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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.

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.

A novel fluorescent sensor based on bovine serum albumin stabilized gold/silver nanoclusters (BSA-Au/Ag NCs) was developed for sensitive and facile detection of alkaline phosphatase (ALP) activity. For this fluorescent sensor, ascorbic acid 2-phosphate (AAP) was decomposed into ascorbic acid (AA) and phosphate by catalysis with ALP. The initial red fluorescence of the BSA-Au/Ag NCs was effectively quenched by KMnO4 and then the fluorescence was recovered by addition of AA. The mechanism of interaction between BSA-Au/Ag NCs and KMnO4 and AA was studied with use of the fluorescence lifetime and UV-vis absorption spectra. The results indicated that the oxidation/reduction modulated by KMnO4/AA led to surface structure destruction/restoration of the BSA-Au/Ag NCs, resulting in fluorescence quenching/recovery. The proposed fluorescence-based method based on a dark background was used to detect ALP and had excellent sensitivity, with a detection limit of 0.00076 U/L. Moreover, the method was applied to the determination of added analytes, with satisfactory recoveries (97.0–105.0 %). In a simulated eutrophic water body, this method successfully detected ALP in actual water samples and could monitor the dynamic changes of ALP activity through visual observation. More importantly, the proposed fluorescent sensor not only has the advantages of simple operation and high sensitivity but has also been successfully used on filter paper to establish a rapid and visual test paper for ALP.

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.

Zearalenone (ZEN) is a prevalent mycotoxin that needs intensive monitoring. A semi-quantitative and quantitative immunochromatographic assay (ICA) was assembled for investigating ZEN contamination in 187 samples of cereal and their products from China in 2019. The semi-quantitative detection model had a limit of detection (LOD) of 0.50 ng/mL with visual judgment and could be completely inhibited within 5 min at 3.0 ng/mL ZEN. The quantitative detection model had a lower LOD of 0.25 ng/mL, and ZEN could be accurately and digitally detected from 0.25&ndash, 4.0 ng/mL. The ICA method had a high sensitivity, specificity, and accuracy for on-site ZEN detection. For investigation of the authentic samples, the ZEN-positive rate was 62.6%, and the ZEN-positive levels ranged from 2.7 to 867.0 ng/g, with an average ZEN-positive level being 85.0 ng/g. Of the ZEN-positive samples, 6.0% exceeded the values of the limit levels. The ZEN-positive samples were confirmed to be highly correlated using LC-MS/MS (R2 = 0.9794). This study could provide an efficiency and accuracy approach for ZEN in order to achieve visual and digitized on-site investigation. This significant information about the ZEN contamination levels might contribute to monitoring mycotoxin occurrence and for ensuring food safety.

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

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.

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...

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.

A fast and ultrasensitive indirect competitive time-resolved fluoroimmunoassay (TRFIA) was developed for the analysis of paclobutrazol in environmental water and soil samples. Paclobutrazol hapten was synthesized and conjugated to bovine serum albumin (BSA) for producing polyclonal antibodies. Under optimal conditions, the 50% inhibitory concentration (IC50 value) and limit of detection (LOD, IC20 value) were 1.09μgL(-1) and 0.067μgL(-1), respectively. The LOD of TRFIA was improved 30-fold compared to the already reported ELISA. There was almost no cross-reactivity of the antibody with the other structural analogues of triazole compounds, indicating that the antibody had high specificity. The average recoveries from spiked samples were in the range from 80.2% to 104.7% with a relative standard deviation of 1.0-9.5%. The TRFIA results for the real samples were in good agreement with that obtained by high-performance liquid chromatography analyses. The results indicate that the established TRFIA has potential application for screening paclobutrazol in environmental samples.

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.

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.

