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Purpose: To determine whether miR-452 regulates osteoblast differentiation (OD) in human periodontal ligament stem cells (hPDLSCs) by targeting polycomb-group protein BMI1. Methods: hPDLSCs were stimulated to differentiate upon treatment with mineralization liquid. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting were used to measure mRNA and protein expressions, respectively. Alkaline phosphatase (ALP) activity and Alizarin red staining were used to determine the osteogenic differentiation (OD) of hPDLSCs. The bioinformatics software, Targetscan, was used to predict the potential target of miR-452, while luciferase assay, qRTPCR, and western blot were employed to verify the target gene of miR-452, BMI1. Results: MiR-452 was downregulated during the OD of hPDLSCs, but miR-452 overexpression inhibited the OD of hPDLSCs. BMI1 was identified as a direct target gene of miR-452 during the OD of hPDLSCs, while miR-452 overexpression correlated inversely with BMI1 expression during OD of hPDLSCs. Conclusion: Overexpression of miR-452 suppresses the OD of hPDLSCs by targeting BMI1.This study may provide potential diagnostic and therapeutic basis for OD in hPDLSCs. [ABSTRACT FROM AUTHOR]

Breast cancer (BC) is the most malignant tumor in women. The molecular mechanisms underlying tumorigenesis still need to be further elucidated. It is necessary to investigate novel candidate genes involved in breast cancer progression and prognosis. In this study, we commit to explore candidate genes that associate with prognosis and therapy in BC by a comprehensive bioinformatic analysis. Four GEO datasets (GSE5764, GSE7904, GSE20711, and GSE29431) and the BC-related transcriptome data in TCGA database were downloaded and used to identify the differently expressed genes (DEGs). The function of DEGs was analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enrichment analysis. The protein–protein interaction (PPI) network of DEGs was constructed to identify hub genes. Prognostic candidate genes were identified through survival analysis. In addition, potential therapeutic targets were identified by constructed gene–drug interaction network through Comparative Toxicogenomics Database. A total of 547 DEGs (302 up and 245 down) were identified. Three core-subnetwork and 25 hub genes were identified in PPI network. Seven genes (namely COL12A1, QPRT, MRPL13, KRT14, KRT15, LAMB3, and MYBPC1) were identified as crucial prognostic candidate genes, which significantly associated with breast cancer overall survival. Furthermore, two representative candidate genes (COL12A1 and LAMB3) were optionally chosen for verification by reverse transcription and quantitative real-time polymerase chain reaction (RT-PCR). What's more, the gene–drugs interaction analysis indicates several antitumor drugs that could affect the expression of these prognostic markers, such as doxorubicin, cisplatin, and tamoxifen. These results identified seven crucial candidate genes that may serve as prognosis biomarkers and novel therapeutic targets of breast cancer, which may facilitate further understanding the molecular pathogenesis and providing potential therapeutic strategies for BC. [ABSTRACT FROM AUTHOR]

Background/Aims: Most of estrogen receptor positive breast cancer patients respond well initially to endocrine therapies, but often develop resistance during treatment with selective estrogen receptor modulators (SERMs) such as tamoxifen. Altered expression and functions of microRNAs (miRNAs) have been reportedly associated with tamoxifen resistance. Thus, it is necessary to further elucidate the function and mechanism of miRNAs in tamoxifen resistance. Methods: Tamoxifen sensitivity was validated by using Cell Counting Kit-8 in tamoxifen- sensitive breast cancer cells (MCF-7, T47D) and tamoxifen-resistant cells (MCF-7/TAM, T47D/ TAM). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to detect the expression level of miR-449a in tamoxifen-sensitive/-resistant cells and patient serums. Dual- luciferase assay was used to identify the binding of miR-449a and predicted gene ADAM22. The expression level of ADAM22 was determined by qRT-PCR and western blotting in miR- 449a +/- breast cancer cells. Subsequently, rescue experiments were carried out to identify the function of ADAM22 in miR-449a-reduced tamoxifen resistance. Finally, Gene ontology (GO) and Protein-protein interaction analyses were performed to evaluate the potential mechanisms of ADAM22 in regulating tamoxifen resistance. Results: MiR-449a levels were downregulated significantly in tamoxifen-resistant breast cancer cells when compared with their parental cells, as well as in clinical breast cancer serum samples. Overexpression of miR-449a re-sensitized the tamoxifen-resistant breast cancer cells, while inhibition of miR-449a conferred tamoxifen resistance in parental cells. Luciferase assay identified ADAM22 as a direct target gene of miR- 449a. Additionally, silencing of ADAM22 could reverse tamoxifen resistance induced by miR- 449a inhibition in ER-positive breast cancer cells. GO analysis results showed ADAM22 was mainly enriched in the biological processes of cell adhesion, cell differentiation, gliogenesis and so on. Protein-protein interaction analyses appeared that ADAM22 might regulate tamoxifen resistance through PPARG, LGI1, KRAS and LYN. Conclusion: Decreased miR-449a causes the upregulation of ADAM22, which induces tamoxifen resistance of breast cancer cells. These results suggest that miR-449a, functioning by targeting ADAM22, contributes to the mechanisms underlying breast cancer endocrine resistance, which may provide a potential therapeutic strategy in ER-positive breast cancers. [ABSTRACT FROM AUTHOR]

Introduction: Early and accurate diagnosis of imported malaria cases in clusters is crucial for protecting the health of patients and local populations, especially confirmed parasitic persons who are asymptomatic. Methodology: A total of 226 gold miners who had stayed in highly endemic areas of Ghana for more than six months and returned in clusters were selected randomly. Blood samples from them were tested with microscopy, nest polymerase chain reaction, and rapid diagnostic test (RDT). The sensitivity, specificity, predictive values, agreement rate, and Youden's index of each of three diagnostic methods were calculated and compared with the defined gold standard. A quick and efficient way to respond to screening such a clustered mobile population was predicted and analyzed by evaluating two assumed results of combining microscopy and RDT with or without symptoms of illness. Results: The rate of the carriers of malaria parasites in the populations of gold miners was 19.47%, including 39 P. falciparum. Among the three diagnostic methods, the microscopy method showed the highest specificity, while the RDT method showed the highest sensitivity but the lowest specificity in detecting P. falciparum. The assumed results of combining RDT and microscopy with symptoms showed the best results among all the test results in screening P. falciparum. Conclusions: It was too complex and difficult to catch all parasite carriers in a short period of time among populations with such a complicated situation as that in Shanglin County. A strategy of combing microscopy and RDT for diagnosis is highly recommended. [ABSTRACT FROM AUTHOR]

Background: The role of type I collagen, composed of collagen type I alpha 1 (COL1A1) and collagen type I alpha 2 (COL1A2), has been studied in several cancers. However, the expression of COL1A1 and COL1A2 in malignant, premalignant, and normal gastric tissues and their clinical significances in gastric cancer have not been elucidated. Methods: Real-time quantitative PCR was performed in 55 malignant, 27 premalignant, and 19 normal tissues to measure COL1A1 and COL1A2 messenger RNA (mRNA) expression, and the correlations between COL1A1 and COL1A2 expression and clinicopathological parameters and patients' survival rate were analyzed. Results: We found that COL1A1 mRNA expression was significantly upregulated in premalignant and malignant tissues than in normal tissues, whereas COL1A2 mRNA expression was significantly higher in malignant tissues than in premalignant and normal tissues. Moreover, COL1A1 expression was unrelated to clinicopathological parameters, while COL1A2 expression was positively related to tumor size and depth of invasion. Besides, higher COL1A1 and COL1A2 expression levels were related to lower overall survival. Conclusions: We find that COL1A1 might have its potential as a monitoring factor to screen early gastric cancer, and COL1A1 and COL1A2 might predict poor clinical outcomes in gastric cancer patients. [ABSTRACT FROM AUTHOR]

Objective(s): Although low-dose radiotherapy (RT) that involves low collateral damage is more suitable for hepatocellular carcinoma (HCC) than traditional high-dose RT, but to achieve satisfactory therapeutic effect with low-dose RT, it is necessary to sensitize HCC cells to irradiation. This study was aimed to determine whether radiosensitivity of HCC cells can be enhanced using miR-26b by targeting erythropoietin producing human hepatocelluar A2 (EphA2). Materials and Methods: The levels of miR-26b and EphA2 expression in multiple HCC cell lines were assessed by qPCR and western blotting, respectively, and compared with those in a hepatic cell line. HCC 97H cells were transfected with miR-26b mimics, EphA2-ShRNA or EphA2 overexpression vector before exposure to low-dose irradiation. Results: Different degrees of miR-26b down-regulation and EphA2 up-regulation were observed in all HCC cell lines, among which the HCC 97H cell line expressed the lowest level of miR-26b and highest level of EphA2. EphA2 was verified as the target of miR-26b by dual luciferase reporter assay. HCC 97H cells transfected with miR-26b mimics or EphA2-ShRNA reduced the expression of EphA2 protein, with significantly lower cell proliferation rate and cell invasion ability and higher apoptosis rate in response to low-dose irradiation than those in the non-transfected cells. These results were reversed after EphA2 was overexpressed by transfection with the EphA2 overexpression vector. Co-transfection with miR-26b mimics and EphA2 overexpression vector barely altered EphA2 expression level and cell response to low-dose irradiation. Conclusion: These data suggest that miR-26b enhances radiosensitivity of HCC 97H cells by targeting EphA2 protein. [ABSTRACT FROM AUTHOR]

