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.     

Wing-dimorphism and population expansion of Pterostichus melanarius (Illiger, 1798) at small and large scales in central Alberta, Canada (Coleoptera, Carabidae, Pterostichini)

A study spanning ten years revealed changes in wing-morph ratios corroborating the hypothesis that the wing-dimorphic introduced carabid, Pterostichus melanarius Ill.,is spreading through flight, from the city of Edmonton, Canada and establishing populations in natural aspen forest of more rural areas 45-50 km to the East. Comparison of wing-morph ratios between Pterostichus melanarius and the native wing dimorphic species Agonum retractum LeConte suggests that the spatial variation in ratios for Pterostichus melanarius does not reflect underlying environmental variation, but instead the action of selective forces on this wing-dimorphic species. About ten years after its earliest detection in some rural sites the frequency of macropterous individuals in Pterostichus melanarius has decreased c. five-fold, but it is still above the level seen in European populations in which the two wing-morphs are thought to exist in equilibrium. Pterostichus melanarius is expanding its range in native aspen forest much faster than three other introduced species Clivina fossor L.), Carabus granulatus O.F. Müllerand Clivina fossor L also encountered in this study. The two Carabus species are flightless, but Carabus fossor can be dimorphic. Although these four non-native ground beetle species comprise >85% of the carabids collected at sites in urban Edmonton, activity-density of native carabids was similar across the urban-rural gradient, suggesting little direct impact of introduced species on the local abundance of native species. In a second study conducted at a smaller scale near George Lake, Alberta, macropterous individuals of Pterostichus melanarius have penetrated furthest and most rapidly into native aspen forest. Furthermore, the percentage of micropterous individuals has increased markedly in areas first colonized a decade previously. Overall, these studies support the idea that macropterous beetles in wing-d dimorphic species are important vanguards for early colonization of unexploited territory, but that flightless individuals replace the flying morph relatively rapidly once populations are established.     

Smart phone, smart science: how the use of smartphones can revolutionize research in cognitive science

Investigating human cognitive faculties such as language, attention, and memory most often relies on testing small and homogeneous groups of volunteers coming to research facilities where they are asked to participate in behavioral experiments. We show that this limitation and sampling bias can be overcome by using smartphone technology to collect data in cognitive science experiments from thousands of subjects from all over the world. This mass coordinated use of smartphones creates a novel and powerful scientific "instrument" that yields the data necessary to test universal theories of cognition. This increase in power represents a potential revolution in cognitive science.     

Low temperature-dependent salmonid alphavirus glycoprotein processing and recombinant virus-like particle formation

Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish. Outbreaks of SAV are associated with large economic losses of ~17 to 50 million $/year. Current control strategies rely on vaccination with inactivated virus formulations that are cumbersome to obtain and have intrinsic safety risks. In this research we were able to obtain non-infectious virus-like particles (VLPs) of SAV via expression of recombinant baculoviruses encoding SAV capsid protein and two major immunodominant viral glycoproteins, E1 and E2 in Spodoptera frugiperda Sf9 insect cells. However, this was only achieved when a temperature shift from 27°C to lower temperatures was applied. At 27°C, precursor E2 (PE2) was misfolded and not processed by host furin into mature E2. Hence, E2 was detected neither on the surface of infected cells nor as VLPs in the culture fluid. However, when temperatures during protein expression were lowered, PE2 was processed into mature E2 in a temperature-dependent manner and VLPs were abundantly produced. So, temperature shift-down during synthesis is a prerequisite for correct SAV glycoprotein processing and recombinant VLP production.     

Inflammatory and coagulation biomarkers and mortality in patients with HIV infection

Background

In the Strategies for Management of Anti-Retroviral Therapy trial, all-cause mortality was higher for participants randomized to intermittent, CD4-guided antiretroviral treatment (ART) (drug conservation [DC]) than continuous ART (viral suppression [VS]).We hypothesized that increased HIV-RNA levels following ART interruption induced activation of tissue factor pathways, thrombosis, and fibrinolysis.