Forkhead box C2 (Foxc2) protein is a transcription factor in regulation of development, metabolism, and immunology. However, the regulatory mechanisms of Foxc2 on proliferation and apoptosis of preadipocytes are unclear. In this study, we found that high-fat-diet-induced obesity elevated the expression of Foxc2 and cyclin E after 6 weeks. Additionally, Foxc2 suppressed preadipocyte differentiation, increased cell counts and augmented G1-S transition of preadipocytes, along with the elevation of cyclin E expression and the reduction levels of p27 and p53. Furthermore, Foxc2 knockdown reduced early apoptotic cells with accompanying reduction of mitochondrial membrane potential and increased fragmentation of genomic DNA. We show that Foxc2 reduces the expression of Bax, caspase-9, and caspase-3 in both serum-starved and palmitic acid-induced cell apoptotic models, which confirms the anti-apoptotic role of Foxc2. Moreover, the protein kinase B (Akt)/mammalian target of rapamycin (mTOR)C1 signaling pathway and the ERK/mTORC1 signaling pathway were activated along with preadipocyte proliferation in response to Foxc2 overexpression, whereas apoptosis marker genes were downregulated during this process. Those effects were blocked by the interference of Foxc2 or signal pathways specific inhibitors. These data collectively reveal that Foxc2 enhances proliferation of preadipocytes and inhibits apoptosis of preadipocytes by activating the Akt/mTORC1 and ERK/mTORC1 signaling pathways.

The suppressor of cytokine signaling 3 (SOCS3) is an established negative feedback regulation transcription factor associated with leptin, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and growth hormone (GH). However, the regulatory mechanism of SOCS3 on inflammation and apoptosis of adipocyte is still not clear. In this study, we found an increased expression of adipocyte inflammatory cytokines TNF-α and IL-6 due to leptin treatment. Meanwhile Caspase3, a key executioner of apoptosis, was also elevated in this process. In addition, we observed that SOCS3 could promote inflammation whereas SOCS3 interference reversed this effect in LPS-induced adipocytes inflammatory model. Moreover, the expression of apoptosis-associated genes Bax, cleaved-Caspase9 and cleaved-Caspase3 were elevated along with decreased Bcl-2 expression as detected with Western blot and ELISA assay. The phosphorylation level of JAK2/STAT3 signal was inhibited by SOCS3 along with the elevated expression of IL-6, TNF-α and Caspase3. We also demonstrated that stable knockdown of SOCS3 along with SD1008, a specific inhibitor of JAK2/STAT3 signaling pathway, could significantly inhibit inflammation and apoptosis of adipocyte. Altogether, these results inferred that SOCS3 promotes adipocyte apoptosis by both aggravating inflammation and inhibiting the activity of JAK2/STAT3 signaling pathway.

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 Information Microtubule affinity-regulating kinase 4 (MARK4) deficiency has been reported to negatively regulate diet-induced obesity and to mitigate insulin resistance in knockout mice, and thus may play a role in metabolic syndrome. However, the details of the molecular mechanism have yet to be revealed and the impacts of MARK4 on apoptosis remain unexplored. This study investigated the role of Mark4 in the regulation of lipid accumulation and apoptosis in adipocytes and analysed signalling pathways involved. Results We found that Mark4 significantly up-regulated the expression of gene sterol regulatory element binding protein-1c (SREBP-1c), fatty acid synthase (FAS), acetyl-CoA carboxylase-α (ACCα) and peroxisome proliferator activated receptor-γ (PPARγ); and reduced the protein contents of adipose triglyceride lipase (ATGL), as evidenced by the dramatic increasing lipid droplet accumulation in 3T3-L1 cells. Furthermore, a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) apoptosis assay showed that Mark4 triggered apoptosis of adipocytes; and apoptosis was confirmed by the decreased protein contents of B-cell lymphoma-2 (Bcl-2), full-length caspase-3 and full-length caspase-9, as well as the increased expression of Bax, cleaved caspase-3 and cleaved caspase-9. Analysis of special inhibitors allowed us to offer the following explanation for these impacts of Mark4: activation of Jun N-terminal kinase1 (JNK1) promoted both apoptosis and adipogenesis, whereas inhibition of the p38 mitogen-activated protein kinase (p38MAPK) pathway contributed to lipid accumulation alone. Conclusions Mark4 promotes adipogenesis in 3T3-L1 adipocytes by activating the JNK1 and inhibiting the p38MAPK pathway, and triggers apoptosis by activating the JNK1 pathway. We conclude that anti-Mark4 therapy targetted to inhibit lipid accumulation and apoptosis of adipocytes shows potential as a novel therapeutic strategy for treatment of obesity-associated metabolic complications.

