Cancer cells migration: a laser scanning confocal microscopy study

Introduction: Nowadays, everyone or almost everyone has seen a love one lose the “battle” against cancer. Even worth than that is to closely watch the pain and agony that current anti-cancer therapies cause to the patient, with a known outcome... Typical cancer therapies target the inhibition of the epidermal growth factor receptor, EGFR, a membrane receptor that plays a key role in regulating normal cellular processes such as cell survival, proliferation and migration. High expression of EGFR is generally associated with invasion, metastasis, late- stage disease, chemotherapy resistance, hormonal therapy resistance and poor general therapeutic outcome. In order to develop new approaches for cancer treatment it is necessary to understand it is necessary to study the morphology of cancer cells when exposed to different stimuli. As in the case of this study human cancer lung cells were stimulated with EGF and its behavior was monitored over time using Laser scanning confocal microscope (Zeiss; LSM780). Results: A set of human cancer lung cells were stimulated with its ligand EGF and another set of human cancer lung cells were not stimulated. The morphological changes were followed Figure 1 – Human lung cancer cells stimulated with of EGF. over time using confocal laser scanning microscopy (CLSM) with time elapse. As it can be seen on figure 1 the cancer cells morphology did significantly changed its phenotype. On the other hand when these cells were stimulated the morphological changes were significant as it can be seen on figure 2. The addition of EGF to the culture medium induce significant morphological changes, namely of loss of cell-cell junctions (b), formation of filipodia (b) and tissue disaggregation (c). Additionally, 3D reconstructions of singles cells were performed which allowed the confirmation of the internalization the EGFR dimers (data not shown). With time elapse laser scanning confocal microscopy it was possible to follow of the events that lead to cell migration, loss of cell-cell junction, filipodia formation and migration in real time

Biochemical characterization of highly stable endolysins with a powerful and broad anti-Gram-negative lytic activity in the presence of weak acids

EMBO Conference on Viruses of Microbes III: Structure and Function - from Molecules to Communities (Programme and Abstract Book)info:eu-repo/semantics/publishedVersio

A thermostable salmonella phage endolysin, Lys68, with broad bactericidal properties against gram-negative pathogens in presence of weak acids

Resistance rates are increasing among several problematic Gram-negative pathogens, a fact that has encouraged the development of new antimicrobial agents. This paper characterizes a Salmonella phage endolysin (Lys68) and demonstrates its potential antimicrobial effectiveness when combined with organic acids towards Gram-negative pathogens. Biochemical characterization reveals that Lys68 is more active at pH 7.0, maintaining 76.7% of its activity when stored at 4°C for two months. Thermostability tests showed that Lys68 is only completely inactivated upon exposure to 100°C for 30 min, and circular dichroism analysis demonstrated the ability to refold into its original conformation upon thermal denaturation. It was shown that Lys68 is able to lyse a wide panel of Gram-negative bacteria (13 different species) in combination with the outer membrane permeabilizers EDTA, citric and malic acid. While the EDTA/Lys68 combination only inactivated Pseudomonas strains, the use of citric or malic acid broadened Lys68 antibacterial effect to other Gram-negative pathogens (lytic activity against 9 and 11 species, respectively). Particularly against Salmonella Typhimurium LT2, the combinatory effect of malic or citric acid with Lys68 led to approximately 3 to 5 log reductions in bacterial load/CFUs after 2 hours, respectively, and was also able to reduce stationary-phase cells and bacterial biofilms by approximately 1 log. The broad killing capacity of malic/citric acid-Lys68 is explained by the destabilization and major disruptions of the cell outer membrane integrity due to the acidity caused by the organic acids and a relatively high muralytic activity of Lys68 at low pH. Lys68 demonstrates good (thermo)stability properties that combined with different outer membrane permeabilizers, could become useful to combat Gram-negative pathogens in agricultural, food and medical industry.This work was supported by the projects FCOMP-01-0124-FEDER-019446, FCOMP-01-0124-FEDER-027462 and PEst-OE/EQB/LA0023/2013 from "Fundacao para a Ciencia e Tecnologia" (FCT), Portugal. The authors thank the Project "BioHealth - Biotechnology and Bioengineering approaches to improve health quality", Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON. 2 - O Novo Norte), QREN, FEDER. Hugo Oliveira acknowledges the FCT grant SFRH/BD/63734/2009. Maarten Walmagh held a PhD scholarship of the IWT Vlaanderen. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript