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.

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.

Structure of Self-assembled Peptide Determines the Activity of Aggregation-Induced Emission Luminogen-Peptide Conjugate for Detecting Alkaline Phosphatase.

The unique property of turning on their fluorescence after aggregation or assembly makes aggregation-induced emission luminogens (AIEgens) ideal luminescent molecules for the construction of self-assembled peptide-based nanoprobes. However, the characteristic highly twisted or propeller-shaped molecular conformation of AIEgens tends to prevent the assembly of AIEgen-peptides. Here, we show that (i) the distance between tetraphenylethene (TPE) and assembled peptides should not be too far (less than five glycines), otherwise the self-assembly of peptides cannot limit the intramolecular rotation of conjugated TPE and the luminous efficiency of TPE-peptide to alkaline phosphatase (ALP) will decrease; (ii) properly increasing the number of amino acids with self-assembly ability (three phenylalanines) can improve their ALP-responsive self-assembly and luminescence ability; (iii) the strategy of co-assembly with a non-AIEgen-capped self-assembled peptide is a simple and effective way to realize the efficient assembly and luminescence of AIEgen-peptides; and (iv) the hydrophilic and hydrophobic balance of the probe should always be considered in the construction of an efficient AIEgen-peptide probe. In addition, AIEgen-peptide probes show good selectivity and sensitivity for ALP detection both in vitro and in live bacteria. These insights illustrated here are crucial for guiding the design of AIEgen-conjugated supramolecular materials, especially for the construction of AIEgen-peptides, for enzymes detection, biomarker imaging, diseases therapy, and other biomedical fields.

Hypertoxic self-assembled peptide with dual functions of glutathione depletion and biosynthesis inhibition for selective tumor ferroptosis and pyroptosis.

Abundant glutathione (GSH) is a biological characteristic of lots of tumor cells. A growing number of studies are utilizing GSH depletion as an effective adjuvant therapy for tumor. However, due to the compensatory effect of intracellular GSH biosynthesis, GSH is hard to be completely exhausted and the strategy of GSH depletion remains challenging. Herein, we report an L-buthionine-sulfoximine (BSO)-based hypertoxic self-assembled peptide derivative (NSBSO) with dual functions of GSH depletion and biosynthesis inhibition for selective tumor ferroptosis and pyroptosis. The NSBSO consists of a hydrophobic self-assembled peptide motif and a hydrophilic peptide derivative containing BSO that inhibits the synthesis of GSH. NSBSO was cleaved by GSH and thus experienced a morphological transformation from nanoparticles to nanofibers. NSBSO showed GSH-dependent cytotoxicity and depletion of intracellular GSH. In 4T1 cells with medium GSH level, it depleted intracellular GSH and inactivated GSH peroxidase 4 (GPX4) and thus induced efficient ferroptosis. While in B16 cells with high GSH level, it exhausted GSH and triggered indirect increase of intracellular ROS and activation of Caspase 3 and gasdermin E, resulting in severe pyroptosis. These findings demonstrate that GSH depletion- and biosynthesis inhibition-induced ferroptosis and pyroptosis strategy would provide insights in designing GSH-exhausted medicines.

Research on Nonlinear Compensation of the MEMS Gyroscope under Tiny Angular Velocity.

The Micro-Electro-Mechanical System (MEMS) gyroscope has been widely used in various fields, but the output of the MEMS gyroscope has strong nonlinearity, especially in the range of tiny angular velocity. This paper proposes an adaptive Fourier series compensation method (AFCM) based on the steepest descent method and Fourier series residual correction. The proposed method improves the Fourier series fitting method according to the output characteristics of the MEMS gyroscope under tiny angular velocity. Then, the optimal weights are solved by the steepest descent method, and finally the fitting residuals are corrected by Fourier series to further improve the compensation accuracy. In order to verify the effectiveness of the proposed method, the angle velocity component of the earth's rotation is used as the input of the MEMS gyroscope to obtain the output of the MEMS gyroscope under tiny angular velocities. Experimental characterization resulted in an input angular velocity between -0.0036°/s and 0.0036°/s, compared with the original data, the polynomial compensation method, and the Fourier series compensation method, and the output nonlinearity of the MEMS gyroscope was reduced from 1150.87 ppm, 641.13 ppm, and 250.55 ppm to 68.89 ppm after AFCM compensation, respectively, which verifies the effectiveness and superiority of the proposed method.

