HLP could regulate intestinal flora to treat loperamide-induced constipation. Constipated mice showed signs of prolonged defecation (up by 60.79 min) and a reduced number of bowel movements and pellet water content (decreased by 12.375 and 11.77%, respectively). The results showed that HLP treatment reduced these symptoms, reversed the changes in related protein expression levels in the colon, and regulated the levels of active peptides associated with the gastrointestinal tract in constipated mice, which significantly improved water-electrolyte metabolism and enhanced gastrointestinal motility. Meanwhile, it was found that intestinal barrier damage was reduced and the inflammatory response was inhibited through histopathology and immunohistochemistry. As a means to further relieve constipation symptoms, treatment with low, medium, and high HLP concentrations increased the total short-chain fatty acid (SCFA) content in the intestine of constipated mice by 62.60 µg/g, 138.91 µg/g, and 126.51 µg/g, respectively. Moreover, an analysis of the intestinal flora's gene for 16S rRNA suggested that the intestinal microbiota was improved through HLP treatment, which is relevant to the motivation for the production of SCFAs. In summary, it was demonstrated that HLP reduced loperamide-induced constipation in mice.

Enzymatic Hydrolysis Optimization for Preparation of Sea Cucumber (Holothuria scabra) Hydrolysate with an Antiproliferative Effect on the HepG2 Liver Cancer Cell Line and Antioxidant Properties.

The sea cucumber body wall was subjected to enzymatic hydrolysis using papain. The relationship between the enzyme concentration (1-5% w/w protein weight) and hydrolysis time (60-360 min) and the degree of hydrolysis (DH), yield, antioxidant activities, and antiproliferative activity in a HepG2 liver cancer cell line was determined. The surface response methodology showed that the optimum conditions for the enzymatic hydrolysis of sea cucumber were a hydrolysis time of 360 min and 4.3% papain. Under these conditions, a 12.1% yield, 74.52% DH, 89.74% DPPH scavenging activity, 74.92% ABTS scavenging activity, 39.42% H2O2 scavenging activity, 88.71% hydroxyl radical scavenging activity, and 9.89% HepG2 liver cancer cell viability were obtained. The hydrolysate was produced under optimum conditions and characterized in terms of its antiproliferative effect on the HepG2 liver cancer cell line.

Fatty Acid-Rich Extract from Holothuria atra for Hyperuricemia via Expressions Modulation of GLUT9a and GLUT9b in Rat Model.

An edible sea cucumber Holothuria atra has been hypothesized to have medicinal benefits against hyperuricemia owing to its bioactive compounds, including mono- and poly-unsaturated fatty acids. Herein, we aimed to investigate the fatty acids-rich extract produced from H. atra to treat hyperuricemic rats (Rattus novergicus). The extraction was carried out using n-hexane solvent and then administered to potassium oxonate-induced hyperuricemic rats, with allopurinol acting as a positive control. The extract (50, 100, 150 mg/kg body weight) and allopurinol (10 mg/kg) were administered QD through an oral route using a nasogastric tube. Serum uric acid, creatinine, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and blood urea nitrogen of the abdominal aortic blood were investigated. Our results suggested that the extract was rich in polyunsaturated (arachidonic acid) and monounsaturated fatty acids (oleic acid), in which its administration of 150 mg/kg could significantly reduce serum uric acid (p < 0.001), AST (p = 0.001), and ALT (p = 0.0302). The anti-hyperuricemic activity could be associated with the modulation of GLUT9 by the H. atra extract. In conclusion, the n-hexane extract from H. atra is a potential serum uric acid-lowering agent targeting GLUT9, where further investigations are crucially warranted.

Wnt/ß-catenin signaling pathway regulates cell proliferation but not muscle dedifferentiation nor apoptosis during sea cucumber intestinal regeneration.

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/ß-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 ß-catenin transcript on gut rudiment explants. Neither cell dedifferentiation nor apoptosis were affected by the reduction of ß-catenin transcripts in the gut rudiment explants. Yet, the number of proliferating cells decreased significantly following the interference, suggesting that the Wnt/ß-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.

Post-autotomy regeneration of respiratory tree in sea cucumber Holothuria parva.

The respiratory trees present only in the class Holothuroidea and the rest of the echinoderms lack it. Only some holothurian species have the ability to regenerate their respiratory trees after autotomy. Therefore, respiratory trees could be considered as a suitable model to assess the regeneration mechanisms in animals. In the present study, the respiratory tree regeneration after posterior evisceration were examined in Holothuria parva during 75 days. Since autotomy reduces antioxidant defense in the organisms, in the present study alterations of antioxidant enzymes were also evaluated during the experiment. H. parva is the dominant intertidal species distributed in the north of the Persian Gulf. In the present study, H. parva ejected the left respiratory tree, the digestive tract and supportive mesenteries from the anus, about 1-2 min after potassium chloride injection. The closure of the opening at the posterior ends of the body was the first reaction to the injury. Seven days after evisceration, the small bud formed on the dorsal side of the cloaca which was covered with the coelomic epithelium of cloaca. The coelomic epithelium started to proliferate to undifferentiated cells on the apex of the buds. The primary respiratory tree consisted of the luminal cuboidal epithelium and thin connective tissue surrounded by the slender coelomic epithelium. This preliminary organ was observed at the apex of the buds, 13 days after evisceration. Gradually, myoepithelial cells arranged around a longitudinal axis and formed a circular muscle. The primitive branches of primary respiratory tree started to form 18 days after evisceration. Forty days after evisceration, the luminal epithelium of the respiratory tree had the same appearance as the intact luminal epithelium. The regenerated respiratory tree was histomorphologically very similar to an intact respiratory tree 56 days postevisceration, but unlike that, it was not yet wrapped around the intestine and was completely separate from it. Despite the development of the regenerating respiratory tree, no wrapping around the intestine was observed until the end of the experiment. According to the results, the activity of the catalase (CAT) and superoxide dismutase (SOD) in the muscle homogenate was significantly higher than the control 5 days after evisceration. The CAT and SOD levels gradually decreased in eviscerated animals. The lipid peroxidation level followed a decreasing trend in the eviscerated animals during the experiment. However, its value reduced to the control level at the end of the experiment.