Exposure to Intermittent Environmental Hypoxia Promotes Vascular Remodeling through Angiogenesis in the Liver of Largemouth Bass (Micropterus salmoides).

In this study, we explored the effects of 4 weeks of intermittent hypoxic exposure (IHE) on liver angiogenesis and related regulatory mechanisms in largemouth bass (Micropterus salmoides). The results indicated that the O2 tension for loss of equilibrium (LOE) decreased from 1.17 to 0.66 mg/L after 4 weeks of IHE. Meanwhile, the red blood cell (RBC) and hemoglobin concentrations significantly increased during IHE. Our investigation also found that the observed increase in angiogenesis was correlated with a high expression of related regulators, such as Jagged, phosphoinositide-3-kinase (PI3K), and mitogen-activated protein kinase (MAPK). After 4 weeks of IHE, the overexpression of factors related to angiogenesis processes mediated by HIF-independent pathways (such as nuclear factor kappa-B (NF-κB), NADPH oxidase 1 (NOX1), and interleukin 8 (IL8)) was correlated with the accumulation of lactic acid (LA) in the liver. The addition of cabozantinib, a specific inhibitor of VEGFR2, blocked the phosphorylation of VEGFR2 and downregulated the expression of downstream angiogenesis regulators in largemouth bass hepatocytes exposed to hypoxia for 4 h. These results suggested that IHE promoted liver vascular remodeling by the regulation of angiogenesis factors, presenting a potential mechanism for the improvement of hypoxia tolerance in largemouth bass.

Chronic hypoxia and Cu2+ exposure induce gill remodeling of largemouth bass through endoplasmic reticulum stress, mitochondrial damage and apoptosis.

Hypoxia and Cu2+ pollution often occur simultaneously in aquatic ecosystems and jointly affect physiology of fish. As the respiratory and ion exchange tissue of fish, how gill responds to the stress induced by these two abiotic environmental factors is still unclear. We have conducted a study by exposing largemouth bass (Micropterus salmoides) to hypoxia (2.0 mg·L-1) and/or Cu2+ (0.5 mg·L-1) for 28 days to answer this question. We subsequently studied respiratory rate, Cu2+ transport, endoplasmic reticulum (ER) stress, mitochondrial damage, and morphology in gill tissue on day 7, 14, 21 and 28. We found that hypoxia exposure increased the respiratory rate of largemouth bass, reflecting the response of largemouth bass to cope with hypoxia. Of note, Cu2+ entered gill by specifically binding to CTR1 and its accumulation dramatically in gill disrupted the response of largemouth bass to hypoxia. Hypoxia and/or Cu2+ exposure led to ER stress and mitochondrial damage in gills of largemouth bass. ER stress and mitochondrial damage induced apoptosis by activating caspase-8 and caspase-9 signaling pathways, respectively. Apoptosis induced by hypoxia and Cu2+ exposure had a positive and synergistic effect on gill remodeling by reducing interlamellar cell masses. In addition, Cu2+ exposure induced hypoxia-like remodeling to gill morphology through mechanisms similar to hypoxia exposure. Most of gene expression changed mainly within 21 days and recovered to the control level on day 28, reflecting the acclimation of largemouth bass to hypoxia and/or Cu2+ exposure at gene expression level. Overall, our research suggests that chronic hypoxia and Cu2+ exposure could induce gill remodeling of largemouth bass through ER stress, mitochondrial damage and apoptosis. The outcomes could provide an insight for fish environmental adaptation and environmental toxicology.

Chromosome-level genome assembly of largemouth bass (Micropterus salmoides) using PacBio and Hi-C technologies.

The largemouth bass (Micropterus salmoides) has become a cosmopolitan species due to its widespread introduction as game or domesticated fish. Here a high-quality chromosome-level reference genome of M. salmoides was produced by combining Illumina paired-end sequencing, PacBio single molecule sequencing technique (SMRT) and High-through chromosome conformation capture (Hi-C) technologies. Ultimately, the genome was assembled into 844.88 Mb with a contig N50 of 15.68 Mb and scaffold N50 length of 35.77 Mb. About 99.9% assembly genome sequences (844.00 Mb) could be anchored to 23 chromosomes, and 98.03% assembly genome sequences could be ordered and directed. The genome contained 38.19% repeat sequences and 2693 noncoding RNAs. A total of 26,370 protein-coding genes from 3415 gene families were predicted, of which 97.69% were functionally annotated. The high-quality genome assembly will be a fundamental resource to study and understand how M. salmoides adapt to novel and changing environments around the world, and also be expected to contribute to the genetic breeding and other research.

