Norepinephrine regulates TNF expression via the A1AR-p38 MAPK-Relish pathway in granulocytes of oyster Crassostrea gigas.

Norepinephrine (NE) is involved in regulating cytokine expression and phagocytosis of immune cells in the innate immunity of vertebrates. In the present study, the modulation mechanism of NE on the biosynthesis of TNFs in oyster granulocytes was explored. The transcripts of CgTNF-1, CgTNF-2 and CgTNF-3 were highly expressed in granulocytes, and they were significantly up-regulated after LPS stimulation, while down-regulated after NE treatment. The phagocytic rate and apoptosis index of oyster granulocytes were also triggered by LPS stimulation and suppressed by NE treatment. The mRNA expressions of CgMAPK14 and CgRelish were significantly induced after NE treatment, and the translocation of CgRelish from cytoplasm to nucleus was observed. The concentration of intracellular Ca2+ in granulocytes was significantly up-regulated upon NE incubation, and this trend reverted after the treatment with DOX (specific antagonist for NE receptor, CgA1AR-1). No obvious significance was observed in intracellular cAMP concentrations in the PBS, NE and NE + DOX groups. Once CgA1AR-1 was blocked by DOX, the mRNA expressions of CgMAPK14 and CgRelish were significantly inhibited, and the translocation of CgRelish from cytoplasm to nucleus was also dramatically suppressed, while the mRNA expression of CgTNF-1 and the apoptosis index increased significantly to the same level with those in LPS group, respectively. These results collectively suggested that NE modulated TNF expression in oyster granulocyte through A1AR-p38 MAPK-Relish signaling pathway.

An IRF2BP member (CgIRF2BP) involved in negative regulation of CgIFNLP expression in oyster Crassostrea gigas.

The IRF2BP family of transcription regulators act as corepressor molecules by inhibiting both enhancer-activated and basal transcription involving in many biological contexts. In the present study, an IRF2BP homologue (CgIRF2BP) was identified from oyster C. gigas. Its open reading frame is of 1809 bp encoding a polypeptide of 602 amino acids, which contains an IRF-2BP1_2 domain and a RING domain. The mRNA transcripts of CgIRF2BP were detected in all tested tissues with highest level in haemocytes (28.99-fold of that in mantle, p < 0.05). After poly (I:C) stimulation, the expression level of CgIRF2BP was significantly down-regulated at 3 h (0.50-fold of that in control group, p < 0.001) and gradually increased from 6 h to 48 h (2.69-fold of that in control group, p < 0.01). The recombinant protein of CgIRF2BP (rCgIRF2BP) showed high affinity to both rCgIRF1 and rCgIRF8 with Kd value of 1.02 × 10-7 and 2.09 × 10-7, respectively. In CgIRF2BP-RNAi oysters, the mRNA expression of CgIFNLP, CgMx1, CgViperin and CgIFI44L were significantly increased after poly (I:C) stimulation, which were 2.88 (p < 0.01), 1.83 (p < 0.05), 2.47 (p < 0.05), and 1.99-fold (p < 0.01) of that in EGFP group, respectively. These findings suggested that CgIRF2BP negatively regulated CgIFNLP expression by binding with CgIRF1 and CgIRF8.

The revised-risk analysis index as a predictor of major morbidity and mortality in older patients after abdominal surgery: a retrospective cohort study.

