Evaluation of Blood Glucose Monitoring System in Screening for Neonatal Hypoglycemia: Tighter Accuracy Standard.

Neonatal hypoglycemia may cause severe neurological damages; therefore, tight glycemic control is crucial to identify neonate at risk. Previous blood glucose monitoring system (BGMS) failed to perform well in neonates; there are calls for the tightening of accuracy requirements. It remains a need for accurate BGMS for effective bedside diabetes management in neonatal care within a hospital population. A total of 300 neonates were recruited from local hospitals. Accuracy performance of a commercially available BGMS was evaluated against reference instrument in screening for neonatal hypoglycemia, and assessment was made based on the ISO15197:2013 and a tighter standard. At blood glucose level < 47 mg/dl, BGMS assessed met the minimal accuracy requirement of ISO 15197:2013 and tighter standard at 100% and 97.2%, respectively.

Protein phosphatase Mg2+/Mn2+ dependent 1F promotes smoking-induced breast cancer by inactivating phosphorylated-p53-induced signals.

We previously demonstrated that the activation of α9-nicotinic acetylcholine receptor (α9-nAchR) signaling by smoking promotes breast cancer formation. To investigate the downstream signaling molecules involved in α9-nAChR-induced breast tumorigenesis, we used real-time polymerase chain reactions and Western blotting to assess expression of protein phosphatase Mg2+/Mn2+ dependent 1F (PPM1F), a Ser/Thr protein phosphatase, in human breast cancer samples (n=167). Additionally, stable PPM1F-knockdown and -overexpressing cell lines were established to evaluate the function of PPM1F. The phosphatase activity of PPM1F in nicotine-treated cells was assessed through Western blotting, confocal microscopy, and fluorescence resonance energy transfer. Higher levels of PPM1F were detected in the breast cancer tissues of heavy smokers (n=7, 12.8-fold) greater than of non-smokers (n= 28, 6.3-fold) (**p=0.01). In vitro, nicotine induced PPM1F expression, whereas α9-nAChR knockdown reduced the protein expression of PPM1F. A series of biochemical experiments using nicotine-treated cells suggested that the dephosphorylation of p53 (Ser-20) and BAX (Ser-184) by PPM1F is a critical posttranslational modification, as observed in breast cancer patients who were heavy smokers. These observations indicate that PPM1F may be a mediator downstream of α9-nAChR that activates smoking-induced carcinogenic signals. Thus, PPM1F expression could be used for prognostic diagnosis or inhibited for cancer prevention and therapy.

Luteolin sensitises drug-resistant human breast cancer cells to tamoxifen via the inhibition of cyclin E2 expression.

Luteolin is a flavonoid that has been identified in many plant tissues and exhibits chemopreventive or chemosensitising properties against human breast cancer. However, the oncogenic molecules in human breast cancer cells that are inhibited by luteolin treatment have not been identified. This study found that the level of cyclin E2 (CCNE2) mRNA was higher in tumour cells (4.89-fold, (∗)P=0.005) than in normal paired tissue samples as assessed using real-time reverse-transcriptase polymerase chain reaction (RT-PCR) analysis (n=257). Further, relatively high levels of CCNE2 protein expression were detected in tamoxifen-resistant (TAM-R) MCF-7 cells. These results showed that the level of CCNE2 protein expression was specifically inhibited in luteolin-treated (5µM) TAM-R cells, either in the presence or absence of 4-OH-TAM (100nM). Combined treatment with 4-OH-TAM and luteolin synergistically sensitised the TAM-R cells to 4-OH-TAM. The results of this study suggest that luteolin can be used as a chemosensitiser to target the expression level of CCNE2 and that it could be a novel strategy to overcome TAM resistance in breast cancer patients.

Long-term ethanol exposure-induced hepatocellular carcinoma cell migration and invasion through lysyl oxidase activation are attenuated by combined treatment with pterostilbene and curcumin analogues.

