Molecular evolution of the Drosophila olfactory system

The olfactory system is an attractive model to study the genetic mechanisms underlying evolution of the nervous system. This sensory system mediates the detection and behavioural responses to an enormous diversity of volatile chemicals in the environment and displays rapid evolution, as species acquire, modify and discard olfactory receptors and circuits to adapt to new olfactory stimuli. Drosophilids provide an attractive model to study these processes. The availability of 12 sequenced genomes of Drosophila species occupying diverse ecological niches provides a rich resource for genomic analyses. Moreover, one of these species, Drosophila melanogaster, is amenable to a powerful combination of genetic and electrophysiological analyses. D. melanogaster has two distinct families of olfactory receptors to detect odours, the well-characterised Odorant Receptors (ORs) and the recently identified lonotropic Receptors (IRs). In my thesis, I have provided new insights into the genetic mechanisms underlying olfactory system evolution through three distinct, but interrelated projects. First, I performed a comparative genomic analysis of the IR repertoire in 12 sequenced Drosophila species, which has revealed that the olfactory IRs are highly conserved across species. By contrast, a large fraction of IRs that are not expressed in the olfactory system - and which may be gustatory receptors - are much more variable in sequence and gene copy number. Second, to identify ligands for IR expressing olfactory sensory neurons, I have performed an electrophysiological screen in D. melanogaster using a panel of over 160 odours. I found that the IRs respond to a number of amines, aldehydes and acids, contrasting with the chemical specificity of the OR repertoire, which is mainly tuned to esters, alcohols and ketones. Finally, the identification of ligands for IRs in this species allowed me to investigate in detail the molecular and functional evolution of a tandem array of IRs, IR75a/IR75b/IR75c, in D. sechellia. This species is endemic to the Seychelles archipelago and highly specialised to breed on the fruits of Morinda citrifolia, which is repulsive and toxic for other Drosophila species. These studies led me to discover that receptor loss, changes in receptor specificity and changes in receptor expression have likely played an important role during the evolution of these IRs in D. sechellia. These changes may explain, in part, the unique chemical ecology of this species. - Le système olfactif est un excellent modèle pour étudier les mécanismes génétiques impliqués dans l'étude de l'évolution du système nerveux. Ce système sensoriel permet la détection de nombreux composés volatils présents dans l'environnement et est à la base des réponses comportementales. Il est propre à chaque espèce et évolue rapidement en modifiant ou en éliminant des récepteurs et leurs circuits olfactifs correspondants pour s'adapter à de nouvelles odeurs. Pour étudier le système olfactif et son évolution, nous avons décidé d'utiliser la drosophile comme modèle. Le séquençage complet de 12 souches de drosophiles habitant différentes niches écologiques permet une analyse génomique conséquente. De plus, l'une de ces espèces Drosophila melanogaster permet la combinaison d'analyses génétiques et électrophysiologiques. En effet, D. melanogaster possède 2 familles distinctes de récepteurs olfactifs qui permettent la détection d'odeurs: les récepteurs olfactifs (ORs) étant les mieux caractérisés et les récepteurs ionotropiques (IRs), plus récemment identifiés. Au cours de ma thèse, j'ai apporté des nouvelles connaissances qui m'ont permis de mieux comprendre les mécanismes génétiques à la base de l'évolution du système olfactif au travers de trois projets différents, mais interdépendants. Premièrement, j'ai réalisé une analyse génomique comparative de l'ensemble des IRs dans les 12 souches de drosophiles séquencées jusqu'à présent. Ceci a montré que les récepteurs olfactifs IRs sont hautement conservés parmi l'ensemble de ces espèces. Au contraire, une grande partie des IRs qui ne sont pas exprimés dans le système olfactif, et qui semblent être des récepteurs gustatifs, sont beaucoup plus variables dans leur séquence et dans le nombre de copie de gènes. Deuxièmement, pour identifier les ligands des récepteurs IRs exprimés par les neurones sensoriels olfactifs, j'ai réalisé une étude électrophysiologique chez D. melanogaster e η testant l'effet de plus de 160 composés chimiques sur les IRs. J'ai trouvé que les IRs répondent à un nombre d'amines, d'aldéhydes et d'acides, contrairement aux récepteurs olfactifs ORs qui eux répondent principalement aux esthers, alcools et cétones. Finalement, l'identification de ligands pour les IRs dans ces espèces m'a permis d'étudier en détail l'évolution fonctionnelle et moléculaire des IR75a/IR75b/IR75c dans D. sechellia. Cette espèce est endémique de l'archipel des Seychelles et se nourrit spécifiquement du fruit Morinda citrifolia qui est répulsif et toxique pour d'autres souches de drosophiles. Ces études m'ont poussé à découvrir que, la perte de IR75a, le changement dans la spécificité de IR75b ainsi que le changement dans l'expression de IR75c ont probablement joué un rôle important dans l'évolution des IRs chez D. sechellia. Ces changements peuvent expliquer, en partie, l'écologie chimique propre à cette espèce. Résumé français large public Le système olfactif permet aux animaux de détecter des milliers de molécules odorantes, les aidant ainsi à trouver de la nourriture, à distinguer si elle est fraîche ou avariée, à trouver des partenaires sexuels, ainsi qu'à éviter les prédateurs. Selon l'environnement et le mode de vie des espèces, le système olfactif doit détecter des odeurs très diverses ; en effet, un moustique qui recherche du sang humain pour se nourrir doit détecter des odeurs bien différentes d'une abeille qui recherche des fleurs. Dans ma thèse, j'ai essayé de comprendre comment les systèmes olfactifs d'une espèce évoluent pour s'adapter aux exigences induites par son environnement. Un très bon modèle pour étudier cela est la drosophile dont les différentes espèces se nichent dans des habitats très divers. Pour ce faire, j'ai étudié les récepteurs olfactifs de différentes espèces de la drosophile. Ces récepteurs sont des protéines qui se lient à des odeurs spécifiques. Lorsqu'ils se lient, ils activent un neurone qui envoie un signal électrique au cerveau. Ce signal est ensuite traité par ce dernier qui indique à la mouche si l'odeur est attractive ou répulsive. J'ai identifié les récepteurs olfactifs de plusieurs espèces de drosophile et étudié s'il y avait des différences entre elles. La plupart des récepteurs sont similaires entre les espèces, cependant dans l'une d'entre elles, certains récepteurs sont différents. Ce fait est particulièrement intéressant car cette espèce de drosophile se nourrit de fruits que les autres espèces n'apprécient pas. Comme nous ne savons pas quels récepteurs se lient à quelles odeurs, j'ai testé un grand nombre de composants odorants. Ceci m'a permis de constater que, effectivement, certains changements produits dans ces récepteurs expliquent pourquoi cette espèce aime particulièrement ces fruits. En outre, mes résultats contribuent à mieux comprendre les changements génétiques qui sont impliqués dans l'évolution du système olfactif.

