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The influences of constituent layers and interfaces on the mechanical and tribological behaviors of metal (Cr, Zr)/ceramic (CrN, ZrN) multilayer coatings were systematically investigated in the present study. Four sets of multilayer coatings (Cr/CrN, Cr/ZrN, Zr/CrN and Zr/ZrN) with the same number of interfaces were synthesized by multi-arc ion plating technique. The crystalline structures of the multilayer coatings were determined by X-ray diffraction and morphologies were observed by scanning electron microscope and transmission electron microscope. Furthermore, the nanoindenter, Vicker indenter, scratch tester and ball-on-disk tribometer were employed to evaluate the hardness, indentation toughness, adhesion strength and tribological properties of as-prepared coatings, respectively. Results revealed that the multilayer coatings with different constitute layers and interfaces presented different microstructures and properties. Based on the analysis of the cross-sectional indentation topographies, Zr interlayers exhibited higher crack resistances compared to Cr interlayers. Zr/CrN multilayer coating showed the highest hardness of 28 GPa, the strongest strength adhesion (>100 N) and the lowest wear rate. The excellent tribological performance of Zr/CrN multilayer coating was mainly ascribed to the high crack resistance of Zr layer, high wear-resistant layer of CrN as well as the local coherent interface between Zr and CrN sublayers.

Bacteria are the most abundant organisms in natural environment and dominant drivers of multiple geochemical functions. Drawing a global picture of microbial community structure and understanding their ecological status remain a grand challenge. As a typical artificial process, aquaculture provides a large amount of foods and creates great economic benefits for human beings. However, few studies are aimed at the microbial community in the aquaculture environment of aquatic plants. We analyzed microbial communities from 21 water samples in a coastal aquaculture area during the whole cultural process of Undaria pinnatifida by using high-throughout sequencing of 16S rRNA gene. The progression of U. pinnatifida aquaculture can be divided into three stages, named Seeding, Growth, and Maturity, respectively. Microbial community structures in water of the aquaculture area were significantly changed during the progression of U. pinnatifida aquaculture. The relative abundance of Flavobacteriia and Thaumarchaeota classes increased in Growth stage, and β-proteobacteria and Acidimirobiia classes decreased with the growth of U. pinnatifida. Meanwhile, environmental factors shaping the microbial community structures were uncovered during the U. pinnatifida aquaculture by using canonical correspondence analysis and Mantel test, in which temperature, dissolved oxygen, pH and nitrogen could be the major influencing factors. In addition, the microbial functions based on KEGG pathways were predicted from the microbial community compositions by PICRUSt. The comparison of predicted functions suggested that Environmental Information Processing and Genetic Information Processing were the functional categories with the most obvious shift in abundance among different stages of U. pinnatifida aquaculture. The findings of this study allowed us to better understand the microbial community in coastal aquaculture systems and the impact of seaweed cultivation on coastal ecosystems.

A new duplex PDMS/CrN coating was developed and the corrosion resistance of the system under seawater conditions was investigated. CrN coatings in four thicknesses were fabricated using a multi-arc ion plating system, and then the thin PDMS layers were covered on the coating surfaces by dip coating to fabricate the duplex PDMS/CrN coatings. Corrosion tests of the coatings were carried out using potentiodynamic polarization and electrochemical impedance spectroscopy in artificial seawater conditions. In a cross sectional image the PDMS layer was well adhered to the CrN layer. The corrosion current density for the duplex PDMS/CrN coating decreased by more than two orders of magnitude compared to the bare substrate, presenting the best corrosion protection efficiency. Furthermore, the impedance value was high to 107 Ω cm2, which improved by about three orders of magnitude compared to bare substrate. The excellent corrosion resistance of the duplex PDMS/CrN coating was attributed to the compact structure of the CrN coating combined with the good sealing barrier effect of the PDMS layer. Such duplex PDMS/CrN coating systems with superior corrosion resistance are considered as potential protective materials for mechanical assemblies for long-term service in marine applications.

In this paper, we are concerned with the Cauchy problem to the multi-dimensional (N?4$N\geq4$) chemotaxis model. We prove the optimal convergence rates of the strong solutions to the system for initial data close to a stable equilibrium state in critical Besov spaces. Our main ideas are based on the low-high frequency decomposition and the smooth effect of dissipative operator.

The complement system plays a crucial role in the innate immune system of animals. It can be activated by distinct yet overlapping classical, alternative and lectin pathways. In the alternative pathway, complement factor B (Bf) serves as the catalytic subunit of complement component 3 (C3) convertase, which plays the central role among three activation pathways. In this study, the Bf gene in sea cucumber (Apostichopus japonicus), termed AjBf, was obtained by rapid amplification of cDNA ends (RACE). The full-length cDNA of AjBf was 3231 bp in length barring the poly (A) tail. It contained an open reading frame (ORF) of 2742 bp encoding 913 amino acids, a 105 bp 5’-UTR (5’-terminal untranslated region) and a 384 bp 3’-UTR. AjBf was a mosaic protein with six CCP (complement control protein) domains, a VWA (von Willebrand factor A) domain, and a serine protease domain. The deduced molecular weight of AjBf protein was 101 kDa. Quantitative real time PCR (qRT-PCR) analysis indicated that the expression level of AjBf in A. japonicus was obviously higher at larval stage than that at embryonic stage. Expression detection in different tissues showed that AjBf expressed higher in coelomocytes than in other four tissues. In addation, AjBf expression in different tissues was induced significantly after LPS or PolyI:C challenge. These results indicated that AjBf plays an important role in immune responses to pathogen infection.

Cr/Cr 2 N nano-multilayer coatings with varied individual thickness of Cr 2 N layer were fabricated on 316L stainless steel substrates by multi-arc ion plating system. The evolutions of microstructures and mechanical properties were investigated by scanning electron microscopy, X-ray diffraction, nanoindentation and scratch testing. The corrosion behaviors and tribological properties in seawater conditions were evaluated using polarization resistance and reciprocating-sliding friction tests, respectively. Results showed that the multilayer coatings with thicker individual Cr 2 N layer presented higher hardness at the expense of toughness, and the coating with thickness ratio of Cr and Cr 2 N layers at 0.45 possessed both high hardness and toughness. The dominant feature influencing the corrosion resistance of deposited multilayer coating was the porosity and interface effect. While the excellent tribological performances of coatings with high load bearing capacity were attributed to the optimization of thickness ratio presenting high mechanical properties. Furthermore, the tribolayer formed during wear process, which small debris particles of chromium nitride embedded into the lubricative matrix and well adhered in the wear track, played a significant role in the anti-wear property. The superior anti-corrosion and anti-wear performances of nano-multilayer coatings made them good candidate protective materials in engineering applications in seawater environments.

