The role of pleural fluid lactate dehydrogenase-to-adenosine deaminase ratio in differentiating the etiology of pleural effusions.

Exudative pleural effusion includes tuberculous pleural effusion (TPE), parapneumonic pleural effusion (PPE), and malignant pleural effusion (MPE). An elevated pleural fluid adenosine deaminase (ADA) typically implies TPE, but the rule may not apply to every individual case. Recent studies proposed that the pleural fluid lactate dehydrogenase (LDH)-to-ADA ratio showed a higher diagnostic power than pleural fluid ADA alone in differentiating the etiology of pleural effusion. Hence, we aimed to investigate the performance of pleural fluid LDH-to-ADA ratio as a biomarker in assistance with the diagnosis of TPE, PPE, and MPE. All patients who underwent thoracentesis for the first time with a pleural fluid ADA >40 U/L were included in this retrospective study. The clinical data including pleural fluid ADA and LDH-to-ADA ratio were analyzed. A total of 311 patients were enrolled during the study interval. The pleural fluid LDH-to-ADA ratio <14.2 (sensitivity: 74.2%; specificity: 90.4%) favored TPE, while the pleural fluid LDH-to-ADA ratio >14.5 (sensitivity: 79.9%; specificity: 78.5%) favored PPE. Besides, the pleural fluid LDH-to-ADA ratio >46.7 (sensitivity: 56.3%; specificity: 78.3%) favored MPE owing to primary lung cancers. In conclusion, the pleural fluid LDH-to-ADA ratio was an effective indicator in differentiating the etiology of pleural effusions in the cases of high ADA level in the pleural fluid.

Calcium-containing crystals enhance receptor activator of nuclear factor κB ligand/macrophage colony-stimulating factor-mediated osteoclastogenesis via extracellular-signal-regulated kinase and p38 pathways.

OBJECTIVE: Diseases associated with calcium-containing crystal deposition can lead to local bone erosion. We aimed to determine whether calcium-containing crystal-hydroxyapatite, ß-tricalcium phosphate and CPPD enhanced osteoclastogenesis and to define underlying mechanisms of action. METHODS: Osteoclastogenesis was studied by culturing murine RAW 264.7 osteoclast precursor cells with RANK ligand (RANKL)/ M-CSF and/or calcium-containing crystals, and observing the tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and TRAP activity. Resorption pit formation was used to evaluate osteoclast activity. Real-time RT-PCR analysis revealed osteoclast marker genes, including TRAP, cathepsin K and calcitonin receptor (CTR). Western blotting was used to analyse the phosphorylation levels of signal transduction molecules. RESULTS: Three kinds of calcium-containing crystal significantly enhanced RANKL/M-CSF-induced osteoclastogenesis in RAW 264.7 cells, as evidenced by the increased number of TRAP-positive multinucleated cells, TRAP activity and resorption pit formation in a dose-dependent manner. Hydroxyapatite, ß-tricalcium phosphate and CPPD treatments significantly enhanced RANKL/M-CSF-induced mRNA expression of TRAP, cathepsin K and CTR. Moreover, the three kinds of calcium-containing crystal enhanced the phosphorylation of extracellular-signal-regulated kinase and p38 in RANKL/M-CSF-treated cells. CONCLUSION: We concluded that calcium-containing crystals can promote osteoclastogenesis and bone resorption through the extracellular-signal-regulated kinase and p38 pathways. Together with synovial activation, this mechanism may be important in the pathogenesis of destructive arthropathies triggered by calcium-containing crystals.

Activin A induction of erythroid differentiation sensitizes K562 chronic myeloid leukemia cells to a subtoxic concentration of imatinib.

