Nanopore detection of single molecule RNAP-DNA transcription complex.

In the past decade, a number of single-molecule methods have been developed with the aim of investigating single protein and nucleic acid interactions. For the first time we use solid-state nanopore sensing to detect a single E. coli RNAP-DNA transcription complex and single E. coli RNAP enzyme. On the basis of their specific conductance translocation signature, we can discriminate and identify between those two types of molecular translocations and translocations of bare DNA. This opens up a new perspectives for investigating transcription processes at the single-molecule level.

Clinical usefulness of FDG-PET/CT scan imaging in the management of posttransplant lymphoproliferative disease.

BACKGROUND: Posttransplant lymphoproliferative disease (PTLD) is, aside skin cancer, the most common malignancy occurring after solid organ transplant in adults. Fluorodeoxyglucose (FDG) positron emission tomography (PET) has proved useful in the management of lymphomas. METHODS: We report our experience with the use of FDG-PET inline with computed tomography (CT) scanning in the management of four transplant recipients with histologically confirmed PTLD, including three monomorphic PTLDs and one polymorphic PTLD. RESULTS: FDG-PET/CT scan at diagnosis showed increased FDG uptake in all examined PTLD lesions, and the disease was upstaged on the basis of FDG-PET/CT scan results over conventional CT scanning in one patient. At the end of treatment, PET/CT scans no longer demonstrated FDG uptake in the original PTLD lesions in all patients. Complete remission of disease persisted for at least 1 year after diagnosis in all. CONCLUSIONS: Our results strongly support that FDG-PET scanning is highly specific for diagnosis and follow-up of PTLD. The clinical relevance of including FDG-PET/CT scanning in the management of PTLD should be evaluated in a larger prospective cohort study.

Imaging spectrum of the operated bladder : P41

Multiple complex procedures involving the bladder have been developed by surgeons in both children and adults, with their own advantages and drawbacks. A greater knowledge of the anatomic changes induced by each category of procedure is mandatory in the follow-up of patients and detection of early and late postoperative complications. A retrospective review of 50 pediatric and adult patients referred or treated in our institution is presented. The combined use of plain films, IVP, MCUG, US, Doppler US, CT, 3DCT and MRI is described. The imaging features are compared with surgical and anatomopathological findings. The purpose of this pictorial essay is to present a wide spectrum of diagnostic imaging findings in children and adult patients with operated bladder. The imaging features of surgical procedures with preserved, partially replaced and totally replaced bladder are described, with emphasis on a systematic multimodality approach. Learning objectives: 1) To provide an in-depth review of bladder surgical procedures with their indications, anatomopathological and imaging features. 2) To describe optimal multimodality imaging techniques for evaluating the operated bladder.

Lymphangioléiomyomatose pulmonaire [Lymphangioleiomyomatosis]

Lymphangioleiomyomatosis is a rare pulmonary disease encountered almost exclusively in women of reproductive age. Pulmonary involvement is characterized by multiple thin-walled cysts in the lungs, recurrent pneumothorax, obstructive lung disorders, and progression to chronic respiratory failure over a mean period of 10 years. Certainty of diagnosis requires a lung biopsy, but international criteria have been proposed for a diagnosis without such a biopsy. International recommendations were recently issued for the diagnosis and treatment of lymphangioleiomyomatosis. Treatment is principally symptomatic and relies on the management of bronchial obstruction by bronchodilators; of hypoxemia by oxygen therapy; of pleural complications by pleurodesis, most often surgical; and of renal angiomyolipomas by percutaneous embolization in cases of hemorrhagic risk. Hormone treatment is not recommended. Hopes are high for mTor inhibitors (sirolimus and everolimus) and treatment trials are currently underway. Lung transplantation must be considered when chronic respiratory failure occurs in patients younger than 60 years.

Performance comparison of two commercial BGO-based PET/CT scanners using NEMA NU 2-2001.

Combined positron emission tomography and computed tomography (PET/CT) scanners play a major role in medicine for in vivo imaging in an increasing number of diseases in oncology, cardiology, neurology, and psychiatry. With the advent of short-lived radioisotopes other than 18F and newer scanners, there is a need to optimize radioisotope activity and acquisition protocols, as well as to compare scanner performances on an objective basis. The Discovery-LS (D-LS) was among the first clinical PET/CT scanners to be developed and has been extensively characterized with older National Electrical Manufacturer Association (NEMA) NU 2-1994 standards. At the time of publication of the latest version of the standards (NU 2-2001) that have been adapted for whole-body imaging under clinical conditions, more recent models from the same manufacturer, i.e., Discovery-ST (D-ST) and Discovery-STE (D-STE), were commercially available. We report on the full characterization both in the two- and three-dimensional acquisition mode of the D-LS according to latest NEMA NU 2-2001 standards (spatial resolution, sensitivity, count rate performance, accuracy of count losses, and random coincidence correction and image quality), as well as a detailed comparison with the newer D-ST widely used and whose characteristics are already published.

