In this preclinical model, TRPC6 channels were essential for glioma development via regulation of G2/M phase transition. This study suggests that TRPC6 might be a new target for therapeutic intervention of human glioma.

Background: Broad-range PCR provides valuable information for detecting bacterial infections. This study assesses the combined use of broad-range real-time PCR and high-resolution melting analysis for rapid detection and identification of clinically important bacteria. Methods: We subjected 46 bacterial culture colonies representing 25 clinically important bacterial species to LightCycler real-time PCR amplification of the 16S rRNA gene in the presence of LCGreen I fluorescent dye. We performed high-resolution melting analysis of the PCR products with the HR-1 instrument and used melting profiles as molecular fingerprints for bacterial species identification. We validated this method via assessment of 54 consecutive bacteria culture colonies obtained from a clinical microbiology laboratory. Results: The 16S rRNA gene of all 25 bacterial species was amplifiable by this method, with PCR product

Ischemic brain injury is acute local inflammation, leading to accumulation of pro-inflammatory cytokines. Cytokines influence the recruitment of leucocytes and play a key role in the inflammatory injury processes. Recently, a number of studies have demonstrated a close relationship between brain ischemia and cytokines. Interleukin-17 (IL-17) is a newly identified T-cell-specific cytokine. In this study, we evaluated the source and the action of IL-17 over the course of cerebral ischemia in rats (Sprague-Dawley) and humans. The levels of IL-17 in the ischemic hemisphere of the human brain, which was removed at necropsy, were assayed immunohistochemically. In rats, permanent middle cerebral artery occlusion (pMCAO) was obtained by inserting nylon monofilament into the right external carotid artery, occluding the right middle cerebral artery. The expression of IL-17 mRNA in rat was assayed using oligoprobe in situ hybridization. IL-17 production by neuroglial cells was assayed by double-staining using antibody glial fibrillary acidic protein (GFAP) and antibody IL-17. Levels of IL-17 were elevated in the ischemic hemispheres of human brain compared with the opposite normal hemispheres and peaked at days 3-5 after brain ischemia. The IL-17-positive cells were found in the ischemic lesion region. IL-17 mRNA was also elevated in ischemic hemispheres of pMCAO-operated rats, which were slightly elevated after 1 h and peaked at 6 days. IL-17 and GFAP double-stained were extensive in rat ischemic hemisphere. The ischemia-induced IL-17 expression in human brain reported here for the first time was very similar to that in rat model except that the peak was slightly earlier. We found for the first time that IL-17 was involved in an intense inflammatory reaction of brain ischemic injury in human. In pMCAO-operated rats, our findings suggest that IL-17 is produced by the neuroglial cells in the brain region undergoing ischemic insult. We suggest that in additional to T cells the neuroglial cell may be another cellular origin of IL-17 in later progression of brain ischemia.

BackgroundHepatocellular carcinoma (HCC) is the one of the most common cancers and lethal diseases in the world. DNA methylation alteration is frequently observed in HCC and may play important roles in carcinogenesis and diagnosis.MethodsUsing the TCGA HCC dataset, we classified HCC patients into different methylation subtypes, identified differentially methylated and expressed genes, and analyzed cis- and trans-regulation of DNA methylation and gene expression. To find potential diagnostic biomarkers for HCC, we screened HCC-specific CpGs by comparing the methylation profiles of 375 samples from HCC patients, 50 normal liver samples, 184 normal blood samples, and 3780 samples from patients with other cancers. A logistic regression model was constructed to distinguish HCC patients from normal controls. Model performance was evaluated using three independent datasets (including 327 HCC samples and 122 normal samples) and ten newly collected biopsies.ResultsWe identified a group of patients with a CpG island methylator phenotype (CIMP) and found that the overall survival of CIMP patients was poorer than that of non-CIMP patients. Our analyses showed that the cis-regulation of DNA methylation and gene expression was dominated by the negative correlation, while the trans-regulation was more complex. More importantly, we identified six HCC-specific hypermethylated sites as potential diagnostic biomarkers. The combination of six sites achieved ~ 92% sensitivity in predicting HCC, ~ 98% specificity in excluding normal livers, and ~ 98% specificity in excluding other cancers. Compared with previously published methylation markers, our markers are the only ones that can distinguish HCC from other cancers.ConclusionsOverall, our study systematically describes the DNA methylation characteristics of HCC and provides promising biomarkers for the diagnosis of HCC.Electronic supplementary materialThe online version of this article (10.1186/s13073-018-0548-z) contains supplementary material, which is available to authorized users.