Chronic myeloid leukemia (CML) is a hematopoietic stem/progenitor cell disorder in which Bcr-Abl oncoprotein inhibits cell differentiation. Differentiation induction is considered an alternative strategy for treating CML. Activin A, a member of the transforming growth factor-ß superfamily, induces erythroid differentiation of CML cells through the p38 MAPK pathway. In this study, treatment of the K562 CML stem/progenitor cell line with activin A followed by a subtoxic concentration of the Bcr-Abl inhibitor imatinib strongly induced growth inhibition and apoptosis compared with simultaneous treatment with activin A and imatinib. Imatinib-induced growth inhibition and apoptosis following activin A pretreatment were dose- and time-dependent. Imatinib-induced growth inhibition and apoptosis were also dependent on the pretreatment dose of activin A. More than 90% of the activin A-induced increases in glycophorin A-positive cells were sensitive to imatinib. However, only some of original glycophorin A-positive cells in the activin A treatment group were sensitive to imatinib. Sequential treatment with activin A and imatinib decreased Bcr-Abl, procaspase-3, Mcl-1, and Bcl-xL and also induced cleavage of procaspase-3/poly(ADP-ribose)polymerase. The reduction of erythroid differentiation in p38 MAPK dominant-negative mutants or by short hairpin RNA knockdown of p38 MAPK decreased the growth inhibition and apoptosis mediated by sequential treatment with activin A and imatinib. Furthermore, the same inhibition level of multidrug resistance 1 expression was observed in cells treated with activin A alone, treated sequentially with activin A and imatinib, or treated simultaneously with activin A and imatinib. The p38 MAPK inhibitor SB-203580 can restore activin A-inhibited multidrug resistance 1 expression. Taken together, our results suggest that a subtoxic concentration of imatinib could exhibit strong cytotoxicity against erythroid-differentiated K562 CML cells.

Interferon-γ suppresses activin A/NF-E2 induction of erythroid gene expression through the NF-κB/c-Jun pathway.

Interferon (IFN)-γ is a proinflammatory cytokine that is linked to erythropoiesis inhibition and may contribute to anemia. However, the mechanism of IFN-γ-inhibited erythropoiesis is unknown. Activin A, a member of the transforming growth factor (TGF)-ß superfamily, induces the erythropoiesis of hematopoietic progenitor cells. In this study, a luciferase reporter assay showed that IFN-γ suppressed activin A-induced ζ-globin promoter activation in K562 erythroblast cells in a dose-dependent manner. Activin A reversed the suppressive effect of IFN-γ on the luciferase activity of ζ-globin promoter in a dose-dependent manner. IFN-γ also suppressed the activation of activin A-induced α-globin promoter. IFN-γ reduced the mRNA expression of α-globin, ζ-globin, NF-E2p45, and GATA-1 induced by activin A. The results also showed that IFN-γ induced c-Jun expression when NF-κBp65 and c-Jun bound to two AP-1-binding sites on the c-Jun promoter. The luciferase activity of α-globin and ζ-globin promoters were enhanced by wild-type c-Jun and eliminated by dominant-negative (DN) c-Jun. The suppressive effects of IFN-γ on the mRNA expression of α-globin and ζ-globin were absent in cells expressing DN c-Jun. The ability of NF-E2 to enhance activin A-induced ζ-globin promoter activation decreased when c-Jun was present, and IFN-γ treatment further enhanced the decreasing effect of c-Jun. Chromatin immunoprecipitation revealed that NF-E2p45 bound to the upstream regulatory element (HS-40) of the α-globin gene cluster in response to activin A, whereas c-Jun eliminated this binding. These results suggest that IFN-γ modulates NF-κB/c-Jun to antagonize activin A-mediated NF-E2 transcriptional activity on globin gene expression.

Screening of flavonoids for effective osteoclastogenesis suppression.

Flavonoids are natural compounds derived from plants and some of them have been shown to inhibit osteoclast formation, implicating their potential use for the treatment of osteoporosis. Conventionally, the screening of antiosteoclastic agents is a tedious process that requires visual counting of the number of osteoclasts produced. The purpose of this study was to establish an easier and faster method for screening the antiosteoclastogenic flavonoids by using an enzyme assay. Tartrate-resistant acid phosphatase (TRAP) is a marker enzyme of the osteoclast. Results obtained demonstrated that cellular TRAP activity tended to correlate with the number of osteoclasts formed. However, the secreted TRAP activity was actually responsible for the resorption activities of the functional osteoclasts. Consequently, the effectiveness of antiosteoclastogenic agents was screened for by assessing their inhibition on receptor activator of NF-κB ligand (RANKL)-induced TRAP secretion. The half-inhibitory concentrations of flavonoids on TRAP secretion were employed as indices to compare the effectiveness of various flavonoids. The effective flavonoids also exhibited similar inhibitory potencies in the pit-formation analysis. This protocol provides a rapid analysis to screen for effective antiosteoclastogenic agents.

