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Due to the complex diagenesis process, basalt usually contains defects in the form of amygdales formed by diagenetic bubbles, which affect its mechanical properties. In this study, a synthetic rock mass method (SRM) based on the combination of discrete fracture network (DFN) and finite-discrete element method (FDEM) is applied to characterizing the amygdaloidal basalt, and to systematically exploring the effects of the development characteristics of amygdales and sample sizes on the mechanical properties of basalt. The results show that with increasing amygdale content, the elastic modulus (E) increases linearly, while the uniaxial compressive strength (UCS) shows an exponential or logarithmic decay. When the orientation of amygdales is between 0° and 90°, basalt shows a relatively pronounced strength and stiffness anisotropy. Based on the analysis of the geometric and mechanical properties, the representative element volume (REV) size of amygdaloidal basalt blocks is determined to be 200 mm, and the mechanical properties obtained on this scale can be regarded as the properties of the equivalent continuum. The results of this research are of value to the understanding of the mechanical properties of amygdaloidal basalt, so as to guide the formulation of engineering design schemes more accurately.

Background Ochratoxin A (OTA) is a harmful mycotoxin and secondary metabolite produced by several species of Aspergillus and Penicillium. It is of vital importance to establish a sensitive method to detect OTA in various grains. Objective A novel, highly sensitive chemiluminescence immunoassay was developed to determine OTA in wheat, corn, and poultry feed. Methods The immunomagnetic beads were used as solid phase carriers and separators in the competitive chemiluminescence method. Anti-OTA monoclonal antibodies coated on the magnetic beads were applied as the capturing antibodies, in which OTA would compete with OTA-alkaline phosphatase in binding with OTA antibodies. Results The proposed method exhibited good linearity (R2 ≥ 0.999), adequate OTA recovery rate (83.60–102.50%), and good repeatability. The sensitivity of the proposed method was 2.05 pg/mL. In addition, the method was highly selective and showed no cross-reactions with other mycotoxins (e.g., AFB1, DON, ZEN). The developed method was applied in the analysis of wheat, corn, and two animal feeds successfully. The results obtained by the chemiluminescence method showed a high correlation with those obtained by the HPLC method (correlation coefficient 0.9958). Conclusion This strategy shows great potential for other toxic and harmful mycotoxin detection. Highlights A novel highly rapid and sensitive chemiluminescence immunoassay was established for quantitation of OTA.

Background The ultrasensitive monitoring strategy of zearalenone (ZEN) is essential and desirable for food safety and human health. In the present study, a coupling of gold nanoparticles-DNA barcode and direct competitive immunoassay-based real-time polymerase chain reaction signal amplification (RT-IPCR) for ZEN close to the sensitivity of PCR-like levels is described and evaluated. Results The RT-IPCR benefited from the use of a DNA barcode and RT-PCR detection strategy, thus resulting in ultrasensitive and simple detection for ZEN. Under the optimal RT-IPCR, the linear range of detection was from 0.5 to 1000 pg mL-1 and the limit of detection was 0.5 pg mL-1 , which was 400-fold lower than the enzyme-linked immunosorbent assay. The detection procedure was simplified and the detection time was shortened. The specificity, accuracy and precision of the RT-IPCR confirmed a high performance. ZEN-positive contamination levels were from 0.056 to 152.12 ng g-1 by the RT-IPCR, which was demonstrated to be highly reliable by liquid chromatography-tandem mass spectrometry. Conclusion The proposed RT-IPCR could be used as an alternative for detecting ZEN with satisfactory ultrasensitivity, simplicity, low cost and high-throughput. The present study could provide a strategy for the ultrasensitive detection of the small molecule with a simple and practical approach, which has significant appeal and application prospects.

Lysosomes have fundamental physiological roles and have previously been implicated in Parkinson’s disease1–5. However, how extracellular growth factors communicate with intracellular organelles to control lysosomal function is not well understood. Here we report a lysosomal K+ channel complex that is activated by growth factors and gated by protein kinase B (AKT) that we term lysoKGF. LysoKGF consists of a pore-forming protein TMEM175 and AKT: TMEM175 is opened by conformational changes in, but not the catalytic activity of, AKT. The minor allele at rs34311866, a common variant in TMEM175, is associated with an increased risk of developing Parkinson’s disease and reduces channel currents. Reduction in lysoKGF function predisposes neurons to stress-induced damage and accelerates the accumulation of pathological α-synuclein. By contrast, the minor allele at rs3488217—another common variant of TMEM175, which is associated with a decreased risk of developing Parkinson’s disease—produces a gain-of-function in lysoKGF during cell starvation, and enables neuronal resistance to damage. Deficiency in TMEM175 leads to a loss of dopaminergic neurons and impairment in motor function in mice, and a TMEM175 loss-of-function variant is nominally associated with accelerated rates of cognitive and motor decline in humans with Parkinson’s disease. Together, our studies uncover a pathway by which extracellular growth factors regulate intracellular organelle function, and establish a targetable mechanism by which common variants of TMEM175 confer risk for Parkinson’s disease. The identification of a lysosomal K+ channel complex—comprising AKT and the pore-forming TMEM175—provides insights into the mechanisms through which variants of the pore-forming protein affect the development of Parkinson’s disease.

The present study evaluated risk factors related to persistent atrial fibrillation (AF) at discharge (AF-d) and recurrent atrial fibrillation (rAF) and all-cause death after the maze IV procedure. Two hundred nineteen patients (63 female, aged 52.5±8.8 years) with valve disease and persistent AF undergoing valve surgery and the maze IV procedure in our center between 2015 and 2016 were included. Baseline demographic and clinical data were obtained by review of medical records. The median follow-up period was 27 months (interquartile range 21–34 months) in our patient cohort. The primary end point was all-cause death. The secondary end point was AF-d or rAF. rAF is defined as AF recurrence at 3 months or later after the procedure. Twenty-eight patients (12.8%) died during follow-up. Multiple logistic regression analysis showed that thrombocytopenia, elevated serum total bilirubin level, a larger right atrium, AF-d, and rAF were independent determinants for all-cause death after the maze IV procedure after adjustment for age, sex, and clinical covariates, including New York Heart Association class III/IV disease, hypertension, and aortic regurgitation, while valvular disease duration and left atrial diameter greater than 80.5 mm were independent determinants for AF-d, and thrombocytopenia, elevated serum total bilirubin level, higher mean pulmonary artery pressure, and AF-d were independent predictors for rAF. In conclusion, thrombocytopenia, elevated serum total bilirubin level, an enlarged right atrium, AF-d, and rAF are independent predictors of all-cause death in patients undergoing the maze IV procedure.

