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Fast and accurate malicious domain detection is an essential research theme to prevent cybercrime, and machine learning is an attractive approach for detecting unseen malicious domains in the past decade. In this paper, we present MADMAX (MAchine learning-baseD MAlicious domain eXhauster), a browser-based application leveraging extreme learning machine (ELM) for malicious domain detection. In contrast to the existing work of ELM-based domain detection, MADMAX newly introduces two methods, i.e., selection of optimized features to provide higher accuracy and throughput based on permutation importance and real-time training to retrain a model with an updated malicious dataset for continuous malicious domain detection. We demonstrate that MADMAX fairly outperforms the existing work with respect to accuracy and throughput by virtue of the selection of optimized features. Moreover, we also confirm a model with real-time training stably detects even unseen malicious domains, whereas accuracy of a model without the real-time training decreases due to the unseen domains. The source codes of MADMAX is publicly available via GitHub.

Background Podoplanin (PDPN) is a transmembrane glycoprotein that mediates tumor cell-induced platelets aggregation in different cancer types. Emerging data indicate that PDPN is a marker for poor prognosis of human oral squamous cell carcinoma (OSCC). However, the functional impacts of PDPN on cancer formation and disease progression of OSCC remain to be elucidated. Methods The sublines of the OECM-1 oral cancer cells with PDPN knockdown or overexpression were established. The cellular characteristics and the ability to induce platelet aggregation of these cells lines were analyzed. An ectopic xenograft animal model by inoculating cancer cells into the anterior neck region of nude mice was established to investigate the functional impact of PDPN on disease progression and cancer-associated thrombosis of OSCC. Results PDPN promoted OSCC cell migration and invasion, but had no effect on cell proliferation in vitro and tumor growth in vivo. Co-incubation of PDPN-positive (PDPN+) OSCC cells with platelets induced platelet activation and aggregation. The mice bearing PDPN+ tumor had a decrease in overall survival despite that there was no gross appearance of distant metastasis. A speckled immunofluorescence staining pattern of platelet marker mCD41 was defined in the PDPN+ tumor sections and the intensity was greater than in the PDPN-low or negative tumor sections. Co-immunofluorescence staining of the tumor sections with mCD41 and the endothelial cell marker mCD31 further demonstrated that platelet aggregates were located in the lumen of blood vessel and were also distributed intratumorally in the mice bearing PDPN+ tumors. Conclusions These data demonstrated that PDPN expression in the cancer cells is associated with high risk of thrombosis, leading to unfavorable overall survival of the mice. This study provides new insights into the functions of PDPN in cancer-associated thrombosis and in the pathophysiology of OSCC.

Two rearranged terpenoids with a rare 3,4,5-trimethyl-cyclohexa-2,5-dien-1-one moiety, namely leucocephins A (1) and B (2), and a megastigmane, namely leucocephin C (3), as well as three known compounds, hollongdione (4), 3-acetoxy-lup-12,20(29)-diene (5), and β-amyrin acetate (6) were isolated from the leaves of Euphorbia leucocephala. Their structures and absolute configurations were determined by spectroscopic methods and comparing with literature data. Compounds 4–6 exhibited potent anti-influenza A virus activity comparable to the positive control, betulinic acid.

Torque teno virus (TTV) is one of the most common human viruses and can infect an individual with multiple genotypes chronically and persistently. TTV group 6 is a recently discovered phylogenetic group first isolated from eastern Taiwan indigenes, but whether the TTV group 6 was also prevalent in the general population still unknown. One hundred and three randomly collected blood samples from general population and 66 TTV positive DNA samples extracted from Taiwan indigenes were included. A group-6-specific PCR was developed for re-screen over TTV positive samples. Two TTV group 6 positive samples from general population were cloned and sequenced for identifying mix-infected TTVs and confirming their classification by maximum-likelihood and Bayesian inference phylogeny. TTV group 6 can be detected in 4.5% (4/89) and 7.6% (5/66) of TTV positive samples from Taiwanese general population and eastern Taiwan indigenes, respectively. Sample VC09 was mix-infected with TTV groups 3 and 6. Sample VC99 was mix-infected with TTV groups 3, 4 and 6. A highly diverse triple overlapping region was observed, which may represent a unique phenomenon of TTV. The group-6-specific PCR can successfully detect TTV group 6. TTV group 6 may be prevalent worldwide regardless of the geographic region and/or ethnic groups.

Eradicating influenza A virus (IAV) is difficult, due to its genetic drift and reassortment ability. As the infectious cycle is initiated by the influenza glycoprotein, hemagglutinin (HA), which mediates the binding of virions to terminal sialic acids moieties, HA is a tempting target of anti-influenza inhibitors. However, the complexity of the HA structure has prevented delineation of the structural characterization of the HA protein–ligand complex. Our computational strategy efficiently analyzed > 200,000 records of compounds held in the United States National Cancer Institute (NCI) database and identified potential HA inhibitors, by modeling the sialic acid (SA) receptor binding site (RBS) for the HA structure. Our modeling revealed that compound NSC85561 showed significant antiviral activity against the IAV H1N1 strain with EC50 values ranging from 2.31 to 2.53 µM and negligible cytotoxicity (CC50 > 700 µM). Using the NSC85561 compound as the template to generate 12 derivatives, robust bioassay results revealed the strongest antiviral efficacies with NSC47715 and NSC7223. Virtual screening clearly identified three SA receptor binding site inhibitors that were successfully validated in experimental data. Thus, our computational strategy has identified SA receptor binding site inhibitors against HA that show IAV-associated antiviral activity.