BACKGROUND: The revised-Risk Analysis Index (RAI-rev) can accurately predict postoperative mortality risk. However, the association of RAI-rev with composite outcome of major morbidity and mortality (MMM) among older surgical patients is largely unknown. This study investigated the association between RAI-rev and postoperative MMM in older patients undergoing abdominal surgery. It also assessed the predictive value of RAI-rev combined with other preoperative risk factors. METHODS: This retrospective cohort study reviewed the medical records of all patients aged 65 and older who underwent abdominal surgery between January 2018 and December 2019. The primary outcome was the postoperative MMM during hospitalization, and its association with preoperative RAI-rev scores was assessed using multivariable logistic regression analysis. The prediction of postoperative outcomes was used the receiver-operating characteristic curve analysis. RESULTS: A total of 2225 older patients were analyzed, and 258 (11.6%) developed postoperative MMM. After adjusting for confounders, each unit increase in RAI-rev scores resulted in a 2.3% increase in the MMM risk and a 3.0% increase in the odds of life-threatening complications and mortality (both P < 0.05). The area under the curves (AUCs) of RAI-rev scores in predicting MMM and life-threatening complications and mortality was 0.604 (95% CI: 0.567 to 0.640) and 0.633 (95% CI: 0.592 to 0.675), respectively (both P < 0.001); when the RAI-rev was combined with age, gender, American Society of Anesthesiologists (ASA) classification, operative stress, and urgency status of surgery (emergency or elective), the AUCs were 0.694 (95% CI: 0.659 to 0.729) and 0.739 (95% CI: 0.702 to 0.777), respectively (both P < 0.001). CONCLUSIONS: Higher RAI-rev scores were independently associated with increased risk of MMM. When combined with age, gender, ASA classification, operative stress, and urgency status of surgery, RAI-rev had improved performance in predicting the risk of MMM, particularly the life-threatening complications and mortality.

Effectiveness of intraoperative cell salvage combined with a modified leucocyte depletion filter in metastatic spine tumour surgery.

BACKGROUND: To compare the effectiveness of intraoperative cell salvage (IOCS) combined with a modified leucocyte depletion filter (MLDF) with IOCS combined with a regular leucocyte depletion filter (RLDF) in eliminating tumour cells from blood salvage during metastatic spine tumour surgery (MSTS). METHODS: Patients with a known primary epithelial tumour who underwent MSTS were recruited for this study. Blood samples were collected in 5 stages: from the patients' vein before anaesthesia induction (S1), from the operative field at the time of maximum tumour manipulation (S2), and from the operative blood after IOCS processing (S3) and after IOCS+RLDF (S4) and IOCS+MLDF (S5) processing. The polyploids of tumour cells in the blood samples were collected and counted with immunomagnetic separation enrichment and fluorescence in situ hybridization. RESULTS: We recruited 20 patients. Tumour cells were detected in 14 patients (70%) in S1, 16 patients (80%) in S2, 13 patients (65%) in S3, and 12 patients (60%) in S4. MLDF was added in 8 patients. Tumour cells were detected in only 1 of 8 patients in S5 (12.5%). There were significantly fewer tumour cells in the samples collected after MLDF processing (S5) than in the samples collected after RLDF (S4) and around the tumour (S2) (P = 0.016 and P = 0.039, respectively). Although no significant difference was observed between S4 and S1, a downward trend was observed after IOCS+RLDF processing. CONCLUSIONS: Tumour cells could be removed by IOCS combined with RLDF from blood salvaged during MSTS, but residual tumour cells remained. The findings support the notion that MLDF eliminates tumour cells more effectively than RLDF. Hence, this technique can be applied to MSTS. TRIAL REGISTRATION: ChiCTR1800016162 Chinese Clinical Trial Registry.

Inhibition of visfatin alleviates sepsis-induced intestinal damage by inhibiting Hippo signaling pathway.

BACKGROUND: The aim of this study is to investigate role of Visfatin, one of the pro-inflammatory adipokines, in sepsis-induced intestinal injury and to clarify the potential mechanism. METHODS: C57BL/6 mice underwent cecal ligation and puncture (CLP) surgery to establish sepsis model in vivo. Intestinal epithelial cells were stimulated with LPS to mimic sepsis-induced intestinal injury in vitro. FK866 (the inhibitor of Visfatin) with or without XMU-MP-1 (the inhibitor of Hippo signaling) was applied for treatment. The expression levels of Visfatin, NF-κB and Hippo signaling pathways-related proteins were detected by western blot or immunohistochemistry. The intestinal cell apoptosis and intestinal injury were investigated by TUNEL staining and H&E staining, respectively. ELISA was used to determine the production of inflammatory cytokines. RESULTS: The expression of Visfatin increased in CLP mice. FK866 reduced intestinal pathological injury, inflammatory cytokines production, and intestinal cell apoptosis in sepsis mice. Meanwhile, FK866 affected NF-κB and Hippo signaling pathways. Additionally, the effects of FK866 on inflammatory response, apoptosis, Hippo signaling and NF-κB signaling were partly abolished by XMU-MP-1, the inhibitor of Hippo signaling. In vitro experiments also revealed that FK866 exhibited a protective role against LPS-induced inflammatory response and apoptosis in intestinal cells, as well as regulating NF-κB and Hippo signaling, whereas addition of XMU-MP-1 weakened the protective effects of FK866. CONCLUSION: In short, this study demonstrated that inhibition of Visfatin might alleviate sepsis-induced intestinal injury through Hippo signaling pathway, supporting a further research on Visfatin as a therapeutic target.