Human cytomegalovirus (CMV) is a ubiquitous, persistent beta herpesvirus. CMV infection contributes to the accumulation of functional antigen‐specific CD8(+) T‐cell pools with an effector‐memory phenotype and enrichment of these immune cells in peripheral organs. We review here this ‘memory T‐cell inflation’ phenomenon and associated factors including age and sex. ‘Collateral damage’ due to CMV‐directed immune reactivity may occur in later stages of life – arising from CMV‐specific immune responses that were beneficial in earlier life. CMV may be considered an age‐dependent immunomodulator and a double‐edged sword in editing anti‐tumour immune responses. Emerging evidence suggests that CMV is highly prevalent in patients with a variety of cancers, particularly glioblastoma. A better understanding of CMV‐associated immune responses and its implications for immune senescence, especially in patients with cancer, may aid in the design of more clinically relevant and tailored, personalized treatment regimens. ‘Memory T‐cell inflation’ could be applied in vaccine development strategies to enrich for immune reactivity where long‐term immunological memory is needed, e.g. in long‐term immune memory formation directed against transformed cells.

Zearalenone (ZEN) has a potential hazard to human health, and is frequently found in agro-products. To minimize ZEN exposure to consumers, a novel metal-organic framework-based immunoassay system using zeolitic imidazolate framework-encapsulated horseradish peroxidase and goat anti-mouse IgG (HRP/Ab@ZIF-L) as labels was proposed for rapid and ultrasensitive detection of ZEN in agro-products. The HRP/Ab@ZIF-L not only maintained recognition ability of antibody and catalytic activity of enzyme, but also protected encapsulated proteins against high temperature, organic solvents and long term storage. Under optimal conditions, the detection limit of HRP/Ab@ZIF- L -based immunoassay reached 0.5 ng/L for ZEN, which was approximately 126-fold lower than that of conventional HRP-based immunoassay. Moreover, the proposed method showed an excellent selectivity, and a good dynamic linear detection for ZEN in the range of 0.5 ng/L to 0.476 μg/L. The recoveries of ZEN from spiked corn and wheat samples ranged from 84.50% to 96.70% with the relative standard deviation under 8.9%. In brief, the proposed immunoassay method has potential application for rapid and ultrasensitive detection of ZEN in agro-products.

Background Patients with brain tumor or pancreatic cancer exhibit the poorest prognosis, while immune fitness and cellular immune exhaustion impacts their survival immensely. This work identifies differences in the immune reactivity to the common human pathogens cytomegalovirus (CMV) and Epstein–Barr virus (EBV) between patients with brain tumor in comparison to those with pancreatic cancer and healthy individuals. Methods We characterized the humoral and cellular immune responses of patients with brain tumor or pancreatic cancer to cytomegalovirus structural protein pp65 (CMV-pp65) as well as Epstein–Barr nuclear antigen-1 (EBNA-1) by whole-blood assay and ELISA. Results Anti-CMV-pp65 plasma immunoglobulin gamma (IgG) titers were significantly lower in patients with brain tumor compared to healthy donors and patients with pancreatic cancer. Among the responding patients with GBM, those with a weak anti-CMV IgG response also had a decreased median overall survival (p = 0.017, 667 vs 419 days) while patients with brain tumor showed a generally suppressed anti-CMV immune-reactivity. Patients with brain tumor exhibited a significantly lower interferon gamma (IFNγ) response to EBNA-1 and CMV-pp65 compared to patients with pancreatic cancer or healthy donors. This antigen-specific response was further amplified in patients with brain tumor upon conditioning of whole blood with IL-2/IL-15/IL-21. Exclusively in this setting, among the responding patients with GBM, those exhibiting a EBV-specific cellular immune response above the median also displayed an increased median overall survival pattern compared to weak responders (753 vs 370 days, p

Background Previous studies have shown that acetylation plays a critical role in regulating the progress of cardiovascular diseases by acetylated histone and non-histones protein. However, the global lysine acetylome during atrial fibrillation (AF) were not fully understood. Purpose The aim of the present study was to identify the underlying mechanisms of AF via profiling of the quantitative changes of global proteomics and lysine acetylome in the left atrial appendage (LAA) tissues from valvular heart disease patients with AF. Methods This study obtained LAA specimens from patients undergoing cardiac surgery for severe valvular heart disease. The LAA specimens were obtained from both 9 patients with AF and with sinus rhythm (SR). The changes of proteome and acetylome in the AF-LAA vs SR-LAA tissues were studied using dimethyl-labeling, HPLC fractionation, affinity enrichment, LC-MS/MS analysis, database Search and bioinformatic analysis. The acetylated levels of each lysine acetylated site were normalized on the basis of the corresponding protein abundance. Results The bioinformatic analysis indicates 294 up-regulated (AF/SR ratio >1.3) proteins and 169 down-regulated (AF/SR ratio Interaction network of Ac-proteins Conclusions This study details and expands our understanding of the changes of proteome and lysine acetylome in the LAA tissues from valvular heart disease patients with AF. The data suggest important expression differences of acetylated proteins related to energy metabolism and cardiac contraction which may be involved in the matrix of AF formation and maintainence. Acknowledgement/Funding This work was supported by the grants from the National Natural Science Foundation of China (no. 81600273, no. 81570310, no.81770337 and no.81870258)

Pulmonary artery-derived ventricular arrhythmia is gradually being recognized, which in a clinical context is recognized as an arterial ectopic beat. Our study aimed to provide new insights on the epidemiological characteristics, origin site, electrocardiogram (ECG) characteristics, intracardiac electrophysiological characteristics and radiofrequency catheter ablation (RFCA) strategies for pulmonary artery-derived ventricular arrhythmia. Patients with a distance between the origin site and the pulmonary valve of >10 mm have what is known as pulmonary trunk-derived ventricular arrhythmia, while patients with a distance between the origin site and the pulmonary valve of ≤10 mm have what is known as pulmonary sinus cusp-derived ventricular arrhythmia. It is very difficult to differentiate pulmonary artery-derived ventricular arrhythmia from right ventricular outflow tract-derived ventricular arrhythmia on ECGs as both share similar anatomical features, but pulmonary artery-derived ventricular arrhythmia shows obvious intracardiac electrophysiological characteristics. Currently, conclusions based on the epidemiological characteristics of pulmonary artery-derived ventricular arrhythmia, relationship between the origin site and the pulmonary valve, electrophysiological characteristics, and RFCA strategies are controversial and still need further study.

