Identification of novel functional TBP-binding sites and general factor repertoires.

Our current knowledge of the general factor requirement in transcription by the three mammalian RNA polymerases is based on a small number of model promoters. Here, we present a comprehensive chromatin immunoprecipitation (ChIP)-on-chip analysis for 28 transcription factors on a large set of known and novel TATA-binding protein (TBP)-binding sites experimentally identified via ChIP cloning. A large fraction of identified TBP-binding sites is located in introns or lacks a gene/mRNA annotation and is found to direct transcription. Integrated analysis of the ChIP-on-chip data and functional studies revealed that TAF12 hitherto regarded as RNA polymerase II (RNAP II)-specific was found to be also involved in RNAP I transcription. Distinct profiles for general transcription factors and TAF-containing complexes were uncovered for RNAP II promoters located in CpG and non-CpG islands suggesting distinct transcription initiation pathways. Our study broadens the spectrum of general transcription factor function and uncovers a plethora of novel, functional TBP-binding sites in the human genome.

Functional diversity of CD8+ T-cell subsets in chronic infection

Certains pathogènes tels que le virus de l'hépatite C ainsi que le virus de l'immunodéficience humaine sont capables de détourner les mécanismes de défense du système immunitaire adaptatif afin d'établir des infections chroniques chez l'homme. La souche clone-13 du virus de la chorioméningite lymphocytaire est utilisée comme modèle d'études d'infection virale chronique chez la souris. Les raisons qui expliqueraient la persistance de certains virus ne sont pas encore bien définies. Toutefois, il a été montré qu'une exposition prolongée à un environnement inflammatoire ainsi que la présence prolongée de l'antigène sont des facteurs qui vont déclencher un procédé de différentiation particulier des lymphocytes T CD8+. Ces cellules sur-expriment alors des récepteurs inhibiteurs tels que PD-1 tandis que leur capacité à produire des cytokines diminue. Ce procédé de différentiation est appelé « exhaustion » et serait à l'origine de la génération de réponses lymphocytaires non ou peu fonctionnelles entraînant de ce fait la persistance de l'infection. Néanmoins, il a également été démontré que ces lymphocytes maintiennent des fonctions effectrices et qu'ils permettent de limiter la réplication du virus. Afin d'étudier la fonction effectrice résiduelle de ces lymphocytes T, nous avons transféré des cellules provenant de souris infectées chroniquement dans des souris receveuses naïves qui ont à leur tour été infectées avec le virus. Grâce à ces expériences, nous avons démontré que les cellules transférées contiennent des cellules qui sont capables de i) re-proliférer, ii) de protéger les souris contre une infection virale, et de iii) survivre en l'absence d'antigène. Nous avons remarqué que les cellules stimulées de façon chronique maintiennent le même phénotype lorsqu'elles sont transférées dans des souris naïves soumises à une infection virale aiguë. Nous avons de ce fait conclu que les cellules stimulées chroniquement contiennent une sous-population de cellules qui comporte des attributs de cellules T mémoire. D'autre part, nous avons pu identifier le facteur de transcription Tcf-1 comme l'élément essential pour la génération des cellules T ressemblant à des cellules mémoires. D'autre part, nous avons étudié l'impact du niveau de stimulation via le récepteur des cellules T (TCR) sur le phénotype adopté par les lymphocytes T au cours d'une infection chronique. Dans ce but, nous avons généré des souches de virus recombinants qui expriment un épitope modifié de manière à réduire le niveau de stimulation via le TCR. D'autre part, nous avons utilisé un mélange de deux souches virales de manière à moduler spécifiquement la quantité d'un épitope tout en conservant la même charge virale. Nous avons montré que la quantité d'antigène avait plus d'influence sur le phénotype des lymphocytes T que la force d'interaction entre le complexe peptide-CMH et le TCR. De plus, l'apparition de ce phénotype ne semble pas avoir d'impact sur la prolifération des cellules en réponse à une infection primaire ou secondaire. Ainsi, nous proposons un modèle par lequel le procédé d'« exhaustion » des cellules T correspond à une différentiation cellulaire particulière qui est indépendante de la capacité de prolifération des cellules. De manière générale, ces découvertes apportent de nouvelles connaissances sur les sous-catégories de lymphocytes T CD8+ qui sont générés pendant une infection virale chronique. Nous pensons que la réponse effectrice du système immunitaire est maintenue pendant de longues périodes grâce à la présence de cellules par partagent certaines caractéristiques avec des cellules mémoires. L'étude approfondie de ces cellules peut avoir des implications importantes sur l'optimisation des stratégies utilisant l'immunothérapie pour combattre les infections chroniques et cancers.