CTCs detection from intraoperative salvaged blood in RCC-IVC thrombus patients by negative enrichment and iFISH identification: a preliminary study.

BACKGROUND: Intra-operative cell salvage (IOCS) and leukocyte-depleted filter (LDF) are widely used and effective in saving blood. However, the safety issue concerning reinfusion of IOCS-LDF processed blood to renal cell carcinoma (RCC) patients with inferior vena cava (IVC) thrombus were inconclusive for fear of increased risk of cancer metastases. This study intends to analyze the circulating tumor cell (CTC) eliminating effect of IOCS-LDF in 5 RCC-IVC thrombus patients. METHODS: A novel strategy integrating negative enrichment by immunomagnetic beads and immunostaining-fluorescence in situ hybridization with probes identifying aneuploid of 8 and/or 7 were used to detect CTCs from salvages blood. Blood samples were collected from 4 stages in each patient. RESULTS: Of the 5 RCC patients, the number of CTCs decreased (from 3, 4, 10, 7, 3, respectively, to all zero) after IOCS-LDF treatment. The triploid of chromosome 7 and/or chromosome 8 were most common karyotype for RCC patients with IVC thrombus. Tetraploid of chromosome 8 occurred in only one sample and no polypoid (number of chromosome > 4) were found. CONCLUSION: IOCS-LDF might be a promising way of reducing of allogeneic product transfusion based on current preliminary outcome. More convincing conclusions are to be drawn with enlarged sample size and long-term follow-up for patients prognosis.

A novel tumor necrosis factor in the Pacific oyster Crassostrea gigas mediates the antibacterial response by triggering the synthesis of lysozyme and nitric oxide.

Tumor necrosis factors (TNFs) are a group of multifunctional inflammatory cytokines involved in various pathological and immune processes. Recently, a few primitive TNFs have been characterized from molluscs, which play important roles in modulating cell apoptosis, phagocytosis and production of immune-related enzymes. In the present study, a novel TNF (named as CgTNF-2) with the activity to mediate antibacterial response was identified from the Pacific oyster Crassostrea gigas. The open reading frame of CgTNF-2 was of 783 bp encoding a putative polypeptide of 261 amino acids with a typical TNF domain. The deduced amino acid sequence of CgTNF-2 shared high identity with that of TNFs previously identified from other molluscs, such as 96.1% identity with that in oyster C. hongkongensis, 33.7% identity with that in scallop Mizuhopecten yessoensis and 33.0% identity with CgTNF-1 in oyster C. gigas. There were two distinct TNF branches of vertebrate and invertebrate in the phylogenetic tree, and CgTNF-2 was firstly clustered with TNF-14 from C. hongkongensis, and then clustered with other molluscan TNFs. The mRNA transcripts of CgTNF-2 were widely expressed in various oyster tissues, with the highest expression level in hemocytes. The expression level of CgTNF-2 increased significantly at 6 h (2.45-fold and 6.20-fold, respectively, p < 0.05) after peptidoglycan and lipopolysaccharides treatments, and peaked at 12 h (31.86-fold and 7.90-fold, respectively, p < 0.05). The recombinant protein of CgTNF-2 (rCgTNF-2) inhibited the growth of human alveolar basal epithelial (A549) cells at a concentration of 800 ng/mL. After the oysters received an injection of rCgTNF-2, the serum from those oysters exhibited significantly higher antibacterial activity compared to that from control group, evidenced by inhibiting the growth of Vibrio splendidus. Moreover, the lysozyme activity as well as the contents of nitric oxide in the oyster serum also increased significantly. The above results collectively suggested that CgTNF-2 was a novel member of bivalve TNF-α family, which could prompt the antibacterial activity by inducing the lysozyme activity and the production of nitric oxide in the innate immune response of oyster.