Apoptosis-Inducing Factor 2 (AIF-2) Mediates a Caspase-Independent Apoptotic Pathway in the Tropical Sea Cucumber (Holothuria leucospilota).

Apoptosis, also known as programmed cell death, is a biological process that is critical for embryonic development, organic differentiation, and tissue homeostasis of organisms. As an essential mitochondrial flavoprotein, the apoptosis-inducing factor (AIF) can directly mediate the caspase-independent mitochondrial apoptotic pathway. In this study, we identified and characterized a novel AIF-2 (HlAIF-2) from the tropical sea cucumber Holothuria leucospilota. HlAIF-2 contains a conserved Pyr_redox_2 domain and a putative C-terminal nuclear localization sequence (NLS) but lacks an N-terminal mitochondrial localization sequence (MLS). In addition, both NADH- and FAD-binding domains for oxidoreductase function are conserved in HlAIF-2. HlAIF-2 mRNA was ubiquitously detected in all tissues and increased significantly during larval development. The transcript expression of HlAIF-2 was significantly upregulated after treatment with CdCl2, but not the pathogen-associated molecular patterns (PAMPs) in primary coelomocytes. In HEK293T cells, HlAIF-2 protein was located in the cytoplasm and nucleus, and tended to transfer into the nucleus by CdCl2 incubation. Moreover, there was an overexpression of HlAIF-2-induced apoptosis in HEK293T cells. As a whole, this study provides the first evidence for heavy metal-induced apoptosis mediated by AIF-2 in sea cucumbers, and it may contribute to increasing the basic knowledge of the caspase-independent apoptotic pathway in ancient echinoderm species.

Integrative Application of Transcriptomics and Metabolomics Provides Insights into Unsynchronized Growth in Sea Cucumber (Stichopus monotuberculatus).

Ever-increasing consumer demand for sea cucumbers mainly leads to huge damage to wild sea cucumber resources, including Stichopus monotuberculatus, which in turn exerts negative impacts on marine environments due to the lack of ecological functions performed by sea cucumbers. Aquaculture of sea cucumbers is an effective way to meet consumer demand and restore their resources. Unsynchronous growth is a prominent problem in the aquaculture of sea cucumbers which has concealed unelucidated molecular mechanisms until now. In this study, we carried out an integrative analysis of transcriptomics and metabolomics on fast-growing (SMF) and slow-growing (SMS) groups of S. monotuberculatus cultured in the same environmental conditions. The results revealed that a total of 2054 significantly differentially expressed genes (DEGs) were identified, which are mainly involved in fat digestion and absorption, histidine metabolism, arachidonic acid metabolism, and glutathione metabolism. 368 differential metabolites (DMs) were screened out between the SMF group and the SMS group; these metabolites are mainly involved in glycerophospholipid metabolism, purine metabolism, biosynthesis of unsaturated fatty acids, pyrimidine metabolism, arachidonic acid metabolism, and other metabolic pathways. The integrative analysis of transcriptomics and metabolomics of S. monotuberculatus suggested that the SMF group had a higher capacity for lipid metabolism and protein synthesis, and had a more frequent occurrence of apoptosis events, which are likely to be related to coping with environmental stresses. The results of this study provide potential values for the aquaculture of sea cucumbers which may promote their resource enhancement.

Transcriptomic analysis of sea cucumber (Holothuria leucospilota) coelomocytes revealed the echinoderm cytokine response during immune challenge.

BACKGROUND: The sea cucumber Holothuria leucospilota belongs to echinoderm, which is evolutionally the most primitive group of deuterostomes. Sea cucumber has a cavity between its digestive tract and the body wall that is filled with fluid and suspended coelomic cells similar to blood cells. The humoral immune response of the sea cucumber is based on the secretion of various immune factors from coelomocytes into the coelomic cavity. The aim of this study is to lay out a foundation for the immune mechanisms in echinoderms and their origins in chordates by using RNA-seq. RESULTS: Sea cucumber primary coelomocytes were isolated from healthy H. leucospilota and incubated with lipopolysaccharide (LPS, 10 µg/ml), polyinosinic-polycytidylic acid [Poly (I:C), 10 µg/ml] and heat-inactived Vibrio harveyi (107 cell/ml) for 24 h, respectively. After high-throughput mRNA sequencing on an Illumina HiSeq2500, a de novo transcriptome was assembled and the Unigenes were annotated. Thirteen differentially expressed genes (DEGs) were selected randomly from our data and subsequently verified by using RT-qPCR. The results of RT-qPCR were consistent with those of the RNA-seq (R2 = 0.61). The top 10 significantly enriched signaling pathways and immune-related pathways of the common and unique DEGs were screened from the transcriptome data. Twenty-one cytokine candidate DEGs were identified, which belong to 4 cytokine families, namely, BCL/CLL, EPRF1, IL-17 and TSP/TPO. Gene expression in response to LPS dose-increased treatment (0, 10, 20 and 50 µg/ml) showed that IL-17 family cytokines were significantly upregulated after 10 µg/ml LPS challenge for 24 h. CONCLUSION: A de novo transcriptome was sequenced and assembled to generate the gene expression profiling across the sea cucumber coelomocytes treated with LPS, Poly (I:C) and V. harveyi. The cytokine genes identified in DEGs could be classified into 4 cytokine families, in which the expression of IL-17 family cytokines was most significantly induced after 10 µg/ml LPS challenge for 24 h. Our findings have laid the foundation not only for the research of molecular mechanisms related to the immune response in echinoderms but also for their origins in chordates, particularly in higher vertebrates.

