Nicole Boggetto

Deciphering the mechanisms of cellular uptake of engineered nanoparticles by accurate evaluation of internalization using imaging flow cytometry.

BackgroundThe uptake of nanoparticles (NPs) by cells remains to be better characterized in order to understand the mechanisms of potential NP toxicity as well as for a reliable risk assessment. Real NP uptake is still difficult to evaluate because of the adsorption of NPs on the cellular surface.ResultsHere we used two approaches to distinguish adsorbed fluorescently labeled NPs from the internalized ones. The extracellular fluorescence was either quenched by Trypan Blue or the uptake was analyzed using imaging flow cytometry. We used this novel technique to define the inside of the cell to accurately study the uptake of fluorescently labeled (SiO2) and even non fluorescent but light diffracting NPs (TiO2). Time course, dose-dependence as well as the influence of surface charges on the uptake were shown in the pulmonary epithelial cell line NCI-H292. By setting up an integrative approach combining these flow cytometric analyses with confocal microscopy we deciphered the endocytic pathway involved in SiO2 NP uptake. Functional studies using energy depletion, pharmacological inhibitors, siRNA-clathrin heavy chain induced gene silencing and colocalization of NPs with proteins specific for different endocytic vesicles allowed us to determine macropinocytosis as the internalization pathway for SiO2 NPs in NCI-H292 cells.ConclusionThe integrative approach we propose here using the innovative imaging flow cytometry combined with confocal microscopy could be used to identify the physico-chemical characteristics of NPs involved in their uptake in view to redesign safe NPs.

G4 motifs affect origin positioning and efficiency in two vertebrate replicators

DNA replication ensures the accurate duplication of the genome at each cell cycle. It begins at specific sites called replication origins. Genome‐wide studies in vertebrates have recently identified a consensus G‐rich motif potentially able to form G‐quadruplexes (G4) in most replication origins. However, there is no experimental evidence to demonstrate that G4 are actually required for replication initiation. We show here, with two model origins, that G4 motifs are required for replication initiation. Two G4 motifs cooperate in one of our model origins. The other contains only one critical G4, and its orientation determines the precise position of the replication start site. Point mutations affecting the stability of this G4 in vitro also impair origin function. Finally, this G4 is not sufficient for origin activity and must cooperate with a 200‐bp cis‐regulatory element. In conclusion, our study strongly supports the predicted essential role of G4 in replication initiation.

Interactions between Magnetic Nanowires and Living Cells : Uptake, Toxicity and Degradation

We report on the uptake, toxicity, and degradation of magnetic nanowires by NIH/3T3 mouse fibroblasts. Magnetic nanowires of diameters 200 nm and lengths between 1 and 40 μm are fabricated by controlled assembly of iron oxide (γ-Fe(2)O(3)) nanoparticles. Using optical and electron microscopy, we show that after 24 h incubation the wires are internalized by the cells and located either in membrane-bound compartments or dispersed in the cytosol. Using fluorescence microscopy, the membrane-bound compartments were identified as late endosomal/lysosomal endosomes labeled with lysosomal associated membrane protein (Lamp1). Toxicity assays evaluating the mitochondrial activity, cell proliferation, and production of reactive oxygen species show that the wires do not display acute short-term (<100 h) toxicity toward the cells. Interestingly, the cells are able to degrade the wires and to transform them into smaller aggregates, even in short time periods (days). This degradation is likely to occur as a consequence of the internal structure of the wires, which is that of a noncovalently bound aggregate. We anticipate that this degradation should prevent long-term asbestos-like toxicity effects related to high aspect ratio morphologies and that these wires represent a promising class of nanomaterials for cell manipulation and microrheology.

Metal-organic compounds: a new approach for drug discovery. N1-(4-methyl-2-pyridyl)-2,3,6-trimethoxybenzamide copper(II) complex as an inhibitor of human immunodeficiency virus 1 protease.