Thyroid cysts remain a common clinical problem. Although simple aspiration, ethanol ablation, and radiofrequency ablation are effective, they have some limitations. There is therefore a need to identify a new and effective sclerosant for resolving these problems. The aim of this study was to test the efficacy as an active compound for sclerotherapy of thyroid cysts.Eight subjects whose thyroid cysts were recurrent despite repeated simple aspiration treatments were included in this study. The cysts were aspirated and then subjected to AHI. Lack of a significant reduction in cyst size (i.e.50%) at follow-up resulted in further AHIs. Treatment success was defined as complete disappearance or a marked (i.e.50%) reduction in the size of the cystic portion of the thyroid nodule on follow-up ultrasonography at least 6 months after the final AHI.A marked size reduction of50% was ultimately found in all of the patients (100%), with a reduction varying from 73.68% to 99.07% (P0.029). Six of the patients received a single AHI treatment, one received two AHIs, and one received three AHIs. None of the patients suffered from a recurrence during the follow-up period. Three patients experienced tolerable pain and local tenderness. No serious side effects were reported.Intracystic AHI may be a safe and effective treatment for benign thyroid cysts.

The potential epitope of a recombinant food allergen protein, cashew Ana o 1, reactive to monoclonal antibody, mAb 2G4, has been mapped by solution-phase amide backbone H/D exchange (HDX) monitored by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Purified mAb 2G4 was incubated with recombinant Ana o 1 (rAna o 1) to form antigen:monoclonal antibody (Ag:mAb) complexes. Complexed and uncomplexed (free) rAna o 1 were then subjected to HDX-MS analysis. Five regions protected from H/D exchange upon mAb binding are identified as potential conformational epitope-contributing segments.

RATIONALE Paired Lys-N and Lys-C proteases produce peptides of identical mass and similar retention time, but different tandem mass spectra. Data from these parallel experiments provide constraints that are applied before data analysis. With this approach, we can find matched spectra before analysis, distinguish ion type, and determine residue level confidence. METHODS Aliquots are digested separately by Lys-N and Lys-C peptidases, and analyzed by reversed-phase nano-flow liquid chromatography, collision-induced dissociation, and 14.5 T Fourier transform ion cyclotron resonance mass spectrometry. Matched pairs of fragmentation spectra with equal precursor mass and similar retention times from each digestion are compared, leveraging single-residue transposed information with independent interferences to confidently identify fragment ion type, residues, and peptides. The paired spectra are solved together as a single de novo sequencing problem. RESULTS Two pairs of spectra of a de novo sequenced 18-mer are presented. In one example, the 18-mer has coverage of all residues except the N- and C- terminal lysines and their adjacent residues. The confidence level is high due to six pairs of transposed ions. In the other example, the coverage is incomplete. Nonetheless, nine pairs of transposed ions facilitate identification of two trimer sequence tags with high confidence, one with medium confidence, and additional sequence information with residue-by-residue confidence, thus demonstrating the value of residue-by-residue confidence. CONCLUSIONS Sequence identity and variability, such as post-translational modifications (PTMs), are essential to understanding biological function and disease. The present method facilitates discovery of new peptides with multiple levels of confidence, promises potential characterization of PTMs, and validates peptides from databases. Independent validation may be of interest for a number of applications. Copyright © 2015 John Wiley & Sons, Ltd.

Early steps of gene expression are a composite of promoter recognition, promoter activation, RNA synthesis and RNA processing, and it is known that SUMOylation, a post-translational modification, is involved in transcription regulation. We previously found that SUMO-1 marks chromatin at the proximal promoter regions of some of the most active housekeeping genes during interphase in human cells, but the SUMOylated targets on the chromatin remained unclear. In this study, we found that SUMO-1 marks the promoters of ribosomal protein genes via modification of the Scaffold Associated Factor B (SAFB) protein, and the SUMOylated SAFB stimulated both the binding of RNA polymerase to promoters and pre-mRNA splicing. Depletion of SAFB decreased RNA polymerase II binding to promoters and nuclear processing of the mRNA, though mRNA stability was not affected. This study reveals an unexpected role of SUMO-1 and SAFB in the stimulatory coupling of promoter binding, transcription initiation and RNA processing.

In this paper, we consider the regularity of weak solutions to the 3D incompressible generalized Navier-Stokes (GNS) equations with fractional dissipative operator (−∆)αu in the Triebel-Lizorkin spaces. Let u be a weak solution in R3×(0, T ), w = curlu, and w = (w1, w2, 0). Employing the Littlewood-Paley decomposition, we prove that u becomes a classical solution if w ∈ Lq(0, T ; Ḟ 0 r,σ) with 2α q + 3 r = 2α, 3 2α < r ≤ ∞, and σ ≤ 2αr 3 .

The immune-related enzymes in marine animals are very sensitive to divalent metal ions. To investigate the roles divalent metal ions play in the influence on the immunity of sea cucumber Apostichopus japonicus, one of the most important commercial species in Asian countries, the effects of eight divalent metal ions at concentrations of 2.5, 5, 10, 15, 20, 25 and 30 mmol L−1 on the activities of superoxide dismutase (SOD), phenoloxidase (PO), acid phosphatase (ACP), alkaline phosphatase (AKP) and myeloperoxidase (MPO) in coelomic fluid were determined with the nitro blue tetrazolium chloride (NBT) method, dopachrome formation method, p-nitrophenyl phosphate (pNPP) method and 3,3′,5,5′-tetramethyl benzidine (TMB) method. The results indicated that Mg2+ enhanced the activities of SOD, PO, ACP and AKP significantly and showed no obvious effect on MPO activity; Zn2+ increased the activities of SOD, ACP and AKP, and showed no obvious effect on the activities of PO and MPO; Cu2+ enhanced the activities of ACP, AKP and MPO and activated SOD and PO at a certain concentration range; Ca2+ and Mn2+ inhibited the activities of ACP and AKP; Fe2+ had strong inhibitory effect on SOD activity; Pb2+ showed inhibitions on the activities of SOD, PO, ACP and AKP; and Cd2+ inhibited MPO activity greatly. The data obtained in this study collectively suggest that Mg2+, Zn2+ and Cu2+ have potential in promotion of A. japonicus immunity, while Ca2+, Fe2+, Pb2+, Cd2+ and Mn2+ might be limiting factors to the immune response of A. 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.