Diverse effects of type II collagen on osteogenic and adipogenic differentiation of mesenchymal stem cells.

Type II collagen is known to modulate chondrogenesis of mesenchymal stem cells (MSCs). In this study, MSCs from human bone marrow aspirates were used to study the modulating effects of type II collagen on MSC differentiation during the early stages of osteogenesis and adipogenesis. With osteogenic induction, MSCs cultured on the type II collagen-coated surface showed an enhanced calcium deposition level with increasing mRNA expressions of RUNX2, osteocalcin, and alkaline phosphatase. A synthetic integrin binding peptide, which specifically interacts with the I-domain of α(1)ß(1)/α(2)ß(1) integrins significantly blocks the mineralization-enhancing effect of type II collagen. MSCs attached on the type II collagen-coated plates exhibited expanded cell morphology with increasing spreading area, and the pretreatment of cells with integrin α(1)ß(1) or α(2)ß(1)-blocking antibody reduced the effect. The phosphorylation levels of FAK, ERK, and JNK significantly increased in the MSCs that attached on the type II collagen-coated plates. On the contrary, the mineralization-enhancing effect of type II collagen was diminished by JNK and MEK inhibitors. Furthermore, type II collagen blocked the adipogenic differentiation of MSCs, and this effect is rescued by JNK and MEK inhibitors. In conclusion, type II collagen facilitates osteogenesis and suppresses adipogenesis during early stage MSC differentiation. Such effects are integrin binding-mediated and conducted through FAK-JNK and/or FAK-ERK signaling cascades. These results inspire a novel strategy encompassing type II collagen in bone tissue engineering.

Notch2-induced COX-2 expression enhancing gastric cancer progression.

Gastric carcinoma is one of the most common and mortal types of malignancy worldwide. To date, the mechanisms controlling its aggressiveness are not yet fully understood. Notch signal pathway can function as either an oncogene or a tumor suppressor in tumorigenesis. Four members (Notch1-4) of Notch receptors were found in mammals and each exhibits distinct roles in tumor progression. Previous study showed that the activated Notch1 receptor promoted gastric cancer progression through cyclooxygenase-2 (COX-2). This study addressed whether Notch2 signal pathway is also involved in gastric cancer progression. Constitutive expression of Notch2 intracellular domain (N2IC), the activated form of Notch2 receptor, promoted both cell proliferation and xenografted tumor growth of human stomach adenocarcinoma SC-M1 cells. The colony formation, migration, invasion, and wound-healing abilities of SC-M1 cells were enhanced by N2IC expression, whereas these abilities were suppressed by Notch2 knockdown. Similarly, Notch2 knockdown inhibited cancer progressions of AGS and AZ521 gastric cancer cells. Expression of N2IC also caused epithelial-mesenchymal transition in SC-M1 cells. Furthermore, N2IC bound to COX-2 promoter and induced COX-2 expression through a CBF1-dependent manner in SC-M1 cells. The ability of N2IC to enhance tumor progression in SC-M1 cells was suppressed by knockdown of COX-2 or treatment with NS-398, a COX-2 inhibitor. Moreover, the suppression of tumor progression by Notch2 knockdown in SC-M1 cells was reversed by exogenous COX-2 or its major enzymatic product PGE(2) . Taken together, this study is the first to demonstrate that the Notch2-COX-2 signaling axis plays an important role in controlling gastric cancer progression.

Selective activation of specific PKC isoforms dictating the fate of CD14(+) monocytes towards differentiation or apoptosis.

