The Leaching Kinetics of Iron from Titanium Gypsum in a Citric Acid Medium and Obtain Materials by Leaching Liquid.

In this study, the effect of citric acid on iron leaching from titanium gypsum (TiG) was systematically investigated. The conditions for the leaching of valuable metals were optimized while varying such parameters as the leaching time, citric acid mass fraction, leaching temperature, and the liquid-solid ratio. It was found that under the conditions of a citric acid mass fraction of 10%, at a 80 °C leaching temperature, a leaching duration of 80-90 min and a liquid-solid ratio of 8, the whiteness of titanium gypsum (TiG) increased from 8.1 to 36.5, and the leaching efficiencies of iron reached 84.37%. The kinetic analysis indicated that the leaching process of iron from TiG was controlled by the reaction product layer from 0-20 min, while the leaching process of iron from TiG was controlled by internal diffusion from 20-90 min. The apparent activation energy of the leaching reactions was 33.91 kJ/mol and 16.59 kJ/mol, respectively. High-value-added calcium oxalate and ferrous oxalate were prepared from the calcium and iron in the filtrate of the oxalic acid extraction. The leaching liquid could be recycled, which will provide a new way to utilize titanium gypsum.

The Expanded Vermiculite Was Quickly Prepared by the Catalytic Action of Manganese Dioxide on Hydrogen Peroxide and Its Adsorption Properties to Cd.

The structure and activity of vermiculite can be maintained by expanding vermiculite (Vrm) with hydrogen peroxide. However, it is time-consuming. In past studies, little attention has been paid to the catalytic properties of manganese dioxide on hydrogen peroxide to improve the swelling efficiency of vermiculite. In this experiment, this catalytic effect was utilized to swell Vrm in a short time. The samples were then used to adsorb Cd from the solution. Through a series of characterization tests. The results showed that the exothermic rate was 1960.42-2089.164 J/min and the total exothermic heat was 39,208.4-41,783.28 J when expanding 10 gVrm, which could have a good expansion effect. The expansion was completed in about 40 min. Compared with Vrm, the adsorption of Cd is enhanced by about 30%. It is consistent with the proposed secondary kinetic adsorption model. This study provides a new perspective and theoretical guidance for improving the efficiency of Vrm stripping by hydrogen peroxide. A kind of expanded Vrm with better Cd adsorption efficiency was also prepared.

Exploiting the gut microbiota to predict the origins and quality traits of cultured sea cucumbers.

Nowadays, the true economic and nutritional value of food is underpinned by both origin and quality traits, more often expressed as increased quality benefits derived from the origin source. Gut microbiota contribute to food metabolism and host health, therefore, it may be suitable as a qualifying indicator of origin and quality of economic species. Here, we investigated relationships between the gut microbiota of the sea cucumber (Apostichopus japonicus), a valuable aquaculture species in Asia, with their origins and quality metrics. Based on data from 287 intestinal samples, we generated the first biogeographical patterns for A. japonicus gut microbiota from origins across China. Importantly, A. japonicus origins were predicted using the random forest model that was constructed using 20 key gut bacterial genera, with 97.6% accuracy. Furthermore, quality traits such as saponin, fat and taurine were also successfully predicted by random forest models based on gut microbiota, with approximately 80% consistency between predicted and true values. We showed that substantial variations existed in the gut microbiota and quality variables in A. japonicus across different origins, and we also demonstrated the great potential of gut microbiota to track A. japonicus origins and predict their quality traits.

Functions of lysin motif (LysM)-containing protein in antibacterial responses of sea cucumbers, Apostichopus japonicus.

