The use of real-time cell analyzer technology in drug discovery: defining optimal cell culture conditions and assay reproducibility with different adherent cellular models

The use of impedance-based label-free technology applied to drug discovery is nowadays receiving more and more attention. Indeed, such a simple and noninvasive assay that interferes minimally with cell morphology and function allows one to perform kinetic measurements and to obtain information on proliferation, migration, cytotoxicity, and receptor-mediated signaling. The objective of the study was to further assess the usefulness of a real-time cell analyzer (RTCA) platform based on impedance in the context of quality control and data reproducibility. The data indicate that this technology is useful to determine the best coating and cellular density conditions for different adherent cellular models including hepatocytes, cardiomyocytes, fibroblasts, and hybrid neuroblastoma/neuronal cells. Based on 31 independent experiments, the reproducibility of cell index data generated from HepG2 cells exposed to DMSO and to Triton X-100 was satisfactory, with a coefficient of variation close to 10%. Cell index data were also well reproduced when cardiomyocytes and fibroblasts were exposed to 21 compounds three times (correlation >0.91, p < 0.0001). The data also show that a cell index decrease is not always associated with cytotoxicity effects and that there are some confounding factors that can affect the analysis. Finally, another drawback is that the correlation analysis between cellular impedance measurements and classical toxicity endpoints has been performed on a limited number of compounds. Overall, despite some limitations, the RTCA technology appears to be a powerful and reliable tool in drug discovery because of the reasonable throughput, rapid and efficient performance, technical optimization, and cell quality control.     

Imaging the subcellular structure of human coronary atherosclerosis using micro-optical coherence tomography

Progress in understanding, diagnosis, and treatment of coronary artery disease (CAD) has been hindered by our inability to observe cells and extracellular components associated with human coronary atherosclerosis in situ. The current standards for microstructural investigation, histology and electron microscopy are destructive and prone to artifacts. The highest-resolution intracoronary imaging modality, optical coherence tomography (OCT), has a resolution of ~10 μm, which is too coarse for visualizing most cells. Here we report a new form of OCT, termed micro-optical coherence tomography (μOCT), whose resolution is improved by an order of magnitude. We show that μOCT images of cadaver coronary arteries provide clear pictures of cellular and subcellular features associated with atherogenesis, thrombosis and responses to interventional therapy. These results suggest that μOCT can complement existing diagnostic techniques for investigating atherosclerotic specimens, and that μOCT may eventually become a useful tool for cellular and subcellular characterization of the human coronary wall in vivo.     

Cloning and optimization of induction conditions for mature PsaA (pneumococcal surface adhesin A) expression in Escherichia coli and recombinant protein stability during long-term storage

The gene corresponding to mature PsaA from Streptococcus pneumoniae serotype 14 was cloned into a plasmid with kanamycin resistance and without a purification tag in Escherichia coli to express high levels of the recombinant protein for large-scale production as a potential vaccine candidate or as a carrier for polysaccharide conjugation at Bio-Manguinhos/Fiocruz. The evaluation of induction conditions (IPTG concentration, temperature and time) in E. coli was accomplished by experimental design techniques to enhance the expression level of mature recombinant PsaA (rPsaA). The optimization of induction process conditions led us to perform the recombinant protein induction at 25°C for 16 h, with 0.1mM IPTG in Terrific Broth medium. At these conditions, the level of mature rPsaA expression obtained in E. coli BL21 (DE3) Star by pET28a induction with IPTG was in the range of 0.8 g/L of culture medium, with a 10-fold lower concentration of inducer than usually employed, which contributes to a less expensive process. Mature rPsaA expressed in E. coli BL21 (DE3) Star accounted for approximately 30-35% of the total protein. rPsaA purification by ion exchange allowed the production of high-purity recombinant protein without fusion tags. The results presented in this work confirm that the purified recombinant protein maintains its stability and integrity for long periods of time in various storage conditions (temperatures of 4 or -70°C using different cryoprotectors) and for at least 3 years at 4 or -70°C in PBS. The conformation of the stored protein was confirmed using circular dichroism. Mature rPsaA antigenicity was proven by anti-rPsaA mouse serum recognition through western blot analysis, and no protein degradation was detected after long periods of storage.     

