IL-17/IL-17 Receptor Pathway-Mediated Inflammatory Response in Apostichopus japonicus Supports the Conserved Functions of Cytokines in Invertebrates.

Inflammation participates in host defenses against infectious agents and contributes to the pathophysiology of many diseases. IL-17 is a well-known proinflammatory cytokine that contributes to various aspects of inflammation in vertebrates. However, the functional role of invertebrate IL-17 in inflammatory regulation is not well understood. In this study, we first established an inflammatory model in the Vibrio splendidus-challenged sea cucumber Apostichopus japonicus (Echinodermata). Typical inflammatory symptoms, such as increased coelomocyte infiltration, tissue vacuoles, and tissue fractures, were observed in the V. splendidus-infected and diseased tissue of the body wall. Interestingly, A. japonicus IL-17 (AjIL-17) expression in the body wall and coelomocytes was positively correlated with the development of inflammation. The administration of purified recombinant AjIL-17 protein also directly promoted inflammation in A. japonicus Through genome searches and ZDOCK prediction, a novel IL-17R counterpart containing FNIII and hypothetical TIR domains was identified in the sea cucumber genome. Coimmunoprecipitation, far-Western blotting, and laser confocal microscopy confirmed that AjIL-17R could bind AjIL-17. A subsequent cross-linking assay revealed that the AjIL-17 dimer mediates the inflammatory response by the specific binding of dimeric AjIL-17R upon pathogen infection. Moreover, silencing AjIL-17R significantly attenuated the LPS- or exogenous AjIL-17-mediated inflammatory response. Functional analysis revealed that AjIL-17/AjIL-17R modulated inflammatory responses by promoting A. japonicus TRAF6 ubiquitination and p65 nuclear translocation and evenly mediated coelomocyte proliferation and migration. Taken together, our results provide functional evidence that IL-17 is a conserved cytokine in invertebrates and vertebrates associated with inflammatory regulation via the IL-17-IL-17R-TRAF6 axis.

Macrocycle Self-Assembly Hydrogel for High-Efficient Oil-Water Separation.

Supramolecular hydrogels involved macrocycles have been explored widely in recent years, but it remains challenging to develop hydrogel based on solitary macrocycle with super gelation capability. Here, the construction of lantern[33 ]arene-based hydrogel with low critical gelation concentration (0.05 wt%), which can be used for efficient oil-water separation, is reported. The lantern[33 ]arenes self-assemble into hydrogen-bonded organic nanoribbons, which intertwine into entangled fibers to form hydrogel. This hydrogel which exhibits reversible pH-responsiveness characteristics can be coated on stainless-steel mesh by in situ sol-gel transformation. The resultant mesh exhibits excellent oil-water separation efficiency (>99%) and flux (>6 × 104 L m-2 h-1 ). This lantern[33 ]arene-based hydrogel not only sheds additional light on the gelation mechanisms for supramolecular hydrogels, but also extends the application of macrocycle-based hydrogels as functional interfacial materials.

Phenotypic and functional characterization of two coelomocyte subsets in Apostichopus japonicus.

The hemocytes of invertebrates are composed of different cell subsets with different morphologies and structures. Different cell subsets have different immune functions, which play an important role in innate immune response against pathogens. However, the understanding of the classification of Apostichopus japonicus coelomocytes and the molecular basis of immune function of different cell subsets is very limited. In this study, two coelomocyte subpopulations of A. japonicus were isolated by Percoll density gradient centrifugation. They were identified from their morphological and structural characteristics, namely, spherical cells with a size of 10-12 µm spherical in shape and a large number of small granules inside; lymphocyte-like cells with a size of 4-5 µm spherical or oval in shape, and 1-3 filopodia. Functionally, the phagocytic capacity and lysosomal activity in spherical cells were significantly greater than those in lymphocyte-like cells. The results suggest that spherical cells may play a more critical role in the immune responses. Meanwhile, transcriptome sequencing analysis was performed to further clarify the functional differences between the two cell subsets. The data indicated significantly different gene expression patterns in them. Spherical cells tend to participate in immune defense, whereas lymphocyte-like cells tend to participate in energy metabolism. In addition, lymphocyte-like cells may convert oxidative phosphorylation to glycolysis by changing the manner of energy metabolism to quickly adapt to the energy demand of external stimuli. Spherical cells may respond to LPS stimulation through phagocytosis, and their response time is slower than that of lymphocyte-like cells. The expression of genes involved in endocytosis, phagocytosis, and lysosomal and humoral immunity in spherical cells was significantly higher than that in lymphocyte-like cells. These data provide valuable information for understanding the molecular basis of cellular and humoral immunity in A. japonicus.