Shedding light on proteolytic cleavage of CD44: the responsible sheddase and functional significance of shedding.

CD44 is the major cell-surface receptor for hyaluronan, which is implicated in cell-cell and cell-matrix adhesion, cell migration, and signaling. Studies have shown that CD44-dependent migration requires CD44 to be shed from the cell surface and that matrix metalloproteinase-mediated cleavage may provide an underlying mechanism. However, the full spectrum of proteases that may participate in CD44 shedding has yet to be defined. In this issue, Anderegg et al. demonstrate that ADAM10, but not ADAM17 or MMP14, mediates constitutive shedding of CD44 in human melanoma cells and that knockdown of ADAM10 blocks the antiproliferative activity of the soluble proteolytic cleavage product of CD44.

Identification of HIV integration sites in infected host genomic DNA.

The integration of the Human Immunodeficiency Virus (HIV) genetic information into the host genome is fundamental for its replication and long-term persistence in the host. Isolating and characterizing the integration sites can be useful for obtaining data such as identifying the specific genomic location of integration or understanding the forces dictating HIV integration site selection. The methods outlined in this article describe a highly efficient and precise technique for identifying HIV integration sites in the host genome on a small scale using molecular cloning techniques and standard sequencing or on a massive scale using 454 pyrosequencing.

Visual activity predicts auditory recovery from deafness after adult cochlear implantation.

Modern cochlear implantation technologies allow deaf patients to understand auditory speech; however, the implants deliver only a coarse auditory input and patients must use long-term adaptive processes to achieve coherent percepts. In adults with post-lingual deafness, the high progress of speech recovery is observed during the first year after cochlear implantation, but there is a large range of variability in the level of cochlear implant outcomes and the temporal evolution of recovery. It has been proposed that when profoundly deaf subjects receive a cochlear implant, the visual cross-modal reorganization of the brain is deleterious for auditory speech recovery. We tested this hypothesis in post-lingually deaf adults by analysing whether brain activity shortly after implantation correlated with the level of auditory recovery 6 months later. Based on brain activity induced by a speech-processing task, we found strong positive correlations in areas outside the auditory cortex. The highest positive correlations were found in the occipital cortex involved in visual processing, as well as in the posterior-temporal cortex known for audio-visual integration. The other area, which positively correlated with auditory speech recovery, was localized in the left inferior frontal area known for speech processing. Our results demonstrate that the visual modality's functional level is related to the proficiency level of auditory recovery. Based on the positive correlation of visual activity with auditory speech recovery, we suggest that visual modality may facilitate the perception of the word's auditory counterpart in communicative situations. The link demonstrated between visual activity and auditory speech perception indicates that visuoauditory synergy is crucial for cross-modal plasticity and fostering speech-comprehension recovery in adult cochlear-implanted deaf patients.