Cellular dimensions and cell dynamics, or the difficulty over capturing time and space in the era of electron microscopy

The introduction of electron microscopy profoundly altered biomedical research, providing a tool for a more detailed but at the same time a spatially and temporally more restricted visual analysis. Examining the case study of Golgi apparatus research in the 1950s and 1960s, it will be shown how microscopists handled these challenges, and how these confrontations modified the general concept of cellular organization. This will also shed light on the artifact debate and on the question of scientific realism in the field of microscopy.     

The proteolipid protein promoter drives expression outside of the oligodendrocyte lineage during embryonic and early postnatal development

The proteolipid protein (Plp) gene promoter is responsible for driving expression of one of the major components of myelin--PLP and its splice variant DM-20. Both products are classically thought to express predominantly in oligodendrocytes. However, accumulating evidence suggests Plp expression is more widespread than previously thought. In an attempt to create a mouse model for inducing oligodendrocyte-specific gene deletions, we have generated transgenic mice expressing a Cre recombinase cDNA under control of the mouse Plp promoter. We demonstrate Plp promoter driven Cre expression is restricted predominantly to mature oligodendrocytes of the central nervous system (CNS) at postnatal day 28. However, crosses into the Rosa26(LacZ) and mT/mG reporter mouse lines reveal robust and widespread Cre activity in neuronal tissues at E15.5 and E10.5 that is not strictly oligodendrocyte lineage specific. By P28, all CNS tissues examined displayed high levels of reporter gene expression well outside of defined white matter zones. Importantly, our study reinforces the emerging idea that Plp promoter activity is not restricted to the myelinating cell lineage, but rather, has widespread activity both during embryonic and early postnatal development in the CNS. Specificity of the promoter to the oligodendrocyte cell lineage, as shown through the use of a tamoxifen inducible Plp-CreER(t) line, occurs only at later postnatal stages. Understanding the temporal shift in Plp driven expression is of consequence when designing experimental models to study oligodendrocyte biology.     

Treatment of poor-risk myelodysplastic syndromes and acute myeloid leukemia with a combination of 5-azacytidine and valproic acid

5-azacytidine (AZA) has become standard treatment for patients with higher-risk myelodysplastic syndrome (MDS). Response rate is about 50% and response duration is limited. Histone deactylase (HDAC) inhibitors are attractive partners for epigenetic combination therapy. We treated 24 patients with AZA (100?mg/m(2), 5?days) plus valproate (VPA; continuous dosing, trough serum level 80-110?μg/ml). According to WHO classification, 5 patients had MDS, 2 had MDS/MPD, and 17 had acute myeloid leukemia (AML). Seven patients (29%) had previously received intensive chemotherapy, and five had previous HDAC inhibitor treatment. The overall response rate was 37% in the entire cohort but significantly higher (57%) in previously untreated patients, especially those with MDS (64%). Seven (29%) patients achieved CR (29%) and two PR (8%), respectively. Hematological CR was accompanied by complete cytogenetic remission according to conventional cytogenetics in all evaluable cases. Some patients also showed complete remission according to FISH on bone marrow mononuclear cells and CD34(+) peripheral blood cells, as well as by follow-up of somatic mitochondrial DNA mutations. Four additional patients achieved at least marrow remissions. Factors influencing response were AML (vs. MDS), marrow blast count, pretreatment, transfusion dependency, concomitant medication with hydroxyurea, and valproic acid (VPA) serum level. This trial is the first to assess the combination of AZA plus VPA without additional ATRA. A comparatively good CR rate, relatively short time to response, and the influence of VPA serum levels on response suggest that VPA provided substantial additional benefit. However, the importance of HDAC inhibitors in epigenetic combination therapy can only be proven by randomized trials.     