Mirror extreme BMI phenotypes associated with gene dosage at the chromosome 16p11.2 locus.

Both obesity and being underweight have been associated with increased mortality. Underweight, defined as a body mass index (BMI) ≤ 18.5 kg per m(2) in adults and ≤ -2 standard deviations from the mean in children, is the main sign of a series of heterogeneous clinical conditions including failure to thrive, feeding and eating disorder and/or anorexia nervosa. In contrast to obesity, few genetic variants underlying these clinical conditions have been reported. We previously showed that hemizygosity of a ∼600-kilobase (kb) region on the short arm of chromosome 16 causes a highly penetrant form of obesity that is often associated with hyperphagia and intellectual disabilities. Here we show that the corresponding reciprocal duplication is associated with being underweight. We identified 138 duplication carriers (including 132 novel cases and 108 unrelated carriers) from individuals clinically referred for developmental or intellectual disabilities (DD/ID) or psychiatric disorders, or recruited from population-based cohorts. These carriers show significantly reduced postnatal weight and BMI. Half of the boys younger than five years are underweight with a probable diagnosis of failure to thrive, whereas adult duplication carriers have an 8.3-fold increased risk of being clinically underweight. We observe a trend towards increased severity in males, as well as a depletion of male carriers among non-medically ascertained cases. These features are associated with an unusually high frequency of selective and restrictive eating behaviours and a significant reduction in head circumference. Each of the observed phenotypes is the converse of one reported in carriers of deletions at this locus. The phenotypes correlate with changes in transcript levels for genes mapping within the duplication but not in flanking regions. The reciprocal impact of these 16p11.2 copy-number variants indicates that severe obesity and being underweight could have mirror aetiologies, possibly through contrasting effects on energy balance.

