Whole genome sequencing analysis of Limosilactobacillus reuteri from the intestinal tract of mice recovering from ulcerative colitis and preliminary study on anti-inflammatory effects of its derived peptides.

Limosilactobacillus reuteri is an indigenous inhabitant of the animal gut known for its probiotic effects on the host. In our previous study, a large number of L. reuteri strains were isolated from the gastrointestinal tract of mice recovering from ulcerative colitis, from which we randomly selected L. reuteri RE225 for whole genome sequencing to explore its probiotic properties. The results of next-generation sequencing and third-generation single molecule sequencing showed that L. reuteri RE225 contained many genes encoding functional proteins associated with adhesion, anti-inflammatory and pathogen inhibition. And compared to other L. reuteri strains in NCBI, L. reuteri RE225 has unique gene families with probiotic functions. In order to further explore the probiotic effect of the L. reuteri RE225, the derived peptides were identified by LC-MS/MS, and the peptides with tumor necrosis factor-α binding ability were screened by reverse molecular docking and microscale thermophoresis. Finally, cell experiments demonstrated the anti-inflammatory ability of the peptides. Western blotting and qPCR analyses confirmed that the selected peptides might alleviate LPS-induced inflammation in NCM460 cells by inhibiting JAK2/STAT3 pathway activation.

Analysis of antimicrobial biological activity of a marine Bacillus velezensis NDB.

A new strain of Bacillus velezensis NDB was isolated from Xiangshan Harbor and antibacterial test revealed antibacterial activity of this strain against 12 major pathogenic bacteria. The whole genome of the bacterium was sequenced and found to consist of a 4,214,838 bp circular chromosome and a 7410 bp circular plasmid. Furthermore, it was predicted by AntiSMASH and BAGEL4 to have 12 clusters of secondary metabolism genes for the synthesis of the inhibitors, fengycin, bacillomycin, macrolactin H, bacillaene, and difficidin, and there were also five clusters encoding potentially novel antimicrobial substances, as well as three bacteriocin biosynthesis gene clusters of amylocyclicin, ComX1, and LCI. qRT-PCR revealed significant up-regulation of antimicrobial secondary metabolite synthesis genes after 24 h of antagonism with pathogenic bacteria. Furthermore, MALDI-TOF mass spectrometry revealed that it can secrete surfactin non-ribosomal peptide synthase and polyketide synthase to exert antibacterial effects. GC-MS was used to analyze methanol extract of B. velezensis NDB, a total of 68 compounds were identified and these metabolites include 16 amino acids, 17 acids, 3 amines, 11 sugars, 11 alcohols, 1 ester, and 9 other compounds which can inhibit pathogenic bacteria by initiating the antibiotic secretion pathway. A comparative genomic analysis of gene families showed that the specificity of B. velezensis NDB was mainly reflected in environmental adaptability. Overall, this research on B. velezensis NDB provides the basis for elucidating its biocontrol effect and promotes its future application as a probiotic.

A roadmap to artificial intelligence (AI): Methods for designing and building AI ready data to promote fairness.

OBJECTIVES: We evaluated methods for preparing electronic health record data to reduce bias before applying artificial intelligence (AI). METHODS: We created methods for transforming raw data into a data framework for applying machine learning and natural language processing techniques for predicting falls and fractures. Strategies such as inclusion and reporting for multiple races, mixed data sources such as outpatient, inpatient, structured codes, and unstructured notes, and addressing missingness were applied to raw data to promote a reduction in bias. The raw data was carefully curated using validated definitions to create data variables such as age, race, gender, and healthcare utilization. For the formation of these variables, clinical, statistical, and data expertise were used. The research team included a variety of experts with diverse professional and demographic backgrounds to include diverse perspectives. RESULTS: For the prediction of falls, information extracted from radiology reports was converted to a matrix for applying machine learning. The processing of the data resulted in an input of 5,377,673 reports to the machine learning algorithm, out of which 45,304 were flagged as positive and 5,332,369 as negative for falls. Processed data resulted in lower missingness and a better representation of race and diagnosis codes. For fractures, specialized algorithms extracted snippets of text around keywork "femoral" from dual x-ray absorptiometry (DXA) scans to identify femoral neck T-scores that are important for predicting fracture risk. The natural language processing algorithms yielded 98% accuracy and 2% error rate The methods to prepare data for input to artificial intelligence processes are reproducible and can be applied to other studies. CONCLUSION: The life cycle of data from raw to analytic form includes data governance, cleaning, management, and analysis. When applying artificial intelligence methods, input data must be prepared optimally to reduce algorithmic bias, as biased output is harmful. Building AI-ready data frameworks that improve efficiency can contribute to transparency and reproducibility. The roadmap for the application of AI involves applying specialized techniques to input data, some of which are suggested here. This study highlights data curation aspects to be considered when preparing data for the application of artificial intelligence to reduce bias.

