Inexpensive and fast pathogenic bacteria screening using field-effect transistors.

While pathogenic bacteria contribute to a large number of globally important diseases and infections, current clinical diagnosis is based on processes that often involve culturing which can be time-consuming. Therefore, innovative, simple, rapid and low-cost solutions to effectively reduce the burden of bacterial infections are urgently needed. Here we demonstrate a label-free sensor for fast bacterial detection based on metal-oxide-semiconductor field-effect transistors (MOSFETs). The electric charge of bacteria binding to the glycosylated gates of a MOSFET enables quantification in a straightforward manner. We show that the limit of quantitation is 1.9×10(5) CFU/mL with this simple device, which is more than 10,000-times lower than is achieved with electrochemical impedance spectroscopy (EIS) and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-ToF) on the same modified surfaces. Moreover, the measurements are extremely fast and the sensor can be mass produced at trivial cost as a tool for initial screening of pathogens.

Tetranectin positive expression in tumour tissue leads to longer survival in Danish women with ovarian cancer. Results from the 'Malova' ovarian cancer study.

The primary objective of this study was to analyse Tetranectin (TN) expression in tumour tissues and TN serum concentration in 758 women with epithelial ovarian tumours. The second was to evaluate, whether TN tissue expression levels correlate with clinico-pathological parameters and prognosis of the disease. Using tissue arrays we analysed the expression levels in tissues from 166 women with borderline ovarian tumours (BOTs) and 592 women with ovarian cancer (OC). A panel of three antibodies was used for immunohistochemistry: a polyclonal and two monoclonal antibodies. Serum TN was measured using the polyclonal antibody A-371. Univariate survival analyses stratified for chemotherapy showed that positive tissue TN as demonstrated by the polyclonal antibody indicated a significantly longer overall survival (OS) (p = 0.0001) as well as cancer specific survival (CSS) (p < 0.0001). High serum TN was likewise found to imply longer OS (p < 0.0001) and CSS (p < 0.0001), whereas tissue staining with the two monoclonal antibodies failed to demonstrate any significant correlation with either survival type. Univariate Kaplan-Meier survival analysis performed on all OC cases showed a significantly longer OS (p = 0.0009) and CSS (p = 0.0006) for women with TN positive tumour tissue and in women with high serum TN levels (p < 0.0001 for both). However, in the multivariate Cox regression analysis, only serum TN was found to be an independent prognostic factor for OS (p = 0.01) and not for CSS (p = 0.08). In conclusion, our results predict that a positive TN expression of both tumour tissue and serum points to a more favourable outcome for OC patients.

Prognostic value of tissue protein expression levels of MIB-1 (Ki-67) in Danish ovarian cancer patients. From the 'MALOVA' ovarian cancer study.

The primary objective of this study was to assess the expression of MIB-1 (Ki-67) in tumour tissues from 808 patients with epithelial ovarian tumours. The second was to evaluate, whether MIB-1 (Ki-67) tissue expression levels correlate with clinicopathological parameters and prognosis of the disease. Using tissue arrays (TA), we analysed the MIB-1 (Ki-67) expression levels in tissues from 202 women with borderline ovarian tumours (BOT) (177 stage I, 5 stage II, 19 stage III, 1 stage IV) and 606 ovarian cancer (OC) patients (177 stage I, 64 stage II, 311 stage III, 54 stage IV). Using a 10% cut-off level for MIB-1 (Ki-67) overexpression, 12% of the BOTs and 51% of the OCs were positive for MIB-1 (Ki-67) expression. The frequency of MIB-1 (Ki-67) expression-positive OC increased with increasing FIGO stage (p = 0.003), increasing histological grade (p ≤ 0.0001), and a significantly different distribution of MIB-1 (Ki-67) positive and negative tumours were found in adenocarcinoma NOS, serous adenocarcinomas, mucinous adenocarcinomas, endometrioid adenocarcinomas, non-epithelial and clear-cell carcinomas (p = 0.016). Univariate Kaplan-Meier survival analysis performed on all OC cases showed a significant shorter disease specific survival in patients with positive MIB-1 (Ki-67) expression in the tumour tissue (p ≤ 0.0001). In a Cox survival analysis including 606 FIGO stages I to IV OC cases, FIGO stage (II vs I: HR = 3.00, 95% CI: 1.81-4.99, III-I: HR = 6.41, 95% CI: 3.90-10.50, IV vs I: HR = 12.69, 95% CI: 7.21-22); age at diagnosis pr.10 years (HR = 1.27, 95% CI: 1.15-1.40), residual tumour after surgery (HR = 1.95, 95% CI: 1.40-2.73) and MIB-1 (Ki-67) expression (HR = 1.31, 95% CI: 1.08-1.60) had a significant independent impact on survival. Histological grade (p = 0.14) and histological tumour type (p = 0.35) had no significant independent impact on survival. In conclusion, our results predict that an increased level of MIB-1 (Ki-67) expression in tumour tissue, points to a less favourable outcome for OC patients.

Is tissue CA125 expression in epithelial ovarian adenocarcinoma heterogenic?

