Recurrent deletions and reciprocal duplications of 10q11.21q11.23 including CHAT and SLC18A3 are likely mediated by complex low-copy repeats.

We report 24 unrelated individuals with deletions and 17 additional cases with duplications at 10q11.21q21.1 identified by chromosomal microarray analysis. The rearrangements range in size from 0.3 to 12 Mb. Nineteen of the deletions and eight duplications are flanked by large, directly oriented segmental duplications of >98% sequence identity, suggesting that nonallelic homologous recombination (NAHR) caused these genomic rearrangements. Nine individuals with deletions and five with duplications have additional copy number changes. Detailed clinical evaluation of 20 patients with deletions revealed variable clinical features, with developmental delay (DD) and/or intellectual disability (ID) as the only features common to a majority of individuals. We suggest that some of the other features present in more than one patient with deletion, including hypotonia, sleep apnea, chronic constipation, gastroesophageal and vesicoureteral refluxes, epilepsy, ataxia, dysphagia, nystagmus, and ptosis may result from deletion of the CHAT gene, encoding choline acetyltransferase, and the SLC18A3 gene, mapping in the first intron of CHAT and encoding vesicular acetylcholine transporter. The phenotypic diversity and presence of the deletion in apparently normal carrier parents suggest that subjects carrying 10q11.21q11.23 deletions may exhibit variable phenotypic expressivity and incomplete penetrance influenced by additional genetic and nongenetic modifiers.

FAM161A, associated with retinitis pigmentosa, is a component of the cilia-basal body complex and interacts with proteins involved in ciliopathies.

Retinitis pigmentosa (RP) is a retinal degenerative disease characterized by the progressive loss of photoreceptors. We have previously demonstrated that RP can be caused by recessive mutations in the human FAM161A gene, encoding a protein with unknown function that contains a conserved region shared only with a distant paralog, FAM161B. In this study, we show that FAM161A localizes at the base of the photoreceptor connecting cilium in human, mouse and rat. Furthermore, it is also present at the ciliary basal body in ciliated mammalian cells, both in native conditions and upon the expression of recombinant tagged proteins. Yeast two-hybrid analysis of binary interactions between FAM161A and an array of ciliary and ciliopathy-associated proteins reveals direct interaction with lebercilin, CEP290, OFD1 and SDCCAG8, all involved in hereditary retinal degeneration. These interactions are mediated by the C-terminal moiety of FAM161A, as demonstrated by pull-down experiments in cultured cell lines and in bovine retinal extracts. As other ciliary proteins, FAM161A can also interact with the microtubules and organize itself into microtubule-dependent intracellular networks. Moreover, small interfering RNA-mediated depletion of FAM161A transcripts in cultured cells causes the reduction in assembled primary cilia. Taken together, these data indicate that FAM161A-associated RP can be considered as a novel retinal ciliopathy and that its molecular pathogenesis may be related to other ciliopathies.

Dissociation of thymic positive and negative selection in transgenic mice expressing major histocompatibility complex class I molecules exclusively on thymic cortical epithelial cells.

Thymic positive and negative selection of developing T lymphocytes confronts us with a paradox: How can a T-cell antigen receptor (TCR)-major histocompatibility complex (MHC)/peptide interaction in the former process lead to transduction of signals allowing for cell survival and in the latter induce programmed cell death or a hyporesponsive state known as anergy? One of the hypotheses put forward states that the outcome of a TCR-MHC/peptide interaction depends on the cell type presenting the selecting ligand to the developing thymocyte. Here we describe the development and lack of self-tolerance of CD8(+) T lymphocytes in transgenic mice expressing MHC class I molecules in the thymus exclusively on cortical epithelial cells. Despite the absence of MHC class I expression on professional antigen-presenting cells, normal numbers of CD8(+) cells were observed in the periphery. Upon specific activation, transgenic CD8(+) T cells efficiently lysed syngeneic MHC class I(+) targets in vitro and in vivo, indicating that thymic cortical epithelium (in contrast to medullary epithelium and antigen-presenting cells of hematopoietic origin) is incapable of tolerance induction. Thus, compartmentalization of the antigen-presenting cells involved in thymic positive selection and tolerance induction can (at least in part) explain the positive/negative selection paradox.

Caveolin-1 interacts with the chaperone complex TCP-1 and modulates its protein folding activity.