Ethanol consumption induces hepatocellular carcinoma (HCC) cell metastasis by changing the extracellular matrix (ECM). Lysyl oxidase (LOX) catalyzes the cross-linkage of collagen or elastin in the ECM. LOX protein and mRNA overexpression (>21-fold compared with controls, n = 6) was detected in cirrhotic HCC patients with a history of alcoholism. LOX protein expression was induced in HCC cells after long-term treatment with ethanol (10 mM) for 20-40 passages (denoted E20-E40 cells). Pterostilbene (PSB, 1 µM) displayed significant potency to reduce LOX-mediated activity in E40 cells when combined with curcumin and its analogues. The ability of E40 cells to form colonies in soft agar was reduced by both genetic depletion of LOX and by chemical inhibitors of LOX expression. This study suggests that targeting LOX expression with food components such as PSB and curcumin may be a novel strategy to overcome ethanol-induced HCC cell metastasis in liver cancer patients.

Use of immunomagnetic reduction for C-reactive protein assay in clinical samples.

BACKGROUND: Magnetic nanoparticles biofunctionalized with antibodies are able to recognize and bind to the corresponding antigens. In this work, anti-C-reactive protein (CRP) antibody was covalently conjugated onto the surface of magnetic nanoparticles to label CRP specifically in serum. METHODS: The level of serum CRP was detected by immunomagnetic reduction (IMR) assay, which identifies the changes in the magnetic signal representing the level of interaction between antibody-conjugated magnetic nanoparticles and CRP proteins. To investigate the feasibility of IMR for clinical application, pure CRP solutions and 40 human serum samples were tested for IMR detection of CRP to characterize sensitivity, specificity, and interference. RESULTS: In comparison with the immunoturbidimetry assay, the results of the IMR assay indicated higher sensitivity and had a high correlation with those of the current immunoturbidimetry assay. CONCLUSION: We have developed a novel and promising way to assay CRP in human serum using immunomagnetic reduction in clinical diagnosis.

Human breast cancer cell metastasis is attenuated by lysyl oxidase inhibitors through down-regulation of focal adhesion kinase and the paxillin-signaling pathway.

The extracellular matrix (ECM) plays a critical role in the development and invasion of primary breast tumors. Lysyl oxidase (LOX), which is an ECM remodeling enzyme, appears to play roles in promoting cancer cell motility and invasion. To ascertain whether LOX overexpression in breast tumor tissues from Asian patients is associated with decreases in metastasis-free and overall survival in breast cancer patients, the mRNA levels of LOX were examined in paired tumor/normal tissue samples using real-time RT-PCR analysis (n = 246 pair-matched samples). To test whether specifically targeting LOX by inhibiting its activity (using beta-aminopropionitrile (ß-APN), a LOX inhibitor), mRNA expression (using siRNA), or protein expression (using 25 µM magnolol) attenuates the invasion of MDA-MB-231 breast cancer cells, a cancer cell migration assay was performed. Interestingly, only 78.5% (n = 193) of the breast cancer tumors displayed detectable LOX expression. Nearly 60% (n = 120) of the cases fell into Group 1 (tumor > normal, T > N); in this group, the mean LOX expression in the tumor cells was 20.2-fold greater than in normal cells. However, in Group 2 (normal > tumor, N > T), the LOX expression level in most of the normal tissues examined (80%, 59/73) was less than fivefold greater than in the tumor tissues. The increased level of active LOX in the invasive breast cancer cell line MDA-MB-231 was accompanied by the increased phosphorylation of focal adhesion kinase at Tyr-576 and of paxillin at Tyr-118. We also found that the addition of ß-APN (300 µM) and magnolol (25 µM), synergistically inhibited the migration and invasion of MDA-MB-231 cells. In this article, we describe, for the first time, higher expression of a LOX protein in breast tumors compared with normal tissues from Asian patients. Moreover, the results indicate that the inhibition of LOX using magnolol may represent a more desirable strategy for breast cancer therapy than the use of ß-APN.

In vivo antitumor effects of 4,7-dimethoxy-5-methyl-1,3-benzodioxole isolated from the fruiting body of Antrodia camphorata through activation of the p53-mediated p27/Kip1 signaling pathway.

