Roles of ß-catenin in innate immune process and regulating intestinal flora in Qi river crucian carp (Carassius auratus).

In mammals, ß-catenin participates in innate immune process through interaction with NF-κB signaling pathway. However, its role in teleost immune processes remains largely unknown. We aimed to clarify the function of ß-catenin in the natural defense mechanism of Qi river crucian carp (Carassius auratus). ß-catenin exhibited a ubiquitous expression pattern in adult fish, as indicated by real-time PCR analysis. Following lipopolysaccharide (LPS), Polyinosinic-polycytidylic acid (polyI: C) and Aeromonas hydrophila (A. hydrophila) challenges, ß-catenin increased in gill, intestine, liver and kidney, indicating that ß-catenin likely plays a pivotal role in the immune response against pathogen infiltration. Inhibition of the ß-catenin pathway using FH535, an inhibitor of Wnt/ß-catenin pathway, resulting in pathological damage of the gill, intestine, liver and kidney, significant decrease of innate immune factors (C3, defb3, LYZ-C, INF-γ), upregulation of inflammatory factors (NF-κB, TNF-α, IL-1, IL-8), and downregulation of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) activities, increase of Malondialdehyde (MDA) content. Following A. hydrophila invasion, the mortality rate in the FH535 treatment group exceeded that of the control group. In addition, the diversity of intestinal microflora decreased and the community structure was uneven after FH535 treatment. In summary, our findings strongly suggest that ß-catenin plays a vital role in combating pathogen invasion and regulating intestinal flora in Qi river crucian carp.

Effective dose of propofol combined with intravenous esketamine for smooth flexible laryngeal mask airway insertion in two distinct age groups of preschool children.

BACKGROUND: There is limited research on the combined use of propofol and esketamine for anesthesia induction during flexible laryngeal mask airway (FLMA) in pediatric patients, and the effective dosage of propofol for FLMA smooth insertion remains unclear. We explored the effective dose of propofol combined with intravenous esketamine for the smooth insertion of FLMA in two distinct age groups of preschool children. METHODS: This is a prospective, observer-blind, interventional clinical study. Based on age, preschool children scheduled for elective surgery were divided into group A (aged 1-3 years) and group B (aged 3-6 years). Anesthesia induction was started with intravenous administration of esketamine (1.0 mg.kg- 1) followed by propofol administration. The FLMA was inserted 2 min after propofol administration at the target dose. The initial dose of propofol in group A and group B was 3.0 mg.kg- 1 and 2.5 mg.kg- 1, respectively. The target dose of propofol was determined with Dixon's up-and-down method, and the dosing interval of propofol was 0.5 mg.kg- 1. If there was smooth insertion of FLMA in the previous patient, the target dose of propofol for the next patient was reduced by 0.5 mg.kg- 1; otherwise, it was increased by 0.5 mg.kg- 1. The median 50% effective dose (ED50) for propofol was estimated using Dixon's up-and-down method and Probit analysis, while the 95% effective dose (ED95) was estimated through Probit analysis. Vital signs and adverse events during induction were recorded. RESULTS: Each group included 24 pediatric patients. Using Dixon's up-and-down method, the ED50 of propofol combined with esketamine for smooth insertion of FLMA in group A was 2.67 mg.kg- 1 (95%CI: 1.63-3.72), which was higher than that in group B (2.10 mg. kg- 1, 95%CI: 1.36-2.84) (p = 0.04). Using Probit analysis, the ED50 of propofol was calculated as 2.44 (95% CI: 1.02-3.15) mg.kg- 1 in group A and 1.93 (95% CI: 1.39-2.32) mg.kg- 1 in group B. The ED95 of propofol was 3.72 (95%CI: 3.07-15.18) mg.kg- 1 in group A and 2.74 (95%CI: 2.34-5.54) mg.kg- 1 in group B. In Group B, one pediatric patient experienced laryngospasm. CONCLUSION: The effective dose of propofol when combined with intravenous esketamine for smooth insertion of FLMA in children aged 1-3 years is 2.67 mg.kg- 1, which is higher than that in children aged 3-6 years (2.10 mg. kg- 1). TRIAL REGISTRATION: Chinese Clinical Trial Registry Center (Registration Number: ChiCTR2100044317; Registration Date: 2021/03/16).