Context: Axillary vein puncture is an alternative method for pacemaker leads implantation. However, the safety and effectiveness of axillary vein puncture become an issue for debate now. Aims: The aim of this study was to investigate the safety, feasibility, and efficacy of a modified axillary vein technique for pacemaker implantation. Settings and Design: This modified axillary vein technique refers to a novel axillary vein puncture method combined with caudal 35° fluoroscopy and surface anatomic landmarks including deltopectoral groove, coracoid process, and the first rib. Subjects and Methods: This technique was applied to 139 consecutive patients referred for pacemaker implantation between March 1, 2016, and December 31, 2016, at our department. Statistical Analysis Used: None. Results: Success rate of pacemaker implantation was 96.4% (134/139). Five patients (3.60%) were switched to an alternate approach for leads implantation. There were no procedure-related complications such as pneumothorax, hemopneumothorax, major bleeding, and significant pocket hematoma. Conclusion: This modified axillary vein puncture strategy combining caudal 35° fluoroscopy and surface anatomic landmarks is safe and feasible for pacemaker implantation.

The potential homogeneous assay employing immunomagnetic beads (IMB) has been receiving attention as a screening tool in food-safety control; the method is simple, efficient, and does not require long incubation times or complex separation steps. In this study, a homogeneous immunoassay has been successfully developed and applied in the determination of aflatoxin B1 (AFB1) contamination in agricultural products by coupling IMB and the biotin-streptavidin (BSA) (BSA-IMB) system. Under optimal conditions, the limit of detection (LOD, IC10), half-maximal inhibition concentration (IC50) and detection range (IC20–IC80) of BSA-IMB are 0.00579, 0.573 and 0.0183–17.9 ng mL−1, respectively, for AFB1. The detection of AFB1 by BSA-IMB can be achieved in 40 min (ELISA needs at least 180 min). The cross-reactivities of BSA-IMB with its analogues are negligible (

The prognosis for patients with glioblastoma is grim. Ex vivo expanded tumor-associated antigen (TAA)-reactive T-cells from patients with glioma may represent a viable source for anticancer-directed cellular therapies. Immunohistochemistry was used to test the survivin (n = 40 samples) and NY-ESO-1 (n = 38 samples) protein expression in tumor specimens. T-cells from peripheral blood were stimulated with TAAs (synthetic peptides) in IL-2 and IL-7, or using a combination of IL-2, IL-15 and IL-21. CD4+ and CD8+ T-cells were tested for antigen-specific proliferation by flow cytometry, and IFN-γ production was tested by ELISA. Twenty-eight out of 38 cancer specimens exhibited NY-ESO-1 protein expression, 2/38 showed a strong universal (4+) NY-ESO-1 staining, and 9/40 cancer lesions exhibited a strong (4+) staining for survivin. We could detect antigen-specific IFN-γ responses in 25% blood samples for NY-ESO-1 and 30% for survivin. NY-ESO-1-expanded T-cells recognized naturally processed and presented epitopes. NY-ESO-1 or survivin expression in glioma represents viable targets for anticancer-directed T-cells for the biological therapy of patients with glioma. Electronic supplementary material The online version of this article (doi:10.1007/s00262-017-2066-z) contains supplementary material, which is available to authorized users.

Tetrabromobisphenol A bis(2-hydroxyethyl) ether (TBBPA DHEE), as one of the TBBPA derivatives, is frequently applied as a flame retardant. To date, however, little evidence has been gathered regarding its environmental behavior and fate. This is mainly attributed to the lack of an analytical approach available to measure it. Mass spectrometry cannot determine levels of TBBPA DHEE. LC-MS could not ionize TBBPA DHEE to yield enough fragments, while GC-MS could not be applied for the analysis either, due to the thermal degradation of TBBPA DHEE. We here designed a novel hapten, then conjugated it with a carrier protein and produced a type of polyclonal antibody capable of recognizing TBBPA DHEE. Based on this approach, a sensitive and high-throughput indirect competitive enzyme-linked immunosorbent assay (ELISA) was established. The respective detection limit (LOD, based on 90% B/B0) and IC50 calculated from the standard curve were 0.702 and 9.868 ng/mL for TBBPA DHEE. And the established method showed good tolerance to various matrix interferents. Using our ELISA, we measured the concentrations of these substances from environmental water samples with no pretreatment other than filtration. We found the presence of TBBPA DHEE in environmental water samples collected in Jiangsu province, and the detected concentrations ranging from 1.5 to 7.7 ng/mL. Our proposed method here could be applied to completely and systematically investigate TBBPA DHEE and TBBPA MHEE concentrations in environmental samples.

The objective of this study is to characterize the relationship between forkhead box C2 protein (Foxc2) and leptin under adipose inflammatory response.Lipopolysaccharide (LPS)-induced inflammatory model was conducted. Data from wild-type and ob/ob mice were used to compare the alternative role of leptin on Foxc2-mediated inflammation and browning. Transcriptional regulation and protein-protein interaction were analyzed by bioinformatics and proved by chromatin immunoprecipitation and co-immunoprecipitation experiment.Foxc2 and leptin correlated with inflammation and browning of white adipose tissue (WAT) in LPS-treated mice. Moreover, Foxc2-mediated inhibition of inflammation involved downstream activation of leptin signal and promoted WAT browning. We then determined CREB, the potential transcriptional factor of leptin, was required for Foxc2-mediated inflammation in the regulation of WAT browning. Foxc2 alleviated adipocyte inflammation by reducing leptin-mediated Janus-activated kinase 2/signal transducer and activator of transcription 3 (STAT3) pathway. Importantly, STAT3 physically interacted with PRDM16 and formed a complex to promote WAT browning. Exogenous Foxc2 overexpression also ameliorated inflammation and promoted adipose browning in high fat diet (HFD)-induced obese mice.Our results indicated that Foxc2 inhibited inflammation and promoted browning of WAT through positive regulation of leptin signal and the STAT3-PRDM16 complex. These findings identify a new potential means to prevent and treat obese caused metabolic syndrome of mammals.