Combined functional and viability cardiac MR imaging in a single breathhold

The combination of cardiac viability and functional information enhances the identification of different heart tissues in the setting of ischemic heart disease. A method has recently been proposed for obtaining black-blood delayed-enhancement (DE) viability images using the stimulated-echo acquisition mode (STEAM) MRI pulse sequence in a single short breathhold. The method was validated against conventional inversion-recovery (IR) DE images for identifying regions of myocardial infarction (MI). The method was based on the acquisition of three consecutive images of the same anatomical slice. One image has T(1)-weighted contrast in which infarction appears bright. The two other images are used to construct an anatomical image of the heart, which is combined with the first image to produce a black-blood viability image. However, using appropriate modulation and demodulation frequencies, the latter two images bear useful information about myocardial deformation that results in a cardiac strain-encoding (SENC) functional image. In this work, a method is proposed for obtaining three consecutive SENC images in a single acquisition that can be combined to produce a composite image of the heart, which shows both functional and viability information. The proposed technique reduces scan time by one-half, compared with separate acquisitions of functional and viability images, and alleviates misregistration problems caused by separate breathholds.

Dynamic changes in brain functional connectivity during concurrent dual-task performance.

This study investigated the spatial, spectral, temporal and functional proprieties of functional brain connections involved in the concurrent execution of unrelated visual perception and working memory tasks. Electroencephalography data was analysed using a novel data-driven approach assessing source coherence at the whole-brain level. Three connections in the beta-band (18-24 Hz) and one in the gamma-band (30-40 Hz) were modulated by dual-task performance. Beta-coherence increased within two dorsofrontal-occipital connections in dual-task conditions compared to the single-task condition, with the highest coherence seen during low working memory load trials. In contrast, beta-coherence in a prefrontal-occipital functional connection and gamma-coherence in an inferior frontal-occipitoparietal connection was not affected by the addition of the second task and only showed elevated coherence under high working memory load. Analysis of coherence as a function of time suggested that the dorsofrontal-occipital beta-connections were relevant to working memory maintenance, while the prefrontal-occipital beta-connection and the inferior frontal-occipitoparietal gamma-connection were involved in top-down control of concurrent visual processing. The fact that increased coherence in the gamma-connection, from low to high working memory load, was negatively correlated with faster reaction time on the perception task supports this interpretation. Together, these results demonstrate that dual-task demands trigger non-linear changes in functional interactions between frontal-executive and occipitoparietal-perceptual cortices.

Stem cell populations derived from heart and pancreas show a with a unique phenotype and give rise to functional cardiomyocytes and insulin-producing cells, respectively

Introduction: Recently, mesenchymal stem cells (MSC) of perivascular origin have been identified in several organs not including the heart. Using a novel cell isolation protocol, we have isolated cells sharing common characteristics from mouse hearts and pancreas. The aim of the present study was to characterize these cells in vitro.Methods: Cells were isolated from neonatal and adult mouse hearts and pancreas and cultured for more than 6 months. Surface marker expression was analyzed by flow cytometry and immunocytochemistry. Cell differentiation was tested using multiple differentiation media. Insulin production by pancreas-derived cells was tested by dithizone staining.Results: Cells showing a similar, distinctive morphology were obtained from the heart and pancreas after 4-8 weeks of culture. Cells from the two organs also showed a very similar immunophenotype, characterized by expression of c-kit (stem cell factor receptor), CD44, the common leukocyte marker CD45, and the monocytic markers CD11b and CD14. A significant proportion of cardiac and pancreatic cells expressed NG2, a marker for pericytes and other vascular cells. A significant proportion of cardiac, but not of pancreatic cells expressed stem cell antigen-1 (Sca-1). However, cells did not express T, B or dendritic cell markers. Cells of both cardiac and pancreatic origin spontaneously formed "spheres" (spherical cell aggregates similar to "neurospheres" formed by neural stem cells) in vitro. Cardiosphere formation was enhanced by TNF-alpha. Several cardiospheres (but no "pancreatospheres") derived from neonatal (but not adult) cells showed spontaneous rhythmic contractions, thus demonstrating cardiac differentiation (this was confirmed by immunostaining for alpha-sarcomeric actinin). Beating activity was enhanced by low serum conditions. Cells from both organs formed adipocytes, osteocytes and osteocytes under appropriate conditions, the typical differentiation pattern of MSCs. Pancreas-derived cells also formed dithizonepositive insulin-producing cells.Conclusions: We have defined cardiac and pancreatic cell populations that share a common morphology, growth characteristics, and a unique immunophenotype. Expression of perivascular and monocytic markers, along with stem/priogenitor cell markers by these cells suggests a relationship with pericytes-mesoangioblasts and so-called multipotent monocytes. Cells show MSC-typical growth and differentiation patterns, together with tissue-specific differentiation potential: cardiomyocytes for cardiac-derived cells and insulinproducing cells for pancreas-derived cells.

