High-throughput single-molecule analysis of DNA–protein interactions by tethered particle motion

Tethered particle motion (TPM) monitors the variations in the effective length of a single DNA molecule by tracking the Brownian motion of a bead tethered to a support by the DNA molecule. Providing information about DNA conformations in real time, this technique enables a refined characterization of DNA–protein interactions. To increase the output of this powerful but time-consuming single-molecule assay, we have developed a biochip for the simultaneous acquisition of data from more than 500 single DNA molecules. The controlled positioning of individual DNA molecules is achieved by self-assembly on nanoscale arrays fabricated through a standard microcontact printing method. We demonstrate the capacity of our biochip to study biological processes by applying our method to explore the enzymatic activity of the T7 bacteriophage exonuclease. Our single molecule observations shed new light on its behaviour that had only been examined in bulk assays previously and, more specifically, on its processivity

Proton MR spectroscopy and diffusion MR imaging monitoring to predict tumor response to interstitial photodynamic therapy for glioblastoma

International audienceDespite recent progress in conventional therapeutic approaches, the vast majority of glioblastoma recur locally, indicating that a more aggressive local therapy is required. Interstitial photodynamic therapy (iPDT) appears as a very promising and complementary approach to conventional therapies. However, an optimal fractionation scheme for iPDT remains the indispensable requirement. To achieve that major goal, we suggested following iPDT tumor response by a non-invasive imaging monitoring. Nude rats bearing intracranial glioblastoma U87MG xenografts were treated by iPDT, just after intravenous injection of AGuIX® nanoparticles, encapsulating PDT and imaging agents. Magnetic Resonance Imaging (MRI) and Magnetic Resonance Spectroscopy (MRS) allowed us an original longitudinal follow-up of post-treatment effects to discriminate early predictive markers. We successfully used conventional MRI, T2 star (T2*), Diffusion Weighted Imaging (DWI) and MRS to extract relevant profiles on tissue cytoarchitectural alterations, local vascular disruption and metabolic information on brain tumor biology, achieving earlier assessment of tumor response. From one day post-iPDT, DWI and MRS allowed us to identify promising markers such as the Apparent Diffusion Coefficient (ADC) values, lipids, choline and myoInositol levels that led us to distinguish iPDT responders from non-responders. All these responses give us warning signs well before the tumor escapes and that the growth would be appreciated

DNA-Guided Delivery of Single Molecules into Zero-Mode Waveguides

Zero-mode waveguides (ZMWs) are powerful analytical tools corresponding to optical nanostructures fabricated in a thin metallic film capable of confining an excitation volume to the range of attoliters. This small volume of confinement allows single-molecule fluorescence experiments to be performed at physiologically relevant concentrations of fluorescently labelled biomolecules. Exactly one molecule to be studied must be attached at the floor of the ZMW for signal detection and analysis; however, the massive parallelism of these nanoarrays suffers from a Poissonian-limited distribution of these biomolecules. To date, there is no method available that provides full single molecule occupancy of massively arrayed ZMWs. Here we report the performance of a DNA-guided method that uses steric exclusion properties of large DNA molecules to bias the Poissonian-limited delivery of single molecules. Non-Poissonian statistics were obtained with DNA molecules that contain a free-biotinylated extremity for efficient binding to the floor of the ZMW, which resulted in a decrease of accessibility for a second molecule. Both random coil and condensed DNA conformations drove non-Poissonian single-molecule delivery into ZMWs arrays. The results suggest that an optimal balance between rigidity and flexibility of the macromolecule is critical for favourable accessibility and single occupancy. The optimized method provides means for full exploitation of these massively parallelized analytical tools

Calcitonin-derived carrier peptide plays a major role in the membrane localization of a peptide–cargo complex

AbstractBilayers made of dioleoylphosphatidylcholine (DOPC)/dipalmitoylphosphatidylcholine (DPPC) mixture containing or not cholesterol (Chl) were used to investigate the interaction of a carrier peptide with membranes. Atomic force microscopy revealed that the C-terminal 9-32 fragment of human calcitonin (hCT (9-32)), free or coupled to enhanced green fluorescent protein (hCT-eGFP) cargo forms aggregates in the DOPC fluid phase in absence of Chl and in the DPPC enriched liquid-ordered phase when Chl is present. The data show that hCT (9-32) plays a determinant role in the membrane localization of the peptide–cargo complex. They suggest that carpet-like mechanism for membrane destabilization may be involved in the carrier function of hCT (9-32)

Visualisation of sentinel lymph node with indium-based near infrared emitting Quantum Dots in a murine metastatic breast cancer model.