The lysin motif (LysM)-containing protein is one of widespread pattern-recognition receptors in prokaryotes and eukaryotes. Numerous LysM-containing gene sequences are present in gene databases; however, few have been well characterized, especially in echinoderms. In this study, the full-length cDNA of a novel LysM-containing gene was obtained from the sea cucumber Apostichopus japonicus, named AjLysM-1, using polymerase chain reaction (PCR) combined with rapid amplification of cDNA ends. We prepared and expressed recombinant AjLysM-1 protein (rAjLysM-1) and determined its pathogen-recognition ability by enzyme-linked immunosorbent and immunofluorescence assays. We also analyzed the tissue expression pattern and response to immune challenges of AjLysM-1 using quantitative real-time reverse transcription-PCR and in situ hybridization. The AjLysM-1 protein was predicted to be an intracellular non-secreted LysM-containing protein, highly homologous to the same protein in other marine echinoderms. AjLysM-1 transcripts were highest expressed in coelomocytes and were strikingly induced by challenge with representative bacterial and fungal polysaccharides. rAjLysM-1 showed weak binding to mannan, Pseudoalteromonas nigrifaciens, and Shewanella baltica, implying that AjLysM-1 might provide inadequate defense against Gram-negative bacteria and fungi. Notably, rAjLysM-1 also interacted with tyrosine protein kinase and filamin-B, indicating that it could be involved in focal adhesion in A. japonicus. These findings improve our understanding of the functions of LysM-containing proteins in marine echinoderms.

Cloning and characterization of a phosphomevalonate kinase gene that is involved in saponin biosynthesis in the sea cucumber Apostichopus japonicus.

The sea cucumber Apostichopus japonicus is one of the most dominant and economically important aquaculture species in China. Saponin, which possesses notable biological and pharmacological properties, is a key determinant of the nutritional and health value of A. japonicus. In the present study, we amplified the full-length cDNA of a phosphomevalonate kinase (PMK) gene (named AjPMK) using rapid amplification of cDNA ends (RACE). Subsequently, we engineered a recombinant AjPMK (rAjPMK) protein and assessed its enzymatic activity by enzyme-linked immunosorbent assay (ELISA). Proteins that interact with rAjPMK were screened and identified via pull-down assay combined with liquid chromatography with tandem mass spectrometry (LC-MS/MS). We found that the full-length cDNA of AjPMK contained 1354 bp and an open reading frame (ORF) of 612 bp. The AjPMK protein was predicted not to contain a signal peptide but to contain a phosphonolate kinase domain seen in higher eukaryotes and a P-loop with a relatively conserved nucleoside triphosphate hydrolase domain. The molecular weight of the AjPMK protein was estimated to be 23.81 kDa, and its isoelectric point was predicted to be 8.72. Phylogenetic analysis showed that AjPMK had a closer evolutionary relationship with genes from starfish than with those of other selected species. Besides, we found that rAjPMK synthesized mevalonate-5-diphosphate, interacted either directly or indirectly with crucial pattern recognition receptors (PRRs) and was regulated by immune-related processes, including antioxidative reactions, stress resistance responses and enzyme hydrolysis. Moreover, AjPMK also interacted with farnesyl pyrophosphate synthase, an enzyme reported to be involved in saponin biosynthesis. Together, our findings implied that AjPMK may be directly involved in saponin biosynthesis and the regulation of various innate immune processes.

Comprehensive evaluation of environmental availability, pollution level and leaching heavy metals behavior in non-ferrous metal tailings.

Amounts of abandoned non-ferrous metal tailings(NMT) piled in the open air are released under geochemistry and migrated to the surrounding environment, causing severe harm to the environment and human health. It is essential to evaluate the heavy metal pollution of NMT. In this study, RAC, Igeo, EF, and RI were used to evaluate the heavy metal pollution risk of NMT. To uniformly simplify the four evaluation results into a comprehensive evaluation result that can reflect the degree of heavy metal pollution risk. Assuming heavy metals' concentration, occurrence, and mobility make the same contribution to the degree of heavy metal pollution. Score the above four evaluation results according to the pollution level, and then weigh the scores to obtain a complete integral result: CRSMo (17) > CRSCd (13) > CRSPb (11) > CRSSr(8) > CRSMn(7) > CRSCu(5) > CRSNi(4) > CRSCr(3) = CRSZn(3). Five higher risk heavy metal elements Mo, Cd, Pb, Sr, and Mn, were found. Cu, Ni, Cr, and Zn are at lower risk. The results showed that Mo, Mn, and Sr's evaluation is more accurate. Pb and Cd have not reached the detection limit for the time being, indicating that the release of heavy metal elements in tailings is not only related to the total concentration, occurrence state, and mobility of heavy metals but also affected by the pH of the tailings. This study's most significant finding is to propose a comprehensive integration result of pollution risk levels based on RAC, Igeo, EF, and RI as the comprehensive evaluation result of heavy metal pollution risk. Simultaneously, this research is also a valuable supplement to the existing risk assessment of heavy metal pollution.