An EORTC phase I itudy of bortezomib in combination with oxaliplatin, leucovorin and 5-fluorouracil in patients with advanced colorectal cancer.

The combination of oxaliplatin, leucovorin and 5-fluorouracil (FOLFOX-4) is still a reference regimen in advanced colorectal cancer; however, the addition of new biologic compounds represents a significant way forward. Bortezomib is an inhibitor of proteasome, a multicatalytic enzyme complex that degrades several intracellular proteins. In this study, escalating doses of Bortezomib were administered along with the standard FOLFOX-4 doses, in order to evaluate the dose-limiting toxicity (DLT), toxicity profile and activity of the combination. Patients with advanced colorectal cancer, unpretreated for metastatic disease, were enroled in the study. Bortezomib starting dose was 1.3mg/m(2), which was to be escalated in the subsequent steps according to the toxicities observed after first cycle. Exploratory pharmacogenetics research was conducted by analysing the association between clinical outcomes and polymorphisms in candidate genes for response to each of the used drugs. Correlation between tumour marker changes and response was also investigated. One mg/m(2) (DL-1) was defined as being the maximum tolerated dose since only 1 DLT was observed in 6 patients. The main toxicities were haematologic, neuropathy, diarrhoea and fatigue. Amongst 13 evaluable patients, five had a partial response, five had a stable disease and three patients progressed. Two patients are long-term survivors after a combined chemosurgical approach. Further trials of the current combination may be justified.

Identification of Genes Affecting Vacuole Membrane Fragmentation in Saccharomyces cerevisiae.

The equilibrium of membrane fusion and fission influences the volume and copy number of organelles. Fusion of yeast vacuoles has been well characterized but their fission and the mechanisms determining vacuole size and abundance remain poorly understood. We therefore attempted to systematically characterize factors necessary for vacuole fission. Here, we present results of an in vivo screening for deficiencies in vacuolar fragmentation activity of an ordered collection deletion mutants, representing 4881 non-essential genes of the yeast Saccharomyces cerevisiae. The screen identified 133 mutants with strong defects in vacuole fragmentation. These comprise numerous known fragmentation factors, such as the Fab1p complex, Tor1p, Sit4p and the V-ATPase, thus validating the approach. The screen identified many novel factors promoting vacuole fragmentation. Among those are 22 open reading frames of unknown function and three conspicuous clusters of proteins with known function. The clusters concern the ESCRT machinery, adaptins, and lipases, which influence the production of diacylglycerol and phosphatidic acid. A common feature of these factors of known function is their capacity to change membrane curvature, suggesting that they might promote vacuole fragmentation via this property.

Assessing the Functionality and Biocompatibility of a Wireless Contact Lens Sensor for 24 hour-intraocular Pressure Monitoring in Volunteers: Preliminary Results

Purpose: In vitro studies in porcine eyes have demonstrated a good correlation between induced intraocular pressure variations and corneal curvature changes, using a contact lens with an embedded microfabricated strain gauge. Continuous 24 hour-intraocular pressure (IOP) monitoring to detect large diurnal fluctuation is currently an unmet clinical need. The aims of this study is to evaluate precision of signal transmission and biocompatibility of 24 hour contact lens sensor wear (SENSIMED Triggerfish®) in humans. Methods: After full eye examination in 10 healthy volunteers, a 8.7 mm radius contact lens sensor and an orbital bandage containing a loop antenna were applied and connected to a portable recorder. Best corrected visual acuity and position, lubrication status and mobility of the sensor were assessed after 5 and 30 minutes, 4, 7 and 24 hours. Subjective comfort was scored and activities documented in a logbook. After sensor removal full eye examination was repeated, and the registration signal studied. Results: The comfort score was high and did not fluctuate significantly, except at the 7 hour-visit. The mobility of the contact lens was minimal but its lubrication remained good. Best corrected visual acuity was significantly reduced during the sensor wear and immediately after its removal. Three patients developed mild corneal staining. In all but one participant we obtained a registration IOP curve with visible ocular pulse amplitude. Conclusions: This 24 hour-trial confirmed the functionality and biocompatibility of SENSIMED Triggerfish® wireless contact lens sensor for IOP-fluctuation monitoring in volunteers. Further studies with a range of different contact lens sensor radii are indicated.

