48 research outputs found
Coupling of importin beta binding peptide on plasmid DNA: transfection efficiency is increased by modification of lipoplex's physico-chemical properties
BACKGROUND: Non-viral vectors for gene transfer are less immunogenic than viral vectors but also less efficient. Significant effort has focused on enhancing non-viral gene transfer efficiency by increasing nuclear import of plasmid DNA, particularly by coupling nuclear localization peptidic sequences to plasmid DNA. RESULTS: We have coupled a 62-aminoacid peptide derived from hSRP1α importin beta binding domain, called the IBB peptide to plasmid DNA by using the heterobifunctional linker N-(4-azido-2,3,5,6 tetrafluorobenzyl)-6-maleimidyl hexanamide (TFPAM-6). When covalently coupled to plasmid DNA, IBB peptide did not increase the efficiency of cationic lipid mediated transfection. The IBB peptide was still able to interact with its nuclear import receptor, importin β, but non-specifically. However, we observed a 20-fold increase in reporter gene expression with plasmid DNA / IBB peptide complexes under conditions of inefficient transfection. In which case, IBB was associated with plasmid DNA through self assembling ionic interaction. CONCLUSIONS: The improvement of transfection activity was not due to an improved nuclear import of DNA, but rather by the modification of physicochemical properties of IBB peptide / plasmid complexes. IBB peptide increased lipoplex size and these larger complexes were more efficient for gene transfer
Housekeeping while brain's storming Validation of normalizing factors for gene expression studies in a murine model of traumatic brain injury
Fate of Tableted Freeze-Dried siRNA Lipoplexes in Gastrointestinal Environment
The incorporation of siRNA into nanocarriers is mandatory to facilitate its intracellular delivery, as siRNA itself cannot enter cells. However, the incorporation of these nanocarriers into oral, solid dosage forms and their fate in the gastrointestinal environment is yet to be explored. In the present work, the fate of, (i) naked siRNA, (ii) freshly prepared siRNA lipoplexes, and (iii) tableted siRNA lipoplexes, in simulated gastric and intestinal fluids was studied. The siRNA, either released from or protected within the lipoplexes, was quantified by gel electrophoresis and siRNA efficacy was assessed in cell transfection. The freshly prepared lipoplexes kept their siRNA load and transfection efficiency totally preserved during 1 h of incubation in simulated gastric fluid at 37 °C. However, in simulated intestinal fluid, despite no release of siRNA from lipoplexes after 6 h of incubation, gene silencing efficacy was dramatically decreased even after 1 h of exposure. The lipoplexes obtained from tablets efficiently protected siRNA in simulated gastric fluid, thus preserving the gene silencing efficacy, whereas their incubation in simulated intestinal fluid resulted in a marked siRNA release and decreased gene silencing efficacy. These results provided a detailed explanation for understanding the fate of siRNA in gastrointestinal conditions, when simply loaded in lipoplexes or formulated in the form of tablets
Targeted delivery to inflammatory monocytes for efficient RNAi-mediated immuno-intervention in auto-immune arthritis
Poster presentationInternational audienc
Low seroprevalence of COVID-19 in Lao PDR, late 2020
Background
In 2020 Lao PDR had low reported COVID-19 cases but it was unclear whether this masked silent transmission. A seroprevalence study was done August - September 2020 to determine SARS-CoV-2 exposure.
Methods
Participants were from the general community (n=2433) or healthcare workers (n=666) in five provinces and bat/wildlife contacts (n=74) were from Vientiane province. ELISAs detected anti- SARS-CoV-2 Nucleoprotein (N; n=3173 tested) and Spike (S; n=1417 tested) antibodies. Double-positive samples were checked by IgM/IgG rapid tests. Controls were confirmed COVID-19 cases (n=15) and pre-COVID-19 samples (n=265). Seroprevalence for the general community was weighted to account for complex survey sample design, age and sex.
Findings
In pre-COVID-19 samples, 5·3%, [95% CI=3·1-8·7%] were anti-N antibody single-positive and 1·1% [0·3-3·5%] were anti-S antibody single positive. None were double positive. Anti-N and anti-S antibodies were detected in 5·2% [4·2-6·5%] and 2·1% [1·1-3·9%] of the general community, 2·0% [1·1-3·3%] and 1·4% [0·5-3·7%] of healthcare workers and 20·3% [12·6-31·0%] and 6·8% [2·8-15·3%] of bat/wildlife contacts. 0·1% [0·02-0·3%] were double positive for anti-N and anti-S antibodies (rapid test negative).
Interpretation
We find no evidence for significant SARS-CoV-2 circulation in Lao PDR before September 2020. This likely results from early decisive measures taken by the government, social behavior, and low population density. High anti-N /low anti-S seroprevalence in bat/wildlife contacts may indicate exposure to cross-reactive animal coronaviruses with threat of emerging novel viruses.