The aim of the present study was to examine the role of eIF2α in cardiomyocyte apoptosis and evaluate the cardioprotective role of salubrinal in a rat myocardial infarction (MI) model. Rat left anterior descending coronary arteries were ligated and the classical proteins involved in the endoplasmic reticulum stress (ERS)-induced apoptotic pathway were analyzed using quantitative polymerase chain reaction and western blot analysis. Salubrinal was administered to the rats and cardiomyocyte apoptosis and infarct size were evaluated by a specific staining method. Compared with the sham surgery group, the rate of cardiomyocyte apoptosis in the MI group was increased with the development of the disease. It was also demonstrated that the mRNA and protein levels of GRP78, caspase-12, CHOP and the protein expression of p-eIF2α were increased in the MI group. Furthermore, the results showed that treatment with salubrinal can decrease cardiomyocyte apoptosis and infarct size by increasing eIF2α phosphorylation and decreasing the expression of caspase-12 and CHOP. The present study suggests that salubrinal protects against ER stress-induced rat cadiomyocyte apoptosis via suppressing the dephosphorylation of eIF2α in the ERS-associated pathway. [ABSTRACT FROM AUTHOR]

Introduction: Rapid dissemination of plasmid-mediated quinolone resistance (PMQR) has been reported in clinical isolates. Methodology: A total of 149 clinical isolates of Enterobacteriaceae were collected in Beijing and screened for PMQR genes usingpolymerase chain reaction (PCR). Real-time quantitative PCR was used to study the expression of qnrS. Results: The rates of qnr and aac(6')-Ib-cr genes were 7.4% and 8.1%, respectively. The higher basal expression of qnrS was observed in transconjugant strains, which had higher minimum inhibitory concentrations (MICs) of quinolones. Furthermore, qnrS expression levels increased in all three isolates when a quinolone was present. Conclusions: Our data suggest that the level of qnrS expression was associated with quinolone resistance. [ABSTRACT FROM AUTHOR]

Aim: Several methods can be used to detect gene mutations associated with β-thalassemia, but it is difficult to achieve reliable and reproducible results. In this study, we introduce a new method, a single-tube multiplex polymerase chain reaction (PCR) assay, to detect both CD17 (A>T) and IVS-II nt-654 (C>T) homozygous mutations. Materials and methods: This new method designs specific primers to diagnose homozygous mutations and normal controls. Results: After PCR amplification, homozygous mutations produce different fragments from normal controls. Conclusion: This study represents an important step towards the development of a novel protocol to diagnose β-thalassemia and other diseases that target numerous mutations. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: Interleukin-12 (IL-12) plays an important role in antitumor immunity. Interleukin-27 (IL-27) is a novel IL-12 family member. The present studies demonstrate that IL-27 mediates potent antitumor activity. However, No studies have examined the association of these polymorphism with colorectal cancer (CRC). Therefore, we investigated the relationship of IL-12 and IL-27 gene polymorphisms and CRC. Design and methods: We analyzed polymorphisms of IL-12 gene 16974 A/C and IL-27 gene −964 A/G, 2905 T/G, 4730 T/C in 410 patients with CRC and 450 controls, using PCR-RFLP method. Results: There were no significant differences in the genotype and allele frequencies of IL-12 and IL-27 gene polymorphisms between the group of patients with CRC and the controls. Furthermore, no association was found between IL-12 family gene polymorphisms and different clinical stages in patients with CRC. Conclusion: These findings suggest that IL-12 and IL-27 gene polymorphisms may not be involved in susceptibility to CRC. [Copyright &y& Elsevier]

Background: Schistosoma mansoni tetraspanin 2 (Sm-TSP-2) has been shown to be strongly recognized by IgG1 and IgG3 antibodies from individuals putatively resistant to schistosome infection, but not chronically infected people, and to induce high levels of protection against challenge infection in the murine model of schistosomiasis. Amplification by PCR of homologous sequences from male and female S. japonicum worms showed the presence of 7 different clusters or subclasses of S. japonicum TSP-2. We determined the protective efficacy of one subclass - Sj-TSP-2e. Methodology/Principal Findings: Following the alignment of 211 cDNAs, we identified 7 clusters encoding S. japonicum TSP-2 (Sj-TSP-2) based on sequence variation in the large extracellular loop (LEL) region with differing frequency of transcription in male and female worms. Quantitative PCR analysis revealed elevated expression of Sj-TSP-2 in adult worms compared with other life cycle stages. We expressed in E. coli the LEL region of one of the clusters which exhibited a high frequency of transcription in female worms, and showed the purified recombinant protein (Sj-TSP-2e) was recognised by 43.1% of sera obtained from confirmed schistosomiasis japonica patients. Vaccination of mice with the recombinant protein induced high levels of IgG1 and IgG2 antibodies, but no consistent protective efficacy against challenge infection was elicited in three independent trials. Conclusions/Significance: The highly polymorphic nature of the Sj-TSP-2 gene at the transcriptional level may limit the value of Sj-TSP-2 as a target for future S. japonicum vaccine development. [ABSTRACT FROM AUTHOR]

Expressed sequence tag (EST) analysis of the pulp of the red-fleshed mutant 'Cara Cara' navel orange provided a starting point for gene discovery and transcriptome survey during citrus fruit maturation. Interpretation of the EST datasets revealed that the mutant pulp transcriptome held a high section of stress responses related genes, such as the type III metallothionein-like gene (6.0%), heat shock protein (2.8%), Cu/Zn superoxide dismutase (0.8%), late embryogenesis abundant protein 5 (0.8%), etc. 133 transcripts were detected to be differentially expressed between the red mutant and its orange-color wild genotype 'Washington' via digital expression analysis. Among them, genes involved in metabolism, defense/stress and signal transduction were statistical overrepresented. Fifteen transcription factors, composed of NAM, ATAF, and CUC transcription factor (NAC); myeloblastosis (MYB); myelocytomatosis (MYC); basic helix-loop-helix (bHLH); basic leucine zipper (bZIP) domain members, were also included. The data reflected the distinct expression profile and the unique regulatory module associated with these two genotypes. Eight differently expressed genes analyzed in digital were validated by quantitative real-time polymerase chain reaction. For structural polymorphism, both simple sequence repeats and single nucleotide polymorphisms (SNP) loci were surveyed; dinucleotide presentation revealed a bias toward AG/GA/TC/CT repeats (52.5%), against GC/CG repeats (0%). SNPs analysis found that transitions (73%) outnumbered transversions (27%). Seventeen potential cultivar-specific and 387 heterozygous SNP loci were detected from 'Cara Cara' and 'Washington' EST pool. [ABSTRACT FROM AUTHOR]

Minocycline is neuroprotective in clinical and experimental stroke studies, due in part to its ability to inhibit poly (ADP-ribose) polymerase. Previous preclinical data have shown that interference with poly (ADP-ribose) polymerase signaling leads to sex-specific neuroprotection, reducing stroke injury only in males. In this study, we show that minocycline is ineffective at reducing ischemic damage in females after middle cerebral artery occlusion, likely due to effects on poly (ADP-ribose) polymerase signaling. Clinical trials must consider possible sex differences in the response to neuroprotective agents, if we hope to translate promising therapies to stroke patients of both sexes.Journal of Cerebral Blood Flow & Metabolism (2009) 29, 670–674; doi:10.1038/jcbfm.2009.3; published online 4 February 2009 [ABSTRACT FROM AUTHOR]

A cDNA clone encoding a putative β-ketoacylacyl carrier protein (ACP) synthase III (KAS III) was isolated from Jatropha curcas L., a woody oil plant. The cDNA clone (named JcKAS III ) contained a 1203-bp open reading frame coding for 400 amino acids with a predicted molecular mass of about 42 kDa. The deduced amino acid sequence of the cDNA clone shares about 80% identity to KAS III from other plants, and contains a conserved Cys176 in the active site and the amino acid motif G355NTSAAS361 which is responsible for binding regulatory acyl-ACPs. Southern blotting analysis indicated that JcKAS III is a single copy gene in the J. curcas genome. Quantitative real-time PCR analysis showed that JcKAS III was expressed in all tissues examined with highest expression in roots, and that expression of JcKAS III increased as seeds developed. [ABSTRACT FROM AUTHOR]

Abstract Objectives  This study investigated whether integrin-linked kinase (ILK) is involved in the pathogenesis of diabetic retinopathy, by analyzing the expression and activity of ILK in the retina from a streptozotocin (STZ)-induced rat model of diabetes. Methods  ILK expression in the retina from both control and STZ-induced diabetic rats was measured by reverse transcription polymerase chain reaction, immunohistochemistry and Western blot analysis. The expressions of Akt and FOXO1A, the downstream molecules of ILK, were also examined. Results  The present study showed that the STZ-induced diabetes was associated with the increase in the vascular permeability in the retina. This elevated vascular permeability increased with the progression of diabetic retinopathy. Meanwhile, the results also showed that the expression of ILK increased in protein and mRNA levels in the retina of STZ-induced diabetic rats. Immunohistochemistry showed that immunostaining of ILK was localized in the outer plexiform layer (OPL), the inner nuclear layer (INL), the inner plexiform layer (IPL), the ganglion cell layer (GCL) and the retinal microvasculature of rats. However, the expression of Akt was reduced in the retinas at 8 and 12 weeks and increased in the retinas at 4 weeks after induction of diabetes. Meanwhile, the expression of the FOXO1A protein increased in the retinas at 8 and 12 weeks and decreased in the retinas at 4 weeks after induction of diabetes. The FOXO1A immunostaining was also observed in the retinal microvasculature and in the OPL, INL, IPL and GCL of rat retinas. Conclusion  These results indicate that diabetes affects the expression of ILK in the retina. ILK may be involved in the diabetes-induced damage and/or alterations of neural and microvascular structures. [ABSTRACT FROM AUTHOR]