BackgroundBidirectional integrin αIIbβ3 signaling is essential for platelet activation. The platelet adaptor protein Disabled-2 (Dab2) is a key regulator of integrin signaling and is phosphorylated at serine 24 in eukaryotic cells. However, the mechanistic insight and function of Dab2-serine 24 phosphorylation (Dab2-pSer24) in platelet biology are barely understood. This study aimed to define whether and how Dab2 is phosphorylated at Ser24 during platelet activation and to investigate the effect of Dab2-pSer24 on platelet function.ResultsAn antibody with confirmed specificity for Dab2-pSer24 was generated. By using this antibody as a tool, we showed that protein kinase C (PKC)-mediated Dab2-pSer24 was a conservative signaling event when human platelets were activated by the platelet agonists such as thrombin, collagen, ADP, 12-O-tetradecanoylphorbol-13-acetate, and the thromboxane A2 activator U46619. The agonists-stimulated Dab2-pSer24 was attenuated by pretreatment of platelets with the RGDS peptide which inhibits integrin outside-in signaling by competitive binding of integrin αIIb with fibrinogen. Direct activation of platelet integrin outside-in signaling by combined treatment of platelets with manganese dichloride and fibrinogen or by spreading of platelets on fibrinogen also resulted in Dab2-pSer24. These findings implicate that Dab2-pSer24 was associated with the outside-in signaling of integrin. Further analysis revealed that Dab2-pSer24 was downstream of Src-PKC-axis and phospholipase D1 underlying the integrin αIIbβ3 outside-in signaling. A membrane penetrating peptide R11-Ser24 which contained 11 repeats of arginine linked to the Dab2-Ser24 phosphorylation site and its flanking sequences (RRRRRRRRRRR19APKAPSKKEKK29) and the R11-S24A peptide with Ser24Ala mutation were designed to elucidate the functions of Dab2-pSer24. R11-Ser24 but not R11-S24A inhibited agonists-stimulated Dab2-pSer24 and consequently suppressed platelet spreading on fibrinogen, with no effect on platelet aggregation and fibrinogen binding. Notably, Ser24 and the previously reported Ser723 phosphorylation (Dab2-pSer723) occurred exclusively in a single Dab2 molecule and resulted in distinctive subcellular distribution and function of Dab2. Dab2-pSer723 was mainly distributed in the cytosol of activated platelets and associated with integrin inside-out signaling, while Dab2-pSer24 was mainly distributed in the membrane fraction of activated platelets and associated with integrin outside-in signaling.ConclusionsThese findings demonstrate for the first time that Dab2-pSer24 is conservative in integrin αIIbβ3 outside-in signaling during platelet activation and plays a novel role in the control of cytoskeleton reorganization and platelet spreading on fibrinogen.

MicroRNA let-7b expression is induced by infection of hepatitis C virus (HCV) and is involved in the regulation of HCV replication by directly targeting the HCV genome. The current study demonstrated that let-7b directly targets negative regulators of type I interferon (IFN) signaling thereby limiting HCV replication in the early stage of HCV infection. Let-7b-regulated genes which are involved in host cellular responses to HCV infection were unveiled by microarray profiling and bioinformatic analyses, followed by various molecular and cellular assays using Huh7 cells expressing wild-type (WT) or the seed region-mutated let-7b. Let-7b targeted the cytokine signaling 1 (SOCS1) protein, a negative regulator of JAK/STAT signaling, which then enhanced STAT1-Y701 phosphorylation leading to increased expression of the downstream interferon-stimulated genes (ISGs). Let-7b augmented retinoic acid-inducible gene I (RIG-I) signaling, but not MDA5, to phosphorylate and nuclear translocate IRF3 leading to increased expression of IFN-β. Let-7b directly targeted the ATG12 and IκB kinase alpha (IKKα) transcripts and reduced the interaction of the ATG5-ATG12 conjugate and RIG-I leading to increased expression of IFN, which may further stimulate JAK/STAT signaling. Let-7b induced by HCV infection elicits dual effects on IFN expression and signaling, along with targeting the coding sequences of NS5B and 5′ UTR of the HCV genome, and limits HCV RNA accumulation in the early stage of HCV infection. Controlling let-7b expression is thereby crucial in the intervention of HCV infection. IMPORTANCE HCV is a leading cause of liver disease, with an estimated 71 million people infected worldwide. During HCV infection, type I interferon (IFN) signaling displays potent antiviral and immunomodulatory effects. Host factors, including microRNAs (miRNAs), play a role in upregulating IFN signaling to limit HCV replication. Let-7b is a liver-abundant miRNA that is induced by HCV infection and targets the HCV genome to suppress HCV RNA accumulation. In this study, we demonstrated that let-7b, as a positive regulator of type I IFN signaling, plays dual roles against HCV replication by increasing the expression of IFN and interferon-sensitive response element (ISRE)-driven interferon-stimulated genes (ISGs) in the early stage of HCV infection. This study sheds new insight into understanding the role of let-7b in combatting HCV infection. Clarifying IFN signaling regulated by miRNA during the early phase of HCV infection may help researchers understand the initial defense mechanisms to other RNA viruses.

Additional file 1: Figure S1. Pre-incubation of platelets with R11-S24 suppresses platelet spreading on fibrinogen during thrombin stimulation. Platelets were pre-incubated with R11, R11-S24 or R11-S24A peptide then stimulated with thrombin (0.05 U/ml) and were spread on coverslips. After spreading on coverslips, the platelets were fixed, permeabilized, stained for F-actin (green) and observed by phase contrast microscopy.

Additional file 2: Figure S2. Pre-incubation of platelets with R11-S24 had no effect on thrombin-induced fibrinogen binding and platelet aggregation. Platelets were pre-incubated with R11, R11-S24 or R11-S24A peptide then stimulated with thrombin (0.05 U/ml). Fibrinogen binding was determined by flow cytometry. Platelet aggregation was recorded by a platelet aggregometer.

