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In vivo Gene electrotransfer: optimisation and mechanisms.

In vivo Gene electrotransfer: optimisation and mechanisms.

Biotechnological and biomedical applications of in vivo delivery of short high voltage pulses, like in vivo DNA electrotransfer, also termed electrogenetherapy, are rapidly developing [1-5]. For efficient in vivo gene transfer, it is necessary to inject DNA into the tissue and to achieve cell plasma membrane permeabilization [6]. Increased membrane permeability results from supraphysiological transmembrane voltages induced by external electric pulses [7-9]. Mechanisms of DNA electrotransfer in vivo have recently been described [6; 10]. The two key steps are the permeabilization of the target cells plasma membrane by electroporation and the electrophoresis of the DNA within the tissue. These two effects can be obtained separately using the appropriate sequence of electric pulses: short (100 μs) square-wave high voltage pulses (HV) that permeabilize the cells without substantial DNA transport to the cells and long (100 ms) low voltage pulses (LV), that are instrumental in facilitating the DNA transfer into the cells [6]. Even though gene transfer efficacy, measured by gene expression level, depends on the characteristics of the electrophoretic long low voltage pulse, target cell permeabilization is mandatory for efficient gene transfer. Moreover, for a safe gene transfer, electropermeabilization, also termed electroporation, must be reversible, that is not excessive, in order to avoid permanent cell damage. Optimal parameters for in vivo electroporation can be determined using in vivo tests for cell permeabilization [11] after the pulse, like the one based on 51Cr-EDTA uptake [12], and by using mathematical modeling to determine electric field distribution [13; 14]. However, it would be much better to control cell permeabilization during the pulse delivery in order to ascertain that (reversible) cell permeabilization will be actually achieved at the end of the pulse, as well as to prevent excessive (irreversible) permeabilization [11; 13; 15]. Real time control of electroporation appears thus critical for this non viral gene transfection method that has many advantages with respect to viral methods.
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Noninvasive Gene Electrotransfer in Skin

Noninvasive Gene Electrotransfer in Skin

ABSTRACT The skin is considered as well suited for gene therapy and vaccination. DNA vaccines elicit both broad humoral and cellular immune responses when injected in the skin. Physical and chemical methods are needed to boost the expression. Gene electrotransfer (GET) is one of the most effective approaches. This step by step protocol describes the procedures to get an efficient GET targeted to the skin by using easy-to-use non-invasive electrodes after intradermal plasmid injection (ID GET). A specific pulse sequence is reported. Expression is observed by in vivo fluorescence imaging during more than 2 weeks as the plasmid was coding for tdtomato. The protocol is efficient for the transient expression of clinical proteins.
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Electrotransfer of CpG free plasmids enhances gene expression in skin

Electrotransfer of CpG free plasmids enhances gene expression in skin

Reviewer 1 This manuscript attempts to confirm, using electroporation, the observations of Yew et al 2002 that CpG motifs in plasmids reduce gene expression. However, some data are missing, some lack significance, and some statistical results are not included, limiting the value of this submission. Yew’s paper that was quoted in our contribution was “N.S. Yew, H. Zhao, M. Przybylska, I.H. Wu, J.D. Tousignant, R.K. Scheule, S.H. Cheng, CpG depleted plasmid DNA vectors with enhanced safety and long-term gene expression in vivo, Mol Ther, 5 (2002) 731-738.” The result was that systemic delivery of cationic lipid-plasmid DNA (pDNA) complexes induces an acute inflammatory response with adverse hematologic changes and liver damage. Our approach was on a targeted delivery (not systemic) by a physical method to the skin with a localized expression making it different from Yew’s results. The role of CpG was first illustrated in a previous paper of the same group in Mol Ther. 2000 Mar;1(3):255-62. The conclusion was that the use of a less immunostimulatory pDNA vector or inhibitors of CpG immunostimulation can reduce significantly the toxicity associated with cationic lipid:pDNA complexes. No cationic lipids are used with GET.
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Gene Electrotransfer in 3D Reconstructed Human Dermal Tissue