Hip circumference has independent association with the risk of hyperuricemia in middle-aged but not in older male patients with type 2 diabetes mellitus.

BACKGROUND: Obesity and type 2 diabetes mellitus (T2DM) are risk factors for hyperuricemia. However, which anthropometric indices can better predict incident hyperuricemia in patients with T2DM remains inconsistent. This study aimed to examine the associations between hyperuricemia and different anthropometric indices in middle-aged and older male patients with T2DM. METHODS: In this retrospective study, a total of 1447 middle-aged (45-65 years, n = 791) and older (≥ 65 years, n = 656) male patients with T2DM were collected from December 2015 to January 2020 at Shanghai Xinhua Hospital. Hyperuricemia was defined as a serum uric acid level above 7.0 mg/dL. Weight, height, waist circumference (WC) and hip circumference (HC) were measured by trained nurses at visit. RESULTS: The median uric acid level of subjects was 5.6 (interquartile ranges: 4.7-6.7) mg/dl, and 279 (19.3%) were hyperuricemia, with 146 (18.5%) in the middle-aged group, and 133 (20.3%) in the older group. After adjusting for age, duration of T2DM, fasting plasma glucose and insulin, homeostasis model assessment-ß, aspartate aminotransferase, triglycerides, high-density lipoprotein cholesterol and estimated glomerular filtration rate, body mass index (BMI), WC, HC, and waist-to-height ratio (WHtR) were associated with a higher risk of hyperuricemia in both middle-aged and older group (P < 0.05). After further adjusting for BMI and WC, HC still showed a positive relationship with the risk of hyperuricemia (Odds Ratio = 1.51, 95% confidence intervals: 1.06-2.14) in the middle-aged group, but such relationship was not found in the older group. Moreover, according to receiver operating characteristic analysis, the optimal cutoff value was 101.3 cm of HC for hyperuricemia screening in the middle-aged male patients with T2DM. CONCLUSION: In middle-aged male patients with T2DM, more attention should be paid to HC with the cutoff value of 101.3 cm in clinical practice for early recognition of individuals with a high risk of hyperuricemia for targeted guidance on disease prevention, such as community screening.

Src kinase mediates coelomocytes phagocytosis via interacting with focal adhesion kinase in Vibrio splendidus challenged Apostichopus japonicus.

Immune cells have many efficient ways to participate in the host immunity, including phagocytosis, which is an important pathway to eliminate pathogens. Only ß-integrin-mediated phagocytosis pathways have been confirmed in Apostichopus japonicus. The Src family kinases (SFKs), a class of non-receptor tyrosine kinases plays an important role in the regulation of phagocytic signals in invertebrates. However, the SFK-mediated phagocytic mechanism is largely unknown in A. japonicus. In this study, a novel SFK homologue (AjSrc) with a conservative SH3 domain, an SH2 domain, and a tyrosine kinase domain was identified from A. japonicus. Both gene and protein expression of AjSrc and phosphorylation levels increased under Vibrio splendidus challenged, reaching the highest level at 24 h. Knock-down of AjSrc could depress coelomocytes' phagocytosis by 25% compared to the control group. To better understand the mechanism of AjSrc-mediated phagocytosis, focal adhesion kinase (FAK) was identified by a Co-immunoprecipitation experiment to be verified as an interactive protein of AjSrc. The phagocytosis rates of coelomocytes were decreased by 33% and 37% in AjFAK and AjSrc + AjFAK interference groups compared with the control group, respectively. Furthermore, the phosphorylation level of AjFAK was increased and reached the maximum level at 24 h post V. splendidus infection, as the same as that of AjSrc. Our results suggested that AjSrc could mediate V. splendidus-induced coelomocytes' phagocytosis via interacting with AjFAK and co-phosphorylation. This study enriched the mechanism of phagocytosis in echinoderm and provided the new theoretical foundation for disease control of sea cucumber.

Genome-wide identification m6A modified circRNAs revealed their key roles in skin ulceration syndrome disease development in Apostichopus japonicus.