OmpU and OmpC are the key OMPs for Litopenaeus vannamei hemocyanin recognizes Vibrio parahaemolyticus.

Hemocyanin is a multifunctional protein present in arthropods and mollusks, responsible for oxygen transport and participating in multiple roles of immune defense including antibacterial activity. However, the molecular basis of how hemocyanin recognizes pathogens and exerts antibacterial activity remains poorly understood. In the present study, the pull-down assay was used to isolate Vibrio parahaemolyticus outer membrane proteins (OMPs) that bind to Litopenaeus vannamei hemocyanin. Two interacting OMPs bands were determined as OmpC and OmpU, and the heterogeneous interaction between hemocyanin and the two OMPs was further confirmed by far-Western blot. After construction of ompC and ompU deletion mutants, we found that the agglutinating activity and antibacterial activity of hemocyanin significantly decreased compared to the wild-type strain. After hemocyanin treatment, we identified four intracellular proteins of V. parahaemolyticus, including fructose-bisphosphate aldolase and ribosomal proteins could interact with rOmpC and rOmpU, respectively. Furthermore, we found that the mRNA levels of ompC, ompU, fbaA, rpsB and rpsC significantly decreased after hemocyanin treatment. These findings indicated that OmpC and OmpU are the key targets for L. vannamei hemocyanin recognize pathogens and exert its antibacterial activity.

The Yin Yang 1 of Penaeus vannamei regulates transcription of the small subunit hemocyanin gene during Vibrio parahaemolyticus infection.

Hemocyanin is a respiratory protein, it is also a multifunctional immune molecule that plays a vital role against pathogen invasion in shrimp. However, the regulation of hemocyanin gene expression in shrimp hemocytes and the mechanisms involved during pathogen infection remains unclear. Here, we used DNA pull-down followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the Yin Yang 1 transcription factor homolog in Penaeus vannamei (PvYY1) as a key factor that modulates transcription of the small subunit hemocyanin gene of P. vannamei (PvHMCs) in hemocytes during Vibrio parahaemolyticus AHPND (VPAHPND) infection. Bioinformatics analysis revealed that the core promoter region of PvHMCs contains two YY1 motifs. Mutational and oligoprecipitation analyses confirmed that PvYY1 could bind to the YY1 motifs in the PvHMCs core promoter region, while truncation of PvYY1 revealed that the N-terminal domain of PvYY1 is essential for the transactivation of PvHMCs core promoter. Besides, the REPO domain of PvYY1 could repress the activity of the PvHMCs core promoter. Overexpression of PvYY1 significantly activates the promoter activity of PvHMCs core promoter, while PvYY1 knockdown significantly decreases the expression level of PvHMCs in shrimp hemocytes and survival rate of shrimp upon infection with VPAHPND. Our present study provides new insights into the transcriptional regulation of PvHMCs by PvYY1 in shrimp hemocytes during bacteria (VPAHPND) infection.

Role of HIF in fish inflammation.

The hypoxia-inducing factor (HIF) is a central transcription factor in cellular oxygen sensing and regulation. It is common that the inflammation always appears in many diseases, like infectious diseases in fishes, and the inflammation is often accompanied by hypoxia, as a hallmark of inflammation. Besides coordinating cellular responses to low oxygen, HIF-mediated hypoxia signaling pathway is also crucial for immune responses such as the regulations of innate immune cell phenotype and function, as well as metabolic reprogramming under the inflammation. However, the understanding of the molecular mechanisms by which HIFs regulate the inflammatory response in fish is still very limited. Here, we review the characteristics of HIF as well as its roles in innate immune cells and the infections caused by bacteria and viruses. The regulatory effects of HIF on the metabolic reprogramming of innate immune cells are also discussed and the future research directions are outlooked. This paper will serve as a reference for elucidating the molecular mechanism of HIF regulating inflammation and identifying treatment strategies to target HIF for fish disease.

Functional roles of CcGSDMEa-like in common carp (Cyprinus carpio) after Aeromonas hydrophila infection.