Gene Electrotransfer in 3D Reconstructed Human Dermal Tissue

4 Current Gene Therapy, 2015, Vol. 0, No. 0 Madi et al. interactions in a healthy tissue. Interestingly, by DNA spot patterns we were able to recognize fibroblast typical morphology. Indeed, these stromal cells are elongated cells growing oriented in parallel. Analysis of tissue cross- sectional views revealed that fluorescent plasmid DNA was confined to the tissue surface. It has been reported that DNA plasmid electrophoretic transport is inversely correlated with the amount of collagen in tumor tissue [36]. Thus, tumors containing only some proteoglycans and collagens were found to be more efficiently electrotransfected in vivo than solid tumors with a rich extracellular matrix [37,38]. Collagen content modulates tumor tissue resistance to macromolecule diffusion by linking and stabilizing glycosaminoglycans [39]. ]. Since the reconstructed human dermal tissue is highly rich in collagens [29], it seems that this observation is also correct for solid healthy tissue.
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Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells

Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells

Toulouse, France; and b Université de Toulouse, UPS (Université Paul Sabatier), IPBS (Institut de Pharmacologie et de Biologie Structurale), F-31077 Toulouse, France Edited by Richard Heller, Old Dominion University, Norfolk, VA, and accepted by the Editorial Board May 16, 2011 (received for review March 3, 2011) The RNA interference-mediated gene silencing approach is pro- mising for therapies based on the targeted inhibition of disease- relevant genes. Electropermeabilization is one of the nonviral methods successfully used to transfer siRNA into living cells in vitro and in vivo. Although this approach is effective in the field of gene silencing by RNA interference, very little is known about the basic processes supporting siRNA transfer. In this study, we investigated, by direct visualization at the single-cell level, the delivery of Alexa Fluor 546-labeled siRNA into murine melanoma cells stably expres- sing the enhanced green fluorescent protein (EGFP) as a target gene. The electrotransfer of siRNA was quantified by time lapse fluorescence microscopy and was correlated with the silencing of egfp expression. A direct transfer into the cell cytoplasm of the negatively charged siRNA was observed across the plasma mem- brane exclusively on the side facing the cathode. When added after electropulsation, the siRNA was inefficient for gene silencing because it did not penetrate the cells. Therefore, we report that an electric field acts on both the permeabilization of the cell plasma membrane and on the electrophoretic drag of the negatively charged siRNA molecules from the bulk phase into the cytoplasm. The transfer kinetics of siRNA are compatible with the creation of nanopores, which are described with the technique of synthetic nanopores. The mechanism involved was clearly specific for the physico-chemical properties of the electrotransferred molecule and was different from that observed with small molecules or plasmid DNA.
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Electroporation-Induced Stress Response and Its Effect on Gene Electrotransfer Efficacy: In Vivo Imaging and Numerical Modeling

Electroporation-Induced Stress Response and Its Effect on Gene Electrotransfer Efficacy: In Vivo Imaging and Numerical Modeling

The differences regarding unipolar and bipolar pulses cannot be explained by our numerical model, since both parameters, EP and electrophoresis, depend only on the norm of the electric field. However, we can stipulate that the reason that a better transfection efficacy of bipolar pulses which was shown previously in vitro [29], was not achieved, as was also reported for in vivo experiments [30], may lie in different sizes of electroporated areas on each side of the cell. The side of the cell facing the anode is hyperpolarized, meaning it reaches the threshold transmembrane potential for EP before the other side of the cell, which is depolarized [58]. By increasing the electric field intensity, the threshold is also reached at the other side of the cell, but the area being electroporated is always smaller than the one on the hyperpolarized side [59], [60]. When using unipolar pulses, plasmid DNA encounters the depolarized side of the cell. The bipolar pulses, on the other hand, enable the contact with the larger electroporated area on the hyperpolarized side, therefore improving the transfection efficacy. We have to keep in mind, however, that changing the orientation of electric field can also have a negative impact on the transfection efficacy. The action of electrophoretic forces in the opposite direction can, namely, lead to partial removal of the DNA from the cell membrane. Due to this effect, unipolar pulses proved to be better choice than bipolar at delays shorter than 100 μs between subsequent pulses [11]. The lag between the electrophoretic MV pulse and the next HV pulse, which is close to 1 s in our experiments, is sufficient for formation of a stable plasmid-membrane complex, but still enables a partial loss of DNA-cell membrane interactions [11].
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In Vivo Evaluation of a New Recombinant Hyaluronidase to Improve Gene Electro-Transfer Protocols for DNA-Based Drug Delivery against Cancer