Circular RNAs (circRNAs) are novel endogenous non-coding RNAs (ncRNAs) and can be acted as competing endogenous RNAs (ceRNAs) to regulate microRNA (miRNA) and downstream gene expression. Recently, m6A modification has been found in circRNA, and m6A circRNAs also play important roles in various biological processes and a variety of diseases. Our previous study had been demonstrated that circRNAs were differentially expressed in skin ulceration syndrome (SUS) diseased sea cucumber Apostichopus japonicus. However, whether the function of circRNAs are dependent on m6A levels are largely unknown. Here, we firstly investigated the genome-wide map of m6A circRNAs in sea cucumbers with different stages of Vibrio splendidus challenge, that's Control group, SUS-diseased group, and SUS-resistant group. MeRIP-seq revealed that m6A abundances were enriched in circRNAs in all three groups, especially for SUS-resistant group. Among them, more than 62% of modified circRNAs harbor only a single m6A peak and about 55% of m6A sites in circRNAs were derived from sense overlapping in each group. After V. splendidus infection, we found that most of m6A peaks in circRNAs were upregulated and less were downregulated in both SUS-diseased and SUS-resistant groups when compared with Control. Furthermore, GO analysis indicated that the host genes of circRNAs with dysregulated m6A peaks in SUS-diseased and SUS-resistant groups were both mainly enriched in the adhesion pathway. More importantly, we discovered that more than 50% m6A circRNAs showed a positive correlation between the circRNAs expression and m6A methylation levels both in SUS-diseased and SUS-resistant groups. Therefore, a core circRNA-miRNA-mRNA (ceRNA) network whether influenced by m6A modification was constructed based on conjoint analysis. Our results indicated that several selected m6A circRNAs bind with miRNAs were mainly targeting to ubiquitylation system and adhesion pathway. What's more, three candidate m6A circRNAs and three target genes were validated by MeRIP-qPCR and qPCR, whose m6A levels in circRNA and mRNA expressions were consistent with disease occurrence or disease resistance. All of our current findings suggested that m6A circRNAs could play important roles during pathogen infection and might be served as a new molecular biomarker in SUS disease diagnose of A. japonicus.

Comparing human milk macronutrients measured using analyzers based on mid-infrared spectroscopy and ultrasound and the application of machine learning in data fitting.

OBJECTIVE: Fat, carbohydrates (mainly lactose) and protein in breast milk all provide indispensable benefits for the growth of newborns. The only source of nutrition in early infancy is breast milk, so the energy of breast milk is also crucial to the growth of infants. Some macronutrients composition in human breast milk varies greatly, which could affect its nutritional fulfillment to preterm infant needs. Therefore, rapid analysis of macronutrients (including lactose, fat and protein) and milk energy in breast milk is of clinical importance. This study compared the macronutrients results of a mid-infrared (MIR) analyzer and an ultrasound-based breast milk analyzer and unified the results by machine learning. METHODS: This cross-sectional study included breastfeeding mothers aged 22-40 enrolled between November 2019 and February 2021. Breast milk samples (n = 546) were collected from 244 mothers (from Day 1 to Day 1086 postpartum). A MIR milk analyzer (BETTERREN Co., HMIR-05, SH, CHINA) and an ultrasonic milk analyzer (HonÉ¡yanÉ¡ Co,. HMA 3000, Hebei, CHINA) were used to determine the human milk macronutrient composition. A total of 465 samples completed the tests in both analyzers. The results of the ultrasonic method were mathematically converted using machine learning, while the Bland-Altman method was used to determine the limits of agreement (LOA) between the adjusted results of the ultrasonic method and MIR results. RESULTS: The MIR and ultrasonic milk analyzer results were significantly different. The protein, fat, and energy determined using the MIR method were higher than those determined by the ultrasonic method, while lactose determined by the MIR method were lower (all p < 0.05). The consistency between the measured MIR and the adjusted ultrasound values was evaluated using the Bland-Altman analysis and the scatter diagram was generated to calculate the 95% LOA. After adjustments, 93.96% protein points (436 out of 465), 94.41% fat points (439 out of 465), 95.91% lactose points (446 out of 465) and 94.62% energy points (440 out of 465) were within the LOA range. The 95% LOA of protein, fat, lactose and energy were - 0.6 to 0.6 g/dl, -0.92 to 0.92 g/dl, -0.88 to 0.88 g/dl and - 40.2 to 40.4 kj/dl, respectively and clinically acceptable. The adjusted ultrasonic results were consistent with the MIR results, and LOA results were high (close to 95%). CONCLUSIONS: While the results of the breast milk rapid analyzers using the two methods varied significantly, they could still be considered comparable after data adjustments using linear regression algorithm in machine learning. Machine learning methods can play a role in data fitting using different analyzers.