Insights into the DNA methylation of sea cucumber Apostichopus japonicus in response to skin ulceration syndrome infection.

DNA methylation is an important epigenetic modification that regulates gene expression in many biological processes, including immune response. In this study, whole-genome bisulfite sequencing (WGBS) was carried out on healthy body wall (HB) and skin ulceration syndrome (SUS) infected body wall (SFB) to gain insights into the epigenetic regulatory mechanism in sea cucumber Apostichopus japonicus. After comparison, a total of 116,522 differentially methylated regions (DMRs) were obtained including 67,269 hyper-methylated and 49,253 hypo-methylated DMRs (p < 0.05, FDR < 0.001). GO enrichment analysis indicated that regulation of DNA-templated transcription (GO: 0006355), where DNA methylation occurred, was the most significant term in the biology process. The integration of methylome and transcriptome analysis revealed that 10,499 DMRs were negatively correlated with 496 differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these DEGs were enriched in the phosphoinositide 3-kinase-protein kinase B (PI3K/Akt)/mammalian target of rapamycin (mTOR) signaling pathway. Interestingly, two serine/threonine-protein kinases, nemo-like kinase (NLK) and mTOR, were highlighted after functional analysis. The variations of methylation in these two genes were associated with SUS infection and immune regulation. They regulated gene expression at different levels and showed interaction during response process. The validation of methylation sites showed high consistency between pyrosequencing and WGBS. WGBS analysis not only revealed the changes of DNA methylation, but also presented important information about the regulation of key genes after SUS infection in A. japonicus.

Effects of glycyrrhizin on the pharmacokinetics of paeoniflorin in rats and its potential mechanism.

Context: Paeoniflorin is reported to possess numerous pharmacological activities. Paeoniflorin and glycyrrhizin are always used together for the treatment of disease in China clinics; however, the drug-drug interaction between glycyrrhizin and paeoniflorin is still unknown. Objective: This study investigates the effects of glycyrrhizin on the pharmacokinetics of paeoniflorin in rats. Materials and methods: The pharmacokinetics of orally administered paeoniflorin (20 mg/kg) with or without glycyrrhizin pre-treatment (at a dose of 100 mg/kg/day for 7 days) were investigated in male Sprague-Dawley rats using LC-MS/MS. Additionally, Caco-2 cell transwell model and rat liver microsome incubation experiments were also conducted to investigate its potential mechanism. Results: The results showed that when the rats were pre-treated with glycyrrhizin, the Cmax of paeoniflorin decreased from 59.57 ± 10.24 to 45.36 ± 8.61 ng/mL, and AUC0-inf also decreased from 282.02 ± 35.06 to 202.29 ± 28.28 µg·h/L. The t1/2 value of paeoniflorin decreased from 8.48 ± 2.01 to 5.88 ± 1.15 h (p < 0.05). The Caco-2 cell transwell experiments indicated that glycyrrhizin could increase the efflux ratio of paeoniflorin from 2.71 to 3.52, and the rat liver microsome incubation experiments showed that glycyrrhizin could significantly increase its intrinsic clearance rate from 53.7 ± 4.6 to 85.6 ± 7.1 µL/min/mg protein. Conclusions: These results indicated that glycyrrhizin could affect the pharmacokinetics of paeoniflorin, and it might work through decreasing the absorption of paeoniflorin by inducing the activity of P-gp or through increasing the clearance rate in rat liver by inducing the activity of CYP450 enzyme.

Comparative expression analysis of immune-related factors in the sea cucumber Apostichopus japonicus.

