Deviation of eyes and head in acute cerebral stroke

Background  

It is a well-known phenomenon that some patients with acute left or right hemisphere stroke show a deviation of the eyes (Prévost's sign) and head to one side. Here we investigated whether both right- and left-sided brain lesions may cause this deviation. Moreover, we studied the relationship between this phenomenon and spatial neglect. In contrast to previous studies, we determined not only the discrete presence or absence of eye deviation with the naked eye through clinical inspection, but actually measured the extent of horizontal eye-in-head and head-on-trunk deviation. In further contrast, measurements were performed early after stroke onset (1.5 days on average).     

Comparison of five commercial extraction kits for subsequent membrane protein profiling

Membrane proteins account for 70–80% of all pharmaceutical targets emphasizing their clinical relevance. Identification of new, differentially expressed membrane proteins reflecting distinct disease properties is thus of high importance. Unfortunately, isolation and analysis of membrane-bound proteins is hampered by their relative low abundance in total cell lysates, their frequently large size and their hydrophobic properties. We thus aimed to identify protocols that allow for highly efficient isolation and purification of membrane-bound proteins for subsequent protein profiling. We present a comparative study of different membrane protein extraction methods that vary in total protein yield between 0.02 and 4.8 mg using constant cell pellets of the colorectal carcinoma cell line SW620. We also demonstrate by means of polyacrylamide gel electrophoresis (SDS–PAGE) and Western blot analysis that the majority of commercial membrane extraction kits harbor a substantial cytosolic contamination of their membranous fraction. Based on purity of membranous fraction, protein yield, time and costs, we show superiority of two commercial extraction kits for downstream proteome analyses of membrane proteins.     

Correction: deviation of eyes and head in acute cerebral stroke

This is a correction article     

Sensitive Detection of Colorectal Cancer in Peripheral Blood by Septin 9 DNA Methylation Assay

Background

Colorectal cancer (CRC) is the second leading cause of cancer deaths despite the fact that detection of this cancer in early stages results in over 90% survival rate. Currently less than 45% of at-risk individuals in the US are screened regularly, exposing a need for better screening tests. We performed two case-control studies to validate a blood-based test that identifies methylated DNA in plasma from all stages of CRC.

Methodology/Principal Findings

Using a PCR assay for analysis of Septin 9 (SEPT9) hypermethylation in DNA extracted from plasma, clinical performance was optimized on 354 samples (252 CRC, 102 controls) and validated in a blinded, independent study of 309 samples (126 CRC, 183 controls). 168 polyps and 411 additional disease controls were also evaluated. Based on the training study SEPT9-based classification detected 120/252 CRCs (48%) and 7/102 controls (7%). In the test study 73/126 CRCs (58%) and 18/183 control samples (10%) were positive for SEPT9 validating the training set results. Inclusion of an additional measurement replicate increased the sensitivity of the assay in the testing set to 72% (90/125 CRCs detected) while maintaining 90% specificity (19/183 for controls). Positive rates for plasmas from the other cancers (11/96) and non-cancerous conditions (41/315) were low. The rate of polyp detection (>1 cm) was ∼20%.

Conclusions/Significance

Analysis of SEPT9 DNA methylation in plasma represents a straightforward, minimally invasive method to detect all stages of CRC with potential to satisfy unmet needs for increased compliance in the screening population. Further clinical testing is warranted.     