The effect of different adiposity factors on insulin resistance in obese children and adolescents.

CONTEXT: Insulin resistance (IR) has been common in obese children, but the effect of different adiposity factors on IR is still unclear. OBJECTIVE: To evaluate the associations between IR with body mass index (BMI), waist circumference (WC), body fat mass (BFM), and body fat percentage (BFP) in obese children and adolescents. METHODS: A total of 224 simple obese children were included in this study, including 150 boys and 74 girls, aged 3-18 who were seen in the clinical nutrition outpatient of Xinhua Hospital from September 2012 to December 2019. Basic information, body composition and laboratory tests were collected. RESULTS: Compared with girls, boys had higher height, weight, BMI, WC, and BFM (P < 0.05), but on the contrary, boys' FINS and HOMA- IR were lower than girls' (P > 0.05). With the age increasing, height, weight, BMI, BFM, WC, HC, WHtR, FINS and HOMA-IR increased accordingly (P < 0.05). The results from univariate analysis and multiple linear regression analysis showed that the impact of BMI on IR was slightly lower than BFM, WC and HC, but higher than BFP, with adjusting for the effects of age, sex and lipid metabolism (P < 0.01). CONCLUSION: Overall adipose tissue, especially abdominal adipose tissue, is a powerful marker in inducing IR in obese children and adolescents. In addition, more attention should be paid to WC and BFM than BMI in obese people with IR.

Near-infrared emission Cu, N-doped carbon dots for human umbilical vein endothelial cell labeling and their biocompatibility in vitro.

Quantum dots (QDs) are luminescent semiconductor nanomaterials (NMs) with various biomedical applications, but the high toxicity associated with traditional QDs, such as Cd-based QDs, limits their uses in biomedicine. As such, the development of biocompatible metal-free QDs has gained extensive research interests. In this study, we synthesized near-infrared emission Cu, N-doped carbon dots (CDs) with optimal emission at 640 nm and a fluorescence quantum yield of 27.1% (in N,N-dimethylformamide [DMF]) by solvothermal method using o-phenylenediamine and copper acetate monohydrate. We thoroughly characterized the CDs and showed that they were highly fluorescent and stable under different conditions, although in highly acidic (pH = 1-2) or alkaline (pH = 12-13) solutions, a redshift or blueshift of fluorescence emission peak of Cu, N-doped CDs was also observed. When exposed to human umbilical vein endothelial cells (HUVECs), Cu, N-doped CDs only significantly induced cytotoxicity at very high concentrations (100 or 200 µg/ml), but their cytotoxicity appeared to be comparable with carbon black (CB) nanoparticles (NPs) at the same mass concentrations. As the mechanisms, 200 µg/ml Cu, N-doped CDs and CB NPs promoted endoplasmic reticulum (ER) stress proteins IRE1α and chop, leading to increased cleaved caspase 3/pro-caspase 3 ratio, but CB NPs were more effective. At noncytotoxic concentration (50 µg/ml), Cu, N-doped CDs successfully labeled HUVECs. In summary, we successfully prepared highly fluorescent and relatively biocompatible CDs to label HUVECs in vitro.

Relationship between energy intake, prognosis and related indicators in adults after cardiac, thoracic, and vascular surgery: A prospective observational study.

BACKGROUND AND OBJECTIVES: The study aimed to explore the association between energy intake (EI), the proportion of enteral nutrition intake (EN%), and prognostic-related indicators. METHODS AND STUDY DESIGN: This was a prospective observational study. Patients aged 18-80 years old, who had undergone cardiothoracic surgery, were enrolled between January 2017 and January 2018. The measured REE (mREE) was evaluated by indirect calorimetry (IC). The observational data on EI, EN% and EI/mREE% were collected following admission to ICU, ICU discharge, and prior to discharge. RESULTS: A total of 160 patients (60.6% male) were studied. The prealbumin and total protein were positively correlated with EN% at the time of ICU discharge; liver function index levels were negatively correlated with EI/mREE% at discharge (p<0.05). Multiple linear regression indicated that ALT levels as well as EI/mREE% were related to the duration of mechanical ventilation; ALT, AST, APACHE II were related to the ICU duration; EN% and EI/mREE% were related to the length of stay (LOS) following ICU discharge. EN% was related to the LOS in the hospital. CONCLUSIONS: The patients treated cardiothoracic surgery demonstrated associations of EN% with LOS in the hospital. Increased EN% and EI/mREE% were associated with higher serum protein levels and maintain normal liver function.