In order to preliminarily explore the joint involvement of different immune-related factors during the same immune process in Apostichopus japonicus, the transcriptional expression of Cu/Zn superoxide dismutase (Cu/Zn-SOD), catalase (CAT), c-type lysozyme (c-LYZ), i-type lysozyme (i-LYZ), cathepsin D, melanotransferrin (MTF), Toll, c-type lectin (c-LCT) and complement 3 (C3) during the development from fertilized eggs to juveniles and after challenging the juveniles with Vibrio splendidus, Pseudoalteromonas nigrifaciens, Shewanella baltica and Bacillus cereus, respectively, was measured using the method of quantitative real-time PCR (qRT-PCR), and then the correlations among different immune-related factors were analyzed. The results showed that the selected immune-related factors were expressed at all of the determined developmental stages and significantly up-regulated at doliolaria stage, suggesting the selected factors are indispensable immune components and the immune system might be broadly activated at doliolaria stage in A. japonicus. After challenged with four pathogenic bacteria, Cu/Zn-SOD, CAT, i-LYZ, cathepsin D, MTF, Toll, C3 were all significantly down-regulated at 4 h, indicating that some components of A. japonicus immune system might be inhibited at the beginning of pathogenic bacteria invasion. The immune-responsive analysis also showed that the significant regulation in Toll after challenged with four tested bacteria, that in MTF after challenged with S. baltica and that in C3 after challenged with P. nigrifaciens were all minus, suggesting Toll, MTF and C3 are probably the primary targets of pathogenic bacteria attack. Furthermore, the correlation analysis indicated that, all of the selected immune-related factors except cathepsin D might be in the same immune regulatory network during A. japonicus development, while all of the selected immune-related factors except c-LYZ might be in the same responsive regulatory network after challenged with four pathogenic bacteria. Altogether, A. japonicus immune system exhibited high complexity in regulation during organism development and after bacterial challenges.

In-depth profiling of miRNA regulation in the body wall of sea cucumber Apostichopus japonicus during skin ulceration syndrome progression.

MicroRNAs (miRNAs) are small non-coding RNAs that mediate mRNA degradation or translation repression. Previous study showed that the expression of miRNAs was significantly changed in the body wall of sea cucumber Apostichopus japonicus after skin ulceration syndrome (SUS) infection, which is a dynamic process. However, the critical miRNAs from body wall that involved in different infection stages of SUS remain unknown. In this study, four cDNA libraries were constructed with the body wall from healthy and three SUS-infected stages of A. japonicus. A total of 248 conserved miRNAs and five novel miRNAs were identified through Illumina HiSeq 2000 platform. Compared to the control, 238 miRNAs showed significant differential expression at three stages of SUS progression. Totally, 3149 miRNA-mRNA pairs were identified by target prediction and 314 miRNA-mRNA pairs showed negative correlation. It is noteworthy that 15 miRNAs and four mRNAs were located at the crucial positions of the network built with the anti-correlated miRNA-mRNA pairs. GO and KEGG enrichment analysis indicated that the predicted targets were involved in many immune-related processes. Deep analysis of miR-31c-5p, miR-29b-3p, NF-kB, mucin 2 and titin showed that they may play important roles in the pathogens attachment and recognition, signaling transduction and lesions repair of A. japonicus after SUS infection. These results would be useful for further investigating the potential roles of critical miRNAs and mRNAs in A. japonicus immune regulation.

Postoperative Outcomes of Magnetic Resonance Imaging (MRI)-Guided Laser Interstitial Thermal Therapy (LITT) in the Treatment of Drug-Resistant Epilepsy: A Meta-Analysis.

BACKGROUND Drug-resistant epilepsy is a common neurological disease in adults and children. This study aimed to undertake a systematic review of the literature with meta-analysis of the data from published studies to assess the effectiveness of magnetic resonance imaging (MRI)-guided laser interstitial thermal therapy (LITT) in treatment-resistant epilepsy. MATERIAL AND METHODS The study was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. PubMed, MEDLINE, and EMBASE databases were systematically searched for indexed publications in the English language up to May 2018. Data on the prevalence, outcome using the Engel Epilepsy Surgery Outcome Scale (Class I to IV), and postoperative complications were analyzed with 95% confidence intervals (CIs). The Methodological Index for Non-Randomized Studies (MINORS) was used to assess the risk of bias in the included studies. RESULTS Sixteen published studies that included a total of 269 patients with treatment resistant epilepsy were identified. The prevalence of Engel Class I, II, III and IV were 61% (95% CI, 0.54-0.68; I²=14.5%; P=0.302), 12% (95% CI, 0.07-0.16; I²=86.8%; P=0.000), 16% (95% CI, 0.10-0.22; I²=3.0%; P=0.397), and 15% (95% CI, 0.08-0.22; I²=13.2%; P=0.330), respectively. The prevalence of postoperative complications was 24% (95% CI, 0.16-0.32; I2=0%; P=0.629). CONCLUSIONS Meta-analysis of data from 16 studies that included 269 patients with treatment-resistant epilepsy showed that MRI-guided LITT significantly reduced the frequency of seizures and reduced postoperative complications, supporting the safety and effectiveness of MRI-guided LITT in the treatment of drug-resistant epilepsy.