In this study, phorbol-12-myristate-13-acetate (PMA) at low concentrations (<10 nM; L-PMA) induces the differentiation of CD14(+) monocytes into monocyte-derived macrophages (MDMs) while PMA at high concentrations (>100 nM; H-PMA) causes the apoptosis of these cells. The pre-treatment with Go6976 (a PKC-α/ß(1) selective inhibitor), not anilinemonoindolylmaleimide [a PKC-ß inhibitor (PKC-ß inh.)], significantly (P < 0.05) reduces the L-PMA-induced generation of MDMs in the cultured CD14(+) monocytes. On the other hand, either of the above two PKC inhibitors is capable of suppressing the H-PMA-induced apoptosis of CD14(+) monocytes. However, only the inclusion of PKC-ß inh., not Go6976, prevents the cells from serum deprivation-induced cell apoptosis. Although the membrane translocation of conventional PKC-α, ß(1), and ß(2) isoforms was observed in the H-PMA-treated CD14(+) monocytes, only PKC-ß(2) exhibits a mitochondrial translocation activity among those PKCs responsive to H-PMA treatment. Moreover, the activation of DEVD-dependent caspases (DEVDase) was also detected in the H-PMA-treated CD14(+) monocytes, indicating the involvement of a caspase-dependent signaling pathway in the H-PMA-induced cell apoptosis of CD14(+) monocytes. Together with our previous findings that the selective activation of PKC-α or PKC-ß(1) induces the differentiation of CD14(+) monocytes into MDMs or dendritic cells (MoDCs), respectively, the results in this study further demonstrate that PKC-ß(2) activation is responsible for relaying the apoptotic signal to intrinsic mitochondria-dependent caspase signaling cascades in the CD14(+) monocytes. It is likely that the selective activation of specific PKC isoforms provides a new strategy to manipulate the differential cell fate commitment of multipotent CD14(+) monocytes towards apoptosis or differentiation into MDMs, MoDCs, and other cell types.

Differential effect of ECM molecules on re-expression of cartilaginous markers in near quiescent human chondrocytes.

The limited source of healthy primary chondrocytes restricts the clinical application of tissue engineering for cartilage repair. Therefore, method to maintain or restore the chondrocyte phenotype during in vitro expansion is essential. The objective of this study is to establish the beneficial effect of ECM molecules on restoring the re-expression of cartilaginous markers in primary human chondrocytes after extensive monolayer expansion. During the course of chondrocyte serial expansion, COL2A1, SOX9, and AGN mRNA expression levels, and GAG accumulation level were reduced significantly in serially passaged cells. Exogenous type II collagen dose-dependently elevated GAG level and induced the re-expression of cartilaginous marker mRNAs in P7 chondrocytes. Chondroitin sulfate did not show significant effect on P7 chondrocytes, while hyaluronic acid inhibited the expression of SOX9 and AGN mRNAs. Upon treatment with type II collagen, FAK, ERK1/2, and JNK were activated via phosphorylation in P7 chondrocytes within 15 min. Furthermore, GFOGER integrin blocking peptide, MEK inhibitor and JNK inhibitor, not p38 inhibitor, significantly reduced the type II collagen-induced GAG deposition level. Finally, in the presence of TGF-ß1 and IGF-I, P7 chondrocytes cultured in 3D type II collagen matrix exhibited better cartilaginous features than those cells cultured in the type I collagen matrix. In conclusion, type II collagen alone can effectively restore cartilaginous features of expanded P7 human chondrocytes. It is probably mediated via the activation of FAK-ERK1/2 and FAK-JNK signaling pathways. The potential application of type II collagen in expanding a scarcity of healthy chondrocytes in vitro for further tissue engineering is implicated.

Reduction of monocyte chemoattractant protein-1 and interleukin-8 levels by ticlopidine in TNF-alpha stimulated human umbilical vein endothelial cells.

Atherosclerosis and its associated complications represent major causes of morbidity and mortality in the industrialized or Western countries. Monocyte chemoattractant protein-1 (MCP-1) is critical for the initiating and developing of atherosclerotic lesions. Interleukin-8 (IL-8), a CXC chemokine, stimulates neutrophil chemotaxis. Ticlopidine is one of the antiplatelet drugs used to prevent thrombus formation relevant to the pathophysiology of atherothrombosis. In this study, we found that ticlopidine dose-dependently decreased the mRNA and protein levels of TNF-alpha-stimulated MCP-1, IL-8, and vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs). Ticlopidine declined U937 cells adhesion and chemotaxis as compared to TNF-alpha stimulated alone. Furthermore, the inhibitory effects were neither due to decreased HUVEC viability, nor through NF-kB inhibition. These results suggest that ticlopidine decreased TNF-alpha induced MCP-1, IL-8, and VCAM-1 levels in HUVECs, and monocyte adhesion. Therefore, the data provide additional therapeutic machinery of ticlopidine in treatment and prevention of atherosclerosis.

Follicle-stimulating hormone-induced Galphah/phospholipase C-delta1 signaling mediating a noncapacitative Ca2+ influx through T-type Ca2+ channels in rat sertoli cells.

