Chemical vectors for gene delivery: uptake and intracellular trafficking.

Chemical vectors for non-viral gene delivery are based on engineered DNA nanoparticles produced with various range of macromolecules suitable to mimic some viral functions required for gene transfer. Many efforts have been undertaken these past years to identify cellular barriers that have to be passed for this issue. Here, we summarize the current status of knowledge on the uptake mechanism of DNA nanoparticles made with polymers and liposomes, their endosomal escape, cytosolic diffusion, and nuclear import of pDNA.

Selective gene delivery in dendritic cells with mannosylated and histidylated lipopolyplexes.

We report for the first time preparation of mannosylated and histidylated lipopolyplexes (Man-LPD100) with uptake and transfection selectivity for dendritic cells (DCs). Man-LPD100 were prepared by addition of mannosylated and histidylated liposomes (Man-Lip100) on preformed PEGylated histidylated polylysine/DNA polyplexes. Man-Lip100 comprised a cationic [O,O-dioleyl-N-(3N-(N-methylimidazolium iodide)propylene) phosphoramidate)] lipid, a neutral [O,O-dioleyl-N-histamine Phosphoramidate] co-lipid and β-D-mannopyranosyl-N-dodecylhexadecanamide (Man-lipid).

Transient transmembrane release of green fluorescent proteins with sonoporation.

Microbubbles under ultrasound (US) activation are assumed to induce pore formation in the plasma membrane, causing its permeabilization and hence molecule incorporation from the extracellular environment. In this study, we investigated whether this permeabilization also engenders a transient release of small molecules from the cytosol of mammalian eukaryotic cells under the combined action of US and microbubbles. Using Hela cells stably expressing the enhanced green fluorescent protein (EGFP) gene, the release of EGFP was evaluated by flow cytometry in terms of the percentage of EGFP-positive cells (EGFP + cells) and the mean cell fluorescence intensity (MFI).

Molecular structure of tail tendon fibers in TIEG1 knockout mice using synchrotron diffraction technology.

The purpose of this study was to characterize the effect of TIEG1 on the molecular structure of collagen within tail tendon fibers using 3-mo-old female C57BL/6 wild-type (WT) and TIEG1 KO mice. Synchrotron X-ray microdiffraction experiments were carried out on single tendon fibers extracted from the WT and TIEG1 KO dorsal tail tendon. The fibers were scanned in the radial direction, and X-ray patterns were obtained.

Uptake of functionalized mesoporous silica nanoparticles by human cancer cells.

Mesoporous silica nanoparticles (MSN) were functionalised by aminofluorescein (AMF) with diethylenetriaminepentaacetic acid spacer molecules which provide free carboxylic groups for binding cell-specific ligands such as folate. AMF allowed the exploration of cellular uptake by HeLa cells using confocal microscopy and flow cytometry. The functionalized nanoparticles (MSN-AMF) penetrated efficiently into HeLa cell cytoplasm through a clathrin dependent endocytosis mechanism.

Accelerated Achilles tendon healing by PDGF gene delivery with mesoporous silica nanoparticles.

We report the ability of amino- and carboxyl-modified MCM-41 mesoporous silica nanoparticles (MSN) to deliver gene in vivo in rat Achilles tendons, despite their inefficiency to transfect primary tenocytes in culture. We show that luciferase activity lasted for at least 2 weeks in tendons injected with these MSN and a plasmid DNA (pDNA) encoding the luciferase reporter gene. By contrast, in tendons injected with naked plasmid, the luciferase expression decreased as a function of time and became hardly detectable after 2 weeks.

Comparative gene transfer between cationic and thiourea lipoplexes.

Background: We have previously developed lipopolythiourea lipids as neutral DNA condensing agents for systemic gene delivery. Optimization of the lipopolythiourea structure led to efficient transfecting agents. To further evaluate these lipids, we investigated the internalization process of the thiourea lipoplexes and their intracellular mechanism of transfection versus that of cationic lipoplexes.

Ultrasound-assisted microbubbles gene transfer in tendons for gene therapy.

Our study aimed at evaluating the use of ultrasound-assisted microbubbles gene transfer in mice Achilles tendons. Using a plasmid encoding luciferase gene, it was found that an efficient and stable gene expression for more than two weeks was obtained when tendons were injected with 10 microg of plasmid in the presence of 5x10(5) BR14 microbubbles with the following acoustic parameters: 1 MHz, 200 kPa, 40% duty cycle and 10 min of exposure time. The rate of gene expression was 100-fold higher than that obtained with naked plasmid injected alone without ultrasound or with ultrasound in absence of microbubbles.