Comprehensive evaluation and analysis of the salinity stress response mechanisms based on transcriptome and metabolome of Staphylococcus aureus.

Staphylococcus aureus possesses an extraordinary ability to deal with a wide range of osmotic pressure. This study performed transcriptomic and metabolomic analyses on the potential mechanism of gradient salinity stress adaptation in S. aureus ZS01. The results revealed that CPS biosynthetic protein genes were candidate target genes for directly regulating the phenotypic changes of biofilm. Inositol phosphate metabolism was downregulated to reduce the conversion of functional molecules. The gluconeogenesis pathway and histidine synthesis were downregulated to reduce the production of endogenous glucose. The pyruvate metabolism pathway was upregulated to promote the accumulation of succinate. TCA cycle metabolism pathway was downregulated to reduce unnecessary energy loss. L-Proline was accumulated to regulate osmotic pressure. Therefore, these self-protection mechanisms can protect cells from hypertonic environments and help them focus on survival. In addition, we identified ten hub genes. The findings will aid in the prevention and treatment strategies of S. aureus infections.

Arginase plays an important role in ammonia detoxification of yellow catfish Pelteobagrus fulvidraco.

A two-stage study was carried out to test the mechanism of arginase in ammonia detoxification of yellow catfish. At stage 1, fish was injected lethal half concentration ammonium acetate and 0.9% sodium chloride respectively every 12 h in six replicates for 72 h. The result found that no significant different in serum ammonia contents of fish in ammonium acetate group at hours 12, 24, 36, 48, 60 and 72. At stage 2, ammonium acetate group was split in two, one continued to injected with ammonium acetate (NH3 group) and the other with ammonium acetate and valine (an inhibitor of arginase; Val group); Sodium chloride group also was split in two, one continued to injected with sodium chloride (NaCl group) and the other with sodium chloride and valine (NaCl + Val group). The experiment continued for 12 h. Serum ammonia and liver arginine contents of fish in Val group were higher than those of fish in NH3 group; Compared with NaCl group, arginase activity and ARG 1 expression in liver of fish in Val group were lower; Fish in NaCl and NaCl + Val groups had the lowest serum superoxide dismutase activities, malondialdehyde, tumor necrosis factor-α, interleukin 1 and 8 contents, TNF-α, IL-1 and IL-8 expressions than fish in NH3 and Val groups, and had the higher lysozyme activities, complement 3 and 4 contents. This study indicates that ammonia poisoning would lead to oxidative damage, immunosuppression and inflammation in yellow catfish; Arginase may be an important target of ammonia toxicity in yellow catfish; Exogenous arginine supplementation might alleviate the symptoms of ammonia poisoning in yellow catfish.

Ammonia stress disrupts intestinal microbial community and amino acid metabolism of juvenile yellow catfish (Pelteobagrus fulvidraco).