Cloning and characterization of the virulence factor Hop from Vibrio splendidus.

BACKGROUND: Vibrio splendidus is an aquaculture pathogen that can cause skin ulcer syndrome (SUS) in Apostichopus japonicus. HopPmaJ is a type III system effector (T3SE) that has been reported to be an important virulence factor. In this study, a gene named hop, which encodes HopPmaJ in V. splendidus was cloned and its cytotoxicity to coelomocytes and its effects on the expression of immune-related genes in A. japonicus were characterized. METHODS: Real time reverse transcription PCR (RT-PCR) was used to determine the expression of the hop gene under various conditions. To obtain the purified Hop, hop gene was conditionally expressed in Escherichia coli BL21(DE3) and was purified by GST tag. The cytotoxicity of Hop to coelomocyte was determined using MTT method, and the effect of Hop on the expression of immune-related genes was determined using real time RT-PCR. RESULTS: The deduced amino acid sequence of Hop from V. splendidus shared 84%-96% homology with those of Hops from other Vibrio spp. The expression of hop gene was induced not only by host-pathogen contact but also by high cell density. Purified recombinant Hop (rHop) showed cytotoxicity to the coelomocyte of A. japonicus. The cell viability decreased to approximately 42%, 26%, 32%, 30% and 20%, when 30, 50, 60, 80 and 100 µL of purified rHop was added, respectively. After being injected with rHop, the expression levels of immune-related genes that encode complement component (C1q) and caspase were significantly increased, and the production of reactive oxygen species were also increased in A. japonicus. CONCLUSION: Our results indicated that Hop not only contributed to the cytotoxicity to coelomocyte, but also caused immune response in A. japonicus.

Minimally invasive surgery for paravertebral or psoas abscess with spinal tuberculosis - a long-term retrospective study of 106 cases.

BACKGROUND: Minimally invasive surgery (MIS) is a common treatment option for paravertebral or psoas abscesses (PAs) in patients with spinal tuberculosis (ST). However, its efficacy remains controversial. The aim of the study was to evaluate the efficacy of MIS for PA with ST combined with anti-tuberculous chemotherapy. METHODS: A total of 106 consecutive patients who underwent MIS for ST with PA from January 2002 to Oct 2012 were reviewed. The MIS involved computed tomography (CT)-guided percutaneous catheter drainage and percutaneous catheter infusion chemotherapy. Clinical outcomes were evaluated based on the changes observed on preoperative and postoperative physical examination, inflammatory marker testing, and magnetic resonance imaging (MRI). RESULTS: The mean follow-up period was 7.21 ± 3.15 years. All surgeries were successfully completed under CT-guidance without intraoperative complications and all patients experienced immediate relief of their symptoms, which included fever and back pain. The preoperatively elevated erythrocyte sedimentation rate and C-reactive protein values returned to normal at a mean period of 3 months postoperatively. Solid bony union was observed in 106 patients and no abscesses were found on MRI examination. CONCLUSION: MIS carries advantages in terms of less invasiveness, precise drainage, and enhanced local drug concentration. While the technique has not been fully characterized and clinically prove, its use in addition to conservative chemotherapy and open debridement and instrumental fixation may be recommended for patients with ST and PA.

Indole contributes to tetracycline resistance via the outer membrane protein OmpN in Vibrio splendidus.