GSDMs could punch holes in cell membrane and participate in the immune response to bacterial infections. In current study, the molecular and structural characteristics of CcGSDMEa-like were analyzed, and the role of CcGSDMEa-like in the inflammatory response against Aeromonas hydrophila was studied. The results showed that the CcGSDMEa-like shared the conserved structural characteristics with GSDMEs of other teleosts. The CcGSDMEa-like mRNA and protein expression levels were significantly affected by A. hydrophila challenge. When the CcGSDMEa-like was overexpressed, the expression of CcIL-1ß were significantly increased in fish and EPC cells, and bacterial contents were significantly decreased in fish tissues. While, when the CcGSDMEa-like was knocked down, the expression and secretion of CcIL-1ß were significantly decreased in vivo and in vitro, and the bacterial contents were increased in vivo after A. hydrophila infection 12 h and 24 h. In brief, CcGSDMEa-like could regulate the content of bacteria in fish through mediating the expression and secretion of CcIL-1ß. Bactericidal assay and cytotoxicity assay showed that CcGSDMEa-like had no bactericidal activity to Escherichia coli, and did not disrupt cytomembrane integrity of HEK293T cells. This study suggested that CcGSDMEa-like could play roles in the antibacterial and inflammatory processes in fish.

The role of CcPTGS2a in immune response against Aeromonas hydrophila infection in common carp (Cyprinus carpio).

Prostaglandin-endoperoxide synthase 2 (PTGS2), a crucial enzyme in prostaglandin synthesis, catalyzes the conversion of arachidonic acid to prostaglandins and plays a significant role in the inflammatory response. This investigation aimed to determine the regulatory role of PTGS2a in the innate immune response to bacterial infection in fish. To achieve this objective, the CcPTGS2a gene was identified and characterized in common carp (Cyprinus carpio), and its function in immune defense was investigated. According to the sequence and structural analysis results, CcPTGS2a had an open reading frame of 1806 bp that encoded 602 amino acids. It was estimated that the protein's theoretical molecular weight was 69.0 kDa, and its isoelectric point was 8.10. The structure of CcPTGS2a was observed to be conserved, with an epidermal growth factor domain and a peroxidase domain present. Moreover, the amino acid sequence of CcPTGS2a exhibited significant homology with the amino acid sequences of several fish species. CcPTGS2a mRNA was detected in the healthy tissues of common carp, with higher expression in the head kidney, spleen, gills, and liver. Following the challenges with Aeromonas hydrophila and lipopolysaccharide, CcPTGS2a mRNA showed unique geographic and temporal expression patterns, with significant increases detected in the head kidney, gills, spleen, and liver. Additionally, the recombinant CcPTGS2a protein exhibited detectable bacterial binding to various bacteria. As determined by subcellular localization analysis, CcPTGS2a was predominantly localized in the nucleus and cytoplasm. Furthermore, it was discovered that the overexpression of CcPTGS2a stimulated the up-regulation of ferroptosis-related genes and inflammatory cytokine mRNA expression in fish and EPC (Epithelioma papulosum cyprinid) cells while concurrently reducing the bacterial load of A. hydrophila. In contrast, the interference of CcPTGS2a decreased the mRNA expression of ferroptosis-related genes and inflammatory cytokines in fish and EPC cells and increased the bacterial load of A. hydrophila. Notably, A. hydrophila stimulation resulted in the up-regulation of CcPTGS2a protein expression in EPC cells. These results suggested that CcPTGS2a was involved in the immune response to bacterial infections in common carp.

Two CcCCL19bs orchestrate an antibacterial immune response in Yellow River carp (Cyprinus carpio haematopterus).

Chemokines are a group of chemotactic cytokines with an essential role in homeostasis as well as immunity via specific G protein-coupled receptors and atypical receptors. In our study, two Yellow River carp (Cyprinus carpio haematopterus) CCL19b genes (CcCCL19bs), tentatively named CcCCL19b_a and CcCCL19b_b, were cloned. The open reading frames (ORFs) of CcCCL19b_a and CcCCL19b_b were both 333 bp that encoded a 12 kDa protein with 110 amino acid residues. CcCCL19bs contained a signal peptide and a SCY domain with four typical conserved cysteine residues. The two CcCCL19b proteins shared high similarities with each other in both secondary and three-dimensional structure. Phylogenetic analysis showed that CcCCL19bs and other CCL19bs from tetraploid cyprinid fish were clustered into one clade. CcCCL19bs were highly expressed in gill and intestine in healthy fish, and a significant up-regulation of gene expression after Aeromonas hydrophila infection and poly(I:C) stimulation was observed in gill, liver, and head kidney. Furthermore, chemotaxis and antibacterial activity of CcCCL19bs were studied. The results indicated that recombinant CcCCL19b_a and CcCCL19b_b protein (rCcCCL19b_a and rCcCCL19b_b) exhibited significant attraction to primary head kidney leukocytes (HKLs). Meanwhile, both of rCcCCL19bs could promote the proliferation of HKLs, and significantly up-regulate the expressions of IL-1ß, CCR7, and IL-6, and down-regulate the expression of IL-10 in primary HKLs. In vitro, rCcCCL19bs could bind and aggregate A. hydrophila and Staphylococcus aureus. The rCcCCL19bs exhibited significant antibacterial activity against A. hydrophila, but not S. aureus. Moreover, they inhibited the growth of A. hydrophila and S. aureus. In vivo, overexpression of CcCCL19bs contributed to the bacterial clearance. These studies suggested that CcCCL19bs orchestrate an antibacterial immune response.