Hepatocellular carcinoma (HCC) is among the most common causes of cancer death in men. Whether or not a longitudinal follow-up of circulating tumor cells (CTCs) before and at different time points during systemic/targeted therapy is useful for monitoring the treatment response of patients with locally advanced or metastatic HCC has been evaluated in this study. Blood samples (n = 104) were obtained from patients with locally advanced or metastatic HCC (n = 30) for the enrichment of CTCs by a negative selection method. Analysis of the blood samples from patients with defined disease status (n = 81) revealed that those with progressive disease (PD, n = 37) had significantly higher CTC counts compared to those with a partial response (PR) or stable disease (SD, n = 44 for PR + SD, p = 0.0002). The median CTC count for patients with PD and for patients with PR and SD was 50 (interquartile range 21&ndash, 139) and 15 (interquartile range 4&ndash, 41) cells/mL of blood, respectively. A longitudinal analysis of patients (n = 17) after a series of blood collections demonstrated that a change in the CTC count correlated with the patient treatment response in most of the cases and was particularly useful for monitoring patients without elevated serum alpha-fetoprotein (AFP) levels. Sequential CTC enumeration during treatment can supplement standard medical tests and benefit the management of patients with locally advanced or metastatic HCC, in particular for the AFP-low cases.

Identification of host factors involved in viral replication is critical for understanding the molecular mechanism of viral replication and pathogenesis. Genes differentially expressed in HuH-7 cells with or without a hepatitis C virus (HCV) sub-genomic replicon were screened by microarray analysis. SERPINE1/PAI-1 was found to be down-regulated after HCV infection in this analysis. Down-regulation of SERPINE1/PAI-1 expression at the transcriptional level was verified by the real-time reverse transcriptase (RT)-PCR assay. Reduced SERPINE1/PAI-1 protein secretion was detected in the supernatant of HCV replicon cells and in sera from HCV-infected patients. SERPINE1 gene expression was down-regulated by HCV NS3/4A and NS5A proteins through the transforming growth factor-β (TGF-β) signalling pathway at the transcriptional level. Down-regulated genes in HCV replicon cells could be the factors supressing HCV replication. Indeed, over-expressed PAI-1 inhibited HCV replication but the mechanism is unknown. It has been demonstrated that HCV induces the expression of TGF-β, and TGF-β enhances HCV replication by a not-yet-defined mechanism. SERPINE1/PAI-1 is also known to be potently induced by TGF-β at the transcriptional level through both Smad-dependent and Smad-independent pathways. The exogenously expressed SERPINE1/PAI-1 suppressed the expression of the endogenous SERPINE1 gene at the transcriptional level through the TGF-β signalling but not the Smad pathway. Thus, SERPINE1/PAI-1 could suppress HCV replication possibly by negatively regulating TGF-β signalling. A model is proposed for the interplay betweenthe TGF-β signalling pathway, HCV and SERPINE1/PAI-1 to keep the homeostasis of the cells.

As a part of our continuing search for anti-HCV agents from a medicinal plant, Flueggea virosa, we identified fourteen new ingredients, including 13-methyl-3,4-seco-ent-podocarpanes (1–13) and a new naphthalene derivative (14). Their structures were determined by analysis of 2D NMR spectroscopic data, while their absolute configurations were determined by comparison of specific optical rotations with related analogues in the literature. The therapeutic potential against hepatitis C virus (HCV) infection was evaluated for the isolates. Compound 8 showed the most potent anti-HCV activity with a therapeutic index (TI = IC50/EC50) of 100.5.

Glaulactams A–C (1–3), which possess a novel skeleton, as well as the known compound daphmanidin B (4), were isolated from the leaves of Daphniphyllum glaucescens and separated using ion-exchange chromatography aided by NMR fingerprinting. Their structures, including their absolute configurations, were elucidated by spectroscopic analyses and time-dependent density-functional-theory-calculated electronic circular dichroism spectra; the data were subsequently analyzed to gain insight into the respective biogenetic relationships between the isolates, which exhibited anti-H1N1 and immunosuppressive activities.

Podoplanin (PDPN) enhances tumor metastases by eliciting tumor cell-induced platelet aggregation (TCIPA) through activation of platelet C-type lectin-like receptor 2 (CLEC-2). A novel and non-cytotoxic 5-nitrobenzoate compound 2CP was synthesized that specifically inhibited the PDPN/CLEC-2 interaction and TCIPA with no effect on platelet aggregation stimulated by other platelet agonists. 2CP possessed anti-cancer metastatic activity in vivo and augmented the therapeutic efficacy of cisplatin in the experimental animal model without causing a bleeding risk. Analysis of the molecular action of 2CP further revealed that Akt1/PDK1 and PKCμ were two alternative CLEC-2 signaling pathways mediating PDPN-induced platelet activation. 2CP directly bound to CLEC-2 and, by competing with the same binding pocket of PDPN in CLEC-2, inhibited PDPN-mediated platelet activation. This study provides evidence that 2CP is the first defined platelet antagonist with CLEC-2 binding activity. The augmentation in the therapeutic efficacy of cisplatin by 2CP suggests that a combination of a chemotherapeutic agent and a drug with anti-TCIPA activity such as 2CP may prove clinically effective.

The UV-guided isolation of polyacetylenes from the crude extract of Codonopsis pilosula has successfully led to the characterization of five new polyynes, pilosulynes A–E (1–5), and two new polyenes, pilosulynes F and G (6 and 7), as well as five known analogues (8–12). Their structures were determined by spectroscopic methods, including ICD and 1D/2D NMR experiments. The absolute configurations of the 6,7-diol moiety of the isolated compounds were determined by the Snatzke's method, which revealed an induced circular dichroism after the addition of dimolybdenum tetraacetate in DMSO. Compound 6 exhibited anti-HCV activity in the HCVcc infection assay with an EC50 value of 47.2 μM.