In this study, 4,7-dimethoxy-5-methyl-1,3-benzodioxole (SY-1) was isolated from three different sources of dried Antrodia camphorata (AC) fruiting bodies. AC is a medicinal mushroom that grows on the inner heartwood wall of Cinnamomum kanehirai Hay (Lauraceae), which is an endemic species that is used in Chinese medicine for its antitumor properties. We demonstrated that SY-1 [given as a 1-30 mg/kg body weight intraperitoneal (ip) injection three times per week] profoundly decreased the growth of COLO-205 human colon cancer cell tumor xenografts in an athymic nude mouse model. We further demonstrated that significant AC extract-mediated antitumor effects were observed at the highest concentration (5 g/kg body weight/day). No gross toxicity signs were observed (i.e., body weight changes, general appearance, or individual organ effects). Frozen COLO-205 xenograft tumors were pulverized in liquid N(2), and the expression of cell cycle regulatory proteins was detected by immunoblotting. We found that the p53-mediated p27/Kip1 protein was significantly induced in the low-dose (1 mg/kg body weight) SY-1-treated tumors, whereas the p21/Cip1 protein levels did not change. The G0/G1 phase cell cycle regulators induced by SY-1 were also associated with a significant decrease in cyclins D1, D3, and A. These results provide further evidence that SY-1 may have significance for cancer chemotherapy.

The in vivo antitumor effects on human COLO 205 cancer cells of the 4,7-dimethoxy-5-(2-propen-1-yl)-1,3-benzodioxole (apiole) derivative of 5-substituted 4,7-dimethoxy-5-methyl-l,3-benzodioxole (SY-1) isolated from the fruiting body of Antrodia camphorate.

CONTEXT: The compound 4,7-dimethoxy-5-(2-propen-1-yl)-1,3-benzodioxole (apiole) has been isolated from several different plant species, including Petroselinum sativum. Our recent study found that apiole is a chemical derivative of 4,7-dimethoxy-5-methyl-l,3-benzodioxole (SY-1), which has been isolated from dried Antrodia camphorata (AC ) fruiting bodies, a traditional Chinese medicine with antitumor properties. AIMS: Our previous in vitro study demonstrated that apiole inhibits the growth of human colon (COLO 205) cancer cells through the arrest of the cell cycle in G0/G1 phase. The in vivo antitumor effects of apiole were evaluated in this study. SETTING AND DESIGN: Apiole was administered to mice at 1-30 mg/kg body weight through intraperitoneal (I.P.) injection three times per week (defined as a dosage of 1×-30×). MATERIALS AND METHODS: The in vivo antitumor effects of apiole were evaluated in mice with xenografts of COLO 205 cells. STATISTICAL ANALYSIS: All of the data are reported as the means ± S.E. Comparisons were performed with a one-way analysis of variance (ANOVA) followed by a Fisher's least significant difference test. Significance was defined as P < 0.05. RESULTS: Apiole (> 1×) markedly decreased the growth of COLO 205 human colon cancer cell tumor xenografts in an athymic nude mouse model system through the up-regulation of cell cycle regulators, such as p53, p21/Cip1, and p27/Kip1. The apiole-induced increase in G0/G1 phase cell cycle regulators was also associated with a significant decrease in the expression of cyclins D1 and D3. Surprisingly, statistically significantly higher tumor volumes were observed in mice that received 5× apiole compared with 30× apiole-treated mice (P < 0.05). No gross signs of toxicity were observed (e.g., body weight changes, general appearance, or individual organ effects) in any group. CONCLUSIONS: Our results show, for the first time, the promising antitumor effects of apiole against colon tumors in an in vivo xenograft model.

Tea polyphenol (-)-epigallocatechin-3-gallate inhibits nicotine- and estrogen-induced α9-nicotinic acetylcholine receptor upregulation in human breast cancer cells.

SCOPE: The aim of this research was to explore whether the tea-polyphenol (-)-epigallocatechin-3-gallate (EGCG) could be used as a potential agent for blocking smoking (nicotine, Nic)- or hormone (estradiol, E2)-induced breast cancer cell proliferation through inhibition of a common signaling pathway. METHODS AND RESULTS: To explore whether Nic (>0.1 µM, 24 h) and E2 (>1 nM, 24 h) significantly increased α9-nicotinic acetylcholine (α9-nicotinic acetylcholine receptor (nAChR)) mRNA and protein expression levels, real-time PCR and immunoblotting analysis experiments were performed in human breast cancer (MCF-7) cells. Luciferase promoter activity experiment was performed to test the α9-nAChR promoter activity affected by Nic, E2 or EGCG. The results indicate that treatment with EGCG (1 µM) profoundly decreases Nic- and E2-induced MCF-7 proliferation by down regulating α9-nAChR expression. The α9-nAChR promoter activity is significantly induced by 24-h treatment with Nic (10 µM) or E2 (10 nM) (>1.8 and ∼2.3-fold, respectively) in MCF-7 cells. Pretreatment with EGCG eliminated the Nic- and E2-induced α9-nAChR promoter-dependent luciferase activity. We further demonstrate that combined treatment with EGCG profoundly inhibits [3H]-Nic/ α9-nAChR binding activity in breast cancer cells. CONCLUSIONS: We found that the EGCG could be used as an agent for blocking smoking (Nic)- or hormone (E2)-induced breast cancer cell proliferation by inhibiting of α9-nAChR signaling pathway. This study reveals the novel antitumor mechanisms of EGCG, and these results may have significant applications for chemopreventive purposes in human breast cancer.

