Objective To establish a method for detecting K-rag gene point mutation in pancreatic adenocarcinoma based on the pyrosequencing and to compare its performance with that of Sanger sequencing.Methods Genomic DNA was extracted from formalin-fixed,paraffin-embedded pancreatic tissues including 49 pancreatic adenocarcinoma,10 normal pancreas,11 chronic pancreatitis,18 benign pancreatic tumor,7 insulin carcinoma,9 ampullary carcinoma,7 bile duct carcinoma and 7 duodenum papillary adenocarcinoma tissues.K-rag gene point mutations at codon 12 were analyzed by pyrosequencing and Sanger sequencing,respectively.Results No mutant K-ras gene Wag detected in normal pancreas,chronic pancreatitis,benign pancreatic tumor,insulin carcinoma,ampullary carcinoma,bile duct carcinoma and duodenum papillary adenocarcinoma tissues by either pyrosequencing or Sanger sequencing.K-rag gene point mutation was detection rate in pancreatic adenocarcinoma tissues was 71.4%(35/49)by pyrosequencing and 61.2%(30/49)by Sanger sequencing,respectively.Conclusions Pyrosequencing is more sensitive than Sanger sequencing and is also accurate,rapid and of high throushput in detecting mutant K-ras gene.It may serve as a practical method for fast batch analysis of clinical samples.

Adolescent ; Adult ; Aged ; Calcaneus ; injuries ; surgery ; Female ; Fractures, Bone ; surgery ; Humans ; Male ; Middle Aged ; Skin Diseases ; etiology ; prevention & control ; Young Adult

Child ; Humans ; Plasma Exchange ; Thrombotic Microangiopathies ; therapy

AIMDesign, synthesis and activity study of novel antifungal triazoles.

METHODSThe structures of two lead-compounds miconazole and itrconazole were modified on the basis of SAR studied by our group and reported in the literature and their antifungal activities in vitro were tested by standard program.

RESULTSTwelve 1-(1H-1, 2, 4-triazole-1-yl) -2-( 2, 4-difluorophenyl)-3-substituted amino-2-propanol compounds and thirteen 2-substituted phenyl-5-(1H-1, 2, 4-triazole-1-methyl ) 5-( 2, 4-difluorophenyl)-N-substituted oxazolidine compounds were synthesized and confirmed by 1HNMR and MS. In vitro inhibitory tests showed that most of them have more or less inhibitory effects on C. albicans and some inhibit S. cerevisiae also. Especially the effects of A10, A12 and A13 on C. albicans were more potent than (or equal to) that of fluconazole or itraconazole.

CONCLUSIONCompounds A10, A12 and A13 are worthy to be intensively studied.

Antifungal Agents ; chemical synthesis ; chemistry ; pharmacology ; Aspergillus niger ; drug effects ; Candida albicans ; drug effects ; Cryptococcus neoformans ; drug effects ; Microbial Sensitivity Tests ; Saccharomyces cerevisiae ; drug effects ; Triazoles ; chemical synthesis ; chemistry ; pharmacology

Objective To investigate transcriptional expression and promoter CpG methylation status of A-kinase anchoring protein 12 (AKAP12) gene and analyze their correlation with clinical pathological stage in bladder transitional cell carcinoma. Methods AKAP12 mRNA expression level and promoter CpG metbylation status was measured by fluorescent quantitative RT-PCR (FQ-RT-PCR) and methylation specific PCR (MSP) in 30 bladder transitional cell carcinoma and adjacent normal tissues. The products of PCR were cloned and bisulfite sequenced. Results Decreased AKAP12 mRNA expression was demonstrated in 22 carcinomas (73. 3% ) and was significantly associated with turnout grade (P =0. 02).The frequency of promoter methylation of AKAP12 gene was 53. 3 % (16/30) and correlated with the tumor stage(r =0.52,Pn =0.03)and grade(r =0.61,Pn =0.01). Conclusion Aberrant promoter methylation of AKAP12 can result in the loss of gene expression and may association with bladder transitional cell carcinoma.

Objective To detect quantitatively AKAP12 methylation and evaluate its clinical significance in peripheral blood in colorectal cancer. Methods MS-HRM technology was used to detect quantitatively AKAP12 methylation in peripheral blood from 80 colorectal cancer patients and 20 healthy volunteers. They also validated the reproducibility and compared with MSP. Results Thirty-eight of the 80 colorectal cancer samples (47. 5% ) were found to be methylated at the AKAP12 promoter region by MS-HRM (the methylation levels of 24 cancer samples ranged between 1 % and 20% , the methylation levels of 12 cancer samples ranged between 20% and 60% , the methylation levels of 2 cancer samples ranged between 60% and 100% ). The methylation levels of 2 health samples were less than 10% . They also compared the results generated by MS-HRM with a traditional MSP assay. The AKAP12 MS-HRM assay was able to reproducibly detect 1% AKAP12 methylated DNA, whereas the MSP method was unable to detect less than 10% methylation. No significant correlation was observed between the AKAP12 methylation levels and patients' age and gender. However, AKAP12 methylation was significantly higher in DNA from colorectal cancer patients with high Dukes stage and differentiation (x2 =5. 93 or 8. 41, P = 0.01). Conclusions The authors demonstrate here for the first time, the utility of quantitative AKAP12 MS-HRM analysis of promoter methylation in peripheral blood samples. AKAP12 MS-HRM quantitative methods have many promising applications in the detection of colorectal cancer.