Disabled-2 (DAB2) is an adapter protein that is up-regulated during megakaryocytic differentiation of hematopoietic cells and is abundantly expressed in platelets. In this study, the role of DAB2 in integrin ␣ IIb ␤ 3 -mediated matrix protein fibrinogen adhesion and cell signaling was investigated. In K562 cells differentiating to the megakaryocytic lineage, down-regulation of DAB2 by DAB2 small interfering RNA augmented integrin ␣ IIb ␤ 3 activation and resulted in an increase in cell adhesion to fibrinogen. Ectopic expression of DAB2 reversed the DAB2 small interfering RNA effect or, by itself, decreased fibrinogen adhesion of K562 cells. Mutational analysis revealed that a DAB2 Ser 24 phosphorylation mutant (S24A) abrogated the inhibitory function of DAB2. The spatial and temporal association/interaction of DAB2 and platelet integrin ␣ IIb ␤ 3 (CD61) in both megakaryocytic cells and platelets led us to examine the effect of Ser 24 phosphorylation on the interaction between DAB2 and integrin ␤ 3 . Through cellular localization and co-immunoprecipitation analysis, we demonstrate for the first time that Ser 24 phosphorylation promotes membrane translocation of DAB2 and its subsequent interaction with integrin ␤ 3 , thereby defining a mechanism for DAB2 in regulating integrin ␣ IIb ␤ 3 activation and inside-out signaling. Consistent with the effect on fibrinogen adhesion, Ser 24 phosphorylation of DAB2 was also involved in the negative regulation of ␣ IIb ␤ 3 -induced T cell factor transcriptional activity. In contrast, the S24A mutant acted like wild-type DAB2 and inhibited both ␤-catenin-and plakoglobin-mediated T cell factor transactivation. Hence, DAB2 elicits distinct regulatory mechanisms in ␣ IIb ␤ 3 and ␤-catenin/plakoglobin signaling in a Ser 24 phosphorylation-dependent and -independent manner, respectively. These findings indicate Ser 24 phosphorylation as a molecular basis for DAB2 acting as a negative regulator in ␣ IIb ␤ 3 inside-out signaling and contribute to our understanding of DAB2 in megakaryocytic differentiation and platelet function.Disabled-2 (DAB2) is an adapter protein that has been implicated in growth factor signaling (1, 2), endocytosis (3-5), cell adhesive function (6, 7), and hematopoietic cell differentiation (8). Like other adapter proteins, DAB2 elicits its function through interaction with other cellular proteins. DAB2 interacts with Grb2, myosin VI, SMAD2/3, DIP1/2, Dvl-3, the integrin ␤ subunit, and c-Src through the N-terminal phosphotyrosine-binding (PTB) 1 domain and the C-terminal proline-rich region (1, 4, 5, 9 -13). These interactions have been shown to modulate cytoskeleton organization, transcriptional activity, and cell signaling of various receptor protein-tyrosine kinases. DAB2 thus plays in a pivotal role in the control of cellular homeostasis.In cells, protein phosphorylation of DAB2 modulates its functional activity during growth factor signaling, megakaryocytic differentiation, macrophage spreading, and cell cycle progression. To date, protein kinase C (PKC) and...

Platelet aggregation plays a pivotal role in the haemostatic process and is involved in the pathological counterpart of arterial thrombosis. We have shown that the adapter protein disabled-2 (DAB2) is expressed abundantly in platelets. In this study, DAB2 was found to distribute in the platelet ␣ ␣-granules and was released from the granular compartment upon platelet activation. The secreted DAB2 binds to the extracellular region of ␣ ␣IIb␤ ␤3 integrin on the platelet surface through the phosphotyrosine-binding domain. The DAB2-platelet interactions result in the inhibition of agonist-induced platelet aggregation with the exception of thrombin, a DAB2 protease that renders DAB2 inactive. Biochemical and mutational analysis revealed that the DAB2 cell-adhesion Arg-Gly-Asp (RGD) motif (amino acid residues 64-66) and the ␣ ␣IIb-integrin-fibrinogenbinding region (amino acid residues 171-464) are important for the DAB2-platelet interactions. Such interactions compete for the binding of ␣ ␣IIb integrin with fibrinogen and provide a mechanism for DAB2 to inhibit platelet aggregation. Accordingly, the synthetic RGD-motifcontaining DAB2 peptide PDARGDKM also elicited antiplatelet aggregation activity. These findings demonstrate for the first time that DAB2 is an ␣ ␣IIb-integrin-binding protein that plays a novel role in the control of plateletfibrinogen interactions and platelet aggregation.Key words: Disabled-2, Fibrinogen, ␣IIb␤3 Integrin, Platelet aggregation, RGD motif SummaryDisabled-2 is a novel ␣ ␣IIb-integrin-binding protein that negatively regulates platelet-fibrinogen interactions and platelet aggregation

Previous studies have shown that Disabled-2 (DAB2) is up-regulated during megakaryocytic di¡erentiation of human K562 cells. To delineate the consequences of DAB2 induction, a DNA vector-based small interfering RNA (siRNA) was designed to intervene in DAB2 expression. We found that DAB2 siRNA speci¢cally inhibited DAB2 induction, resulting in the modulation of cell^cell adhesion and mitogen-activated protein kinase (MAPK) phosphorylation. The morphological changes and L L3 integrin expression associated with megakaryocytic differentiation were not a¡ected. Since the MAPK pathway has been shown to involve DAB2 induction [Tseng et al., Biochem. Biophys. Res. Commun. 285 (2001) 129^135], our results suggest a reciprocal regulation between DAB2 and MAPK in the di¡erentiation of K562 cells. In addition, we have demonstrated for the ¢rst time that DAB2 siRNA is a valuable tool for unveiling the biological consequences of DAB2 expression.

We assessed the use of high-resolution melting (HRM) analysis for the rapid identification of influenza