Crosstalk between nicotine and estrogen-induced estrogen receptor activation induces α9-nicotinic acetylcholine receptor expression in human breast cancer cells.

The primary aim of this study was to elucidate the role of the estrogen receptor (ER), a transcription factor involved in the nicotine- and 17ß-estradiol (E2)-mediated up-regulation of α9-nAChR gene expression. A real-time polymerase chain reaction (PCR) assay was used to quantify the α9-nAChR mRNA expression levels of surgically isolated (n=339) and laser-capture microdissected tissues (ER+ versus ER-, n= 6 per group). Chromatin immunoprecipitation (ChIP) and luciferase-promoter activity assays were used to investigate the ER-mediated transcriptional regulation of α9-nAChR gene expression. We observed that breast tumors with higher α9-nAChR mRNA expression levels (i.e., a mean fold ratio in the tumor/normal-paired samples of greater than tenfold) were associated with the lowest 5-year disease-specific survival rate (50%, dead/alive= 4/4, total = 8 patients, P= 0.006), in contrast to breast tumors with low levels (i.e., a mean fold ratio of less than onefold) of α9-nAChR expression (88%, dead/alive= 3/22, total= 25 patients). Furthermore, higher α9-nAChR mRNA expression levels were preferentially detected in ER+ tumor tissues in comparison to ER- tumor tissues (ER+ versus ER- patients: n=160 vs. 72; mean fold ratios of α9-nAChR expression = 11 ± 3 vs. 6.7 ± 2.3 fold, respectively). In vitro promoter-binding assays demonstrated that the ER is a major transcription factor that mediates nicotine- and E2-induced up-regulation of α9-nAChR gene expression in MCF-7 cells. In conclusion, our data indicate that the ER plays a central role in mediating α9-nAChR gene up-regulation in response to either nicotine or E2 stimulation.

Long-term ethanol exposure causes human liver cancer cells to become resistant to mitomycin C treatment through the inactivation of bad-mediated apoptosis.

The aim of this study was to test whether long-term ethanol consumption confers therapeutic resistance to human liver cancer patients infected with hepatitis B virus (HBV). Chronic ethanol-treated cells were established by consecutively culturing a human hepatocellular carcinoma cell line, Hep 3B, which contains integrated HBV sequences, for 20-40 passages with or without 10 mM ethanol (designated as E20-E40 and C20-C40, respectively). Flow cytometry analysis demonstrated that a growth promoting effect of long-term ethanol treatment was induced in the E40 cells through preferential acceleration of S-phase in these cells. Lower protein expression levels of p16, p21/Cip1, and p27/Kip1 were detected in the ethanol-treated E40 cells. We further demonstrated that long-term ethanol-treated E40 cells develop drug resistance in response to mitomycin C (MMC) treatment (>8 microM). Immunoblot analysis revealed that caspase-8-mediated mitochondrial apoptotic signals (such as Bad) were inactivated in the MMC-resistant E40 cells. Immunoprecipitation experiments demonstrated that the sequestration of phosphorylated Bad (Ser-112) through its binding with 14-3-3 was detected more profoundly in the MMC-resistant E40 cells. Next, we examined the therapeutic efficacy of MMC (10 mg MMC/kg body weight, three times per week) in severe combined immunodeficient (SCID) mice bearing E40- and C40-xenografted tumors. Significant reductions (>3-fold) in tumor growth were detected in MMC-treated C40-xenografted mice. In vivo and in vitro studies demonstrated that AKT- and extracellular signal-regulated kinase (ERK)-mediated survival factors inhibited the Bad-induced mitochondrial apoptotic signals that were involved in E40 tumor cells and that conferred resistance to MMC.

Combination treatment with luteolin and quercetin enhances antiproliferative effects in nicotine-treated MDA-MB-231 cells by down-regulating nicotinic acetylcholine receptors.

