Optimisation de la cryo microscopie électronique pour les études des minicercles d'ADN

RESUME : Bien que les propriétés physiques de la structure de l'ADN aient été intensivement étudiées pendant plus de 50 ans il y a encore beaucoup de questions importantes qui attendent des réponses. Par exemple, qu'arrive-t-il à la structure de la double hélice d'ADN nue (sans protéines liées) lorsqu'elle est fortement courbée, de la même manière que dans les nucléosomes? Cet ADN nu est-il facilement plié (il reste dans le régime élastique) ou réduit-il la contrainte de flexion en formant des sites hyperflexibles «kinks» (il sort du régime élastique en cassant l'empilement des paires de bases à certains endroits) ? La microscopie électronique peut fournir une réponse à cette question par visualisation directe des minicercles d'ADN de la longueur d'un tour de nucléosome (environ 90 paires de bases). Pour que la réponse soit scientifiquement valide, on doit observer les molécules d'ADN lorsqu'elles sont en suspension dans la solution d'intérêt et sans que des colorations, produits chimiques ou fixatifs n'aient été ajoutés, étant donné que ceux-ci peuvent changer les propriétés de l'ADN. La technique de la cryo-microscopie électronique (cryo-EM) développée par le groupe de Jacques Dubochet au début des années 80, permet la visualisation directe des molécules d'ADN suspendues dans des couche minces vitrifiées de solutions aqueuses. Toutefois, le faible contraste qui caractérise la cryo-EM combinée avec la très petite taille des minicercles d'ADN rendent nécessaire l'optimisation de plusieurs étapes, aussi bien dans la préparation des échantillons que dans le processus d'acquisition d'images afin d'obtenir deux clichés stéréo qui permettent la reconstruction 3-D des minicercles d'ADN. Dans la première partie de ma thèse, je décris l'optimisation de certains paramètres pour la cryoEM et des processus d'acquisition d'image utilisant comme objets de test des plasmides et d'autres molécules d'ADN. Dans la deuxième partie, je .décris comment j'ai construit les minicercles d'ADN de 94 bp et comment j'ai introduit des modifications structurelles comme des coupures ou des lacunes. Dans la troisième partie, je décris l'analyse des reconstructions des rninicercles d'ADN. Cette analyse, appuyée par des tests biochimiques, indique fortement que des molécules d'ADN sont capables de former de petites molécules circulaires de 94 bp sans dépasser les limites d'élasticité, indiquant que les minicercles adoptent une forme circulaire régulière où la flexion est redistribuée le long la molécule. ABSTRACT : Although physical properties of DNA structure have been intensively studied for over 50 years there are still many important questions that need to be answered. For example, what happens to protein-free double-stranded DNA when it is strongly bent, as in DNA forming nucleosomes? Is such protein-free DNA smoothly bent (i.e. it remains within elastic limits of DNA rigidity) or does it release its bending stress by forming sharp kinks (i.e. it exits the elastic regime and breaks the stacking between neighbouring base-pairs in localized regions)? Electron microscopy can provide an answer to this question by directly visualizing DNA minicircles that have the size of nucleosome gyres (ca 90 bp). For the answer to be scientifically valid, one needs to observe DNA molecules while they are still suspended in the solution of interest and no staining chemicals or fixatives have been added since these can change the properties of the DNA. CryoEM techniques developed by Jacques Dubochet's group beginning in the 1980's permit direct visualization of DNA molecules suspended in cryo-vitrified layers of aqueous solutions. However, a relatively weak contrast of cryo-EM preparations combined with the very small size of the DNA minicircles made it necessary to optimize many of the steps and parameters of the cryo-EM specimen preparation and image acquisition processes in order to obtain stereo-pairs of images that permit the 3-D reconstruction of the observed DNA minicircles. In the first part of my thesis I describe the optimization of the cryo-EM preparation and the image acquisition processes using plasmid size DNA molecules as a test object. In the second part, I describe how I formed the 94 by DNA minicircles and how I introduced structural modifications like nicks or gaps. In the third part, I describe the cryo-EM analysis of the constructed DNA minicircles. That analysis, supported by biochemical tests, strongly indicates that DNA minicircles as small as 94 by remain within the elastic limits of DNA structure, i.e. the minicircles adopt a regular circular shape where bending is redistributed along the molecules.