The flagella of an atypical enteropathogenic Escherichia coli strain are required for efficient interaction with and stimulation of interleukin-8 production by enterocytes in vitro.

The ability of some typical enteropathogenic Escherichia coli (EPEC) strains to adhere to, invade, and increase interleukin-8 (IL-8) production in intestinal epithelial cells in vitro has been demonstrated. However, few studies regarding these aspects have been performed with atypical EPEC (aEPEC) strains, which are emerging enteropathogens in Brazil. In this study, we evaluated a selected aEPEC strain (1711-4) of serotype O51:H40, the most prevalent aEPEC serotype in Brazil, in regard to its ability to adhere to and invade Caco-2 and T84 cells and to elicit IL-8 production in Caco-2 cells.

Chemical vectors for gene delivery: a current review on polymers, peptides and lipids containing histidine or imidazole as nucleic acids carriers.

DNA/cationic lipid (lipoplexes), DNA/cationic polymer (polyplexes) and DNA/cationic polymer/cationic lipid (lipopolyplexes) electrostatic complexes are proposed as non-viral nucleic acids delivery systems. These DNA-nanoparticles are taken up by the cells through endocytosis processes, but the low capacity of DNA to escape from endosomes is regarded as the major limitations of their transfection efficiency. Here, we present a current report on a particular class of carriers including the polymers, peptides and lipids, which is based on the exploitation of the imidazole ring as an endosome destabilization device to favour the nucleic acids delivery in the cytosol.

Involvement of histidines 11, 15 and 19 in the binding of zinc to the fusogenic H5WYG peptide.

The histidine-rich GLFHAIAHFIHGGWHGLIHGWYG peptide (H5WYG) coordinates a Zn2+ ion and forms a stable peptide-metal complex promoting membrane fusion at physiologic pH. In our previous article titled 'Histidine-rich peptide: evidence for a single zinc-binding site on H5WYG peptide that promotes membrane fusion at neutral pH' in Journal of Mass Spectrometry (2009, 44, 81-89), tandem mass spectrometry experiments have provided evidence for the binding of a single Zn2+ ion to H5WYG and suggested that this binding is shared between H11, H19 and probably H15 residues. To clarify the involvement of these histidine residues in the binding to the Zn2+ ion and especially to remove the doubt about the implication of the H15 residue, here we have used three H5WYG mutants termed H5WYGH11A, H5WYGH15A and H5WYGH19A, whose H11, H15 and H19 residues were replaced with an alanine residue.

An optimized extended DNA kappa B site that enhances plasmid DNA nuclear import and gene expression.

Background: The nuclear factor kappa B (NF kappaB) transcription factor, which shuttles between the cytoplasm and the nucleus under specific conditions, is a suitable intracellular target to increase the nuclear import of plasmid DNA. We report the design of an optimized and extended NF kappaB DNA binding sequence that promotes an efficient plasmid nuclear import.

Methods: On the basis of structural studies, the 5'-CTGGGGACTTTCCAGCTGGGGACTTTCCAGCTGGGGACTTTCCAGG-3' segment (termed 3NF) comprising three 10-bp kappaB sites (GGGACTTTCC) separated by a 5-bp optimized spacer (AGCTG) was selected for its capacity to ensure the best structural fit with NF kappaB and to fix simultaneously three proteins.

The cooperative induction of hypoxia-inducible factor-1 alpha and STAT3 during hypoxia induced an impairment of tumor susceptibility to CTL-mediated cell lysis.

Hypoxia is an essential component of tumor microenvironment. In this study, we investigated the influence of hypoxia (1% PO(2)) on CTL-mediated tumor cell lysis. We demonstrate that exposure of target tumor cells to hypoxia has an inhibitory effect on the CTL clone (Heu171)-induced autologous target cell lysis.

In situ XRD, XAS, and magnetic susceptibility study of the reduction of ammonium nickel phosphate NiNH4PO4 x H2O into nickel phosphide.