The Increased Expression of an Engrailed to Sustain Shell Formation in Response to Ocean Acidification.

Engrailed is a transcription factor required in numerous species for important developmental steps such as neurogenesis, segment formation, preblastoderm organization, and compartment formation. Recent study has proved that engrailed is also a key gene related to shell formation in marine bivalves. In the present study, the expression pattern of an engrailed gene (Cgengrailed-1) in Pacific oyster Crassostrea gigas under CO2-driven acidification was investigated to understand its possible role in the regulation of shell formation and adaptation to ocean acidification (OA). The open reading frame (ORF) of Cgengrailed-1 was obtained, which was of 690 bp encoding a polypeptide of 229 amino acids with a HOX domain. Phylogenetic analysis indicated that the deduced amino acid sequence of Cgengrailed-1 shared high homology with other engraileds from Drosophila melanogaster, Mizuhopecten yessoensi, and Crassostrea virginica. The mRNA transcripts of Cgengrailed-1 were constitutively expressed in various tissues with the highest expression levels detected in labial palp and mantle, which were 86.83-fold (p < 0.05) and 75.87-fold (p < 0.05) higher than that in hepatopancreas. The mRNA expression of Cgengrailed-1 in mantle decreased dramatically after moderate (pH 7.8) and severe (pH 7.4) acidification treatment (0.75- and 0.15-fold of that in control group, p < 0.05). The results of immunofluorescence assay demonstrated that the expression level of Cgengrailed-1 in the middle fold of mantle increased significantly upon moderate and severe acidification treatment. Moreover, after the oyster larvae received acidification treatment at trochophore stage, the mRNA expression levels of Cgengrailed-1 increased significantly in D-shape larvae stages, which was 3.11- (pH 7.8) and 4.39-fold (pH 7.4) of that in control group (p < 0.05). The whole-mount immunofluorescence assay showed that Cgengrailed-1 was mainly expressed on the margin of shell gland, and the periostracum in trochophore, early D-shape larvae and D-shape larvae in both control and acidification treatment groups, and the intensity of positive signals in early D-shape larvae and D-shape larvae increased dramatically under acidification treatment. These results collectively suggested that the expression of Cgengrailed-1 could be triggered by CO2-driven acidification treatment, which might contribute to induce the initial shell formation in oyster larvae and the formation of periostracum in adult oyster to adapt to the acidifying marine environment.

ATG10 (autophagy-related 10) regulates the formation of autophagosome in the anti-virus immune response of pacific oyster (Crassostrea gigas).

Autophagy, a highly conserved intracellular degradation system, is involved in numerous processes in vertebrate and invertebrate, such as cell survival, ageing, and immune responses. However, the detailed molecular mechanism of autophagy and its immune regulatory role in bivalves are still not well understood. In the present study, an autophagy-related protein ATG10 (designated as CgATG10) was identified from Pacific oyster Crassostrea gigas. The open reading frame of CgATG10 cDNA was of 621 bp, encoding a polypeptide of 206 amino acid residues with an Autophagy_act_C domain (from 96 to 123 amino acid), which shared high homology with that from C. virginica and Octopus bimaculoides. The mRNA transcripts of CgATG10 were widely expressed in all the tested tissues including mantle, gonad, gills, hemocytes and hepatopancreas, with the highest expression level in mantle. After the stimulation with poly (I:C), the mRNA expression level of CgATG10 in the mantle of oysters was significantly up-regulated (4.92-fold of that in Blank group, p < 0.05), and the LC3-conversion from LC3-I to LC3-II (LC3-II/LC3-I) also increased. After an additional injection of dsRNA to knock-down the expression of CgATG10 (0.33-fold and 0.10-fold compared respectively with Blank group and dsGFP group, p < 0.05), the downstream conversion of CgLC3 was inhibited significantly compared with that of the control dsGFP group, while the expression level of autophagy-initiator CgBeclin1 did not change significantly. In addition, the mRNA transcripts of interferon regulatory factor CgIRF-1 increased significantly in CgATG10-knockdown oysters at 12 h post poly (I:C) stimulation. All the results indicated that CgATG10 might participate in the immune response against poly (I:C) by regulating autophagosome formation and interferon system in oysters.