The Linnan Sag is one of the main oil-producing units in the Huimin Depression, Eastern China, and the pore pressure gradients obtained from drill stem tests (DSTs) range from 9.0 to 16.0 MPa/km. Uncertainty about the origin and distribution of abnormally high pressures in the Linnan Sag has led to different interpretations of hydrocarbon accumulation and resource assessments, and it interferes with safe drilling. In the Linnan Sag, mudstone compaction curves are substantially affected by several non-compaction factors, and the normal trend of the compaction curve is difficult to determine. The determination of the origin and distribution of overpressure in the Linnan Sag is a challenge. In this study, the factors that may affect mudstone compaction&mdash, such as the shale volume, higher calcareous, and organic matter content&mdash, were carefully examined and processed. The pressures in the mudstones were estimated by the corrected mudstone compaction curves, which were compiled from acoustic, density, and neutron logs, and calibrated using DST and mud weight data. The log response&ndash, vertical effective stress and acoustic velocity-density crossplots were used to identify the mechanisms that generate overpressure. The comprehensive compaction curve shows that the mudstones in the overpressured layer exhibit clear disequilibrium compaction characteristics. The logging response crossplots demonstrate that those overpressured points were consistent with the loading curve. The findings suggest that, the fundamental mechanism resulting in overpressures is the disequilibrium compaction of thick Paleocene mudstones. Hydrocarbon generation and vertical transfer of overpressure may be the main unloading mechanisms, which corresponds to the overpressure points that deviate from the loading curves. Since organic matter cracking may occur in formations at depths greater than 4000 m (Ro &gt, 1.0%), the contribution of hydrocarbon generation to overpressuring is expected to be limited. The transfer of overpressure through opening faults is therefore considered to be the main cause of higher overpressure in local sandstones. The overpressures in the mudstones are characterized by a gradual decrease from the center to the margin in the Linnan Sag. The pressure in the isolated sand bodies are generally similar to that in the surrounding mudstones, whereas it can be lower or higher when the overpressure in the sand bodies are vertically transferred by faults to other pressure systems. The results of this analysis provide an indication of the magnitude, mechanism, and distribution of overpressure in the Linnan Sag. This insight can be used to guide further exploration of the Linnan Sag and similar geological basins.

Agricultural drainage plays an effective role in preventing waterlogging and salinity disasters and also is the main transport pathway for agricultural non-point source pollutants into rivers and lakes. Hence, the water quality of agricultural drainage should be a point of focus. In this paper, nitrogen and phosphorus loss under improved subsurface drainage with different filter materials (gravel, layered sand-gravel, mixed sand-gravel, straw) were studied by a three-year field experiment (2016&ndash, 2018) compared with the conventional subsurface drainage. The pH values, total nitrogen, ammonia nitrogen, nitrate nitrogen, total phosphorus and soluble reactive phosphate were considered. The results showed that the nitrogen and phosphorus concentrations of drain outflow under improved subsurface drainage with gravel filter were larger than that with layered sand-gravel filter and mixed sand-gravel filter. The improved subsurface drainages with layered sand-gravel filter and mixed sand-gravel filter had an effect on reducing the ammonia nitrogen, total phosphorus and soluble reactive phosphate concentrations of the outflow. Meanwhile, the characteristics of nitrogen and phosphorus loss under the improved subsurface drainage with straw filter were different from that with layered sand-gravel filter and mixed sand-gravel filter. For the improved subsurface drainage with layered sand-gravel filter outflow, the ammonia nitrogen, total phosphorus, and soluble reactive phosphate concentrations were about 13%&ndash, 78%, 38%&ndash, 63%, 40%&ndash, 68% less, and total nitrogen, nitrate nitrogen concentrations were 24%&ndash, 80%,18%&ndash, 96% more than that under conventional subsurface drainage. Meanwhile, for the improved subsurface drainage with straw filter outflow, compared with conventional subsurface drainage outflow, the percentage changes of the total nitrogen, nitrate nitrogen, ammonia nitrogen, total phosphorus and the soluble reactive phosphate concentrations were about &minus, 76%&ndash, 62%, &minus, 77%&ndash, 78%, &minus, 152%&ndash, &minus, 274%, &minus, 103%&ndash, 400% and &minus, 221%&ndash, 291%, respectively. Additionally, in the outflow of all subsurface drainage patterns, there were much higher total nitrogen and nitrate nitrogen concentrations which should be focused on and the agricultural water management should be adopted.

This paper describes an algorithm to assist in relative quantitation of peptide post-translational modifications using stable isotope labeling by amino acids in cell culture (SILAC). The described algorithm first determines the normalization factor and then calculates SILAC ratios for a list of target peptide masses using precursor ion abundances. Four yeast histone mutants were used to demonstrate the effectiveness of this approach for quantitation of peptide post-translational modifications changes. The details of the algorithm's approach for normalization and peptide ratio calculation are described. The examples demonstrate the robustness of the approach as well as its utility to rapidly determine changes in peptide post-translational modifications within a protein.

The best-characterized acetylation of newly synthesized histone H4 is the diacetylation of the NH2-terminal tail on lysines 5 and 12. Despite its evolutionary conservation, this pattern of modification has not been shown to be essential for either viability or chromatin assembly in any model organism. We demonstrate that mutations in histone H4 lysines 5 and 12 in yeast confer hypersensitivity to replication stress and DNA-damaging agents when combined with mutations in histone H4 lysine 91, which has also been found to be a site of acetylation on soluble histone H4. In addition, these mutations confer a dramatic decrease in cell viability when combined with mutations in histone H3 lysine 56. We also show that mutation of the sites of acetylation on newly synthesized histone H4 results in defects in the reassembly of chromatin structure that accompanies the repair of HO-mediated double-strand breaks. This defect is not due to a decrease in the level of histone H3 lysine 56 acetylation. Intriguingly, mutations that alter the sites of newly synthesized histone H4 acetylation display a marked decrease in levels of phosphorylated H2A (γ-H2AX) in chromatin surrounding the double-strand break. These results indicate that the sites of acetylation on newly synthesized histones H3 and H4 can function in nonoverlapping ways that are required for chromatin assembly, viability, and DNA damage response signaling.