Methods and Findings

Stored samples were used to measure six biomarkers: high sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), amyloid A, amyloid P, D-dimer, and prothrombin fragment 1+2. Two studies were conducted: (1) a nested case–control study for studying biomarker associations with mortality, and (2) a study to compare DC and VS participants for biomarker changes. For (1), markers were determined at study entry and before death (latest level) for 85 deaths and for two controls (n = 170) matched on country, age, sex, and date of randomization. Odds ratios (ORs) were estimated with logistic regression. For each biomarker, each of the three upper quartiles was compared to the lowest quartile. For (2), the biomarkers were assessed for 249 DC and 250 VS participants at study entry and 1 mo following randomization. Higher levels of hsCRP, IL-6, and D-dimer at study entry were significantly associated with an increased risk of all-cause mortality. Unadjusted ORs (highest versus lowest quartile) were 2.0 (95% confidence interval [CI], 1.0–4.1; p = 0.05), 8.3 (95% CI, 3.3–20.8; p < 0.0001), and 12.4 (95% CI, 4.2–37.0; p < 0.0001), respectively. Associations were significant after adjustment, when the DC and VS groups were analyzed separately, and when latest levels were assessed. IL-6 and D-dimer increased at 1 mo by 30% and 16% in the DC group and by 0% and 5% in the VS group (p < 0.0001 for treatment difference for both biomarkers); increases in the DC group were related to HIV-RNA levels at 1 mo (p < 0.0001). In an expanded case–control analysis (four controls per case), the OR (DC/VS) for mortality was reduced from 1.8 (95% CI, 1.1–3.1; p = 0.02) to 1.5 (95% CI, 0.8–2.8) and 1.4 (95% CI, 0.8–2.5) after adjustment for latest levels of IL-6 and D-dimer, respectively.

Conclusions

IL-6 and D-dimer were strongly related to all-cause mortality. Interrupting ART may further increase the risk of death by raising IL-6 and D-dimer levels. Therapies that reduce the inflammatory response to HIV and decrease IL-6 and D-dimer levels may warrant investigation. Trial Registration: ClinicalTrials.gov ({"type":"clinical-trial","attrs":{"text":"NCT00027352","term_id":"NCT00027352"}}NCT00027352).     

Structural recognition mechanisms between human Src homology domain 3 (SH3) and ALG-2-interacting protein X (Alix)

The functions of Src family kinases are tightly regulated through Src homology (SH) domain-mediated protein-protein interactions. We previously reported the biophysical characteristics of the apoptosis-linked gene 2-interacting protein X (Alix) in complex with the haemopoietic cell kinase (Hck) SH3 domain. In the current study, we have combined ITC, NMR, SAXS and molecular modeling to determine a 3D model of the complex. We demonstrate that Hck SH3 recognizes an extended linear proline-rich region of Alix. This particular binding mode enables Hck SH3 to sense a specific non-canonical residue situated in the SH3 RT-loop of the kinase. The resulting model helps clarify the mechanistic insights of Alix-Hck interaction.     

Genetic Diversity of African and Worldwide Strains of Ralstonia solanacearum as Determined by PCR-Restriction Fragment Length Polymorphism Analysis of the hrp Gene Region

The genetic diversity among a worldwide collection of 120 strains of Ralstonia solanacearum was assessed by restriction fragment length polymorphism (RFLP) analysis of amplified fragments from the hrp gene region. Five amplified fragments appeared to be specific to R. solanacearum. Fifteen different profiles were identified among the 120 bacterial strains, and a hierarchical cluster analysis distributed them into eight clusters. Each cluster included strains belonging to a single biovar, except for strains of biovars 3 and 4, which could not be separated. However, the biovar 1 strains showed rather extensive diversity since they were distributed into five clusters whereas the biovar 2 and the biovar 3 and 4 strains were gathered into one and two clusters, respectively. PCR-RFLP analysis of the hrp gene region confirmed the results of previous studies which split the species into an “Americanum” division including biovar 1 and 2 strains and an “Asiaticum” division including biovar 3 and 4 strains. However, the present study showed that most of the biovar 1 strains, originating from African countries (Reunion Island, Madagascar, Zimbabwe, and Angola) and being included in a separate cluster, belong to the “Asiaticum” rather than to the “Americanum” division. These African strains could thus have evolved separately from other biovar 1 strains originating from the Americas.