Our previous study demonstrated that FSH-induced immediate Ca(2+) influx in rat Sertoli cells (SCs) is mediated by the Galphah/phospholipase C-delta1 (PLC-delta1) signaling pathway. As to which Ca(2+) channel is responsible for such Ca(2+) influx was not understood. In this study, thapsigargin triggered an in-store calcium release and evoked a 1.5-fold elevation of intracellular Ca(2+) in Ca(2+)-free media, whereas FSH exhibited no effect. The readdition of CaCl(2) (2.5 mm) to FSH-pretreated or thapsigargin-sensitized SCs in Ca(2+)-free media immediately elicited a rapid Ca(2+) influx or a 2-fold increase of second intracellular Ca(2+) elevation, respectively. The addition of Ca(2+) chelator EGTA (0.2 mm) reduced the FSH-induced elevation of intracellular Ca(2+) in SCs incubated with CaCl(2). However, pretreatment with dantrolene (25 microM), which inhibits in-store calcium release, did not affect the FSH-induced elevation of intracellular Ca(2+). NiCl(2) (10 microM), a T-type calcium channel blocker, abolished the FSH-induced SC Ca(2+) influx. Furthermore, mibefradil (10 and 100 microm), another specific blocker for T-type Ca(2+) channels, dose-dependently suppressed the FSH-induced Ca(2+) influx. In contrast, nifedipine (10 and 50 microm) or omega-conotoxin GVIA (100 and 500 nm), blocker of L- or N-type Ca(2+) channels, respectively, did not affect the FSH-induced SC Ca(2+) influx. On the other hand, FSH-induced Ca(2+) influx was significantly reduced by pretreatment of SCs with myristoylated synthetic peptide (0.1 and 1 microm) of PLC-delta1 fragment TIPWNSLKQGYRHVHLL but not affected by 2',5'-dideoxyadenosine (3 and 15 microm), a selective inhibitor of adenylate cyclase. In conclusion, the FSH-induced Galphah/PLC-delta1 pathway-dependent Ca(2+) influx of rat SCs is mediated by T-type Ca(2+) channels and independent of in-store calcium release.

Characterization of and mechanism for copper-induced thioureation of serum albumin.

Thioureas (Tus) are widely used in chemical and pharmaceutical industries. This study demonstrated that copper induced the disulfide-linkage between Tus, such as alpha-naphthylthiourea (ANTU) and fluorescein-5-isothiocyanate cadaverine (FTC), with albumin (Alb), a major carrier protein in plasma with multiple functions. This reaction was absolutely copper-dependent, whereas cobalt, nickel, calcium, magnesium, zinc, iron, and manganese ions could not induce the same reaction. The reaction was substrate dose-dependent, and occurred optimally at pH 6.5. The resulting conjugated product was heat-labile, but stable in pH 6.0-8.0 buffer at 25 degrees C. The linkage could be reduced by Cu(I) (in acidic pH) and thiol-reducing agents. The mechanism of albumin thioureation was concluded: (i) the binding of Cu(II) with albumin is not necessary for the reaction, while the formation of Tus-Cu(II) complex is essential; (ii) thioureation resulted from the attack of Tus-Cu(II) at Alb-Cys(34)-SH to form the Alb-Cys(34)-S-S-Tus complex accompanied by the release of Cu(I); (iii) the released Cu(I) would back inhibit the reaction because of its competition with Cu(II) for Tus binding. These phenomenons may have important implications for the pharmacokinetics of thiourea-based drugs in plasma.

Screening, purification, and identification of a copper-dependent FITC-binding protein in human plasma: albumin.

In this study, a protein purified by fluorescein isothiocyanate (FITC)-affinity chromatography from human plasma was identified as albumin by MALDI-TOF-MS. Albumin was found to conjugate with FITC-labeled molecules through a copper-dependent reaction. The formation of this complex was confirmed by methods including a newly developed "charcoal-based fluorescence assay" (CFA), gel-filtration, affinity chromatography, and ultrafiltration. The binding was identified as disulfide bridge formation. This is the first to demonstrate that copper induces a covalent binding of FITC-labeled molecules with albumin. In addition, the developed CFA method facilitates the screening of small fluorescent dyes binding to macromolecules.

Optical imaging of ovarian cancer using a matrix metalloproteinase-3-sensitive near-infrared fluorescent probe.