Preparation of Graphite Oxide Containing Different Oxygen-Containing Functional Groups and the Study of Ammonia Gas Sensitivity.

A series of graphite oxide samples were prepared using the modified Hummers method. Flake graphite was used as the raw material and the reaction temperature of the aqueous solution was changed (0 °C, 30 °C, 50 °C, 60 °C, 70 °C, 80 °C, and 100 °C). X-ray diffraction, Fourier-transform infrared spectroscopy, Raman spectral analysis, X-ray photoelectron spectroscopy, and contact angle tests were performed to characterize the structure, chemical bonding, type, and content of oxygen-containing functional groups of the graphite oxide samples. The results showed that the type and content of each oxygen-containing functional group could be controlled by changing the reaction temperature with the addition of water. As the temperature of the system increased, the degree of oxidation of the graphite oxide samples first increased and then decreased. Too high a temperature (100 °C) of the system led to the formation of epoxy groups by the decomposition of some hydroxyl groups in the samples, causing the reduction of oxygen-containing functional groups between the graphite layers, poor hydrophilic properties, and low moisture content. When the system temperature was 50 °C, the interlayer spacing of the graphite oxide samples was at its highest, the graphite was completely oxidized (C/O = 1.85), and the oxygen-containing functional groups were mainly composed of hydroxyl groups (accounting for approximately 28.88% of the total oxygen-containing functional groups). The high content of hydroxyl and carboxyl groups had good hydrophilic ability and showed the highest moisture content. The sample at 50 °C had better sensitivity to ammonia because of its high hydroxyl group and carboxyl group content, with the sample showing an excellent profile when the ammonia concentration was 20⁻60 ppm.

Comparative analysis of immunocompetence between females and males in the sea cucumber Apostichopus japonicus.

In order to preliminarily understand the immune difference between females and males in the sea cucumber Apostichopus japonicus, the activities assay of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), phenoloxidase (PO), acid phosphatase (ACP) and alkaline phosphatase (ALP) with biochemical methods, the detection of PO isozymes with native-PAGE and catechol staining, and the test of antibacterial activities with bacterial growth curve determination method were performed in this study using cell-free coelomic fluid (CCF) and coelomocyte lysate supernatant (CLS) from females and males as the samples. The PO activities were not detected in the CLS and showed no significant difference between the CCF from females and males. However, totally five PO isozyme bands were detected in the CLS of females while only four were detected in the CLS of males after zymogram analysis. These results implied that the PO isozymes in the coelomocytes of viripotent A. japonicus were inactive under natural condition and may be activated by some certain treatments during native-PAGE, and PO might play different immune and physiological roles between females and males. In addition, the activities of SOD, CAT, POD and ALP in the CCF and the activities of CAT, POD, ACP and ALP in the CLS from males were all significantly higher than those from females. The results collectively suggested that in viripotent A. japonicus, the gender had a remarkable effect on the immunity, and the immunocompetence of males might have an advantage over that of females. Furthermore, the activities of all determined enzymes except PO and the number of detected PO isozymes showed higher values in CLS than in CCF, implying that in viripotent A. japonicus, the coelomocytes might take more immune responsibility in comparison with CCF.

Comparative study of three phenoloxidases in the sea cucumber Apostichopus japonicus.