As an interspecies and interkingdom signaling molecule, indole has recently received attention for its diverse effects on the physiology of both bacteria and hosts. In this study, indole increased the tetracycline resistance of Vibrio splendidus. The minimal inhibitory concentration of tetracycline was 10 µg/mL, and the OD600 of V. splendidus decreased by 94.5% in the presence of 20 µg/mL tetracycline; however, the OD600 of V. splendidus with a mixture of 20 µg/mL tetracycline and 125 µM indole was 10- or 4.5-fold higher than that with only 20 µg/mL tetracycline at different time points. The percentage of cells resistant to 10 µg/mL tetracycline was 600-fold higher in the culture with an OD600 of approximately 2.0 (higher level of indole) than that in the culture with an OD600 of 0.5, which also meant that the level of indole was correlated to the tetracycline resistance of V. splendidus. Furthermore, one differentially expressed protein, which was identified as the outer membrane porin OmpN using SDS-PAGE combined with MALDI-TOF/TOF MS, was upregulated. Consequently, the expression of the ompN gene in the presence of either tetracycline or indole and simultaneously in the presence of indole and tetracycline was upregulated by 1.8-, 2.54-, and 6.01-fold, respectively, compared to the control samples. The combined results demonstrated that indole enhanced the tetracycline resistance of V. splendidus, and this resistance was probably due to upregulation of the outer membrane porin OmpN.

A sigma factor RpoD negatively regulates temperature-dependent metalloprotease expression in a pathogenic Vibrio splendidus.

Vibrio splendidus is an important aquatic pathogen that can cause typical symptoms of skin ulceration syndrome (SUS) in sea cucumber Apostichopus japonicus. A metalloprotease Vsm from V. splendidus, has been reported to be an important virulence factor for SUS outbreak. In the present study, the mRNA expression level of vsm was found to correlate to temperature with a peak expression at 28 °C. In contrast, the expression of a sigma factor rpoD, was significantly repressed at 28 °C. A predicted RpoD binding site in the promoter region of vsm revealed the potential regulation of RpoD on vsm expression. Electrophoretic mobility shift assay showed that the purified recombinant RpoD could specifically bind to the promoter region of vsm. Co-transfection of vsm promotor and pT3-rpoD into E. coli significantly inhibited the ß-Galactosidase activities in a temperature-dependent manner, and the activities were 0.41-, 0.89- and 0.18-fold at 10 °C, 28 °C and 37 °C compared to the control DH5α/pT3. A rpoD overexpression strain Vs/JRTcrpoD was constructed to further examine the effect of RpoD on the expression of vsm in vivo. By real time RT-PCR analysis, vsm expression level was 0.47-fold in Vs/JRTcrpoD compared to that in Vs/JRTc. Consistently, the metalloprotease activities in Vs/JRTcrpoD was decreased by 18% compared to that in Vs/JRTc. All the results suggested that the sigma factor RpoD, showed a negative regulation on expression of vsm gene by directly interacting with the promoter region of vsm.

Low vitamin D is associated with diabetes peripheral neuropathy in older but not in young and middle-aged patients.

BACKGROUND: The relationship between vitamin D and diabetes peripheral neuropathy (DPN) is not consistent among epidemiologic studies. Thus, we aimed to investigate this relationship in different age groups. METHODS: In this cross-sectional study, 1461 patients admitted to the Department of Endocrinology at Xinhua Hospital from June 2016 to September 2017 were divided into three age groups: a Youth group (24-44 years, n = 127), a Middle-age group (45-64 years, n = 779), and an Elderly group (≥65 years, n = 555). Basic information and laboratory results were collected from medical records. RESULTS: Among the patients, 32.72% had DPN, with 12.59% in the Youth group, 33.63% in the Middle-age group, and 36.04% in the Elderly group. For the total sample and the Elderly group, serum 25(OH)D concentrations in DPN patients were lower than in non-DPN patients (P < .05). The results of multivariate logistic regression indicated a low vitamin D concentration to be a risk factor for DPN in the Elderly group (P < .05), but such relationship was not found in the Youth or Middle-age groups. Moreover, according to ROC analysis, a serum 25(OH)D level < 34.87 nmol/L suggests the occurrence of DPN in elderly patients with type 2 diabetes (P < .001). CONCLUSIONS: This study is the first to report that a low vitamin D level is associated with DPN in diabetic patients over 65 years of age and might be used as a predictor of DPN in this population. The interaction between vitamin D and age in the development of DPN and its underlying mechanisms need to be further explored.

Major yolk protein and HSC70 are essential for the activation of the TLR pathway via interacting with MyD88 in Apostichopus japonicus.