// Liu Zhenjiang 1 , Martin Rao 1 , Thomas Poiret 1,2 , Silvia Nava 1 , Qingda Meng 1 , Anna von Landenberg 1 , Jiri Bartek Jr. 3,4,5 , Shanshan Xie 6 , Georges Sinclair 3 , Inti Peredo 3 , Ernest Dodoo 3,* and Markus Maeurer 1,2,* 1 Department of Laboratory Medicine, Division of Therapeutic Immunology, Karolinska Institutet, Stockholm, Sweden 2 Centre for Allogeneic Stem Cell Transplantation, Karolinska University Hospital Huddinge, Stockholm, Sweden 3 Department of Neurosurgery, Karolinska University Hospital, Stockholm, Sweden 4 Department of Clinical Neuroscience and Department of Medicine, Karolinska Institutet, Stockholm, Sweden 5 Department of Neurosurgery, Copenhagen University Hospital Rigshospitalet, Copenhagen, Denmark 6 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden * These authors have contributed equally to this work Correspondence to: Markus Maeurer, email: // Keywords : glioblastoma, mesothelin, T-cell response, interferon gamma, immunotherapy, Immunology and Microbiology Section, Immune response, Immunity Received : February 25, 2017 Accepted : June 11, 2017 Published : August 16, 2017 Abstract Glioblastoma multiforme (GBM) presents the most malignant form of glioma, with a 5-year survival rate below 3% despite standard therapy. Novel immune-based therapies in improving treatment outcomes in GBM are therefore warranted. Several molecularly defined targets have been identified mediating anti-GBM cellular immune responses. Mesothelin is a tumor-associated antigen (TAA) which is expressed in several solid tumors with different histology. Here, we report the immunological significance of mesothelin in human malignant glioma. Expression of mature, surface-bound mesothelin protein was found to bein human GBM defined by immunofluorescence microscopy, and on freshly isolated, single cell suspension of GBM tumor cells and GBM tumor cell lines, determined by based on flow cytometric analysis. Peripheral blood (PB) from patients with GBM, stimulated with mesothelin peptides and IL-2, IL-15 and IL-21, exhibited increased antigen-specific IFN-γ and TNF-α production. Anti-mesothelin directed T-cell responses could also be detected in tumor - infiltrating lymphocytes (TIL) isolated from GBM speciments. Furthermore, T cells cultured in the presence of IL-2, IL-15 and IL-21 displayed enhanced mesothelin-specific CD4+ and CD8+ subset proliferation, based on ELISA and flow cytometric readouts. Mesothelin-specific IgG antibodies as well as (shed) mature mesothelin protein were detected in plasma samples from patients with GBM by indirect ELISA. Finally yet importantly, we identified distinct immune recognition hotspots within the mature mesothelin component, defined by peptide-specific IFN-γ responses from peripheral T-cells from patients with GBM. Mesothelin may therefore qualify as a viable target for immunotherapeutic approaches for patients with GBM.

On the basis of H2O2-mediated growth of gold nanoparticle (AuNPs), a novel plasmonic enzyme-linked immunosorbent assay (pELISA) was developed with a polyclonal antibody for the ultrasensitive simultaneous naked-eye detection of tetrabromobisphenol A bis(2-hydroxyetyl) ether (TBBPA DHEE) and tetrabromobisphenol A mono(hydroxyethyl) ether (TBBPA MHEE), one of the major derivatives and byproducts of tetrabromobisphenol A (TBBPA), respectively. In this modified indirect competitive pELISA, glucose oxidase (GOx) played an important role leading to the growth of AuNPs through a reaction between GOx and glucose to produce hydrogen peroxide (H2O2). In addition, further signal amplification was achieved via a large number of GOx molecules, which were immobilized on silica nanoparticles carrying poly brushes (SiO2@PAA) to increase the enzyme load, and the whole complex was conjugated on the second antibody. Under the optimized conditions, 10–3 μg/L TBBPA DHEE can be distinguished via the observation of a colored so...

Lanthanide ions, the excellent fluorescence markers, have the potential to simplify the performance of multi-analytes analysis. In this work, a powerful time-resolved fluoroimmunoassay (TRFIA) based on dual-labeled format were performed for detecting clenbuterol and salbutamol simultaneously in water and swine feed samples. Europium (Eu3+)-labeled goat anti-mouse IgG and Samarium (Sm3+)-labeled goat anti-rabbit IgG were prepared and used as fluorescent probes. Under optimal conditions, the half-maximal inhibition concentration (IC50) and the limit of detection (LOD, IC10) of the TRFIA were 0.263 and 0.007 ng/mL for clenbuterol, and 0.179 and 0.014 ng/mL for salbutamol, respectively. The cross-reactivities of the TRFIA between these two β2-agonists and with their analogues were negligible, thus indicated high specificity and promoted the simultaneous determination. The spiked recoveries were 72.2%–102.2% with relative standard deviations (RSDs) of 3.4–13.8% for clenbuterol and salbutamol in water and swine feed samples. Furthermore, the results of TRFIA correlated well with those obtained by HPLC. The proposed dual-labeled TRFIA was a satisfactory tool for simultaneous, sensitive, efficient and labour-saving detection of clenbuterol and salbutamol in water and swine feed samples.

Background Real-time radiofrequency (RF) ablation lesion assessment is a major unmet need in cardiac electrophysiology. Objective The purpose of this study was to assess whether improved temperature measurement using a novel thermocoupling (TC) technology combined with information derived from impedance change, contact force (CF) sensing, and catheter orientation allows accurate real-time prediction of ablation lesion formation. Methods RF ablation lesions were delivered in the ventricles of 15 swine using a novel externally irrigated-tip catheter containing 6 miniature TC sensors in addition to force sensing technology. Ablation duration, power, irrigation rate, impedance drop, CF, and temperature from each sensor were recorded. The catheter "orientation factor" was calculated using measurements from the different TC sensors. Information derived from all the sources was included in a mathematical model developed to predict lesion depth and validated against histologic measurements. Results A total of 143 ablation lesions were delivered to the left ventricle (n = 74) and right ventricle (n = 69). Mean CF applied during the ablations was 14.34 ± 3.55g, and mean impedance drop achieved during the ablations was 17.5 ± 6.41 Ω. Mean difference between predicted and measured ablation lesion depth was 0.72 ± 0.56 mm. In the majority of lesions (91.6%), the difference between estimated and measured depth was ≤1.5 mm. Conclusion Accurate real-time prediction of RF lesion depth is feasible using a novel ablation catheter-based system in conjunction with a mathematical prediction model, combining elaborate temperature measurements with information derived from catheter orientation, CF sensing, impedance change, and additional ablation parameters.

Glioblastoma multiforme (GBM) is the most common and most severe form of malignant gliomas. The prognosis is poor with current combinations of pharmaceutical, radiotherapy, and surgical therapy. A continuous search for new treatments has therefore been ongoing for many years. Therapy with tumor-infiltrating lymphocytes (TILs) is a clinically promising strategy to treat various cancers, including GBM. An endovascular intraarterial injection of TILs as a method of delivery may, instead of intravenous infusion, result in better retention of effector cells within the tumor. Prior to clinical trials of intra-arterial injections with any cells, preclinical safety data with special emphasis on embolic–ischemic events are necessary to obtain. We used native rabbits as a model for intra-arterial injections with routine clinical catheter material and a clinical angiography suite. We selectively infused a total dose of 20 million activated T cells at a cell concentration of 4,000 cells/ml over 8 min of injection time. The rabbits were evaluated for ischemic lesions by 9.4 T magnetic resonance imaging (MRI) ( n = 6), and for tracking of injected cells, single-photon emission computed tomography/computed tomography (SPECT/CT) was used ( n = 2). In this study, we show that we can selectively infuse activated T cells to a CNS volume of 3.5 cm3 (estimated from the volumetric MRI) without catastrophic embolic– ischemic adverse events. We had one adverse event with a limited basal ganglia infarction, probably due to catheter-induced mechanical occlusion of one of the lateral lenticulostriatal arteries. The cells pass through the native brain without leaving SPECT signals. The cells then, over the first hours, end up in the liver to a large extent and to a lesser degree by the spleen, pancreas, and kidneys. Virtually no uptake could be detected in the lungs. This indicates a difference in biodistribution as opposed to other cell types when infused intravenously.