In Vivo Evaluation of a New Recombinant Hyaluronidase to Improve Gene Electro-Transfer Protocols for DNA-Based Drug Delivery against Cancer

Apart from this, the treatment with rHyal-sk was able to activate an adequate response which could efficiently recruit APCs and macrophages in the site of injection for a suitable immunotherapy strategy. Importantly, cytokines release was not affected by rHyal-sk alone. Since rHyal-sk is a Hyal obtained by a recombinant pathway in the non-pathogenic microorganism S. koganeiensis, it has low homology with the human enzyme. Consequently, we evaluated the level of its immunogenicity in vivo. We found that when repeatedly administered subcutaneously in rats, the presence of nADA could be detected following the highest dose and remains measurable for at least one month afterward. We also investigated the immune response of rHyal-sk when injected intramuscularly associated to GET and we did not observe any antibody titer against rHyal-sk at day 7 (Figure 4). More importantly, no particular toxicological findings were recorded in any of the treated animals. Therefore, while the induction of nADA could limit the use of rHyal-sk in treatments requiring repeated administrations for long periods, this enzyme can find use as an adjuvant to potentiate the immune response of DNA electrotransfer, which needs a single injection, and in all the “single treatments” into the clinics.
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Optical imaging of luminescence for in vivo quantification of gene electrotransfer in mouse muscle and knee.

Optical imaging of luminescence for in vivo quantification of gene electrotransfer in mouse muscle and knee.

High and long term (> 2 months) level of luciferase expression after intramuscular electrotransfer has been well documented. Here, we confirm by optical imaging the stability of luciferase expression for at least 62 days in tibial cranial muscle after electrotransfer of 0.3 or 3 µg of luciferase-encoding plasmid. In contrast, a decrease of luciferase activity occurred three weeks after electrotrans- fer of the higher dose of 30 µg of plasmid, concomitantly to the production of antibodies against luciferase. Similar immune responses were also observed by others, after pC1luc plasmid DNA transfer into the liver of C57Bl6 mouse [40]. After electrotransfer of 60 µg of plasmid into the mouse knee joint, luciferase activity reached a peak between 3 to 6 days and returned to the control level two weeks after electrotransfer. A similar kinetic profile was also observed by Ohashi et al by in vitro evaluation of the transgene expression in the synovium [29]. In contrast Grossin et al observed expression of an electrotransfered plasmid encoding for GFP for a longer period and local- ized mainly in the knee patellae [28]. These differences may be related to the use of different electrotransfer con- ditions (injection site, electric conditions and electrodes). In the present study, by using optical imaging, we have detected the luminescence of the whole knee and it was
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JMJD6 participates in the maintenance of ribosomal DNA integrity in response to DNA damage

JMJD6 participates in the maintenance of ribosomal DNA integrity in response to DNA damage

rDNA transcription we used the DIvA cell line in which DSBs are produced across the genome by the AsiSI endonuclease [ 29 ] one of which being located within rDNA and potentially gener- ating one DSB per rDNA repeat [ 30 ]. As expected, control cells showed a decrease in rDNA transcription following DSB induction, as measured by the incorporation of 5-FUrd metabolic labelling ( Fig 7A and 7B ). Interestingly, rDNA transcription was further decreased in JMJD6- depleted cells compared to control cells ( Fig 7 ) while rDNA transcription in the absence of DNA damage remained largely unaffected (although in some experiments such as the one shown in Fig 7C , we could observe a slight decrease of basal rDNA transcription upon JMJD6 knock-down). Similar effects on rDNA transcription repression were observed 1h post IR (8 Gy), with a further reduction of transcription in JMJD6 depleted cells ( Fig 7C and 7D ). Inter- estingly, rDNA transcription levels had fully recovered 6 hours following IR in both control and JMJD6-depleted cells ( Fig 7C and 7D ), indicating that the rDNA transcription decrease observed upon JMJD6 depletion was transient. We confirmed these results in the MRC5 cell line in which we generated specific DNA damage in rDNA using CRISPR-Cas9 ( S9 Fig ). Alto- gether, these data show that JMJD6 expression defect leads to a slightly more efficient tran- scriptional repression upon DNA breaks induction. This is consistent with the increase in TCOF1/NBS1 interaction we observed in the absence of JMJD6, given that this complex medi- ates rDNA transcriptional repression. Note however that we cannot rule out the possibility that this higher transcriptional repression is due to higher levels of unrepaired DNA damage in the absence of JMJD6.
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Efficiency of high- and low-voltage pulse combinations for gene electrotransfer in muscle, liver, tumor, and skin.

