Development and application of a CNT-Ag-Cu-Al/PS-based paper electrode for detecting diverse analytes in complex matrices.

Electrochemical sensors play a crucial role in the detection of different analytes in complex matrices, and their performance is highly dependent on the electrode capacity. However, most of the available electrodes can only be used for single-component detection, so it is urgent to develop electrodes with high sensitivity and selectivity for different components. Herein, we report an amphiprotic amino-bonded carbon nanotube-Ag/Cu/Al nanoparticle/polystyrene-coated paper electrode (CNT-Ag-Cu-Al/PS electrode), which can be used for the measurement of glucose (Glc), oxytetracycline (OTC), and hydroquinone (HQ), respectively. The results showed that the analytical sensitivity and selectivity of the CNT-Ag-Cu-Al/PS electrode were comparable to those of single metal-coated paper substrate. The developed electrode also exhibited excellent linear responses for Glc, OTC, and HQ in the ranges of 1.0-1000.0 µM, 1.0 × 10-2 to 10.0 µM, and 5.0 × 10-3 to 50.0 µM, and the limits of detection (LODs) were 0.2055 µM (Glc), 0.0074 µM (OTC), and 0.0048 µM (HQ). Owing to the characteristics of good selectivity, anti-interference, stability, and reproducibility, the CNT-Ag-Cu-Al/PS paper electrode has been successfully applied to the detection of these analytes in complex human body fluids, food, and environmental waters. The paper electrode is promising for the detection of target compounds in complex matrices.

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.

Full-length transcriptome analysis of a bloom-forming dinoflagellate Prorocentrum shikokuense (Dinophyceae).

Prorocentrum shikokuense (formerly P. donghaiense) is a pivotal dinoflagellate species associating with the HABs in the East China Sea. The complexity of its large nuclear genome hindered us from understanding its genomic characteristics. Full-length transcriptome sequencing offers a practical solution to decipher the physiological mechanisms of a species without the reference genome. In this study, we employed single-molecule real-time (SMRT) sequencing technology to sequence the full-length transcriptome of Prorocentrum shikokuense. We successfully generated 41.73 Gb of clean SMRT sequencing reads and isolated 105,249 non-redundant full-length non-chimeric reads. Our trial has led to the identification of 11,917 long non-coding RNA transcripts, 514 alternative splicing events, 437 putative transcription factor genes from 17 TF gene families, and 34,723 simple sequence repeats. Additionally, a total of 78,265 open reading frames were identified, of them 15,501 were the protein coding sequences. This dataset is valuable for annotating P. shikokuense genome, and will contribute significantly to the in-depth studies on the molecular mechanisms underlining the dinoflagellate bloom formation.

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.

Enhanced stability of M1 protein mediated by a phospho-resistant mutation promotes the replication of prevailing avian influenza virus in mammals.

Avian influenza virus (AIV) can evolve multiple strategies to combat host antiviral defenses and establish efficient infectivity in mammals, including humans. H9N2 AIV and its reassortants (such as H5N6 and H7N9 viruses) pose an increasing threat to human health; however, the mechanisms involved in their increased virulence remain poorly understood. We previously reported that the M1 mutation T37A has become predominant among chicken H9N2 isolates in China. Here, we report that, since 2010, this mutation has also been found in the majority of human isolates of H9N2 AIV and its emerging reassortants. The T37A mutation of M1 protein enhances the replication of H9N2 AIVs in mice and in human cells. Interestingly, having A37 instead of T37 increases the M1 protein stability and resistance to proteasomal degradation. Moreover, T37 of the H9N2 M1 protein is phosphorylated by protein kinase G (PKG), and this phosphorylation induces the rapid degradation of M1 and reduces viral replication. Similar effects are also observed in the novel H5N6 virus. Additionally, ubiquitination at K187 contributes to M1-37T degradation and decreased replication of the virus harboring T37 in the M1 protein. The prevailing AIVs thereby evolve a phospho-resistant mutation in the M1 protein to avoid viral protein degradation by host factors, which is advantageous in terms of replication in mammalian hosts.

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.

p21 restricts influenza A virus by perturbing the viral polymerase complex and upregulating type I interferon signaling.