Electrochemiluminescent Ion-Channeling Framework for Membrane Binding and Transmembrane Activity Assays.

Recent upgrades in the electrochemiluminescence (ECL) technique showcased its brilliant knack in probing microscopic biointerfacial events, many of which were actually underlain by the ionotropic membrane processes, yet not being ostensive. Here, by modeling an artificial lipoid-supported porin ensemble, we explore and establish the ECL potency in profiling ion-channel activities. A lipophilic hollowed construct dubbed ZnPC was made out of the dynamic covalent chemistry, and its unique geometry was characterized that configured stoichiometric ECL-emissive units in a cubic stance; while the aliphatic vertices of ZnPC helped it safely snorkel and steadily irradiate in a biofilm fusion. After expounding basic ECL properties, the brightness was traced out in response to halogen contents that was lit up by F-/Cl- but down by Br-/I-. The overall pattern fitted the Langmuir isotherm, from which the membrane-binding strengths of the four were analyzed, compared, and collaterally examined in impedimetrics. On the other hand, one could derive anionic transmembrane kinetics from the time-dependent ECL statistics that pinpointed the ECL signaling via the nanocage-directed mass-transfer pathway. More data mining unveiled an ECL-featured Hofmeister series and the thermodynamic governing force behind all scenes. Finally, combining with halide-selective fluorometry, the synthetic conduit was identified as an ECL symporter. In short, this work develops a novel ECL model for the evaluation of life-mimicking membrane permeation. It might intrigue the outreach of ECL applications in the measurement of diverse surface-confined transient scenarios, e.g., in vitro gated ion or molecule trafficking, which used to be handled by nanopore and electrofluorochromic assays.

Functional miR-142a-3p Induces Apoptosis and Macrophage Polarization by Targeting tnfaip2 and glut3 in Grass Carp (Ctenopharyngodon idella).

In the process of microbial invasion, the inflammation reaction is induced to eliminate the pathogen. However, un-controlled or un-resolved inflammation can lead to tissue damage and death of the host. MicroRNAs (miRNAs) are the signaling regulators that prevent the uncontrolled progress of an inflammatory response. Our previous work strongly indicated that miR-142a-3p is related to the immune regulation in grass carp. In the present study, we found that the expression of miR-142a-3p was down-regulated after infection by Aeromonas hydrophila. tnfaip2 and glut3 were confirmed as be the target genes of miR-142a-3p, which were confirmed by expression correlation analysis, gene overexpression, and dual luciferase reporter assay. The miR-142a-3p can reduce cell viability and stimulate cell apoptosis by targeting tnfaip2 and glut3. In addition, miR-142a-3p also regulates macrophage polarization induced by A. hydrophila. Our results suggest that miR-142a-3p has multiple functions in host antibacterial immune response. Our research provides further understanding of the molecular mechanisms between miRNAs and their target genes, and provides a new insights for the development of pro-resolution strategies for the treatment of complex inflammatory diseases in fish.

Conjoint applications of meta-analysis and bioinformatic data toward understanding the effect of nitrate on fish.