Background: Hepatitis B virus (HBV) infection is a severe health problem in the world; however, there is still no satisfactory therapeutic strategy for the HBV infection. In search for new anti-HBV agents with higher efficiency and less side effects, the anti-HBV activities of traditional Chinese medicine Rheum palmatum L. ethanol extract (RPE) and isolated anthraquinones were evaluated. Methods: The anti-HBV activities of RPE and isolated anthraquinones were demonstrated in a stable HBV-producing cell line HepG2 2.2.15 by using real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and Southern blot analysis. Results: RPE could inhibit HBV-DNA production and HBsAg expression in a dose-dependent manner. The concentration of 50% HBV-DNA inhibition (IC50) of RPE was calculated at 212.36 ± 11 μg/ml. Six anthraquinones were isolated from RPE by using RP-HPLC. Five free anthraquinones showed weakly or slightly inhibitory activities against HBV. The only combined anthraquinone chrysophanol 8-O-β-D-glucoside exhibited significant activity against HBV DNA production and antigens expression with an IC50 value of 36.98 ± 2.28 μg/ml on HBV DNA inhibition. Endogenous HBV DNA polymerase activity assay indicated that chrysophanol 8-O-β-D-glucoside might be a potential inhibitor of the HBV DNA polymerase. Conclusions: The results suggested that RPE could effectively inhibit HBV. The combined anthraquinone chrysophanol 8-O-β-D-glucoside is the major active compound in RPE and could be a promising candidate for the development of new anti-HBV drugs in the treatment of HBV infection. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been used clinically to treat inflammatory diseases clinically. However, the adverse effects of NSAIDs cannot be ignored. Therefore, it is critical for us to find alternative anti-inflammatory drugs that can reduce adverse reactions to herbal medicine, such as Iris tectorum Maxim., which has therapeutic effects and can treat inflammatory diseases and liver-related diseases. This study aimed to isolate active compounds from I. tectorum and investigate their anti-inflammatory effects and action mechanisms. Fourteen compounds were isolated from I. tectorum using silica gel column chromatography, Sephadex LH-20, ODS and high performance liquid chromatography, and their structures were identified by examining physicochemical properties, ultraviolet spectroscopy, infrared spectroscopy, mass spectrometry, and nuclear magnetic resonance spectroscopy. Classical inflammatory cell models were established using lipopolysaccharide (LPS)-stimulated RAW264.7 cells and rat primary peritoneal macrophages to examine the effect of these compounds. To examine the action mechanisms, the nitric oxide (NO) levels were measured by Griess reagent and the levels of inflammatory cytokines in the supernatant were measured by ELISA; The expressions of major proteins in prostaglandin E 2 (PGE 2) synthesis and the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways were examined by Western blotting, and the mRNA expression levels were measured by quantitative real-time polymerase chain reaction; and the nuclear translocation of p65 was examined by high content imaging. Molecular docking was used to predict the binding of active compound to target protein. Our findings revealed that Iristectorigenin C (IT24) significantly inhibited the levels of NO and PGE 2 without affecting cyclooxygenase (COX)-1/COX-2 expression in LPS-induced RAW264.7 cells and rat peritoneal macrophages. Furthermore, IT24 was shown to decrease the expression of microsomal prostaglandin synthetase-1 (mPGES-1) in LPS-induced rat peritoneal macrophages. IT24 did not suppress the phosphorylation and nuclear translocation of proteins in the NF-κB pathway, but it inhibited the phosphorylation of p38/JNK in LPS-stimulated RAW264.7 cells. Additionally, molecular docking analysis indicated that IT24 may directly bind to the mPGES-1 protein. IT24 might inhibit mPGES-1 and the p38/JNK pathway to exert its anti-inflammatory effects and could be also developed as an inhibitor of mPGES-1 to prevent and treat mPGES-1-related diseases, such as inflammatory diseases, and holds promise for further research and drug development. [Display omitted] • Iristectorigenin C is a new compound isolated from Iris tectorum Maxim. • Iristectorigenin C exerts an anti-inflammatory effect by regulating mPGES-1 expression and p38/JNK pathway. • Targeting mPGES-1 without affecting COX-1 and COX-2 is a promising approach for controlling different diseases. [ABSTRACT FROM AUTHOR]

Introduction: Urotensin II (UII) is an important vasoactive peptide involved in the pathogenesis of atherosclerosis. Monocytes/macrophages play important roles in every step of atherosclerosis. Although UII has a chemoattractant effect on monocytes, it is unclear whether UII regulates inflammatory responses in macrophages. The present study sought to explore whether UII can promote leukotriene B4 (LTB4) production by macrophages.Material and Methods: The mRNA expression level of LTB4 and 5-lipoxygenase were determined by real-time polymerase chain reaction. The protein level of LTB4 and 5-lipoxygenase expression was assayed by enzyme-linked immunosorbent assay and Western blot, respectively. Western blot analysis was also employed to determine the phosphorylated forms of Akt. Reactive oxygen species (ROS) level was detected by the fluorescent probe 2',7'-dichlorofluorescin diacetate and fluorescence intensity was measured with a multiwell fluorescence plate reader.Results: Urotensin II promoted LTB4 release and increased 5-lipoxygenase expression in a concentration- and time-dependent manner in RAW264.7 cells. Leukotriene B4 production and 5-lipoxygenase expression were decreased by blocking the UII receptor (UT) with urantide, eliminating ROS with N-acetylcysteine and diphenyliodonium, and inhibiting Akt phosphorylation with LY294002. UII significantly elevated ROS production, whereas urantide, N-acetylcysteine and diphenyliodonium substantially attenuated this effect. UII also enhanced Akt phosphorylation significantly, and this effect was potently inhibited by urantide, N-acetylcysteine, diphenyliodonium and LY294002.Conclusions: Urotensin II may promote 5-lipoxygenase expression and LTB4 release in RAW264.7 macrophages via UT-ROS-Akt pathways. These results indicate that UII may participate in macrophage activation and suggest a potential new mechanism underlying atherosclerosis. [ABSTRACT FROM AUTHOR]

Objectives: This study aimed at characterizing 23 Escherichia coli isolates from various sources and their respective bla SHV-12 -carrying plasmids and sequencing one of these plasmids completely.Methods: Isolates were typed by XbaI-PFGE, MLST and PCR-based phylotyping. Transformed bla SHV-12 -carrying plasmids were examined by replicon typing, S1-nuclease, conjugation, EcoRI-HindIII-BamHI digests and plasmid MLST. Co-located resistance genes and integrons as well as the bla SHV-12 genetic environment were analysed by PCR and sequencing. One IncI1 plasmid was sequenced completely using HiSeq 2500 and gap closure by PCRs and Sanger sequencing.Results: Among the 23 SHV-12-positive E. coli , some isolates from different sources showed the same characteristics: ST23/phylogroup A (human, dog, livestock), ST57/D (wild bird, chicken meat) and ST117/D (chicken meat, chicken). All bla SHV-12 genes were horizontally transferable via 30-120 kb plasmids of incompatibility groups IncI1 ( n  = 17), IncK ( n  = 3), IncF ( n  = 1), IncX3 ( n  = 1) and a non-typeable plasmid. IncK plasmids, indistinguishable in size and restriction patterns, were found in isolates from different sources (ST57/D, meat; ST131/B2, meat; ST57/B1, dog). The IncI1- bla SHV-12 -carrying plasmids were mostly assigned to plasmid ST (pST) 26 and pST3. Three plasmids showed novel pSTs (pST214, pST215). The majority of the IncI1 transformants exhibited resistance to β-lactams, chloramphenicol and streptomycin (in relation with a class 1 integron containing an estX - psp - aadA2 - cmlA1 - aadA1 - qacI gene cassette array), and to tetracycline. A novel bla SHV-12 environment was detected and whole plasmid sequencing revealed a Tn 21 -derived- bla SHV12 -ΔTn 1721 resistance complex.Conclusions: Results from this study suggest that the dissemination of bla SHV-12 genes occurs by vertical (clonal) and horizontal transfer, the latter mainly mediated through IncI1 multidrug-resistance plasmids. [ABSTRACT FROM AUTHOR]

Purpose: Polymorphism in miR-146a (rs2910164) has been reported to be associated with gastric cancer risk in the Chinese population. We aimed at evaluating the relationship between rs2910164 and the clinical characteristics and outcomes in stage IB-III gastric cancer patients treated with adjuvant chemotherapy after surgery. Materials and methods: Ninety-eight patients with stage IB-III gastric cancer treated with surgical resection followed by adjuvant chemotherapy of oxaliplatin and fluoropyrimidines were included in the analysis. Genomic DNA was extracted from peripheral blood sample of all patients. Polymerase chain reaction-based restriction fragment length polymorphism assay was used to determine the genotypes. Results: The 2-year disease-free survival rate was 63%, and the 3-year overall survival (OS) rate was 73.4%. In dominant model, we found that rs2910164 GC + CC (G: guanine, C: cytosine) genotype carriers were less likely to develop lymph node metastasis (P=0.059). The 3-year OS was significantly different for patients with or without lymph node metastasis (89.3% vs 63.7%, P=0.015) and for patients with stage I-III disease (100.0%, 88.6%, and 56.9%; P=0.018). The 3-year OS for GC + CC carriers was significantly higher than for GG carriers (75.1% vs 66.7%, P=0.041). After the multivariant Cox regression analysis, histological grade (P=0.033, relative risk: 5.116, 95% confidence interval: 1.145-22.865) and lymph node status (P=0.031, relative risk: 6.648, 95% confidence interval: 1.191-37.118) were found to be independent prognostic factors for these patients. Conclusion: rs2910164 could be associated with the lymph node metastasis and prognosis of Chinese gastric cancer patients treated with oxaliplatin and fluoropyrimidines after surgical resection. [ABSTRACT FROM AUTHOR]