The reduction of the ammonium nickel phosphate NiNH(4)PO(4) x H(2)O precursor into nickel phosphide (Ni(2)P), a highly active phase in hydrotreating catalysis, was studied using a combination of magnetic susceptibility and in situ X-ray diffraction and X-ray absorption spectroscopy (XAS) techniques. The transformation of NiNH(4)PO(4) x H(2)O into Ni(2)P could be divided into three distinguishable zones: (1) from room temperature to 250 degrees C, the NiNH(4)PO(4) x H(2)O structure was essentially retained; (2) from 300 to 500 degrees C, only an amorphous phase was observed; (3) above 500 degrees C, a crystallization process occurred with the formation of Ni(2)P. An in situ XAS study and magnetic susceptibility measurements clearly revealed for the first time that the amorphous region corresponds to the nickel pyrophosphate phase alpha-Ni(2)P(2)O(7).

Organised genome dynamics in the Escherichia coli species results in highly diverse adaptive paths.

The Escherichia coli species represents one of the best-studied model organisms, but also encompasses a variety of commensal and pathogenic strains that diversify by high rates of genetic change. We uniformly (re-) annotated the genomes of 20 commensal and pathogenic E. coli strains and one strain of E.

Cold streams in early massive hot haloes as the main mode of galaxy formation.

Massive galaxies in the young Universe, ten billion years ago, formed stars at surprising intensities. Although this is commonly attributed to violent mergers, the properties of many of these galaxies are incompatible with such events, showing gas-rich, clumpy, extended rotating disks not dominated by spheroids. Cosmological simulations and clustering theory are used to explore how these galaxies acquired their gas.

Uropathogenic Escherichia coli AL511 requires flagellum to enter renal collecting duct cells.

Escherichia coli is the leading cause of urinary tract infections, but the mechanisms governing renal colonization by this bacterium remain poorly understood. We investigated the ability of 13 E. coli strains isolated from the urine of patients with pyelonephritis and cystitis and normal stools to invade collecting duct cells, which constitute the first epithelium encountered by bacteria ascending from the bladder.

Olfactory behavior and physiology are disrupted in prion protein knockout mice.

The prion protein PrP(C) is infamous for its role in disease, but its normal physiological function remains unknown. Here we found a previously unknown behavioral phenotype of Prnp(-/-) mice in an odor-guided task. This phenotype was manifest in three Prnp knockout lines on different genetic backgrounds, which provides strong evidence that the phenotype is caused by a lack of PrP(C) rather than by other genetic factors.

Small RNA gene identification and mRNA target predictions in bacteria.

Bacterial small ribonucleic acids (sRNAs) that are not ribosomal and transfer or messenger RNAs were initially identified in the sixties, whereas their molecular functions are still under active investigation today. It is now widely accepted that most play central roles in gene expression regulation in response to environmental changes. Interestingly, some are also implicated in bacterial virulence.

Octa- and nonanuclear nickel(II) polyoxometalate clusters: synthesis and electrochemical and magnetic characterizations.

Three high-nuclearity NiII-substituted polyoxometalate compounds functionalized by exogenous ligands have been synthesized and characterized. The octanuclear complexes in Na15[Na{(A-R-SiW9O34)Ni4(CH3COO)3(OH)3}2] . 4NaCl .

Polymer-based gene delivery: a current review on the uptake and intracellular trafficking of polyplexes.

Lipoplexes and polyplexes, electrostatic complexes between a plasmid DNA and cationic lipids or polymers are chemical systems that are developed for gene delivery. Considerable efforts have been done to delineate the exact knowledge of their entry mechanisms and the intracellular routing of the plasmid DNA that are of major importance for the designing of these gene delivery systems. While the uptake of lipoplexes made with several types of cationic lipids proceeds mainly by the clathrin-dependent pathway, it appears that for polyplexes the uptake pathway is more dependent on the polymer and the cell types.

Low dose beta-blocker prevents ovariectomy-induced bone loss in rats without affecting heart functions.

Findings from animal studies have suggested that bone remodeling is under beta-adrenergic control. However, the level of adrenergic inhibition required to achieve the most favorable effects on the skeleton remains unknown. To address this question, we compared the effects of low (0.

Histidine-rich peptide: evidence for a single zinc-binding site on H5WYG peptide that promotes membrane fusion at neutral pH.

The histidine-rich peptide H5WYG (GLFHAIAHFIHGGWHGLIHGWYG) was found to induce membrane fusion at physiologic pH in the presence of zinc chloride. In this study, we examined the ion selectivity of the interaction of Zn(2+) with H5WYG. This investigation was conducted by using adsorption at air/water interface and mass spectrometry.

Novel neutral imidazole-lipophosphoramides for transfection assays.

New helper lipids, possessing an imidazole polar head, have been synthesized and included in formulations for transfection assays; these new helper lipids can improve the transfection by a factor of up to 100 compared to the use of DOPE as co-lipid.