Characterization of hepcidin gene and protection of recombinant hepcidin supplemented in feed against Aeromonas hydrophila infection in Yellow River carp (Cyprinus carpio haematopterus).

Hepcidin is a small peptide of defensins with antibacterial activity, and plays an important role in innate immunity against pathogenic microorganisms, which can also participate in the regulation of iron metabolism. The hepcidin gene in Yellow River carp (Cyprinus carpio haematopterus) (CcHep) was cloned and identified. The total length of CcHep cDNA was 480 bp, containing an open reading frame (ORF) that encoded 91 amino acids (aa), which contained a 24-aa signal peptide, a 42-aa propeptide, and a 25-aa mature peptide. The mature peptide had a typical RX (K/R) R motif and eight conserved cysteine residues forming four pairs of disulfide bonds. Homology and phylogenetic tree analysis showed that CcHep had the closest relationship with that of crucian carp. The expression levels of hepcidin mRNA in healthy and Aeromonas hydrophila stimulated fish were measured by real-time fluorescence quantitative PCR. The results showed that CcHep mRNA was expressed in different tissues of healthy fish with the highest relative expression level in liver, followed by kidney and intestine, and the lowest expression level was observed in heart. The hepcidin gene was extremely significantly up-regulated in head kidney, intestine, liver, skin, spleen, and gill at 6 h and 12 h after A. hydrophila infection. Furthermore, the immunoregulation effect of dietary recombinant protein was evaluated. The recombinant hepcidin protein (rCcHep) was successfully expressed by Pichia pastoris X-33 and showed strong antibacterial activity against A. hydrophila, Escherichia coli, Vibrio anguillarum and Bacillus subtilis in vitro. In order to evaluate the preventive effect of rCcHep, fish were fed with basal diet or diet supplemented with different doses of rCcHep, and then challenged with A. hydrophila. The results showed that immune genes were up-regulated to varying degrees, and feed additive groups exhibited a significantly improved up-regulation expressions of Lysozyme, Toll-like receptor 5 (TLR 5), Major histocompatibility complex classⅡ (MHCⅡ), while inhibited up-regulation expressions of Interleukin 1ß (IL-1ß), Interleukin 8 (IL-8), and Tumor necrosis factor α (TNF-α) in liver and spleen compared to the control. Meanwhile, the relative immune protection rate in 120 mg/kg feed additive group was 28%, and the bacterial clearance rate in tissues of this group was higher than that of the control. Collectively, these results indicated that rCcHep had antibacterial activity and showed an immune protection effect against A. hydrophila, and could be considered as a dietary supplement to apply in aquaculture.

Structural Change and Radical Generation of a Cadmium-Based Metal-Organic Framework Induced by an Electric Field.

Herein we report the structural change and radical generation of a cadmium-based metal-organic framework (Cd-MOF) induced by external electric fields. Under a weaker single electric field, different coordination modes of Cd-L lead to 3D → 2D structural change. Under stronger superposed electric fields, Cd-MOF was excited to produce a stable free radical. This study will provide a new avenue for the controlled assembly of MOFs.

Characterization of CD3γ/δ gene and its immune response in Qihe crucian carp Carassius auratus after challenged by Aeromonas veronii and Poly(I:C).