Systematized stenting of Baerveldt shunts: techniques to reduce early post-operative hypotony

Purpose: To investigate the effect of the systematized use of intraluminal stents in Baerveldt shunts (BS) on early postoperative IOP control and complication rates. Methods: One hundred and twenty eyes with medically uncontrolled glaucoma were prospectively recruited to undergo BS implantation at Jules Gonin Eye Hospital, Switzerland. Baerveldt shunts were stented (full-length of the intraluminal tube) using a Supramid® 3.0 suture. A minority of shunts (37%) were also ligated intraoperatively and laser suture lysis performed postoperatively. Stent removals, either partial (retraction of 5mm) or complete, were carried out according to a predetermined protocol. Surgery was considered a success when IOP was ≤ 21mmHg and a minimum of 20% reduction from baseline was achieved with/without glaucoma medication (GMs). Hypotony related complications were defined as: choroidal effusions, shallow AC, hypotonous maculopathy or IOP≤5mmHg for over 2 weeks. Results: Mean age was 61.8 years (± standard deviation; ±21.5). Mean follow-up was 17.1 (±7.9) months. Mean preoperative IOP was 26.9 mmHg; mean IOP on the last visit 13.2 mmHg (p<0.001). At year one, the success rate was 87%. In 90% of eyes, IOP was ≤18 mmHg at last visit. Mean number of preoperatively GMs was 3.1; postoperatively 1.4 (p<0.001). Stent removals were performed in 87% of eyes (24% partial; 61% complete). 13% of eyes required no stent removal to reach target IOP. Complications were minor and infrequent (16%) and only 7% were hypotony related. Conclusions: Systematized use of intraluminal stents with Baerveldt aqueous shunts resulted in gradual and controlled IOP lowering with minimal hypotony-related complications. This may have important implications on clinical practice, given the rising rates of aqueous shunt implantation.

Optimization of spatial resolution for peripheral magnetic resonance angiography.

RATIONALE AND OBJECTIVES: To determine optimum spatial resolution when imaging peripheral arteries with magnetic resonance angiography (MRA). MATERIALS AND METHODS: Eight vessel diameters ranging from 1.0 to 8.0 mm were simulated in a vascular phantom. A total of 40 three-dimensional flash MRA sequences were acquired with incremental variations of fields of view, matrix size, and slice thickness. The accurately known eight diameters were combined pairwise to generate 22 "exact" degrees of stenosis ranging from 42% to 87%. Then, the diameters were measured in the MRA images by three independent observers and with quantitative angiography (QA) software and used to compute the degrees of stenosis corresponding to the 22 "exact" ones. The accuracy and reproducibility of vessel diameter measurements and stenosis calculations were assessed for vessel size ranging from 6 to 8 mm (iliac artery), 4 to 5 mm (femoro-popliteal arteries), and 1 to 3 mm (infrapopliteal arteries). Maximum pixel dimension and slice thickness to obtain a mean error in stenosis evaluation of less than 10% were determined by linear regression analysis. RESULTS: Mean errors on stenosis quantification were 8.8% +/- 6.3% for 6- to 8-mm vessels, 15.5% +/- 8.2% for 4- to 5-mm vessels, and 18.9% +/- 7.5% for 1- to 3-mm vessels. Mean errors on stenosis calculation were 12.3% +/- 8.2% for observers and 11.4% +/- 15.1% for QA software (P = .0342). To evaluate stenosis with a mean error of less than 10%, maximum pixel surface, the pixel size in the phase direction, and the slice thickness should be less than 1.56 mm2, 1.34 mm, 1.70 mm, respectively (voxel size 2.65 mm3) for 6- to 8-mm vessels; 1.31 mm2, 1.10 mm, 1.34 mm (voxel size 1.76 mm3), for 4- to 5-mm vessels; and 1.17 mm2, 0.90 mm, 0.9 mm (voxel size 1.05 mm3) for 1- to 3-mm vessels. CONCLUSION: Higher spatial resolution than currently used should be selected for imaging peripheral vessels.

Gamma Knife, CyberKnife, TomoTherapy: gadgets or useful tools?

Purpose of reviewThis review provides information and an update on stereotactic radiosurgery (SRS) equipment, with a focus on intracranial lesions and brain neoplasms.Recent findingsGamma Knife radiosurgery represents the gold standard for intracranial radiosurgery, using a dedicated equipment, and has recently evolved with a newly designed technology, Leksell Gamma Knife Perfexion. Linear accelerator-based radiosurgery is more recent, and originally based on existing systems, either adapted or dedicated to radiosurgery. Equipment incorporating specific technologies, such as the robotic CyberKnife system, has been developed. Novel concepts in radiation therapy delivery techniques, such as intensity-modulated radiotherapy, were also developed; their integration with computed tomography imaging and helical delivery has led to the TomoTherapy system. Recent data on the management of intracranial tumors with radiosurgery illustrate the trend toward a larger use and acceptance of this therapeutic modality.SummarySRS has become an important alternative treatment for a variety of lesions. Each radiosurgery system has its advantages and limitations. The 'perfect' and ubiquitous system does not exist. The choice of a radiosurgery system may vary with the strategy and needs of specific radiosurgery programs. No center can afford to acquire every technology, and strategic choices have to be made. Institutions with large neurosurgery and radiation oncology programs usually have more than one system, allowing optimization of the management of patients with a choice of open neurosurgery, radiosurgery, and radiotherapy. Given its minimally invasive nature and increasing clinical acceptance, SRS will continue to progress and offer new advances as a therapeutic tool in neurosurgery and radiotherapy.