Hepatitis C virus (HCV) infection of hepatocytes promotes liver fibrosis by activation of hepatic stellate cells (HSCs) and excessive deposition of extracellular matrix in liver tissue. Whether or not host factors released from the HCV-infected hepatocytes play role in HSCs activation is unclear. In this study, HSCs were activated by the conditioned medium derived from HCV replicon cells. Secretomic profiling of HCV replicon cells and the parental Huh7 cells revealed ubiquitin carboxy-terminal hydrolase L1 (UCHL1) as a novel secreted protein from HCV-infected hepatocytes. UCHL1 expression in hepatocytes was induced by HCV infection. UCHL1 was expressed in the liver and found in the plasma of patients with chronic hepatitis C. Molecular analysis by use of the anti-UCHL1 neutralization antibody and purified UCHL1 protein showed that secreted UCHL1 protein was bound to the cell surface of HSCs and activated JNK signaling leading to overexpression of alpha-smooth muscle actin and the activation of HSCs. These results provide further for understanding the underlying mechanism in HCV-mediated hepatic fibrogenesis.

Essentials Disabled-2 (Dab2) phosphorylation status in thrombin signaling of human platelet was investigated. Ser723 was the major Dab2 phosphorylation site in human platelets stimulated by thrombin. Dab2 S723 phosphorylation (pS723) caused the dissociation of Dab2-CIN85 protein complex. Dab2-pS723 regulated ADP release and integrin αIIbβ3 activation in thrombin-treated platelets. SummaryBackground Disabled-2 (Dab2) is a platelet protein that is functionally involved in thrombin signaling in mice. It is unknown whether or not Dab2 undergoes phosphorylation during human platelet activation. Objectives To investigate the phosphorylation status of Dab2 and its functional consequences in thrombin-stimulated human platelets. Methods Dab2 was immunoprecipitated from resting and thrombin-stimulated platelet lysates for differential isotopic labeling. After enrichment of the phosphopeptides, the phosphorylation sites were analyzed by mass spectrometry. The corresponding phospho-specific antibody was generated. The protein kinases responsible for and the functional significance of Dab2 phosphorylation were defined by the use of signaling pathway inhibitors/activators, protein kinase assays, and various molecular approaches. Results Dab2 was phosphorylated at Ser227, Ser394, Ser401 and Ser723 in thrombin-stimulated platelets, with Ser723 phosphorylation being the most significantly increased by thrombin. Dab2 was phosphorylated by protein kinase C at Ser723 in a Gαq-dependent manner. ADP released from the stimulated platelets further activated the Gβγ-dependent pathway to sustain Ser723 phosphorylation. The Cbl-interacting protein of 85 kDa (CIN85) bound to Dab2 at a motif adjacent to Ser723 in resting platelets. The consequence of Ser723 phosphorylation was the dissociation of CIN85 from the Dab2–CIN85 complex. These molecular events led to increases in fibrinogen binding and platelet aggregation in thrombin-stimulated platelets by regulating αIIbβ3 activation and ADP release. Conclusions Dab2 Ser723 phosphorylation is a key molecular event in thrombin-stimulated inside-out signaling and platelet activation, contributing to a new function of Dab2 in thrombin signaling.

Objective— The essential role of platelet activation in hemostasis and thrombotic diseases focuses attention on unveiling the underlying intracellular signals of platelet activation. Disabled-2 (Dab2) has been implicated in platelet aggregation and in the control of clotting responses. However, there is not yet any in vivo study to provide direct evidence for the role of Dab2 in hemostasis and platelet activation. Approach and Results— Megakaryocyte lineage-restricted Dab2 knockout (Dab2 −/− ) mice were generated to delineate in vivo functions of Dab2 in platelets. Dab2 −/− mice appeared normal in size with prolonged bleeding time and impaired thrombus formation. Although normal in platelet production and granule biogenesis, Dab2 −/− platelets elicited a selective defect in platelet aggregation and spreading on fibrinogen in response to low concentrations of thrombin, but not other soluble agonists. Investigation of the role of Dab2 in thrombin signaling revealed that Dab2 has no effect on the expression of thrombin receptors and the outside-in signaling. Dab2 −/− platelets stimulated by low concentrations of thrombin were normal in G αq -mediated calcium mobilization and protein kinase C activation, but were defective in G α12/13 -mediated RhoA-ROCKII activation. The attenuated G α12/13 signaling led to impaired ADP release, Akt-mammalian target of rapamycin and integrin αIIbβ3 activation, fibrinogen binding, and clot retraction. The defective responses of Dab2 −/− platelets to low concentrations of thrombin stimulation may contribute to the impaired hemostasis and thrombosis of Dab2 −/− mice. Conclusions— This study sheds new insight in platelet biology and represents the first report demonstrating that Dab2 is a key regulator of hemostasis and thrombosis by functional interplay with G α12/13 -mediated thrombin signaling.