We have addressed the friction and wear behaviors of multilayer Cr/CrN/GLC (graphite-like carbon) coatings sliding against different rubbers in water environment. Stribeck curves and start–stop tests were presented to analyze the water-lubrication mechanism and tribological properties of coating/rubber tribopairs. Results exhibited that applied loads had a minor influence on the tribological performances of coating/rubber tribopairs compared to sliding speeds. The friction coefficients fluctuated during the test process, including in the start–stop process, which related to the viscoelasticity of rubbers. Furthermore, the different tribological performances of tribopairs were attributed to the hardness, surface wettability and tensile strength as well as viscoelasticity.

Soil salinization has random characteristics because of the influences of natural and anthropogenic factors. Therefore, a study on the dynamic prediction model of soil salinity is important for irrigation water management in salinization irrigation districts. In the present paper, the theory of supporting vector machine was introduced in the dynamic prediction of soil electrical conductivity (EC) values. Based on groundwater depth, irrigation water volume, and evaporation data from 1991 to 2010 in the Shahaoqu subdistrict of the Hetao irrigation district in Inner Mongolia, a dynamic prediction model of soil EC value was developed. The results show that the fitted values, tested values and the predicted values of the model have little difference from the real values. The absolute value of the fitting mean relative error is 2.14%, that of the testing mean relative error is 3.48%, and that of the predicting mean relative error is 6.37%. Compared with the Artificial Neural Network (ANN) model, the results show that SVM predictors perform better in forecasting soil EC values than the ANN model. The feasibility of the application of SVMs to soil salinity forecasting was demonstrated and their performance in soil salinity data analysis was proven.

Vaccinia VH1-related (VHR) is a dual specificity phosphatase that consists of only a single catalytic domain. Although several protein substrates have been identified for VHR, the elements that control the in vivo substrate specificity of this enzyme remain unclear. In this work, the in vitro substrate specificity of VHR was systematically profiled by screening combinatorial peptide libraries. VHR exhibits more stringent substrate specificity than classical protein-tyrosine phosphatases and recognizes two distinct classes of Tyr(P) peptides. The class I substrates are similar to the Tyr(P) motifs derived from the VHR protein substrates, having sequences of (D/E/φ)(D/S/N/T/E)(P/I/M/S/A/V)pY(G/A/S/Q) or (D/E/φ)(T/S)(D/E)pY(G/A/S/Q) (where φ is a hydrophobic amino acid and pY is phosphotyrosine). The class II substrates have the consensus sequence of (V/A)P(I/L/M/V/F)X1–6pY (where X is any amino acid) with V/A preferably at the N terminus of the peptide. Site-directed mutagenesis and molecular modeling studies suggest that the class II peptides bind to VHR in an opposite orientation relative to the canonical binding mode of the class I substrates. In this alternative binding mode, the Tyr(P) side chain binds to the active site pocket, but the N terminus of the peptide interacts with the carboxylate side chain of Asp164, which normally interacts with the Tyr(P) + 3 residue of a class I substrate. Proteins containing the class II motifs are efficient VHR substrates in vitro, suggesting that VHR may act on a novel class of yet unidentified Tyr(P) proteins in vivo. Background: Factors that determine the in vivo substrate specificity of dual specificity phosphatases are currently unknown. Results: Specificity profiling of VHR through peptide library screening identified two distinct classes of peptide substrates, which bind to VHR in opposite orientations. Conclusion: VHR may act on a previously unrecognized class of protein substrates. Significance: The results should help identify new VHR substrates and elucidate its biological function.

A gene encoding hsc70 was cloned from the sea cucumber Apostichopus japonicus and named AjHsc70. The full-length cDNA sequence was 2,508 bp, containing a 5'-UTR of 77 bp, an ORF of 2,010 bp encoding 670 amino acids, and a 3'-UTR of 421 bp. Quantitative RT-PCR analysis revealed that AjHsc70 was expressed constitutively in all of the tested tissues with respiratory tree tissue showing the highest expression level. AjHsc70 expression was significantly induced by lipopolysaccharide, and the expression levels peaked at different sampling times in the body wall (24 h), coelomocytes (12 h), and intestine and respiratory tree tissues (6 h). After heat stress, AjHsc70 expression in intestine, coelomocytes, and body wall decreased acutely at first and then increased slightly. AjHsc70 expression patterns indicated that hsc70 plays an important role in mediating the responses of A. japonicus to bacterial challenge and heat stress.

Toll-like receptors (TLRs) are a family of type I integral membrane glycoproteins which play pivotal roles in innate immunity. In this study, two TLRs named AjTLR3 and AjToll were cloned from sea cucumber (Apostichopus japonicus). The full-length cDNA sequences of AjTLR3 and AjToll are 3484 bp and 4211 bp, with an open reading frame (ORF) of 2679 bp and 2853 bp, encoding 892 and 950 amino acids, respectively. Both AjTLR3 and AjToll are composed of a leucine-rich repeat (LRR) domain, a transmembrane (TM) domain and an intracellular Toll/interleukin-1 receptor (TIR) domain. Evolution analysis revealed that AjTLR3 and AjToll were clustered with the vertebrate-like TLRs (V-TLRs) and the protostome-like TLRs (P-TLRs), respectively. These two genes were widely expressed in all five tested tissues (body wall, coelomocytes, tube feet, intestine and respiratory tree), but showed different expression patterns. The significantly up-regulated expressions of AjTLR3 and AjToll after peptidoglycan (PGN), lipopolysaccharides (LPS), Zymosan A and polyinosinic–polycytidylic acid (PolyI:C) challenges suggested that they were functionally involved in the immune responses to the Cram-positive bacteria, Gram-negative bacteria, fungi and double-stranded RNA (dsRNA) viruses, respectively.