Subgroup-specific structural variation across 1,000 medulloblastoma genomes.

Medulloblastoma, the most common malignant paediatric brain tumour, is currently treated with nonspecific cytotoxic therapies including surgery, whole-brain radiation, and aggressive chemotherapy. As medulloblastoma exhibits marked intertumoural heterogeneity, with at least four distinct molecular variants, previous attempts to identify targets for therapy have been underpowered because of small samples sizes. Here we report somatic copy number aberrations (SCNAs) in 1,087 unique medulloblastomas. SCNAs are common in medulloblastoma, and are predominantly subgroup-enriched. The most common region of focal copy number gain is a tandem duplication of SNCAIP, a gene associated with Parkinson's disease, which is exquisitely restricted to Group 4α. Recurrent translocations of PVT1, including PVT1-MYC and PVT1-NDRG1, that arise through chromothripsis are restricted to Group 3. Numerous targetable SCNAs, including recurrent events targeting TGF-β signalling in Group 3, and NF-κB signalling in Group 4, suggest future avenues for rational, targeted therapy.

Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfection.

Transfection with polyethylenimine (PEI) was evaluated as a method for the generation of recombinant Chinese hamster ovary (CHO DG44) cell lines by direct comparison with calcium phosphate-DNA coprecipitation (CaPO4) using both green fluorescent protein (GFP) and a monoclonal antibody as reporter proteins. Following transfection with a GFP expression vector, the proportion of GFP-positive cells as determined by flow cytometry was fourfold higher for the PEI transfection as compared to the CaPO4 transfection. However, the mean level of transient GFP expression for the cells with the highest level of fluorescence was twofold greater for the CaPO4 transfection. Fluorescence in situ hybridization on metaphase chromosomes from pools of cells grown under selective pressure demonstrated that plasmid integration always occurred at a single site regardless of the transfection method. Importantly, the copy number of integrated plasmids was measurably higher in cells transfected with CaPO4. The efficiency of recombinant cell line recovery under selective pressure was fivefold higher following PEI transfection, but the average specific productivity of a recombinant antibody was about twofold higher for the CaPO4-derived cell lines. Nevertheless, no difference between the two transfection methods was observed in terms of the stability of protein production. These results demonstrated the feasibility of generating recombinant CHO-derived cell lines by PEI transfection. However, this method appeared inferior to CaPO4 transfection with regard to the specific productivity of the recovered cell lines.

Validation of real-time methylation-specific PCR to determine O6-methylguanine-DNA methyltransferase gene promoter methylation in glioma.

Epigenetic silencing of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) by promoter methylation predicts successful alkylating agent therapy, such as with temozolomide, in glioblastoma patients. Stratified therapy assignment of patients in prospective clinical trials according to tumor MGMT status requires a standardized diagnostic test, suitable for high-throughput analysis of small amounts of formalin-fixed, paraffin-embedded tumor tissue. A direct, real-time methylation-specific PCR (MSP) assay was developed to determine methylation status of the MGMT gene promoter. Assay specificity was obtained by selective amplification of methylated DNA sequences of sodium bisulfite-modified DNA. The copy number of the methylated MGMT promoter, normalized to the beta-actin gene, provides a quantitative test result. We analyzed 134 clinical glioma samples, comparing the new test with the previously validated nested gel-based MSP assay, which yields a binary readout. A cut-off value for the MGMT methylation status was suggested by fitting a bimodal normal mixture model to the real-time results, supporting the hypothesis that there are two distinct populations within the test samples. Comparison of the tests showed high concordance of the results (82/91 [90%]; Cohen's kappa = 0.80; 95% confidence interval, 0.82-0.95). The direct, real-time MSP assay was highly reproducible (Pearson correlation 0.996) and showed valid test results for 93% (125/134) of samples compared with 75% (94/125) for the nested, gel-based MSP assay. This high-throughput test provides an important pharmacogenomic tool for individualized management of alkylating agent chemotherapy.