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) that acts as a key player in the HCV replication complex. Understanding the interplay between the viral and cellular components of the HCV replication complex could provide new insight for prevention of the progression of HCV-associated hepatocellular carcinoma (HCC). In this study, the NS5B protein was used as the bait in a pulldown assay to screen for NS5B-interacting proteins that are present in Huh7 hepatoma cell lysates. After mass spectrophotometric analysis, fatty acid synthase (FASN) was found to interact with NS5B. Coimmunoprecipitation and double staining assays further confirmed the direct binding between NS5B and FASN. The domain of NS5B that interacts with FASN was also determined. Moreover, FASN was associated with detergent-resistant lipid rafts and colocalized with NS5B in active HCV replication complexes. In addition, overexpression of FASN enhanced HCV expression in Huh7/Rep-Feo cells, while transfection of FASN small interfering RNA (siRNA) or treatment with FASN-specific inhibitors decreased HCV replication and viral production. Notably, FASN directly increased HCV NS5B RdRp activity in vitro . These results together indicate that FASN interacts with NS5B and modulates HCV replication through a direct increase of NS5B RdRp activity. FASN may thereby serve as a target for the treatment of HCV infection and the prevention of HCV-associated HCC progression.

The severity of an influenza epidemic season may be influenced not only by variability in the surface glycoproteins, but also by differences in the internal proteins of circulating influenza viruses. To better understand viral antigenic evolution, all eight gene segments from 44 human H3N2 epidemic strains isolated during 2004–2008 in Taiwan were analyzed to provide a profile of protein variability. Comparison of the evolutionary profiles of the HA, NA and PB2 genes of influenza A (H3N2) viruses indicated that they were derived from a group of H3N2 isolates first seen in 2004. However, the PA, M and PB1 genes were derived from a different group of H3N2 isolates from 2004. Tree topology revealed the NP and NS genes could each be segregated into two groups similar to those for the polymerase genes. In addition, new genetic variants occurred during the non-epidemic period and become the dominant strain after one or two seasons. Comparison of evolutionary patterns in consecutive years is necessary to correlate viral genetic changes with antigenic changes as multiple lineages co-circulate.

Patients with thyroid cancer usually have good prognosis. However, 15-20% of patients ultimately develop recurrence and disease-related death. In this study, we investigate whether the number of circulating tumor cells (CTCs) expressing either epithelial cell adhesion molecule (EpCAM), podoplanin (PDPN, a marker related to poor prognosis in patients with thyroid cancer), or thyroid-stimulating hormone receptor (TSHR, a marker for thyroid cells) is a prognostic marker for non-remission and disease-specific mortality (DSM) of patients with thyroid cancer. Blood samples were collected from patients (n = 128) after thyroidectomy or radioactive iodide therapy. After enrichment of CTCs by a negative selection PowerMag system, enumeration and subtyping of the CD45-depleted cells were performed by immunofluorescence staining using the antibodies aginst EpCAM, TSHR, and PDPN, respectively. Our data revealed that the number of EpCAM+-CTCs (p < 0.001) and PDPN+-CTCs (p < 0.001), and TSHR+-CTCs (p < 0.001) for patients in the non-remission group (n = 43) was significantly higher when compared to the remission group (n = 85). At the cutoff of 40, 14, and 47 cells/mL for EpCAM+-CTCs, TSHR+-CTCs, and PDPN+-CTCs, the accuracy of the assay was equivalent to 80.4%, 76.6%, and 77.3%, respectively. On the other hand, the number of EpCAM+-CTCs (p < 0.001), PDPN+-CTCs (p = 0.013), and TSHR+-CTCs (p < 0.001) for patients in the DSM group (n = 17) was significantly higher when compared to the patients who survived (n = 111). At the cutoff of 27, 25, and 9 cells/mL for EpCAM+-CTCs, TSHR+-CTCs, and PDPN+-CTCs, the accuracy of the assay was equivalent to 69.5%, 67.2%, and 68.5%, respectively. These data together indicate that CTC testing is worthy to be considered as a routine clinical test to benefit clinical care of patients with thyroid cancer. Citation Format: Ching-Ping Tseng, Jen-Der Lin, Miaw-Jene Liou, Wei-Shan Hung, Hsueh-Ling Hsu, Kong-Kit Leong, Yu-Ting Chen, Ju-Chien Cheng. Circulating tumor cells as the prognostic marker for non-remission and disease-specific mortality of patients with thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5586.

Current methods for clinical early detection and disease monitoring of patients with colorectal cancer (CRC) are immunochemical fecal occult blood test (iFOBT) and serum levels of carcinoembryonic antigen (CEA), which are known with low sensitivity. There is an unmet need to seek for additional molecular tools for early detection and disease monitoring of patients with CRC. Whether enumeration of circulating tumor cells (CTCs) supplements current clinical tests and facilitates early detection and monitoring disease progression was investigated. A total of 109 pre-operative patients with CRC (including early and advanced stages) and 65 non-cancerous controls were enrolled in this study. CTCs were enriched from the peripheral blood of these individuals by using the PowerMag negative selection system. Immunofluorescence staining of epithelial cell adhesion molecule (EpCAM), and podoplanin (PDPN, a marker related to poor cancer progression) was performed to define the two distinct subtypes of CTCs. Heterogeneous CTCs expressing PDPN or EpCAM are defined in the peripheral blood of patients with CRC. Regardless clinical stages in patients, the number of both EpCAM+-CTCs and PDPN+-CTCs explicitly differentiated non-cancerous controls and patients with CRC, and were also correlated with clinicopathological diagnosis. Receiver operating characteristic analysis demonstrated that the sensitivity and specificity of EpCAM+-CTCs was 86.32% and 78.46%, respectively, when the cutoff value was 23 EpCAM+-CTCs/mL. Using 7 PDPN+-CTCs/mL as the cutoff value, the sensitivity and specificity of PDPN+-CTCs was 77.98% and 75.38%, respectively. 84.2% and 80.3% of patients with serum CEA < 5 ng/mL were found to have higher EpCAM+-CTCs and PDPN+-CTCs counts, respectively. In addition, simultaneous measurements of iFOBT and CTCs could compromise the false-positive rate of iFOBT, while measurements of CEA and CTCs are applicable to monitor cancer recurrence after surgery. This study implicates that CTC testing supplements the current clinical methods to facilitate early detection and disease monitoring in CRC. Citation Format: Wei-Shan Hung, Wen-Sy Tsai, Tzu-Min Wang, Hsueh-Ling Hsu, Hsuan Liu, Chia-Yu Yang, Ching-Ping Tseng, Ju-Chien Cheng. Combined analysis of circulating tumor cells, fecal occult blood test and serum carcinoembryonic antigen for early detection and monitoring recurrence of patients with colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4583.