Objective Effects of melatonin on the proliferation and differentiation of human dental pulp cells (hDPCs) remain unclear. The purpose of this study was to investigate the effect of melatonin on the proliferation and differentiation of the hDPCs. Design Primary hDPCs were obtained from the third molar of volunteer aged from 18 to 25. CCK8 assay evaluated the effect of melatonin upon cell proliferation at day 1, 2, 3, 4, 5. After 7 days’ osteogenic induction with melatonin or vehicle, alkaline phosphatase (ALP) activity was measured with a commercial kit. Then levels of dentin sialophosphoprotein (DSPP) were determined by immunocytochemical staining and western blot analysis, followed by quantitative real-time reverse transcription-Polymerase chain reaction (qRT-PCR) to analyse mRNA levels of ALP and DSPP. Finally hDPCs exposed to osteogenic medium containing melatonin or vehicle for 14 days were stained with alizarin red to detect mineralization nodules formation. Results Melatonin significantly inhibited the proliferative ability of the hDPCs in a concentration- and time-dependent manner. The hDPCs cultured in osteogenic induction medium with melatonin presented an increase of ALP activity, expression of DSPP, mRNA levels of ALP and DSPP, and mineralization nodules formation. Conclusions These findings indicate that melatonin at physiological concentrations can inhibit proliferation and promote the differentiation of hDPCs, which might give some new insights into the mechanism of regulating DPCs to achieve dentine regeneration.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2810-3) contains supplementary material, which is available to authorized users.

The plant hormone auxin plays a vital role in regulating many aspects of plant growth and development. Small auxin up-regulated RNAs (SAURs) are primary auxin response genes hypothesized to be involved in auxin signaling pathway, but their functions remain unclear. Here, a genome-wide search for SAUR gene homologues in Solanaceae species identified 99 and 134 members of SAUR gene family from tomato and potato, respectively. Phylogenetic analysis indicated that the SAUR proteins from Arabidopsis, rice, sorghum, tomato and potato were divided into four major groups with 16 subgroups. Among them, 25 histidine-rich SAURs genes with metal-binding characteristics were found in Arabidopsis, sorghum and Solanaceae species, but not in rice. Using tomato as a model, a comprehensive overview of SAUR gene family is presented, including the gene structures, phylogeny and chromosome locations. Quantitative real-time PCR analysis indicated that 11 randomly selected SlSAUR genes in tomato could be expressed at least in one of the tomato organs/tissues tested. However, different SlSAUR genes displayed distinctive expression levels. SlSAUR16 and SlSAUR71 exhibited highly tissue-specific expression patterns. Almost all of the detected SlSAURs showed an accumulating pattern of mRNA along tomato flower and fruit development. Some of them displayed differential response to exogenous IAA treatment. The abiotic (cold, salt and drought) stresses significantly modified transcript levels of SlSAURs genes. Most of them were down-regulated in response to abiotic stresses (drought, heat and salinity), but SlSAUR58, as a histidine-rich SAUR gene, was up-regulated after salt treatment, indicating that it may play a specific role in the salt signaling transduction pathway. Our comparative analysis provides some basic genomic information for the SAUR genes in the Solanaceae species and will pave the way for deciphering their function during plant development.

Hymeniacidon sinapium (De Laubenfels, 1930) (Demospongiae, Halichondrida, Halichondriidae) collected from China's Yellow Sea has been a model sponge species for extensive studies of in vitro cultures of sponge cells and explants in our group since 2002. To better understand its reproductive cycle, the aim of this study is to elucidate the temporal changes in microbial community, the spiculogenesis activity (in terms of silicatein gene expression) and the stress response (in terms of hsp70 gene expression) during its annual life cycle. Histological studies by light microscopy revealed that H. sinapium is a characteristic seasonal species with four distinctive developmental stages of resuscitation, bloom, decline and dormancy during its annual life cycle. The sexual reproductive processes were observed from August to October (summer), with all the reproductive elements (oocytes, sperm cysts, embryos and larvae) documented. There is no direct correlation between seawater temperature and gametogenesis. The gene expression of silicatein and hsp70 was investigated by quantitative real-time RT-PCR during its annual life cycle. Silicatein expression correlated with the histogenesis and reached the highest value in late July (bloom stage). While hsp70 expression correlated with the seawater temperature and also reached the highest value at the same time. We hypothesize that after reaching the maximum biomass growth, sponge should adjust energy distribution to meet the need of gametogenesis and embryogenesis in the reproductive season. Bacterial community variability in a life cycle ranged from 18% to 98% (dice similarity of each two samples) using DGGE, and formed four distinctive clusters consistent with seasons. After sequencing, twelve typical bands from DGGE were compared to GenBank and basically clustered into five phyla: Actinobacteria, alpha-Proteobacteria, gamma-Proteobacteria, Bacteroidetes and Cyanobacteria. This study reported, for the first time, high temporal variability in silicatein, hsp70 and bacterial community during the annual life cycle of H. sinapium. This knowledge is significant for the development of sponge explants culture under controlled laboratory conditions and large-scale sponge seed-rearing technology. Crown Copyright (C) 2012 Published by Elsevier B.V. All rights reserved.

p12(CDK2-AP1) is a growth suppressor that negatively regulates cyclin-dependent kinase 2 (CDK2) activities and shows to interfere in DNA replication. Here, we aim to elucidate the role of p12(CDK2-AP1) in breast cancer progression.Expression of p12(CDK2-AP1) protein was examined in 60 pairs of breast cancer specimens and adjacent non-tumor tissues using immunohistochemistry assay. Loss-of-function and gain-of-function analysis was performed on MCF-7 and MDA-MB-231 breast cancer cells. Routine assays including MTT, colony formation, flow cytometry, and tumorigenesis in nude mice were performed and cell cycle regulators were analyzed.p12(CDK2-AP1) was found to be significantly downregulated in 60 breast cancer tissues compared to corresponding non-tumorous tissues. The proliferation and colony formation ability was inhibited in cells that transduced with p12(CDK2-AP1) over-expression lentivirus, but enhanced in cells that transduced with p12(CDK2-AP1) RNAi lentivirus. p12(CDK2-AP1) over-expression led to G0/G1 phase arrest in the cell cycle and caused expression changes of cell cycle-related genes (CDK2, CDK4, p16(Ink4A), p21(Cip1/Waf1)). Furthermore, p12(CDK2-AP1) over-expression inhibited in vivo tumor growth in immunodeficiency mice, supporting an inhibitory role for p12(CDK2-AP1) in breast cancer development.As a cell cycle regulator, p12(CDK2-AP1) is involved in the development of breast cancer and maybe a potential therapeutic candidate to suppress tumorigenicity in breast cancer.