Detection of Bacterial Alkaline Phosphatase Activity by Enzymatic In Situ Self-Assembly of the AIEgen-Peptide Conjugate.

Abnormal levels of alkaline phosphatase (ALP) activity are associated with various diseases, and many ALP probes have been developed to date. However, the development of ALP-sensitive probes for living cells, especially for the detection of bacterial ALP, remains challenging because of the complex and dynamic context. In this study, we constructed the first fluorescent probe (TPEPy-pY) for sensing bacterial ALP activity. TPEPy-pY is an AIEgen-peptide conjugate with property of aggregation-induced emission (AIE) and could turn on its fluorescence by ALP-catalyzed in situ self-assembly of the probe. The probe shows excellent selectivity and sensitivity for ALP activity, with a detection limit of 6.6 × 10-3 U mL-1. TPEPy-pY performs well in detection and in situ imaging of bacterial ALP activity against E. coli. Also, the detection does not require tedious washing steps and takes approximately 1 h, which is advantageous over commercial ALP kits. Therefore, the proposed strategy paved a new avenue for bacterial ALP detection, and we envision that more self-assembling fluorescent probes will be designed with higher sensitivity in the near future.

Molecular characterization, inducible expression and functional analysis of an IKKß from the tropical sea cucumber Holothuria leucospilota.

The inhibitory kappa B kinase (IKK) is a critical regulator for the nuclear factor-κB (NF-κB) pathway. In this study, an IKKß named as HLIKKß was identified from the sea cucumber Holothuria leucospilota. The full-length cDNA of HLIKKß is 4246 bp in size, containing a 132 bp 5'-untranslated region (UTR), a 1783 bp 3'-UTR and a 2331 bp open reading frame (ORF) encoding a protein of 776 amino acids with a deduced molecular weight of 89.66 kDa. HLIKKß contains a kinase domain (KD) at its N-terminal, a leucine zipper (LZ) and a helix-loop-helix (HLH) motif at its C-terminal. In the KD, a conserved active loop (SXXXS) were identified. The results of luciferase reporter assay and ELISA assay showed that over-expressed HLIKKß in HEK293T cells could activate the nuclear factor-κB (NF-κB) and induce the secretion of proinflammatory cytokines TNF-α and IL-1ß. When HLIKKß was silenced by siRNA, the apoptosis rate of sea cucumber coelomocytes was increased significantly, indicating the anti-apoptotic function of HLIKKß. Moreover, the up-regulation of HLIKKß mRNA was observed in the sea cucumber coelomocytes after polyriboinosinic polyribocytidylic acid [Poly (I:C)] or lipopolysaccharides (LPS) challenge, suggesting that the HLIKKß might play important roles in the innate immune defense of sea cucumber against the viral and bacterial infections.

Two Distinct C-Type Lysozymes in Goldfish: Molecular Characterization, Antimicrobial Potential, and Transcriptional Regulation in Response to Opposing Effects of Bacteria/Lipopolysaccharide and Dexamethasone/Leptin.