The halophilic actinomycete genus Streptomonospora, forming a distinct branch in the 16S rRNA gene phylogenetic tree adjacent to the genera Nocardiopsis and Thermobifida, was first proposed by Cui et al. to accommodate the species type Streptomonospora salina. During a biodiversity and taxonomic study on halophilic filamentous actinomycetes from a saline lake in western China, numerous new halophilic actinomycetes strains were isolated. To confirm whether they are members of the genus Streptomonospora, one set of genus-specific oligonucleotides was designed which allows rapid detection of members of the genus Streptomonospora by means of PCR amplification. The genus specificity of these primers was validated with reference strains as well as with wild-type isolates, which exhibited morphological characteristics common to this genus. [ABSTRACT FROM AUTHOR]

Background: Transcription factor 21 (TCF21) is identified as a tumor suppressor in a variety of human tumors. The purpose of the study was to examine its expression tendency and prognostic value in hepatocellular carcinoma (HCC). Methods: Relative expression of TCF21 mRNA in tissue samples from HCC patients and healthy volunteers were detected through quantitative real-time polymerase chain reaction (qRT-PCR) while its protein level was examined via immunohistochemistry analysis. Chi-square test was adopted to assess the association of TCF21 expression with the clinicopathological characteristic of the patients. Then Kaplan–Meier analysis was employed to analyze the function of TCF21 expression on overall survival among HCC patients. Results: Both the mRNA and protein levels of TCF21 were significantly reduced in HCC tissue samples compared with healthy controls (p <.05). Also, its expression was obviously affected by the classification of tissue pathology, metastasis, T stage, N stage and pathological grading. According to Kaplan–Meier analysis, patients with higher expression of TCF21 experienced dramatically longer overall survival time than those with lower expression (log rank test, p <.001). Conclusions:TCF21 expression was decreased in HCC patients and it could act as a prognostic marker. [ABSTRACT FROM AUTHOR]

Introduction: Pseudomonas aeruginosa, an important opportunistic pathogen, carries multiple virulence factors which contribute to its adaptation and pathogenicity. The goal of this study was to characterize the virulence factors among extensively drug-resistant P. aeruginosa. Methodology: In this study, 63 non-duplicated extensively drug-resistant P. aeruginosa clinical isolates were collected from December 2013 to July 2015. Polymerase chain reaction (PCR) was used to analyze the homogeneity and the type III secretion system. Microtiter plate method was performed to evaluate the ability to form biofilms associated to twitching and swimming motilities. Results: High percentage (96.8%) of isolates was sensitive to polymyxin B, while the resistance rate to other antibiotics (amikacin, aztreonam, ceftazidime, ciprofloxacin, gentamicin, imipenem, levofloxacin, meropenem, piperacillin-tazobactam) ranged from 80.9% to 100%. Enterobacterial repetitive intergenic consensus-PCR detected seven major groups with minimal genetic variation. All the isolates carried exoT gene, 96.8% carried exoY, 69.8% carried exoS, and 31.7% carried exoU gene. Biofilm formation was confirmed in all strains, out of which 41.3% formed strong biofilm. Motilities analysis showed heterogeneous diameters ranging from 6.02 to 26.09 mm for swimming and from 7.60 to 23.34 mm for twitching motilities. Conclusions: Our findings revealed that the clinical P. aeruginosa isolates tested are the major invasive types in nature and multiple virulence factors were commonly carried in the extensively drug-resistant strains. [ABSTRACT FROM AUTHOR]

Background: Radiotherapy may represent an alternative treatment modality for cystic echinococcosis (CE), but there is no adequate evidence for it up to now. In this study, we aim to investigate the parasiticidal effects of X-ray on the metacestodes of Echinococcus granulosus in vitro.Methods: Protoscoleces obtained from sheep naturally infected with CE were cultivated in RPMI 1640 medium containing 10% fetal bovine serum (FBS) at 37 °C in 5% CO2. Upon encystation on day 14, the metacestodes were subjected to various intensities of X-ray. Metacestode structures were observed using light microscope and transmission electron microscopy (TEM), and Real-Time PCR was carried out to determine the expression of EgTPX, EgHSP70, EgEPC1 and Caspase-3.Results: On day 14, encystation was noticed in the majority of protoscoleces in the control group. In the X-ray groups, the encystation rate showed significant decrease compared with that of the control group (P < 0.05), especially the groups subjected to a dose of ≥40 Gy (P < 0.01). Light microscope findings indicated the hooklets on the rostellum were deranged in the irradiation group, and malformation was noticed in the suckers in a dose dependent manner. For the TEM findings, the cellular structure of the germinal layer of the cysts was completely interrupted by X-ray on day 7. The expression of EgTPX, EgHSP70, EgEPC1 and Caspase-3 was up-regulated after irradiation, especially at a dose of ≥45Gy (P < 0.05).Conclusions: X-ray showed parasiticidal effects on the metacestodes of E. granulosus. Irradiation triggered increased expression of EgTPX, EgHSP70, EgEPC1 and Caspase-3. [ABSTRACT FROM AUTHOR]

Background: The prognosis of human astrocytoma is poor, and the molecular alterations underlying its pathogenesis still needed to be elucidated. Nucleolar and spindle associated protein 1 (NUSAP1) was observed in several types of cancers, but its role in astrocytoma remained unknown. Methods: The expression of NUSAP1 in astrocytoma cell lines and tissues were measured with western blotting and Real-Time PCR. Two hundred and twenty-one astrocytoma tissue samples were analyzed by immunochemistry to demonstrate the correlation between the NUSAP1 expression and clinicopathological characteristics. 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay, colony formation, transwell matrix penetration assay, wound healing assay and anchorage-independent growth assay were used to investigate the biological effect of NUSAP1 in astrocytoma. An intracranial brain xenograft tumor model was used to confirm the oncogenic role of NUSAP1 in human astrocytoma. Luciferase reporter assay was used to investigate the effect of NUSAP1 on Hedgehog signaling pathway. Results: NUSAP1 was markedly overexpressed in astrocytoma cell lines and tissues compared with normal astrocytes and brain tissues. NUSAP1 was found to be overexpressed in 152 of 221 (68.78%) astrocytoma tissues, and was significantly correlated to poor survival. Further, ectopic expression or knockdown of NUSAP1 significantly promoted or inhibited, respectively, the invasive ability of astrocytoma cells. Moreover, intracranial xenografts of astrocytoma cells engineered to express NUSAP1 were highly invasive compared with the parental cells. With regard to its molecular mechanism, upregulation of NUSAP1 in astrocytoma cells promoted the nuclear translocation of GLI family zinc finger 1 (GLI1) and upregulated the downstream genes of the Hedgehog pathway. Conclusion: These findings indicate that NUSAP1 contributes to the progression of astrocytoma by enhancing tumor cell invasiveness via activation of the Hedgehog signaling pathway, and that NUSAP1 might be a potential prognostic biomarker as well as a target in astrocytoma. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the effects of neuropeptide Y (NPY) on the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor glutamate receptor 2 (GluR2) subunit in epileptiform discharge hippocampal neurons. Hippocampal neurons were harvested from neonatal Sprague-Dawley rats aged <24 h and primarily cultured in vitro. At day 12 following culture, hippocampal neurons were divided into the following groups: Control, Mg2+-free, NPY+Mg2+-free and BIBP3226+NPY+Mg2+-free. The action potential of neurons was measured using the whole cell patch clamp technique in the control, Mg2+-free and NPY+Mg2+-free groups. AMPA current (IAMPA) was detected and peak current density was calculated in each group. Alterations in total protein and phosphorylation of the GluR2 subunit were detected by western blot analysis, and GluR2 mRNA expression levels were detected by reverse transcription-quantitative polymerase chain reaction, in each group. The whole cell patch clamp technique demonstrated an abnormal action potential in the Mg2+-free group. The frequency and amplitude of the action potential were significantly greater in the Mg2+-free group compared with the control group, and significantly reduced in the NPY+Mg2+-free group compared with the Mg2+-free group (P<0.05). In the Mg2+-free group, compared with the control group, peak current density was significantly reduced (P<0.05), GluR2 subunit protein content was slightly reduced (P>0.05), phosphorylation levels of GluR2 subunit were significantly greater (P<0.05) and GluR2 mRNA was significantly reduced (P<0.05). In the NPY+Mg2+-free group, compared with the Mg2+-free group, peak current density was significantly greater (P<0.05), phosphorylation levels of GluR2 subunit were significantly reduced (P<0.05) and GluR2 mRNA expression was significantly greater (P<0.05). In the BIBP3226+NPY+Mg2+-free group, compared with the NPY+Mg2+-free group, peak current density was significantly reduced (P<0.05), phosphorylation levels of GluR2 subunit were significantly greater (P<0.05) and GluR2 mRNA expression was significantly reduced (P<0.05). After 3 h of treatment with Mg2+-free extracellular fluid, epileptiform discharge was detected in the cells. NPY inhibited the discharge and its underlying mechanism may be that epileptiform discharge suppressed the function of the AMPA receptor GluR2 subunit. NPY relieved the inhibition of the GluR2 subunit via the Y1 receptor. This may provide a novel direction for future studies on the pathogenesis and treatment of epilepsy. [ABSTRACT FROM AUTHOR]