Efficiency of high- and low-voltage pulse combinations for gene electrotransfer in muscle, liver, tumor, and skin.

F.M. André, 1,2 J. Gehl, 3 G. Sersa, 4 V. Préat, 5 P. Hojman, 3,6 J. Eriksen, 3 M. Golzio, 6,7 M. Cemazar, 4 N. Pavselj, 5,8 M.-P. Rols, 6 D. Miklavcic, 8 E. Neumann, 9 J. Teissié, 6,7 and L.M. Mir 1,2 Abstract Gene electrotransfer is gaining momentum as an efficient methodology for nonviral gene transfer. In skeletal muscle, data suggest that electric pulses play two roles: structurally permeabilizing the muscle fibers and elec- trophoretically supporting the migration of DNA toward or across the permeabilized membrane. To investi- gate this further, combinations of permeabilizing short high-voltage pulses (HV; hundreds of V/cm) and mainly electrophoretic long low-voltage pulses (LV; tens of V/cm) were investigated in muscle, liver, tumor, and skin in rodent models. The following observations were made: (1) Striking differences between the various tissues were found, likely related to cell size and tissue organization; (2) gene expression is increased, if there was a time interval between the HV pulse and the LV pulse; (3) the HV pulse was required for high electrotransfer to muscle, tumor, and skin, but not to liver; and (4) efficient gene electrotransfer was achieved with HV field strengths below the detectability thresholds for permeabilization; and (5) the lag time interval between the HV and LV pulses decreased sensitivity to the HV pulses, enabling a wider HV amplitude range. In conclusion, HV plus LV pulses represent an efficient and safe option for future clinical trials and we suggest recommen- dations for gene transfer to various types of tissues.
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Melanopsin-dependent nonvisual responses: evidence for photopigment bistability in vivo.: melanopsin bistability in vivo

Melanopsin-dependent nonvisual responses: evidence for photopigment bistability in vivo.: melanopsin bistability in vivo

light independent conversion of all-trans- back to 11- cis- retinal (retinoid cycle) a process resident in non-photoreceptor cells of retinal pigment epithelium (RPE) and in Muller cells ( McBee et al., 2001 Arshavsky, 2002 Mata et al., 2002 ; ; ). In contrast, invertebrate photopigments are characterised by a photosensory 11-cis- retinal bound state and a photoisomerase function capable of regenerating all trans to 11-cis- retinal. An essential feature of invertebrate photoreception, this bistable property has been documented by a long history of in vitro investigations (using absorption spectrophotometry, electrophysiology, HPLC) in species such as bee, fly, squid and Limulus (see ( Hillman et al., 1983 ) for review) in which photoisomerase excitation at one wavelength restores photosensory responses at the peak wavelength of the 11-cis- retinal bound photopigment. The invertebrate visual cycle thus requires photon absorption to drive both activation and regeneration pathways, whereas the vertebrate cycle only exploits photons for the activation pathway ( Kiselev and Subramaniam, 1994 )..
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Hydrodynamic interactions in DNA thermophoresis

Hydrodynamic interactions in DNA thermophoresis

measured S T and D [17] as described in Fig. [29]. The theoretical curves are calculated with the permittivity- gradient only ( ˆ S = 0). The initial increase of the data up to n = 22 agree quantitatively with the relation (7), thus providing strong evidence for the role of hydrodynamic interactions. The maximum and the subsequent decrease are well described by counterion condensation according to (9). Adding a significant thermoelectric contribution would not improve the quality of the fit, quite on the contrary. This suggests that the Seebeck field in NaCl solution is small, confirming a previous analysis of Soret data for polystyrene beads [16].
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DNA Replication Restart in Archaea