Rationale : Hyperhomocysteinemia contributes to vascular dysfunction and risks of cardiovascular diseases. Stromal cell–derived factor (SDF)-1, a chemokine expressed by endothelial cells (ECs), is highly expressed in advanced atherosclerotic lesions. The interplays among homocysteine, chemokines, and shear stress in regulating vascular endothelial function are not clearly understood. Objective : To investigate the mechanisms for modulations of EC SDF-1 expression by homocysteine and shear stress. Methods and Results : Homocysteine stimulation induced dose- and time-dependent SDF-1 expression and phosphorylation of mitogen-activated protein kinases extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. By using specific inhibitors, small interfering (si)RNA, and dominant negative mutants, we demonstrated that activation of JNK pathway is critical for the homocysteine-induced SDF-1 expression. Transcription factor ELISA and chromatin immunoprecipitation assays showed that homocysteine increased Sp1- and AP-1–DNA binding activities in ECs. Inhibition of Sp1 and AP-1 activations by specific siRNA blocked the homocysteine-induced SDF-1 promoter activity and expression. Preshearing of ECs for 1 to 4 hours at 20 dyn/cm 2 inhibited the homocysteine-induced JNK phosphorylation, Sp1 and AP-1 activation, and SDF-1 expression. The homocysteine-induced SDF-1 expression was suppressed by NO donor. Inhibitor or siRNA for endothelial NO synthase abolished the shear inhibition of SDF-1 expression. Conclusions : Our findings serve to elucidate the molecular mechanisms underlying the homocysteine induction of SDF-1 expression in ECs and the shear stress protection against this induction.

A novel hepatotropic virus, named NV-F virus, was recently identified. The clinical information for this virus is still scarce. Using PCR assay, NV-F viral DNA (NV-F-DNA) was detected in 12 of 50 (24%) hepatitis C virus (HCV)-infected patients (HCV-coinfected [HCVCI] group), 34 of 250 (13.6%) hepatitis B virus (HBV)-infected patients (HBV-coinfected [HBVCI] group), and 28 of 100 (28%) non-A-to-E (NAE) hepatitis patients. Basic clinical parameters were not significantly different among the three groups. By use of a PCR-based quantitative assay, the NV-F-DNA concentration was found to be above the detection limit (1.2 × 10 5 copies/ml) in 12/12 (100%) HCVCI patients, 14/34 (41.2%) HBVCI patients, and 4/28 (14.3%) NAE patients. The median serum NV-F-DNA concentration was 9.3 × 10 5 copies/ml in HCVCI patients, but it was below the detection limit in HBVCI and NAE patients ( P values were 0.0045 and 0.0001, respectively). Stepwise multiple regression analysis identified the presence of anti-HCV as an independent factor for NV-F-DNA concentrations (β = 6.2 × 10 9 ; P = 0.0245). In HBVCI patients, the NV-F-DNA concentration was inversely correlated with the HBV DNA concentration. The median NV-F-DNA concentration was below the detection limit in patients with HBV DNA concentrations above 1.4 × 10 5 copies/ml, but it was 1.58 × 10 6 copies/ml in patients with HBV DNA concentrations below 1.4 × 10 5 copies/ml ( P = 0.030). In conclusion, NV-F-DNA concentrations were higher in HCVCI patients. A reciprocal relationship was found between NV-F-DNA and HBV DNA concentrations in HBVCI patients, indicating the presence of viral interference between these two DNA viruses.

With the ability to exponentially amplify regions of DNA, two different PCR-based strategies have been developed for nonculture diagnosis of bacteremia. The first approach targets the species-specific genes for amplification (1), whereas the second approach involves universal PCR amplification of conserved bacterial DNA sequences, such as the 16S rRNA, the 23S rRNA, and the 16S-23S rRNA interspace regions (2)(3)(4). Although universal PCR is not able to distinguish bacteria to the species level, numerous studies have shown that this method provides valuable information complementary to the results of time-consuming and subjective phenotypic tests used in detection of bacterial infection (5) and can be used to differentiate bacterial from viral or other infections (6)(7). In clinical applications, real-time PCR for broad-range amplification of bacterial DNA sequences could offer additional benefits: it is less labor-intensive, less time-consuming, and reduces the risk of PCR carryover contamination. A major obstacle for broad-range PCR amplification is the presence of bacterial DNA in the Taq DNA polymerase and real-time PCR master mixture (8)(9)(10). The contaminating DNA is effectively amplified, giving rise to false-positive results. Efforts have been taken to eliminate contaminating DNA, including the use of Sau 3AI restriction endonuclease (11), DNase I (12)(13), and ultraviolet irradiation (7)(8). Although these strategies may be effective for conventional PCR, the study by Corless et al. (14) indicated that the contamination issue could not be avoided without affecting sensitivity when TaqMan probe-based real-time PCR is performed. Therefore, an appropriate method for removal of contaminating bacterial DNA and subsequent real-time amplification is required. In this study, we address this issue and report optimized conditions for broad-range amplification of the bacterial 16S rRNA gene by real-time PCR using SYBR Green I dye (15) as the fluorescent signal. Bacterial …