Alpha-melanocyte stimulating hormone (αMSH) has an important role in the regulation of body weight and energy expenditure. Nevertheless, the molecular mechanisms of circulating αMSH on preadipocyte proliferation remain elusive. We found αMSH was reduced by high fat diet (HFD) while leptin was elevated in adipose tissue. Leptin resistance and endoplasmic reticulum (ER) stress of adipose tissue were increased in obese mice. αMSH increased leptin sensitivity and alleviated ER stress along with increased p-STAT3 level and reduced SOCS3, GRP78, CHOP, ATF4, p27 and p53 levels. αMSH and leptin co-treatment alleviated ER stress through decreasing the levels of GRP78 and CHOP. Tunicamycin (TM) or thapsigargin (Tg) induced ER stress blunted leptin sensitivity and inhibited preadipocyte proliferation. αMSH and leptin co-treatment increased the cell number, augmented G1-S transition, elevated leptin sensitivity, and reduced ER stress; it also activated Notch1 signal and stimulated preadipocyte proliferation, whereas ER stress marker genes were decreased during this process. However, the effects of αMSH and leptin were blocked by the specific inhibitor of Notch1 signal. In summary, our data revealed αMSH enhanced leptin sensitivity and preadipocyte proliferation, meanwhile inhibited ER stress of preadipocytes by activating Notch1 signal.

Increased awareness of phthalic acid esters (PAEs) toxicity has given rise to a dramatic increase in concern about the determination of these contaminations in the environment. In this paper, a sensitive, selective and rapid enzyme immunoassay of ELISA based on polyclonal antibody for detecting butyl benzyl phthalate (BBP) was developed and applied in the environmental water and soil samples. The hapten of BBP was synthesized, then applied to prepare artificial antigen and produce polyclonal antibody capable of specific recognizing BBP. From the optimal standard curve of ELISA for BBP, the values of LOD (limit of detection, IC10) and IC50 were 2.5 and 79.4 ng/mL, respectively. The ELISA showed high specificity, with the cross-reactivity toward BBP analogs

An indirect competitive enzyme-linked immunosorbent assay (ELISA) for flonicamid was developed based on a polyclonal antibody. 4-Trifluoronicotinic acid (TFNA) was used as flonicamid hapten and conjugated to bovine serum albumin (BSA) to produce an immunogen and ovalbumin (OVA) to produce a coating antigen. Polyclonal antibodies against flonicamid were successfully produced by immunizing New Zealand white rabbits. Under the optimal conditions, the 50% inhibitory concentration (IC50 value) of standard curves was 7.89 mg L−1 and the limit of detection (IC10) was 0.063 mg L−1. There was almost no cross-reactivity of the antibody with three structurally related metabolites, indicating that the antibody had high specificity. The recoveries from spiked water, soil, tomato and apples were in the range of 82.4–113.4% with relative standard deviations of 2.9–10.3%. Moreover, the ELISA for spiked samples showed reliability and high correlation with gas chromatography. These results suggest that the proposed ELISA method has potential application for screening flonicamid in agricultural and environmental samples.

Cytomegalovirus (CMV) infection remains a life-threatening condition in individuals with a suppressed immune system. CMV may also represent a clinically relevant target for immune responses in CMV-positive malignancies. We established a protocol to expand CMV-specific T cells (CMV-T) using peripheral blood mononuclear cells (PBMCs). PBMCs from 16 HLA-A*0201 donors were cultured with a cytokine cocktail comprising IL-2/IL-15/IL-21 along with overlapping peptides from CMV-pp65. Ten days later, T cells were stimulated with anti-CD3 (OKT3) and irradiated autologous PBMCs. CMV-T were detected by HLA-A*0201 CMV-pp65NLVPMVATV wild type and q226a mutant tetramers (for high-affinity T cells), intracellular cytokine staining, a CD107a mobilization assays as well as IFN-γ and TNF-α production in cell culture supernatants. We reliably obtained 50.25 ± 27.27% of CD8+ and 22.08 ± 21.83% of CD4+ T cells post-CMV-pp65 stimulation of PBMCs with a Th1-polarized phenotype and decreased Th2/Th17 responses. Most CD3 + CD8 + tetramer+ T cells were effector-memory cells, particularly among high-affinity CMV-T (q226a CMV-tetramer+). High-affinity CMV-T cells, compared to WT-tetramer+ cells, expressed higher IL-21R and lower FasL post-stimulation with CMV-pp65. The IL-2/IL-15/IL-21 cocktail also promoted CCR6 and CXCR3 expression necessary for T-cell migration into tissues. We have optimized methods for generating high-affinity CMV-specific T cells that can be used for adoptive cellular therapy in clinical practice.

Leptin is an adipocyte-derived hormone and maintains adipose function under challenged conditions. Autophagy is also essential to maintain cellular homeostasis and regulate characteristics of adipose tissue. However, the effects of leptin on autophagy of adipocyte remain elusive. Here, we demonstrated endoplasmic reticulum (ER) stress and leptin were correlated with autophagy and inflammation by transcriptome sequencing of adipose tissue. Leptin-mediated inhibition of autophagy was involved in upstream reduction of ER stress proteins such as Chop, GRP78, and Atf4, since blockage of autophagy using pharmacological approach had no effect on tunicamycin-induced ER stress. Moreover, we determined KLF4, the potential transcriptional factor of Atf4, was required for the leptin-mediated autophagy in the regulation of adipocyte inflammation. Importantly, ATF4 physically interacted with ATG5 and subsequently formed a complex to promote adipocyte autophagy. Further analysis revealed that Atg5, a core component of autophagosome, was the target for leptin-mediate autophagy. In addition, leptin alleviated ER stress-induced inflammation by reducing autophagy-mediated degradation of IκB in adipocytes. Exogenous leptin treatment also ameliorated autophagy and inflammation of white adipose tissue in ob/ob mice. Taken together, our results indicated that leptin inhibited ER stress-mediated autophagy and inflammation through the negatively regulation of Atf4/Atg5 complex in adipocytes. These findings identify a new potential means for intervention of autophagy to prevent or treat obese caused metabolic syndrome of mammals.