DNA Replication Restart in Archaea

nuclease domain to allow subsequent cleavage of the DNA substrate. The DNA was bent by around 90° upon interaction, suggesting that XPF binding causes distortion at double-strand/ single-strand DNA junctions. The nuclease activity of XPF from Sulfulobus solfataricus has been studied in more details. The replication factor PCNA (Proliferating Cell Nuclear Antigen) was required in vitro for nucle‐ ase activity of this “short” XPF [43]. In the cell, the trimeric PCNA ring encircles double- strand DNA (dsDNA) and firmly attaches the replicative polymerase to the template strand, enhancing its processivity. PCNA is a central protein as it also interacts with various proteins involved in replication and/or repair like Fen1. Interaction with PCNA often involves a conserved motif known as PCNA-Interacting Protein (PIP) motif conserved in XPF proteins. Indeed it was shown that SsoXPF interacts with PCNA through its conserved PIP motif [43, 44]. Intramolecular FRET experiments showed that the binding of SsoXPF to a 3’-flap indeed bent the DNA as observed in ApeXPF structure, but that the interaction with PCNA allowed SsoXPF to distort the DNA structure in a proper conformation for efficient cleavage [45, 46]. SsoXPF preferentially cleaved 3’-flap and processed them into gapped duplex products. It was also observed that SsoXPF can act on substrates containing a variety of DNA damages or modifica‐ tions [47, 48].
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Safe and efficient novel approach for non-invasive gene electrotransfer to skin

Safe and efficient novel approach for non-invasive gene electrotransfer to skin

Lise Pasquet 1 , Sophie Chabot 1 , Elisabeth Bellard 1 , Bostjan Markelc 1 , Marie-Pierre Rols 1 , Jean-Paul Reynes 2 , Gérard Tiraby 2 , Franck Couillaud 3 , Justin Teissie 1 & Muriel Golzio 1 Gene transfer into cells or tissue by application of electric pulses (i.e. gene electrotransfer (GET)) is a non-viral gene delivery method that is becoming increasingly attractive for clinical applications. In order to make GET progress to wide clinical usage its efficacy needs to be improved and the safety of the method has to be confirmed. Therefore, the aim of our study was to increase GET efficacy in skin, by optimizing electric pulse parameters and the design of electrodes. We evaluated the safety of our novel approach by assaying the thermal stress effect of GET conditions and the biodistribution of a cytokine expressing plasmid. Transfection efficacy of different pulse parameters was determined using two reporter genes encoding for the green fluorescent protein (GFP) and the tdTomato fluorescent protein, respectively. GET was performed using non-invasive contact electrodes immediately after intradermal injection of plasmid DNA into mouse skin. Fluorescence imaging of transfected skin showed that a sophistication in the pulse parameters could be selected to get greater transfection efficacy in comparison to the standard ones. Delivery of electric pulses only mildly induced expression of the heat shock protein Hsp70 in a luminescent reporting transgenic mouse model, demonstrating that there were no drastic stress effects. The plasmid was not detected in other organs and was found only at the site of treatment for a limited period of time. In conclusion, we set up a novel approach for GET combining new electric field parameters with high voltage short pulses and medium voltage long pulses using contact electrodes, to obtain a high expression of both fluorescent reporter and therapeutic genes while showing full safety in living animals.
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La maturation de l'ovocyte canin in vivo et in vitro

La maturation de l'ovocyte canin in vivo et in vitro

41 D. But de la thèse L’ovocyte canin est donc une cellule rare, différente du modèle mammifère et complexe à étudier. Au vu des spécificités de la biologie du développement canin, de l’état actuel des connaissances sur la maturation ovocytaire chez la chienne, tant in vivo qu’in vitro, et du retard des biotechnologies disponibles dans cette espèce, il semble clair que la première étape clef à maîtriser soit la maturation ovocytaire. Or nous manquons d’éléments de connaissance sur la maturation in vivo. L’objectif de ce travail de thèse a donc été, dans un premier temps, de mieux décrire la maturation ovocytaire canine in vivo. Pour cela, nous avons cherché à établir précisément les évènements intracellulaires de la maturation ovocytaire in vivo, en les replaçant dans le contexte physiologique si particulier de la chienne. Nous avons ainsi tenté de relier chronologiquement les principales étapes de la maturation ovocytaire canine avec le moment du pic de LH et de l’ovulation. Ces deux événements sont capitaux dans l’étude de la physiologie de la reproduction puisqu’ils sont intimement reliés au moment optimal de la fécondation, aux stades du développement embryonnaire et à la date de la mise-bas (Luvoni, 2000 ; Reynaud et al., 2006). Il était donc indispensable pour décrire précisément la maturation ovocytaire d’utiliser le pic de LH et l’ovulation comme références temporelles. De plus, bien que la maturation in vivo nucléaire ait déjà été décrite (voir plus haut), peu d’informations sont disponibles sur la maturation cytoplasmique. Le choix de la microscopie électronique à transmission, permettant une description précise et simultanée des organites cytoplasmiques, du noyau et de la membrane, s’est donc imposé malgré sa lourdeur.
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A Chemical Genetics Analysis of the Roles of Bypass Polymerase DinB and DNA Repair Protein AlkB in Processing N[superscript 2]-Alkylguanine Lesions In Vivo