CD3γ/δ found in non-mammalian vertebrates is a CD3 homolog with structural characteristics similar to both mammalian CD3γ and CD3δ, and plays important roles in T cell recognization and immune response in fish. In this study, the full-length of CD3γ/δ from Qihe crucian carp (named CaCD3γ/δ) was cloned and characterized, then the expression response profiles and potential immune functions was explored after Aeromonas veronii and Poly(I:C) challenge. The results showed that the full-length of CaCD3γ/δ was 819 bp including a 5'-UTR of 141 bp, a 3'-UTR of 168 bp, and an ORF of 510 bp encoding a putative 169-aa protein with an estimated MW of 18.71 kD and a theoretical pI of 8.77. The protein sequence of CaCD3γ/δ contained a Leu-Leu and a CXXXC motif in the extracellular domain, and an ITAM and a Leu-Ile motif in the cytoplasm, and a residue of Asn in the transmembrane. CaCD3γ/δ was constitutively expressed in the spleen, liver, gill, and blood of Qihe crucian carp. After the carp were challenged with Poly(I:C) and Aeromonas veronii, the mRNA expression levels of CaCD3γ/δ were significantly changed in the spleen, head kidney, intestine and gill, according to the results of qPCR. However, compared with A. veronii, Poly(I:C) challenge can rapidly induce the CaCD3γ/δ expression levels in head kidney, intestine and spleen, which suggested CaCD3γ/δ may be differentially modulated by different pathogens. Moreover, the results of immunohistochemical analysis showed that the CaCD3γ/δ+ secreted cells in the spleen and gills of Qihe crucian were increased after challenged with Poly(I:C), as well as the spleen challenged with A. veronii, but at different levels. Combined with the fact that vascular congestion, necrosis of parenchymal cells, and inflammatory cells including lymphocytes infiltration were also observed in the gill and spleen of Qihe crucian carp treated with A. veronii and Poly(I:C) revealed by pathological analysis, it was predicted that CaCD3γ/δ+ T lymphocytes may participated in the immune response against pathogens. This study will contribute to understand the important role of CaCD3γ/δ+ T lymphocytes in the immune response of Qihe crucian carp, and provide new insights for the prevention and treatment of the diseases of Qihe crucian carp.

Contribution of flagellar cap gene in virulence and pathogenicity of Aeromonas veronii.

Aeromonas veronii is an important zoonotic and aquatic pathogen that causes a number of illnesses in both humans and animals. It is related to gastroenteritis, skin and soft tissue infections and bacteremia in humans, as well as causing significant economic losses in aquaculture owing to fish sepsis. Here, we constructed the flagellar cap gene (fliD) mutant strain of A. veronii by suicide plasmid-mediated homologous recombination system and analysed its characteristics. It was found that the deletion of fliD had no effect on growth and biochemical properties and could be inherited stably. However, the motility of A. veronii ΔfliD was significantly reduced, the flagellum was defective and the biofilm formation was attenuated compared with that of A. veronii wild-type strain. In vivo experiments revealed that the colonization capacity of ΔfliD was significantly lower than that of the wild-type strain in the period of first 24 h, and the median lethal dose (LD50 ) was 56 times higher than that of the wild-type strain. The Cyprinus carpio infected with the wild-type strain indicated faster death speed and more severe clinical signs compared to ΔfliD strain. These results suggest that fliD is closely related to the virulence of A. veronii and plays an important role in pathogenicity, providing the foundation for pathogenic mechanism studies of A. veronii.

Induction and potential molecular mechanism of the enhanced immune response in Procambarus clarkii after secondary encountered with Aeromonas veronii.

For a long time, it was believed that invertebrates do not possess acquired immunity and mainly rely on innate immunity for protection against pathogens infection. However, an increasing number of studies have suggested that some form of "immune memory" can be initiated in invertebrates after primary exposure to the pathogen, which was defined as "specific immune priming". In the present study, two experiments were carried out to determine whether specific immune priming can be induced in crayfish (Procambarus clarkii) by Aeromonas veronii, if so, to identify the underlying mechanism. Once being "preimmunization" by formalin-killed A. veronii, the survival rate, in vitro antibacterial activity and haemocyte phagocytosis rate of crayfish were enhanced, which indicated that better immune protection was obtained. Furthermore, at some time points, the expression of antimicrobial peptide (AMP) and Down syndrome cell adhesion molecule (Dscam) genes was significantly higher in P. clarkii individuals that underwent stimulation twice than in those that were only stimulated once. Taken together, the results suggest that enhanced specific immune protection can be obtained in primed crayfish and that the Dscam molecule, haemocyte phagocytosis function, and AMPs may be involved in this immune priming. The present study provides a better understanding of the molecular mechanism of immune priming in invertebrates.

