Mek2 is dispensable for mouse growth and development

MEK is a dual-specificity kinase that activates the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase upon agonist binding to receptors. The ERK/MAP kinase cascade is involved in cell fate determination in many organisms. In mammals, this pathway is proposed to regulate cell growth and differentiation. Genetic studies have shown that although a single Mek gene is present in Caenorhabditis elegans, Drosophila melanogaster, and Xenopus laevis, two Mek homologs, Mek1 and Mek2, are present in the mammalian cascade. The inactivation of the Mek1 gene leads to embryonic lethality and has revealed the unique role played by Mek1 during embryogenesis. To investigate the biological function of the second homolog, we have generated mice deficient in Mek2 function. Mek2 mutant mice are viable and fertile, and they do not present flagrant morphological alteration. Although several components of the ERK/MAP kinase cascade have been implicated in thymocyte development, no such involvement was observed for MEK2, which appears to be nonessential for thymocyte differentiation and T-cell-receptor-induced proliferation and apoptosis. Altogether, our findings demonstrate that MEK2 is not necessary for the normal development of the embryo and T-cell lineages, suggesting that the loss of MEK2 can be compensated for by MEK1.     

Detection of a decrease in green fluorescent protein fluorescence for the monitoring of cell death: an assay amenable to high-throughput screening technologies.

BACKGROUND: Reliable assessment of cell death is now pivotal to many research programs aiming at generating new anti-tumor compounds or at screening cDNA libraries. Such approaches need to rely on reproducible, easy-to-handle, and rapid microplate-based cytotoxicity assays that are amenable to high-throughput screening (HTS) technologies. We describe a method for the direct measurement of cell death, based on the detection of a decrease in fluorescence observed following death induction in cells expressing enhanced green fluorescent protein (EGFP). METHODS: Cell death was induced by a variety of apoptotic stimuli in various EGFP-expressing mammalian cell lines, including those routinely used in anti-cancer drug screening. Decrease in fluorescence was assessed either by flow cytometry (and compared with other apoptotic markers) or by a fluorescence microplate reader. RESULTS: Cells expressing EGFP exhibited a decrease in fluorescence when treated by various agents, such as chemotherapeutic drugs, UV irradiation, or caspase-independent cell death inducers. Kinetics and sensitivity of this EGFP-based assay were comparable to those of traditional apoptosis markers such as annexin-V binding, propidium iodide incorporation, or reactive oxygen species production. We also show that the decrease in EGFP fluorescence is directly quantifiable in a fluorescence-based microplate assay. Furthermore, analysis of EGFP protein content in cells undergoing cell death demonstrates that the decrease in fluorescence does not arise from degradation of the protein. CONCLUSIONS: This novel GFP-based microplate assay combines sensitivity and rapidity, is easily amenable to HTS setups, making it an assay of choice for cytotoxicity evaluation.     

Gabapentin for the Management of Chronic Pelvic Pain in Women (GaPP1): A Pilot Randomised Controlled Trial

Chronic pelvic pain (CPP) affects 2.1–24% of women. Frequently, no underlying pathology is identified, and the pain is difficult to manage. Gabapentin is prescribed for CPP despite no robust evidence of efficacy. We performed a pilot trial in two UK centres to inform the planning of a future multicentre RCT to evaluate gabapentin in CPP management. Our primary objective was to determine levels of participant recruitment and retention. Secondary objectives included estimating potential effectiveness, acceptability to participants of trial methodology, and cost-effectiveness of gabapentin. Women with CPP and no obvious pelvic pathology were assigned to an increasing regimen of gabapentin (300-2700mg daily) or placebo. We calculated the proportion of eligible women randomised, and of randomised participants who were followed up to six months. The analyses by treatment group were by intention-to-treat. Interviews were conducted to evaluate women’s experiences of the trial. A probabilistic decision analytical model was used to estimate cost-effectiveness. Between September 2012–2013, 47 women (34% of those eligible) were randomised (22 to gabapentin, 25 to placebo), and 25 (53%) completed six-month follow-up. Participants on gabapentin had less pain (BPI difference 1.72 points, 95% CI:0.07–3.36), and an improvement in mood (HADS difference 4.35 points, 95% CI:1.97–6.73) at six months than those allocated placebo. The majority of participants described their trial experience favorably. At the UK threshold for willingness-to-pay, the probabilities of gabapentin or no treatment being cost-effective are similar. A pilot trial assessing gabapentin for CPP was feasible, but uncertainty remains, highlighting the need for a large definitive trial.