Lysozymes are key antimicrobial peptides in the host innate immune system that protect against pathogen infection. In this study, the full-length cDNAs of two c-type lysozymes (gfLyz-C1 and gfLyz-C2) were cloned from goldfish (Carassius auratus). The structural domains, three-dimensional structures, and amino acid sequences of gfLyz-C1 and gfLyz-C2 were highly comparable, as the two proteins shared 89.7% sequence identity. The gfLyz-C1 and gfLyz-C2 recombinant proteins were generated in the insoluble fractions of an Escherichia coli system. Based on the results of lysoplate and turbidimetric assays, gfLyz-C1 and gfLyz-C2 showed broad-spectrum antimicrobial properties with high levels of activity against Micrococcus lysodeikticus, Vibrio parahemolyticus, and Edwardsiella tarda, and relatively low activity against E. coli. Both gfLyz-C1 and gfLyz-C2 mRNAs were mainly expressed in the trunk kidney and head kidney, and gfLyz-C1 was expressed at much higher levels than gfLyz-C2 in the corresponding tissues. The expression of the gfLyz-C1 and gfLyz-C2 transcripts in the trunk kidney and head kidney was induced in these tissues by challenge with heat-inactivated E. coli and lipopolysaccharides (LPS), and the transcriptional responses of gfLyz-C1 were more intense. In goldfish primary trunk kidney cells, the levels of the gfLyz-C1 and gfLyz-C2 transcripts were upregulated by heat-inactivated E. coli, V. parahemolyticus, and E. tarda, as well as LPS, and downregulated by treatment with dexamethasone and leptins. Overall, this study may provide new insights that will improve our understanding of the roles of c-type lysozymes in the innate immunity of cyprinid fish, including the structural and phylogenetic characteristics, antimicrobial effects, and regulatory mechanism.

The first cloned echinoderm tumor necrosis factor receptor from Holothuria leucospilota: Molecular characterization and functional analysis.

In this study, an echinoderm tumor necrosis factor receptor named HLTNFR-16 was first cloned from the tropical sea cucumber Holothuria leucospilota. The full-length cDNA of HLTNFR-16 is 3675 bp in size, containing a 415 bp 5'-untranslated region (UTR), a 2024 bp 3'-UTR and a 1236 bp open reading frame (ORF) encoding a protein of 411 amino acids with a deduced molecular weight of 45.63 kDa. The HLTNFR-16 protein contains a signal peptide, four TNFR domains (the last three were identified as extracellular cysteine-rich domains), a transmembrane region and a death domain. Phylogenetic analysis showed that HLTNFR-16 was clustered into a clade with TNFR-16s in other species, indicating that this echinoderm TNFR may be a new member of the TNFR-16 subfamily. The results of TUNEL assay showed that the over expression of HLTNFR-16 could induce apoptosis in HEK293T cells. When HLTNFR-16 was silenced by siRNA, the apoptosis of sea cucumber coelomocytes induced by inactivated Vibrio harveyi was suppressed significantly, indicating that HLTNFR-16 is important for apoptosis induction. Additionally, luciferase reporter assay exhibited that the over-expressed HLTNFR-16 in HEK293T cells could activate the transcription factors nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). Moreover, the secretion of proinflammatory cytokines interleukin (IL)-1ß, IL-6 and IL-18 in HEK293T cells was increased by the over-expression of HLTNFR-16. This study provides evidences for the potential roles of sea cucumber TNFR in the innate immunity.

The first cloned sea cucumber FADD from Holothuria leucospilota: Molecular characterization, inducible expression and involvement of apoptosis.

In this study, a sea cucumber Fas-associated death domain (FADD) named HLFADD was first cloned from Holothuria leucospilota. The full-length cDNA of HLFADD is 2137 bp in size, containing a 116-bp 5'-untranslated region (UTR), a 1334-bp 3'-UTR and a 687-bp open reading frame (ORF) encoding a protein of 228 amino acids with a deduced molecular weight of 26.42 kDa. HLFADD protein contains a conserved death effector domain at its N-terminal and a conserved death domain at its C-terminal, structurally similar to its counterparts in vertebrates. The over-expressed HLFADD protein could induce apoptosis in HEK293 cells, suggesting a possible death receptor-mediated apoptosis pathway in echinoderms adapted with FADD. Moreover, HLFADD mRNA is ubiquitously expressed in all examined tissues, with the highest transcript level in the coelomocytes, followed by intestine. In vitro experiments performed in the H. leucospilota coelomocytes, the expression of HLFADD mRNA was significantly up-regulated by lipopolysaccharides (LPS) or polyriboinosinic-polyribocytidylic acid [poly (I:C)] challenge, suggesting that HLFADD might play important roles in the innate immune defense of sea cucumber against the invasion of bacteria and viruses.

Suppression of leptin-AI/AII transcripts by insulin in goldfish liver: A fish specific response of leptin under food deprivation.