In order to preliminarily illustrate the functional differences of phenoloxidases (POs) in Apostichopus japonicus, the full-length cDNAs of two POs (named as AjPOⅡ and AjPOⅢ, respectively) were cloned from the coelomocytes of A. japonicus using 3'- and 5'-rapid amplification of cDNA ends method, and combined with the previously acquired full-length cDNA of a laccase-type PO from A. japonicus (Accession No. KF040052, named as AjPOⅠ), the sequence structure and phylogenic status of POs from A. japonicus (AjPOs) were comparatively analyzed, and the transcriptional expression of AjPOs in different tissues, at different developmental stages and after different bacterial challenges was determined with quantitative real-time PCR method. Sequence analysis indicated AjPOⅡ and AjPOⅢ were both laccase-type POs, coincident to the results of phylogenic analysis. Sequence analysis also showed that AjPOⅠ had a transmembrane domain (J. Jiang et al., 2014), AjPOⅡ contained a signal peptide, and AjPOⅢ possessed a signal peptide and a transmembrane domain, implying that three AjPOs might play different roles in immune and physiological processes. Transcriptional expression analysis showed that AjPOⅡ and AjPOⅢ were most abundant in tube feet, while AjPOⅠhad the highest expression level in coelomocytes (J. Jiang et al., 2014), suggesting that AjPOⅠ may be mainly involved in immune response, while AjPOⅡ and AjPOⅢ are probably responsible for other physiological processes in addition to immune response. Besides, three AjPOs were determined to have different expression patterns during organism development and different spectrums of response against bacteria, which further indicated that there might be immune and physiological functional differentiation among three AjPOs.

RNA sequencing analysis to capture the transcriptome landscape during skin ulceration syndrome progression in sea cucumber Apostichopus japonicus.

BACKGROUND: Sea cucumber Apostichopus japonicus is an important economic species in China, which is affected by various diseases; skin ulceration syndrome (SUS) is the most serious. In this study, we characterized the transcriptomes in A. japonicus challenged with Vibrio splendidus to elucidate the changes in gene expression throughout the three stages of SUS progression. RESULTS: RNA sequencing of 21 cDNA libraries from various tissues and developmental stages of SUS-affected A. japonicus yielded 553 million raw reads, of which 542 million high-quality reads were generated by deep-sequencing using the Illumina HiSeq™ 2000 platform. The reference transcriptome comprised a combination of the Illumina reads, 454 sequencing data and Sanger sequences obtained from the public database to generate 93,163 unigenes (average length, 1,052 bp; N50 = 1,575 bp); 33,860 were annotated. Transcriptome comparisons between healthy and SUS-affected A. japonicus revealed greater differences in gene expression profiles in the body walls (BW) than in the intestines (Int), respiratory trees (RT) and coelomocytes (C). Clustering of expression models revealed stable up-regulation as the main pattern occurring in the BW throughout the three stages of SUS progression. Significantly affected pathways were associated with signal transduction, immune system, cellular processes, development and metabolism. Ninety-two differentially expressed genes (DEGs) were divided into four functional categories: attachment/pathogen recognition (17), inflammatory reactions (38), oxidative stress response (7) and apoptosis (30). Using quantitative real-time PCR, twenty representative DEGs were selected to validate the sequencing results. The Pearson's correlation coefficient (R) of the 20 DEGs ranged from 0.811 to 0.999, which confirmed the consistency and accuracy between these two approaches. CONCLUSIONS: Dynamic changes in global gene expression occur during SUS progression in A. japonicus. Elucidation of these changes is important in clarifying the molecular mechanisms associated with the development of SUS in sea cucumber.

Expression analysis of microRNAs related to the skin ulceration syndrome of sea cucumber Apostichopus japonicus.

MicroRNAs (miRNAs) are small non-coding RNAs that are involved in many biological processes. To investigate the miRNAs related to skin ulceration syndrome (SUS) of Apostichopus japonicus, small RNA libraries of body wall, intestine, respiratory tree and coelomocytes from healthy and diseased A. japonicus were sequenced on Illumina Hiseq 2000 platform. A total of 247 conserved and 10 novel miRNAs were identified across all libraries. After pair-wise comparisons, 215 miRNAs in body wall, 36 in intestine, 2 in respiratory tree and 38 in coelomocytes showed significant expression differences. Further analyses were conducted on some tissue-specific differentially expressed miRNAs: miR-8 and miR-486-5p in body wall, miR-200-3p, let-7-5p and miR-125 in intestine, miR-278a-3p and bantam in respiratory, miR-10a and miR-184 in coelomocytes. Notably, these miRNAs in some species were reported to function in various physiological or pathological processes associated with immune regulations. Using stem-loop quantitative real time PCR, six representative miRNAs in four tissues were selected to validate the sequencing results. The Pearson's correlation coefficient (R) of the six miRNAs ranged from 0.777 to 0.948, which confirmed the consistency and accuracy between these two approaches. This study provides comprehensive expression and regulation patterns of functional miRNAs in different tissues and gives insights into the tissue-specific immune response mechanisms in SUS-infected A. japonicus.

