Objective: To establish a multiplex PCR-microarray method for detecting important enteropahogens.Methods: Uniplex and multiplex PCR were performed to obtain the best primer sets for identifying the target bacteria at species and multi-species level.Fluorescent dyes were mixed into PCR reaction to determine whether it can affect the efficiency of amplification.To improve the efficiency of microarray,a 35 pairs primer-labeling system was optimized based on the hybridization results to find the best combination to avoid false negative results.Results: Specific PCR products were all obtained using species-specific primer sets.More preferential amplification may happen when more primer pairs were added to the reaction.The hybridization results showed a positive association between the efficiency of multiplex-PCR and signal intensity.Conventional PCR yielded more products than fluorescent dyes labeled PCR.Thirty-five primers were divided into three different combinations to label target respectively,hybridization results showed a high specificity.Conclusion: Mixing fluorescent dyes into PCR may reduce the efficiency of amplification and hybridization,but may have no effect on the analysis of hybridization results.The hybridization efficiency of microarray depends on the amplification efficiency of multiplex PCR.For microarray target labeling,three primer sets could be used to avoid negative hybridization led by preferential amplification of multiplex-PCR.It indicates that the multiplex PCR-microarray method is an attractive diagnosis tool for the high-throughput identification of enteropathogenic organisms especially for multiple causative agents and epidemiological investigations.

ObjectiveTo investigate the expression and clinical value of coiled-coil domain-containing protein 34 (CCDC34) in hepatocellular carcinoma (HCC), and to predict the role of CCDC34 in the development and progression of HCC. MethodsThe datasets of HCC were downloaded from The Cancer Genome Atlas (TCGA) to obtain the expression profile and clinical information of the CCDC34 gene. The bioinformatics method was used to analyze the expression of CCDC34 in HCC, its correlation with clinicopathological parameters, and its influence on prognosis. The gene set enrichment analysis (GSEA) was used to predict the possible pathways regulated by the CCDC34 gene in HCC. The independent samples t-test and the paired t-test were used for comparison of continuous data between two groups; the Kaplan-Meier method and the log-rank test were used for survival analysis; the Cox proportional-hazards regression model analysis was used to investigate the influencing factors for prognosis. P＜0.01 was the standard for judging significant enrichment in GSEA, and the false discovery rate was ＜0.05. ResultsIn TCGA database, CCDC34 was highly expressed in tumor tissue, and there was a significant difference in the expression of CCDC34 between patients with different TNM stages and tumor grades (t=2.118 and 3.622, P=0.035 and P＜0.001). The patients with high expression of CCDC34 had a significantly shorter overall survival time than those with low expression (χ2=21.716, P＜005). The multivariate Cox regression analysis showed that the expression of CCDC34 （HR＝2.287,95%CI:1.312-3.987）and TNM stage(HR=1.943,95%CI:1.101-3.429) were independent risk factors for the overall survival time of patients with HCC (all P＜0.05). The enrichment of 8 pathway gene sets, including base excision repair and spliceosome, was observed in the samples with high expression of CCDC34 (P＜0.01, FDR ＜0.05). ConclusionCCDC34 may play a vital role in the development and progression of HCC and thus become a new prognostic indicator and a potential therapeutic target.

ObjectiveTo investigate the expression and clinical value of ZNF580 mRNA in hepatocellular carcinoma (HCC), as well as the role of ZNF580 mRNA in the development and progression of HCC. MethodsHCC dataset was downloaded from The Cancer Genome Atlas (TCGA) to obtain the gene expression profile and clinical information of ZNF580 mRNA. The bioinformatics method was used to analyze the association between the expression of ZNF580 mRNA in HCC and clinicopathological indices and the influence of ZNF580 mRNA on prognosis. A gene enrichment analysis was used to predict the possible pathways of ZNF580 mRNA involved in the regulation of HCC. The t-test was used for comparison of continuous data between two groups, the chi-square test was used for comparison of categorical data between two groups. The Cox proportional-hazards regression model was used to investigate the risk factors for prognosis, and hazard ratio (HR) and 95% confidence interval (CI) were calculated. ResultsIn the TCGA dataset, the expression of ZNF580 mRNA in tumor tissue was significantly higher than that in adjacent tissue (8.440±0.705 vs 7.736±0.387, P＜0.05), and there was a significant difference in the expression of ZNF580 mRNA between the patients with different tumor grades (t=-2.068, P＜0.05). The patients with high expression of ZNF580 mRNA had a significantly shorter overall survival time than those with low expression (χ2=5.456, P＜0.05). The multivariate Cox regression analysis showed that ZNF580 mRNA expression ［HR(95%CI)=1.600(1.079~2.372)］ and TNM stage ［HR(95%CI)=2.006(1.394~2.888)］ were independent risk factors for overall survival time of HCC patients (both P＜0.05). The samples with high expression of ZNF580 mRNA showed enrichment of genes in the pathways of ribosome and Huntington’s disease (all P＜0.05, FDR=0.198,0.243). ConclusionZNF580 mRNA may act as an oncogene in HCC and is expected to become an indicator for prognostic evaluation of HCC and a potential therapeutic target for HCC.