A novel globular C1q domain containing protein (C1qDC-7) from Crassostrea gigas acts as pattern recognition receptor with broad recognition spectrum.

The globular C1q domain containing (C1qDC) proteins are a family of versatile pattern recognition receptors (PRRs) to bind various ligands by their globular C1q (gC1q) domain. In the present study, a novel globular C1qDC (CgC1qDC-7) was characterized from Pacific oyster Crassostrea gigas. The open reading frame of CgC1qDC-7 was of 555 bp, encoding a polypeptide of 185 amino acids. Phylogenetic analysis indicated that CgC1qDC-7 shared high homology with C1qDCs from Crassostrea virginica, Mytilus galloprovincialis, and Mizuhopecten yessoensis. The mRNA transcripts of CgC1qDC-7 were widely expressed in all the tested tissues including mantle, gonad, gills, adductor muscle, hemocytes, hepatopancreas and labial palps, with the highest expression level in hemocytes and gills. The recombinant protein of CgC1qDC-7 (rCgC1qDC-7) exhibited binding activity towards Gram-negative bacteria (Vibrio splendidus, V. anguillarum, Escherichia coli, V. alginolyticus, and Aeromonas hydrophila), Gram-positive bacteria (Micrococcus luteus and Staphylococcus aureus) and fungi (Pichia pastoris and Yarrowia lipolytica), and displayed strongest binding affinity towards Gram-negative bacteria V. splendidus and V. anguillarum. It also exhibited affinity to vital pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS), peptidoglycan (PGN), mannan (MAN) and Poly (I:C) with high affinity towards LPS and PGN, and low affinity to MAN and Poly (I:C). These results collectively indicated that CgC1qDC-7 was a novel PRR in C. gigas with high binding affinity towards LPS and PGN as well as Gram-negative bacteria.

Co-expression network analysis of Down's syndrome based on microarray data.

Down's syndrome (DS) is a type of chromosome disease. The present study aimed to explore the underlying molecular mechanisms of DS. GSE5390 microarray data downloaded from the gene expression omnibus database was used to identify differentially expressed genes (DEGs) in DS. Pathway enrichment analysis of the DEGs was performed, followed by co-expression network construction. Significant differential modules were mined by mutual information, followed by functional analysis. The accuracy of sample classification for the significant differential modules of DEGs was evaluated by leave-one-out cross-validation. A total of 997 DEGs, including 638 upregulated and 359 downregulated genes, were identified. Upregulated DEGs were enriched in 15 pathways, such as cell adhesion molecules, whereas downregulated DEGs were enriched in maturity onset diabetes of the young. Three significant differential modules with the highest discriminative scores (mutual information>0.35) were selected from a co-expression network. The classification accuracy of GSE16677 expression profile samples was 54.55% and 72.73% when characterized by 12 DEGs and 3 significant differential modules, respectively. Genes in significant differential modules were significantly enriched in 5 functions, including the endoplasmic reticulum (P=0.018) and regulation of apoptosis (P=0.061). The identified DEGs, in particular the 12 DEGs in the significant differential modules, such as B-cell lymphoma 2-associated transcription factor 1, heat shock protein 90 kDa beta member 1, UBX domain-containing protein 2 and transmembrane protein 50B, may serve important roles in the pathogenesis of DS.

[Mutation analysis of 35 Wilson's disease pedigrees].

OBJECTIVE: To analyze the features of genetic mutations underlying Wilson's disease and provide prenatal and presymptomatic diagnosis. METHODS: For 35 pedigrees affected with the disease, the exons and exon-intron boundaries of the ATP7B gene were amplified with polymerase chain reaction and subjected to Sanger sequencing. After the genotypes of parents of the probands were determined, prenatal diagnosis were performed through chorionic villus sampling. RESULTS: The overall rate for mutation detection was 92.9%. A total of 24 distinct mutations were detected, which included 7 novel mutations, i.e., c.3871G>A(p.A1291T), c.2593_2594insGTCA, c.2790_2792delCAT, c.3661_3663delGGG, c.3700delG, c.4094_4097delCTGT, and IVS6+1G>A. Three mutations, including R778L (c.2333G>T)(45.7%), A874V (c.2621C>T)(7.1%) and P992L (c.2975C>T)(7.1%), were relatively common. Two presymptomatic patients were detected through familial screening, for whom treatment was initiated. Prenatal genetic diagnosis has verified three healthy fetuses and one carrier. CONCLUSION: In this study the most popular mutation ofATP7B gene is R778L and 7 novel mutations have been identified in this gene. For pedigrees of Wilson's disease, genetic counseling in addition with prenatal and presymptomatic diagnosis should be provided through Sanger sequencing and haplotype analysis.