A Chemical Genetics Analysis of the Roles of Bypass Polymerase DinB and DNA Repair Protein AlkB in Processing N[superscript 2]-Alkylguanine Lesions In Vivo

Lesion bypass and mutagenesis assays The relative bypass of each lesion was measured using the CRAB assay; mutational analysis was performed by using the REAP assay [57]. Briefly, the constructed viral genomes were first normalized using an established protocol [57]. Each lesion-containing genome was then mixed with the ‘‘+3’’ competitor genome in a 75:25 ratio (ratio empirically determined, see Supporting Methods S1 in File S1) and then electroporated into E. coli strains of all combinations of AlkB and DinB proficiency and deficiency. After 6 h incubation at 37 uC, the progeny phage were isolated and amplified by infecting SCS110 wild-type cells, to dilute out any lesion- containing genomes that did not electroporate and replicate in cells. Single-stranded M13 DNA was then isolated from the amplified progeny, using the M13 Qiaprep columns (Qiagen). The region of interest was then PCR amplified using the CRAB primers for the lesion bypass assay, or the REAP primers for the mutagenesis assay. The PCR products were subsequently digested with BbsI, HaeIII and radiolabeled to yield an 18-mer DNA fragment that contains at its 59 end the specific site that initially contained the lesion of interest. The ‘‘+3’’ competitor genome was only amplified by the CRAB primers and yielded a 21-mer fragment. To quantitate the lesion bypass, the ratio between the intensities of the 18-mer and 21-mer fragments was determined and normalized to the ratio of the same bands for the unmodified ‘‘G’’ control, considered 100% bypass. To analyze the mutagenicity of a lesion, the radiolabeled 18-mer band was cut out from the gel and digested to single nucleotide monophosphates with nuclease P1. The nucleotides were then separated on PEI- TLC plates using a saturated solution of ammonium phosphate (pH = 5.8), and the radioactive signals quantitated using phosphorimagery. An approximately equimolar of GATC control genome mixture, which yielded four distinct TLC spots corresponding to the four normal nucleotides, was used as a mixture of standards. The detailed protocols for the CRAB and REAP assays are included in the Supporting Methods S1 in File S1.
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Wireless In vivo Biofuel Cell Monitoring

Wireless In vivo Biofuel Cell Monitoring

Keywords — RFID tags, biofuel cell, wearable sensors, in vitro, in vivo. I. I NTRODUCTION 1 HE research of a suitable alternative to bulky batteries to power Electronic Medical Devices (EMDs) is nowadays one of the most important research topic in bioelectronics, in particular to power Implanted Medical Devices (IMDs) [1-5]. Batteries are energy inefficient, unsustainable and need to be recharged/replaced, demanding surgery in case of implants. Moreover, batteries often occupy a larger volume than the sensor itself, limiting the device miniaturization, critical to place the implant at the right location of the body with minimum biological tissue displacement [6]. A possible solution consists in transferring the power wirelessly from a source placed outside of the body, commonly using optical [7, 8], ultrasonic [9, 10] or Radio Frequency (RF) [11-15] links. However, these systems need the constant presence of an external power source, which has to be coupled to the implant, implying permanent additional stress for the patient. To avoid the continuous presence of the transmitter,
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Un nouveau marqueur fluorescent pour l’imagerie in vivo