Molecular characteristics and the roles of CaASC and its restriction to Aeromonas hydrophila in Carassius auratus.

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), as a critical adaptor molecule in inflammasome complexes, plays an important role in mediating inflammation reaction. In this study, the complete cDNA of ASC gene with 891 bp was cloned in Qihe crucian carp Carassius auratus (named as CaASC), which was composed of a 5'-UTR of 36 bp, a 3'-UTR of 252 bp, and an ORF of 603 bp encoded 200 amino acids with a predicted isoelectric point of 5.61 and a molecular mass of 22.0 kDa. Multiple sequence alignment and motif analysis revealed that CaASC contained a conserved N-terminal Pyrin domain (PYD) and a C-terminal Caspase recruitment domain (CARD). CaASC mRNA and protein expressions were detected in selected tissues, with the highest mRNA level in the spleen. Meanwhile, CaASC gene expressions were clearly altered in intestine, gill, skin, spleen, liver and head kidney of fish challenged by Aeromonas hydrophila, LPS, and polyI:C, respectively. The recombined proteins of CaASC with fluorescent tag were over-expressed in transfected 293T cells, and the green specks were observed obviously and located in the cytoplasm. Furthermore, knockdown of CaASC reduced the expression of IL-1ß and promoted the bacterial colonization in fish tissues, while overexpression of CaASC increased the expression of IL-1ß and hampered the bacterial colonization in these tissues. Taken together, these results identified the molecular characteristics of CaASC in C. auratus, and revealed its role in regulating IL-1ß expression and restricting bacterial infection in vivo.

Amine-Linked Flavonoids as Agents Against Cutaneous Leishmaniasis.

We have designed, synthesized, and characterized a library of 38 novel flavonoid compounds linked with amines. Some of these amine-linked flavonoids have potent in vitro activity against parasites that cause cutaneous leishmaniasis, a tropical disease endemic in 80 countries worldwide. The most promising candidate, FM09h, was highly active with IC50 of 0.3 µM against L. amazonensis, L. tropica and L. braziliensis amastigotes. It was metabolically stable (39% and 66% of FM09h remaining after 30-minute incubation with human and rat liver microsomes respectively). In L. amazonensis LV78 cutaneous leishmaniasis mouse model, intralesional injection of FM09h (10 mg/kg, once every 4 days for 8 times) demonstrated promising effect in reducing the footpad lesion thickness by 72%, displaying an efficacy comparable to SSG (63%).

Sedative effect and safety of different doses of S-ketamine in combination with propofol during gastro-duodenoscopy in school-aged children: a prospective, randomized study.

BACKGROUND: Propofol combined with opioids can reduce the dosage of propofol and improve the safety of endoscopy. However, there are few studies on propofol combined with S-ketamine in children undergoing gastro-duodenoscopy. We aim to determine the sedative effect and safety of different doses of S-ketamine in combination with propofol in school-aged children undergoing gastro-duodenoscopy. METHODS: This is a prospective, randomized trial. Totally, 120 school-aged children who underwent gastro-duodenoscopy were randomly allocated into Group P, Group S0.3, Group S0.5 and Group S0.7. During induction, children in Group P, Group S0.3, Group S0.5 and Group S0.7 received 0, 0.3 mg.kg-1, 0.5 mg.kg-1 and 0.7 mg.kg-1 S-ketamine, respectively, following 3 mg.kg-1 propofol injection. During gastro-duodenoscopy, 1 mg.kg-1 of propofol was added according to the condition of the children and the BIS (bispectral index) value. The primary outcome was smooth placement rate of the first endoscope insertion. The secondary outcome was the times of additional propofol, the total amount of propofol, adverse events, recovery time, length of PACU (post anesthesia care unit) stay and endoscopist satisfaction. RESULTS: The smooth placement rate of the first endoscope insertion in Group P, Group S0.3 and Group S0.5 was significantly lower than that in Group S0.7 (16.70%, 34.50%, 50.00% vs. 83.30%, respectively, P < 0.001). The times of additional propofol in Group S0.3 (P = 0.018), Group S0.5 (P = 0.014) and Group S0.7 (P = 0.001) were significantly less than Group P. The total amount of propofol in Group S0.7 was significantly less than Group P (P < 0.001). The incidence of intraoperative hypotension in Group S0.5 and Group S0.7 was low. Group S0.7 had significantly higher incidence of postoperative dizziness (P = 0.003), longer PACU stay (P = 0.018) and higher endoscopist satisfaction (P = 0.001) than Group P. There was no difference in the recovery time among groups. CONCLUSION: S-ketamine (0.7 mg.kg-1) in combination with propofol can provide satisfactory sedative effect and reduce the dosage of propofol in school-aged children undergoing gastro-duodenoscopy, but there are higher incidence of postoperative dizziness and longer PACU stay.