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.

The Haoyaoerhudong gabbro is a mafic intrusion located in the Haoyaoerhudong gold deposit, which is a giant gold deposit (148 t Au) hosted in Proterozoic strata on the northern margin of the North China Craton. In this paper, we present integrated SHRIMP U&ndash, Pb, geochemical and Sr&ndash, Nd isotopic data from gabbro of the Haoyaoerhudong gold deposit to reveal the magmatic processes behind its origin. SHRIMP zircon U&ndash, Pb dating constrains the timing of crystallization of the Haoyaoerhudong gabbro to 278.8 &plusmn, 0.81 Ma. Whole-rock geochemical results indicate that the Haoyaoerhudong gabbro has calc-alkaline features with enrichments of large-ion lithophile elements (LILE) and light rare-earth elements (REE) as well as depletions of high-field strength elements (HFSE). The relatively high (87Sr/86Sr)i (0.7053 to 0.7078) and low &epsilon, Nd(t) (&minus, 4.6 to &minus, 15.1) values of the gabbro indicate the involvement of crustal materials. Low Ce/Pb ratios (1.35 to 7.38), together with nearly constant La/Sm and Th/Yb ratios and variable Ba/Th and Sr/Nd ratios, suggest that the ancient mantle was modified by slab dehydration fluids. Based on new geochemical data and regional geological investigations, we propose that both the Haoyaoerhudong gold deposit and the Haoyaoerhudong gabbro formed in a post-orogenic extensional setting.

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.

An enzyme-linked immunosorbent assay (ELISA) was developed based on a polyclonal antibody for the analysis of thiacloprid in agricultural samples. Thiacloprid hapten was synthesized and conjugated to bovine serum albumin to produce an immunogen and ovalbumin to produce a coating antigen. Polyclonal antibodies were obtained from immunized New Zealand white rabbits. Under optimal conditions (5 % methanol, 0.1 mol/L Na+, pH 5.5), the ELISA showed a 50 % inhibitory concentration (IC50) value of 0.01 mg/L and a limit of detection (IC10) of 0.47 μg/L. No obvious cross-reaction with the other structural analogs of neonicotinoid insecticides showed that the polyclonal antibodies had a high specificity for thiacloprid. The average recoveries from spiked water, soil, pear, and tomato were in the range of 80 to 119 %. The results of the ELISA were confirmed by high-performance liquid chromatography, and the correlation of the results from the two methods had a high correlation coefficient of 0.99 (n = 3) in spiked samples (soil, pear, and tomato). The proposed ELISA could successfully be applied to the determination of thiacloprid residues in agricultural samples.

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.

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for the herbicide haloxyfop-P-methyl is reported. Two haptens of haloxyfop-P-methyl with different spacer-arm lengths were synthesized, to which polyclonal antibodies were obtained by immunizing New Zealand rabbits. The most sensitive combination of antibody/coating antigen was selected by homologous and heterologous assays. The results indicated that the spacer-arm length of hapten can affect assay sensitivity. The optimized ic-ELISA showed that the 50% inhibitory concentration (IC(50)) was 0.0419 mg/L, and the limit of detection (LOD) was 0.0019 mg/L. Recoveries were obtained from the agricultural samples, including water, soil, cucumbers, cabbages, tomatoes, and potatoes; all were in the range of 87.4%-111.8%, well within the requirements of residue detection.