The Toll cascade plays important functions in innate immunity against infectious pathogens in animals. Toll cascade as an ancient immune defender were conserved among different species. The activation of the TLR pathway between different species often involves different interacting proteins. The core members of this pathway have been well established in a wide range of organisms, including the marine invertebrate sea cucumber. However, these proteins do not function as single isolated entities but are engaged in a dynamic physical network with other proteins in the biomolecular context of a cell. To fill the knowledge gap in this context, two novel members of major yolk protein (MYP) and heat shock cognate protein 70 (HSC70) were identified as myeloid differentiation factor 88 (MyD88) interacting proteins by GST pull-down and mass spectrometry assays in Apostichopus japonicus. Their interactions were further confirmed by a co-immunoprecipitation analysis. Confocal microscopy analysis revealed that these three proteins were co-localized in the cytoplasm. A functional experiment indicated that each protein alone could suppress NF-κB translocated in the nucleus in cultured coelomocytes via a siRNA interference assay, suggesting that the three proteins functioned as a complex. To better address these interactions, we used the ZDOCK docking platform to mock the structure of the MyD88-HSC70-MYP complex. The death domain of MyD88 bound to HSC70 and MYP in separate spatial positions. The extent of interaction between MyD88 and HSC70 were K574, D591, E592 and E619 in HSC70 and E75, R76, K197 and R203 in MyD88. In the MYP-MyD88 model, K260, K452, K467 and E839 of MYP and D29, R40 and E62 of MyD88 were considered essential sites. Site-specific mutagenesis of these sites showed that most residues were key sites for their interaction with distinctly reduced binding constants relative to those of their native counterparts by biolayer interferometry assays, in which only K197 and R203 of MyD88 mutants displayed no effect on these interactions. Our results provide the first evidence of the roles of HSC70 and MYP in immune regulation via interacting with MyD88 and activating the TLR pathway in Apostichopus japonicus.

Macrophage migration inhibitory factor is involved in inflammation response in pathogen challenged Apostichopus japonicus.

Macrophage migration inhibitory factor (MIF) is a cytokine and plays critical roles in inflammatory and immune responses in vertebrates. However, its functional role in inflammation has not been well studied in invertebrates. In the present study, we cloned and characterized MIF gene from Apostichopus japonicus by RNA-seq and RACE approaches (designated as AjMIF). A 1047 bp fragment representing the full-length cDNA of AjMIF was obtained, including a 5' UTR of 100 bp, an open reading frame (ORF) of 366 bp encoding a polypeptide of 121 amino acids residues with the molecular weight of 13.43 kDa and theoretical isoelectric point of 5.63 and a 3' UTR of 580 bp. SMART analysis showed that AjMIF has conserved MIF domain (2-117aa) similar to its mammalian counterparts. The amino terminal proline residue (P2) and invariant lysine residue (K33) which are critical active sites of tautomerase activity in mammalian MIF were also detected. Phylogenic analysis and multiple alignments have shown that AjMIF shared higher degree of structural conservation and sequence identities with other counterparts from invertebrates and vertebrates. For Vibrio splendidus challenged sea cucumber, the peak expression of AjMIF mRNAs in coelomocytes were detected at 6 h (23.5-fold) and remained at high levels until 24 h (4.01-fold), and returned to normal level at 48 h in comparison with that of the control group. Similarly, a significant increase in the relative mRNA levels of AjMIF was also found in 10 µg mL-1 LPS-exposed primary cultured coelomocytes. Functional analysis indicated that recombinant AjMIF incubation could promote inflammatory response related genes of Ajp105, AjVEGF, AjMMP1 and AjHMGB3 expression by 1.35-fold, 1.36-fold, 1.83-fold and 1.27-fold increase, respectively, which was consistent with the findings in vertebrate MIFs. All these results collectively suggested that AjMIF had a similar function to MIFs in higher animals and might serve as a candidate cytokine in inflammatory regulation in sea cucumber.

Novel Ca2+-independent C-type lectin involved in immune defense of the razor clam Sinonovacula constricta.

C-type lectins (CTLs) are important pattern recognition molecules that participate in bacterial binding and agglutination by specific recognition of carbohydrates from pathogens. In this study, a full-length cDNA of CTL was cloned from Sinonovacula constricta (designated ScCTL-2). ScCTL-2 has a length of 981 bp, a 5'-untranslated region (UTR) of 47 bp, a short 3'-UTR of 37 bp, and an open reading frame (ORF) of 894 bp, which encodes a polypeptide of 298 amino acid residues. The deduced amino acid of ScCTL-2 possesses a conserved carbohydrate-recognition domain (CRD) similar to that of C31-E171. Spatial distribution analysis demonstrated that ScCTL-2 was constitutively expressed in all tested tissues, with dominant expression in foot and siphon and weak expression in hepatopancreas. The mRNA expression level of ScCTL-2 in gills and hepatopancreas was significantly upregulated at 6 and 12 h after challenge with the pathogen Vibrio parahaemolyticus. The recombinant ScCTL-2 showed specific binding and agglutinate capacities to all examined Gram-negative bacterial species, namely, Escherichia coli, Vibro anguillarum, and V. parahaemolyticus in a Ca2+-independent manner. However, these binding activities were not detected in Gram-positive Micrococcus luteus. Our results indicated that ScCTL-2 could be a novel pattern recognition receptor that can specifically recognize Gram-negative microorganisms in the innate immunity of S. constricta.