Molecular characterization of NLRC3 and its interaction with other inflammasome components and regulation on the bacterial colonization in Qihe crucian carp Carassius auratus.

The NLRC as a very unique subfamily of Nod like receptors (NLRs), is believed to play an important role in the bacterial recognition of animals. However, the molecular characterization and immunological role of NLRC3 in Carassius auratus is little known. In this study, we identified and achieved a complete cDNA sequence of NLRC3 gene in Qihe crucian carp Carassius auratus (named as CaNLRC3). The full-length cDNA sequence of CaNLRC3 was composed of 3823 bp, which contained a 5'-UTR of 251 bp, a 3'-UTR of 158 bp, and an open reading frame (ORF) of 3414 bp encoded 1137 amino acids with a predicted isoelectric point of 8.25 and a molecular mass of 124.1 kDa, characterized with a caspase recruitment domain (CARD) at N-terminus. The mRNA expression of CaNLRC3 was detected to be constitutive in all the examined tissues, with the high expression levels in spleen, skin and intestine. After challenges with bacteria or pathogenic analogue, expression levels of CaNLRC3 gene were strongly induced. Co-localization and co-immunoprecipitation results found that CaNLRC3 can assemble CaASC through CARD domain interaction, then CaASC associated with CaCaspase-1a, presumably to assemble the NLRC3 inflammasome complex. The overexpression of CaNLRC3 could significantly increase the mRNA expression of IL-1ß, and promote the bacterial elimination and result in the decrease of bacterial loading in liver, spleen and kidney after bacterial infection. Vice versa, the knockdown of CaNLRC3 could obviously reduce IL-1ß expression at mRNA level, and bacterial loading was significantly increased in tissue. Taken together, CaNLRC3 is proved to be a pivotal cytosolic innate immune receptor in this study, which is acted as the potential component of inflammasome to regulate inflammation reaction, and could modify bacterial loading in tissue and restrict bacterial infection in teleost.

Genome-wide identification, evolutionary analysis, and antimicrobial activity prediction of CC chemokines in allotetraploid common carp, Cyprinus carpio.

Chemokines are a group of secreted small molecules which are essential for cell migration in physiological and pathological conditions by binding to specific chemokine receptors. They are structurally classified into five groups, namely CXC, CC, CX3C, XC and CX. CC chemokine group is the largest one among them. In this study, we identified and characterized 61 CC chemokines from allotetraploid common carp (Cyprinus carpio). The sequence analyses showed that the majority of CC chemokines had an N-terminal signal peptide, and an SCY domain, and all CC chemokines were located in the extracellular region. Phylogenetic, evolutionary and syntenic analyses confirmed that CC chemokines were annotated as 11 different types (CCL19, CCL20, CCL25, CCL27, CCL32, CCL33, CCL34, CCL35, CCL36, CCL39, and CCL44), which exhibited unique gene arrangement pattern and chromosomal location respectively. Furthermore, genome synteny analyses between common carp and four representative teleost species indicated expansion of common carp CC chemokines resulted from the whole genome duplication (WGD) event. Additionally, the continuous evolution of gene CCL25s in teleost afforded a novel viewpoint to explain the WGD event in teleost. Then, we predicted the three-dimensional structures and probable function regions of common carp CC chemokines. All the CC chemokines core structures were constituted of an N-loop, a three-stranded ß-sheet, and a C-terminal helix. Finally, 43 CC chemokines were predicted to have probable general antimicrobial activity. Their tertiary structures, cationic and amphiphilic physicochemical property supported the viewpoint. To verify the prediction, six recombinant CCL19s proteins were prepared and the antibacterial activity against Escherichia coli and Aeromonas hydrophila were verified. The results supported our prediction that rCCL19a.1s (rCCL19a.1_a, rCCL19a.1_b) and rCCL19bs (rCCL19b_a, rCCL19b_b), especially rCCL19bs, exhibited extremely significant inhibition to the growth of both E. coli and A. hydrophila. On the contrary, two rCCL19a.2s had no significant inhibitory effect. These studies suggested that CC chemokines were essential in immune system evolution and not monofunctional during pathogen infection.