The Waddlia genome: a window into chlamydial biology.

Growing evidence suggests that a novel member of the Chlamydiales order, Waddlia chondrophila, is a potential agent of miscarriage in humans and abortion in ruminants. Due to the lack of genetic tools to manipulate chlamydia, genomic analysis is proving to be the most incisive tool in stimulating investigations into the biology of these obligate intracellular bacteria. 454/Roche and Solexa/Illumina technologies were thus used to sequence and assemble de novo the full genome of the first representative of the Waddliaceae family, W. chondrophila. The bacteria possesses a 2'116'312 bp chromosome and a 15'593 bp low-copy number plasmid that might integrate into the bacterial chromosome. The Waddlia genome displays numerous repeated sequences indicating different genome dynamics from classical chlamydia which almost completely lack repetitive elements. Moreover, W. chondrophila exhibits many virulence factors also present in classical chlamydia, including a functional type III secretion system, but also a large complement of specific factors for resistance to host or environmental stresses. Large families of outer membrane proteins were identified indicating that these highly immunogenic proteins are not Chlamydiaceae specific and might have been present in their last common ancestor. Enhanced metabolic capability for the synthesis of nucleotides, amino acids, lipids and other co-factors suggests that the common ancestor of the modern Chlamydiales may have been less dependent on their eukaryotic host. The fine-detailed analysis of biosynthetic pathways brings us closer to possibly developing a synthetic medium to grow W. chondrophila, a critical step in the development of genetic tools. As a whole, the availability of the W. chondrophila genome opens new possibilities in Chlamydiales research, providing new insights into the evolution of members of the order Chlamydiales and the biology of the Waddliaceae.

Towards the identification of a genetic basis for Landau-Kleffner syndrome.

OBJECTIVE: To establish the genetic basis of Landau-Kleffner syndrome (LKS) in a cohort of two discordant monozygotic (MZ) twin pairs and 11 isolated cases. METHODS: We used a multifaceted approach to identify genetic risk factors for LKS. Array comparative genomic hybridization (CGH) was performed using the Agilent 180K array. Whole genome methylation profiling was undertaken in the two discordant twin pairs, three isolated LKS cases, and 12 control samples using the Illumina 27K array. Exome sequencing was undertaken in 13 patients with LKS including two sets of discordant MZ twins. Data were analyzed with respect to novel and rare variants, overlapping genes, variants in reported epilepsy genes, and pathway enrichment. RESULTS: A variant (cG1553A) was found in a single patient in the GRIN2A gene, causing an arginine to histidine change at site 518, a predicted glutamate binding site. Following copy number variation (CNV), methylation, and exome sequencing analysis, no single candidate gene was identified to cause LKS in the remaining cohort. However, a number of interesting additional candidate variants were identified including variants in RELN, BSN, EPHB2, and NID2. SIGNIFICANCE: A single mutation was identified in the GRIN2A gene. This study has identified a number of additional candidate genes including RELN, BSN, EPHB2, and NID2. A PowerPoint slide summarizing this article is available for download in the Supporting Information section here.

Late Cretaceous to Eocene geology of the South Central American forearc area (southern Costa Rica and western Panama) : initiation and evolution of an intra-oceanic convergent margin