Ammonia has adverse effects on aquatic animals, which is also widely distributed in natural aquatic environments and intensive aquaculture systems. The intestine is a primary defensive line for aquatic animals, the accumulation of ammonia in the aquatic environment can cause irreversible damage to intestinal function. In this study, we investigated the effects of acute ammonia stress on the reaction characteristics of digestive function, amino acid metabolism, and the variation in the intestinal microbiota of juvenile yellow catfish (Pelteobagrus fulvidraco). Thus, the yellow catfish was placed in water with the addition of ammonia at 0 (control), 14.6, and 146 mg/L total ammonia nitrogen for 96-h. The present study observed that ammonia accumulated in the intestine and muscle (ammonia contents in the intestine and muscle increased) and induced the activities of protein digestive enzymes dysfunction (pepsin increased while trypsin decreased). Ammonia stress changed various amino acids composition (proline, arginine, lysine, histidine, phenylalanine, tyrosine, leucine, isoleucine, valine, alanine, glutamic acid, tyrosine, and aspartic acid contents were increased in muscle) and increased the activities of alanine aminotransferase and aspartate aminotransferase in muscle. Furthermore, through 16 S rRNA gene analysis, ammonia stress-induced reduction in diversity, richness, and evenness and structure of microbiota alteration in the intestine. At the phylum level, the abundance of Fusobacteria increased while Firmicutes and Actinobacteria decreased significantly. At the genus level, the abundance of beneficial microbiota Cetobacterium significantly increased after ammonia stress. In conclusion, activation of amino acid synthesis in muscle may be involved in ammonia detoxification after severe ammonia stress. The accumulation of ammonia can disrupt the intestinal digestive function and intestinal microbiota community. The Cetobacterium may be a new potential positive factor in the resistance of ammonia toxicity.

A Novel Framework for Anomaly Detection for Satellite Momentum Wheel Based on Optimized SVM and Huffman-Multi-Scale Entropy.

The health status of the momentum wheel is vital for a satellite. Recently, research on anomaly detection for satellites has become more and more extensive. Previous research mostly required simulation models for key components. However, the physical models are difficult to construct, and the simulation data does not match the telemetry data in engineering applications. To overcome the above problem, this paper proposes a new anomaly detection framework based on real telemetry data. First, the time-domain and frequency-domain features of the preprocessed telemetry signal are calculated, and the effective features are selected through evaluation. Second, a new Huffman-multi-scale entropy (HMSE) system is proposed, which can effectively improve the discrimination between different data types. Third, this paper adopts a multi-class SVM model based on the directed acyclic graph (DAG) principle and proposes an improved adaptive particle swarm optimization (APSO) method to train the SVM model. The proposed method is applied to anomaly detection for satellite momentum wheel voltage telemetry data. The recognition accuracy and detection rate of the method proposed in this paper can reach 99.60% and 99.87%. Compared with other methods, the proposed method can effectively improve the recognition accuracy and detection rate, and it can also effectively reduce the false alarm rate and the missed alarm rate.

The protective effects of selenium on chronic ammonia toxicity in yellow catfish (Pelteobagrus fulvidraco).

Ammonia is toxic to most fish, and its negative effects can be eliminated by nutritional manipulation. In this study, triplicate groups of yellow catfish (0.58 ± 0.03 g) were fed diets supplemented with 0, 0.30 and 0.60 mg selenium (Se) kg-1 diet for 56 days under three ammonia contents (0.00, 5.70 and 11.40 mg L-1 total ammonia nitrogen). The results showed that ammonia toxicity could affects growth (weight gain, feed efficiency ratio, Se contents in muscle and whole body declined) and survival, leads to oxidative stress (total antioxidant capacity, superoxide dismutase, catalase and glutathione peroxidase activities declined and malondialdehyde accumulation), immunosuppression (lysozyme activity, 50% hemolytic complement, immunoglobulin M, respiratory burst and phagocytic index declined) and cytokines release (TNF, IL 1 and IL 8 elevated), induces up-regulation of antioxidant enzymes (Cu/Zn-SOD, Mn-SOD, CAT and GPx), cytokines (TNFα, IL 1 and IL 8) and pro-apoptotic genes (p53, Bax, Cytochrome c, Caspase 3 and Caspase 9) transcription, and down-regulation of anti-apoptotic gene Bcl2 transcription. The dietary Se supplementation could mitigate the adverse effect of ammonia poisoning on fish growth, oxidative damage, immunosuppression and apoptotic.

Ammonia toxicity in the yellow catfish (Pelteobagrus fulvidraco): The mechanistic insight from physiological detoxification to poisoning.

