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We previously demonstrated that circulating extracellular vesicles (EVs) from patients with valvular heart disease (VHD; vEVs) contain inflammatory components and inhibit endothelium-dependent vasodilation. Neutrophil chemotaxis plays a key role in renal dysfunction, and dexmedetomidine (DEX) can reduce renal dysfunction in cardiac surgery. However, the roles of vEVs in neutrophil chemotaxis and effects of DEX on vEVs are unknown. Here, we investigated the impact of vEVs on neutrophil chemotaxis in kidneys and the influence of DEX on vEVs. Circulating EVs were isolated from healthy subjects and patients with VHD. The effects of EVs on chemokine generation, forkhead box protein O3a (FOXO3a) pathway activation and neutrophil chemotaxis on cultured human umbilical vein endothelial cells (HUVECs) and kidneys in mice and the influence of DEX on EVs were detected. vEVs increased FOXO3a expression, decreased phosphorylation of Akt and FOXO3a, promoted FOXO3a nuclear translocation, and activated the FOXO3a signaling pathway in vitro. DEX pretreatment reduced vEV-induced CXCL4 and CCL5 expression and neutrophil chemotaxis in cultured HUVECs via the FOXO3a signaling pathway. vEVs were also found to suppress Akt phosphorylation and activate FOXO3a signaling to increase plasma levels of CXCL4 and CCL5 and neutrophil accumulation in kidney. The overall mechanism was inhibited in vivo with DEX pretreatment. Our data demonstrated that vEVs induced CXCL4-CCL5 to stimulate neutrophil infiltration in kidney, which can be inhibited by DEX via the FOXO3a signaling. Our findings reveal a unique mechanism involving vEVs in inducing neutrophils chemotaxis and may provide a novel basis for using DEX in reducing renal dysfunction in valvular heart surgery.

Acute heart failure (AHF) is a severe complication after cardiac surgery with cardiopulmonary bypass (CPB). Although some AHF biomarkers have been used in clinic, they have limitations when applied in the prediction and diagnosis of AHF after cardiac surgery with CPB, and there are still no effective and specific biomarkers. We and other researchers have shown that circulating microparticles (MPs) increased in a variety of cardiovascular diseases. However, whether the concentration of circulating MPs could be a new biomarker for AHF after cardiac surgery remains unknown. Here, 90 patients undergoing cardiac surgery with CPB and 45 healthy subjects were enrolled. Patients were assigned into AHF (n=14) or non-AHF (n=76) group according to the diagnosis criteria of AHF. The concentrations of circulating MPs were determined before, as well as 12 h and 3 days after operation with nanoparticle tracking analysis technique. MPs concentrations in patients before surgery were significantly higher than those of healthy subjects. Plasma levels of MPs were significantly elevated at 12 h after surgery in patients with AHF, but not in those without AHF, and the circulating MPs concentrations at 12 h after surgery were higher in AHF group compared with non-AHF group. Logistic regression analysis indicated that MPs concentration at postoperative 12 h was an independent risk factor for AHF. The area under receiver operating characteristic curve for MPs concentration at postoperative 12 h was 0.81 and the best cut-off value is 5.20×108 particles mL-1 with a sensitivity of 93% and a specificity of 10%. These data suggested that the concentration of circulating MPs might be a new biomarker for the occurrence of AHF after cardiac surgery with CPB.

In previous studies, we identified a putative 38-nucleotide stem-loop structure (zipcode) in the 3' untranslated region of the cytochrome c oxidase subunit IV (COXIV) mRNA that was necessary and sufficient for the axonal localization of the message in primary superior cervical ganglion (SCG) neurons. However, little is known about the proteins that interact with the COXIV-zipcode and regulate the axonal trafficking and local translation of the COXIV message. To identify proteins involved in the axonal transport of the COXIV mRNA, we used the biotinylated 38-nucleotide COXIV RNA zipcode as bait in the affinity purification of COXIV zipcode binding proteins. Gel-shift assays of the biotinylated COXIV zipcode indicated that the putative stem-loop structure functions as a nucleation site for the formation of ribonucleoprotein complexes. Mass spectrometric analysis of the COXIV zipcode ribonucleoprotein complex led to the identification of a large number RNA binding proteins, including fused in sarcoma/translated in liposarcoma (FUS/TLS), and Y-box protein 1 (YB-1). Validation experiments, using western analyses, confirmed the presence of the candidate proteins in the COXIV zipcode affinity purified complexes obtained from SCG axons. Immunohistochemical studies show that FUS, and YB-1 are present in SCG axons. Importantly, RNA immunoprecipitation studies show that FUS, and YB-1 interact with endogenous axonal COXIV transcripts. siRNA-mediated downregulation of the candidate proteins FUS and YB-1 expression in the cell-bodies diminishes the levels of COXIV mRNA in the axon, suggesting functional roles for these proteins in the axonal trafficking of COXIV mRNA.

NEL-like protein 1 (NELL1) is a newly identified secreted protein involved in craniosynostosis and has been found to promote osteogenic differentiation of mesenchymal stem cells. The objective of this study was to investigate the effect of NELL1 on osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and the potential underlying mechanism. hPDLSCs underwent lentivirus-mediated NELL1 transfection (Lenti-NELL1) and markers of osteogenesis were assessed [alkaline phosphate (ALP), osteocalcin (OCN) and calcium deposition] to evaluate the effect of NELL1 on the differentiation of these cells. Quantitative polymerase chain reaction (qPCR) was employed to measure the mRNA expression of Msx2 and Runx2, and Lenti-enhanced green fluorescent protein (EGFP) served as a control. Western blot analysis and qPCR analyses confirmed that Lenti-NELL1-transfected hPDLSCs could express NELL1. Compared with the Lenti-EGFP group, ALP, OCN, calcium deposition and Msx2 mRNA expression were markedly increased (P0.01), but there was no significant difference in Runx2 mRNA expression between the two groups (P0.01). hPDLSCs can be transfected by Lenti-NELL1 and can stably express NELL1. NELL1 is able to promote the osteogenic differentiation of hPDLSCs, which may be related to the downregulation of Msx2 expression. Lenti-NELL1 transfection can be used during in vitro gene therapy for periodontal regeneration.