Psychological balance in high level athletes: gender-based differences and sport-specific patterns

Objectives

Few epidemiological studies have focused on the psychological health of high level athletes. This study aimed to identify the principal psychological problems encountered within French high level athletes, and the variations in their prevalence based on sex and the sport practiced.

Methods

Multivariate analyses were conducted on nationwide data obtained from the athletes'' yearly psychological evaluations.

Results

A representative sample of 13% of the French athlete population was obtained. 17% of athletes have at least one ongoing or recent disorder, generalized anxiety disorder (GAD) being the most prevalent (6%), followed by non-specific eating disorders (4.2%). Overall, 20.2% of women had at least one psychopathology, against 15.1% in men. This female predominance applied to anxiety and eating disorders, depression, sleep problems and self-harming behaviors. The highest rates of GAD appeared in aesthetic sports (16.7% vs. 6.8% in other sports for men and 38.9% vs. 10.3% for women); the lowest prevalence was found in high risk sports athletes (3.0% vs. 3.5%). Eating disorders are most common among women in racing sports (14% vs. 9%), but for men were found mostly in combat sports (7% vs. 4.8%).

Discussion

This study highlights important differences in psychopathology between male and female athletes, demonstrating that the many sex-based differences reported in the general population apply to elite athletes. While the prevalence of psychological problems is no higher than in the general population, the variations in psychopathology in different sports suggest that specific constraints could influence the development of some disorders.     

Role of the PLA2-independent peroxiredoxin VI activity in the survival of immortalized fibroblasts exposed to cytotoxic oxidative stress

Peroxiredoxin VI (PrxVI) is a bifunctional enzyme with non-selenium glutathione peroxidase and Ca2+-independent acidic phospholipase A2 activities. We demonstrate that transfection-mediated PrxVI overexpression protects immortalized human WI-38 and murine NIH3T3 fibroblasts against cytotoxic doses of tert-butylhydroperoxide and H2O2. Mutants for either glutathione peroxidase or phospholipase A2 activity show that glutathione peroxidase but not phospholipase A2 activity is required to promote cell survival after stress. Also, ectopic PrxVI overexpression does not protect telomerase-stabilized WI-38 fibroblasts against stress-induced premature senescence.     

A high-throughput method to measure the sensitivity of yeast cells to genotoxic agents in liquid cultures

The sensitivity of yeast Saccharomyces cerevisiae to DNA damaging agents is better represented when cells are grown in liquid media than on solid plates. However, systematic assessment of several strains that are grown in different conditions is a cumbersome undertaking. We report an assay to determine cell growth based on automatic measurements of optical densities of very small (100 microl) liquid cell cultures. Furthermore, an algorithm was elaborated to analyze large data files obtained from the cell growth curves, which are described by the growth rate--that starts at zero and accelerates to the maximal rate (mu(m))--and by the lag time (lambda). Cell dilution spot test for colony formation on solid media and the growth curve assay were used in parallel to analyze the phenotypes of cells after treatments with three different classes of DNA damaging agents (methyl methanesulfonate, bleomycin, and ultraviolet light). In these experiments the survival of the WT (wild type) and a number of DNA repair-deficient strains were compared. The results show that only the cell growth curve assay could uncover subtle phenotypes when WT cells, or mutant strains that are only weakly affected in DNA repair proficiency, were treated with low doses of cytotoxic compounds. The growth curve assay was also applied to establish whether histone acetyltransferases and deacetylases affect the resistance of yeast cells to UV irradiation. Out of 20 strains tested the sir2delta and rpd3delta cells were found to be more resistant than the WT, while gcn5delta and spt10delta cells were found to be more sensitive. This new protocol is sensitive, provides quantifiable data, offers increased screening capability and speed compared to the colony formation test.