Synergistic Effect of Quercetin on Antibacterial Activity of Florfenicol Against Aeromonas hydrophila In Vitro and In Vivo.

The overuse or abuse of antimicrobial drugs in aquaculture, aggravates the generation of drug-resistant bacteria, which has caused potential risks to human health and the aquaculture industry. Flavonoid-antibiotic combinations have been shown to suppress the emergence of resistance in bacteria, and sometimes even reverse it. Here, the antibacterial activity of florfenicol in combination with quercetin, a potential drug to reverse multidrug resistance, was tested against Aeromonas hydrophila (A. hydrophila). Of eleven selected antimicrobial agents, quercetin and florfenicol showed the strongest bactericidal effect, and fractional inhibitory concentration (FIC) indices were 0.28, showing a highly synergistic effect. Then, the antibacterial activities of quercetin and florfenicol against A. hydrophila were further tested in vitro and in vivo. Bacterial viability of A. hydrophila decreased in a florfenicol dose-dependent manner, about 16.3-191.4-fold lower in the presence of 15 µg/mL quercetin and 0.156 to 1.25 µg/mL florfenicol than in the absence of quercetin, respectively. The cell killing was maximum at 45 µg/mL quercetin in the dose range tested plus 0.156 µg/mL florfenicol. The viability decreased over time during the combined treatment with quercetin and florfenicol by 60.5- and 115-fold in 0.156 µg/mL florfenicol and 0.625 µg/mL florfenicol, respectively. Additionally, the synergistic effect was confirmed by the bacterial growth curve. Furthermore, quercetin and florfenicol had an obvious synergistic activity in vivo, reducing the bacterial load in the liver, spleen and kidney tissues of Cyprinus carpio up to 610.6-fold compared with the florfenicol group, and improving the survival rate of infected fish from 10% in the control group to 90% in drug combinations group. These findings indicated that quercetin could potentiate the antibacterial activity of florfenicol against A. hydrophila infection and may reduce the use of antimicrobial drugs and improve the prevention and control capability of bacterial resistance.

Molecular characterization of Rab7 and its involvement in innate immunity in red swamp crayfish Procambarus clarkii.

Rab7 is a member of the Rab GTPases protein family, and it plays an essential role in regulating trafficking organelles in higher animals. However, recent studies showed that it also participated in the immune response and cytophagy against pathogens in invertebrates. In the present study, the full-length of Rab7 from Procambarus clarkii (PcRab7) was cloned, and its function during pathogen infection and phagocytosis of haemocytes was also explored. The results showed that the full-length of PcRab7 was 3639 bp, containing a 618 bp open reading frame encoding 155 amino acids. The predicted molecular weight and isoelectric point of PcRab7 were 23.2 kDa and 5.77, respectively. PcRab7 was widely expressed in various tissues including haemocytes, intestine, muscle, gill, and hepatopancreas, and the highest expression level was in haemocytes. The mRNA transcripts of PcRab7 in the main organs (gill, intestine, and hepatopancreas, and haemocytes) were significantly affected by white spot syndrome virus (WSSV) and Aeromonas veronii infection. Subsequently, the prokaryotic and eukaryotic expression vectors were successfully constructed, and polyclonal antibodies, which could specifically recognize the endogenous Rab7 protein, were also obtained. Furthermore, the phagocytosis rate of haemocytes against FITC-labeled A. veronii was significantly decreased when the PcRab7 was silenced, while the over-expression of Rab7 increased the phagocytosis rate of haemocytes. The abnormal expression of Rab7 protein could also affect the survival rate of P. clarkii infected with WSSV or A. veronii. These results could provide a basis for further study on the immunological function of PcRab7.