The fifth most common cancer worldwide is hepatocellular carcinoma (HCC), which has an annual mortality rate of ~800,000. Although surgical procedures for HCC, such as hepatic resection and liver transplantation, have progressed and the outcomes of patients have improved, HCC is still characterized by frequent recurrence, even after liver transplantation. In the present study the expression of the protein coding gene, S100 calcium binding protein A3 (S100A3), was observed in 62 HCC tissues and tumor-surrounding tissues. The present study indicated that S100A3 activation was involved in tumorigenesis and tumor aggressiveness. The protein and mRNA expression levels of S100A3 in the human HCC cell line (HepG2) were investigated using western blotting and reverse transcription-quantitative polymerase chain reaction analysis, respectively. The function of sodium cantharidinate in inducing HCC cell apoptosis was also investigated. Sodium cantharidinate inhibited the protein and gene expression of S100A3 in HepG2 cells in vitro. These data suggested that S100A3 has an important role in human HCC. The present study indicates that the functional properties of sodium cantharidinate are promising for the development of a novel drug that may control the expression of S100A3 and improve the treatment of human HCC in the near future. [ABSTRACT FROM AUTHOR]

Diabetic cardiomyopathy is a major cause of mortality in patients with diabetes, but specific strategies for preventing or treating diabetic cardiomyopathy have not been clarified yet. MICU1 is a key regulator of mitochondrial Ca2+ uptake, which plays important roles in regulating mitochondrial oxidative phosphorylation and redox balance. To date, however, the significance of MICU1 in diabetic hearts has not been investigated. Here, we demonstrate that MICU1 was downregulated in db/db mouse hearts, which contributes to myocardial apoptosis in diabetes. Importantly, the reconstitution of MICU1 in diabetic hearts significantly inhibited the development of diabetic cardiomyopathy, as evidenced by enhanced cardiac function and reduced cardiac hypertrophy and myocardial fibrosis in db/db mice. Moreover, our in vitro data show that the reconstitution of MICU1 inhibited the apoptosis of cardiomyocytes, induced by high glucose and high fat, through increasing mitochondrial Ca2+ uptake and subsequently activating the antioxidant system. Finally, our results indicate that hyperglycemia and hyperlipidemia induced the downregulation of MICU1 by inhibiting Sp1 expression in diabetic cardiomyocytes. Collectively, our findings provide the first direct evidence that upregulated MICU1 preserves cardiac function in diabetic db/db mice, suggesting that increasing the expression or activity of MICU1 may be a pharmacological approach to ameliorate cardiomyopathy in diabetes. [ABSTRACT FROM AUTHOR]

Background: Although chemotherapy represents a predominant anti-cancer therapeutic modality, drug treatment efficacy is often limited due to the development of resistant tumor cells. The pregnane X receptor (PXR) affects chemotherapeutic effects by regulating targets involved in drug metabolism and transportation, but the regulatory mechanism is poorly understood. Methods: Oxaliplatin (L-OHP) content in tumor cells was analyzed by mass cytometry. The roles of PXR on cancer cell proliferation, apoptosis and tumor growth with L-OHP-treated were investigated by MTS, colony formation, flow cytometry and xenograft tumor assays. Luciferase reporter, Chromatin-immunoprecipitation and Site-directed mutagenesis were evaluated the mechanisms. The PXR and multidrug resistance-related protein 3 (MRP3) expressions were examined by western blot, RT-PCR or immunohistochemistry of TMA. Kaplan-Meier and Cox regression were adopted to analyze the prognostic value of PXR in colorectal cancer (CRC). Results: PXR over-expression significantly increased oxaliplatin (L-OHP) transport capacity with a reduction of its content and repressed the effects of L-OHP on tumour cell proliferation and apoptosis. Conversely, PXR knockdown augments L-OHP-mediated cellular proliferation and apoptosis. Moreover, PXR significantly reduced the therapeutic effects of L-OHP on tumor growth in nude mice. Further studies indicated a positive correlation between PXR and MRP3 expression and this finding was confirmed in two independent cohorts. Significantly increased MRP3 expression was also found in PXR over-expressing cell lines. Mechanistically, PXR could directly bind to the MRP3 promoter, activating its transcription. The PXR binding sites were determined to be at -796 to -782bp (CTGAAGCAGAGGGAA) and the key binding sites were the "AGGGA" (-787 to -783bp) on the MRP3 promoter. Accordingly, blockade of MRP3 diminishes the effects on drug resistance of PXR. In addition, PXR expression is significantly associated with poor overall survival and represents an unfavorable and independent factor for male or stage I + II CRC patient prognosis. Conclusions: PXR is a potential biomarker for predicting outcome and activates MRP3 transcription by directly binding to its promoter resulting in an increased L-OHP efflux capacity, and resistance to L-OHP or platinum drugs in CRC. Our work reveals a novel and unique mechanism of drug resistance in CRC. [ABSTRACT FROM AUTHOR]

Clubroot caused by Plasmodiophora brassicae is an increasingly important soilbome disease in China. The host range of P. brassicae was investigated with 30 cruciferous plants, including 16 crop species, 9 ornamentals, and 5 weeds in field and pot-cultured conditions. In the field, 17 species from five genera produced visible galls, and these included radish, Capsella bursa-pastoris, Orychophragmus violaceus, Sinapis alba, and 13 Brassica crops. In pot-cultured conditions, an additional 13 plant species (11 genera) were detemiined to be hosts of P. brassicae. Five common weeds were found to be hosts of P. brassicae, including C. bursa-pastoris, Lepidium apetalum, Descurainia sophia, S. alba, and Thellungiella salsuginea. The infection of these plants was confirmed via polymerase chain reaction (PCR) with primers specific to P. brassicae. No galls were found on Mattliiola incana roots in the field or in pots and no resting spores of P. brassicae were observed in M. incana roots, although P. brassicae was detected in M. incana roots via PCR. Microscopic examination revealed infection only in the root hairs of M. incana roots. These results suggested that M. incana was highly resistant to P. brassicae in China and could be developed as a bait crop. In total, 297 accessions of oilseed rape were tested in the field, and 3 accessions of Brassica napus and 1 accession of B. juncea were found to be highly resistant to clubroot disease. These resistant resources provide options for managing clubroot in P. brassicae-infested fields. [ABSTRACT FROM AUTHOR]

Aim: The aimof this studywas to explore the correlation between geneticmutations in matrix metalloproteinase-10 (MMP-10) and susceptibility to pelvic organ prolapse (POP). Material and Methods: From September 2011 to December 2013, 263 subjects were recruited, including 91 patients with POP (case group) and 172 non-POP patients (control group). Total MMP-10 concentrations in serum were measured by enzyme-linked immunosorbent assay. The genotyping ofMMP-10 was achieved by quantitative real-time polymerase chain reaction. All data were analyzed with SPSS 18.0. Results: We found that parity, menopause, history of total hysterectomy, and family history of POP were all significantly higher in the POP group than in the control group (P=0.017, P=0.046, P=0.0029 and P<0.001, respectively). Serum MMP-10 levels were obviously higher in the POP group than in the control group (P<0.05). In addition, there was a statistically significant difference between the two groups in the distribution frequency of the MMP-10 (rs17435959G/C) genotype (P<0.05). However, the distribution frequency of the MMP-10 (rs17293607C/T) genotype between the two groups showed no significant differences (P>0.05). Furthermore, the patients with parity>2 and postmenopausal women had elevated serum MMP-10 levels, and the patients with parity>2 and postmenopausal women who carried the G/C+C/C genotype in the MMP-10 gene had an increased risk of POP. Conclusion: We support the view that the rs17435959 polymorphism of the MMP-10 gene may be associated with an increased risk of POP. [ABSTRACT FROM AUTHOR]

Background: Malaria is one of the most serious vector-borne diseases in the world. Vector control is an important measure for malaria prevention and elimination. However, this strategy is under threat as disease vectors are developing resistance to insecticides. Therefore, it is important to monitor mechanisms responsible for insecticide resistance. In this study, the presence of G119S mutation in the acetyl cholinesterase-encoding gene (ace-1) was investigated in nine Anopheles sinensis populations sampled across Guangxi Zhuang Autonomous Region China. Methods: PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method was used to genotype each individual adult of An. sinensis. Direct sequencing of PCR products was performed to verify the accuracy of PCR-RFLP genotyping result. Population genetics analysis was conducted using Genepop programme. Results: The frequencies of susceptible homozygotes, heterozygotes and resistant homozygotes in the nine populations ranged between 0-0.296, 0.143-0.500 and 0.333-0.857, respectively. Overall, a high frequency (0.519-0.929) of mutant 119S allele was observed and the genotype frequency of the ace-1 gene of An. sinensis was at Hardy-Weinberg equilibrium in each of the nine examined populations. Conclusion: The G119S mutation has become fixed and is widespread in An. sinensis field populations in Guangxi, China. These findings are useful in helping design strategies for An. sinensis control. [ABSTRACT FROM AUTHOR]