Many cellular genes and networks induced in human lung epithelial cells infected with the influenza virus remain uncharacterized. Here, we find that p21 levels are elevated in response to influenza A virus (IAV) infection, which is independent of p53. Silencing, pharmacological inhibition or deletion of p21 promotes virus replication in vitro and in vivo, indicating that p21 is an influenza restriction factor. Mechanistically, p21 binds to the C-terminus of IAV polymerase subunit PA and competes with PB1 to limit IAV polymerase activity. Besides, p21 promotes IRF3 activation by blocking K48-linked ubiquitination degradation of HO-1 to enhance type I interferons expression. Furthermore, a synthetic p21 peptide (amino acids 36 to 43) significantly inhibits IAV replication in vitro and in vivo. Collectively, our findings reveal that p21 restricts IAV by perturbing the viral polymerase complex and activating the host innate immune response, which may aid the design of desperately needed new antiviral therapeutics.

Lentinan Attenuates Damage of the Small Intestinal Mucosa, Liver, and Lung in Mice with Gut-Origin Sepsis.

This study is aimed at exploring the effects of lentinan on small intestinal mucosa as well as lung and liver injury in mice with gut-origin sepsis. Cecal ligation and perforation (CLP) were used to construct a mouse model of gut-origin sepsis. The mice were randomly divided into six groups: sham operation group (sham), gut-origin sepsis model group (CLP), ulinastatin-positive drug control group (UTI), lentinan low concentration group (LTN-L, 5 mg/kg), lentinan medium concentration group (LTN-M, 10 mg/kg), and lentinan high concentration group (LTN-H, 20 mg/kg). H&E staining was used to detect the pathological damage of the small intestine, liver, and lung. The serum of mice in each group was collected to detect the expression changes of inflammatory cytokines, oxidative stress biomarkers, and liver function indexes. In vitro assessment of bacterial translocation was achieved through inoculated culture media. Western blot and RT-qPCR were used to detect the expression of molecules related to the NF-κB signaling pathway in the small intestine tissues of mice. The results showed that compared with the CLP group, the injury degree of the small intestine, liver, and lung in mice with gut-origin sepsis was improved with the increase of lentinan concentration. In addition, TNF-α, IL-1ß, IL-6, and HMGB1 were decreased with the increase of lentinan concentration, but the expression of IL-10 was increased. Lentinan could also reduce the expression of oxidative stress injury indexes and liver function indexes and inhibit bacterial translocation to liver and lung tissues. Further mechanism investigation revealed that lentinan downregulated the expression of the NF-κB signaling pathway molecules (NF-κB, TLR4, and Bax) and upregulated the expression of occludin and Bcl-2. In conclusion, lentinan inhibits the activity of the NF-κB signaling pathway, thus attenuating injuries of small intestinal mucosa and liver and lung in mice with gut-origin sepsis and reducing the inflammatory response in the process of sepsis.

Increased Expression of Extracellular Vesicles Is Associated With the Procoagulant State in Patients With Established Rheumatoid Arthritis.

This study sought to identify different subpopulations of extracellular vesicles (EVs) in plasma from female patients with established rheumatoid arthritis (RA) in relation to the activation of coagulation and fibrin formation in these patients. Forty women were included in the study, 20 patients and 20 age-matched healthy controls. The mean disease duration in patients was 13.0 (5.0-25.0) years, with medium to high disease activity despite ongoing treatment with low-dose prednisolone and methotrexate. There were no differences between the investigated groups regarding the presence of traditional cardiovascular risk factors. The concentration of phosphatidylserine-positive (PS+) EVs; platelet (CD42a+), leucocyte (CD45+), monocyte (CD14+), and endothelial (CD144+)-derived EVs; and EVs-expressing tissue factor (CD142+), P-selectin (CD62P+), and E-selectin (CD62E+) were determined by flow cytometry analysis. Overall hemostasis potential (OHP) was assessed to follow the hemostatic disturbances, including the parameters for overall coagulation potential (OCP) and overall fibrinolytic potential (OFP). Fibrin clot turbidity was measured together with clot lysis time, and scanning electron microscopy was performed. Increased concentrations of PS+, CD42a+, CD142+, CD45+, CD14+, and CD62P+ EVs were found in plasma from patients with RA compared to healthy controls, and the concentrations of PS+, CD42a+, CD14+, and CD62P+ EVs were positively correlated with the inflammatory parameters in RA patients. Positive correlations were also found between the levels of PS+ and CD42a+ EVs and OCP as well as between the levels of PS+, CD42a+, and CD62P+EVs and OHP. The levels of PS+, CD42a+, CD14+, CD62P+, and CD62E+ EVs were negatively correlated with OFP. Elevated levels of circulating EVs of different cell origins were found in patients with established RA, in relation to the inflammatory burden and coagulation activation in the disease.

A novel CgIFNLP receptor involved in regulating ISG expression in oyster Crassostrea gigas.

