Near-Infrared Optical Imaging of Nucleic Acid Nanocarriers In Vivo.

Noninvasive, real-time optical imaging methods are well suited to follow the in vivo distribution of nucleic acid nanocarriers, their dissociation and the resulting gene expression or inhibition. Indeed, most small animal imaging devices are performing bioluminescence and fluorescence measurements without moving the animal, allowing a simple, rapid, and cost-effective method of investigation of several parameters at a time, in longitudinal experiments that can last for days or weeks.Here we help the reader in choosing adapted near-infrared (NIR) fluorophores or pairs of fluorophores for FRET assays, imaging of reporter genes as well as nanocarriers for in vivo gene and siRNA delivery.

Sticky siRNAs targeting survivin and cyclin B1 exert an antitumoral effect on melanoma subcutaneous xenografts and lung metastases.

Background: Melanoma represents one of the most aggressive and therapeutically challenging malignancies as it often gives rise to metastases and develops resistance to classical chemotherapeutic agents. Although diverse therapies have been generated, no major improvement of the patient prognosis has been noticed. One promising alternative to the conventional therapeutic approaches currently available is the inactivation of proteins essential for survival and/or progression of melanomas by means of RNA interference.

Systemic delivery of sticky siRNAs targeting the cell cycle for lung tumor metastasis inhibition.

RNA interference allows the design of new inhibitors that target deregulated pathways in cancer. However systemic delivery of siRNA for the treatment of solid tumors still remains an issue. In our study, in order to suppress the progression of lung cancer metastasis in mice, we developed sticky siRNA (ssiRNA) to inhibit survivin and cyclin B1, two candidates involved in cell survival and proliferation.

Inhibition of Sox2 Expression in the Adult Neural Stem Cell Niche In Vivo by Monocationic-based siRNA Delivery.

RNA interference (RNAi) is a major tool for basic and applied investigations. However, obtaining RNAi data that have physiological significance requires investigation of regulations and therapeutic strategies in appropriate in vivo settings. To examine in vivo gene regulation and protein function in the adult neural stem cell (NSC) niche, we optimized a new non-viral vector for delivery of siRNA into the subventricular zone (SVZ).

Conjugating phosphospermines to siRNAs for improved stability in serum, intracellular delivery and RNAi-mediated gene silencing.

siRNAs are usually formulated with cationic polymers or lipids to form supramolecular particles capable of binding and crossing the negatively charged cell membrane. However, particles hardly diffuse through tissues when administered in vivo. We therefore are developing cationic siRNAs, composed of an antisense sequence annealed to an oligophosphospermine-conjugated sense strand.

Near-infrared optical imaging of nucleic acid nanocarriers in vivo.

Noninvasive, real-time optical imaging methods are well suited to follow the in vivo distribution of nucleic acid nanocarriers, their dissociation, and the resulting gene expression or inhibition. Indeed, most small animal imaging devices perform bioluminescence and fluorescence measurements without moving the animal, allowing a simple, rapid, and cost-effective method of investigation of several parameters at a time, in longitudinal experiments that can last for days or weeks.Here we help the reader in choosing adapted near-infrared (NIR) fluorophores or pairs of fluorophores for Förster resonance energy transfer assays, imaging of reporter genes, as well as nanocarriers for in vivo gene and siRNA delivery.

An amphiphilic dendrimer for effective delivery of small interfering RNA and gene silencing in vitro and in vivo.

An amphiphilic dendrimer bearing a hydrophobic alkyl chain and hydrophilic poly(amidoamine) dendrons is able to combine the advantageous features of lipid and dendrimer vectors to deliver a heat shock protein 27 siRNA and produce potent gene silencing and anticancer activity in vitro and in vivo in a prostate cancer model. This dendrimer can be used alternatively for treating various diseases.

Abnormal Wnt and PI3Kinase signaling in the malformed intestine of lama5 deficient mice.

Laminins are major constituents of basement membranes and are essential for tissue homeostasis. Laminin-511 is highly expressed in the intestine and its absence causes severe malformation of the intestine and embryonic lethality. To understand the mechanistic role of laminin-511 in tissue homeostasis, we used RNA profiling of embryonic intestinal tissue of lama5 knockout mice and identified a lama5 specific gene expression signature.