The extensively accumulation of nitrate in different water resources is currently regarded as one of the most predominant threats facing aquatic organisms on worldwide scale. In recent years, a growing body of evidences have been attempting to uncover the influences of nitrate on fish growth and health, thereby evaluating its environment security. However, the systematic assessment and intrinsic mechanism of such influences are apparently devoid. Hence, this investigation employed systematic analysis, meta-analysis and bioinformatic analysis to evaluate the nitrate biotoxicity. We first speculated two levels of nitrate concentration according to forty-four published bibliographies. Systematic analysis indicated that the broad variations of fish sensitivity to chronic and acute nitrate exposures were found in juvenile and larval stage, respectively, comparing to egg. Meta-analysis further revealed that survival rate, CF and SGR were significantly improved in low nitrate concentration during chronic exposure. Such improvements were reflected by Total mean differences (TMD) and 95% CIs (Confidence Intervals): Survival rate (-4.06 [-7.67, -0.45]), Fulton's condition factor (CF) (-0.03 [-0.03, -0.02]) and Specific growth rate (SGR) (-0.10 [-0.16, -0.04]). To trace the impact, the alternations of molecular expression and histology in brain, gill, liver, intestine, and blood suggested that the chronic and acute nitrate exposures could result in abnormal tissue structures and molecular dynamics. Moreover, omics analysis via integrating intestinal microbiome (microbial composition; %) and liver transcriptome (Gene Ontology: biological processes) revealed that the low concentration exposure induced a weakly immune response in fish liver and it matched to the intestinal immune response. Overall, current study has filled the gaps in the field of nitrate toxicity. It could also provide a novel insight for the evaluation of pollutant toxicity on aquatic species.

Tracing and exploring the evolutionary origin and systematic function of fish complement C9.

Complement C9, as a member of terminal complement component (TCC) protein, plays important roles in innate immunity. However, some complement components appear to show difference and evolutionary complexity between higher and lower vertebrates. Hence, it is essential to carry on a study of evolutionary origin and systematic function of C9 in fish and non-fish vertebrates. This study aims to explore the complement gene evolution and potential function in fish based on molecular and structural biology. Herein, we found complete divergence of C9 throughout the gene evolution. The optimal codons of C9 sequences tended to be closer to the genomes of lower vertebrates compared to higher vertebrates. Further, conserved amino acids in the C9 TMH1 region were identified, implying their potential functional association with MAC growth and pore formation. Transposons and simple repeats, as gene elements, exhibited a differential distribution in the genomic regions in different animal groups but were sparsely scattered around the sixth exon (TMH1 region). Notably, this demonstrated the regulatory complexity of the C9 gene in higher vertebrates. The negative selection pressures on fish and non-fish groups improved both the sequence conservation and similarity. Through gene/protein regulatory network and pathway analyses, the systematic function of C9 protein was showcased; thus, we could reveal the divergence of the systematic function of C9 across species from different evolutionary positions. In addition, more complicated functions of C9 in higher vertebrates could established by the altered spatial conformation of the protein. Collectively, the present study illustrates the C9 gene evolutionary process and the difference in its systematic function across multiple species. Such advances provide new insights for understanding the evolutionary and potential functions of complement C9.

The evolutionary analysis of complement component C5 and the gene co-expression network and putative interaction between C5a and C5a anaphylatoxin receptor (C5AR/CD88) in human and two Cyprinid fish.

The complement system is a complex network of soluble and membrane-associated serum proteins that regulate immune response. Activation of the complement C5 generates C5a and C5b which generate chemoattractive effect on myeloid cells and initiate the membrane attack complex (MAC) assembly. However, the study of evolutionary process and systematic function of C5 are still limited. In this study, we performed an evolutionary analysis of C5. Phylogeny analysis indicated that C5 sequences underwent complete divergence in fish and non-fish vertebrate. It was found that codon usage bias improved and provided evolution evidence of C5 in species. Notably, the codon usage bias of grass carp was evolutionarily closer to the zebrafish genome compared with humans and stickleback. This suggested that the zebrafish cell line may provide an alternative environment for heterologous protein expression of grass carp. Sequence comparison showed a higher similarity between human and mouse, grass carp, and zebrafish. Moreover, selective pressure analysis revealed that the C5 genes in fish and non-fish vertebrates exhibited different evolutionary patterns. To study the function of C5, gene co-expression networks of human and zebrafish were built which revealed the complexity of C5 function networks in different species. The protein structure simulation of C5 indicated that grass carp and zebrafish are more similar than to human, however, differences between species in C5a proteins are extremely smaller. Spatial conformations of C5a-C5AR (CD88) protein complex were constructed, which showed that possible interaction may exist between C5a and CD88 proteins. Furthermore, the protein docking sites/residues were measured and calculated according to the minimum distance for all atoms from C5a and CD88 proteins. In summary, this study provides insights into the evolutionary history, function and potential regulatory mechanism of C5 in fish immune responses.