Interferons (IFNs) are the key coordinators of antiviral immunity by binding to their receptors to orchestrate a complex transcriptional network in vertebrates. Recently, the existence of molluscan IFN-like system has been certified by the identification of important components in IFN system, such as IFN-like protein (CgIFNLP) from oyster Crassostrea gigas. In the present study, a novel CgIFNLP receptor (designed CgIFNLPR-1) was identified from C. gigas. The open reading frame (ORF) of CgIFNLPR-1 cDNA was of 1962 bp encoding a peptide of 653 amino acid residues with five fibronectin type III (FNIII) domains and one transmembrane helix region. The mRNA transcripts of CgIFNLPR-1 were constitutively distributed in all the tested tissues, with the highest level in gonad. After Poly (I:C) stimulation, the mRNA expression of CgIFNLPR-1 in haemocytes was significantly up-regulated to the highest level at 48 h (4.54-fold of that in control group, p < 0.05). CgIFNLPR-1 protein was mainly distributed in the cytoplasm and membrane of oyster haemocytes. CgIFNLP and CgIFNLPR-1 were able to interact with each other in vitro. After the CgIFNLPR-1 was knocked down by RNAi, the mRNA expression of IFN-stimulated genes (ISGs), including CgMx, CgViperin and CgIFNIP-44, were significantly inhibited after Poly (I:C) stimulation, which was 0.17, 0.31 and 0.53-fold of that in EGFP group, respectively (p < 0.01). These findings suggested that CgIFNLPR-1 was a novel CgIFNLP receptor in the oyster to recognize CgIFNLP and regulate the expressions of CgISGs.

The cGAS/STING-TBK1-IRF Regulatory Axis Orchestrates a Primitive Interferon-Like Antiviral Mechanism in Oyster.

Interferon (IFN) system is considered as the first defense line against viral infection, and it has been extensively studied in vertebrates from fish to mammals. In invertebrates, Vagos from arthropod and IFN-like protein (CgIFNLP) from Crassostrea gigas appeared to function as IFN-like antiviral cytokines. In the present study, the CgIFNLP protein in hemocytes was observed to increase after Poly (I:C) stimulation. After CgIFNLP was knocked down by RNAi, the mRNA expression of IFN-stimulated genes (CgISGs) was significantly inhibited. Both cyclic GMP-AMP synthase (CgcGAS) and stimulator of interferon gene (CgSTING) identified from oyster were able to recognize the double-stranded nucleic acid [Poly (I:C) and dsDNA] and expressed at high level after Poly (I:C) stimulation. The expression of CgIFNLP and interferon regulatory factors (CgIRF1/8) and the nuclear translocation of CgIRF8 were all suppressed in CgcGAS-RNAi or CgSTING-RNAi oysters after Poly (I:C) stimulation. The expression level of CgSTING and TANK binding kinase1 (CgTBK1) did not decrease in CgcGAS-RNAi oysters. After CgSTING was knocked down, the high expression of CgTBK1 induced by Poly (I:C) was prevented significantly. These results indicated that there was a primitive IFN-like antiviral mechanism dependent on the cGAS/STING-TBK1-IRFs regulatory axis in mollusks, which was different from the classic cGAS-STING-TBK1 signal pathway in mammals.

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.

Reassortment with dominant chicken H9N2 influenza virus contributed to the fifth H7N9 virus human epidemic.

H9N2 Avian influenza virus (AIV) is regarded as a principal donor of viral genes through reassortment to co-circulating influenza viruses that can result in zoonotic reassortants. Whether H9N2 virus can maintain sustained evolutionary impact on such reassortants is unclear. Since 2013, avian H7N9 virus had caused five sequential human epidemics in China; the fifth wave in 2016-2017 was by far the largest but the mechanistic explanation behind the scale of infection is not clear. Here, we found that, just prior to the fifth H7N9 virus epidemic, H9N2 viruses had phylogenetically mutated into new sub-clades, changed antigenicity and increased its prevalence in chickens vaccinated with existing H9N2 vaccines. In turn, the new H9N2 virus sub-clades of PB2 and PA genes, housing mammalian adaptive mutations, were reassorted into co-circulating H7N9 virus to create a novel dominant H7N9 virus genotype that was responsible for the fifth H7N9 virus epidemic. H9N2-derived PB2 and PA genes in H7N9 virus conferred enhanced polymerase activity in human cells at 33°C and 37°C, and increased viral replication in the upper and lower respiratory tracts of infected mice which could account for the sharp increase in human cases of H7N9 virus infection in the 2016-2017 epidemic. The role of H9N2 virus in the continual mutation of H7N9 virus highlights the public health significance of H9N2 virus in the generation of variant reassortants of increasing zoonotic potential.IMPORTANCEAvian H9N2 influenza virus, although primarily restricted to chicken populations, is a major threat to human public health by acting as a donor of variant viral genes through reassortment to co-circulating influenza viruses. We established that the high prevalence of evolving H9N2 virus in vaccinated flocks played a key role, as donor of new sub-clade PB2 and PA genes in the generation of a dominant H7N9 virus genotype (G72) with enhanced infectivity in humans during the 2016-2017 N7N9 virus epidemic. Our findings emphasize that the ongoing evolution of prevalent H9N2 virus in chickens is an important source, via reassortment, of mammalian adaptive genes for other influenza virus subtypes. Thus, close monitoring of prevalence and variants of H9N2 virus in chicken flocks is necessary in the detection of zoonotic mutations.