In vitro antibacterial analysis of phenoloxidase reaction products from the sea cucumber Apostichopus japonicus.

Three phenoloxidases (POs) of Apostichopus japonicus, AjPOs (AjPO1, AjPO2 and AjPO3), were partially purified from the coelomocytes with an electrophoretic method, and then employed for the in vitro antibacterial analysis. Using L-3,4-dihydroxyphenylalanine (L-DOPA) as a substrate, AjPO1 and AjPO2-derived compounds inhibited the growth of Vibrio splendidus and Staphylococcus aureus, while AjPO3-derived compounds only inhibited the growth of V. splendidus. When dopamine was used as a substrate, AjPO1 and AjPO3-derived compounds inhibited the growth of V. splendidus and Vibrio harveyi, while AjPO2-derived compounds only inhibited the growth of V. splendidus. Moreover, AjPO1-derived compounds showed stronger inhibition in V. harveyi than AjPO3-derived compounds did. However, all of the three AjPO reaction products showed no inhibitions on the growth of Pseudoalteromonas nigrifaciens, Shewanella baltica, Micrococcus lysodeikticus, Streptococcus dysgalactiae and Nocardiopsis sp. with L-DOPA or dopamine as a substrate. Scanning electron microscope (SEM) observation of V. harveyi treated by AjPOs and dopamine showed that AjPO1-derived compounds resulted in massive bacteriolysis, AjPO2-derived compounds caused no obvious alteration on bacterial morphology, and AjPO3-derived compounds increased the ratio of spheroidal bacteria. All these results suggested that AjPO reaction products derived by L-DOPA and dopamine had different but limited antibacterial spectrum, and the different antibacterial effects observed among three AjPOs resulted from the different reaction products generated by AjPOs with the same substrate.

Phenoloxidase from the sea cucumber Apostichopus japonicus: cDNA cloning, expression and substrate specificity analysis.

Phenoloxidase (PO) is a crucial component of the immune system of echinoderms. In the present study, the full-length cDNA of PO (AjPO) was cloned from coelomocytes of the sea cucumber Apostichopus japonicus using 3'- and 5'-rapid amplification of cDNA ends (RACE) PCR method, which is 2508 bp, with an open reading frame (ORF) of 2040 bp encoding 679 amino acids. AjPO contains a transmembrane domain, and three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine respectively. Phylogenetic analysis revealed that AjPO was clustered with laccase-type POs of invertebrates. Using the isolated membrane proteins as crude AjPO, the enzyme could catalyze the substrates catechol, L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine. The results described above collectively proved that AjPO was a membrane-binding laccase-type PO. The quantitative real-time PCR (qRT-PCR) analysis revealed that AjPO mRNA was expressed in muscle, body wall, coelomocytes, tube feet, respiratory tree and intestine with the highest expression level in coelomocytes. AjPO could be significantly induced by lipopolysaccharide (LPS), peptidoglycan (PGN), Zymosan A and polyinosinic-polycytidylic acid (PolyI:C), suggesting AjPO is closely involved in the defense against the infection of bacteria, fungi and double-stranded RNA viruses.

Expression analysis of immune related genes identified from the coelomocytes of sea cucumber (Apostichopus japonicus) in response to LPS challenge.

The sea cucumber (Apostichopus japonicus) occupies a basal position during the evolution of deuterostomes and is also an important aquaculture species. In order to identify more immune effectors, transcriptome sequencing of A. japonicus coelomocytes in response to lipopolysaccharide (LPS) challenge was performed using the Illumina HiSeq™ 2000 platform. One hundred and seven differentially expressed genes were selected and divided into four functional categories including pathogen recognition (25 genes), reorganization of cytoskeleton (27 genes), inflammation (41 genes) and apoptosis (14 genes). They were analyzed to elucidate the mechanisms of host-pathogen interactions and downstream signaling transduction. Quantitative real-time polymerase chain reactions (qRT-PCRs) of 10 representative genes validated the accuracy and reliability of RNA sequencing results with the correlation coefficients from 0.88 to 0.98 and p-value <0.05. Expression analysis of immune-related genes after LPS challenge will be useful in understanding the immune response mechanisms of A. japonicus against pathogen invasion and developing strategies for resistant markers selection.