Fish lysozyme gene family evolution and divergent function in early development.

Lysozymes are an ancient group of antimicrobial enzymes of the innate immune system. Here we provide a comparative analysis of the evolution and function of lysozymes during early development in fish, the most speciose vertebrate group. In fishes, lineage and species-specific evolution of both C-type (chicken or conventional) and G-type (goose type) genes occurred. Phylogenetic analysis revealed that the teleost lysozyme G-type members group with the tetrapod homologues but the teleost C-type form three different clusters with the tetrapods. Most of the teleost C-type cluster with tetrapod Lyz but there are some that group with the mammalian Lyzl1/2 and LALBA. This suggests that early in gnathostome evolution these genes already existed and that lyzl1/2 and lalba genes are present in fish and tetrapods. Gene synteny analysis to confirm sequence orthologies failed to identify conserved genome regions between teleosts and other vertebrates lysozyme gene regions suggesting that in the ancestral bony fish genome lyz, lyzl1/2, lalba and lyg precursor genes were transposed to different chromosome regions. The homologue of the mammalian lactalbumin (LALBA) gene was identified for the first time in teleosts and was expressed in skin and during egg and larval development. Lysozyme activity was detected in teleost eggs and varied between species and in the gilthead sea bream lyg and lalba transcript abundance differed in eggs and larvae from different brood stock suggesting differences exist in maternal innate immune protection.

Fish complement C8 evolution, functional network analyses, and the theoretical interaction between C8 alpha chain and CD59.

Complement C8, as a main component of the membrane attack complex, has only been identified in vertebrates. C8 comprises three subunits encoded by individual genes: C8a (alpha chain), C8b (beta chain), and C8g (gamma chain). However, in fish, there have been limited studies on the evolutionary history and systematic function of C8. In the present study, phylogenetic analysis indicated the complete divergence of C8 genes in different fish species. Codon usage bias analysis revealed the evolutionary complexity of C8 genes. Selective pressure analysis found that C8 genes have been affected by negative selection during evolution. Sequence alignment identified the sites that are under selective pressure. The systematic functions of C8 were revealed by gene co-expression and protein-protein interaction (PPI) network analyses. Notably, gene ontology enrichment analysis suggested that C8 proteins in zebrafish function mainly in the neuroendocrine system. Protein structural comparisons showed that putative functional residues and domains were conserved between the C8 subunits of human and grass carp. A preliminary study on the theoretical interaction between C8a and CD59 was performed according to the simulated protein stereo structure. The first functionally-related site was absent in the simulated conformation of the grass carp (Ctenopharyngodon idella) C8a-CD59 protein complex. We speculated that Tyr63 is involved in the functional loss of CD59 binding. The docking of CD59 to four potential sites (Met390, Ser391, Leu392, and Val405) in grass carp C8a was analyzed. The results of the present study provide a deeper understanding of the evolution and function of fish complement C8.

Fish complement C4 gene evolution and gene/protein regulatory network analyses and simulated stereo conformation of C4-MASP-2 protein complex.

Complement C4 is a central protein by acting as pivotal molecule in the activation of the complement system. More than a decade ago, C4 gene duplication had been found in several species including fish, revealing the evolutionary origin of C4 gene. However, the evolutionary pattern and systematic function of C4 are still limited. In this study, C4 D and H types in different species groups were completely diverged. The codon usage of C4 H type in higher vertebrates were much closer to their own genome environment, in contrast to lower vertebrates, suggesting that the evolution may provide the dynamic for homogeneous codon usage between specific gene and genome. Multiple C4 sequence alignment showed that the sequences were conserved among different species. However, sequence similarity was obviously different between species C4 D and H type. Negative selection pressure was found on C4 gene evolution and it may be one of the possible reasons for the sequence broad similarity and conservation among interspecies. Proteins from C4 protein-protein interaction (PPI) network were enriched in more hematopoiesis, infections, diseases and immune-related pathways in human than zebrafish. The result suggested that the functional complexities of C4 isotypes are distinct in species from different evolutionary positions. The simulated C4 protein structures between human and grass carp shared structural similarity and the stereo structures of grass carp C4-MASP-2 protein complexes were further simulated according to a study of human. These results suggested that the interaction between C4 and MASP-2 proteins may also exist in grass carp. Our results can provide an insight for the evolutionary process of C4 and better understanding to the potential mechanism of interaction between C4 and MASP-2 in fish species.

