Lipotoxic stress alters the membrane lipid profile of extracellular vesicles released by Huh-7 hepatocarcinoma cells.

Extracellular vesicles (EVs) are well-known mediators in intercellular communication playing pivotal roles in promoting liver inflammation and fibrosis, events associated to hepatic lipotoxicity caused by saturated free fatty acid overloading. However, despite the importance of lipids in EV membrane architecture which, in turn, affects EV biophysical and biological properties, little is known about the lipid asset of EVs released under these conditions. Here, we analyzed phospholipid profile alterations of EVs released by hepatocarcinoma Huh-7 cells under increased membrane lipid saturation induced by supplementation with saturated fatty acid palmitate or Δ9 desaturase inhibition, using oleate, a nontoxic monounsaturated fatty acid, as control. As an increase of membrane lipid saturation induces endoplasmic reticulum (ER) stress, we also analyzed phospholipid rearrangements in EVs released by Huh-7 cells treated with thapsigargin, a conventional ER stress inducer. Results demonstrate that lipotoxic and/or ER stress conditions induced rearrangements not only into cell membrane phospholipids but also into the released EVs. Thus, cell membrane saturation level and/or ER stress are crucial to determine which lipids are discarded via EVs and EV lipid cargos might be useful to discriminate hepatic lipid overloading and ER stress.

Evidence of DMSO-Induced Protein Aggregation in Cells.

We report on a study of protein aggregation induced on different cell samples by dimethyl sulfoxide (DMSO) addition. DMSO is the most commonly used cryoprotectant because it is supposed to readily diffuse across lipid bilayers, thus reducing water activity within cells; despite its large use, the mechanism of penetration and even the main interaction features with cell components are far from being understood. In the present work, infrared absorption spectroscopy is successfully applied to real time detection of chemical and structural changes occurring in cells during dehydration from water and water/DMSO suspensions. As a most interesting result, DMSO is observed to favor protein aggregation both in cellular model systems, as cultured lymphocytes and fibroblasts, and in human samples for clinic use, as hematopoietic stem cells from cord blood. This effect is evidenced at low water content, analogously to what is observed for protein solutions. Such tendency is not specific of the type of protein and suggests one possible origin of DMSO toxicity.

Cryopreservation of cells: FT-IR monitoring of lipid membrane at freeze-thaw cycles.

In the present study, FTIR spectroscopy was used to monitor the freeze-thaw cycle of two cellular lines (HuDe and Jurkat) suspended in three different media: phosphate buffer solution (PBS); dimethylsulfoxide (DMSO)/PBS solution at 0.1 DMSO molar fraction; and CryoSure (0.1 DMSO molar fraction PBS solution+dextran 5% w/v) solution. The Trypan Blue test was also applied before freezing and after thawing each cell sample to estimate the recovery of membrane integrity after thermal treatment, and correlate this datum with spectroscopic results. By following the temperature evolution of two different spectral components (the libration and bending combination mode νc(H2O) at 2000-2500 cm(-1), and the methylene symmetric stretching vibration νsym(CH2) at about 2850 cm(-1)) in the -120÷28°C range, we evidenced the main transition of lipid membrane in connection with cell dehydration, as induced by ice formation in the extracellular medium. In particular, in DMSO/PBS and CryoSure samples we observed a transition to a more rigid state of the lipid membrane together with an increased amount of non-freezable water in the extracellular medium; these results are connected to the role of DMSO as a cryoprotective agent irrespective of the nature of cell type.

Raman micro-spectroscopy study of living SH-SY5Y cells adhering on different substrates.

In this paper we test the ability of Raman micro-spectroscopy and Raman mapping to investigate the status of cells grown in adhesion on different substrates. The spectra of immortalized SH-SY5Y cells, grown on silicon and on metallic substrates are compared with those obtained for the same type of cells adhering on organic polyaniline (PANI), a memristive substrate chosen to achieve a living bio-hybrid system. Raman spectra give information on the status of the single cell, its local biochemical composition, and on the modifications induced by the substrate interaction. The good agreement between Raman spectra collected from cells adhering on different substrates confirms that the PANI, besides allowing the cell growth, doesn't strongly affect the general biochemical properties of the cell. The investigation of the cellular state in a label free condition is challenging and the obtained results confirm the Raman ability to achieve this information.

Changes in Lipid Composition During Manganese-Induced Apoptosis in PC12 Cells.

Lipid composition of membranes is fundamental to modulate signaling pathways relying on lipid metabolites and/or membrane proteins, thus resulting in the regulation of important cell processes such as apoptosis. In this case, membrane remodeling is an early event important for the activation of signaling leading to cell death and removal of apoptotic cells. In the present study, we analyzed phospholipid, cholesterol and fatty acid content during apoptosis induced by manganese in PC12 cells. Lipid analysis of whole cells and detergent-resistant membranes was carried out by HPLC/GC. Results showed that apoptosis is associated with changes in lipid composition detectable in whole cell extracts, namely cholesterol, phosphatidylserine and phosphatidylethanolamine decreases. Noteworthy, phosphatidylserine level reduction was detectable before to the detection of apoptosis, in correlation with our previous study carried out by radioactive labelling. By contrast, phosphatidylserine and phosphatidylethanolamine changes were not detected in detergent resistant membranes, which instead showed an altered composition in phosphatidylinositol, phosphatidylcholine and sphingomyelin in apoptotic cells.

