We have previously identified C1 domain-containing phosphatase and TENsin homologue (C1-TEN) as being an intracellular binding partner for Axl receptor tyrosine kinase (RTK). C1-TEN is a tensin-related protein that houses an N-terminal region with predicted structural similarity to PTEN. Here, we report our observations on the effects of ectopic expression of C1-TEN in HEK293 cells, which resulted in profound molecular and phenotypic changes. Stable expression of C1-TEN altered cellular morphology, with less cell spreading and weaker filamentous actin staining. Cells overexpressing C1-TEN were inhibited greatly in their proliferation and migration rates as compared with mock-transfected cells. Furthermore, serum starvation-induced apoptosis caused a twofold increase in caspase 3 activity in C1-TEN-overexpressing cells vs. mock cells. In addition, C1-TEN-overexpressing cells showed a markedly reduced phosphorylation of Akt/PKB kinase and its substrate GSK3, as well as reduced Akt enzymatic activity. No such effects on JNK were observed. Also, serum-stimulated activation of Akt was delayed in C1-TEN-overexpressing cells, while no difference in profile of ERK activation was observed. Furthermore, cells expressing a C1-TEN mutant where the putative phosphatase active site cysteine at position 231 was substituted for a serine displayed full restoration of both cell proliferation and Akt activation. In conclusion, C1-TEN appears to be a novel intracellular phosphatase that negatively regulates the Akt/PKB signaling cascade, and is similar to its relative PTEN in this respect. However, the particular domain organization of C1-TEN may enable it to regulate RTK and other signaling complexes that are linked to Akt/PKB signaling in a unique manner.

A new sensing technology platform integrating magnetic permeability detection and a two-site heterogeneous immunoassay in a one-step analysis is described. As a platform model, measurements of C-reactive protein (CRP), a cardiac and inflammation marker, were performed in a rapid (11.5 min) high-sensitivity (hs) procedure with a low detection limit (0.2 mg/L) and accuracy (CV = 11%). The two-site heterogeneous immunoassay was performed in 1.2-mL disposable reagents vials containing solid phase (polyclonal anti-CRP conjugated silica microparticles), labeling agent (monoclonal anti-CRP conjugated superparamagnetic nanoparticles), and reaction buffer. Whole blood (20 muL) was assayed by introducing the sample into a reagent vial using a glass capillary and mixing its contents by hand for 30 s. After a 11-min sedimentation step, the vial was placed into the coil of the magnetic permeability detector, which measured the enrichment of superparamagnetic nanoparticles in the solid-phase sediment. Magnetic permeability detection and quantification is based on the principle that when paramagnetic materials are placed inside a coil, the inductance of the coil is influenced. Screening of CRP on whole blood patient samples showed good correlation with central hospital measurements for hsCRP (y = 1.018x - 0.021, R(2) = 0.980, n = 103) and normal range CRP (y = 1.02x + 2.53, R(2)= 0.991, n = 33) analyses. The mean differences of the two methods according to the Bland and Altman plots were -0.03 +/- 1.12 mg/L for hsCRP analysis and -3.4 +/- 8.64 mg/L for normal range CRP analysis.

A rapid (5.5 min) one-step whole blood C-reactive protein (CRP) magnetic permeability immunoassay utilizing monoclonal antibody conjugated dextran iron oxide nanoparticles (70 nm) as superparamagnetic labels and mixed fractions (1:1 ratio of 15-40 and 60 microm) of polyclonal anti-CRP conjugated silica microparticles for enhanced sedimentation is described. In this one-step assay procedure, a whole blood sample (4 microl) is applied to an assay glass vial, containing both antibody conjugates, and mixed for 30 s. The target analyte, CRP, forms a sandwich complex between the conjugated nanoparticles and microparticles, and, subsequently, the complex sediments under normal gravitation within 5 min to the bottom of the vial. The magnetic permeability increase of the sediment due to the presence of the complexed superparamagnetic nanoparticles is determined using an inductance-based transducer. Assayed patient whole blood samples were compared with the Abbott Diagnostics Architect reference method. A strong linear correlation was observed for the CRP concentration range 0-260 mg/l in whole blood (y=1.001x+0.42, R2=0.982, n=50). The CRP assay presented showed a limit of detection of 3 mg/l and a total imprecision (coefficient of variation) of 10.5%. On the basis of our observations, we propose a rapid, one-step, CRP assay for near-patient testing.