Biopsy ; methods ; Esophageal Neoplasms ; pathology ; surgery ; Esophagoscopy ; Esophagus ; pathology ; Humans ; Mucous Membrane ; pathology ; Precancerous Conditions ; pathology ; surgery

OBJECTIVETo determine human epidermal growth factor receptor 2 (HER2) status in breast carcinoma by the techniques of a fully automated immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), to compare the concordance of protein expression with gene amplification and to explore the optimization in process quality control.

METHODSA prospective study of invasive breast cancer specimens excised between May 2009 and April 2011 at the Cancer Hospital, Chinese Academy of Medical Sciences was conducted by automated IHC staining with the new 4B5 rabbit monoclonal antibody and FISH. An evaluation was performed according to the ASCO/CAP guidelines (2007) and Chinese guidelines (2009). The gene amplification status of 740 cases were detected by FISH.

RESULTSA total of 2420 cases of breast invasive ductal carcinoma without pre-operation therapy were tested by automated IHC. 551 cases (22.8%) were scored as positive (3+), 664 cases (27.4%) as equivocal (2+), and 1205 cases (49.8%) as negative (1+/0). Gene amplification was detected in 98.0% (242/247) HER2 protein expression positive (3+) cases and in 13.6% (53/389) equivocal (2+) cases. One of 247 (0.4%) HER2 expression 3+ cases and 5 of 389 (1.3%) HER2 expression 2+ cases were equivocal for gene amplification. No gene amplification was detected in expression negative (1+/0) cases by FISH (0/104). The overall concordance between IHC and FISH was 98.6% [(242 + 104)/(247 + 104)].

CONCLUSIONSThere is a high concordance rate between automated IHC with 4B5 rabbit monoclonal antibody and FISH results for assessing the HER2 gene amplification status in surgically-excised breast cancer specimens, suggesting that automated IHC with 4B5 antibody can provide a reliable method to detect HER2 overexpression for eligibility of HER2 targeted therapy.

Adult ; Aged ; Aged, 80 and over ; Breast Neoplasms ; genetics ; metabolism ; pathology ; Carcinoma, Ductal, Breast ; genetics ; metabolism ; pathology ; Female ; Gene Amplification ; Gene Expression Regulation, Neoplastic ; Humans ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Middle Aged ; Prospective Studies ; Quality Control ; Receptor, ErbB-2 ; genetics ; Young Adult

OBJECTIVETo study gene expression of collagen types IX and X in human lumbar intervertebral discs during aging and degeneration and to explore the role of collagen types IX and X in disc degeneration.

METHODSFetal, adult and pathologic specimens were subjected to in situ hybridization with cDNA probes to investigate mRNA-expressions of types IX and X collagen gene.

RESULTSIn fetal intervertebral discs, positive mRNA hybridization signals of type IX collagen were concentrated in the nucleus pulposus and the inner layer of anulus fibrosus. Interstitial matrix of the nucleus pulposus also showed positive type X collagen staining. Positive mRNA hybridization signals of types IX and X were not detected in the middle and outer layers of anulus fibrosus. In adult specimens, expression of type IX collagen mRNA was markedly decreased. No hybridization signals of type X collagen was observed. As for pathological specimens, there was no gene expression of type IX collagen. In severe degenerated discs from adults, there were focal positive expressions of type X collagen.

CONCLUSIONSObvious changes of collagen gene expression occur with aging. Expression of type IX collagen decreases in adult and pathological discs. Results of type X collagen expression suggest that type X collagen is expressed only in older adult and senile discs (i.e., when disc degeneration has already reached a terminal stage), indicating the terminal stage of degeneration.

Adolescent ; Adult ; Collagen Type IX ; metabolism ; Collagen Type X ; metabolism ; Female ; Gene Expression ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Intervertebral Disc ; embryology ; metabolism ; Lumbar Vertebrae ; Male

BACKGROUNDDysregulated metallothionein 2A (MT2A) has been implicated in carcinogenesis. The purpose of this study was to investigate the expression of MT2A in gastric cancer (GC) and its correlation with prognosis.

METHODSReverse transcription-polymerase chain reaction and real-time polymerase chain reaction were used to detect the mRNA expression of MT2A in 12 GC cell lines, normal gastric epithelial GES-1 cells, and 36 GC and adjacent normal tissues. MT2A protein expression was determined in 258 GC tissues and 171 adjacent normal tissues by immunohistochemistry.

RESULTSMT2A mRNA expression was lower in GC cells and primary tumors than in GES-1 cells and adjacent normal tissues, respectively. High protein expression of MT2A was present in 130 of 171 normal tissues (76.0%) and in 56 of 258 GC tissues (21.7%; P < 0.001). MT2A protein expression was higher in well/moderately differentiated GC (22/54; 40.7%) than in poorly differentiated GC (34/204; 16.7%; P < 0.001). Moreover, the protein expression of MT2A was lower in diffuse-type GC (6/82; 7.3%) than in intestinal-type GC (50/176; 28.4%; P = 0.0001). Importantly, MT2A expression was an independent prognostic factor for GC, and decreased MT2A expression was associated with poor clinical outcome (P < 0.001). The expression status of MT2A could predict prognosis in intestinal and diffuse-type GCs.

CONCLUSIONExpression status of MT2A might be a useful prognostic biomarker for GC, especially when used in combination with Lauren's classification.

Adult ; Aged ; Cell Line, Tumor ; Female ; Humans ; Logistic Models ; Male ; Metallothionein ; analysis ; genetics ; MicroRNAs ; analysis ; Middle Aged ; Prognosis ; Proportional Hazards Models ; Stomach Neoplasms ; chemistry ; classification ; pathology