Résumé : L'arc volcanique du sud de l'Amérique Centrale se situe sur la marge SW de la Plaque Caraïbe, au-dessus des plaques subduites de Cocos et Nazca. Il s'agit de l'un des arcs intra-océaniques les plus étudiés au monde, qui est généralement considéré comme s'étant développé à la fin du Crétacé le long d'un plateau océanique (le Plateau Caraïbe ou CLIP) et se trouvant actuellement dans un régime de subduction érosive. Au cours des dernières décennies, des efforts particuliers ont été faits pour comprendre les processus liés à la subduction sur la base d'études géophysiques et géochimiques. Au sud du Costa Rica et à l'ouest du Panama, des complexes d'accrétions et structures à la base de l'arc volcanique ont été exposés grâce à la subduction de rides asismiques et de failles transformantes. Des affleurements, situés jusqu'à seulement 15 km de la fosse, offrent une possibilité unique de mieux comprendre quelques uns des processus ayant lieu le long de la zone de subduction. Nous présentons de nouvelles contraintes sur l'origine de ces affleurements en alliant une étude de terrain poussée, de nouvelles données géochimiques, sédimentaires et paléontologiques, ainsi que des observations structurales effectuées en télédétection. Une nouvelle stratigraphie tectonique entre le Campanien et l'Éocène est définie pour la région d'avant-arc située entre la Péninsule d'Osa (Costa Rica) et la Péninsule d'Azuero (Panama). Nos résultats montrent que la partie externe de la marge est composée d'un arrangement complexe de roches ignées et de séquences sédimentaires de recouvrement qui comprennent principalement le socle de l'arc, des roches d'arc primitif, des fragments de monts sous-marins accrétés et des mélanges d'accrétion. Des preuves sont données pour le développement de l'arc volcanique du sud de l'Amérique Centrale sur un plateau océanique. Le début de la subduction le long de la marge SW de la Plaque Caraïbe a eu lieu au Campanien et a généré des roches d'arc primitif caractérisées par des affinités géochimiques particulières, globalement intermédiaires entre des affinités de plateau et d'arc insulaire. L'arc était mature au Maastrichtien et formait un isthme essentiellement continu entre l'Amérique du Nord et l'Amérique du Sud. Ceci a permis la migration de faunes terrestres entre les Amériques et pourrait avoir contribué à la crise fin Crétacé -Tertiaire en réduisant les courants océaniques subéquatoriaux entre le Pacifique et l'Atlantique. Plusieurs unités composées de fragments de monts sous-marins accrétés sont définies. La nature et l'arrangement structural de ces unités définissent de nouvelles contraintes sur les modes d'accrétion des monts sous-marins/îles océaniques et sur l'évolution de la marge depuis la formation de la zone de subduction. Entre la fin du Crétacé et l'Éocène moyen, la marge a enregistré plusieurs épisodes ponctuels d'accrétion de monts sous-marins alternant avec de la subduction érosive. A l'Éocène moyen, un événement tectonique régional pourrait avoir causé un fort couplage entre les plaques supérieure et inférieure, menant à des taux plus important d'accrétion de monts sous-marins. Durant cette période, la situation le long de la marge était très semblable à la situation actuelle et caractérisée par la présence de monts sous-marins subductants et l'absence d'accrétion de sédiments. L'enregistrement géologique montre qu'il n'est pas possible d'attribuer une nature érosive ou accrétionnaire à la marge dans le passé ou -par analogie- aujourd'hui, parce que (1) les processus d'accrétion et érosifs varient fortement spatialement et temporellement et (2) il est impossible d'évaluer la quantité exacte de matériel tectoniquement enlevé à la marge depuis le début de la subduction. Au sud du Costa Rica, certains fragments de monts sous-marins accrétés sont représentatifs d'une interaction entre une ride et un point chaud dans le Pacifique au Crétacé terminal/Paléocène. L'existence de ces fragments de monts sous-marins et la morphologie du fond de l'Océan Pacifique indiquent que la formation de la ride de Cocos-Nazca s'est formée au moins ~40 Ma avant l'âge proposé par les modèles tectoniques actuels. Au Panama, nous avons identifié une île océanique d'âge début Éocène qui a été accrétée à l'Éocène moyen. L'accrétion a eu lieu à très faible profondeur par détachement de l'île dans la fosse, et a mené à une exceptionnelle préservation des structures volcaniques. Des affleurement comprenant aussi bien des parties basses et hautes de l'édifice volcanique on été étudiées, depuis la phase sous-marine bouclier jusqu'à la phase subaérienne post-bouclier. La stratigraphie nous a permis de différencier les laves de la phase sous-marine de celles de la phase subaérienne. La composition des laves indique une diminution progressive de l'intensité de la fusion partielle de la source et une diminution de la température des laves produites durant les derniers stades de l'activité volcanique. Nous interprétons ces changements comme étant liés à l'éloignement progressif de l'île océanique de la zone de fusion ou point chaud. Abstract The southern Central American volcanic front lies on the SW edge of the Caribbean Plate, inboard of the subducting Cocos and Nazca Plates. It is one of the most studied intra-oceanic convergent margins around the world, which is generally interpreted to