Obesity is associated with macrophage infiltration and metabolic inflammation, both of which promote metabolic disease progression. Melatonin is reported to possess anti-inflammatory properties by inhibiting inflammatory response of adipocytes and macrophages activation. However, the effects of melatonin on the communication between adipocytes and macrophages during adipose inflammation remain elusive. Here, we demonstrated melatonin alleviated inflammation and elevated α-ketoglutarate (αKG) level in adipose tissue of obese mice. Mitochondrial isocitrate dehydrogenase 2 (Idh2) mRNA level was also elevated by melatonin in adipocytes leading to increase αKG level. Further analysis revealed αKG was the target for melatonin inhibition of adipose inflammation. Moreover, sirtuin 1 (Sirt1) physically interacted with IDH2 and formed a complex to increase the circadian amplitude of Idh2 and αKG content in melatonin-inhibited adipose inflammation. Notably, melatonin promoted exosomes secretion from adipocyte and increased adipose-derived exosomal αKG level. Our results also confirmed that melatonin alleviated adipocyte inflammation and increased ratio of M2 to M1 macrophages by transporting of exosomal αKG to macrophages and promoting TET-mediated DNA demethylation. Furthermore, exosomal αKG attenuated signal transducers and activators of transduction-3 (STAT3)/NF-κB signal by its receptor oxoglutarate receptor 1 (OXGR1) in adipocytes. Melatonin also attenuated adipose inflammation and deceased macrophage number in chronic jet-lag mice. In summary, our results demonstrate melatonin alleviates metabolic inflammation by increasing cellular and exosomal αKG level in adipose tissue. Our data reveal a novel function of melatonin on adipocytes and macrophages communication, suggesting a new potential therapy for melatonin to prevent and treat obesity caused systemic inflammatory disease.

Fatty acid binding protein 4 (FABP4), plays key role in fatty acid transportation and oxidation and increases with leptin synergistically during adipose inflammation process. However, the regulation mechanism between FABP4 and leptin on mitochondrial fatty acid oxidation remains unclear. In this study, we found that FABP4 reduced the expression of leptin, CPT-1 and AOX1 in mice adipocytes. Conversely, FABP4 was down-regulated in a time-dependent manner by leptin treatment. Additionally, forced expression of FABP4 attenuated the expression of PGC1-α, UCP2, CPT-1, AOX1 and COX2 compared with leptin incubation. Moreover, mitochondrial membrane potential, fatty acid oxidation enzyme medium-chain acyl-CoA dehydrogenase (MCAD), long-chain acyl-CoA dehydrogenase (LCAD) and Cyt C levels were reduced in response to the overexpression of FABP4. These reductions correspond well with the reduced release of free fatty acid and the inactivation of mitochondrial complexes I and III by FABP4 overexpression. Furthermore, addition of the Akt/mTOR pathway-specific inhibitor (MK2206) blocked the mitochondrial fatty acid oxidation and respiration factors, whereas interference of FABP4 overcame these effects. Taken together, FABP4 could reverse the activation of the leptin-induced mitochondrial fatty acid oxidation and the inhibition of Akt/mTOR signal pathway played a key role in this process.

Pyroptosis is a proinflammatory form of cell death that is associated with pathogenesis of many chronic inflammatory diseases. Melatonin is substantially reported to possess anti-inflammatory properties by inhibiting inflammasome activation. However, the effects of melatonin on inflammasome-induced pyroptosis in adipocytes remain elusive. Here, we demonstrated that melatonin alleviated lipopolysaccharides (LPS)-induced inflammation and NLRP3 inflammasome formation in mice adipose tissue. The NLRP3 inflammasome-mediated pyroptosis was also inhibited by melatonin in adipocytes. Further analysis revealed that gasdermin D (GSDMD), the key executioner of pyroptosis, was the target for melatonin inhibition of adipocyte pyroptosis. Importantly, we determined that nuclear factor κB (NF-κB) signal was required for the GSDMD-mediated pyroptosis in adipocytes. We also confirmed that melatonin alleviated adipocyte pyroptosis by transcriptional suppression of GSDMD. Moreover, GSDMD physically interacted with interferon regulatory factor 7 (IRF7) and subsequently formed a complex to promote adipocyte pyroptosis. Melatonin also attenuated NLRP3 inflammasome activation and pyroptosis, which was induced by LPS or obesity. In summary, our results demonstrate that melatonin alleviates inflammasome-induced pyroptosis by blocking NF-κB/GSDMD signal in mice adipose tissue. Our data reveal a novel function of melatonin on adipocyte pyroptosis, suggesting a new potential therapy for melatonin to prevent and treat obesity caused systemic inflammatory response.