Circular RNA circ_0102049 promotes cell progression as ceRNA to target MDM2 via sponging miR-1304-5p in osteosarcoma.

Osteosarcoma (OS) is known as a tumor that derives from skeletal system with increasing incidence worldwide. This study aimed to explore the effect of a circular RNA (circRNA), circ_0102049, on OS and reveal its potential molecular mechanism. In this work, the expression of circ_0102049 was detected by quantitative real time polymerase chain reaction (qRT-RCR) in both OS specimens and cell lines. The relationship between circ_0102049 level and patients' overall survival was evaluated by Kaplan-Meier curves and Cox regression analysis. Cell proliferation, apoptosis, migration and invasion of U2OS and MG63 cells were measured by cell counting kit-8 (CCK-8), flow cytometry, wound healing and transwell experiments, respectively. In addition, subcellular fractionation, bioinformatics analysis and dual-luciferase reporter assay were utilized to reveal the mechanism of circ_0102049 in OS. Circ_0102049 was overexpressed in both OS specimens and cells. Moreover, the level of circ_0102049 in OS patients was markedly correlated with larger tumor size, pulmonary metastasis and poor prognosis. Circ_0102049 remarkably accelerated cell proliferation, migration and invasion but attenuated cell apoptosis in OS cells analyzed by gain/loss of function experiments. What's more, we identified that circ_0102049 functions as a competing endogenous RNA (ceRNA) to competitively sponge miR-1304-5p to upregulate MDM2 expression at posttranscriptional level, thus mediating the cellular behaviors of OS cells. Collectively, our study provides an innovatively regulatory mechanism of circ_0102049 in OS and points out a new way for OS treatment.

Complement component 3 (C3): An important role in grass carp (Ctenopharyngodon idella) experimentally exposed to Aeromonas hydrophila.

Complement is traditionally recognized as part of the innate immune system, defending the host against the invasion of foreign pathogens. In complement system, C3 (complement component 3) is a central component. Therefore, research into C3 can help us better understand the functions of fish complement system. In this study, we detected the grass carp C3 (gcC3) mRNA expression in all sample tissues from healthy grass carp, which was highest in the liver, followed by the heart and the spleen, and lowest in the muscle, head kidney, trunk kidney, blood and intestine. After infection with Aeromonas hydrophila, gcC3 mRNA expression levels were significantly upregulated in the gill, liver, spleen, intestine, trunk kidney and head kidney. Interestingly, C3 protein levels were downregulated and subsequently upregulated in the liver and serum. Histologically, C3 protein at 24 h pi was over expressed in necrotic liver sites, and the liver index (LI) at this point was significantly higher than that of the control. These findings are indicated that C3 plays an important role in the immune response of grass carp after A. hydrophila infection, and C3 protein may play an assistant role in repairing liver tissues from A. hydrophila injury.

Mannose-binding lectin and its roles in immune responses in grass carp (Ctenopharyngodon idella) against Aeromonas hydrophila.

The complement system is a crucial component of the innate immune system that links innate and adaptive immunity via four pathways. Mannose-binding lectin (MBL), the initiating molecule of the lectin pathway, plays a significant role in the innate immune system in mammals and fish. Herein, we identified an MBL homolog (gcMBL) in grass carp (Ctenopharyngodon idella). The full-length 948 bp gcMBL cDNA includes a 741 bp open reading frame encoding a 246 amino acid protein with a signal peptide, collagen triple helix repeat domain, and a C-type lectin-like/link domain. The gcMBL protein shares low similarity with MBL counterparts in other species, and is most closely related to Cyprinus carpio MBL. Transcription of gcMBL was widely distributed in different tissues, and was induced by Aeromonas hydrophila in vivo and in vitro. Expression of gcMBL was also affected by LPS and flagellin stimulation in vitro. In cells over-expressing gcMBL, transcripts of almost all components except gcC5 were up-regulated, and gcMBL, gcIL1ß, gcTNF-α, gcIFN, gcCD59, gcC5aR and gcITGß-2 were significantly up-regulated following exposure to A. hydrophila or stimulation by bacterial PAMPs. Meanwhile, gcMBL deficiency achieved by RNAi down-regulated transcript levels following A. hydrophila challenge, and gcMBL induced NF-κB signalling. These findings indicate a vital role of gcMBL in innate immunity in grass carp.