Gene identification and antimicrobial activity analysis of a novel lysozyme from razor clam Sinonovacula constricta.

Lysozymes are important immune effectors present in phylogenetically diverse organisms. They play vital roles in bacterial elimination during early immune responses. In the present study, a second invertebrate-type (i-type) lysozyme gene from razor clam Sinonovacula constricta (denoted as ScLYZ-2) was cloned by RACE and nested PCR methods. The full-length cDNA sequences of ScLYZ-2 were 1558 bp, including a 5' untranslated region (UTR) of 375 bp, an open reading frame of 426 bp, and a 3'-UTR of 757 bp with polyadenylation signal sequence (AATAAA) located upstream of the poly(A) tail. SMART analysis showed that ScLYZ-2 contains a signal peptide in the first 16 amino acid (AA) sequences and a destabilase domain located from 24 to 134 AA sequences. The deduced AA sequences of ScLYZ-2 were highly similar (42%-58%) to other known lysozyme genes of bivalve species. Multiple alignments of AA sequences showed that ScLYZ-2 possesses the classical i-type lysozyme family signature of two motifs ["MDVGSLSCGP(Y/F)QIK" and "CL(E/L/R/H)C(I/M)C"] and two catalytic residues (Glu35 and Asp46). Moreover, phylogenetic analysis showed that ScLYZ-2 is a new member of the i-type lysozyme family. In healthy razor clams, ScLYZ-2 was highly expressed in the hepatopancreas, followed by the gills, water pipes, and abdominal foot. Lysozyme activity and ScLYZ-2 expression levels were significantly upregulated in the hepatopancreas and gills after being infected with V. splendidus, V. harveyi, V. parahaemolyticus and S. aureus and M. luteus. Moreover, the recombinant ScLYZ-2 had strong antimicrobial activities against V. splendidus, V. harveyi, and V. parahaemolyticus. Furthermore, the minimal inhibitory concentration of the recombinant ScLYZ-2 against V. parahaemolyticus was 7.2 µmol/mL. Taken together, our results show that ScLYZ-2 plays an important role in the immune defense of razor clam by eliminating pathogenic microorganisms.

Cloning and characterization of the target protein subunit lst8 of rapamycin in Apostichopus japonicus.

Autophagy plays an important role in the immune defense systems of vertebrates through the interaction between the lethal with SEC13 protein 8 (lst8) and the mechanistic target of rapamycin. In the present study, a novel invertebrate lst8 homologue is identified from Apostichopus japonicus (designated as Ajlst8) via polymerase chain reaction. The full-length complementary DNA of Ajlst8 comprises a 5'-untranslated region (UTR) of 78 base pair (bp), a 3'-UTR of 479 bp, and a putative open reading frame of 951 bp; hence, 316 amino acids are encoded. Structural analysis shows that the deduced amino acid of Ajlst8 shares six typical WD40 domains (28 aa-248 aa). Spatial expression analysis indicates that Ajlst8 is ubiquitously expressed in all the examined tissues, with a larger magnitude in coelomocytes. Vibrio splendidus infection in vivo and lipopolysaccharide exposure in vitro can significantly upregulate the messenger RNA expression of Ajlst8 by 2.39-fold and 1.93-fold compared with the control group, respectively. LPS exposure could also significantly induced the protein level of Ajlst8 to 2.38-fold and the autophagy level was markedly increased by 3.08-fold under same condition. The RNA interference of Ajlst8 in primary coelomocytes also reduces the relative expression of autophagy with a 0.71-fold decrease in the ratio of LC3-II/LC3-I compared with that in the control group. These results indicate that Ajlst8 is a novel immune regulator that may be involved in the antibacterial response process of sea cucumber by regulating autophagy.