Melatonin is synthesized in the pineal gland and controls circadian rhythm of peripheral adipose tissue, resulting in changes in body weight. Although core regulatory components of clock rhythmicity have been defined, insight into the mechanisms of circadian rhythm-mediated proliferation in adipose tissue is still limited. Here, we showed that melatonin (20 mg/kg/d) promoted circadian and proliferation processes in white adipose tissue. The circadian amplitudes of brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (Bmal1, P

MicroRNAs are small non-coding RNAs that partially bind to the 3’ untranslated (3’UTR) regions of target genes in animals and regulate protein production of the target transcripts. MiR-103 has been confirmed to play a critical role in lipid metabolism, however, the target genes and signaling pathway regulated by miR-103 is still unclear. In our experiment, we observed a positive function of miR-103 on the adipogenic differentiation of 3T3-L1 pre-adipocyte. Furthermore, we proved that this function of miR-103 worked through activating AKT/mTOR signal pathway and impairing target gene MEF2D. By inhibiting and over-expressing the MEF2D gene, we found that MEF2D had a negative role in regulating adipocyte key genes, and this function of MEF2D could be impaired by miR-103. In conclusion, we found that miR-103 can promote 3T3-L1 cells differentiation by targeting MEF2D and activating AKT/mTOR signal pathway. These results will shed a light on further study of microRNAs.

Adiponectin is a cytokine produced predominantly by adipose tissue and correlates with glucose and lipid homeostasis. However, the effects of adiponectin on endoplasmic reticulum (ER) stress and apoptosis of adipose tissue remain elusive. In this study, we found that tunicamycin-induced ER stress increased serum free fatty acid (FFA) and impaired glucose tolerance, elevated the mRNA levels of GRP78, Chop, ATF2 and caspase 3, but reduced adiponectin mRNA level in white adipose tissue. Moreover, ER stress-triggered adipocyte apoptosis by increasing cellular FFA level and Ca2+ level. Further analysis revealed that adiponectin alleviated ER stress-induced adipocyte apoptosis by elevating peroxisome proliferator-activated receptor alpha (PPARα) mRNA level. Our data also confirmed that adiponectin reduced early apoptotic cells and blocked the mitochondrial apoptosis pathway by activating the AdipoR1/AMP-activated protein kinase (AMPK) signal pathway. In addition, PPARα bound to ATF2 promoter region and inhibited transcription of ATF2. The inhibition of adipocyte apoptosis by adiponectin was correlated with transcriptional suppression of ATF2. Furthermore, adiponectin inhibited ER stress-induced apoptosis by activating the AMPK/PKC pathway. In summary, our data demonstrate adiponectin inhibited ER stress and apoptosis of adipocyte in vivo and in vitro by activating the AMPK/PPARα/ATF2 pathway. Our study establishes that adiponectin is an important adipocytokine for preventing and treating obesity.

BackgroundIn the European Union (EU), the use of diniconazole-M is no longer authorized. However, residues of diniconazole-M occur in various plant commodities.Methodology/principal findingsA selective and simple analytical method for the trace level determination of diniconazole in soil, fruit, vegetables and water samples was developed based on immunoaffinity extraction followed by Enzyme-linked immunosorbent assay (ELISA) and the high-performance liquid chromatography (HPLC) analysis. The ELISA was based on monoclonal antibodies highly specific to diniconazole and was a fast, cost-effective, and selective screening method for the detection of diniconazole. The results of the ELISA correlated well with gas chromatography (GC) results, with the correlation coefficient of 0.9879 (n = 19). A simple gel permeation chromato- graphy clean-up method was developed to purify extracts from matrices containing high amounts of fat and natural pigments, without the need for a large dilution of the sample. The immunoaffinity column (IAC) capacity was 0.180 mg g(-1). The columns could be re-used approximately 20 times with no significant alteration in capacity. The recoveries from complex samples were in the range of 89.2% to 96.1% with a relative standard deviation (RSD) of 0.770%-6.11% by ELISA. The results were in good agreement with those obtained by HPLC method.Conclusion/significanceThe IAC extraction procedure coupled with HPLC and ELISA analysis could be also used as alternative effective analytical methods for the determination of diniconazole concentrations in complex samples.