Ammonia is toxic to fishes. Different fish have different defense strategies against ammonia, so the mechanism of ammonia poisoning is different. In this study, yellow catfish were exposed to three levels of ammonia (0, 5.70 and 57.00 mg L-1) for 96 h. The results showed that ammonia poisoning could lead to free amino acid imbalance (ornithine and citrulline contents declined; arginine content elevated), urea cycle enzymes deficiency (carbamyl phosphate synthetase and arginase contents declined), oxidative stress (superoxide dismutase, catalase and glutathione peroxidase activities declined), immunosuppression (lysozyme activity, 50% hemolytic complement and total immunoglobulin contents and phagocytic index declined) and cytokines release (TNF, IL 1 and IL 8 contents elevated). In addition, ammonia poisoning could induce up-regulation of antioxidant enzymes (Cu/Zn-SOD, Mn-SOD, CAT and GPx), cytokines (TNFα, IL 1 and IL 8) and apoptosis (p53, Bax, cytochrome c, Caspase 3 and Caspase 9) genes transcription. This study suggesting that the urea cycle and glutamine synthesis both were involved in the ammonia detoxification of yellow catfish, and the immunosuppression, inflammation and apoptotic induced by ammonia poisoning in yellow catfish are related to oxidative stress.

Do the toll-like receptors and complement systems play equally important roles in freshwater adapted Dolly Varden char (Salvelinus malma)?

Unlike the normal anadromous lifestyle, Chinese native Dolly Varden char (Salvelinus malma) is locked in land and lives in fresh water lifetime. To explore the effect of freshwater adaption on its immune system, we constructed a pooled cDNA library of hepatopancreas and spleen of Chinese freshwater Dolly Varden char (S. malma). A total of 27,829 unigenes were generated from 31,233 high-quality transcripts and 17,670 complete open reading frames (ORF) were identified. Totally 25,809 unigenes were successfully annotated and it classified more native than adaptive immunity-associated genes, and more genes involved in toll-like receptor signal pathway than those in complement and coagulation cascades (51 vs 3), implying the relative more important role of toll-like receptors than the complement system under bacterial injection for the freshwater Dolly Varden char. These huge different numbers of TLR and complement system identified in freshwater Dolly Varden char probably caused by distinct evolution pressure patterns between fish TLR and complement system, representative by TLR3 and TLR5 as well as C4 and C6, respectively, which were under purifying and positively selecting pressure, respectively. Further seawater adaptation experiment and the comparison study with our library will no doubt be helpful to elucidate the effect of freshwater adaption of Chinese native Dolly Varden char on its immune system.

Modulation of lipid metabolism in juvenile yellow catfish (Pelteobagrus fulvidraco) as affected by feeding frequency and environmental ammonia.

In the intensive culture systems, excessive feeding leads to ammonia accumulation, which results in lipid metabolism disorder. However, little information is available on the modulation of lipid metabolism in fish as affected by feeding frequency and ammonia stress. In this study, weight gain increased as feeding frequency increased from one to four times daily, but feed conversion ratio is opposite. The highest survival was found in ammonia group when fish was fed two times daily. Liver ammonia content increased as feeding frequency increased from one to four times daily, and the highest brain ammonia content was found when fish was fed four times daily. The highest liver 6-phospho-gluconate dehydrogenase (6PGD), fatty acid synthase (FAS), carnitine palmitoyltransferase (CPT), and lipoprotein lipase (LPL) contents were found in control group when fish was fed four times daily; in comparison, the highest liver 6PGD, FAS, CPT, and LPL contents were found in ammonia group when fish was fed two times daily. Liver 6PGD, FAS, CPT 1, SREBP-1, and PPARα mRNA expression in control group increased significantly as feeding frequency increased from one to four times daily, and the highest expression of 6PGD, G6PD, and FAS was observed in ammonia group when fish was fed two times daily. This study indicated that the optimal feeding frequency is two times daily when yellow catfish exposed to ammonia.

Effects of acute ammonia toxicity on oxidative stress, immune response and apoptosis of juvenile yellow catfish Pelteobagrus fulvidraco and the mitigation of exogenous taurine.