Due to its non-invasiveness, high temporal resolution and lower cost, fluorescence imaging is an interesting alternative to the current method (blue dye and radiocolloid) of sentinel lymph node (SLN) mapping in breast cancer. Near-infrared (NIR) emitting cadmium-based Quantum Dots (QDs) could be used for this purpose; however, their wide application is limited because of the toxicity of heavy metals composing the core. Our recent work demonstrated that indium-based QDs exhibit a weak acute local toxicity in vivo compared to their cadmium-based counterparts. In the present study we confirmed the weak toxicity of CuInS(2)/ZnS QDs in different in vitro models. Further in vivo studies in healthy mice showed that In-based QDs could be visualised in SLN in a few minutes after administration with a progressive increase in fluorescence until 8 h. The quantity of indium was assessed in selected organs and tissues by inductively coupled plasma - mass spectroscopy (ICP-MS) as a function of post-injection time. QD levels decrease rapidly at the injection point in the first hours after administration with a parallel increase in the lymph nodes and to a lesser extent in the liver and spleen. In addition, we observed that 3.5% of the injected indium dose was excreted in faeces in the first 4 days, with only trace quantities in the urine. Metastatic spread to the lymph nodes may hamper its visualisation. Therefore, we further performed non-invasive fluorescence measurement of QDs in SLN in tumour-bearing mice. Metastatic status was assessed by immunohistology and molecular techniques and revealed the utmost metastatic invasion of 36% of SLN. Fluorescence signal was the same irrespective of SLN status. Thus, near-infrared emitting cadmium-free QDs could be an excellent SLN tracer

Identification de la période de « normalisation » vasculaire induite par le bevacizumab, in vivo, à l’aide d’un algorithme de traitement d’images

Présentation PosterInternational audienceLes agents anti-angiogènes sont largement utilisés dans la thérapie anti-cancéreuse, en association avec la chimiothérapieou la radiothérapie, pour leurs effets vasculaires. Ils sont supposés induire des modifications morphologiqueset fonctionnelles au sein du réseau vasculaire tumoral qui permettraient d’accroitre l’efficacité de ces thérapies grâceà l’amélioration de la distribution de la chimiothérapie au sein de la tumeur et à l’amélioration de l’oxygénation tumorale.Cependant, il reste difficile de trouver la séquence de traitements permettant d’avoir une efficacité thérapeutique optimalecar il n’y a à ce jour aucun consensus dans la littérature définissant quand le réseau vasculaire est amélioré et pendantcombien de temps.Nous avons développé un algorithme de traitement d’images capable d’analyser les structures vasculaires observéessur des images en microscopie optique du réseau vasculaire et nous avons suivi leurs modifications, in vivo, au coursdu temps, à l’aide du modèle de la chambre dorsale. L’algorithme a été appliqué pour suivre l’évolution de paramètresvasculaires (surface vasculaire, embranchements, bourgeons, longueur) en réponse ou non au bevacizumab (anti-VEGF,10 mg/kg/jour) pour déterminer une période de « normalisation » du réseau vasculaires par comparaison à des réseauxvasculaires sains.Les résultats présentés ici démontrent que le choix de la région d’intérêt au sein du réseau vasculaire doit se faire en dehors des régions présentant une hiérarchie artériole-capillaire-veinule afin de pouvoir déterminer la période de « normalisation » vasculaire. L’analyse à l’aide de l’algorithme de traitement d’images a permit de définir que la période de « normalisation » se situait entre 8 et 12 jours de traitement par bevacizumab et a été confirmé par analyses immunohistochimiques et évaluation de la fonctionnalité vasculaire (perméabilité, perfusion).Mots clés : thérapie anti-angiogène, normalisation vasculaire, quantification algorithmique, imagerie intravitale

Histology of sentinel lymph node sections of tumour-bearing mice.

<p>Sections were subjected to CK19 immunohistochemistry and hematoxylin staining. Black scale corresponds to 100 µm, the arrow indicates the metastatic invasion of the cortical sinus.</p