Large-scale epidemiological cohort studies performed in the United States indicate that breast cancer risk is associated with active and passive smoking. As of yet, however, there is no direct evidence of antitumor effects by agents that block the effect of tobacco compound nicotine (Nic) on relevant nicotinic receptors (nAChR) involved in breast tumorigenesis. In the present study, the expression profiles of different nAChR subunits in the human breast cancer cell line (MDA-MB-231) were characterized by RT-PCR. Nic (>0.1 microM, 6 h) significantly increased alpha9-nAChR mRNA and protein expression levels in human breast cancer cells (MDA-MB-231 cells). On the other hand, combined treatment with luteolin (Lut, 0.5 microM) and quercetin (Que, 0.5 microM) profoundly decreased MDA-MB-231 proliferation by down-regulating alpha9-nAChR expression. MDA-MB-231 cells were cultured in soft agar to evaluate anchorage-independent colony formation; combined treatment of Lut+Que inhibited Nic-induced MDA-MB-231 colony formation. Interestingly, the number of colonies formed was profoundly reduced in alpha9-nAChR knockdown (Si alpha9) cells in the combined (Lut+Que)-treated group as compared to the relevant control groups. Such results show that Lut- or Que-induced antitransforming activities were not limited to specific inhibition of the alpha9-nAChR receptor. Both alpha5- and alpha9-nAChR appear to be important molecular targets for Lut- and Que-induced antitumor effects in human breast cancer cells.

Kawasaki disease presenting with lymphadenopathy and gastrointestinal hemorrhage: report of one case.

We report a 5-year-old boy with Kawasaki disease (KD) initially presenting with cervical lymphadenitis and complicated by gastrointestinal hemorrhage before the use of salicylates (aspirin). A previously well and fully immunized 5-year-old Japanese boy presented with a 5-day history of neck mass and fever. Physical examination showed bilateral lymphadenopathy but not nonexudative conjunctivitis, oral cavity changes, skin rash, or extremity changes. He was admitted with a diagnosis of neck lymphadenitis and treated with parenteral antibiotics. Tarry stool was noted on the second day of admission, before the definitive diagnosis of KD was made, and cimetidine was given to treat the gastrointestinal hemorrhage. On the fourth day of admission, he fulfilled the diagnostic criteria for KD (fever for 9 days plus lymphadenopathy, lip fissure, conjunctivitis, and skin rash). He was treated with intravenous immunoglobulin (2 g/kg/day) and aspirin (80 mg/kg/day), and the fever subsided promptly. Massive gastrointestinal bleeding occurred on the ninth day of admission and was treated with whole blood transfusion, after which the vital signs were stable.

Role of protein kinase C in BSA-AGE-mediated inducible nitric oxide synthase expression in RAW 264.7 macrophages.

In the present study, the roles of protein kinase C (PKC) in BSA-derived advanced glycosylation end products (BSA-AGEs)-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression were investigated. Treatment of RAW 264.7 cells with BSA-AGEs caused dose- and time-dependent increases in NO release and iNOS expression in RAW 264.7 cells, whereas BSA alone had no effect on iNOS induction. The tyrosine kinase inhibitor (genistein), the phosphatidylinositol-specific phospholipase C inhibitor (U-73122), the phosphatidylcholine-specific phospholipase C inhibitor (D-609), and the PKC inhibitors (staurosporine, Ro 31-8220, and Go 6976) all inhibited BSA-AGE-induced NO release and iNOS expression in RAW 264.7 cells. Stimulation of RAW 264.7 cells with BSA-AGEs resulted in the formation of inositol monophosphate; the response was attenuated by U-73122 and genistein. BSA-AGEs stimulated PKC-alpha, -betaI, -delta, and -eta but not -zeta translocation from the cytosol to the membrane. However, incubation of RAW 264.7 cells with BSA-AGEs increased phosphorylation of PKC-zeta at threonine-410, which reflects activation of PKC-zeta, indicating the possible involvement of these PKC isoforms in AGE-mediated effects. Pretreatment of RAW 264.7 cells with U-73122, D-609, and genistein reduced the AGE-stimulated translocation of PKC-alpha, -betaI, -delta, and -eta and activation of PKC-zeta. Taken together, these data suggest that BSA-AGEs might activate PKC and subsequently induce iNOS expression and NO release.