Mesomorphic imidazolium salts: new vectors for efficient siRNA transfection.

The preparation of chloride (1(n)) and bromide (2(n)) derivatives of 1-methyl-3-[3,4-bis(alkoxy)benzyl]-4H-imidazolium with n = 6, 12, 16, 18 is described. The two series of salts possess a rich thermotropic mesomorphism, chain-length dependent. Thus, a lamellar smectic A phase, a bicontinuous cubic Ia3d phase, and a columnar hexagonal liquid crystalline mesophase are induced as a function of increasing chain length.

Tissue recombinants to study extracellular matrix targeting to basement membranes.

Several techniques have been used to study the expression of basement membranes molecules but none of them allow distinguishing the cellular origin of the deposition of a single molecule at the subepithelial basement membrane. For this purpose, we designed an experimental model using recombinants between chick and mouse embryonic intestines. Following constructions of interspecies endodermal/mesenchymal associations in culture, developmental growth was achieved by in vivo transplantation in the chick embryo.

Systemic delivery of DNA or siRNA mediated by linear polyethylenimine (L-PEI) does not induce an inflammatory response.

Purpose: The success of nucleic acid therapies depends upon delivery vehicle's ability to selectively and efficiently deliver therapeutic nucleic acids to target organ with minimal toxicity. The cationic polymer polyethylenimine (PEI) has been widely used for nucleic acid delivery due to its versatility and efficiency. In particular, the last generation of linear PEI (L-PEI) is being more efficient in vivo than the first generation of branched PEI.

Sticky overhangs enhance siRNA-mediated gene silencing.

siRNA delivery to cells offers a convenient and powerful means of gene silencing that bypasses several barriers met by gene delivery. However, nonviral vectors, and especially polymers, form looser complexes with siRNA than with plasmid DNA. As a consequence, exchange of siRNA for larger polymeric anions such as proteoglycans found outside cells and at their surface may occur and lower delivery.

Glycated polyelectrolyte multilayer films: differential adhesion of primary versus tumor cells.

Glycated polymers have already been widely employed for cell transfection studies, as cells possess specific lectins. However, up to now, these glycated polymers have barely been investigated for their cell adhesive properties, save macrophages. In this work, we use polyelectrolyte multilayer films made of poly(L-lysine) and poly(L-glutamic) acid as polymeric substrates to investigate the role of sugar molecules (e.

Antifungal coating by biofunctionalized polyelectrolyte multilayered films.

The surface of medical devices is a common site of bacterial and fungal adhesion, first step to the constitution of a resistant biofilm leading frequently to chronic infections. In order to prevent such complications, several physical and chemical modifications of the device surface have been proposed. Here, we experiment a new type of topical antifungal coating using the layer-by-layer technique.

Molecular cloning and characterization of mouse LITAF cDNA: role in the regulation of tumor necrosis factor-alpha (TNF-alpha) gene expression.

The inflammatory response to bacteria and bacterial products, such as lipopolysaccharides (LPSs), is mediated by a variety of secreted factors, but cytotoxic effects of LPS have been ascribed to the tumor necrosis factor alpha (TNF-alpha) activity. TNF-alpha is probably the most pleiotropic cytokine and, given the deleterious effects to the host of this factor, it has been postulated that its expression must be tightly regulated. Our laboratory has recently isolated, cloned and characterized a novel human transcription factor named LITAF or LPS-induced TNF-alpha factor.

Expression of RNAs encoding for alpha and beta integrin subunits in periodontitis and in cyclosporin A gingival overgrowth.

Background: Variation of integrin expression in healthy and diseased gingiva revealed a potential biological role for these cell matrix receptors during gingival remodeling.

Aim: Here we determined the level of RNA and tissue localization of different integrin subunits in periodontitis and cyclosporin A-induced gingival overgrowth.

