Identification of Novel Pro-Migratory, Cancer-Associated Genes Using Quantitative, Microscopy-Based Screening

Background: Cell migration is a highly complex process, regulated by multiple genes, signaling pathways and external stimuli. To discover genes or pharmacological agents that can modulate the migratory activity of cells, screening strategies that enable the monitoring of diverse migratory parameters in a large number of samples are necessary. Methodology: In the present study, we describe the development of a quantitative, high-throughput cell migration assay, based on a modified phagokinetic tracks (PKT) procedure, and apply it for identifying novel pro-migratory genes in a cancer-related gene library. In brief, cells are seeded on fibronectin-coated 96-well plates, covered with a monolayer of carboxylated latex beads. Motile cells clear the beads, located along their migratory paths, forming tracks that are visualized using an automated, transmitted-light screening microscope. The tracks are then segmented and characterized by multi-parametric, morphometric analysis, resolving a variety of morphological and kinetic features. Conclusions: In this screen we identified 4 novel genes derived from breast carcinoma related cDNA library, whose over-expression induces major alteration in the migration of the stationary MCF7 cells. This approach can serve for high throughput screening for novel ways to modulate cellular migration in pathological states such as tumor metastasis and invasion

Population Dynamics and Diversity of Viruses, Bacteria and Phytoplankton in a Shallow Eutrophic Lake

We have studied the temporal variation in viral abundances and community assemblage in the eutrophic Lake Loosdrecht through epifluorescence microscopy and pulsed field gel electrophoresis (PFGE). The virioplankton community was a dynamic component of the aquatic community, with abundances ranging between 5.5 × 107 and 1.3 × 108 virus-like particles ml−1 and viral genome sizes ranging between 30 and 200 kb. Both viral abundances and community composition followed a distinct seasonal cycle, with high viral abundances observed during spring and summer. Due to the selective and parasitic nature of viral infection, it was expected that viral and host community dynamics would covary both in abundances and community composition. The temporal dynamics of the bacterial and cyanobacterial communities, as potential viral hosts, were studied in addition to a range of environmental parameters to relate these to viral community dynamics. Cyanobacterial and bacterial communities were studied applying epifluorescence microscopy, flow cytometry, and denaturing gradient gel electrophoresis (DGGE). Both bacterial and cyanobacterial communities followed a clear seasonal cycle. Contrary to expectations, viral abundances were neither correlated to abundances of the most dominant plankton groups in Lake Loosdrecht, the bacteria and the filamentous cyanobacteria, nor could we detect a correlation between the assemblage of viral and bacterial or cyanobacterial communities during the overall period. Only during short periods of strong fluctuations in microbial communities could we detect viral community assemblages to covary with cyanobacterial and bacterial communities. Methods with a higher specificity and resolution are probably needed to detect the more subtle virus–host interactions. Viral abundances did however relate to cyanobacterial community assemblage and showed a significant positive correlation to Chl-a as well as prochlorophytes, suggesting that a significant proportion of the viruses in Lake Loosdrecht may be phytoplankton and more specific cyanobacterial viruses. Temporal changes in bacterial abundances were significantly related to viral community assemblage, and vice versa, suggesting an interaction between viral and bacterial communities in Lake Loosdrecht

Life-Cycle and Genome of OtV5, a Large DNA Virus of the Pelagic Marine Unicellular Green Alga Ostreococcus tauri

Large DNA viruses are ubiquitous, infecting diverse organisms ranging from algae to man, and have probably evolved from an ancient common ancestor. In aquatic environments, such algal viruses control blooms and shape the evolution of biodiversity in phytoplankton, but little is known about their biological functions. We show that Ostreococcus tauri, the smallest known marine photosynthetic eukaryote, whose genome is completely characterized, is a host for large DNA viruses, and present an analysis of the life-cycle and 186,234 bp long linear genome of OtV5. OtV5 is a lytic phycodnavirus which unexpectedly does not degrade its host chromosomes before the host cell bursts. Analysis of its complete genome sequence confirmed that it lacks expected site-specific endonucleases, and revealed the presence of 16 genes whose predicted functions are novel to this group of viruses. OtV5 carries at least one predicted gene whose protein closely resembles its host counterpart and several other host-like sequences, suggesting that horizontal gene transfers between host and viral genomes may occur frequently on an evolutionary scale. Fifty seven percent of the 268 predicted proteins present no similarities with any known protein in Genbank, underlining the wealth of undiscovered biological diversity present in oceanic viruses, which are estimated to harbour 200Mt of carbon

Structure and Inhibition of the SARS Coronavirus Envelope Protein Ion Channel

The envelope (E) protein from coronaviruses is a small polypeptide that contains at least one α-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA), but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV) that the transmembrane domain of E protein (ETM) forms pentameric α-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular α-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293) cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA), but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target

Linearized esculentin-2EM shows pH dependent antibacterial activity with an alkaline optimum

Here the hypothesis that linearized esculentin 2EM (E2EM-lin) from Glandirana emeljanovi possesses pH dependent activity is investigated. The peptide showed weak activity against Gram-negative bacteria (MLCs ≥ 75.0 μM) but potent efficacy towards Gram-positive bacteria (MLCs ≤ 6.25 μM). E2EM-lin adopted an α-helical structure in the presence of bacterial membranes that increased as pH was increased from 6 to 8 (↑ 15.5 to 26.9 %), while similar increases in pH enhanced the ability of the peptide to penetrate (↑ 2.3 to 5.1 mN m-1) and lyse (↑ 15.1 to 32.5%) these membranes. Theoretical analysis predicted that this membranolytic mechanism involved a tilted segment, that increased along the α-helical long axis of E2EM-lin (1-23) in the N → C direction, with - increasing overall from circa - 0.8 to - 0.3. In combination, these data showed that E2EM-lin killed bacteria via novel mechanisms that were enhanced by alkaline conditions and involved the formation of tilted and membranolytic, α-helical structure. The preference of E2EM-lin for Gram-positive bacteria over Gram-negative organisms was primarily driven by the superior ability of phosphatidylglycerol to induce α-helical structure in the peptide as compared to phosphatidylethanolamine. These data were used to generate a novel pore-forming model for the membranolytic activity of E2EM-lin, which would appear to be the first, major reported instance of pH dependent AMPs with alkaline optima using tilted structure to drive a pore-forming process. It is proposed that E2EM-lin has the potential for development to serve purposes ranging from therapeutic usage, such as chronic wound disinfection, to food preservation by killing food spoilage organisms