Leptin is primarily considered a peripheral satiety hormone and is also found to perform important roles in energy homeostasis in vertebrates ranging from fish to mammals. The liver is a major source of leptin production in teleost fish. Using goldfish as a model, a previous report by our group illustrated the positive regulation of leptin mRNA levels by treatment with the hyperglycemic hormone glucagon, and our present study provided evidence for the negative regulation of hepatic leptin-AI and leptin-AII transcripts through the administration of the hypoglycemic hormone insulin. This study is the first to demonstrate changes in the hepatopancreatic insulin, glucagon, leptin-AI and leptin-AII mRNA levels in goldfish during fasting and refeeding. Insulin was found to be effective in suppressing leptin-AI and leptin-AII transcript levels in goldfish liver via both in vivo intraperitoneal injection and in vitro cell incubation approaches. Only the insulin receptor, not the IGF-I receptor, was involved in insulin-inhibited leptin mRNA level. The suppression of leptin levels by insulin was caused by the activation of MKK3/6/p38MAPK and MEK1/2/Erk1/2 cascades. Insulin treatment could eliminate the stimulation of glucagon on leptin mRNA level. Our study describes the regulation and signal transduction mechanism of insulin on leptin mRNA levels in the goldfish liver, suggesting that the leptin function in fish is speculated to be not only an anorexigenic factor but also a metabolic mediator. This also supports the hypothesis that the poikilothermal fish use a passive survival strategy during the periods of food deprivation, which is mediated by the fish-specifically high leptin levels induced by the cooperation of insulin and glucagon.

Effective Cycle Slip Detection and Repair for PPP/INS Integrated Systems.

Cycle slip (CS) is a primary error source in Precise Point Positioning/Inertial Navigation System (PPP/INS) integrated systems. In this study, an INS-aided CS detection and repair method is presented. It utilizes high-precision INS information instead of a pseudorange to remove the satellite⁻receiver geometric range in the wide-lane (WL) and ionospheric-free (IF) phase combinations and creates an INS-aided WL (WL-INS) model and an INS-aided IF (IF-INS) model. Since INS information is superior to pseudorange, the INS-aided models have high detection accuracy. However, the effectiveness of INS-aided models cannot persist for a long time because of INS accumulation error. To overcome the disturbance of INS error, improved INS-aided models are proposed. This idea takes advantage of the long wavelength of WL combination and tries to fix WL CS. Once it succeeds, the INS error can be evaluated and removed. The proposed method was tested using land vehicle data, in which simulated cycle slips and signal interruption were introduced. The results show that this method can accurately detect and repair different cycle slips between the continuous Global Positioning System (GPS) epoch. When it comes to the cycle slip after a GPS interruption, the method can also accelerate PPP re-convergence, as it is not affected by the inertial accumulation error.

Molecular characterization and functional analysis of MyD88 from the tropical sea cucumber, Holothuria leucospilota.

In this study, a myeloid differentiation factor 88 (MyD88) named as HLMyD88 was identified from the sea cucumber Holothuria leucospilota. The full-length cDNA of HLMyD88 is 4797 bp in size, containing a 227 bp 5'-untranslated region (UTR), a 3721 bp 3'-UTR and an 849 bp open reading frame (ORF) encoding a protein of 282 amino acids with a deduced molecular weight of 32.25 kDa HLMyD88 contains an N-terminal death domain and a C-terminal Toll/interluekin-1 receptor (TIR) domain with three highly conserved sequence motifs named as Box 1, Box 2 and Box 3. The results of luciferase reporter assay showed that over-expressed HLMyD88 in HEK293T cells could activate the transcription factors nuclear factor-κB (NF-κB) and activator protein 1 (AP-1). Additionally, the secretion of proinflammatory cytokines IL-1ß and TNF-α in the HEK293T cells was increased by over-expressed HLMyD88, indicating the potential role of HLMyD88 in the innate immunity of sea cucumber. Moreover, we further confirmed that over-expressed HLMyD88 could also induce apoptosis.

A novel caspase-6 from sea cucumber Holothuria leucospilota: Molecular characterization, expression analysis and apoptosis detection.