BACKGROUND: The effect of pituitary adenylate cyclase activating polypeptide (PACAP) during traumatic brain injury (TBI) and whether it can modulate secondary injury has not been reported previously. The present study evaluated the potential protective effects of ventricular infusion of PACAP in a rat model of TBI. METHODS: Male Sprague Dawley rats were randomly divided into 3 treatment groups (n=6, each): sham-operated, vehicle (normal saline)+TBI, and PACAP+TBI. Normal saline or PACAP (1g/5L) was administered intracerebroventricularly 20 minutes before TBI. Right parietal cortical contusion was produced via a weight-dropping method. Brains were extracted 24 hours after trauma. Histological changes in brains were examined by HE staining. The numbers of CD4+ and CD8+ T cells in blood and the spleen were detected via flow cytometry. RESULTS: In injured brain regions, edema, hemorrhage, inflammatory cell infiltration, and swollen and degenerated neurons were observed under a light microscope, and the neurons were disorderly arrayed in the hippocampi. Compared to the sham group, average CD4+ CD8– lymphocyte counts in blood and the spleen were significantly decreased in rats that received TBI+vehicle, and CD4– CD8+ were increased. In rats administered PACAP prior to TBI, damage was attenuated as evidenced by significantly increased CD4+, and decreased CD8+, T lymphocytes in blood and the spleen. CONCLUSION: Pretreatment with PACAP may protect against TBI by influencing periphery T cellular immune function.

Objective： A prospective randomized trial was performed to evaluate the contribution of neoadjuvant chemotherapy in the patients with locoregionally advanced nasopharyngeal carcinoma. Methods： The patients with locoregionally advanced nasopharyngeal carcinoma were treated with either radiotherapy alone (RT group) or neoadjuvant chemotherapy plus radiotherapy (CT/RT group). Neoadjuvant chemotherapy consisting of 2-3 cycles of cisplatin (20 mg/m2 on Day 1 to Day 5), bleomycin (7 mg/m2 on Day 1 and Day 5), and 5-FU (500 mg/m2 on Day 1 to Day 5) followed by radiotherapy was given to CT/RT group. All patients were treated in a uniform definitive-intent radiation therapy in two groups. Results： From 1992 to 1993, 457 patients were enrolled and 440 patients (221 in RT group, 219 in CT/RT group) were assessable. The 5-year overall survival (OS) rates were 62％ for CT/RT group and 55％ for RT group (P=0.1335)； The 5-year relapse free survival rate was 48％ versus 58％ , respectively (P=0.0539). The 5-year free local recurrence (FLR) rate was 82％ for CT/RT group, 74％ for RT group (P=0.0412). There was no significant difference in free distant metastasis (FDM) between two treatment groups (CT/RT group, 79％ ; RT group, 75％ ; P=0.4177). Subgroup analyses showed that neoadjuvant chemotherapy improved local control in patients with T3-4 disease, and had no effect in preventing distant metastases in patients with N2-3 disease. Conclusion： Despite improving FLR and RFS, neoadjuvant chemotherapy and radiatherapy failed to gain other survival benefit or reduce distant metastases in patients with locoregionally advanced nasopharyngeal carcinoma. The indication for neoadjuvant chemotherapy is proposed.

Objective To investigate the effects of radicular dentin treatments of sodium hypochlorite (NaOCl) and ethylenediaminetctraacetic acid (EDTA) on the regional root canal bending interface of quartz fiber posts using 2 luting materials with SEM analysis. Methods Nine intact maxillary central incisors were sectioned and endodontically treated. Standardized post space preparations and acid etch were performed . All specimens were randomly divided into 3 groups(n =3). D.T. LIGHT posts were placed into the root canal using one of three radicular dentin treatments(0. 9% NaCl for 60 s, 10% NaOCl for60 s, 17% EDTA for 60 s followed by 5.25% NaOCl for 60 s) in combination of one of two luting materials (DuoLink, LuxaCore) respectively (factorial design). Cervical, middle, apical sections of each teeth are used for SEM study and spectroscopy of dispersion energy (EDS) microanalysis. Results With the radicular dentin treatment with 10% NaOCl alone or with 17% EDTA followed by 5.25% NaOCl, longer and increased number of penetration of resin tags into the dentinal tubules were observed at the resin-dentin interfaces, and adhesive lateral branches could be found easily. EDS microanalysis showed increase in the infiltration behavior of the luting cement. Conclusions Radicular dentin treatments provide good resin infiltration, which can produce a three-dimensional interlocking micronetwork of resin tags in the dentin tubules with multiple lateral branches that penetrate the intertubalar dentin, thus positively influence the adhesion between dentin and the luting materials.