Biological parameters, immune enzymes, and histological alterations in the livers of grass carp infected with Aeromonas hydrophila.

Aeromonas hydrophila is the causative agent of bacterial septicemia that is frequently observed in grass carp, Ctenopharyngodon idellus. In this study, we evaluated the biological parameters and immune enzymes in the liver of grass carp following A. hydrophila infection and quantified the alterations in liver histology using a semi-quantitative system. For the biological parameters, we found that the liver somatic index (LSI) was more sensitive than Fulton's condition factor (CF) and was significantly decreased at three days post-injection (DPI). At the immune enzyme level, the level of peroxidase (POD) in the liver significantly increased at 1 and 3 DPI. The activity of alkaline phosphatase (ALP) significantly increased at 3 DPI. Similarly, acid phosphatase (ACP) activity significantly increased at 1, 3, and 5 DPI. Histologically, the results indicated that the liver index at 3, 5, and 7 DPI was significantly higher than that of control groups. The regressive alterations as the highly variable reactions patterns and its index at 5 DPI was significantly higher than that of 1, 21 DPI, and the control groups. Based on our results, we suggest that grass carp resist A. hydrophila infection via an innate immune mechanism in the liver. The findings of this study will help elucidate the underlying mechanisms of resistance to A. hydrophila infection.

Mannan-binding lectin-associated serine protease-1 (MASP-1) mediates immune responses against Aeromonas hydrophila in vitro and in vivo in grass carp.

The mannan-binding lectin-associated serine protease-1 (MASP-1) gene is a crucial component of the lectin pathway in the complement and coagulation cascade. Although MASP-1 has been found in the immune system of teleosts, its immune functions in response to bacterial infection are unclear. In this study, we identified a MASP-1 homolog (gcMASP-1) in the grass carp (Ctenopharyngodon idella). The full-length 3308-bp gcMASP-1 cDNA includes a 2160-bp open reading frame encoding a protein composed of 719 amino acids with epidermal growth factor-like, complement control protein, and trypsin-like domains. gcMASP-1 shares a high similarity with MASP-1 counterparts in other species, and it is most closely related to Cyprinus carpio MASP-1 and Sinocyclocheilus anshuiensis MASP-1. Transcription of gcMASP-1 was widely distributed in different tissues and induced by Aeromonas hydrophila in vivo and in vitro. Expression of gcMASP-1 was also affected by lipopolysaccharide and flagellin stimulation in vitro. In cells over-expressing gcMASP-1, transcript levels of almost all components, except gcMBL and gcC5, were significantly enhanced, and gcIL1ß, gcTNF-α, gcIFN, gcCD59, gcC5aR1, and gcITGß-2 were significantly upregulated after exposure to A. hydrophila; gcMASP-1 interference downregulated the transcript levels after A. hydrophila challenge. In addition, gcMASP-1 activated NF-κB signaling. These findings indicate the vital role of gcMASP-1 in innate immunity in C. idella.

Identification and functional analysis of the toll-like receptor 20.2 gene in grass carp, Ctenopharyngodon idella.