Graphite-like carbon (GLC) coatings are being increasingly used in mechanical seals, especially under the water-lubricated condition, to improve component durability by providing a low friction coefficient and high wear resistance. We have addressed the friction and wear performances of multilayer Cr/CrN/GLC coatings sliding against polyether–ether–ketone (PEEK), polyimide (PI), and polytetrafluoroethylene (PTFE) in distilled water under various applied loads and sliding speeds using a ring-on-block test rig. Stribeck curves were developed to analyze the water lubrication mechanisms. Start–stop tests were also carried out to evaluate the tribological performances of the coatings. Our results showed that the friction coefficients and wear rates of the polymers decreased with increasing sliding speeds. Compared to the applied load, the sliding speed had a major effect on the friction and wear performance of the polymer. Three coating/polymer tribopairs demonstrated different tribological behaviors, which were attributed to their different molecular structures, the different mechanical properties of the polymers, and the different lubrication mechanisms. The transfer film determined the lubrication mechanism of the three tribopairs and also resulted in the low friction coefficient and wear rate. The initial and steady-state friction coefficient also decreased with increases in the repeated start–stop times. Based on our results, we concluded that the coating/PEEK tribopair presented an excellent tribological performance that we ascribed to the chemical properties of PEEK and the hydrodynamic lubrication regime of the coating/PEEK tribopair at high sliding speeds.

The cratonic region of the Tarim Basin contains two main Lower Paleozoic source rocks–the Middle-Upper Ordovician and Lower-Middle Cambrian. However, few research focus on the relation between the evolution of hydrocarbon kitchens and its role in controlling oil and gas. Based on previous research results, this paper applies geochemical parameters of rock pyrolysis, hydrocarbon expulsion threshold theory and hydrocarbon-generated potential methods to identify the effectiveness of source rocks from the Lower Paleozoic while quantitatively calculating the hydrocarbon expulsion intensities and amounts for the two sets of source rocks. The above result is used to analysis the distribution of the effective hydrocarbon kitchen in each primary history stage and the controls on oil and gas. This study shows multiple phases of oil and gas generation and expulsion from the Cambrian-Ordovician source rocks with four oil-generating peaks with large-scale oil migration and accumulation. According to the changes in hydrocarbon expulsion intensity within different stages, we can elucidate the evolution of the source kitchens, because the hydrocarbon kitchens from the two sets of source rocks migrated in time and space, and there are multiple hydrocarbon expulsion centers. Based on the concept of the oil/gas threshold distribution model, the relation between the quantitative characterization of the hydrocarbon kitchen controlling function, and quantitative prediction of reservoir formation probability is studied, and the result shows that oil and gas reservoirs are located in more than 50% probability of controlling area. However, different periods and different layers of stacked hydrocarbon kitchen is complex and causing the main mixing of oil and gas. All these hydrocarbon expulsion centers had control over the distribution of oil and gas in the basin. From a macro and quantitative point of view, this discovery will provide certain guide for oil and gas exploration.

We consider the 3D fluid system with the linear Soret effect. We obtain a logarithmically improved regularity criterion in the BMO space.

A duplex treatment involving nitrogen ion pre-implantation and gradient interfacial transition was performed to obtain a high-performance graphite-like carbon (GLC) coating on a Ti6Al4V alloy. Characteristics of the as-deposited coating systems were systemically investigated by Raman spectrometry, scanning electron microscopy, atomic force microscopy, nano-indentation, and scratch tests. The friction and wear behaviors in distilled water and sea water environments were evaluated by a ball-on-disk tribometer. The results showed that the GLC multilayer coating on nitrogen ion-implanted Ti6Al4V possessed a greater hardness and adhesion strength than to that on un-implanted Ti6Al4V. The tribological performances of these duplex process systems showed a great improvement in both the distilled water and sea water environments. In particular, the Cr/CrN/GLC coatings on nitrogen ion-implanted substrates demonstrated the best friction and wear behaviors. These striking improvements were attributed to the greatly enhanced interface strength between substrate and coating by the nitrogen ion implantation process and improved adhesion strength between gradient layers by the appropriate gradient interlayers with a similar thermal expansion coefficient.

The regeneration potential of mesenchymal stem cells (MSCs) diminishes with advanced age and this diminished potential is associated with changes in cellular functions. This study compared MSCs isolated from the bone marrow of rhesus monkeys (rBMSCs) in three age groups: young ( 12 years). The effects of aging on stem cell properties and indicators of stem cell fitness such as proliferation, differentiation, circadian rhythms, stress response proteins, miRNA expression, and global histone modifications in rBMSCs were analyzed. rBMSCs demonstrated decreased capacities for proliferation and differentiation as a function of age. The production of heat shock protein 70 (HSP70) and heat shock factor 1 (HSF1) were also reduced with increasing age. The level of a core circadian protein, Rev-erb α, was significantly increased in rBMSCs from old animals. Furthermore, analysis of miRNA expression profiles revealed an up-regulation of mir-766 and mir-558 and a down-regulation of mir-let-7f, mir-125b, mir-222, mir-199-3p, mir-23a, and mir-221 in old rBMSCs compare to young rBMSCs. However, there were no significant age-related changes in the global histone modification profiles of the four histone core proteins: H2A, H2B, H3, and H4 on rBMSCs. These changes represent novel insights into the aging process and could have implications regarding the potential for autologous stem cells therapy in older patients.

The echinoderm immunity system has been extensively investigated in adults in several classes such as echinoid and holothuroidea. However, the defense mechanism in embryos and larvae remains largely unexplored. To profile the immune-related genes expression in embryos and larvae and to monitor the stimulation of the innate immune response by lipopolysaccharides (LPS) challenge, we investigated the expression patterns of nine immune-related genes in embryos and larvae of sea cucumber (Apostichopus japonicus) at eleven developmental stages using quantitative real-time PCR (qRT-PCR). The expression of six encoding proteins including heat shock protein70 (Hsp70), Hsp90, Hsp gp96, thymosin-beta, ferritin and DD104 protein was detected at all eleven development stages according to mRNA expression data. However, the expression of mannan-binding C-type lectin (MBCL) was detected at early auricularia to juvenile stages, while lysozyme and serine proteinase inhibitor (SPI) were detected only at juvenile stage. Out of these nine genes, three (MBCL, lysozyme and SPI) were found to be up-regulated in mRNA expression upon LPS challenge, whereas the other six showed no significant change. Our study presents a first preliminary view into the expression patterns of immune-related genes at different developmental stages of sea cucumber, which increases the available information on echinoderm immunity.