International audience; Here, we examine tRNA-aminoacyl synthetase (ARS) localization in protein synthesis. Proteomics reveals that ten of the twenty cytosolic ARSs associate with ribosomes in sucrose gradients: phenylalanyl-RS (FRS), and the 9 ARSs that form the multi-ARS complex (MSC). Using the ribopuromycylation method (RPM) for localizing intracellular translation, we show that FRS and the MSC, and to a lesser extent other ARSs, localize to translating ribosomes, most strikingly when translation is restricted to poxvirus or alphavirus factories in infected cells. Immunoproximity fluorescence indicates close proximity between MSC and the ribosome. Stress induced-translational shutdown recruits the MSC to stress-granules, a depot for mRNA and translation components. MSC binding to mRNA provides a facile explanation for its delivery to translating ribosomes and stress granules. These findings, along with the abundance of the MSC (9 × 10(6) copies per cell, roughly equimolar with ribosomes), is consistent with the idea that MSC specificity, recently reported to vary with cellular stress (Netzer, N., Goodenbour, J. M., David, A., Dittmar, K. A., Jones, R. B., Schneider, J. R., Boone, D., Eves, E. M., Rosner, M. R., Gibbs, J. S., Embry, A., Dolan, B., Das, S., Hickman, H. D., Berglund, P., Bennink, J. R., Yewdell, J. W., and Pan, T. (2009) Nature 462, 522-526) can be modulated at the level of individual mRNAs to modify decoding of specific gene products.

To investigate the influence of combined thoracoscopic and laparoscopic esophagectomy for early postoperative pulmonary function, and to study the relative factors for postoperative pulmonary complications.From September 2009 to December 2010, 61 patients with esophageal cancer had undergone esophagectomy surgery, of which 32 patients had undergone combined thoracoscopic and laparoscopic esophagectomy (CTLE group), and 29 patients had undergone open three-field esophagectomy (open group). Pulmonary function, including forced vital capacity (FVC), forced expiratory volume in 1 second (FEV(1)) were measured on the 1(th) preoperative day, 5(th) and 10(th) postoperative day, and arterial blood gas analyses were performed during the same period. Meanwhile, pain scores and other potentially relevant factors were recorded as well.Preoperative pulmonary function and arterial blood gas analysis, including FEV(1)%, FVC%, PaO2 in two groups had no significant difference (t = -1.608 to 0.709, P = 0.113 to 0.481). On the 10(th) postoperative day, FEV(1)%, FVC%, PaO2, and SaO2 of two groups were significantly different (FEV(1)%: 77% ± 17% vs. 53% ± 13%, t = 6.241, P = 0.000; FVC%: 78% ± 13% vs. 57% ± 16%, t = 5.549, P = 0.000; PaO2: (87 ± 9) mmHg vs. (79 ± 14) mmHg, t = 2.477, P = 0.017; SaO2: 96% ± 3% vs. 94% ± 2%, t = 2.313, P = 0.024; 1 mmHg = 0.133 kPa). Pain score of CTLE group was lower than open group, and the scores of two groups had significant difference before the 5(th) day after surgery (t = -4.398 to -1.815, P = 0.000 to 0.049). Postoperative pulmonary complications of CTLE group was lower than open group (6/32 vs. 12/29, χ(2) = 3.745, P = 0.049).Combined thoracoscopic and laparoscopic esophagectomy has advantages on early postoperative pulmonary function. It can relatively reduce the incidence of pulmonary complications after surgery.

To demonstrate that mitochondrial morphological and functional changes are an important intermediate link in the course of apoptosis in esophageal carcinoma cells induced by As2O3.The esophageal carcinoma cell line SHEEC1, established in our laboratory, was cultured in 199 growth medium, supplemented with 100mL x L(-1) calf serum and 3 mol x L(-1)As2O3 (the same below). After 2, 4, 6, 12, 24 h of drug adding, the SHEEC1 cells were collected for light-and electron-microscopic examination. The mitochondria were labeled by Rhodamine fluorescence probe and the fluorescence intensity of the mitochondria was measured by flow cytometer and cytofluorimetric analysis. Further,the mitochondrial transmembrane potential (MTP, psim) change was also calculated.The mitochondrial morphological change after adding As2O3 could be divided into three stages. In the early-stage (2-6h) after adding As2O3, an adaptive proliferation of mitochondria appeared; in the mid-stage (6-12 h) a degenerative change was observed; and in the late-stage (12-24 h) the mitochondria swelled with outer membrane broken down and then cells death with apoptotic changes of nucleus. The functional change of the mitochondria indicated by fluorescent intensity, which reflected the MTP status of mitochondria, was in accordance with morphological change of the mitochondria. The fluorescent intensity increased at early-stage, declined in mid-stage and decreased to the lowest in the late-stage. 24 h after As2O3 adding, the cell nucleus showed typical apoptotic changes.Under the inducement of As2O3, the early apoptotic changes of SHEEC1 cells were the apparent morphological and functional changes of mitochondria, afterwards the nucleus changes followed. It is considered that changes of mitochondria are an important intermediate link in the course of apoptosis of esophageal carcinoma cells induced by As2O3.