Integrin α6Bβ4 inhibits colon cancer cell proliferation and c-Myc activity

<p>Abstract</p> <p>Background</p> <p>Integrins are known to be important contributors to cancer progression. We have previously shown that the integrin β4 subunit is up-regulated in primary colon cancer. Its partner, the integrin α6 subunit, exists as two different mRNA splice variants, α6A and α6B, that differ in their cytoplasmic domains but evidence for distinct biological functions of these α6 splice variants is still lacking.</p> <p>Methods</p> <p>In this work, we first analyzed the expression of integrin α6A and α6B at the protein and transcript levels in normal human colonic cells as well as colorectal adenocarcinoma cells from both primary tumors and established cell lines. Then, using forced expression experiments, we investigated the effect of α6A and α6B on the regulation of cell proliferation in a colon cancer cell line.</p> <p>Results</p> <p>Using variant-specific antibodies, we observed that α6A and α6B are differentially expressed both within the normal adult colonic epithelium and between normal and diseased colonic tissues. Proliferative cells located in the lower half of the glands were found to predominantly express α6A, while the differentiated and quiescent colonocytes in the upper half of the glands and surface epithelium expressed α6B. A relative decrease of α6B expression was also identified in primary colon tumors and adenocarcinoma cell lines suggesting that the α6A/α6B ratios may be linked to the proliferative status of colonic cells. Additional studies in colon cancer cells showed that experimentally restoring the α6A/α6B balance in favor of α6B caused a decrease in cellular S-phase entry and repressed the activity of c-Myc.</p> <p>Conclusion</p> <p>The findings that the α6Bβ4 integrin is expressed in quiescent normal colonic cells and is significantly down-regulated in colon cancer cells relative to its α6Aβ4 counterpart are consistent with the anti-proliferative influence and inhibitory effect on c-Myc activity identified for this α6Bβ4 integrin. Taken together, these findings point out the importance of integrin variant expression in colon cancer cell biology.</p

Real-Time Imaging of HIF-1α Stabilization and Degradation

HIF-1α is overexpressed in many human cancers compared to normal tissues due to the interaction of a multiplicity of factors and pathways that reflect specific genetic alterations and extracellular stimuli. We developed two HIF-1α chimeric reporter systems, HIF-1α/FLuc and HIF-1α(ΔODDD)/FLuc, to investigate the tightly controlled level of HIF-1α protein in normal (NIH3T3 and HEK293) and glioma (U87) cells. These reporter systems provided an opportunity to investigate the degradation of HIF-1α in different cell lines, both in culture and in xenografts. Using immunofluorescence microscopy, we observed different patterns of subcellular localization of HIF-1α/FLuc fusion protein between normal cells and cancer cells; similar differences were observed for HIF-1α in non-transduced, wild-type cells. A dynamic cytoplasmic-nuclear exchange of the fusion protein and HIF-1α was observed in NIH3T3 and HEK293 cells under different conditions (normoxia, CoCl2 treatment and hypoxia). In contrast, U87 cells showed a more persistent nuclear localization pattern that was less affected by different growing conditions. Employing a kinetic model for protein degradation, we were able to distinguish two components of HIF-1α/FLuc protein degradation and quantify the half-life of HIF-1α fusion proteins. The rapid clearance component (t1/2 ∼4–6 min) was abolished by the hypoxia-mimetic CoCl2, MG132 treatment and deletion of ODD domain, and reflects the oxygen/VHL-dependent degradation pathway. The slow clearance component (t1/2 ∼200 min) is consistent with other unidentified non-oxygen/VHL-dependent degradation pathways. Overall, the continuous bioluminescence readout of HIF-1α/FLuc stabilization in vitro and in vivo will facilitate the development and validation of therapeutics that affect the stability and accumulation of HIF-1α

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Ostréiculture sous les tropiques

Narrateur: J. GroulxVersion anglaise disponible dans la Bibliothèque numérique du CRDI : Oyster farming in the tropicsVersion espagnole disponible dans la Bibliothèque numérique du CRDI: Cultivo de ostras en los tropicosFilm éducatif sur l'élevage des huîtres

Application of a Simple Method to Study Single-Particle Bioaerosols Including Preferential Aerosolization

<div><p>Bacteria, viruses, fungus, and other biological components (toxins, membranes, spores) can spread in the air through various aerosolization processes (breathing, bubbling, explosion, evaporation) and travel until they reach a surface or a host. Nosocomial diseases are an example of illnesses caused by a human contact with such pathogen vectors in hospital settings. Very little is known about the aerosolization processes of viruses and bacteria and their potential to infect people after their passage in the airborne state and about the microbial burden carried by individual aerosol particles. Here we propose a novel approach to study the aerosolization mechanisms of bacteria in single particles using fluorescence spectroscopy and a homemade system allowing the control of the aerosolization and the impaction of bacteria on a black filter. We validated the concept using <i>P. fluorescence</i> and <i>E. coli</i>. The results show that independently of the amount of <i>P. fluorescens</i> and <i>E. coli</i> aerosolized the average distribution of cells impacted on a black filter is described by a Poisson fit with λ ∼ 0.6 ± 0.2. This means that using this aerosolization process, an aerosol will present no bacterium, but when it does, the number of bacteria per particle in the distributions will more probably be one. We also observed that the aerosolization processes of these two bacterial species allow <i>P. fluorescens</i> to be preferentially aerosolized against <i>E. coli</i>. These results demonstrate that fluorescence spectroscopy is a powerful tool to study bioaerosols in single particles. This technique can be used to study several phenomena like preferential aerosolization.</p><p>Copyright 2015 American Association for Aerosol Research</p></div

Nanoscale aerovirology: An efficient yet simple method to analyze the viral distribution of single bioaerosols

<p>The aerosolization mechanisms of viruses are poorly known, because of the challenges related to their sampling and observation. For example, single particle studies are needed to improve our understanding of bioaerosol enrichment processes. Such studies would help to develop models of airborne disease propagation. We propose a novel approach to study viral aerosols in single particles using a combination of fluorescence and transmission electron microscopy (TEM). This method allows for rapid analysis of labeled bacteriophages aerosolized and captured on a black membrane filter. It also requires performing image analyses on fluorescent spots. TEM is necessary to determine a single bacteriophage dimensions. Thus, the clustering of bacteriophage PP01 in a single aerosol particle was investigated and found to give a comparable number of virions to what was observed with TEM. The impact of the GFP (green fluorescent protein) in the head of PP01 virion compared to wild type (WT) PP01 was also tested by comparing the clustering of similar bioaerosol sizes generated by the aerosolization of PP01 WT, PP01-GFP, and PP01-GFP labeled with syto-red dye. Surprisingly, the PP01 WT bioaerosols were enriched compared to the PP01-GFP ones (64.9 ± 17.5% more). PP01-GFPs were also found to be more numerous compared to those produced by PP01-GFP labeled with syto-red dye (28.9 ± 16.9% more). The aerosolization process might be dependent on the electrochemical properties of the viruses and the environment. Changes of this nature could affect the mechanism of the aerosol formation in natural forming aerosols as demonstrated in this study for artificially generated aerosols.</p> <p>© 2016 American Association for Aerosol Research</p