In this study, a novel caspase-6 named HLcaspase-6 was identified from sea cucumber Holothuria leucospilota. The full-length cDNA of HLcaspase-6 is 2195 bp in size, containing a 126 bp 5'-untranslated region (UTR), a 1043 bp 3'-UTR and a 1026 bp open reading frame (ORF) encoding a protein of 341 amino acids with a deduced molecular weight of 38.57 kDa. HLcaspase-6 contains the common signatures of the caspase family, including the conserved pentapeptide motif QACRG, as well as the P20 and P10 domains. In addition, HLcaspase-6 contains a short pro-domain. HLcaspase-6 mRNA is ubiquitously expressed in all tissues examined, with the highest transcript level in the intestine, followed by coelomocytes. In in vitro experiments, the expression of HLcaspase-6 mRNA in coelomocytes was significantly up-regulated by lipopolysaccharides (LPS) or polyriboinosinic-polyribocytidylic acid [poly (I:C)] challenge, suggesting that HLcaspase-6 might play important roles in the innate immune defense of sea cucumber against bacterial and viral infections. Moreover, we further confirmed that overexpression of HLcaspase-6 could induce apoptosis and activate the p53 signal pathway.

The first tropical sea cucumber caspase-8 from Holothuria leucospilota: Molecular characterization, involvement of apoptosis and inducible expression by immune challenge.

In this study, the first tropical sea cucumber caspase-8 named HLcaspase-8 was identified from Holothuria leucospilota. The full-length cDNA of HLcaspase-8 is 2293 bp in size, containing a 245 bp 5'-untranslated region (UTR), a 521 bp 3'-UTR and a 1527 bp open reading frame (ORF) encoding a protein of 508 amino acids with a deduced molecular weight of 57.47 kDa. Besides the common signatures of caspase family including conserved cysteine active site pentapeptide motif QACQG, P20 domain and P10 domain, HLcaspase-8 also contains a characteristic DED domain. The over-expression of HLcaspase-8 in HEK293T cells showed that HLcaspase-8 protein could induce apoptosis and the apoptosis could be promoted by TNF-α, indicating that the apoptosis induced by HLcaspase-8 might also be triggered via a receptor-mediated pathway. Moreover, the expression of HLcaspase-8 in in vitro experiments performed in coelomocytes was significantly up-regulated by lipopolysaccharides (LPS) or polyriboinosinic-polyribocytidylic Acid [poly (I:C)] challenge, suggesting that the sea cucumber caspase-8 might play some important roles in the innate immune defense against bacterial and viral infections.

Identification and characterization of a locus putatively involved in colanic acid biosynthesis in Vibrio alginolyticus ZJ-51.

Colanic acid (CA) is a group I extracellular polysaccharide (EPS) that contributes to resistance against adverse environments in many members of the Enterobacteriaceae. In the present study, a genetic locus EPSC putatively involved in CA biosynthesis was identified in Vibrio alginolyticus ZJ-51, which undergoes colony morphology variation between translucent/smooth (ZJ-T) and opaque/rugose (ZJ-O). EPSC in ZJ-T carries 21 ORFs and resembles the CA cluster of Escherichia coli K-12. The deletion of EPSC led to decreased EPS and biofilm formation in both genetic backgrounds but no alternation of lipopolysaccharide. The loss of this locus also changed the colony morphology of ZJ-O on the 2216E plate and reduced the motility of ZJ-T. Compared with ZJ-T, ZJ-O lacks a 10-kb fragment (epsT) in EPSC containing homologs of wecA, wzx and wzy that are essential for O-antigen synthesis. However, the deletion or overexpression of epsT resulted in no change of colony morphology, biofilm formation or EPS production. This study reported at the first time a genetic locus EPSC that may be involved in colanic acid synthesis in V. alginolyticus ZJ-51, and found that it was related to EPS biosynthesis, biofilm formation, colony morphology and motility, which may shed light on the environmental adaptation of the vibrios.

Supramolecular "Trojan Horse" for Nuclear Delivery of Dual Anticancer Drugs.

Nuclear delivery and accumulation are very important for many anticancer drugs that interact with DNA or its associated enzymes in the nucleus. However, it is very difficult for neutrally and negatively charged anticancer drugs such as 10-hydroxycamptothecine (HCPT). Here we report a simple strategy to construct supramolecular nanomedicines for nuclear delivery of dual synergistic anticancer drugs. Our strategy utilizes the coassembly of a negatively charged HCPT-peptide amphiphile and the positively charged cisplatin. The resulting nanomaterials behave as the "Trojan Horse" that transported soldiers (anticancer drugs) across the walls of the castle (cell and nucleus membranes). Therefore, they show improved inhibition capacity to cancer cells including the drug resistant cancer cell and promote the synergistic tumor suppression property in vivo. We envision that our strategy of constructing nanomaterials by metal chelation would offer new opportunities to develop nanomedicines for combination chemotherapy.