NS4B Self-Interaction through Conserved C-Terminal Elements Is Required for the Establishment of Functional Hepatitis C Virus Replication Complexes.

Hepatitis C virus (HCV) is an important human pathogen, persistently infecting more than 170 million individuals worldwide. Studies of the HCV life cycle have become possible with the development of cell culture systems supporting the replication of viral RNA and the production of infectious virus. However, the exact functions of individual proteins, especially of nonstructural protein 4B (NS4B), remain poorly understood. NS4B triggers the formation of specific, vesicular membrane rearrangements, referred to as membranous webs, which have been reported to represent sites of HCV RNA replication. However, the mechanism of vesicle induction is not known. In this study, a panel of 15 mutants carrying substitutions in the highly conserved NS4B C-terminal domain was generated. Five mutations had only a minor effect on replication, but two of them enhanced assembly and release of infectious virus. Ten mutants were replication defective and used for selection of pseudoreversions. Most of the pseudoreversions also localized to the highly conserved NS4B C-terminal domain and were found to restore replication competence upon insertion into the corresponding primary mutant. Importantly, pseudoreversions restoring replication competence also restored heterotypic NS4B self-interaction, which was disrupted by the primary mutation. Finally, electron microscopy analyses of membrane alterations induced by NS4B mutants revealed striking morphological abnormalities, which were restored to wild-type morphology by the corresponding pseudoreversion. These findings demonstrate the important role of the C-terminal domain in NS4B self-interaction and the formation of functional HCV replication complexes.

Focal dystonia and the Sensory-Motor Integrative Loop for Enacting (SMILE).

Performing accurate movements requires preparation, execution, and monitoring mechanisms. The first two are coded by the motor system, the latter by the sensory system. To provide an adaptive neural basis to overt behaviors, motor and sensory information has to be properly integrated in a reciprocal feedback loop. Abnormalities in this sensory-motor loop are involved in movement disorders such as focal dystonia, a hyperkinetic alteration affecting only a specific body part and characterized by sensory and motor deficits in the absence of basic motor impairments. Despite the fundamental impact of sensory-motor integration mechanisms on daily life, the general principles of healthy and pathological anatomic-functional organization of sensory-motor integration remain to be clarified. Based on the available data from experimental psychology, neurophysiology, and neuroimaging, we propose a bio-computational model of sensory-motor integration: the Sensory-Motor Integrative Loop for Enacting (SMILE). Aiming at direct therapeutic implementations and with the final target of implementing novel intervention protocols for motor rehabilitation, our main goal is to provide the information necessary for further validating the SMILE model. By translating neuroscientific hypotheses into empirical investigations and clinically relevant questions, the prediction based on the SMILE model can be further extended to other pathological conditions characterized by impaired sensory-motor integration.

Glibenclamide enhances neurogenesis and improves long-term functional recovery after transient focal cerebral ischemia

Glibenclamide is neuroprotective against cerebral ischemia in rats. We studied whether glibenclamide enhances long-term brain repair and improves behavioral recovery after stroke. Adult male Wistar rats were subjected to transient middle cerebral artery occlusion (MCAO) for 90 minutes. A low dose of glibenclamide (total 0.6mg) was administered intravenously 6, 12, and 24 hours after reperfusion. We assessed behavioral outcome during a 30-day follow-up and animals were perfused for histological evaluation. In vitro specific binding of glibenclamide to microglia increased after pro-inflammatory stimuli. In vivo glibenclamide was associated with increased migration of doublecortin-positive cells in the striatum toward the ischemic lesion 72 hours after MCAO, and reactive microglia expressed sulfonylurea receptor 1 (SUR1) and Kir6.2 in the medial striatum. One month after MCAO, glibenclamide was also associated with increased number of NeuN-positive and 5-bromo-2-deoxyuridine-positive neurons in the cortex and hippocampus, and enhanced angiogenesis in the hippocampus. Consequently, glibenclamide-treated MCAO rats showed improved performance in the limb-placing test on postoperative days 22 to 29, and in the cylinder and water-maze test on postoperative day 29. Therefore, acute blockade of SUR1 by glibenclamide enhanced long-term brain repair in MCAO rats, which was associated with improved behavioral outcome.