We characterized and identified the cDNA sequence of Toll-like receptor 20.2 in Ctenopharyngodon idella (gctlr20.2); it consisted of 3197 bp, with an open reading frame of 2835 bp that encoded a 944 amino acid polypeptide. Relatively, high expression levels of gctlr20.2 were observed in the spleen, head kidney, liver and brain tissues, with lower expression levels in the trunk kidney, intestine and heart tissues. In vivo and in vitro, after being challenged with Aeromonas hydrophila or grass carp reovirus (GCRV), gctlr20.2 expression was induced in C. idella kidney cells stimulated with lipopolysaccharide, flagellin or polyinosinic-polycytidylic acid. Overexpression of gctlr20.2 increased the expression of il1ß, il8 and tnf-α, but not ifn, and also increased the activity of the nf-κB signal pathway. Silencing, via siRNA-tlr20.2, inhibited gctlr20.2 transcription by 65.7% and down-regulated the expression of inflammatory cytokine genes, but not tnf-α. This study increases understanding of the immune system in C. idella.

Transcriptome Analysis of the Innate Immunity-Related Complement System in Spleen Tissue of Ctenopharyngodon idella Infected with Aeromonas hydrophila.

The grass carp (Ctenopharyngodon idella) is an important commercial farmed herbivorous fish species in China, but is susceptible to Aeromonas hydrophila infections. In the present study, we performed de novo RNA-Seq sequencing of spleen tissue from specimens of a disease-resistant family, which were given intra-peritoneal injections containing PBS with or without a dose of A. hydrophila. The fish were sampled from the control group at 0 h, and from the experimental group at 4, 8, 12, 24, 48 and 72 h. 122.18 million clean reads were obtained from the normalized cDNA libraries; these were assembled into 425,260 contigs and then 191,795 transcripts. Of those, 52,668 transcripts were annotated with the NCBI Nr database, and 41,347 of the annotated transcripts were assigned into 90 functional groups. 20,569 unigenes were classified into six main categories, including 38 secondary KEGG pathways. 2,992 unigenes were used in the analysis of differentially expressed genes (DEGs). 89 of the putative DEGs were related to the immune system and 41 of them were involved in the complement and coagulation cascades pathway. This study provides insights into the complement and complement-related pathways involved in innate immunity, through expression profile analysis of the genomic resources in C. idella. We conclude that complement and complement-related genes play important roles during defense against A. hydrophila infection. The immune response is activated at 4 h after the bacterial injections, indicating that the complement pathways are activated at the early stage of bacterial infection. The study has improved our understanding of the immune response mechanisms in C. idella to bacterial pathogens.

Hematological and Immunological plasma assays for grass carp (Ctenopharyngodon idella) infected with Aeromonas hydrophila as an immune model in carp aquaculture.

Aeromonas hydrophila is the causative agent of bacterial septicemia, a common disease observed in grass carp, Ctenopharyngodon idella. In our study, C. idella specimens were infected with A. hydrophila, and parameters of Hematological and Immunological plasma parameters were monitored. At blood cell level, levels of red blood cells (RBCs), hematocrit (HCT), and mean corpuscular volume (MCV) showed no differences between the treatment and control groups, but levels of white blood cells (WBCs) increased. The monocyte and neutrophil varied significant according to stimulation by A. hydrophila at 1 DPI, the thrombocyte and lymphocyte at 14 and 21 DPI. At serum level, total protein, lysozyme, and IgM increased at the early infection phase and then decreased at other time points; however, peroxidase levels were significantly lower in the treatment group than that in the control group during the early infection phase. ACH50 was significantly higher in the treatment group than that in the control group during the late infection phase. On the basis of the results, we suggest that innate and adaptive immune mechanisms of C. idella are able to neutralize the virulence factors secreted by A. hydrophila. Our findings would help in understanding the mechanisms underlying resistance to infection by A. hydrophila.

Electrical stimuli improve osteogenic differentiation mediated by aniline pentamer and PLGA nanocomposites.

Electrical stimulation may improve the proliferation of animal cells. In the present study, osteoblasts were cultured on electroactive aniline pentamer (AP)/poly(lactic-co-glycolic acid) (PLGA) copolymer composites, on which electric pulse was imposed. The combination of polymer and electric pulse enhanced the osteogenic differentiation of the osteoblasts, characterized by the upregulated expression of bone morphogenetic protein (BMP)-2, collagen I and osteonectin and the phosphorylation of Samd4, in contrast to polymer or electrical pulse alone. This action occurred in a polymer content-dependent manner. Therefore, the action of the electric pulse, assisted by the electroactive polymer implant, may be promising in the expedition of injured bone repair.