Trial registration

Controlled-Trials.com ISRCTN45178534     

Fatigue after stroke: baseline predictors and influence on survival. Analysis of data from UK patients recruited in the International Stroke Trial

Background and Purpose

Little is known about the associations of post-stroke fatigue or its influence on survival. The vitality component of the Short Form 36 (SF-36) is a valid and reliable measure of post-stroke fatigue. We sought to identify associates of post-stroke fatigue and determine whether fatigue predicted survival.

Methods

We used SF-36 vitality scores obtained by postal questionnaires from 1080 UK patients randomised in the International Stroke Trial, at a mean of 64 weeks after stroke onset. We used logistic regression to explore factors at randomisation which predicted SF-36 vitality at follow-up, and the relationship between SF-36 vitality and both SF-36 mental health and SF-36 emotional role function at follow-up. We used Cox proportional hazards to explore the influence of SF-36 vitality at follow-up on subsequent survival, using four different statistical models for handling missing data.

Results

Female sex, increasing age, lower mental health and lower emotional role function scores were associated with greater degrees of fatigue after stroke (i.e. lower vitality scores) but these factors explained <30% of the variance (R²) in fatigue. In two models, fatigue at follow-up was associated with shorter subsequent survival.

Conclusion

Increasing age, female sex, emotional role function and mental health were associated with increased fatigue at a mean of 64 weeks after stroke onset, but explained less than 30% of the variance. Fatigue was associated with reduced subsequent long-term survival in 2/4 models. Further work is needed to identify the biological substrate of fatigue and to clarify its influence on survival.     

Apoptosis mediated through CD45 is independent of its phosphatase activity and association with leukocyte phosphatase-associated phosphoprotein

Besides the well-recognized role of CD45 as a major player in TCR signaling, we and others have demonstrated that cross-linking of CD45 with mAbs can induce cell death in T lymphocytes. To investigate the role of CD45 phosphatase activity in apoptosis induction, we expressed either wild-type or phosphatase-dead CD45 molecules in a CD45-deficient BW5147 T cell line. We show here that the phosphatase activity of CD45 was not required for apoptosis triggering after cross-linking of the molecule. It is noteworthy that a revertant of the CD45-negative BW5147 cell line, expressing a truncated form of CD45 lacking most of the cytoplasmic domain, was also susceptible to CD45-mediated death. Moreover, we also demonstrate that leukocyte phosphatase-associated phosphoprotein expression is totally dispensable for CD45-mediated apoptosis to occur. Taken together, these results strongly suggest a role for the extracellular and/or the transmembrane portion of CD45 in apoptosis signaling, which contrasts with the previously reported functions for CD45 in T lymphocytes.     

Impact of functional status at six months on long term survival in patients with ischaemic stroke: prospective cohort studies

Objective To estimate the impact on long term survival of functional status at six months after ischaemic stroke.Design Prospective cohort study.Settings Three cohorts: Oxfordshire community stroke project, Lothian stroke register, and the first international stroke trial (in the United Kingdom).Participants 7710 patients with ischaemic stroke registered between 1981 and 2000 and followed up for a maximum of 19 years.Main outcome measures Functional status at six months after stroke assessed with modified Rankin scale or “two simple questions.” Mortality during follow-up. Survival analysis with Kaplan-Meier curves, log rank test, and Cox’s regression model.Results In a combined analysis of all three cohorts, among patients who survived to assessment six months after the index stroke, the subsequent median length of survival among those independent in daily living and those dependent was 9.7 years (95% confidence interval 8.9 to 10.6) and 6.0 years (5.7 to 6.4), respectively. In a combined analysis of the Oxfordshire and Lothian cohorts, subsequent median survival fell progressively from 12.9 years (10.0 to 15.9) for patients with a Rankin score of 0-1 at six months after the stroke to 2.5 years (1.4 to 3.5) for patients with a Rankin score of 5. All previously stated differences in median survival were significant (log rank test P<0.001). The influence of functional outcome on survival remained significant (P<0.05) in each cohort after adjustment for relevant covariates (such as age, presence of atrial fibrillation, visible infarct on computed tomography, subtype of stroke) in a Cox’s regression model.Conclusion Functional status six months after an ischaemic stroke is associated with long term survival. Early interventions that reduce dependency at six months might have positive effects on long term survival.