Background: Subgroups A, B, E and J are the major subgroups of avian leukosis virus (ALV) infecting chickens. ALV infection has become endemic in China and has a significant negative effect on the poultry industry. Consequently, there is an urgent need for a specific, sensitive and rapid method for diagnosis and eradication of ALV. Therefore we developed a simple and rapid real-time loop-mediated isothermal amplification (LAMP) reaction for the timely detection of the common ALV subgroups, whereby the amplification can be obtained in 35 min under isothermal conditions at 63 °C, ability to specific, sensitive and rapid detect all the common ALV subgroups. Methods: A set of four specific primers was designed to target the sequences of the pol gene of ALV, and the loop-mediated isothermal amplification (LAMP) assay were developed and compared with PCR and virus isolation methods. Results: The results from specificity of the LAMP assay showed that only target ALVs DNA was amplified. The LAMP assay demonstrated a sensitivity of 20 copies/reaction of ALV DNA, which was 10 times higher than the conventional PCR measurement. To further evaluate the reliability of the method, the assay was evaluated with ALV DNA from a panel of 81 clinical samples suspected of ALV infection. The results verify that the LAMP method was more sensitive than the conventional PCR and virus isolation method. Conclusion: In conclusion, the developed LAMP assay was a simple, inexpensive, sensitive method for the rapid detection of the most common subgroups of ALV, and it provided a useful and practical tool in the eradication program for ALV in the poultry industry. [ABSTRACT FROM AUTHOR]

STUDY QUESTION Can avian influenza A (H7N9) virus be transmitted between unrelated individuals in a hospital setting? METHODS An epidemiological investigation looked at two patients who shared a hospital ward in February 2015, in Quzhou, Zhejiang Province, China. Samples from the patients, close contacts, and local environments were examined by real time reverse transcriptase (rRT) polymerase chain reaction (PCR) and viral culture. Haemagglutination inhibition and microneutralisation assays were used to detect specific antibodies to the viruses. Primary outcomes were clinical data, infection source tracing, phylogenetic tree analysis, and serological results. STUDY ANSWER AND LIMITATIONS A 49 year old man (index patient) became ill seven days after visiting a live poultry market. A 57 year old man (second patient), with a history of chronic obstructive pulmonary disease, developed influenzalike symptoms after sharing the same hospital ward as the index patient for five days. The second patient had not visited any poultry markets nor had any contact with poultry or birds within 15 days before the onset of illness. H7N9 virus was identified in the two patients, who both later died. Genome sequences of the virus isolated from both patients were nearly identical, and genetically similar to the virus isolated from the live poultry market. No specific antibodies were detected among 38 close contacts. Transmission between the patients remains unclear, owing to the lack of samples collected from their shared hospital ward. Although several environmental swabs were positive for H7N9 by rRT-PCR, no virus was cultured. Owing to delayed diagnosis and frequent hospital transfers, no serum samples were collected from the patients, and antibodies to H7N9 viruses could not be tested. WHAT THIS STUDY ADDS Nosocomial H7N9 transmission might be possible between two unrelated individuals. Surveillance on patients with influenza-like illness in hospitals as well as chickens in live poultry markets should be enhanced to monitor transmissibility and pathogenicity of the virus. FUNDING, COMPETING INTERESTS, DATA SHARING Funding support from the Program of International Science and Technology Cooperation of China (2013DFA30800), Basic Work on Special Program for Science and Technology Research (2013FY114600), National Natural Science Foundation of China (81402730), Special Program for Prevention and Control of Infectious Diseases in China (2013ZX10004218), US National Institutes of Health (1R01-AI108993), Zhejiang Province Major Science and Technology Program (2014C03039), and Quzhou Science and Technology Program (20111084). The authors declare no other interests and have no additional data. [ABSTRACT FROM AUTHOR]

Background: In our study, we investigated whether circulating T follicular helper (Tfh) and the related cytokines are involved in human cystic echinococcosis (CE).Methods: A total of 64 patients with CE and 30 healthy controls were enrolled in this study. Percentages of CCR7(lo)PD-1(hi) cells within CXCR5(+) CD4(+) T cells (circulating Tfh cells) were detected by flow cytometry. Levels of IL-21 and IL-4 in peripheral blood were detected by cytometric bead array. The mRNA expression of IL-21, IL-4, Bcl-6, and Blimp-1 in peripheral blood mononuclear cells (PBMCs) were measured by real-time PCR. Levels of IgG1, IgG2, IgG3, and IgG4 in the patients' sera were measured using enzyme-linked immunosorbent assay.Results: Percentages of circulating Tfh cells were significantly increased in the CE1, CE2, and CE3 groups (p < 0.05). The concentrations of IL-21 and IL-4 in the serum were significantly increased in CE1, CE2, and CE3 groups (p < 0.05). IL-21 was positively correlated with circulating Tfh cells in CE3 group (r = 0.779, p < 0.05). The mRNA levels of IL-21, IL-4, and Bcl-6 were increased in CE1, CE2, and CE3 groups. Levels of IgG1 and IgG4 in patients' sera were increased in CE1, CE2, and CE3 groups. Levels of IgG2 and IgG3 were increased in CE4-5 group. Additionally, after stimulation with hydatid fluid in vitro, the levels of circulating Tfh cells, IL-21 and IL-4 in PBMCs isolated from CE patients were significantly increased (p < 0.05).Conclusions: The levels of circulating Tfh and related cytokines were significantly increased in CE patients, suggesting that they are involved in human CE. [ABSTRACT FROM AUTHOR]

The interleukin (IL)-23/IL-17 pathway is considered to be important in the pathogenesis of Crohn's disease (CD). The present study aimed to evaluate the effects of targeting the IL-23/IL-17 pathway using the anti-IL-23p19 monoclonal antibody (mAb) on 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced CD rats. A total of 60 Sprague-Dawley rats were randomly divided into a control group, model group and an anti-IL-23p19 mAb treatment group (administered intramuscularly every week at a dose of 1 ml/mg). Disease activity index (DAI), colon macroscopic damage index (CMDI) and tissue damage index (TDI) were then evaluated. The mRNA expression of IL-23p19, p40 (IL-23/12), retinoic acid-related orphan receptor-γt (ROR-γt) and IL-17 in colonic tissues were detected by reverse transcription-polymerase chain reaction and levels of serum IL-23p19, p40, ROR-γt and IL-17 were measured using an enzyme-linked immunosorbent assay. Anti-IL-23p19 mAb was found to effectively attenuate colonic inflammation demonstrated by reduced DAI, CMDI and TDI scores, improvement in pathological evaluation and downregulation of expression levels of IL-23p19, p40 (IL-23/12), ROR-γt and the downstream proinflammatory cytokine, IL-17. Anti-IL-23p19 mAb attenuated TNBS-induced CD in model rats. The possible underlying mechanisms may be associated with inhibition of the IL-23/IL-17 pathway by inhibiting the expression of IL-23p19 and downregulating the downstream proinflammatory cytokine IL-17. Targeting the IL-23/IL-17 pathway may be a relevant and realistic therapeutic approach for the development of additive and alternative treatments to the biologics currently available in the treatment of CD. [ABSTRACT FROM AUTHOR]

Macrophages play a crucial role in rheumatoid arthritis (RA). Their activation is the initial step of RA. This study was designed to detect the effects of total flavonoids from Litsea coreana Levl. (TFLC) on the complete Freund's adjuvant-induced (CFA-induced) arthritis (AA) in rats and to explore whether inflammatory cytokines were induced by the IRE1/mTORC1/TNF-α-dependant mechanism in peritoneal macrophages. In vivo, our data indicated that TFLC (100, 200 mg/kg, i.g. x 10 days) could significantly suppress secondary paw swelling and serum levels of TNF-α and IL-1β. Histopathological figures showed that TFLC treatment improved the morphologic changes of articular cartilages and synovium. Results of RT-PCR and western blotting demonstrated that TFLC suppressed expression of 78-KD glucose regulated protein (GRP78), X-box binding protein 1 (XBP1), mTOR complex 1 (mTORC1) and TNF-α in peritoneal macrophages of AA rats. Collectively, these results indicate that TFLC is able to ameliorate adjuvant-induced arthritis in a dose-dependent manner by suppressing the IRE1/mTORC1/TNF-α-regulated inflammatory response initiated in peritoneal macrophages. [ABSTRACT FROM AUTHOR]

Recent studies have shown that DNA methylation in the promoter of microRNA-375 (miR-375) downregulates its expression during tumorigenesis. However, it is not known if CpG methylation of the miR-375 promoter also has a role in the pathogenesis of type 2 diabetes mellitus (T2DM). In this study, the expression level and CpG methylation status of miR-375 in patients with T2DM were analyzed. Plasma samples from 100 patients with T2DM and 100 healthy controls with normal glucose tolerance (NGT) were collected. The plasma levels of miR-375 were examined using quantitative polymerase chain reaction (qPCR) and the methylation status of 17 CpG sites in the promoter of the miR-375 were determined using Mass ARRAY spectrometry. The plasma levels of miR-375 were found to be upregulated in patients with T2DM compared with controls with NGT (P<0.05). Overall, the methylation levels of the miR-375 promoter in patients with T2DM were not significantly different compared with controls with NGT; however, further studies revealed that four of the eight analyzed individual CpG units within the amplicon were significantly hypomethylated in T2DM samples compared with the NGT samples. This study demonstrated for the first time, to the best of our knowledge, that miR-375 is over expressed in plasma in patients with T2DM, and this may be used as a novel biomarker to distinguish between patients with T2DM and healthy individuals. It was also demonstrated in this study that the miR-375 promoter is hypomethylated, in patients with T2DM, which may regulate the expression of miR-375 and contribute to the pathogenesis of T2DM. [ABSTRACT FROM AUTHOR]