Epithelial ovarian cancer (EOC) is the seventh most common cancer among women worldwide. The 5-year survival rate for women with EOC is only 30%-50%, which is largely due to the typically late diagnosis of this condition. EOC is difficult to detect in its early stage because of its asymptomatic nature. Recently, near-infrared fluorescent (NIRF) imaging has been developed as a potential tool for detecting EOC at the molecular level. In this study, a NIRF-sensitive probe was designed to detect matrix metalloproteinase (MMP) activity in ovarian cancer cells. A cyanine fluorochrome was conjugated to the amino terminus of a peptide substrate with enzymatic specificity for MMP-3. To analyze the novel MMP-3 probe, an in vivo EOC model was established by subcutaneously implanting SKOV3 cells, a serous-type EOC cell line, in mice. This novel MMP-3-sensitive probe specifically reacted with only the active MMP-3 enzyme, resulting in a significantly enhanced NIRF emission intensity. Histological analysis demonstrated that MMP-3 expression and activity were enhanced in the stromal cells surrounding the ovarian cancer cells. These studies establish a molecular imaging reporter for diagnosing early-stage EOC. Additional studies are required to confirm the early-stage activity of MMP-3 in EOC and its diagnostic and prognostic significance.

Correction: Effects of collagen matrix and bioreactor cultivation on cartilage regeneration of a full-thickness critical-size knee joint cartilage defects with subchondral bone damage in a rabbit model.

[This corrects the article DOI: 10.1371/journal.pone.0196779.].

Effects of collagen matrix and bioreactor cultivation on cartilage regeneration of a full-thickness critical-size knee joint cartilage defects with subchondral bone damage in a rabbit model.

Cartilage has limited self-repair ability. The purpose of this study was to investigate the effects of different species of collagen-engineered neocartilage for the treatment of critical-size defects in the articular joint in a rabbit model. Type II and I collagen obtained from rabbits and rats was mixed to form a scaffold. The type II/I collagen scaffold was then mixed with rabbit chondrocytes to biofabricate neocartilage constructs using a rotating cell culture system [three-dimensional (3D)-bioreactor]. The rabbit chondrocytes were mixed with rabbit collagen scaffold and rat collagen scaffold to form neoRBT (neo-rabbit cartilage) and neoRAT (neo-rat cartilage) constructs, respectively. The neocartilage matrix constructs were implanted into surgically created defects in rabbit knee chondyles, and histological examinations were performed after 2 and 3 months. Cartilage-like lacunae formation surrounding the chondrocytes was noted in the cell cultures. After 3 months, both the neoRBT and neoRAT groups showed cartilage-like repair tissue covering the 5-mm circular, 4-mm-deep defects that were created in the rabbit condyle and filled with neocartilage plugs. Reparative chondrocytes were aligned as apparent clusters in both the neoRAT and neoRBT groups. Both neoRBT and neoRAT cartilage repair demonstrated integration with healthy adjacent tissue; however, more integration was obtained using the neoRAT cartilage. Our data indicate that different species of type II/I collagen matrix and 3D bioreactor cultivation can facilitate cartilage engineering in vitro for the repair of critical-size defect.

Correction: Optical imaging of ovarian cancer using a matrix metalloproteinase-3-sensitive near-infrared fluorescent probe.

[This corrects the article DOI: 10.1371/journal.pone.0192047.].

A novel follicle-stimulating hormone-induced G alpha h/phospholipase C-delta1 signaling pathway mediating rat sertoli cell Ca2+-influx.