Ammonia can easily form in intensive culture systems due to ammonification of uneaten food and animal excretion, which usually brings detrimental health effects to fish. However, little information is available on the mechanisms of the detrimental effects of ammonia stress and mitigate means in fish. In this study, the four experimental groups were carried out to test the response of yellow catfish to ammonia toxicity and their mitigation through taurine: group 1 was injected with NaCl, group 2 was injected with ammonium acetate, group 3 was injected with ammonium acetate and taurine, and group 4 was injected taurine. The results showed that ammonia poisoning could induce ammonia, glutamine, glutamate and malondialdehyde accumulation, and subsequently lead to blood deterioration (red blood cell, hemoglobin and serum biochemical index reduced), oxidative stress (superoxide dismutase and catalase activities declined) and immunosuppression (lysozyme, 50% hemolytic complement, total immunoglobulin, phagocytic index and respiratory burst reduced), but the exogenous taurine could mitigate the adverse effect of ammonia poisoning. In addition, ammonia poisoning could induce up-regulation of antioxidant enzymes (Cu/Zn-SOD, CAT, GPx and GR), inflammatory cytokines (TNF, IL-1 and IL-8) and apoptosis (p53, Bax, caspase 3 and caspase 9) genes transcription, suggesting that cell apoptotic and inflammation may relate to oxidative stress. This result will be helpful to understand the mechanism of aquatic toxicology induced by ammonia in fish.

Symbolic Important Point Perceptually and Hidden Markov Model Based Hydraulic Pump Fault Diagnosis Method.

Hydraulic pump is a driving device of the hydraulic system, always working under harsh operating conditions, its fault diagnosis work is necessary for the smooth running of a hydraulic system. However, it is difficult to collect sufficient status information in practical operating processes. In order to achieve fault diagnosis with poor information, a novel fault diagnosis method that is the based on Symbolic Perceptually Important Point (SPIP) and Hidden Markov Model (HMM) is proposed. Perceptually important point technology is firstly imported into rotating machine fault diagnosis; it is applied to compress the original time-series into PIP series, which can depict the overall movement shape of original time series. The PIP series is transformed into symbolic series that will serve as feature series for HMM, Genetic Algorithm is used to optimize the symbolic space partition scheme. The Hidden Markov Model is then employed for fault classification. An experiment involves four operating conditions is applied to validate the proposed method. The results show that the fault classification accuracy of the proposed method reaches 99.625% when each testing sample only containing 250 points and the signal duration is 0.025 s. The proposed method could achieve good performance under poor information conditions.

Twenty years of ModelDB and beyond: building essential modeling tools for the future of neuroscience.

Neuron modeling may be said to have originated with the Hodgkin and Huxley action potential model in 1952 and Rall's models of integrative activity of dendrites in 1964. Over the ensuing decades, these approaches have led to a massive development of increasingly accurate and complex data-based models of neurons and neuronal circuits. ModelDB was founded in 1996 to support this new field and enhance the scientific credibility and utility of computational neuroscience models by providing a convenient venue for sharing them. It has grown to include over 1100 published models covering more than 130 research topics. It is actively curated and developed to help researchers discover and understand models of interest. ModelDB also provides mechanisms to assist running models both locally and remotely, and has a graphical tool that enables users to explore the anatomical and biophysical properties that are represented in a model. Each of its capabilities is undergoing continued refinement and improvement in response to user experience. Large research groups (Allen Brain Institute, EU Human Brain Project, etc.) are emerging that collect data across multiple scales and integrate that data into many complex models, presenting new challenges of scale. We end by predicting a future for neuroscience increasingly fueled by new technology and high performance computation, and increasingly in need of comprehensive user-friendly databases such as ModelDB to provide the means to integrate the data for deeper insights into brain function in health and disease.

Growth, blood health, antioxidant status and immune response in juvenile yellow catfish Pelteobagrus fulvidraco exposed to α-ethinylestradiol (EE2).