Regarding oral squamous cell carcinoma (OSCC) development, chewing areca is known to be a strong risk factor in many Asian cultures. Therefore, we established an OSCC induced mouse model by 4-nitroquinoline-1-oxide (4-NQO), or arecoline, or both treatments, respectively. These are the main two components of the areca nut that could increase the occurrence of OSCC. We examined the effects with the noncommercial MCGI (mouse CpG islands) microarray for genome-wide screening the DNA methylation aberrant in induced OSCC mice. The microarray results showed 34 hypermethylated genes in 4-NQO plus arecoline induced OSCC mice tongue tissues. The examinations also used methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing to realize the methylation pattern in collected mouse tongue tissues and human OSCC cell lines of different grades, respectively. These results showed that retinoic acid receptorβ(RARB) was indicated in hypermethylation at the promoter region and the loss of expression during cancer development. According to the results of real-time PCR, it was shown thatde novoDNA methyltransferases were involved in gene epigenetic alternations of OSCC. Collectively, our results showed thatRARBhypermethylation was involved in the areca-associated oral carcinogenesis.

The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) possesses protease, nucleoside triphosphatase, and helicase activities. Although the enzymatic activities have been extensively studied, the ATP- and RNA-binding domains of the NS3 helicase are not well-characterized. In this study, NS3 proteins with point mutations in the conserved helicase motifs were expressed in Escherichia coli , purified, and analyzed for their effects on ATP binding, RNA binding, ATP hydrolysis, and RNA unwinding. UV cross-linking experiments indicate that the lysine residue in the AX 4 GKS motif is directly involved in ATP binding, whereas the NS3(GR1490DT) mutant in which the arginine-rich motif (1486-QRRGRTGR-1493) was changed to QRRDTTGR bound ATP as well as the wild type. The binding activity of HCV NS3 helicase to the viral RNA was drastically reduced with the mutation at Arg1488 (R1488A) and was also affected by the K1236E substitution in the AX 4 GKS motif and the R1490A and GR1490DT mutations in the arginine-rich motif. Previously, Arg1490 was suggested, based on the crystal structure of an NS3-deoxyuridine octamer complex, to directly interact with the γ-phosphate group of ATP. Nevertheless, our functional analysis demonstrated the critical roles of Arg1490 in binding to the viral RNA, ATP hydrolysis, and RNA unwinding, but not in ATP binding.

Summary Background Reelin is a large extracellular glycoprotein that is present in the peripheral blood. That Reelin interacts with the coagulation components and elicits a functional role in hemostasis has not yet been elucidated. Objectives The hemostatic activity of Reelin is investigated and defined in this study. Methods The interplay of Reelin with coagulation components was elucidated by far-Western and liposome/platelet binding assays. In vivo and ex vivo hemostasis-related analyses of Reelin-deficient mice and plasma were also performed. Results Reelin interacted with the liposomes containing phosphatidylserine (PS) or phosphatidylcholine. Instead of interacting with known Reelin receptors (ApoE receptor 2, very low density lipoprotein receptor and integrin β1), Reelin interacted with PS of the activated platelets. The interaction between Reelin and the coagulation factors of thrombin and FXa was also demonstrated with the Kd of 11.7 and 21.2 nm, respectively. Reelin-deficient mice displayed a prolonged bleeding time and an increase in rebleeding rate. Despite the fact that Reelin deficiency had no significant effect on the clotting time of prothrombin and activated partial thromboplastin time, the fibrin clot formation was abnormal and the fibrin clot structure was relatively loosened with reduced clot strength. Abnormal fibrinogen expression did not account for the hemostatic defects associated with Reelin deficiency. Instead, thrombin generation was impaired concomitant with an altered prothrombin cleavage pattern. Conclusions By interacting with platelet phospholipids and the coagulation factors, thrombin and FXa, Reelin plays a selective role in coagulation activation, leading to thrombin generation and formation of a normal fibrin clot.

The evolution and population dynamics of human influenza in Taiwan is a microcosm of the viruses circulating worldwide, which has not yet been studied in detail. We collected 343 representative full genome sequences of human influenza A viruses isolated in Taiwan between 1979 and 2009. Phylogenetic and antigenic data analysis revealed that H1N1 and H3N2 viruses consistently co-circulated in Taiwan, although they were characterized by different temporal dynamics and degrees of genetic diversity. Moreover, influenza A viruses of both subtypes underwent internal gene reassortment involving all eight segments of the viral genome, some of which also occurred during non-epidemic periods. The patterns of gene reassortment were different in the two subtypes. The internal genes of H1N1 viruses moved as a unit, separately from the co-evolving HA and NA genes. On the other hand, the HA and NA genes of H3N2 viruses tended to segregate consistently with different sets of internal gene segments. In particular, as reassortment occurred, H3HA always segregated as a group with the PB1, PA and M genes, while N2NA consistently segregated with PB2 and NP. Finally, the analysis showed that new phylogenetic lineages and antigenic variants emerging in summer were likely to be the progenitors of the epidemic strains in the following season. The synchronized seasonal patterns and high genetic diversity of influenza A viruses observed in Taiwan make possible to capture the evolutionary dynamic and epidemiological rules governing antigenic drift and reassortment and may serve as a “warning” system that recapitulates the global epidemic.