OBJECTIVEIschemic postconditioning effectively minimizes the ischemic/reperfusion injury, and the large series of case reports on its protective effects in cardiac surgery are limited. A randomized trial was conducted to investigate the effect of ischemic postconditioning on cardiopulmonary protection in children undergoing cardiac surgery for tetralogy of Fallot.

METHODSOne hundred and five-children with tetralogy of Fallot undergoing surgery were randomly assigned to control (n=58) and ischemic postconditioning groups (n=47). Ischemic postconditioning was performed by intermittent aortic clamping after reperfusion. After surgery, the duration of intensive care unit (ICU) stay, capacity of blood transfusion, hemodynamics, inotropic scores, respiratory function, and release of blood lactate were assayed.

RESULTSThere was a significant decrease in the ICU stay in the postconditioned group compared with the control group (37+/-21 hrs vs 54+/-26 hrs; P<0.05 ). The capacity of blood transfusion (308+/-230 mL vs 526+/-515 mL; P<0.05) and the inotropic scores (5.9+/-5.0 vs 10.3+/-7.7; P<0.05) in the postconditioned group were significantly reduced compared with those in the control group. Blood lactate contents in the postconditioned group was significantly lower that those in the control group 1, 3, 6, 9, 12 and 20 hrs after surgery. The postconditioned group showed more improved hemodynamics and respiratory function than the control group.

CONCLUSIONSIschemic postconditioning may provide clinical benefits with respects to myocardial and pulmonary protections in children undergoing repair for tetralogy of Fallot.

Adolescent ; Cardiac Surgical Procedures ; methods ; Cardiopulmonary Bypass ; Child ; Child, Preschool ; Female ; Hemodynamics ; Humans ; Infant ; Lactic Acid ; metabolism ; Male ; Myocardial Reperfusion Injury ; prevention & control ; Postoperative Complications ; prevention & control ; Respiration ; Tetralogy of Fallot ; physiopathology ; surgery

BACKGROUNDOsteoarthritis (OA) is a chronic and incurable disease, lacking effective treatment. Gene therapy offers a radical different approach to the treatment of arthritis. Even though the etiology of OA remains unclear, there is now considerable evidence to suggest that interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) are the main mediators in the pathogenesis of OA. The goal of this study was to determine the efficacy of local expression of interleukin-1 receptor antagonist (IL-1Ra) and soluble tumor necrosis factor-alpha receptor type I (sTNF-RI) by direct adenoviral-mediated intra-articular gene delivery in the rabbit model of osteoarthritis.

METHODSAdenoviral vectors containing IL-1Ra or sTNF-RI genes were constructed. OA was induced in both hind knees of 12 New Zealand white rabbits by the excision of the medial collateral ligament plus medial meniscectomy. Five days after surgery, approximately 1 x 10(8) plaque-forming units (pfu) of adenovirus were injected into the joint space of the knee through the patellar tendon. A total of 12 operated rabbits were divided into four groups. Three experimental rabbit groups received 1 x 10(8) pfu of adenovirus encoding either IL-1Ra (3 rabbits), sTNF-RI (3 rabbits) or IL-1Ra and sTNF-RI in combination (3 rabbits), into both knee joints respectively. An inflamed control group of 3 rabbits received approximately 1 x 10(8) pfu of Ad-GFP into both joints. Three days after injection of the adenovirus, both knees of each rabbit were lavaged with 1 ml of saline solution through the patellar tendon. At day 7, the rabbits were sacrificed, and the knees were lavaged, dissected and analyzed for effects of transgene expression. Levels of IL-1Ra and sTNF-RI expression in recovered lavage fluids were measured using a cytokine ELISA kit. Cartilage from the lesion areas of medial femoral condyle and synovium were fixed, embedded, sectioned and stained with hematoxylin and eosin (cartilage and synovium) and toluidine blue (cartilage). The samples were examined by light microscopy and quantitatively evaluated.

RESULTSIntra-articular delivery of IL-1Ra resulted in a significant inhibition of cartilage degradation, but did not affect synovial changes. In contrast, rabbit knee joints receiving sTNF-RI alone showed no detectable reduction in cartilage degradation. However, double gene transfer of IL-1Ra and sTNF-RI resulted in a higher suppression of the cartilage degradation and an observable reduction in synovitis. These data add to and confirm that IL-1Ra has good chondroprotective properties, but TNF-alpha blockade has little effect on joint destruction.