Methods: The level of expression was determined by Reverse Transcriptase Polymerase Chain Reaction in 12 periodontitis-affected patients, four patients exhibiting severe cyclosporin A-induced gingival overgrowth and seven healthy patients as controls.

Kruppel-like factors regulate the Lama1 gene encoding the laminin alpha1 chain.

Laminin-1 (alpha1beta1gamma1), a basement membrane (BM) constituent, has been associated with differentiation processes and also with malignant progression. In the intestinal tissue, the alpha1 chain is expressed and secreted in the subepithelial BM during the developmental period; in the adult rodent tissue, it is restricted to the BM of the dividing cells. To understand how laminin alpha1 chain expression is regulated, we cloned and characterized a 2-kb promoter region of the Lama1 mouse gene.

Laminin alpha5 chain is required for intestinal smooth muscle development.

Laminins (comprised of alpha, beta, and gamma chains) are heterotrimeric glycoproteins integral to all basement membranes. The function of the laminin alpha5 chain in the developing intestine was defined by analysing laminin alpha5(-/-) mutants and by grafting experiments. We show that laminin alpha5 plays a major role in smooth muscle organisation and differentiation, as excessive folding of intestinal loops and delay in the expression of specific markers are observed in laminin alpha5(-/-) mice.

Expression of matrix metalloproteinases in healthy and diseased human gingiva.

Background, Aims: The aim of our study was to investigate the patterns of several metalloproteinases (MMP-1, MMP-2 and MT1-MMP) mRNAs expression using a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and to correlate them with clinical parameters and bacteriological diagnosis in healthy versus diseased human gingiva.

Methods: To identify the cell origin of MMP production, in situ hybridization (ISH) was also performed for the MMPs on the same samples. 17 gingival biopsies were collected (13 affected by advanced periodontitis and 4 healthy used as controls) and plaque index, gingival index, pocket depth and bleeding on probing were measured.

Expression of mRNAs encoding for alpha and beta integrin subunits, MMPs, and TIMPs in stretched human periodontal ligament and gingival fibroblasts.

The biological mechanisms of tooth movement result from the cellular responses of connective tissues to exogenous mechanical forces. Among these responses, the degradation of the extracellular matrix takes place, but the identification of the molecular basis as well as the components implicated in this degradation are poorly understood. To contribute to this identification, we subjected human fibroblasts obtained from the periodontal ligament (PDLs) and from the gingiva (HGFs) to a continuous stretch to quantify the mRNAs encoding for various metalloproteinases (MMPs), their tissue inhibitors (TIMPs), and alpha and beta integrin subunits.

X-twi is expressed prior to gastrulation in presumptive neurectodermal and mesodermal cells in dorsalized and ventralized Xenopus laevis embryos.

Early X-twi expression has been now investigated from egg laying to the early neurulation stages in Xenopus embryos, using both in situ hydridization and the more sensitive techniques of RT-PCR. We show that in unfertilized eggs, a decreasing gradient of X-twi transcript distribution is observed from animal to vegetative caps. X-twi RNA can be weakly detected at stages prior to gastrulation, and with increased intensity from stage 8 onwards.

The variable expressivity and incomplete penetrance of the twist-null heterozygous mouse phenotype resemble those of human Saethre-Chotzen syndrome.

Most targeted gene mutations are recessive and analyses of gene function often focus on homozygous mutant phenotypes. Here we describe parts of the expression pattern of M-twist in the head of developing wild-type mice and present our analysis of the phenotype of heterozygous twist- null animals at around birth and in adults. A number of twist -null heterozygous mice present skull and limb defects and, in addition, we observed other malformations, such as defects in middle ear formation and the xyphoïd process.

Mutations of the TWIST gene in the Saethre-Chotzen syndrome.

Saethre-Chotzen syndrome (acrocephalo-syndactyly type III, ACS III) is an autosomal dominant craniosynostosis with brachydactyly, soft tissue syndactyly and facial dysmorphism including ptosis, facial asymmetry and prominent ear crura. ACS III has been mapped to chromosome 7p21-22. Of interest, TWIST, the human counterpart of the murine Twist gene, has been localized on chromosome 7p21 as well.