Potentiometric platform for the quantification of cellular potassium efflux

Renewed interest in the measurement of cellular K(+) effluxes has been prompted by the observation that potassium plays an active and important role in numerous key cellular events, in particular cell necrosis and apoptosis. Although necrosis and apoptosis follow different pathways, both induce intracellular potassium effluxes. Here, we report the use of potassium-selective microelectrodes located in a microfluidic platform for cell culture to monitor and quantify such effluxes in real time. Using this platform, we observed and measured the early signs of cell lysis induced by a modification of the extracellular osmolarity. Furthermore, we were able to quantify the number of dying cells by evaluating the extracellular potassium concentration. A comparison between the potentiometric measurement with a fluorescent live-dead assay performed under similar conditions revealed the delay between potassium effluxes and cell necrosis. These results suggest that such platforms may be exploited for applications, such as cytotoxicological screening assays or tumor cell proliferation assays, by using extracellular K(+) as cell death marker.

CCN2/CTGF is required for matrix organization and to protect growth plate chondrocytes from cellular stress

CCN2 (connective tissue growth factor (CTGF/CCN2)) is a matricellular protein that utilizes integrins to regulate cell proliferation, migration and survival. The loss of CCN2 leads to perinatal lethality resulting from a severe chondrodysplasia. Upon closer inspection of Ccn2 mutant mice, we observed defects in extracellular matrix (ECM) organization and hypothesized that the severe chondrodysplasia caused by loss of CCN2 might be associated with defective chondrocyte survival. Ccn2 mutant growth plate chondrocytes exhibited enlarged endoplasmic reticula (ER), suggesting cellular stress. Immunofluorescence analysis confirmed elevated stress in Ccn2 mutants, with reduced stress observed in Ccn2 overexpressing transgenic mice. In vitro studies revealed that Ccn2 is a stress responsive gene in chondrocytes. The elevated stress observed in Ccn2-/- chondrocytes is direct and mediated in part through integrin α5. The expression of the survival marker NFκB and components of the autophagy pathway were decreased in Ccn2 mutant growth plates, suggesting that CCN2 may be involved in mediating chondrocyte survival. These data demonstrate that absence of a matricellular protein can result in increased cellular stress and highlight a novel protective role for CCN2 in chondrocyte survival. The severe chondrodysplasia caused by the loss of CCN2 may be due to increased chondrocyte stress and defective activation of autophagy pathways, leading to decreased cellular survival. These effects may be mediated through nuclear factor κB (NFκB) as part of a CCN2/integrin/NFκB signaling cascade.

Cellular Photo Digital Breathalyzer for Monitoring Alcohol Use: A Pilot Study

Background: Monitoring alcohol use is important in numerous situations. Direct ethanol metabolites, such as ethyl glucuronide (EtG), have been shown to be useful tools in detecting alcohol use and documenting abstinence. For very frequent or continuous control of abstinence, they lack practicability. Therefore, devices measuring ethanol itself might be of interest. This pilot study aims at elucidating the usability and accuracy of the cellular photo digital breathalyzer (CPDB) compared to self-reports in a naturalistic setting. Method: 12 social drinkers were included. Subjects used a CPDB 4 times daily, kept diaries of alcohol use and submitted urine for EtG testing over a period of 5 weeks. Results: In total, the 12 subjects reported 84 drinking episodes. 1,609 breath tests were performed and 55 urine EtG tests were collected. Of 84 drinking episodes, CPDB detected 98.8%. The compliance rate for breath testing was 96%. Of the 55 EtG tests submitted, 1 (1.8%) was positive. Conclusions: The data suggest that the CPDB device holds promise in detecting high, moderate, and low alcohol intake. It seems to have advantages compared to biomarkers and other Monitoring devices. The preference for CPDB by the participants might explain the high compliance. Further studies including comparison with biomarkers and transdermal devices are needed.