The use of metal-organic complexes is a potentially fruitful approach for the development of novel enzyme inhibitors. They hold the attractive promise of forming stronger attachments with the target by combining the co-ordination ability of metals with the unique stereoelectronic properties of the ligand. We demonstrated that this approach can be successfully used to inhibit the protease of the human immunodeficiency virus (type 1). Several ligands bearing substituents designed to interact with the catalytic site of the enzyme when complexed to Cu(2+) were synthesised. The inhibition pattern of the resulting copper(II) complexes was analysed. We showed that the copper(II) complex of N1-(4-methyl-2-pyridyl)-2,3,6-trimethoxybenzamide (C1) interacts with the active site of the enzyme leading to competitive inhibition. On the other hand, N2-pyridine-amide ligands and oxazinane carboxamide ligand were found to be poor chelators of the cupric ion under the enzymatic assay conditions. In these cases, the observed inhibition was attributed to released cupric ions which react with cysteine residues on the surface of the protease. While unchelated metal cations are not likely to be useful agents, metal chelates such as C1 should be considered as promising lead compounds for the development of targeted drugs.

REGULATION OF PHOSPHORIBULOKINASE AND GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE IN A FRESHWATER DIATOM, ASTERIONELLA FORMOSA

The regulation of phosphoribulokinase (PRK) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was investigated in a freshwater pennate diatom, Asterionella formosa Hassall, and compared to the well‐studied chlorophyte Chlamydomonas reinhardtii P. A. Dang. As has been reported for a marine centric diatom, in A. formosa, PRK was not regulated by reduction with dithiothreitol (DTT) apart from a weak induction in the presence of NADPH and DTT. However, NADPH‐GAPDH was strongly activated when reduced, in contrast to a previous report on a diatom. Surprisingly, it was inhibited by NADPH, unlike in C. reinhardtii, while NADH‐GAPDH was not affected. NADH‐GAPDH was also strongly activated by DTT in contrast to most other photosynthetic cells. In A. formosa, unlike C. reinhardtii, 1,3‐bisphosphoglycerate, the substrate of GAPDH, activated this enzyme, even in the absence of DTT, when using both NADH and NADPH as cofactors. Some of these kinetic behaviors are consistent with regulation by protein–protein interactions involving CP12, a small protein that links PRK and GAPDH in cyanobacteria, green algae, and higher plants. This conclusion was supported by immunodetection of CP12 in crude extracts of A. formosa, using antibodies raised against CP12 from C. reinhardtii. This is the first report of the existence of CP12 in a diatom, but CP12 may be a common feature of diatoms since a bioinformatic search suggested that it was also present in the Thalassiosira pseudonana Hasle et Heimdal genome v3.0. Despite the presence of CP12, this work provides further support for the differential regulation of Calvin cycle enzymes in diatoms compared to green algae.

USF Binding Sequences from the HS4 Insulator Element Impose Early Replication Timing on a Vertebrate Replicator

A combination of cis-regulatory elements can impose the formation of an early replicating domain in a naturally late replicating region and might constitute the basic unit of early replicating domains.

Thyroxine-derivatives of lipopeptides: bifunctional dimerization inhibitors of human immunodeficiency virus-1 protease

The structure of new lipopeptides targeting the enzymic dimer interface have been rationally improved resulting in dimerization inhibitors of the human immunodeficiency virus 1 protease (K(id)=5nM for the best inhibitor). The contribution of each amino acid in inhibitory 3-mer lipopeptides was analyzed demonstrating that the C-terminal amino acid residue may preferably be replaced by thyroxine and thyronine. The negative charge of Glu is not essential. Lengthening of the peptidic chain may lead to a decrease of efficiency and a change in the mechanism (competitive inhibition instead of dimerization inhibition). The N-terminal blocking group can be replaced by 2-aminopalmitic acid. The mechanism of inhibition has been ascertained using Zhangs kinetic analysis combined with a physical method based on binding of 1-anilino-8-naphtalene sulfonate to enzyme. By targeting the hydrophobic pocket and the interface antiparallel beta-sheet found relatively free of mutations in contrary to the active site, these efficient dimerization inhibitors may provide a way of overcoming the drug resistances observed with therapeutic antiproteases that bind to the active site.