To develop effective alternatives for detecting genetically modified organisms (GMOs), we reported one optimized visual loop-mediated isothermal amplification (LAMP) method for the detection of exogenous DNA targets from two GM soybean events in this study. This isothermal amplification can be performed within 40 min without polymerase chain reaction (PCR) equipment and the derived LAMP products can be directly observed by naked eye employing SybrGreen I dye instead of conventional gel electrophoresis analysis. The limits of detection of these established visual LAMP assays were about 4 copies of haploid soybean genomic DNA, and which were much higher than those of reported conventional PCR assays. Furthermore, the high specificity of LAMP assays was determined. All the results demonstrated that the developed visual LAMP assays are convenient, cost-efficient, and rapid for on spot detection of GMOs.

Sea cucumber ( Apostichopus japonicus ) is one of the most important aquaculture species in China. To profile gene expression in different tissues and to identify immune-related genes, we constructed three independent cDNA libraries that were derived from intestine (Int), body wall (BW) and respiratory tree (RT) of healthy individuals. From these libraries, a total of 5728 high-quality expressed sequence tags (ESTs) of longer than 100 bp were generated. Clustering and assembly of the ESTs identified 495 contigs and 2118 singletons, representing 2613 unigenes. Basic Local Alignment Search Tool (BLAST) analysis showed that 902 unigenes (34.52%) had significant homology ( E value ≤10 − 5 ) to genes with known or putative functions in GenBank. These unigenes were grouped into 18 functional categories. When compared among these three libraries, RT library showed fewer categories (9) while the other two libraries displayed more (17 and 16 for Int and BW libraries respectively). Considering the components among the three cDNA libraries, 655, 259 and 484 tissue-specific unigenes were observed in Int, BW and RT libraries respectively; however, only 95 unigenes were common to all three libraries. The most abundant contigs in the three libraries were identified as genes related to cell and organism defense (551 ESTs), metabolism (415 ESTs), translation/ribosomal structure and biogenesis (342 ESTs), gene/protein expression (178 ESTs), structure and motility (75 ESTs), energy production and conversion (51 ESTs), and cytoskeleton (39 ESTs). The list of 636 ESTs that matched with immune-related genes were categorized as pattern recognition receptors and signaling, complement components, transferrin superfamily members, cytokines and growth factors, and effector genes. The present manuscript presents a fair amount of data regarding the spatial expression pattern of many genes (BW vs. Int vs. RT of A. japonicus ).

Genetically modified (GM) papaya (Carica papaya L.), Huanong No. 1, was approved for commercialization in Guangdong province, China in 2006, and the development of the Huanong No. 1 papaya detection method is necessary for implementing genetically modified organism (GMO) labeling regulations. In this study, we reported the characterization of the exogenous integration of GM Huanong No. 1 papaya by means of conventional polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR strategies. The results suggested that one intact copy of the initial construction was integrated in the papaya genome and which probably resulted in one deletion (38 bp in size) of the host genomic DNA. Also, one unintended insertion of a 92 bp truncated NptII fragment was observed at the 5' end of the exogenous insert. Furthermore, we revealed its 5' and 3' flanking sequences between the insert DNA and the papaya genomic DNA, and developed the event-specific qualitative and quantitative PCR assays for GM Huanong No. 1 papaya based on the 5' integration flanking sequence. The relative limit of detection (LOD) of the qualitative PCR assay was about 0.01% in 100 ng of total papaya genomic DNA, corresponding to about 25 copies of papaya haploid genome. In the quantitative PCR, the limits of detection and quantification (LOD and LOQ) were as low as 12.5 and 25 copies of papaya haploid genome, respectively. In practical sample quantification, the quantified biases between the test and true values of three samples ranged from 0.44% to 4.41%. Collectively, we proposed that all of these results are useful for the identification and quantification of Huanong No. 1 papaya and its derivates.

Two kinds of ZnO@polymer core–shell nanoparticles were prepared through polymerization initiated by the inherent free radicals on the ZnO surface. The as-prepared quantum dots exhibited intense blue fluorescence. They were used as raw materials to synthesize blue-emitting ZnO@polymer core–shell microspheres which were very stable, even in aqueous solutions of strong acids or alkalis. These microspheres, as stable aqueous suspensions, showed moderate quantum yields, and their self-assembly behavior and biomedical application could be expected.

 Abstract—For engineering university, its ultimate goal is to cultivate high-leveled, application-oriented graduate. So, engineering education, engineering ability cultivation, curriculum teaching and curriculum teaching reformation is vital. Firstly, the meaning of engineering educations is discussed in the paper. Then, the history, achievements, problems and weakness of China's engineering education are analyzed. Thirdly, opportunities and challenges of China's engineering education are put forward. Finally, some opinions are given that: Engineering education is very consanguineous relation with China's curriculum. A better curriculum system decides the excellent engineering education. These description or argumentations are in favor of the development of China's engineering education and curriculum reform, be beneficial to the construction of applied university, be useful for cultivating excellent applied graduates.

The potential epitope of a recombinant food allergen protein, cashew Ana o 1, reactive to monoclonal antibody, mAb 2G4, has been mapped by solution-phase amide backbone H/D exchange (HDX) monitored by Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Purified mAb 2G4 was incubated with recombinant Ana o 1 (rAna o 1) to form antigen:monoclonal antibody (Ag:mAb) complexes. Complexed and uncomplexed (free) rAna o 1 were then subjected to HDX-MS analysis. Five regions protected from H/D exchange upon mAb binding are identified as potential conformational epitope-contributing segments.

In this study, we investigated the bioremediation of petrochemical wastewater containing BTEX compounds by immobilized Comamonas sp. JB cells. Three kinds of magnetic nanoparticles were evaluated as immobilization supports for strain JB. After comparison with Fe3O4 and a-Fe2O3 nanoparticles, r-Fe2O3 nanoparticle was selected as the optimal immobilization support. The highest biodegradation activity of r-Fe2O3-magnetically immobilized cells was obtained when the concentration of r-Fe2O3 nanoparticle was 120 mg L(-1). Additionally, the recycling experiments demonstrated that the degradation activity of r-Fe2O3-magnetically immobilized cells was still high and led to less toxicity than untreated wastewater during the eight recycles. qPCR suggested the concentration of strain JB in r-Fe2O3-magnetically immobilized cells was evidently increased after eight cycles of degradation experiments. These results supported developing efficient biocatalysts using r-Fe2O3-magnetically immobilized cells and provided a promising technique for improving biocatalysts used in the bioremediation of not only petrochemical wastewater but also other hazardous wastewater.