Salbutamol (SAL) has potential hazardous effects on health, and has been found in natural water globally. To protect consumers from risks related to SAL residues, a fast and highly sensitive enzyme-linked immunosorbent assay (ELISA) was developed for the detection of SAL in environmental water samples. SAL hapten was synthesized and conjugated to proteins to be used as the immunogen for producing polyclonal antibodies. Under optimized conditions, the ELISA showed a 50% inhibitory concentration (IC50 value) of 0.466μg/L, with a limit of detection (LOD, IC10 value) of 0.021μg/L. The LOD of the ELISA was 5-fold lower than the maximum residue limits. The average recoveries of SAL from environmental samples, including tap water, river water and wastewater, ranged from 80.1 to 115.6%, which is well within the requirements of residue detection. Moreover, the results of the ELISA correlated well with high-performance liquid chromatography results, with a correlation coefficient of 0.988. These results suggest that the proposed ELISA method has potential application for the monitoring of SAL residues in environmental water samples.

// Qingda Meng 1 , Davide Valentini 2 , Martin Rao 1 , Zhenjiang Liu 1 , Shanshan Xie 1 , Ann Morgell 3 , Ernest Dodoo 1 , Matthias Lohr 3 , Elena Rangelova 3 , Marco del Chiaro 3 , Ingemar Ernberg 4 and Markus Maeurer 1, 2 1 Division of Therapeutic Immunology (TIM), Department of Laboratory Medicine (LABMED), Karolinska Institutet, Stockholm, Sweden 2 Centre for Allogeneic Stem Cell Transplantation (CAST), Karolinska University Hospital Huddinge, Stockholm, Sweden 3 Pancreatic Surgery Unit, Division of Surgery, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden 4 Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden Correspondence to: Markus Maeurer, email: markus.maeurer@gmail.com Keywords: pancreatic cancer; mesothelin; interferon gamma; survival; antigen-specific response Received: February 27, 2017 Accepted: June 19, 2017 Published: April 27, 2018 ABSTRACT Most patients with pancreatic cancer present with extensive metastasis at diagnosis, with a 5-year survival rate of approximately 5%, despite chemotherapy and surgery. New treatment modalities are needed to improve survival. Mesothelin is a tumor-associated antigen (TAA) in patients with pancreatic cancer that could be used to gauge cellular immune responses directed against transformed cells since up to 100 percent of pancreatic ductal adenocarcinoma cells have been shown to strongly express mesothelin. A prospective, observational study was carried out in twenty-six, chemotherapy-naive patients with resectable pancreatic ductal adenocarcinoma. Participants were between 48 and 81 years (median age: 64.5 years), 15 males and 11 females. All participants were clinically followed-up between 439 and 853 days post-surgery (n=14) or until death (n=12). Peripheral blood drawn on the day of surgery was stimulated with a mesothelin peptide pool (42 peptides, non-overlapping), individual mesothelin peptides, positive (anti-CD3 antibody, OKT3) and negative controls (medium) with or without adding IL-21. Kaplan-Meier estimators were used to gauge patients’ survival pattern in relation to mesothelin-specific IFN-γ responses. A survival benefit was linked with IFN-γ responses to peptides corresponding to mature mesothelin (p=0.018) and targeted recognition of the mesothelin 601-615 epitope (MQEALSGTPCLLGPG) (p=0.006) in the presence of IL-21. Conversely, production of high levels of IFN-γ to OKT3 stimulation with IL-21 conditioning was associated with reduced survival of patients (p=0.016). Gauging anti-Mesothelin- directed immune responses will aid to identify patients i) in need of a more intensive clinical follow-up and ii) who may benefit from immunotherapeutic approaches targeting mesothelin.

Tumor-infiltrating lymphocytes (TILs) may represent a viable source of T cells for the biological treatment of patients with gliomas. Glioma tissue was obtained from 16 patients, tumor cell lines were established, and TILs were expanded in 16/16 cases using a combination of IL-2/IL-15/IL-21. Intracellular cytokine staining (ICS, IL-2, IL-17, TNFα and IFNγ production) as well as a cytotoxicity assay was used to detect TIL reactivity against autologous tumor cells or shared tumor-associated antigens (TAAs; i.e., NY-ESO-1, Survivin or EGFRvIII). TILs were analyzed by flow cytometry, including T-cell receptor (TCR) Vβ family composition, exhaustion/activation and T-cell differentiation markers (CD45RA/CCR7). IL-2/IL-15/IL-21 expanded TILs exhibited a mixture of CD4+, CD8+, as well as CD3+ CD4−CD8− T cells with a predominant central memory CD45RA−CCR7+ phenotype. TIL showed low frequencies of T cells testing positive for PD-1, TIM-3 and CTLA-4. LAG3 tested positive in up to 30% of CD8+ TIL, with low (1.25%) frequencies in CD4+ T cells. TIL cultures exhibited preferential usage of Vβ families and recognition of autologous tumor cells defined by cytokine production and cytotoxicity. IL-2/IL-15/IL-21 expanded TILs represent a viable source for the cellular therapy of patients with gliomas.

Background The origin of pulmonary sinus cusp (PSC)-derived ventricular arrhythmia (VA) is a highly specialized anatomical structure; therefore, insertion of the radiofrequency ablation catheter tip to the target site to ensure safe ablation is a major challenge for clinicians. Purpose To summarize ablation methods, and provide valuable experience for the treatment of pulmonary sinus cusp-derived VA with ablation. Methods A retrospective analysis and summary of the clinical data of 15 patients undergoing catheter ablation for pulmonary sinus cusp-derived VA in Cardiac Intervention Therapy Center, The Second Xiangya Hospital of Central South University between January 2013 and July 2015 was conducted. Results For the 15 patients, the pulmonary sinus cusp-derived VA originated from the lower regions of the pulmonary sinuses, leading from the right, left and anterior sinuses of the pulmonary sinus cusp in 4, 6 and 5 patients, respectively. Nine patients with pulmonary sinus cusp-derived VAs originating from the right and anterior sinuses underwent successful reversed U-curve catheter ablation, while the other 6 cases with arrhythmias originating from the left sinus underwent successful ablation with the conventional method (non-reversed U-curve catheter ablation). All the patients were followed-up for 6–31 months, and no cases of recurrence or complications occurred. Conclusions Reversed U-curve catheter ablation is suitable for VAs originating from the right and anterior pulmonary sinus cusps, while conventional ablation can also be used for those originating from the left PSCs. This article is protected by copyright. All rights reserved

Suppressor of cytokine signaling 3 (SOCS3), a signal transduction cytokine, is involved in lipid metabolism as well as in cell proliferation, differentiation, apoptosis, and so on. To explore the effects of SOCS3 on apoptosis and lipid metabolism in liver, we used a simple effective method named hydrodynamic tail vein injection to overexpress SOCS3. Then orbital blood was obtained for the assessment of blood lipid after injection. Lipid metabolism related genes were detected by Western blot after the determination of serum lipids. Meanwhile, liver cell apoptosis was observed by Hoechst and TUNEL staining and the expression of apoptosis related proteins Bax, Bcl-2, and Caspase3 were detected as well as the JAK2/STAT3 signaling pathway. In addition, we also demonstrated the effect of SOCS3 in prime hepatocyte by overexpression or interference of SOCS3 along with SD1008, which is a specific inhibitor of the JAK2/STAT3 signaling pathway. Taken together, all the results indicated that SOCS3 promoted lipid synthesis in mice liver and promoted hepatocyte apoptosis by inhibiting the activation of the JAK2/STAT3 signaling pathway, however the detailed regulation mechanism had not yet been fully understood and needs further study.