Design of Novel Frequency Generation Circuit with Application to Ultra-Wideband Distributed Oscillators

Given the urgence of a new paradigm in wireless digital trasmission which should allow for higher bit rate, lower latency and tigher delay constaints, it has been proposed to investigate the fundamental building blocks that at the circuital/device level, will boost the change towards a more efficient network architecture, with high capacity, higher bandwidth and a more satisfactory end user experience. At the core of each transciever, there are inherently analog devices capable of providing the carrier signal, the oscillators. It is strongly believed that many limitations in today's communication protocols, could be relieved by permitting high carrier frequency radio transmission, and having some degree of reconfigurability. This led us to studying distributed oscillator architectures which work in the microwave range and possess wideband tuning capability. As microvave oscillators are essentially nonlinear devices, a full nonlinear analyis, synthesis, and optimization had to be considered for their implementation. Consequently, all the most used nonlinear numerical techniques in commercial EDA software had been reviewed. An application of all the aforementioned techniques has been shown, considering a systems of three coupled oscillator ("triple push" oscillator) in which the stability of the various oscillating modes has been studied. Provided that a certain phase distribution is maintained among the oscillating elements, this topology permits a rise in the output power of the third harmonic; nevertheless due to circuit simmetry, "unwanted" oscillating modes coexist with the intenteded one. Starting with the necessary background on distributed amplification and distributed oscillator theory, the design of a four stage reverse mode distributed voltage controlled oscillator (DVCO) using lumped elments has been presented. All the design steps have been reported and for the first time a method for an optimized design with reduced variations in the output power has been presented. Ongoing work is devoted to model a wideband DVCO and to implement a frequency divider.

Measuring the inflammasome.

Inflammasomes are multiprotein complexes whose activity has been implicated in physiological and pathological inflammation. The hallmarks of inflammasome activation are the secretion of the mature forms of Caspase-1 and IL-1β from cells of the innate immune system. This protocol covers the methods required to study inflammasome activation using mouse bone marrow-derived dendritic cells (BMDCs) as a model system. The protocol includes the generation and handling of BMDCs, the stimulation of BMDCs with established Nlrp3 inflammasome activators, and the measurement of activation by both ELISA and western blot. These methods can be useful for the study of potential inflammasome activators, and of the signaling pathways involved in inflammasome activation. General considerations are provided that may help in the design and optimization of modified methods for the study of other types of inflammasomes and in other cell types.

Optimization of the piggyBac Transposon Using mRNA and Insulators: Toward a More Reliable Gene Delivery System.

Integrating and expressing stably a transgene into the cellular genome remain major challenges for gene-based therapies and for bioproduction purposes. While transposon vectors mediate efficient transgene integration, expression may be limited by epigenetic silencing, and persistent transposase expression may mediate multiple transposition cycles. Here, we evaluated the delivery of the piggyBac transposase messenger RNA combined with genetically insulated transposons to isolate the transgene from neighboring regulatory elements and stabilize expression. A comparison of piggyBac transposase expression from messenger RNA and DNA vectors was carried out in terms of expression levels, transposition efficiency, transgene expression and genotoxic effects, in order to calibrate and secure the transposition-based delivery system. Messenger RNA reduced the persistence of the transposase to a narrow window, thus decreasing side effects such as superfluous genomic DNA cleavage. Both the CTF/NF1 and the D4Z4 insulators were found to mediate more efficient expression from a few transposition events. We conclude that the use of engineered piggyBac transposase mRNA and insulated transposons offer promising ways of improving the quality of the integration process and sustaining the expression of transposon vectors.

Coronary MR angiography: comparison of quantitative and qualitative data from four techniques.

