FRET-Sensing of Multivalent Protein Binding at the Interface of Biomimetic Microparticles Functionalized with Fluorescent Glycolipids.

Cell adhesion is a central process in cellular communication and regulation. Adhesion sites are triggered by specific ligand-receptor interactions inducing the clustering of both partners at the contact point. Investigating cell adhesion using microscopy techniques requires targeted fluorescent particles with a signal sensitive to the clustering of receptors and ligands at the interface. Herein, we report on simple cell or bacterial mimics, based on liquid microparticles made of lipiodol functionalized with custom-designed fluorescent lipids. These lipids are targeted toward lectins or biotin membrane receptors, and the resulting particles can be specifically identified and internalized by cells, as demonstrated by their phagocytosis in primary murine bone marrow-derived macrophages. We also evidence the possibility to sense the binding of a multivalent lectin, concanavalin A, in solution by monitoring the energy transfer between two matching fluorescent lipids on the surface of the particles. We anticipate that these liquid particle-based sensors, which are able to report via Förster resonance energy transfer (FRET) on the movement of ligands on their interface upon protein binding, will provide a useful tool to study receptor binding and cooperation during adhesion processes such as phagocytosis.

EBV infection of primary colonic epithelial cells causes inflammation, DDR and autophagy dysregulation, effects that may predispose to IBD and carcinogenesis.

EBV is a gammaherpesvirus strongly associated to human cancer. The virus has been shown to play a role also in inflammatory diseases, including IBD, in the context of which colon cancer more frequently arise. In this study, we show for the first time that EBV infects primary colonic epithelial cells (HCoEpC), promotes pro-inflammatory cytokine secretion and activates molecular pathways bridging inflammation and cancer, such as ERK1/2. These effects, occurring in the course of the lytic phase of the viral life cycle, led to DDR and autophagy dysregulation. Such cellular responses, playing a key role in the maintenance of proteostasis and genome integrity, are essential to prevent carcinogenesis. Interestingly, we found that the use of the demethylating agent 5-AZA could counteract most of the effects induced by EBV infection in HCoEpC, suggesting that DNA hyper-methylation may strongly contribute to viral-driven inflammation and colon cancer predisposition.

3,4-Dihydroxyphenylethanol (DPE or Hydroxytyrosol) Counteracts ERK1/2 and mTOR Activation, Pro-Inflammatory Cytokine Release, Autophagy and Mitophagy Reduction Mediated by Benzo[a]pyrene in Primary Human Colonic Epithelial Cells.

Understanding the effects induced by carcinogens on primary colonic epithelial cells and how to counteract them might help to prevent colon cancer, which is one of the most frequent and aggressive cancers. In this study, we exposed primary human colonic epithelial cells (HCoEpC) to Benzo[a]pyrene (B[a]P) and found that it led to an increased production of pro-inflammatory cytokines and activated ERK1/2 and mTOR. These pathways are known to be involved in inflammatory bowel disease (IBD), which represents a colon cancer risk factor. Moreover, B[a]P reduced autophagy and mitophagy, processes whose dysregulation has been clearly demonstrated to predispose to cancer either by in vitro or in vivo studies. Interestingly, all the effects induced by B[a]P could be counteracted by 3,4-Dihydroxyphenylethanol (DPE or Hydroxytyrosol, H), the most powerful anti-inflammatory and antioxidant compound contained in olive oil. This study sheds light on the mechanisms that could be involved in colon carcinogenesis induced by a chemical carcinogen and identifies a safe natural product that may help to prevent them.

Lovastatin reduces PEL cell survival by phosphorylating ERK1/2 that blocks the autophagic flux and engages a cross-talk with p53 to activate p21.

Statins are inhibitors of the mevalonate pathway that besides being cholesterol lowering agents, display anti-cancer properties. This is because cholesterol is an essential component of cell membranes but also because the mevalonate pathway controls protein farnesylation and geranylation, processes essential for the activity of GTPase family proteins. In this study, we found that Lovastatin exerted a dose- and time-dependent cytotoxic effect against PEL cells, an aggressive B cell lymphoma strictly associated with the gammaherpesvirus KSHV and characterized by a poor response to conventional chemotherapies. At molecular level, Lovastatin by dephosphorylating STAT3, induced ERK1/2 activation that inhibited autophagy and phosphorylated p53ser15 that in turn maintained ERK1/2 activated and up-regulated p21. However, p21 played a pro-survival role in this setting, as its inhibition by UC2288 further reduced cell survival in PEL cells undergoing Lovastatin treatment. In conclusion, this study suggests that Lovastatin may represent a valid therapeutic alternative against PEL cells, especially if used in combination with p21 inhibitors.

Determination of glyphosate residue in maize and rice using a fast and easy method involving liquid chromatography-mass spectrometry (LC/MS/MS).

A fast and easy method was developed for the determination of glyphosate in maize and rice by using liquid chromatography triple quadrupole mass spectrometry with a Dionex Ion Pack column and phosphate buffer mobile phase. Samples were extracted with an acidified methanol solution. An isotope-labeled internal standard was added to the sample before extraction to ensure accurate tracking and quantification. The method's performance was evaluated through a series of assessments to determine the accuracy, precision, linearity, matrix effect, limit of detection (LOD), and limit of quantification (LOQ). The mean recoveries for both matrices were within 70-105% at three fortification levels, including the LOQ. The precision for replicates was <20% (RSD%) for both matrices. Good linearity (R2=0.9982) was obtained over the concentration range of 0.01-1.5 mg kg-1. The LOD was determined to be 0.002 mg kg-1 for rice and 0.004 mg kg-1 for maize. The LOQ was 0.01 mg kg-1 for both maize and rice. Due to its versatility, the proposed method could be considered useful for the determination of glyphosate in cereals in routine analysis.