Background Reduced beta2-glycoprotein I (beta2-GPI) is a free thiol-containing form of beta2-GPI that displays a powerful effect in protecting endothelial cells from oxidative stress-induced cell death. The present study aims to investigate the effect of beta2-GPI or reduced beta2-GPI on ox-LDL-induced foam cell formation and on cell apoptosis and to determine the possible mechanisms. Methods The RAW264.7 macrophage cell line was selected as the experimental material. Oil red O staining and cholesterol measurement were used to detect cholesterol accumulation qualitatively and quantitatively, respectively. Flow cytometry was used to detect cell apoptosis. Real-time quantitative PCR was used to detect the mRNA expression of the main proteins that are associated with the transport of cholesterol, such as CD36, SRB1, ABCA1 and ABCG1. Western blot analysis was used to detect the protein expression of certain apoptosis-related proteins, such as caspase-9, caspase-3, p38 MAPK/p-p38 MAPK and JNK/p-JNK. Results Beta2-GPI or reduced beta2-GPI decreased ox-LDL-induced cholesterol accumulation (96.45 ± 8.51 μg/mg protein vs. 114.35 ± 10.38 μg/mg protein, p < 0.05;74.44 ± 5.27 μg/mg protein vs. 114.35 ± 10.38 μg/mg protein, p < 0.01) and cell apoptosis (30.00 ± 5.10% vs. 38.70 ± 7.76%, p < 0.05; 20.66 ± 2.50% vs. 38.70 ± 7.76%, p < 0.01), and there are significant differences between beta2-GPI and reduced beta2-GPI (p < 0.05). Reduced beta2-GPI decreased the ox-LDL-induced expression of CD36 mRNA and ABCA1 mRNA (p < 0.05), as well as CD36, cleaved caspase-9, cleaved caspase-3, p-p38 MAPK and p-JNK proteins (p < 0.05 or p < 0.01). Beta2-GPI did not significantly decrease the expression of ABCA1 mRNA and the p-p38 MAPK protein. Conclusions Both beta2-GPI and reduced beta2-GPI inhibit ox-LDL-induced foam cell formation and cell apoptosis, and the latter exhibits a stronger inhibition effect. Both of these glycoproteins reduce the lipid intake of macrophages by downregulating CD36 as well as protein expression. Reduced beta2-GPI inhibits cell apoptosis by reducing the ox-LDL-induced phosphorylation of p38 MAPK and JNK, and the amount of cleaved caspase-3 and caspase-9. Beta2-GPI does not inhibit the ox-LDL-induced phosphorylation of p38 MAPK. [ABSTRACT FROM AUTHOR]

Purpose: In view of the numerous clinical observations and laboratory studies that suggest a critical role for the spleen in immune thrombocytopenia (ITP) pathophysiology, we aimed to characterize Th1-associated chemokine receptors CXCR3 and CCR5 and Th2-associated chemokine receptor CCR3 in spleens of ITP patients and assess the significance of their differential expression in the clinical setting. Methods: The histopathology of spleens was observed using hematoxylin-eosin staining (HE), and the positive rate of CXCR3, CCR5 and CCR3 expression in spleens of 24 ITP patients and 12 patients with traumatic splenic rupture as normal controls was detected by immunohistochemistry using the SP method. CXCR3, CCR5 and CCR3 protein expression was analyzed by Western blot and mRNA levels were investigated by real-time polymerase chain reaction (RT-PCR). Results: Reactive hyperplasia could be seen in follicles of the white pulp, the germinal central zone was enlarged and the marginal zone was thickened, the central arteries were thickened and fibrotic, and the density of the capillary vessel was increased in ITP patients. ITP group displayed a higher rate of expression of Th1-associated chemokine receptors CXCR3 and CCR5 (83.3 % vs. 75 %, 100 % vs. 83.3 %) but lower rate of expression of Th2-associated chemokine receptor CCR3 (50 % vs. 66.7 %) compared with the controls ( P < 0.05, respectively). Western blot analysis revealed that CXCR3 and CCR5 protein expression was significantly increased in ITP patients while CCR3 was significantly reduced ( P < 0.05, respectively). Meanwhile, ITP patients displayed increased mRNA levels of CXCR3 and CCR5 but decreased gene expression of CCR3 ( P < 0.05, respectively). Conclusion: The data suggested that the abnormal expression of Th1/Th2 chemokine receptors may participate in splenic immune disorder in patients with ITP. Using corresponding inhibitors may inhibit Th1-dominant expression and mitigate the progress of the disease. [ABSTRACT FROM AUTHOR]

This study investigated the effect of Angelica sinensis polysaccharides (APS-3c) on rat osteoarthritis (OA) model in vivo and rat interleukin-1-beta- (IL-1β-) stimulated chondrocytes in vitro. APS-3c was administrated into rat OA knee joints and had protective effects on rat OA cartilage in vivo. Primary rat articular chondrocytes were cotreated with APS-3c and IL-1β in vitro. 2-50 μg/mL APS-3c had no effect on chondrocytes viability, whereas it increased the proteoglycans (PGs) synthesis inhibited by IL-1β. Microarray analysis showed that the significant changes were concentrated in the genes which were involved in PGs synthesis. RT-PCR confirmed that treatment with APS-3c increased the mRNA expression of aggrecan and glycosyltransferases (GTs) inhibited by IL-1β but did not affect the mRNA expression of matrix-degrading enzymes. These results indicate that APS-3c can improve PGs synthesis of chondrocytes on rat OA model in vivo and IL-lβ-stimulated chondrocytes in vitro, which is due to the promotion of the expression of aggrecan and GTs involved in PGs synthesis but not the inhibition of the expression of matrix-degrading enzymes. Our findings suggest the clinical relevance of APS-3c in the prospective of future alternative medical treatment for OA. [ABSTRACT FROM AUTHOR]

Background: Findings from previous studies linking brain-derived neurotrophic factor (BDNF) and schizophrenia are inconsistent and few studies have assessed the relationship between BDNF C270T gene polymorphisms and the clinical and cognitive symptoms of schizophrenia. Aim: Compare the prevalence of the BDNF C270T gene polymorphisms between patients with schizophrenia and controls and, in the patients, assess the relationship of genotypes to the severity of symptoms. Methods: BDNF C270T genotype and allele frequency were measured using Polymerase Chain Reaction methods in 224 drug-free patients with schizophrenia and 220 controls. Psychotic symptoms were assessed using the Positive and Negative Syndrome Scale (PANSS), and cognitive functioning was assessed using the Wisconsin Card Sorting Test (WCST) and the Trail Making Test (TMT). In the patient group, differences in severity of symptoms across the three genotypes (i.e., C/C, C/T, and T/T) of C270T were assessed using one-way analysis of variance. Results: The frequency of the T allele was much higher in patients than in controls(15.6% vs. 4.3%, X2=31.47, p<0.001) and the C/T genotype was more common among patients than controls (27.7% vs. 7.7%, X2=34.93, p<0.001). Compared to controls, patients performed poorly on all the cognitive tests, but there were no significant differences in the cognitive measures between patients with the three different genotypes. The total PANSS score, the PANSS negative symptoms subscale score, and the PANSS general psychopathology subscale score were not significantly different between the three groups of patients. However, the PANSS positive symptoms subscale score showed a small, statistically significant elevation in the severity of positive symptoms in the C/T genotype compared to the C/C genotype. Conclusion: We confirm previous findings about differences in the prevalence of the BDNF C270T gene polymorphisms in schizophrenia, but do not find strong evidence of a relationship between different genotypes and the severity of the clinical or cognitive symptoms of schizophrenia. Clinical and cognitive symptoms in schizophrenia fluctuate over the course of the illness and with treatment, so stable, individual-specific measures of these parameters (that is, traits) need to be identified before it will be possible to definitively assess their relationship to different genotypes.. [ABSTRACT FROM AUTHOR]

Endophytic actinobacteria colonize internal tissues of their host plants and are considered as a rich and reliable source of diverse species and functional microorganisms. In this study, endophytic actinobacterial strain YIM 63111 was isolated from surface-sterilized tissue of the medicinal plant Artemisia annua. We identified strain YIM 63111 as a member of the genus Pseudonocardia. A. annua seedlings grown under both sterile and greenhouse conditions were inoculated with strain YIM 63111. The growth of A. annua seedlings was strongly reduced when YIM 63111 was inoculated at higher concentrations under sterile conditions. However, no growth inhibition was observed when A. annua was grown under greenhouse conditions. Using an enhanced green fluorescent protein (EGFP) expressing YIM 63111 strain, we also observed the endophytic colonization of A. annua seedling using confocal laser-scanning microscopy. The transcription levels of the key genes involved in artemisinin biosynthesis were investigated using real time RT-PCR, revealing that cytochrome P450 monooxygenase (CYP71AV1) and cytochrome P450 oxidoreductase (CPR) expression were up-regulated in A. annua upon inoculation with strain YIM 63111 under certain conditions. The up-regulation of these genes was associated with the increased accumulation of artemisinin. These results suggest that endophytic actinobacteria effectively stimulate certain plant defense responses. Our data also demonstrate the use of Pseudonocardia sp. strain YIM 63111 as a promising means to enhance artemisinin production in plants. [ABSTRACT FROM AUTHOR]