Toward standardized high-throughput serum diagnostics: multiplex-protein array identifies IL-8 and VEGF as serum markers for colon cancer

Development and progression of colon cancer may be related to cytokines. Cytokines with diagnostic value have been identified individually but have not been implemented into clinical praxis. Using a multiplex protein array, the authors explore a panel of cytokines simultaneously and compared its performance to carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9). Serum concentrations of 12 cytokines were simultaneously determined by multiplex biochip technology in 50 colon cancer patients and 50 healthy controls. Serum levels of interleukin-8 (IL-8) and CEA were significantly higher in cancer patients than in healthy controls. Areas under the receiver operating characteristic curves (AUCs) were largest for IL-8, followed by CEA, vascular endothelial growth factor (VEGF), and CA 19-9. Analyses regarding marker combinations showed an advantage over single marker performance for CEA, VEGF, and CA 19-9 but not for IL-8. Multiplex biochip array technology represents a practical tool in cytokine and cancer research when simultaneous determination of different biomarkers is of interest. The results suggest that the assessment of IL-8, CEA, VEGF, and possibly CA 19-9 serum levels could be useful for colon cancer screening with the potential of also detecting early stage tumors. Further validation studies using these and additional markers on a multiplex array format are encouraged.     

Polypeptide expression in prostate hyperplasia and prostate adenocarcinoma.

Cells were collected from prostate hyperplasias (n = 6) and prostate carcinomas (n = 6) and subjected to two-dimensional gel electrophoresis (2-DE). The resulting polypeptide patterns were analysed with the PDQUEST computer software. Malignant tumors showed significant increases in the level of expression of proliferating cell nuclear antigen (PCNA), calreticulin, HSP 90 and pHSP 60, oncoprotein 18(v), elongation factor 2, glutathione-S-transferase pi (GST-pi), superoxide dismutase and triose phosphate isomerase. In addition, decreases in the levels of tropomyosin-1 and 2 and cytokeratin 18 were observed in prostate carcinomas compared to prostate hyperplasias. This pattern of alterations is similar to that observed in other carcinomas in our previous studies. All malignant tumors showed simultaneous alterations in 5 or more of 9 markers studied, whereas only one case of benign hyperplasia showed alterations in 5 markers. The EST-data base for prostate tumors available from NCI (CGAP) was searched for the expression of the mRNAs corresponding to proteins identified in our gels. Large differences in the relative expression of mRNAs and proteins were observed. Our data show alterations in the pattem of polypeptide expression in prostate carcinomas which are similar to those observed in other carcinomas.     

Expression of Cyclins A,E and Topoisomerase II α correlates with centrosome amplification and genomic instability and influences the reliability of cytometric S-phase determination

Background

The progression of normal cells through the cell cycle is meticulously regulated by checkpoints guaranteeing the exact replication of the genome during S-phase and its equal division at mitosis. A prerequisite for this achievement is synchronized DNA-replication and centrosome duplication. In this context the expression of cyclins A and E has been shown to play a principal role.

Results

Our results demonstrated a correlation between centrosome amplification, cell cycle fidelity and the level of mRNA and protein expression of cyclins A and E during the part of the cell cycle defined as G1-phase by means of DNA content based histogram analysis. It is shown that the normal diploid breast cell line HTB-125, the genomically relatively stable aneuploid breast cancer cell line MCF-7, and the genomically unstable aneuploid breast cancer cell line MDA-231 differ remarkably concerning both mRNA and protein expression of the two cyclins during G1-phase. In MDA-231 cells the expression of e.g. cyclin A mRNA was found to be ten times higher than in MCF-7 cells and about 500 times higher than in HTB-125 cells. Topoisomerase II α showed high mRNA expression in MDA compared to MCF-7 cells, but the difference in protein expression was small. Furthermore, we measured centrosome aberrations in 8.4% of the MDA-231 cells, and in only 1.3% of the more stable aneuploid cell line MCF-7. MDA cells showed 27% more incorporation of BrdU than reflected by S-phase determination with flow cytometric DNA content analysis, whereas these values were found to be of the same size in both HTB-125 and MCF-7 cells.

Conclusions

Our data indicate that the breast cancer cell lines MCF-7 and MDA-231, although both DNA-aneuploid, differ significantly regarding the degree of cell cycle disturbance and centrosome aberrations, which partly could explain the different genomic stability of the two cell lines. The results also question the reliability of cytometric DNA content based S-phase determination in genomically unstable tumor cell populations.