Complex interplay between LPS and IL-10 in dendritic cells: uncoupling of inflammatory chemokine receptors and positive effects on B cell chemokines

Abstract: Protective immune responses against pathogen invasion and transformed cells requires the coordinated action of distinct leukocyte subsets and soluble factors, overall termed immunological network. Among antigen-presenting cells (APC), a crucial role is played by dendritic cells (DC), which initiate, amplify and determine the outcome of the immune response. Micro-environmental conditions profoundly influence DC in such ways that the resulting immune response ranges from successful immune stimulation to abortive response or immune suppression. For instance, the presence in the milieu of anti-inflammatory cytokine interleukin-10 (IL-10) reverts most of the effects mediated on DC by even strong pro-inflammatory agents such as bacterial Lipopolysaccharide (LPS), in terms of differentiation, activation and functions. In an environment containing both LPS and IL-10, uncoupling of receptors for inflammatory chemokines already occurs after a few hours and in a reversible manner on DC, allowing scavenging of chemokines and, consequently, attenuation of the inflammatory process which could be deleterious to the organism. By studying the effects on DC of concomitant stimulation by LPS and IL-10 from the gene expression point of view, we were able to define four distinct transcriptional programs: A. the inhibition of inflammation and immunity, B. the regulation of tissue remodeling, C. the tuning of cytokine/growth factor receptors and G protein-coupled receptors, D. the stimulation of B cell function and lymphoid tissue neogenesis. Among the latter genes, we further demonstrated that IL-10 synergizes with Toll-like receptor ligands for the production of functionally active B cell attracting chemokine CXCL13. Our data provide evidence that the combined exposure of APC to LPS and IL-10, via the production of CXCL13, involves humoral immunity by attracting antibody-producing cells. It is well known that the persistent release of CXCL13 leads to the development of ectopic lymphoid tissue aggregates and production of high levels of antibodies, thus favoring the induction of auto-immunity. Our findings suggest that the IL-10 produced in chronic inflammatory conditions may promote lymphoid tissue neogenesis through increased release of CXCL13. IL-10 is an anti-inflammatory cytokine inhibiting cellular-mediated TH 1-polarized immune responses. In this study we demonstrate that IL- 10 strongly supports the development of humoral immunity. IL-10 and CXCL13 can thus be targets for specific therapies in auto-immune diseases.

Efficient T cell activation requires an optimal dwell-time of interaction between the TCR and the pMHC complex.

Cytotoxic T cell (CTL) activation by antigen requires the specific detection of peptide-major histocompatibility class I (pMHC) molecules on the target-cell surface by the T cell receptor (TCR). We examined the effect of mutations in the antigen-binding site of a Kb-restricted TCR on T cell activation, antigen binding and dissociation from antigen.These parameters were also examined for variants derived from a Kd-restricted peptide that was recognized by a CTL clone. Using these two independent systems, we show that T cell activation can be impaired by mutations that either decrease or increase the binding half-life of the TCR-pMHC interaction. Our data indicate that efficient T cell activation occurs within an optimal dwell-time range of TCR-pMHC interaction. This restricted dwell-time range is consistent with the exclusion of either extremely low or high affinity T cells from the expanded population during immune responses.

Fission yeast Sec3 and Exo70 are transported on actin cables and localize the exocyst complex to cell poles.

The exocyst complex is essential for many exocytic events, by tethering vesicles at the plasma membrane for fusion. In fission yeast, polarized exocytosis for growth relies on the combined action of the exocyst at cell poles and myosin-driven transport along actin cables. We report here the identification of fission yeast Schizosaccharomyces pombe Sec3 protein, which we identified through sequence homology of its PH-like domain. Like other exocyst subunits, sec3 is required for secretion and cell division. Cells deleted for sec3 are only conditionally lethal and can proliferate when osmotically stabilized. Sec3 is redundant with Exo70 for viability and for the localization of other exocyst subunits, suggesting these components act as exocyst tethers at the plasma membrane. Consistently, Sec3 localizes to zones of growth independently of other exocyst subunits but depends on PIP(2) and functional Cdc42. FRAP analysis shows that Sec3, like all other exocyst subunits, localizes to cell poles largely independently of the actin cytoskeleton. However, we show that Sec3, Exo70 and Sec5 are transported by the myosin V Myo52 along actin cables. These data suggest that the exocyst holocomplex, including Sec3 and Exo70, is present on exocytic vesicles, which can reach cell poles by either myosin-driven transport or random walk.

Direct Fourier Tomographic Reconstruction Image-To-Image Filter

We present an open-source ITK implementation of a directFourier method for tomographic reconstruction, applicableto parallel-beam x-ray images. Direct Fourierreconstruction makes use of the central-slice theorem tobuild a polar 2D Fourier space from the 1D transformedprojections of the scanned object, that is resampled intoa Cartesian grid. Inverse 2D Fourier transform eventuallyyields the reconstructed image. Additionally, we providea complex wrapper to the BSplineInterpolateImageFunctionto overcome ITKâeuro?s current lack for image interpolatorsdealing with complex data types. A sample application ispresented and extensively illustrated on the Shepp-Loganhead phantom. We show that appropriate input zeropaddingand 2D-DFT oversampling rates together with radial cubicb-spline interpolation improve 2D-DFT interpolationquality and are efficient remedies to reducereconstruction artifacts.

A-Kinase Anchoring Protein (AKAP)-Lbc Anchors a PKN-based Signaling Complex Involved in α1-Adrenergic Receptor-induced p38 Activation.

The mitogen-activated protein kinases (MAPKs) pathways are highly organized signaling systems that transduce extracellular signals into a variety of intracellular responses. In this context, it is currently poorly understood how kinases constituting these signaling cascades are assembled and activated in response to receptor stimulation to generate specific cellular responses. Here, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor activity, is critically involved in the activation of the p38α MAPK downstream of α(1b)-adrenergic receptors (α(1b)-ARs). Our results indicate that AKAP-Lbc can assemble a novel transduction complex containing the RhoA effector PKNα, MLTK, MKK3, and p38α, which integrates signals from α(1b)-ARs to promote RhoA-dependent activation of p38α. In particular, silencing of AKAP-Lbc expression or disrupting the formation of the AKAP-Lbc·p38α signaling complex specifically reduces α(1)-AR-mediated p38α activation without affecting receptor-mediated activation of other MAPK pathways. These findings provide a novel mechanistic hypothesis explaining how assembly of macromolecular complexes can specify MAPK signaling downstream of α(1)-ARs.