To evaluate if heterogeneity of tissue cancer antigen 125 (CA125) expression is present in epithelial serous adenocarcinomas. Furthermore, to investigate whether there is a correlation between levels of CA125 tissue expression, serum level of CA125, stage, and grade. A total of 10 patients diagnosed with serous ovarian adenocarcinomas were included. Preoperative blood samples were collected to determine serum CA125 levels. Tumor tissue from primary surgery was collected and processed for immunohistochemical analyses. CA125 was expressed in varying degrees in tumor tissues from all patients. Mean tissue CA125 expression for each patient ranged from 36% to 98%. Intrapatient variations in tissue expression ranged from 10% to 90% point. No significant correlations between levels of CA125 tissue expression, serum level of CA125, stage, and grade were found. We found that the tissue expression of CA125 is heterogenic. Although most patients had a high mean expression, it covers a large intrapatient variation in expression. This suggests that if using CA125 as a tissue marker and anti-CA125 (oregovomab) as immunotherapy treatment in future studies, it will be necessary to take heterogeneity into consideration and examine a larger number of biopsies. Therefore, the study demonstrates the need for heterogeneity studies in future translational research.

Limited prognostic value of tissue protein expression levels of cyclin E in Danish ovarian cancer patients: from the Danish 'MALOVA' ovarian cancer study.

The primary objective of this study was to assess the expression of cyclin E in tumour tissues from 661 patients with epithelial ovarian tumours. The second was to evaluate whether cyclin E tissue expression levels correlate with clinico-pathological parameters and prognosis of the disease. Using tissue arrays (TA), we analysed the cyclin E expression levels in tissues from 168 women with borderline ovarian tumours (BOT) (147 stage I, 4 stage II, 17 stage III) and 493 Ovarian cancer (OC) patients (127 stage I, 45 stage II, 276 stage III, 45 stage IV). Using a 10% cut-off level for cyclin E overexpression, 20% of the BOTs were positive with a higher proportion of serous than mucinous tumours. Sixty-two per cent of the OCs were positive for cyclin E expression with the highest percentage found in clear cell carcinomas. Results based on univariate and multivariate survival analyses with a 10% cut-off value showed that cyclin E had no independent prognostic value. In conclusion, we found cyclin E expression in tumour tissue to be of limited prognostic value to Danish OC patients.

Targeted induction of endoplasmic reticulum stress induces cartilage pathology.

Pathologies caused by mutations in extracellular matrix proteins are generally considered to result from the synthesis of extracellular matrices that are defective. Mutations in type X collagen cause metaphyseal chondrodysplasia type Schmid (MCDS), a disorder characterised by dwarfism and an expanded growth plate hypertrophic zone. We generated a knock-in mouse model of an MCDS-causing mutation (COL10A1 p.Asn617Lys) to investigate pathogenic mechanisms linking genotype and phenotype. Mice expressing the collagen X mutation had shortened limbs and an expanded hypertrophic zone. Chondrocytes in the hypertrophic zone exhibited endoplasmic reticulum (ER) stress and a robust unfolded protein response (UPR) due to intracellular retention of mutant protein. Hypertrophic chondrocyte differentiation and osteoclast recruitment were significantly reduced indicating that the hypertrophic zone was expanded due to a decreased rate of VEGF-mediated vascular invasion of the growth plate. To test directly the role of ER stress and UPR in generating the MCDS phenotype, we produced transgenic mouse lines that used the collagen X promoter to drive expression of an ER stress-inducing protein (the cog mutant of thyroglobulin) in hypertrophic chondrocytes. The hypertrophic chondrocytes in this mouse exhibited ER stress with a characteristic UPR response. In addition, the hypertrophic zone was expanded, gene expression patterns were disrupted, osteoclast recruitment to the vascular invasion front was reduced, and long bone growth decreased. Our data demonstrate that triggering ER stress per se in hypertrophic chondrocytes is sufficient to induce the essential features of the cartilage pathology associated with MCDS and confirm that ER stress is a central pathogenic factor in the disease mechanism. These findings support the contention that ER stress may play a direct role in the pathogenesis of many connective tissue disorders associated with the expression of mutant extracellular matrix proteins.

Characterization of E2F8, a novel E2F-like cell-cycle regulated repressor of E2F-activated transcription.

The E2F family of transcription factors are downstream effectors of the retinoblastoma protein, pRB, pathway and are essential for the timely regulation of genes necessary for cell-cycle progression. Here we describe the characterization of human and murine E2F8, a new member of the E2F family. Sequence analysis of E2F8 predicts the presence of two distinct E2F-related DNA binding domains suggesting that E2F8 and, the recently, identified E2F7 form a subgroup within the E2F family. We show that E2F transcription factors bind the E2F8 promoter in vivo and that expression of E2F8 is being induced at the G1/S transition. Purified recombinant E2F8 binds specifically to a consensus E2F-DNA-binding site indicating that E2F8, like E2F7, binds DNA without the requirement of co-factors such as DP1. E2F8 inhibits E2F-driven promoters suggesting that E2F8 is transcriptional repressor like E2F7. Ectopic expression of E2F8 in diploid human fibroblasts reduces expression of E2F-target genes and inhibits cell growth consistent with a role for repressing E2F transcriptional activity. Taken together, these data suggest that E2F8 has an important role in turning of the expression of E2F-target genes in the S-phase of the cell cycle.