FSH is known to activate Gs/cAMP signaling pathway in Sertoli cells (SCs) to support spermatogenesis. However, the molecular mechanism of FSH-induced Gs/cAMP-independent Ca2+-influx in SCs is not clear. In this study, FSH indeed induced an immediate and dose-dependent intracellular Ca2+-elevation in rat SCs. In the presence of EDTA (2.5 mm) or in the absence of extracellular Ca2+, the FSH-induced intracellular Ca2+-elevation was abolished. The confocal microscopic observation of Ca2+ image revealed that the SC cellular Ca2+ level was gradually increased after 50 sec of FSH treatment. Dantrolene, a blocker of intracellular Ca2+ release, did not affect this FSH-induced intracellular Ca2+ elevation. The pretreatment of rat SCs with phosphatidylinositol-phospholipase C (PLC)-specific inhibitor, U73122 (3 and 10 microm), inhibited the FSH-induced Ca2+-influx in a dose-dependent manner, but treatment with Gs-specific inhibitor, NF449 (0.1 and 0.3 microm), did not. On the other hand, the activation of G alpha h was immediately induced by FSH in the rat SCs within 5 sec of treatment. The translocation of PLC-delta1 from cytosol to cell membrane and the formation of G alpha h /PLC-delta1 complexes occurred within 5 and 10 sec, respectively, of FSH exposure. The intracellular inositol 1,4,5-triphosphate (IP3) production was also detected after 30 sec of FSH treatment. The synthetic peptide of PLC-delta1 (TIPWNSLKQGYRHVHLL), not Gs inhibitor, predominantly inhibited the FSH-induced PLC-delta1 translocation, formation of G alpha h /PLC-delta1 complex, intracellular IP3 production, and Ca2+ influx. In contrast, the peptide did not interfere with FSH-induced intracellular cAMP accumulation. In conclusion, the FSH-induced immediate Ca2+ influx is unambiguously mediated by an alternative G alpha h /PLC-delta1/IP3 pathway that is distinct from the Gs/cAMP pathway in rat SCs.

Taiwan's Travel and Border Health Measures in Response to Zika.

Zika virus has recently emerged as a worldwide public health concern. Travel and border health measures stand as one of the main strategies and frontline defenses in responding to international epidemics. As of October 31, 2016, Taiwan has reported 13 imported cases, 5 of which were detected through routine entry screening and active monitoring at international airports. This article shares Taiwan's disease surveillance activities at designated points of entry and travel and border health measures in response to Zika. The Taiwan government collaborates with its tourism industry to disseminate information about precautionary measures and encourages tour guides to report suspected individuals or events to activate early response measures. Taiwan also engages in vector control activities at points of entry, including targeting aircraft from countries where vector-borne diseases are endemic, implementing mosquito sweep measures, and collecting vector surveillance data. In future emerging and reemerging disease events, entry surveillance at designated points of entry may enable early detection of diseases of international origin and more rapid activation of public health preparedness activities and international collaboration. Taiwan will continue to maximize border and travel health measures in compliance with IHR (2005) requirements, which rely on continued risk assessment, practical implementation activities, and engagement with all stakeholders.

The Gαh-PLCδ1 signaling axis drives metastatic progression in triple-negative breast cancer.

BACKGROUND: Distant metastasis of triple-negative breast cancer (TNBC) to other organs, e.g., the lungs, has been correlated with poor survival rates among breast cancer patients. Therefore, the identification of useful therapeutic targets to prevent metastasis or even inhibit tumor growth of TNBC is urgently needed. Gαh is a novel GTP-binding protein and known as an inactive form of calcium-dependent tissue transglutaminase. However, the functional consequences of transamidating and G-protein activities of tissue transglutaminase in promoting cancer metastasis are still controversial. METHODS: Kaplan-Meier analyses were performed to estimate the prognostic values of Gαh and PLCδ1 by utilizing public databases and performing immunohistochemical staining experiments. Cell-based invasion assays and in vivo lung colony-forming and orthotropic lung metastasis models were established to evaluate the effectiveness of interrupting the protein-protein interaction (PPI) between Gαh and PLCδ1 in inhibiting the invasive ability and metastatic potential of TNBC cells. RESULTS: Here, we showed that the increased level of cytosolic, not extracellular, Gαh is a poor prognostic marker in breast cancer patients and correlates with the metastatic evolution of TNBC cells. Moreover, clinicopathological analyses revealed that the combined signature of high Gαh/PLCδ1 levels indicates worse prognosis in patients with breast cancer and correlates with lymph node metastasis of ER-negative breast cancer. Blocking the PPI of the Gαh/PLCδ1 complex by synthetically myristoylated PLCδ1 peptide corresponding to the Gαh-binding interface appeared to significantly suppress cellular invasiveness in vitro and inhibit lung metastatic colonies of TNBC cells in vivo. CONCLUSIONS: This study establishes Gαh/PLCδ1 as a poor prognostic factor for patients with estrogen receptor-negative breast cancers, including TNBCs, and provides therapeutic value by targeting the PPI of the Gαh/PLCδ1 complex to combat the metastatic progression of TNBCs.