CONCLUSIONThe enhanced therapeutic effects of both antagonists in combination suggest inhibition of multiple inflammatory cytokines may be more efficacious than blockade of either cytokine alone in treating OA.

Adenoviridae ; genetics ; Animals ; Arthritis, Experimental ; genetics ; therapy ; Cartilage ; metabolism ; pathology ; Cartilage, Articular ; metabolism ; pathology ; Cell Line ; Cells, Cultured ; Genetic Therapy ; methods ; Genetic Vectors ; genetics ; Humans ; Interleukin 1 Receptor Antagonist Protein ; Osteoarthritis ; genetics ; therapy ; Rabbits ; Receptors, Tumor Necrosis Factor, Type I ; genetics ; physiology ; Sialoglycoproteins ; genetics ; physiology ; Synovial Fluid ; metabolism ; Synovial Membrane ; cytology ; metabolism ; Transfection ; methods

OBJECTIVETo explore the effect of curcumin (CUR) on cycteinyl aspirate specific protease-12 (Caspase-12) and pneumocyte apoptosis in pulmonary ischemia/reperfusion (I/R) injury mice.

METHODSThe in vivo unilateral in situ pulmonary I/R injury mouse model was established in C57BL/6J mice. Sixty experimental mice were randomly divided into six groups by random digit table, i. e., the sham-operation group (Sham), the I/R group, the I/R + dimethyl sulfoxide group (I/R + DMSO), the I/R + low dose CUR pre-treated group (I/R + CUR-100), the I/R + middle dose CUR pre-treated group (I/R + CUR-150), the I/R + high dose CUR pre-treated group (I/R + CUR-200), 10 in each group. Mice were euthanized and their left lungs were excised. Wet lung weight to dry lung weight (W/D) and the total lung water content (TLW) were tested. The morphological changes of the lung tissue were observed and index of quantitative evaluation for alveolar damage (IQA) detected under light microscope. The ultra-microstructure of the lung tissue was observed under electron microscope. The mRNA and protein expression levels of Caspase-12 and glucose regulated protein (GRP78) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Apoptosis index (AI) of the lung tissue was determined by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) method.

RESULTSCompared with the Sham group, expression levels of Caspase-12, GRP78 mRNA and protein all significantly increased in the I/R group (P < 0.05); W/D, TLW, IQA, and AI were all notably higher (P < 0.05, P < 0.01); the morphological and ultrastructural injury of the lung tissue were notably observed in I/R group. Compared with the I/R + DMSO group, expression levels of GRP78 mRNA and protein were increasingly higher in the I/R + CUR-100 group, the I/R + CUR-150 group, and the I/R +CUR-200 group (P < 0.05), expression levels of Caspase-12 mRNA and protein were lower (P < 0.05); W/D, TLW, IQA, and AI also decreased (P < 0.05, P < 0.01); the morphological and ultrastructural injury of the lung tissue were gradually alleviated in the I/R + CUR groups.

CONCLUSIONCUR had better effect on the lung protection against I/R injury, which might be related to inhibition for pneumocyte apoptosis associated with Caspase-12 in excessive unfolded protein response (UPR).

Animals ; Apoptosis ; drug effects ; Caspase 12 ; metabolism ; Curcumin ; pharmacology ; Heat-Shock Proteins ; metabolism ; Lung ; blood supply ; Male ; Mice ; Mice, Inbred C57BL ; Reperfusion Injury ; metabolism ; pathology ; prevention & control

Intracellular signal transduction plays an important role in the process of cellular metabolism, segmentation, differentiation, biological behaviour and cell death. Overactive signal transduction relates to tumor development and progression. Signaling pathways operated by protein tyrosine kinases (PTKs) will be illuminated here briefly. The Ras/Raf/MAPK and PI-3K/Akt pathways through receptor protein tyrosine kinases (RTKs), the Src, Bcr-Abl and JAK/STAT pathways by non-receptor protein tyrosine kinases (nrPTKs) are shown separately. Antitumor agents targeting the key proteins involved in the above five signalling routes are also summarized in this review.
Animals ; Antineoplastic Agents ; pharmacology ; Humans ; Phosphatidylinositol 3-Kinases ; metabolism ; Protein-Tyrosine Kinases ; metabolism ; Receptor Protein-Tyrosine Kinases ; metabolism ; STAT Transcription Factors ; metabolism ; Signal Transduction ; drug effects ; ras Proteins ; metabolism ; src-Family Kinases ; metabolism