Papillary thyroid carcinoma (PTC) is one type of thyroid cancer and accounts for about 80% of the cases. Routine surveillance by serum thyroglobulin (Tg) and medical imaging is the current practice to monitor disease progression of the patients. However, the presence of anti-Tg antibody or other influence factors may discount the value of serum Tg in disease monitoring. Whether enumeration of circulating epithelial cells (CECs) and its combined analyses with serum Tg help to define disease status of PTC patients was investigated. CECs were first enriched from the peripheral blood of the healthy control subjects (G1, n = 17) and the patients at disease-free status (G2, n = 26) or with distant metastasis (G3, n = 22). The number of CECs expressing epithelial cell adhesion molecule (EpCAM) was determined by immunofluorescence microscopy analyses. The medium number of EpCAM+-CECs was 6 (intequartile range 1-11), 12 (interquartile range 7-16) and 91 (interquartile range 31-206) cells/ml of blood for G1, G2 and G3, respectively. EpCAM+-CEC counts were significantly higher in G3 than in G1 (p Citation Format: Ching-Ping Tseng, Jen-Der Lin, Hung-Chih Lin, Ju-Chien Cheng. Combined analysis of circulating epithelial cell count and serum thyroglobulin for differentiating disease status of the patients with papillary thyroid carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 461.

Reelin is an extracellular glycoprotein that is highly conserved in mammals. In addition to its expression in the nervous system, Reelin is present in erythroid cells but its function there is unknown. We report in this study that Reelin is up-regulated during erythroid differentiation of human erythroleukemic K562 cells and is expressed in the erythroid progenitors of murine bone marrow. Reelin deficiency promotes erythroid differentiation of K562 cells and augments erythroid production in murine bone marrow. In accordance with these findings, Reelin deficiency attenuates AKT phosphorylation of the Ter119+CD71+ erythroid progenitors and alters the cell number and frequency of the progenitors at different erythroid differentiation stages. A regulatory role of Reelin in erythroid differentiation is thus defined.

We investigated the effect of traditional risk factors (hypertension, dyslipidemia and smoking) on the association between angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism and peripheral arterial disease (PAD) in 945 (454 men and 491 women) Taiwanese type 2 diabetic patients with a mean age of 63.5 (SD: 11.4) years. Among them, 81 (31 men and 50 women) had PAD (ankle-brachial index

The non-coding microRNA (miRNA) is involved in the regulation of hepatitis C virus (HCV) infection and offers an alternative target for developing anti-HCV agent. In this study, we aim to identify novel cellular miRNAs that directly target the HCV genome with anti-HCV therapeutic potential. Bioinformatic analyses were performed to unveil liver-abundant miRNAs with predicted target sequences on HCV genome. Various cell-based systems confirmed that let-7b plays a negative role in HCV expression. In particular, let-7b suppressed HCV replicon activity and down-regulated HCV accumulation leading to reduced infectivity of HCVcc. Mutational analysis identified let-7b binding sites at the coding sequences of NS5B and 5'-UTR of HCV genome that were conserved among various HCV genotypes. We further demonstrated that the underlying mechanism for let-7b-mediated suppression of HCV RNA accumulation was not dependent on inhibition of HCV translation. Let-7b and IFNα-2a also elicited a synergistic inhibitory effect on HCV infection. Together, let-7b represents a novel cellular miRNA that targets the HCV genome and elicits anti-HCV activity. This study thereby sheds new insight into understanding the role of host miRNAs in HCV pathogenesis and to developing a potential anti-HCV therapeutic strategy.

Background Many studies concentrate on variation in the hemagglutinin glycoprotein (HA) because of its significance in host immune response, the evolution of this virus is even more complex when other genome segments are considered. Recently, it was found that cytotoxic T lymphocytes (CTL) play an important role in immunity against influenza and most CTL epitopes of human influenza viruses were remarkably conserved. The NP gene has evolved independently in human and avian hosts after 1918 flu pandemic and it has been assigned a putative role as a determinant of host range. Methods and Findings Phylodynamic patterns of the genes encoding nucleoprotein (NP) of influenza A viruses isolated from 1979–2009 were analyzed by applying the Bayesian Markov Chain Monte Carlo framework to better understand the evolutionary mechanisms of these Taiwanese isolates. Phylogenetic analysis of the NP gene showed that all available H3 worldwide isolates collected so far were genetically similar and divided into two major clades after the year 2004. We compared the deduced amino acid sequences of the NP sequences from human, avian and swine hosts to investigate the emergence of potential adaptive mutations. Overall, selective pressure on the NP gene of human influenza A viruses appeared to be dominated by purifying selection with a mean dN/dS ratio of 0.105. Site-selection analysis of 488 codons, however, also revealed 3 positively selected sites in addition to 139 negatively selected ones. Conclusions The demographic history inferred by Bayesian skyline plot showed that the effective number of infections underwent a period of smooth and steady growth from 1998 to 2001, followed by a more recent rise in the rate of spread. Further understanding the correlates of interspecies transmission of influenza A virus genes from other host reservoirs to the human population may help to elucidate the mechanisms of variability among influenza A virus.

The Chinese herb modified Yi Guan Jian (mYGJ) is an effective regimen that is usually used in outpatients with chronic liver diseases such as fibrosis and cirrhosis. However, the mechanism for the action of mYGJ on liver fibrosis is not yet clear. In this study, we found that mYGJ induced hepatic stellate cells (HSCs) apoptosis concomitant with the downregulation of Bcl-2 expression and slight elevation of Bax level. Moreover, the reactive oxygen species (ROS) were generated in the early stages of mYGJ-induced HSCs apoptosis to facilitate calcium and cytochrome c release from the mitochondria to cytosol. Subsequently, caspase 9 and caspase 3 were activated. Furthermore, the activation of ER stress-associated caspase 12 in HSCs was also evaluated. Together, we report the first evidence-based study to demonstrate that mYGJ decoction induces HSCs apoptosis through ROS accumulation and the intrinsic apoptosis pathway. These findings provide rationale for further clinical investigation of traditional Chinese medicine recipes against liver fibrosis.