Synergistic Effect of Bypassing Agents and Sequence Identical Analogue of Emicizumab and Fibrin Clot Structure in the In Vitro Model of Hemophilia A.

Development of inhibitors to factor VIII (FVIII) occurs in approximately 30% of severe hemophilia A (HA) patients. These patients are treated with bypassing agents (activated prothrombin complex concentrate [aPCC] and recombinant activated FVII-rFVIIa). Recently, a bispecific FIX/FIXa- and FX/FXa-directed antibody (emicizumab) has been approved for the treatment of HA patients with inhibitors. However, the data from clinical studies imply that coadministration of emicizumab and bypassing agents, especially aPCC, could have a thrombotic effect. This study was aimed to address the question of potential hypercoagulability of emicizumab and bypassing agents' coadministration, we have investigated fibrin clot formation and structure in the in vitro model of severe HA after adding sequence-identical analogue (SIA) of emicizumab and bypassing agents. Combined overall hemostasis potential (OHP) and fibrin clot turbidity assay was performed in FVIII-deficient plasma after addition of different concentrations of SIA, rFVIIa, and aPCC. Pooled normal plasma was used as control. The fibrin clots were analyzed by scanning electron microscopy (SEM). OHP and turbidity parameters improved with the addition of aPCC, while therapeutic concentrations of rFVIIa did not show substantial improvement. SIA alone and in combination with rFVIIa or low aPCC concentration improved OHP and turbidity parameters and stabilized fibrin network, while in combination with higher concentrations of aPCC expressed hypercoagulable pattern and generated denser clots. Our in vitro model suggests that combination of SIA and aPCC could potentially be prothrombotic, due to hypercoagulable changes in fibrin clot turbidity and morphology. Additionally, combination of SIA and rFVIIa leads to the formation of stable clots similar to normal fibrin clots.

Ocean acidification inhibits initial shell formation of oyster larvae by suppressing the biosynthesis of serotonin and dopamine.

Ocean acidification has severely affected the initial shell formation of marine bivalves during their larval stages. In the present study, it was found that dopamine (DA) content in early D-shape larvae was significantly higher than that in trochophore and D-shape larvae, while the serotonin (5-HT) content in early D-shape larvae and D-shape larvae was obviously higher than that in trochophore. Incubation of trochophore with 5-HT or DA could accelerate the formation of calcified shell, and the treatments with selective antagonists of receptors for 5-HT and DA (Cg5-HTR-1 and CgD1DR-1) obviously inhibited the formation of calcified shells. When oyster larvae were subjected to an experimental acidified treatment (pH 7.4), the biosynthesis of 5-HT and DA was inhibited, while the mRNA expression levels of the components in TGF-ß pathway were significantly up-regulated in D-shape larvae. Moreover, the phosphorylation of TIR and the translocation of smad4 were hindered upon acidification treatments, and the expression patterns of chitinase and tyrosinase were completely reverted. These results collectively suggested that monoamine neurotransmitters 5-HT and DA could modulate the initial shell formation in oyster larvae through TGF-ß smad pathway by regulating the expression of tyrosinase and chitinase to guarantee the chitin synthesis for shell formation. CO2-induced seawater acidification could suppress the biosynthesis of 5-HT and DA, as well as the activation of TGF-ß smad pathway, which would subvert the expression patterns of chitinase and tyrosinase and cause the failure of initial shell formation in oyster early D-shape larvae.

A Signaling Pathway to Mediate the Combined Immunomodulation of Acetylcholine and Enkephalin in Oyster Crassostrea gigas.