We report that caveolin-1, one of the major structural protein of caveolae, interacts with TCP-1, a hetero-oligomeric chaperone complex present in all eukaryotic cells that contributes mainly to the folding of actin and tubulin. The caveolin-TCP-1 interaction entails the first 32 amino acids of the N-terminal segment of caveolin. Our data show that caveolin-1 expression is needed for the induction of TCP-1 actin folding function in response to insulin stimulation. Caveolin-1 phosphorylation at tyrosine residue 14 induces the dissociation of caveolin-1 from TCP-1 and activates actin folding. We show that the mechanism by which caveolin-1 modulates TCP-1 activity is indirect and involves the cytoskeleton linker filamin. Filamin is known to bind caveolin-1 and to function as a negative regulator of insulin-mediated signaling. Our data support the notion that the caveolin-filamin interaction contributes to restore insulin-mediated phosphorylation of caveolin, thus allowing the release of active TCP-1.

Almost routine prophylactic cholecystectomy during laparoscopic gastric bypass is safe

RAPPORT DE SYNTHESE : Introduction : les patients obèses morbides présentent un risque majeur de développer des calculs biliaires en raison d'une sécrétion accrue de cholestérol dans la bile. Ce risque, davantage élevé dans la phase de perte pondérale rapide consécutive à la chirurgie bariatrique ou lors de régimes amaigrissants, est souvent la cause de nombreux symptômes, voire de complications biliaires. Aussi l'association d'une cholécystectomie à la chirurgie bariatrique, notamment le bypass gastrique laparoscopique a-t-elle été proposée afin d'éviter ces complications parfois redoutables dans cette population fragile. Ce concept a cependant fait l'objet de démentis dans de récentes études où ce risque apparaîtrait moins élevé, et la cholécystectomie durant le by- pris gastrique laparoscopique pourrait être grevée de difficultés et présenter des risques opératoires non négligeables pour le patient. Patients et méthodes : notre série comporte 772 patients opérés entre 2000 et 2007 par by-pass gastrique laparoscopique, avec montage d'une anse en Y selon Roux. Ces patients obèses morbides avaient été sélectionnés sur la base d'une anamnèse concluante, d'un examen anthropométrique, d'un bilan sanguin et d'un ultrason abdominal. Une analyse rétrospective des résultats d'ultrason abdominal préopératoire et des rapports histopathologiques des vésicules biliaires en postopératoire a été réalisée chez les patients opérés avant 2004. Résultats : 58 patients (7,5 %) avaient déjà eu une cholécystectomie. L'US abdominal a révélé des calculs ou de la boue biliaire chez 81 patients (11,3 %), un polype chez un patient et une vésicule biliaire normale chez les patients restants. La cholécystectomie a été réalisée concomitamment au by-pass gastrique chez 66S patients (91,7 %) et des calculs biliaires retrouvés à l'examen per-opératoire des vésicules biliaires chez 25 patients (3,9 %), rapportant alors la prévalence de la cholélithiase à 21,2 % dans cette population. L'âge des patients porteurs de calculs biliaires était significativement plus élevé que celui des patients sans calculs biliares (43,5 contre 38,7 ans, P < 0,0001). A l'examen histopathologique, des anomalies ont été décrites dans 81,8 % des vésicules biliaires, consistant pour la plupart en cholécystite chronique et cholestérolose. Aucune complication post-opératoire n'a été associée à la cholécystectomie et le prolongement du temps opératoire était en moyenne de 19 minutes (4 - 45 minutes) sans aucun impact sur le séjour hospitalier. La cholécystectomie n'a pas été réalisée chez 59 patients (8,3 %) en raison de conditions opératoires défavorables, notamment une exposition insuffisante. Un traitement d'acide ursodésoxycholique a été prescrit sur une période de 6 mois et aucun de ces patients n'a manifesté de symptômes biliaires. Conclusion : la cholécystectomie peut être réalisée à titre prophylactique et en toute sécurité au cours du by-pass gastrique laparoscopique. Cet acte opératoire supplémentaire sans conséquence sur le séjour hospitalier, constitue selon la présente étude une forme de prophylaxie recommandable dans la prévention de la formation des calculs biliaires dans la phase de perte pondérale post-opératoire. Sa supériorité ou non par rapport à la prophylaxie médicamenteuse à l'acide ursodésoxycholique n'a pas encore été établie. Des études prospectives randomisées seraient nécessaires afin de confirmer l'avantage de l'une ou l'autre de ces deux alternatives.

Antiretroviral drug toxicity in relation to pharmacokinetics, metabolic profile and pharmacogenetics.