Funding
Agence Française de Développement. Additional; Institut Pasteur du Laos, Institute Pasteur, Paris and Luxembourg Ministry of Foreign and European Affairs (“PaReCIDS II”)
Functionalization of single- and multi-walled carbon nanotubes with cationic amphiphiles for plasmid DNA complexation and transfection
Lipoplexes Strengthened by Anionic Polymers: Easy Preparation of Highly Effective siRNA Vectors Based on Cationic Lipids and Anionic Polymers
International audienc
Junctional Neurulation: A Unique Developmental Program Shaping a Discrete Region of the Spinal Cord Highly Susceptible to Neural Tube Defects
International audienceIn higher vertebrates, the primordium of the nervous system, the neural tube, is shaped along the rostrocaudal axis through two consecutive, radically different processes referred to as primary and secondary neurulation. Failures in neurulation lead to severe anomalies of the nervous system, called neural tube defects (NTDs), which are among the most common congenital malformations in humans. Mechanisms causing NTDs in humans remain ill-defined. Of particular interest, the thoracolumbar region, which encompasses many NTD cases in the spine, corresponds to the junction between primary and secondary neurulations. Elucidating which developmental processes operate during neurulation in this region is therefore pivotal to unraveling the etiology of NTDs. Here, using the chick embryo as a model, we show that, at the junction, the neural tube is elaborated by a unique developmental program involving concerted movements of elevation and folding combined with local cell ingression and accretion. This process ensures the topological continuity between the primary and secondary neural tubes while supplying all neural progenitors of both the junctional and secondary neural tubes. Because it is distinct from the other neurulation events, we term this phenomenon junctional neurulation. Moreover, the planar-cell-polarity member, Prickle-1, is recruited specifically during junctional neurulation and its misexpression within a limited time period suffices to cause anomalies that phenocopy lower spine NTDs in human. Our study thus provides a molecular and cellular basis for understanding the causality of NTD prevalence in humans and ascribes to Prickle-1 a critical role in lower spinal cord formation
Aspects cellulaires et moléculaires de l'émergence des cellules souches hématopoïétiques : implication de RUNX1/AML1
Chez l'embryon de vertébré, le plancher de l'aorte est le site majeur de production des Cellules Souches Hématopoïétiques (CSH) assurant le renouvellement des cellules sanguines chez l'adulte. Les CSH sont produites à partir des Cellules Endothéliales (CE)
via une cascade complexe d'événements moléculaires qui sont, à
l'heure actuelle, encore peu compris. Le facteur de transcription RUNX1/AML1 et son cofacteur CBFβ, impliqués dans 20 % des leucémies myéloïdes aiguës, semblent
contrôler ce processus. L'étude détaillée de l'expression de RUNX1 et des facteurs associés au cours de la production des CSH chez l'embryon d'oiseau, nous permet d'envisager les cascades moléculaires impliquées. Cependant, la fonction de RUNX1 est finement régulée à plusieurs niveaux et les mécanismes moléculaires sousjacents sont difficiles à analyser par les approches génétiques classiques. Afin d'offrir de nouvelles possibilités d'étude, nous avons mis au point une technique permettant de cibler l'arbre vasculaire et les CE hémogéniques in vivo. Le transfert de gène est réalisé par lipofection après inoculation par injection intra-cardiaque chez l'embryon d'oiseau. Cette méthode a été optimisée pour permettre de mener des expériences de gain ou de perte de fonction de manière simple et efficace. Combiné aux avantages expérimentaux de l'embryon d'oiseau, ce nouveau système d'analyse génétique nous permet d'étudier en détail la fonction de RUNX1 dans la production des CSH à partir des CE
Functionalization of single- and multi-walled carbon nanotubes with cationic amphiphiles for plasmid DNA complexation and transfection
International audienceThe possibility of delivering DNA effi ciently to cells represents a crucial issue for the treatment of both genetic and acquired diseases. However, even although the effi ciency of non-viral transfection systems has improved in the last decade, none have yet proven to be suffi ciently effective in vivo. We report herein our results on the functionalization of single-walled carbon nanotubes (SWNT) and multi-walled carbon nanotubes (MWNT) by two cationic amphiphiles (lipid RPR120535 and pyrenyl polyamine), their use for the complexation of plasmid DNA, and their efficiency in transfecting cells in vitro. The experiments have shown that the efficiency of transfection is higher when using SWNT instead of MWNT, and that transfection effi ciency is similar or slightly higher when using nanoplexes (SWNT/lipid RPR120535/DNA) instead of lipoplexes (lipid RPR120535/DNA) and several orders of magnitude higher than that of naked DNA. This study therefore shows both that the transfection is better when using SWNTs and that it is dependent on the nature of the amphiphilic molecules adsorbed on the nanotubes