Triplicate groups of juvenile yellow catfish Pelteobagrus fulvidraco were exposed to three levels of α-ethinylestradiol (EE2) (0, 0.1 and 1 ng L-1) for 56 days. Fish survival rate (>93.33%) was not different among experimental groups. Weight gain and specific growth rate of fish exposed to EE2 were higher than those of control fish. Hepatosomatic index of fish exposed to 1 ng L-1 EE2 was the highest. Serum total protein, albumin, globulin, alanine aminotransferase, aspartate transaminase, cholesterol and triglyceride increased with increasing EE2 exposure levels. Liver total anti-oxidative capacity, malondialdehyde content and lysozyme activity of fish exposed to EE2 were higher than those of control fish. Phagocytic indices of fish exposed to 1 ng L-1 EE2 was lower than that of control fish. This study indicates that although EE2 exposure can promote the growth of yellow catfish in short-term, EE2 exerts its toxic effects by inducing reactive oxygen species generation and malondialdehyde accumulation, leading to blood deterioration and interfering with immune response.

The protective effects of taurine on acute ammonia toxicity in grass carp Ctenopharynodon idellus.

The four experimental groups were carried out to test the response of grass carp Ctenopharyngodon idella to ammonia toxicity and taurine: group 1 was injected with NaCl, group 2 was injected with ammonium acetate, group 3 was injected with ammonium acetate and taurine, and group 4 was injected taurine. Fish in group 2 had the highest ammonia content in the liver and brain, and alanine, arginine, glutamine, glutamate and glycine contents in liver. Brain alanine and glutamate of fish in group 2 were significantly higher than those of fish in group 1. Malondialdehyde content of fish in group 2 was the highest, but superoxide dismutase and glutathione activities were the lowest. Although fish in group 2 had the lowest red cell count and hemoglobin, the highest alkaline phosphatase, complement C3, C4 and total immunoglobulin contents appeared in this group. In addition, superoxide dismutase and glutathione activities, red cell count and hemoglobin of fish in group 3 were significantly higher than those of fish in group 2, but malondialdehyde content is the opposite. This study indicates that ammonia exerts its toxic effects by interfering with amino acid transport, inducing reactive oxygen species generation and malondialdehyde accumulation, leading to blood deterioration and over-activation of immune response. The exogenous taurine could mitigate the adverse effect of high ammonia level on fish physiological disorder.

Complete mitochondrial genome sequence of Tridentiger bifasciatus and Tridentiger barbatus (Perciformes, Gobiidae): a mitogenomic perspective on the phylogenetic relationships of Gobiidae.

The fishes of suborder Gobioidei is the largest group of those in present living Perciformes, which contains about 2,200 species belonging to 270 genera of 9 families in the world. The monophyly and phylogenetic relationships of gobies have been controversial and disputable for a long time. In the present study, the complete mitochondrial genome of the shimofuri goby Tridentiger bifasciatus (T. bifasciatus) and shokihaze goby Tridentiger barbatus (T. barbatus) were firstly determined. The two mitochondrial genomes were both consisted of 2 ribosomal RNA (rRNA) genes, 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and one major control region (CR). They shared similar features with those of other gobies in terms of gene arrangement, base composition, and tRNA structures. The CR was absence of typical conserved blocks (CSB-E, and CSB-F) respectively for the T. bifasciatus and T. barbatus. Phylogenomic analyses, which based on 12 concatenated protein-coding genes and complete mitochondrial genome sequences, revealed that there were two groups within the Gobiidae. A large group consisted of the Amblyopinae, Gobionellinae, Oxudercinae and Sicydiinae, and Amblyopinae was nested in Oxudercinae and they were both paraphyletic to Sicydiinae. The other group was the Gobiinae. As a whole, our phylogenetic data was different from the traditionally classification of Gobiidae, but supported the new phylogenetic taxonomy view of Thacker (Copeia 2009:93-104, 2009).

Comparative genomic of the teleost cathepsin B and H and involvement in bacterial induced immunity of miiuy croaker.