Targeted gene transduction of mammalian cells expressing the HER2/neu receptor by filamentous phage.

Screening a random peptide library displayed on phage as fusion to the major capsid protein pVIII identified a ligand binding the human epidermal growth factor receptor 2 (HER2) specifically. By mutating the sequence of this ligand, a "secondary" library was generated, whose panning on HER2-positive cells isolated a phage-borne peptide with increased specific binding to HER2 (phage NL1.1). The same peptide recognised HER2 specifically when expressed as an N-terminal fusion to the minor coat protein pIII. Phage NL1.1 was engineered to include a mammalian expression cassette for a reporter gene within its genome. This modified phage transduced HER2-expressing cells with very high specificity (more than 1000-fold that of parental HER2-negative cells) and with an efficiency comparable to that of chemical transfection protocols. The gene delivery process was remarkably fast, requiring less than 15 minutes incubation of phage with target cells to generate detectable levels of gene expression.

Phage as gene delivery vectors.

Bacteriophage can be considered as a natural system to efficiently condense and package DNA. They tolerate many different types of mutations, including those that lead to the display of polypeptides as a fusion to any of the structural proteins comprising the phage particle. In addition, they are a powerful biological system for generating and screening mutants with the desired functional properties. It has also been shown that phage vectors can be engineered for receptor-mediated gene transfer to mammalian cells. The attractive features offered by this system have paved the way for various attempts to develop phage as a vector for gene therapy applications.

'In vitro evolution' of ligands for HCV-specific serum antibodies.

We developed a strategy to improve the properties of ligands selected from phage-displayed random peptide libraries. A site-directed mutagenesis protocol that introduces mutations and extends the size of a target sequence has been set up to generate diversity in a single or in a population of clones. The pool of mutants thus created is screened to identify variants with the desired properties. We refer to this strategy as in vitro evolution' of ligands. Here we report the application of this in vitro evolution protocol to the identification of improved ligands for HCV-specific serum antibodies. A single clone or population of clones were processed to generate a secondary library. Screening of these libraries with sera from HCV-infected patients identified peptides with an enhanced and broadened ability to detect HCV-specific serum antibodies.

"Affinity maturation" of ligands for HCV-specific serum antibodies.

We have previously screened a phage-displayed random peptide library using sera from patients and identified ligands binding to antibodies specifically associated with the hepatitis C virus infection. The ability of these peptides to detect HCV-specific antibodies was improved through an in vitro procedure which mimics the natural process of antibody affinity maturation operating in secondary immune response. Libraries were generated by mutating the sequence of the original peptide through a protocol that efficiently introduced substitution, insertion and deletion mutations on a single or population of clones. Screening these libraries isolated mutants that displayed increased specific reactivity with a broader range of sera from HCV-infected patients. Several variants of the original peptide were identified which discriminate between the various components of the specific polyclonal response. This methodology to select artificial ligands from RPL using sera and to enhance their diagnostic properties by affinity maturation makes the development of a diagnostic assay to detect disease-associated antibodies feasible, without requiring the natural antigen.

Differential scanning calorimetry of chromatin at different levels of condensation.

The thermal denaturation of calf thymus total chromatin and of fractions enriched in heterochromatin or euchromatin, has been investigated by differential scanning calorimetry and compared to that of calf thymus DNA and DNA-histone complexes. In our experimental conditions, chromatin melts in three thermal transitions: the main one, assigned to separation of the DNA double helix, occurs at 83 degrees C, while the other two occur at 63 degrees C and 74 degrees C. The data show that: (a) the transition enthalpy for denaturation of DNA in the total chromatin and in DNA-histone complexes is nearly the same as that of DNA in solution; (b) the transition at 63 degrees C is present in the thermogram of the heterocromatin enriched fraction, while it is completely absent in that of the euchromatin enriched one. The results suggest that this transition can be attributed to the higher order structures of heterochromatin.

DNA-based selection and screening of peptide ligands.

Phage display selection strategies rely on the physical link between the displayed heterologous protein ligand and the DNA encoding it. Thus, genes expressing a ligand with a specific binding affinity can be selected rapidly. To improve the specificity and sensitivity of this technology for potential use in identifying ligands to a specific antibody present in a complex mixture, we incorporated a DNA selection step along with the phage display technology. Ligands for hepatitis C virus (HCV) antibodies present in serum were identified by panning a phage-displayed random peptide library against pools of serum HCV antibodies. An additional DNA hybridization screening step using single-stranded DNA isolated from one of the pools increased the specificity and sensitivity, resulting in the selection of an HCV antibody ligand with diagnostic potential.