Introduction: Besides therapeutic effectiveness, drug tolerability is a key issue for treatments that must be taken indefinitely. Given the high prevalence of toxicity in HIV therapy, the factors implicated in drug-induced morbidities should be identified in order to improve the safety, tolerability and adherence to the treatments. Current approaches have focused almost exclusively on parent drug concentrations; whereas recent evidence suggests that drug metabolites resulting from complex genetic and environmental influences can also contribute to treatment outcome. Pharmacogenetic variations have shown to play a relevant role in the variability observed in antiretroviral drug exposure, clinical response and sometimes toxicity. The integration of pharmacokinetic, pharmacogenetic and metabolic determinants will more probably address current therapeutic needs in patients. Areas covered: This review offers a concise description of three classes of antiretroviral drugs. The review looks at the metabolic profile of these drugs and gives a comprehensive summary of the existing literature on the influence of pharmacogenetics on their pharmacokinetics and metabolic pathways, and the associated drug or metabolite toxicity. Expert opinion: Due to the high prevalence of toxicity and the related risk of low adherence to the treatments, association of kinetic, genetic and metabolic markers predictive of therapeutic or toxicity outcomes could represent a more complete approach for optimizing antiretroviral therapy.

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.

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.

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.

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.

A Miocene mud volcano and its plumbing system: A chaotic complex revisited (Monferrato, NW Italy)

Chaotic deposits are frequently reported in the geological literature and are commonly interpreted as olistostromes or tectonic melanges. A chaotic complex in the Cenozoic succession of Monferrato (NW Italy) consists of interbedded mud breccia and burrowed silty clays that are pierced by sheared mud breccias and embed carbonate-cemented blocks. These may be represented by microcrystalline limestones or strongly cemented matrix-supported breccias locally containing remains of chemosymbiotic organisms (lucinid bivalves). Moreover, cylindrical concretions, up to 15 cm in diameter and 1 m long, occur in the chaotic complex and crosscut bedding planes at high angles. The cement of all these lithified portions is mainly dolomite characterized by low delta(13)C values (from -10.3 to -23parts per thousand PDB) and delta(18)O values up to + 7parts per thousand PDB. The delta(13)C values testify to precipitation of carbonates induced by microbial oxidation of methane, whereas the markedly positive delta(18)C signature, ubiquitous in the cylindrical concretions, is the evidence for the presence and destabilization of gas hydrates. The studied section provides a well-exposed example of the geological record of the birth, life, and death of a mud volcano. Unsheared, soft mud breccias represent mud flows along the flanks of the volcano, whereas sheared mud breccias are the result of the injection of unconsolidated overpressured fine-grained sediments, both taking place during ``eruptive'' phases. They were followed by more quiet stages of hemipelagic sedimentation, burrowing, and CH4 seeping. The cylindrical concretions represent the first described ancient example of the chimneys observed in present-day mud-volcano settings. They are the remnants of a cold-seep plumbing network that crosscut the mud volcano edifice. The chimneys were the pathway for the expulsion toward the sea floor of gas- and sediment-charged fluids likely originated from destabilization of methane gas hydrates. The association of mud breccias and methane-derived carbonates may not be due to mass gravity flows but can be primary and, therefore, is a diagnostic criterion for recognizing chaotic deposits due to mud volcano activity in the geological record.

Are invasive marsh frogs (Pelophylax ridibundus) replacing the native P. lessonae /P. esculentus hybridogenetic complex in Western Europe? Genetic evidence from a field study.

The water-frog L-E system, widespread in Western Europe, comprises the pool frog Pelophylax lessonae and the hybridogenetic edible frog P. esculentus, which originated from hybridization between pool frogs and marsh frogs (P. ridibundus). In P. esculentus, the lessonae (L) genome is eliminated during meiosis and has to be gained anew each generation from a P. lessonae partner, while the ridibundus (R') genome is transmitted clonally. It therefore accumulates deleterious mutations, so that R'R' offspring from P. esculentus x P. esculentus crosses are normally unviable. This system is now threatened by invasive P. ridibundus (RR) imported from Eastern Europe and the Balkans. We investigated the genetic interactions between invasive marsh frogs and native water frogs in a Swiss wetland area, and used genetic data collected in the field to validate several components of a recently postulated mechanism of species replacement. We identified neo-ridibundus individuals derived from crosses between invasive ridibundus and native esculentus, as well as newly arisen hybridogenetic esculentus lineages stemming from crosses between invasive ridibundus (RR) and native lessonae (LL). As their ridibundus genomes are likely to carry less deleterious mutations, such lineages are expected to produce viable ridibundus offspring, contributing to species replacement. However, such crosses with invasive ridibundus only occurred at a limited scale; moreover, RR x LL crosses did not induce any introgression from the ridibundus to the lessonae genome. We did not find any ridibundus stemming from crosses between ancient esculentus lineages. Despite several decades of presence on the site, introduced ridibundus individuals only represent 15% of sampled frogs, and their spatial distribution seems shaped by specific ecological requirements rather than history of colonization. We therefore expect the three taxa to coexist stably in this area.