Cathepsins are a family of lysosomal proteases play different roles at physiological and pathological states and present in almost all animals as well as other organisms. Cathepsins B and H are both cysteine proteases of cathepsins. Cathepsin B and H have been studied playing parts in protein degradation/turnover, antigen presentation/processing and hormone maturation in mammals. However, little is known about the structures and functions of cathepsin B and H in teleosts. In the present study, we identified and characterized the full-length miiuy croaker (Miichthys miiuy) cathepsin B and H genes. The sequence analysis results showed that both cathepsin B and H contain the characteristics of papain family with a signal peptide, propeptide and mature peptide regions. The comparison of the genomic organizations and locations indicated the conserved synteny and mild evolution in the cathepsin B and H genes adjacent regions. In addition, the gene synteny analysis showed that miiuy croaker cathepsin B has a closer relationship to stickleback and fugu than to cave fish and zebrafish, and cathepsin H was most similar with the 2 subtype in tilapia and fugu. By phylogenetic analysis, miiuy croaker cathepsin B and H were all assigned to cysteine proteases, and with a close relationship to Salmo salar cathepsin B and Oplegnathus fasciatus cathepsin H, respectively. Quantitative real-time RT-PCR analysis results confirmed that cathepsin B and H genes expressed ubiquitously in all tested healthy tissues from miiuy croaker. Furthermore, up-regulated expression of the cathepsin B and H transcripts in liver, spleen and kidney after exposure upon Vibrio anguillarum suggested that they may play important roles in innate immune response and antigen processing of miiuy croaker.

Three representative subtypes of caspase in miiuy croaker: genomic organization, evolution and immune responses to bacterial challenge.

Caspase proteins are intracellular proteases which function as initiators and effectors of apoptosis. According their difference of functions, the caspases can be divided into apoptosis related caspases and inflammatory mediator, and the former included apoptosis activator and apoptosis executioner. In this study, three different subtype caspases (caspase1, caspase3 and caspase9) from Miichthys miiuy miiuy croaker were analyzed. The caspase1 belongs to the inflammatory mediator, caspase3 belongs to apoptosis executioner, and caspase9 belongs to apoptosis activator. Miichthys miiuy caspase1 (Mmcaspase1) and Mmcaspase9 exhibited three conserved domains including a CARD, a large subunit p20 and a small subunit p10. The Mmcaspase3 encoded two conserved domains including a large subunit p20 and a small subunit p10. Mmcaspase3 and Mmcaspase9 contained a histidine active sequence and a cysteine active sequence. However, Mmcaspase1 only contained the cysteine active sequence. The real-time PCR (RT-PCR) analysis showed these three caspases were expressed constitutively in all examined tissues in miiuy croaker, although the expression levels varied from tissue to tissue. Expression analysis showed that Mmcaspase1 was up-regulated obviously in liver, spleen and kidney, and indicated its positive role in response to Vibrio anguillarum infection, but Mmcaspase3 and Mmcaspase9 showed different expression pattern in liver, spleen and kidney, its showed that different subtypes of caspase having different immune response mechanisms. These results revealed that the organs adjusted the expressions of these three genes with the infected of pathogens, suggesting the immunoprotection of these genes.

Genomic characterization and expression pattern of Bf/C2 and C4 in miiuy croaker and molecular evolution analysis on mammals and fishes.

The complement system plays an important role in both innate and adaptive host defense against the invading microorganisms in vertebrates. It can be activated by three pathways: the classical, alternative and lectin pathways. Bf/C2 and C4, as members of complement, play a pivotal role in the activation of the complement system. In our study, we identified Bf/C2 and C4 genes and genomic structure in miiuy croaker, and expression patterns of Bf/C2 and C4 genes was analyzed. In healthy miiuy croaker tissues, Bf/C2 and C4 genes were found to be ubiquitously expressed in all ten tested tissues. Analysis of expression of Bf/C2 and C4 genes after bacterial infection showed a significant up-regulated in liver. The evolutionary analysis showed that the ancestral lineages of Bf/C2 and C4 genes in mammals and fishes experienced positive selection indicated that the ancestors of mammals and fishes had further evolved to adapt to their environment, respectively. A series of maximum likelihood (ML) methods were used to study the evolution on vertebrates' Bf/C2 and C4 genes. One and five positive selection sites were found in mammals of Bf/C2 and C4 genes, but no positive selection site was found in fishes of Bf/C2 and C4 genes, indicating that Bf/C2 and C4 genes in mammals and fishes underwent different evolutionary patterns.