Un nouveau marqueur fluorescent pour l’imagerie in vivo

sieurs jours, et les embryons traités ont un phénotype identique à celui des embryons qui n’ont pas reçu de QD. L’injection de quantités supérieures de QD (au-delà de 5.10 9 QD-micelles/cellule) entraîne des anomalies du développement embryon- naire. La cause de cette toxicité est pour l’instant inconnue, mais elle est probable- ment liée au changement de pression osmo- tique à l’intérieur de la cellule. (3) Les QD- micelles sont stables in vivo. Après quatre jours de développement embryonnaire, il n’y a pas d’agrégation détectable de QD et leur fluorescence reste stable. (4) Les QD- micelles peuvent être introduits dans tous les types de cellules embryonnaires incluant celles des somites, les neurones et les axones (Figure 1F), l’ectoderme (Figure 1G), la crête neurale (Figure 1H) et l’endoderme (Figure 1I) sans ségrégation visible. (5) La fluorescence des QD peut être observée très tôt pendant le développement (Figure 1B) malgré la pigmentation et la fluorescence intrinsèque importante des cellules. Cela contraste avec le temps limité pendant lequel la GFP, un traceur classique, peut être détectée après l’injection de l’ARN ou de l’ADNc codant pour cette protéine, une fois qu’elle est exprimée à des niveaux détec- tables in vivo. (6) L’examen d’embryons au stade du têtard montre qu’il est possible de détecter la fluorescence émise par les QD- micelles même dans des régions ayant une fluorescence intrinsèque élevée comme les intestins de l’embryon (Figure 1I). (7) Les QD micelles résistent beaucoup mieux au photoblanchiment que les autres fluoro- phores in vivo. La Figure 2 compare le pho- toblanchiment de QD-micelles avec celui du dextran-rhodamine-vert (D-Rv). Les QD- micelles et le D-Rv ont été injectés au même moment dans des embryons au même stade de développement. Après 80 minutes d’illu- mination constante (à 450 nm) sous le microscope, l’intensité de la fluorescence des QD n’a pas changé alors que celle du D- Rv a été complètement éteinte par photo- blanchiment. Des résultats similaires ont été obtenus en comparant la fluorescence des QD-micelles avec celle des GFP mem- branaires.
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Mécanismes de l'activation de la transcription in vivo par le Médiateur

Mécanismes de l'activation de la transcription in vivo par le Médiateur

n’a cependant de spécificité pour l’ADN lésé où elles doivent donc être dirigées via leur interaction avec d’autres protéines. XPG est une 3’ endonucléase qui possède deux domaines nucléases N et I séparés par une région « spacer », ce qui la rattache à la même famille que Fen1 et Exo1 (Scherly et al., 1993) (Figure 21). Les analyses structurales suggèrent que les deux domaines N et I forment le cœur catalytique de l’enzyme (Hosfield et al., 2001). La région « spacer » est importante pour les interactions avec TFIIH et RPA (He et al., 1995; Thorel et al., 2004) et permet le recrutement de XPG sur la lésion (Dunand-Sauthier et al., 2005). Cette région forme une boucle, qui doit subir une modification de structure pour rendre l’enzyme catalytiquement active. XPG possède plusieurs sites d’interaction avec TFIIH (Araújo et al., 2001; Iyer et al., 1996), créant un lien très fort et très stable entre ces deux facteurs. Dans la GGR, c’est cette interaction qui va permettre le recrutement de XPG au sein du complexe de pré-incision (Zotter et al., 2006). Dans la TCR, XPG pourrait être recruté au tout début de la réparation, en même temps que CSB, en coopération avec ce dernier ou indépendamment, et interagirait directement avec la Pol II bloquée (Sarker et al., 2005). Avant d’exercer son rôle d’endonuclease, XPG possède un rôle structural. Arrivé sur la lésion, il forme un complexe très stable avec TFIIH et RPA-XPA. A ce stade son activité endonucléase est nulle mais son recrutement induit un changement de conformation requis pour le recrutement de XPF-ERCC1 (Dunand-Sauthier et al., 2005). Plusieurs facteurs ont été proposés comme régulateurs de l’activité nucléase de XPG. RPA est capable de stimuler l’incision par XPG in vitro (Overmeer et al., 2011) et serait donc un bon candidat pour l’activation de la fonction nucléase de XPG. PCNA interagit avec XPG (Gary et al., 1997) et permet l’incision de l’ADN par Fen1 durant la réplication (Chapados et al., 2004). Un rôle similaire peut donc être imaginé dans l’activation de XPG. Ainsi, les activités de liaison à l’ADN et de clivage de l’ADN sont totalement découplées chez XPG et XPF empêchant ainsi les incisions non spécifiques.
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Analysis of microtububule morphogenesis in vivo

Analysis of microtububule morphogenesis in vivo

One might be a protein with which excess 3-tubulin interacts to cause microtubule disassembly, which might include associated proteins or nucleating ele- ments, but also m[r]

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