[Rocuronium anesthesia induced anaphylactic shock: a case report].

Anaphylaxis is an acute and fatal systemic allergic reaction to an allergen, and it could be an unpredictable and life-threatening cause during anesthesia. The main purpose of this paper is to report a case of anaphylactic shock during the anesthesia induction and to review the prophylaxis and treatment of anaphylactic reactions and anaphylactoid reactions during the anesthesia period. A 63-year-old man, with a mass on his adrenal, was scheduled to a laparoscopic adrenal tumor excision. During the anesthesia induction period, after administrated sulfentanil, propofol and rocuronium, the blood pressure was decreased and the heart rate was increased. Then, the patient had rash on his whole body and developed an anaphylactic shock. After being treated with the anti-allergic agents and norepinephrine, the rash disappeared and the vital sign become stable. The patient felt nothing uncomfortable during the two weeks'follow-up. Anaphylactic reactions and anaphylactoid reactions are not rare during the anesthesia period. The most common inducements are muscle relaxant, latex and antibiotics. Anaphylactic reactions in the perioperative period are often serious and potentially life-threatening conditions, involving multiple organ systems in which the clinical manifestations are the consequence of the release of preformed mediators from mast cells and basophils. Before anesthesia, we should acquire the allergic history. During the anesthesia period, the vital sign and the skin should be observed carefully.

Prenatal diagnosis using genetic sequencing and identification of a novel mutation in MMACHC.

BACKGROUND: Combined methylmalonic aciduria and homocystinuria, cobalamin(cbl)C deficiency, is a rare disorder of intracellular vitamin B12(cbl) metabolism caused by mutations in the MMACHC gene. Both genetic and biochemical approach have been established to diagnose children and fetuses with cblC deficiency, while in China there is no report of prenatal genetic diagnosis of cblC deficiency. The aim of the present study was to characterize the mutational spectrum of cblC deficiency and investigate the feasibility of genetic-sequencing-based prenatal diagnosis for cblC deficiency. METHODS: 10 pedigrees were recruited in this study with the probands clinically and biochemically confirmed combined methymalonic aciduria and homocystinuria. Peripheral blood samples were collected for MMACHC genetic test from the probands and their parents (4 probands had already dead) and 50 control subjects. The entire coding region and adjacent splice sites of MMACHC were sequenced. After the genotypes of the pedigrees were identified, chorionic villi sampling were performed for 3 high-risk pregnant women for prenatal genetic diagnosis. RESULTS: A total of 7 mutations were identified: c.217C > T (R73X), c.394C > T (R132X), c.463G > C (G155R), c.609G > A (W203X), c.616C > T (R206W), c.658-660delAAG (220delK), and c.567dupT (I190YfsX13), as well as 2 polymophsims: c.321G > A(V107V), c.-302G > T. And G155R is a novel mutation that haven't been reported in the literatures. All the 6 probands identified with compound heterozygous mutations or homozygous mutations of MMACHC gene, and all the parents of the probands were found to have one MMACHC mutation at a heterozygous level. Prenatal diagnosis of fetuses from 3 families with a child affected cblC deficiency showed that one fetus had the same compound heterozygous mutations as the proband, one did not have MMACHC mutation, and the third fetus had a mutation at a heterozygous level of MMACHC gene. Results from the follow-ups were consistent with the prenatal diagnosis. CONCLUSION: A novel mutation p.G155R of the MMACHC gene is identified. Genetic diagonsis is an accurate and convenient method for prenatal diagnosis and early intervention of combined methylmalonic aciduria and homocystinuria.