Background The modification of cardiac energy metabolism during atrial fibrillation (AF) has been demonstrated in previous studies, indicating a close association between these 2 processes. The aim of the present study was to identify the underlying mechanisms via profiling of the expression of energy metabolism-related proteins in the left atrial appendage (LAA) of patients with AF. Methods and results Isobaric tag for relative and absolute quantification-coupled 2-D liquid chromatography-tandem mass spectrometry (iTRAQ-coupled 2-D LC-MS/MS) was used to profile the expression of energy metabolism-related proteins in the LAA from valvular disease patients with sinus rhythm (SR; n=6) and AF (n=8). Using ProteinPilot 4.0, 122 energy metabolism-related proteins, consisting of 39 carbohydrate metabolism-related proteins, 22 proteins involved in lipid metabolism, 49 biological oxidation-related proteins and 12 other kinds of proteins, were identified. Most of them were key enzymes involved in energy metabolism. Moreover, most of the proteins that were expressed differently in the LAA between the AF and SR patients, and which were related to energy metabolism, were downregulated. These results were further validated on western blot. Conclusions Atrial myocardium energy production in valvular disease patients is impaired during permanent AF, and this impairment in energy production may be involved in the matrix of AF formation.

In order to investigate the mechanism of suppressor of cytokine signaling 2 (SOCS2) on mitochondrial biogenesis, RNA interference and over-expression plasmid vectors of SOCS2 were used to transfect murine skeletal muscle C2C12 cells. Results demonstrated that over-expression of SOCS2 inhibited the differentiation of C2C12 cells, and reduced the expression of MyHC, MyoD and MyoG while elevated the protein expression of MSTN. Meanwhile the expression of PGC-1α, MDH, CPT-1 were significantly elevated in the RNA interference of SOCS2 group which were decreased in SOCS2 overexpression group. However, there was no change on the expression of UCP1 in both two groups. JC-1 dyeing showed overexpression of SOCS2 decreased the mitochondrial membrane potential and results of immunofluorescence, real-time PCR and western blotting indicated the increase expression of Cyt c, while interference SOCS2 had the opposite effects in C2C12 cells. Moreover, interference of SOCS2 elevated the p38 phosphorylation level then further increased the phosphorylation of ATF2, whereas overexpression of SOCS2 alleviated this phenomenon. Taken together, our observations indicated that SOCS2 could suppress myotube formation, act as an anti-regulator of mitochondria biogenesis via inhibiting p38 MAPK signal pathway.

We conducted a population-based case-control study in Connecticut women to test the hypothesis that genetic variations in DNA repair pathway genes may modify the relationship between body mass index (BMI) and risk of non-Hodgkin lymphoma (NHL). Compared to those with BMI

Alpha melanocyte stimulating hormone (α-MSH) and Forkhead box C2 protein (Foxc2) enhance lipolysis in multiple tissues. However, their relationship in adipose fatty acid oxidation (FAO) remains unclear. Here, we demonstrated that α-MSH and Foxc2 increased palmitate oxidation to CO2 in white (WAT) and brown adipose tissue (BAT). C/EBPβ expression was reduced by α-MSH and Foxc2. FFA level was elevated by α-MSH and pc-Foxc2 treatment along with increased FAO in white and brown adipocytes. The expression of FAO key enzymes, medium-chain acyl-CoA dehydrogenase (MCAD) and long-chain acyl-CoA dehydrogenase (LCAD) were increased in α-MSH and pc-Foxc2 group. Combination of α-MSH and Foxc2 treatment synergistically promoted FAO through increasing the activity of CPT-1 and phosphorylation of ACC. We found C/EBPβ bind to MC5R and Foxc2 promoter regions and inhibited FAO. cAMP level was increased by α-MSH and Foxc2 individually treated or combined treatment. Furthermore, cAMP/PKA pathway-specific inhibitor (H89) blocked the FAO, despite in α-MSH and Foxc2 both added group. While forskolin, the cAMP agonist, promoted FAO and enhanced the effect of α-MSH and Foxc2. Collectively, α-MSH and Foxc2 mutual promote FAO in WAT and BAT via cAMP/PKA signal pathway. And C/EBPβ as a transcription suppressor inhibits α-MSH and Foxc2 expression and FAO.

MAP/Microtubule affinity-regulating kinase 4 (Mark4) plays an important role in the regulation of microtubule organization, adipogenesis and apoptosis. However, the role of Mark4 plays in oxidative stress and inflammation are poorly understood. In this study, we found Mark4 was induced by high fat diet (HFD) while PPARγ was elevated significantly in mice adipocytes. Further analyses revealed Mark4 impaired mitochondrial oxidative respiration and increased reactive oxygen species (ROS) production. At same time, the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) were greatly reduced. By treating cells with H2O2 and vitamin E (VE), Mark4 accentuated oxidative stress along with increased mRNA level of inflammatory factor interleukin-6 (IL-6) and decreased leptin mRNA. Furthermore, we found PPARγ bind to Mark4 promoter region and inhibited Mark4 expression. We showed PPARγ interacted with Mark4 and inhibited the stimulating effect of Mark4 on oxidative stress and inflammation. Finally, we demonstrated that the IKKα/NF-κB signal pathway was involved in Mark4 induced oxidative stress and inflammation, while PTDC, a special inhibitor of NF-κB signal pathway, reduced oxidative stress and inflammation. Thus, our study indicated that Mark4 was a potential drug target for treating metabolic diseases.