Cardiovascular disease (CVD) is still a primary cause of death in most countries. The pathogenesis of CVD can be influenced by multiple risk factors. Recent studies suggested that the innate immune system may be involved in the development of CVD. Pentraxins are critical components of the innate immune system. Recent researches have demonstrated that elevated plasma pentraxin 3 (PTX3) is associated with cardiovascular events and cardiovascular risk factors. Thus, we hypothesized that elevated plasma PTX3 is a potential cardiovascular risk factor evidenced by the following aspects: (1) PTX3 inhibited angiogenesis and promoted restenosis, (2) the expression of PTX3 increased in advanced atherosclerotic lesions and the blood of patients with ischemic heart disorders, (3) elevated plasma PTX3 levels were associated with adverse cardiovascular outcomes. If the hypothesis is confirmed, PTX3 will be an effective target point to the prevention and treatment of CVD.

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been developed for detection of pretilachlor in water and soil. An immunogen was prepared from haptens of pretilachlor conjugated to bovine serum albumin(BSA). The specific polyclonal antibodies were obtained by immunizing New Zealand white rabbits. The influence of parameters including concentrations of methanol, ionic strength and pH values were optimized to improve the sensitivity of the assay. The optimized ELISA was shown to have a high sensitivity and specificity for pretilachlor. Under optimal conditions, the ELISA has demonstrated an 50% inhibitory concentration (IC50) value of 0.0359 mg/L with a limit of detection (LOD, IC10) of 6.9 ng/L. The cross-reactivities to some analogs of pretilachlor (acetochlor, butachlor, metazachlor and metalaxyl) were below 1.5%. The average recoveries of pretilachlor from distilled water, tap water, paddy water and soil were in the range of 77.0–115.2% between 0.005 and 5.0 mg/L. The results of ELISA for spiked samples were confirmed by GC–ECD with a high correlation coefficient of 0.9950(n=11). Thus, the ELISA proven to be a sensitive, specific, inexpensive and quantitative tool for detection of pretilachlor from four kinds of spiked samples.

Neural tube defects (NTDs) are common birth defects, whose specific biomarkers are needed. The purpose of this pilot study is to determine whether protein profiling in NTD-mothers differ from normal controls using SELDI-TOF-MS. ProteinChip Biomarker System was used to evaluate 82 maternal serum samples, 78 urine samples and 76 amniotic fluid samples. The validity of classification tree was then challenged with a blind test set including another 20 NTD-mothers and 18 controls in serum samples, and another 19 NTD-mothers and 17 controls in urine samples, and another 20 NTD-mothers and 17 controls in amniotic fluid samples. Eight proteins detected in serum samples were up-regulated and four proteins were down-regulated in the NTD group. Four proteins detected in urine samples were up-regulated and one protein was down-regulated in the NTD group. Six proteins detected in amniotic fluid samples were up-regulated and one protein was down-regulated in the NTD group. The classification tree for serum samples separated NTDs from healthy individuals, achieving a sensitivity of 91% and a specificity of 97% in the training set, and achieving a sensitivity of 90% and a specificity of 97% and a positive predictive value of 95% in the test set. The classification tree for urine samples separated NTDs from controls, achieving a sensitivity of 95% and a specificity of 94% in the training set, and achieving a sensitivity of 89% and a specificity of 82% and a positive predictive value of 85% in the test set. The classification tree for amniotic fluid samples separated NTDs from controls, achieving a sensitivity of 93% and a specificity of 89% in the training set, and achieving a sensitivity of 90% and a specificity of 88% and a positive predictive value of 90% in the test set. These suggest that SELDI-TOF-MS is an additional method for NTDs pregnancies detection.