OBJECTIVE: To investigate the synergistic effect of transforming growth factor-β(1) (TGF-β(1)) and platelet-derived growth factor-BB (PDGF-BB) on the expression of integrin β(3) in periodontal membrane of rat orthodontic tooth. METHODS: An orthodontic tooth movement model was established. Up to 32 experimental rats were randomly divided into four groups according to a random number table. The four groups were injected with 1%PBS, TGF-β(1) (5 ng), PDGF-BB (10 ng), and combined TGF-β(1) (5 ng) and PDGF-BB (10 ng) in the buccal submucosal, respectively. The volume injected in each group was 0.1 mL. The animals were then sacrificed on the 10th day. The left maxillary first molar and periodontal tissue were taken. Different expressions of integrin β(3) were detected in periodontal tissues through immunohistochemistry. Mean optical density (OD) values of the positive fields were examined. The data obtained were analyzed through ANOVA. The data followed normal distribution, and were compared via t-test. RESULTS: Compared with the control groups, the expression of integrin β(3) was higher in the experimental groupin tension sides (P0.05). CONCLUSION: In terms of local regulatory factors, TGF-β(1) combined with PDGF-BB enhance the expression of integrin β(3) in the periodontal membrane and accelerate periodontal remodeling. The synergistic effect of the two growth factors is better than the single growth factor.

A bacterium designated strain JB, able to degrade six benzene, toluene, ethylbenzene, and o-, m-, and p-xylene (BTEX) compounds, was isolated from petroleum-contaminated soil. Taxonomic analyses showed that the isolate belonged to Comamonas, and until now, the genus Comamonas has not included any known BTEX degraders. The BTEX biodegradation rate was slightly low on the mineral salt medium (MSM), but adding a small amount of yeast extract greatly enhanced the biodegradation. The relationship between specific degradation rate and individual BTEX was described well by Michaelis-Menten kinetics. The treatment of petrochemical wastewater containing BTEX mixture and phenol was shown to be highly efficient by BTEX-grown JB. In addition, toxicity assessment indicated the treatment of the petrochemical wastewater by BTEX-grown JB led to less toxicity than untreated wastewater.

Mitogen-activated protein kinase (MAPK) cascades have important functions in plant growth, development, and response to various stresses. The MAPKK and MAPKKK gene families in tomato have never been systematically analyzed. In this study, we performed a genome-wide analysis of the MAPKK and MAPKKK gene families in tomato and identified 5 MAPKK genes and 89 MAPKKK genes. Phylogenetic analyses of the MAPKK and MAPKKK gene families showed that all the MAPKK genes formed four groups (groups A, B, C, and D), whereas all the MAPKKK genes were classified into three subfamilies, namely, MEKK, RAF, and ZIK. Evolutionary analysis showed that whole genome or chromosomal segment duplications were the main factors responsible for the expansion of the MAPKK and MAPKKK gene families in tomato. Quantitative real-time RT-PCR analysis showed that the majority of MAPKK and MAPKKK genes were expressed in all tested organs with considerable differences in transcript levels indicating that they might be constitutively expressed. However, the expression level of most of these genes changed significantly under heat, cold, drought, salt, and Pseudomonas syringae treatment. Furthermore, their expression levels exhibited significant changes in response to salicylic acid and indole-3-acetic acid treatment, implying that these genes might have important roles in the plant hormone network. Our comparative analysis of the MAPKK and MAPKKK families would improve our understanding of the evolution and functional characterization of MAPK cascades in tomato.

Phenoloxidase (PO) is a crucial immune-related enzyme in invertebrates. In this study, three POs of the sea cucumber Apostichopus japonicus were detected in coelomic fluid using linear-gradient native-PAGE combined with catechol staining and then partially purified by gel excising. The results showed that the three POs had a color of mahogany (AjPO1), yellow (AjPO2) and purple (AjPO3) respectively with molecular weights smaller than 21kDa in native-PAGE after staining with catechol. Enzymatic activities analysis revealed that AjPO1, AjPO2 and AjPO3 had optimal temperature of 45, 95 and 85°C and pH of 5.0, 8.0 and 8.0, respectively. Kinetic analysis showed that the Km values of AjPO1 for catechol, l-DOPA, dopamine and hydroquinone were 3.23, 0.86, 3.98 and 1.20mmol/l, respectively, those of AjPO2 were 0.31, 0.38, 2.05 and 1.30mmol/l, respectively, and those of AjPO3 were 5.95, 1.28, 5.81 and 0.62mmol/l, respectively. These results suggest that the three POs are laccase-type phenoloxidase. The activities of all three A. japonicus POs were significantly promoted by Ca(2+), Mg(2+) and Mn(2+), and strongly inhibited by ethylenediamine tetraacetic acid disodium (EDTA), sodium diethyldithiocarbamate (DETC) and some common antioxidants. The inhibitions by EDTA and DETC suggest that the three A. japonicus POs are copper-containing metalloenzymes. Immune-responsive analysis showed that the total PO activities in coelomocytes (TPAC) increased greatly after lipopolysaccharide (LPS) challenge and declined significantly after polyinosinic-polycytidylic acid (PolyI:C) challenge, implying that A. japonicus PO immune system, which is composed of several isoenzymes with different characteristics, is closely involved in the defense against the infection of Gram-negative bacteria and double-stranded RNA viruses.

Graves' disease (GD) is a common organ-specific autoimmune disease with the prevalence between 0.5 and 2% in women. Several lines of evidence indicate that the shed A-subunit rather than the full-length thyrotropin receptor (TSHR) is the autoantigen that triggers autoimmunity and leads to hyperthyroidism. We have for the first time induced GD in female rhesus monkeys, which exhibit greater similarity to patients with GD than previous rodent models. After final immunization, the monkeys injected with adenovirus expressing the A-subunit of TSHR (A-sub-Ad) showed some characteristics of GD. When compared with controls, all the test monkeys had significantly higher TSHR antibody levels, half of them had increased total thyroxine (T4) and free T4, and 50% developed goiter. To better understand the underlying mechanisms, quantitative studies on subpopulations of CD4+T helper cells were carried out. The data indicated that this GD model involved a mixed Th1 and Th2 response. Declined Treg proportions and increased Th17:Treg ratio are also observed. Our rhesus monkey model successfully mimicked GD in humans in many aspects. It would be a useful tool for furthering our understanding of the pathogenesis of GD and would potentially shorten the distance toward the prevention and treatment of this disease in human.

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.