Molluscs have evolved a primitive but complete neuroendocrine-immune (NEI) system with a vast array of neurotransmitters to conduct both humoral and cellular immunomodulation. Previous studies have illustrated the immune functions of several key neurotransmitters. However, the combined effects of multiple neurotransmitters and the signaling pathway to mediate such immunomodulation have not been well-understood. In the present study, iTRAQ and LC-ESI-MS/MS approaches were employed to investigate the combined immunomodulation functions of two crucial neurotransmitters, acetylcholine (ACh), and [Met5]-enkephalin (ENK), in oyster Crassostrea gigas. A total number of 5,379 proteins were identified from hemocytes of oysters after the treatments with Ach and ENK separately or simultaneously, and 1,475 of them were found to be significantly up-regulated, while 1,115 of them were significantly down-regulated. The protein expression patterns in the groups treated by ACh and ENK separately were quite similar, which were dramatically different from that in the group treated by ACh+ENK. One hundred seventy-two proteins were found to be differentially expressed in all the three neurotransmitter treatment groups. Functional validation suggested that ACh and ENK possibly modulate the immune response in oyster hemocytes by enhancing pathogen recognition, cell apoptosis, and the enzyme activities of superoxide dismutase (SOD). Moreover, GO enrichment and co-expression network analyses implied that the combined immunomodulation of ACh and ENK might be mediated by p53, EGF-R-ErbB, and Fc gamma R (FcγR) signaling pathways. These results collectively indicated that multiple neurotransmitters executed a combined and ordered immune regulation through common signaling cascades in molluscs, which was under delicate control to maintain the homeostasis.

Phosphatidylserine positive microparticles improve hemostasis in in-vitro hemophilia A plasma models.

Circulating microparticles (MPs) are procoagulant due to the surface containing phosphatidylserine (PS), which facilitates coagulation. We investigated if MPs improve hemostasis in HA plasma models. MPs isolated from pooled normal human plasma were added to severe, moderate and mild HA plasma models (0%, 2.5%, 20% FVIII). The MPs' effect on hemostasis was evaluated by calibrated automated thrombogram (CAT) and overall hemostasis potential (OHP) assays, while fibrin structure was imaged by standard confocal, stimulated emission depletion (STED) microscopy and scanning electron microscopy (SEM). MPs partially restored thrombin generation and fibrin formation in all HA plasma models. The procoagulant effect of MPs requires PS exposure, to a less extent of contact pathway activation, but not tissue factor exposure or in vitro stimulation of MPs. MPs partially normalized the fibrin structure, and using super-resolution STED, MPs attached to fibrin were clearly resolved. In summary, our results demonstrate that PS positive MPs could improve hemostasis in HA plasma models.

Metabolomic and transcriptomic profiling reveals the alteration of energy metabolism in oyster larvae during initial shell formation and under experimental ocean acidification.

Marine bivalves secrete calcified shells to protect their soft bodies from predation and damages, which is of great importance for their survival, and for the safety of the coastal ecosystem. In recent years, larval shell formation of marine bivalves has been severely affected by ocean acidification (OA), and previous study indicated that OA might affect such process by disrupting endogenous energy metabolism. Developmental stages from trochophore to D-shape larvae are extremely important for initial shell formation in oyster since a calcified shell was formed to cover the chitin one. In the present study, metabolomic and transcriptomic approaches were employed to investigate the energy metabolism of oyster larvae during initial shell (prodissoconch I, PDI shell) formation and under experimental OA treatment. Totally 230 chemical compounds were identified from the present dataset, most of which were highly expressed in the "middle" stage (early D-shape larvae) which was critical for PDI shell formation since a calcified shell was formed to cover the chitin one. Several compounds such as glucose, glutarylcarnitine (C5), ß-hydroxyisovaleroylcarnitine, 5-methylthioadenosine (MTA), myristoleate (14:1n5) and palmitoleate (16:1n7) were identified, which were involved in energy metabolic processes including amino acid oxidation, glycolysis, pentose phosphate pathway and fatty acid metabolism. In addition, mRNA expressions of genes related to protein metabolism, glycolysis, lipid degradation, calcium transport and organic matrix formation activities were significantly down-regulated upon experimental OA. These results collectively suggested that formation of the initial shell in oyster larvae required endogenous energy coming from amino acid oxidation, glycolysis, pentose phosphate pathway and fatty acid metabolism. These metabolic activities could be severely inhibited by experimental OA, which might alter the allocation of endogenous energy. Insufficient endogenous energy supply then suppressed the mobilization of calcium and resulted in a failure or delay in PDI shell formation.