Generation of T lymphocytes with reactivity against cancer is a prerequisite for effective adoptive cellular therapies. We established a protocol for tumor-infiltrating lymphocytes (TILs) from patients with pancreatic ductal adenocarcinoma. Tumor samples from 17 pancreatic cancer specimens were cultured with cytokines (IL-2, IL-15, and IL-21) to expand TILs. After 10 days of culture, TILs were stimulated with an anti-CD3 antibody (OKT3) and irradiated allogeneic peripheral blood mononuclear cells. Reactivity of TILs against tumor-associated antigens (mesothelin, survivin, or NY-ESO-1) was detected by intracellular cytokine production by flow cytometry. Cytotoxicity was measured using a Chromium 51 release assay, and reactivity of TILs against autologous tumor cells was detected by INF-[gamma] production (ELISA). TIL composition was tested by CD45RA, CCR7, 4-1BB, LAG-3, PD-1, TIM3, and CTLA-4 marker analysis. TCR V[beta] was determined by flow cytometry and TCR clonality was gauged measuring the CDR3 region length by PCR analysis and subsequent sequencing. We could reliably obtain TILs from 17/17 patients with a majority of CD8(+) T cells. CD3(+)CD8(+), CD3(+)CD4(+), and CD3(+)CD4(-)CD8(-)[double-negative (DN) T cells] resided predominantly in central (CD45RA(-)CCR7(+)) and effector (CD45RA-CCR7-) memory subsets. CD8(+) TILs tested uniformly positive for LAG-3 (about 100%), whereas CD4(+) TILs showed only up to 12% LAG-3(+) staining and PD-1 showed a broad expression pattern in TILs from different patients. TILs from individual patients recognized strongly (up to 11.9% and 8.2% in CD8(+)) NY-ESO-1, determined by ICS, or mesothelin, determined respectively by TNF-[alpha] and IFN-[gamma] production. Twelve of 17 of CD8(+) TILs showed preferential expansion of certain TCR V[beta] families (eg, 99.2% V[beta]13.2 in CD8(+) TILs, 77% in the V[beta]1, 65.9% in the V[beta]22, and 63.3% in the V[beta]14 family). TCR CDR3 analysis exhibited monoclonal or oligoclonal TCRs, some of them (eg, CD8(+) V[beta]13.2) reacting strongly against autologous tumor defined by INF-[gamma] production or by cytotoxicity. We have optimized methods for generating pancreatic cancer–specific TILs that can be used for adoptive cellular therapy of patients with pancreatic cancer.

Cardiovascular disease associated with oxidative stress, including atherosclerosis, is the leading cause of mortality worldwide. The accelerated proliferation and migration of vascular smooth muscle cells are the predominant characteristics of atherogenesis, and endothelial dysfunction is a major risk factor for the pathogenesis of atherosclerosis. Podocalyxin (PODXL), a type I member of the cluster of differentiation 34 family of sialomucins, functions as a pro-adhesive molecule. Emerging evidence has revealed the importance of micro (mi)RNAs in the cardiovascular system. The present study demonstrated that there was an inverse association between miRNA (miR)-125b and PODXL in human umbilical vein endothelial cells and human aortic vascular smooth muscle cells (HAVSMCs) treated with oxidized low‑density lipoprotein (LDL) and platelet derived growth factor. Additionally, miR-125b had a suppressive function in cell proliferation and migration, at least partially via targeting PODXL in the HAVSMCs. Furthermore, the data suggested that the functions of miR-125b in arteriosclerosis obliterans may be associated with transgelin, lectin-type oxidized LDL receptor-1, vascular endothelial-cadherin, intercellular adhesion molecule-1, interleukin-6 and monocyte chemotactic protein-1. In conclusion, miR-125b was found to be important in arteriosclerosis obliterans by suppressing the expression of PODXL and may serve as a potential therapeutic target for the treatment of arteriosclerosis obliterans.

A fast and sensitive polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was developed for the analysis of flonicamid in environmental and agricultural samples. Two haptens of flonicamid differing in spacer arm length were synthesized and conjugated to proteins to be used as immunogens for the production of polyclonal antibodies. To obtain most sensitive combination of antibody/coating antigen, two antibodies were separately screened by homologous and heterologous assays. After optimization, the flonicamid ELISA showed that the 50% inhibitory concentration (IC50 value) was 3.86mgL(-1), and the limit of detection (IC20 value) was 0.032mgL(-1). There was no cross-reactivity to similar tested compounds. The recoveries obtained after the addition of standard flonicamid to the samples, including water, soil, carrot, apple and tomato, ranged from 79.3% to 116.4%. Moreover, the results of the ELISA for the spiked samples were largely consistent with the gas chromatography (R(2)=0.9891). The data showed that the proposed ELISA is an alternative tool for rapid, sensitive and accurate monitoring of flonicamid in environmental and agricultural samples.

Sirtuin type 1 (Sirt1) and protein kinase B (Akt2) are associated with development of obesity and inflammation, but the molecular mechanisms of Sirt1 and Akt2 interaction on adipose inflammation remain unclear. To explore these mechanisms, a mouse model was used. Mice were fed with a high-fat diet (HFD) for 8 weeks, with interventions of resveratrol (RES) or nicotinamide (NAM) during the last 15 days. The HFD reduced Sirt1 mRNA in adipose tissue and elevated interleukin-6 (IL-6) expression. RES reduced the adipose tissue weight, increased the Sirt1 mRNA level, and reduced both mRNA and protein levels of IL-6, MCP-1, inducible nitric oxide synthase, and TNF-α by inhibiting phosphorylation of Akt2 in adipose tissue. Additionally, macrophage type I marker genes were reduced while macrophage type II marker genes were elevated by RES addition. Moreover, activation of Akt2 signal by using insulin significantly blunted the inhibitory effect of RES on adipose inflammation. Immunoprecipitation assay demonstrated that RES enhances the protein-protein interaction between Sirt1 and Akt2, but NAM inhibits this interaction. Furthermore, Sirt1 significantly reduced the levels of raptor and inactivated mammalian target of rapamycin (mTOR)C1 signal by interacting with Akt2, and confirmed that RES attenuated adipose inflammation by inhibiting the mTOR/S6K1 pathway via rapamycin.

Rationale: This study aimed to describe the case of a 3-year-old girl with old bilateral facet dislocation on cervical vertebrae 6 and 7, who had spinal cord transection, received surgical treatment, and achieved a relative satisfactory therapeutic effect. Patient concerns: A 3-year-old girl was urgently transferred to the hospital after a car accident. Diagnoses: she was diagnosed with splenic rupture, intracranial hemorrhage, cervical dislocation, spinal transection, and Monteggia fracture of the left upper limb. Interventions: The girl underwent emergency splenectomy and was transferred to the intensive care unit of the hospital 15 days later. One-stage anterior–posterior approach surgery (anterior discectomy, posterior laminectomy, and pedicle screw fixation) was performed when the patient stabilized after 45-day symptomatic treatment. The operation was uneventful. Outcomes: The reduction of lower cervical dislocation was satisfactory, with sufficient spinal cord decompression. The internal fixation position was good, and the spinal sequence was well restored. The girl was discharged 2 weeks later after the operation and followed up for 2 years. The major nerve function of both upper limbs was recovered, with no obvious retardation of the growth of immature spine. Lessons: A satisfactory therapeutic effect was achieved for a pediatric old subaxial cervical dislocation with bilateral locked facets using anterior discectomy, posterior laminectomy, and pedicle screw fixation. The posterior pedicle screw fixation provided a good three-dimensional stability of the spine, with reduced risk and complications caused by anterior internal fixation. The growth of immature spine was not obviously affected during the 2-year follow-up.