Abstract: Ethnopharmacological relevance: Miaoyao Fanggan Sachets (MFS) has long been used as a folk medicine for the prevention of influenza in Southeast of Guizhou Province, China. However, the precise immunological mechanisms by which MFS confers protection have not been defined. Study aim: To explore the effects of MFS on innate immune system responses using a cold stress-induced immune impairment model as a means of examining MFS-mediated influenza prevention. We investigated the effects of MFS on toll-like receptor 2 and 4 (TLR2/4) gene and protein expression levels and on the percentage of NKp46+ cells present in serum. No overt toxicity was observed following continuous administration of MFS at high doses. Methods: Kunming male mice (n=40) were randomly divided into 4 groups consisting of the continuous inhalation Sachet group, Yu-Ping-Feng powder (YPF-P) gavage positive control group, discontinuous inhalation MFS group and untreated controls. After 4 weeks, mice were sacrificed and lungs harvested. The expression of toll-like receptors 2 and 4 (TLR2/4) gene and protein levels was assessed using real-time polymerase chain reaction (RT-PCR) and Western blot analyses, respectively. An additional 60 Kunming mice were randomly divided into 6 groups comprised of a blank control group, continuous MFS inhalation group, an immune-compromised continuous MFS inhalation group, an immuno-compromised group, an immune-compromised MFS discontinuous inhalation group and an immune-compromised positive control group. Immune suppression was induced by cold stress (4°C/4h daily for 3 days) and mice were treated with MFS or YPF-P before cold stress treatments. Immuno-compromised mice were treated with MFS continuously or intermittently, or treated with YPF-P. Blood samples were collected and examined for natural killer cells based on positive NKp46 staining. The isorhamnetin associated with MFS-induced immune modulation was obtained from ‘wo ga le’ which is considered to be a major component of MFS, and analyzed by HPLC. Results: Mice continuously inhaling MFS showed a moderate increase in TLR2/4 mRNA and protein levels compared to mice in the control and discontinuous inhalation groups. MFS significantly increased the TLR2/4 expression in a dose-dependent manner. Furthermore, there was also a slightly significant increase in the number of NKp46+ cells in the continuous inhalation group compared to controls and discontinuous inhalation group. Pretreatment with MFS partially prevented cold stress-induced inhibition of NKp46+ cells. HPLC analysis of the ‘wo ga le’ associated with immune function identified the major component to be isorhamnetin. Conclusions: Taken together, these data suggested that MFS significantly enhanced TLR2/4 expression levels and the number of NKp46+ cells in mice and moderately affected innate immune responses associated with protection against influenza, suggesting that isorhamnetin in the MFS enhanced innate immune potency. The use of MFS for the prevention of various respiratory tract infections can be attributed to its antimicrobial properties. In a pilot study, a large quantity (40g) was administered over a prolonged period did not produce apparent toxicity. [Copyright &y& Elsevier]

Objectives: Seizure is a common presenting symptom of primary brain tumors. There are no published studies regarding the roles of MicroRNA (miRNA) in tumor-related epilepsy. The authors set out to correlate miR-196b expression in low-grade glioma patients with pre-operative seizures and post-operative seizure control. Methods: Twenty-three patients with WHO grade II astrocytomas and 83 similar patients for independent validation were included. Follow-up visits regarding seizure prognosis were scheduled at 6 months. MiRNA profiling was used to identify differentially expressed miRNAs. The most important miRNA was determined by quantitative reverse-transcriptase polymerase chain reaction (q-PCR) in the validation cohort. Gene ontology (GO) analysis and whole genome mRNA profiling was performed to investigate the underlying biological processes. Results: Array results showed that 30 miRNAs were overexpressed and 10 miRNAs were underexpressed (with more than 2 fold change) in patients with pre-operative seizures. MiR-196b was validated in the independent validation cohort. Patients with good seizure prognosis exhibited low levels of miR-196b expression compared with those who had poor seizure prognosis in the group without pre-operative seizures. Biological processes that relate to transcription and cell cycles were over-represented in the miR-196b-associated gene expression signature. MiR-196b-associated gene expression profiling was characterized by enrichment of genes usually involved in cell proliferation. Conclusions: We have provided the first evidence that expression of miR-196b was associated with the occurrence of pre-operative seizures in low-grade gliomas, and may predict seizure prognosis in patients without pre-operative seizures. Targeted treatments that decrease endogenous levels of miR-196b might represent novel therapeutic strategies. [ABSTRACT FROM AUTHOR]

Objective: To determine the role of DNA-binding inhibitors and differentiation protein in the regulation of gastric-cancer cell growth and sensitivity to cisplatin. Methods: An ID1 protein (ID1)-expression vector was constructed for gene transfection. Western blot, methylthiazol tetrazolium (MTT) assay, and flow cytometric testing were used in gastric-cancer cells to assess ID1 expression, cell viability, and distribution of cell-cycle stages, respectively. Results: ID1 complementary DNA (cDNA) and small hairpin RNA (shRNA) effectively induced and reduced ID1 expression, respectively, in gastric cancer cells. Knockdown of ID1 expression suppressed tumor-cell viability and reduced colony formation, whereas overexpression of ID1 increased colony formation. Further, we found that ID1 overexpression antagonized the effects of cisplatin-induced apoptosis in gastric-cancer cells. Conclusion: The data from the current study demonstrate the role of ID1 in inducing gastric cancer-cell proliferation and colony formation, which suggests that ID1 may be a novel target for gastric-cancer treatment. [ABSTRACT FROM AUTHOR]

It has been shown that bridging integrator 1 (BIN1) can interact with c-myelocytomatosis (c-Myc) oncoprotein in cancer. However, the role of BIN1 in hepatocellular carcinoma (HCC) is not clear. In the present study, we investigated the expression and prognostic role of BIN1 in primary HCC and evaluated the function of BIN1 in hepatocarcinogenesis. Using real-time polymerase chain reaction and Western blot analysis, we found significantly decreased expression of BIN1 in primary HCC tumor tissues (n = 42) compared with adjacent normal tissues and in HCC cell lines. Immunohistochemistry analysis also found decreased BIN1 expression in HCC tumor tissues (n = 117). In clinicopathological analysis, loss of BIN1 expression correlated significantly (P < 0.05) with differentiation scores and tumor size. Importantly, decreased expression of BIN1 in tumors was found to be closely associated with a poor prognosis, and we conclude that BIN1 was an independent prognostic factor in a multivariate analysis. In mechanistic studies, restoring BIN1 expression in BIN1-null HCC cells significantly inhibited cell proliferation and colony formation and induced apoptosis of HCC cells. Furthermore, we found that BIN1 overexpression could significantly suppress the motility and invasion of HCC cells in vitro. Our results indicate that BIN1 may function as a potential tumor suppressor and serve as a novel prognostic marker in HCC patients. The BIN1 molecule might play an important role in tumor growth, cell motility and invasion. Modulation of BIN1 expression may lead to clinical applications of this critical molecule in the control of hepatocellular carcinoma as well as in early and effective diagnosis of this aggressive tumor. [ABSTRACT FROM AUTHOR]

Abstract: Thalassemia is the commonest inherited autosomal recessive disorders of hemoglobin in southern China. We developed and evaluated a reverse dot blot (RDB) assay combined with flow-through hybridization technology platform for the rapid and simultaneous identification of 5 types of α-thalassemia and 16 types of β-thalassemia common in Chinese. Reliable genotyping of wild-type and thalassemic genomic DNA samples was achieved by means of a gene chip on which allele-specific oligonucleotide probes were immobilized on a nylon membrane. This method involved two multiplex PCR amplification systems of α-thalassemia and β-thalassemia and one time of hybridization. The whole procedure starting from blood sampling to the identification of thalassemia genotype required less than 4h. The diagnostic reliability of this reverse dot blot assay was evaluated on 427 samples (387 cases of thalassemia and 40 healthy persons) by using direct DNA sequence analysis and gap-PCR in a blind study. These samples included 377 cases of blood, 7 cases of amniotic fluid, 18 cases of chorionic villus, and 25 cases of cord blood. The RDB gene chip was in complete concordance with the reference method. The reverse dot blot assay was a simple, rapid, accurate, and cost-effective method to identify common thalassemia genotypes in the Chinese population. [Copyright &y& Elsevier]

Background: The storage times of formalin-fixed paraffin embedded (FFPE) colorectal cancer (CRC) tissues can differ. In this study we investigated the effects of storage time of CRC tissue on K-ras detection. Methods: Forty fresh tissues and 120 FFPE CRC tissues were selected for this study. The storage time of the FFPE samples was 1 to 2 years, 2 to 5 years, and 5 to 10 years with 40 samples in each group. DNA was extracted from these samples and exon 1 of K-ras was sequenced. Results: The concentration or quantity of DNA extracted from the fresh tissues and FFPE tissues did not change. The K-ras detection rate in fresh tissues, tissues stored for 1 to 2 years, tissues stored for 2 to 5 years, and tissues stored for 5 to 10 years was 100%, 92.5%, 87.5%, and 77.5%, respectively. Conclusions: When the storage time was prolonged, the K-ras detection rate of CRC tissue decreased. [ABSTRACT FROM AUTHOR]

Reg family proteins such as Reg1 and islet neogenesis-associated protein (INGAP) have long been implicated in the growth and/or neogenesis of pancreatic islet cells. Recent reports further suggest similar roles to be played by new members such as Reg2, Reg3αα, and Reg3ββ. We have studied their age-, isoform-, and tissue-specific expressions. RNA and protein were isolated from C57BL/6 mice aged 7, 30, and 90 days. Using real-time polymerase chain reaction, the levels of Reg gene expression in the pancreas were 20--600-fold higher than that in other tissues (≫≫duodenum>stomach>liver); gene expression of Reg2, Reg3αα, and Reg3ββ was age dependent as it was hardly detectable at day 7, increased drastically at day 30, and significantly decreased at day 90; the levels of pancreatic proteins displayed similar age-dependent variations. Using dual-labeled immunofluorescence, Reg2, Reg3αα, and Reg3ββ were abundantly expressed in most acinar cells of the pancreas, in contrast to INGAP which exhibited stepwise increases from day 7 to day 90 and colocalized with the αα-cells. These new Reg genes were mainly expressed in the pancreas, with clear age-dependent and isoform-specific patterns. [ABSTRACT FROM AUTHOR]