OBJECTIVE: The optimal coronary MR angiography sequence has yet to be determined. We sought to quantitatively and qualitatively compare four coronary MR angiography sequences. SUBJECTS AND METHODS. Free-breathing coronary MR angiography was performed in 12 patients using four imaging sequences (turbo field-echo, fast spin-echo, balanced fast field-echo, and spiral turbo field-echo). Quantitative comparisons, including signal-to-noise ratio, contrast-to-noise ratio, vessel diameter, and vessel sharpness, were performed using a semiautomated analysis tool. Accuracy for detection of hemodynamically significant disease (> 50%) was assessed in comparison with radiographic coronary angiography. RESULTS: Signal-to-noise and contrast-to-noise ratios were markedly increased using the spiral (25.7 +/- 5.7 and 15.2 +/- 3.9) and balanced fast field-echo (23.5 +/- 11.7 and 14.4 +/- 8.1) sequences compared with the turbo field-echo (12.5 +/- 2.7 and 8.3 +/- 2.6) sequence (p < 0.05). Vessel diameter was smaller with the spiral sequence (2.6 +/- 0.5 mm) than with the other techniques (turbo field-echo, 3.0 +/- 0.5 mm, p = 0.6; balanced fast field-echo, 3.1 +/- 0.5 mm, p < 0.01; fast spin-echo, 3.1 +/- 0.5 mm, p < 0.01). Vessel sharpness was highest with the balanced fast field-echo sequence (61.6% +/- 8.5% compared with turbo field-echo, 44.0% +/- 6.6%; spiral, 44.7% +/- 6.5%; fast spin-echo, 18.4% +/- 6.7%; p < 0.001). The overall accuracies of the sequences were similar (range, 74% for turbo field-echo, 79% for spiral). Scanning time for the fast spin-echo sequences was longest (10.5 +/- 0.6 min), and for the spiral acquisitions was shortest (5.2 +/- 0.3 min). CONCLUSION: Advantages in signal-to-noise and contrast-to-noise ratios, vessel sharpness, and the qualitative results appear to favor spiral and balanced fast field-echo coronary MR angiography sequences, although subjective accuracy for the detection of coronary artery disease was similar to that of other sequences.

Differentiation of blue ballpoint pen by positive and negative mode LDI-MS

Usually, the differentiation of inks on questioned documents is carried out by optical methods and thin layer chromatography (TLC). Therefore, spectrometric methods were also proposed in forensic literature for the analysis of dyes. Between these techniques, laser desorption/ionization mass spectrometry (LDI-MS) has demonstrated a great versatility thanks to its sensitivity to blue ballpoint ink dyes and minimal sample destruction. Previous researches concentrated mostly on the LDI-MS positive mode and have shown that this analytical tool offers higher discrimination power than high performance TLC (HPTLC) for the differentiation of blue ballpoint inks. Although LDI-MS negative mode has already been applied in numerous forensic domains like the studies of works of art, automotive paints or rollerball pens, its potential for the discrimination of ballpoint pens was never studied before. The aim of the present paper is therefore to evaluate its potential for the discrimination of blue ballpoint inks. After optimization of the method, ink entries from 33 blue ballpoint pens were analyzed directly on paper in both positive and negative modes by LDI-MS. Several cationic and anionic ink components were identified in inks; therefore, pens were classified and compared according to their formulations. Results show that additional information provided by anionic dyes and pigments significantly increases the discrimination power of positive mode. In fact, it was demonstrated that classifications obtained by the two modes were, to some extent, complementary (i.e., inks with specific cationic dyes not necessarily contained the same anionic components).

Techniques of DNA-studies on prehispanic ectoparasites (Pulex sp., Pulicidae, Siphonaptera) from animal mummies of the Chiribaya Culture, Southern Peru

During a paleoparasitological survey of several animal mummies (Cavia aperea f. porcellus and Canis familiaris) from Chiribaya Baja, an archaeological site in Southern Peru, an unexpected find was made. In the well preserved fur, large numbers of mummified fleas (Pulex simulans/irritans)that parasitized the animals during life were encountered. Due to the relative recent event of the host mummification and the outstanding preservation of the fleas, an attempt for the retrieval of DNA was made. A DNA extraction and sequencing protocol for archaeological ectoparasitic remains has been established, taking additional studies for tissue and protein preservation into account. Tissue preservation was assessed with transmission electron microscopy and the protein preservation was tested through the racemisation ratios of aspartic acid. Regions of the 28S rDNA gene were successfully amplified and sequenced. Further research perspectives are outlined.

Combination of enteral and parenteral nutrition in intensive care patients: A new concept of optimization

Undernutrition is a widespread problem in intensive care unit and is associated with a worse clinical outcome. A state of negative energy balance increases stress catabolism and is associated with increased morbidity and mortality in ICU patients. Undernutrition-related increased morbidity is correlated with an increase in the length of hospital stay and health care costs. Enteral nutrition is the recommended feeding route in critically ill patients, but it is often insufficient to cover the nutritional needs. The initiation of supplemental parenteral nutrition, when enteral nutrition is insufficient, could optimize the nutritional therapy by preventing the onset of early energy deficiency, and thus, could allow to reduce morbidity, length of stay and costs, shorten recovery period and, finally, improve quality of life. (C) 2009 Elsevier Masson SAS. All rights reserved.