Molecular and Functional Characterization of Neuroblastoma-Initiating Cells

Neuroblastoma (NB) is the most common extracranial malignant tumor in young children and arises at any site of the sympathetic nervous system. The disease exhibits a remarkable phenotypic diversity ranging from spontaneous regression to fatal disease. Poor outcome results from a rapidly progressive, metastatic and drug-resistant disease. Recent studies have suggested that solid tumors may arise from a minor population of cancer stem cells (CSCs) with stem cell markers and typical properties such as self-renewal ability, asymmetric division and drug resistance. In this model, CSCs possess the exclusive ability to initiate and maintain the tumor, and to produce distant metastases. Tumor cell subpopulations with stem-like phenotypes have indeed been identified in several cancer including leukemia, breast, brain and colon cancers. CSC hypothesis still needs to be validated in the other cancers including NB.NB originates from neural crest-derived malignant sympatho-adrenal cells. We have identified rare cells that express markers in conformity with neural crest stem cells and their derived lineages within primary NB tissue and cell lines, leading us to postulate the existence of CSCs in NB tumors.In the absence of specific markers to isolate CSCs, we adapted to NB tumor cells the sphere functional assay, based on the ability of stem cells to grow as spheres in non-adherent conditions. By serial passages of spheres from bone marrow NB metastases, a subset of cells was gradually selected and its specific gene expression profile identified by micro-array time-course analysis. The differentially expressed genes in spheres are enriched in genes implicated in development including CD133, ABC-transporters, WNT and NOTCH genes, identified in others solid cancers as CSCs markers, and other new markers, all referred by us as the Neurosphere Expression Profile (NEP). We confirmed the presence of a cell subpopulation expressing a combination of the NEP markers within a few primary NB samples.The tumorigenic potential of NB spheres was assayed by in vivo tumor growth analyses using orthotopic (adrenal glands) implantations of tumor cells into immune-compromised mice. Tumors derived from the sphere cells were significantly more frequent and were detected earlier compared to whole tumor cells. However, NB cells expressing the neurosphere-associated genes and isolated from the bulk tumors did not recapitulate the CSC-like phenotype in the orthotopic model. In addition, the NB sphere cells lost their higher tumorigenic potential when implanted in a subcutaneous heterotopic in vivo model.These results highlighted the complex behavior of CSC functions and led us to consider the stem-like NB cells as a dynamic and heterogeneous cell population influenced by microenvironment signals.Our approach identified for the first time candidate genes that may be associated with NB self-renewal and tumorigenicity and therefore would establish specific functional targets for more effective therapies in aggressive NB.

Integration of Pore Features into the Evaluation of Fingerprint Evidence

Fingerprint practitioners rely on level 3 features to make decisions in relation to the source of an unknown friction ridge skin impression. This research proposes to assess the strength of evidence associated with pores when shown in (dis)agreement between a mark and a reference print. Based upon an algorithm designed to automatically detect pores, a metric is defined in order to compare different impressions. From this metric, the weight of the findings is quantified using a likelihood ratio. The results obtained on four configurations and 54 donors show the significant contribution of the pore features and translate into statistical terms what latent fingerprint examiners have developed holistically through experience. The system provides LRs that are indicative of the true state under both the prosecution and the defense propositions. Not only such a system brings transparency regarding the weight to assign to such features, but also forces a discussion in relation to the risks of such a model to mislead.