have developed in the late Cretaceous along an oceanic plateau (the Caribbean Large Igneous Province or CLIP) and to be currently undergoing a regime of subduction erosion. In the last decades a particular effort has been made to understand subduction-related processes on the basis of geophysical and geochemical studies. In southern Costa Rica and western Panama accretionary complexes and structures at the base of the volcanic front have been exposed in response to subduction of aseismic ridges and transforms. Onland exposures are located as close as to 15 km from the trench and provide a unique opportunity to better understand some of the processes occurring along the subduction zone. We provide new constraints on the origins of these exposures by integrating a comprehensive field work, new geochemical, sedimentary and paleontological data, as well as structural observations based on remote imaging. A new Campanian to Eocene tectonostratigraphy is defined for the forearc area located between the Osa Peninsula (Costa Rica) and the Azuero Peninsula (Panama). Our results show that the outer margin is composed of a complicated arrangement of igneous complexes and overlapping sedimentary sequences that essentially comprise an arc basement, primitive island-arc rocks, accreted seamount fragments and accretionary mélanges. Evidences are provided for the development of the southern Central American arc on the top an oceanic plateau. The subduction initiation along the SW edge of the Caribbean Plate occurred in the Campanian and led to formation of primitive island-arc rocks characterized by unusual geochemical affinities broadly intermediate between plateau and arc affinities. The arc was mature in the Maastrichtian and was forming a predominantly continuous landbridge between the North and South Americas. This allowed migration of terrestrial fauna between the Americas and may have contributed to the Cretaceous-Tertiary crisis by limiting trans-equatorial oceanic currents between the Pacific and the Atlantic. Several units composed of accreted seamount fragments are defined. The nature of the units and their structural arrangement provide new constraints on the modes of accretion of seamounts/oceanic islands and on the evolution of the margin since subduction initiation. Between the late Cretaceous and the middle Eocene, the margin recorded several local episodes of seamount accretion alternating with tectonic erosion. In the middle Eocene a regional tectonic event may have triggered strong coupling between the overriding and subducting plates, leading to higher rates of seamount accretion. During this period the situation along the margin was very similar to the present and characterized by subducting seamounts and absence of sediment accretion. The geological record shows that it is not possible to ascribe an overall erosive or accretionary nature to the margin in the past and, by analogy, today, because (1) accretionary and erosive processes exhibit significant lateral and temporal variations and (2) it is impossible to estimate the exact amount of material tectonically eroded from the margin since subduction initiation. In southern Costa Rica, accreted seamount fragments point toward a plume-ridge interaction in the Pacific in the late Cretaceous/Paleocene. This occurrence of accreted seamount fragments and morphology of the Pacific Ocean floor is indicative of the formation of the Cocos-Nazca spreading system at least ~40 Ma prior to the age proposed in current tectonic models. In Panama, we identified a remarkably-well preserved early Eocene oceanic island that accreted in the middle Eocene. The accretion probably occurred at very shallow depth by detachment of the island in the trench and led to an exceptional preservation of the volcanic structures. Exposures of both deep and superficial parts of the volcanic edifice have been studied, from the submarine-shield to subaerial-postshield stages. The stratigraphy allowed us to distinguish lavas produced during the submarine and subaerial stages. The lava compositions likely define a progressive diminution of source melting and a decrease in the temperature of erupted melts in the latest stages of volcanic activity. We interpret these changes to primarily reflect the progressive migration of the oceanic island out of the melting region or hotspot.

Estimating traveltimes and groundwater flow patterns using 3D time-lapse crosshole ERT imaging of electrical resistivity fluctuations induced by infiltrating river water

The infiltration of river water into aquifers is of high relevance to drinking-water production and is a key driver of biogeochemical processes in the hyporheic and riparian zone, but the distribution and quantification of the infiltrating water are difficult to determine using conventional hydrological methods (e.g., borehole logging and tracer tests). By time-lapse inverting crosshole ERT (electrical resistivity tomography) monitoring data, we imaged groundwater flow patterns driven by river water infiltrating a perialpine gravel aquifer in northeastern Switzerland. This was possible because the electrical resistivity of the infiltrating water changed during rainfall-runoff events. Our time-lapse resistivity models indicated rather complex flow patterns as a result of spatially heterogeneous bank filtration and aquifer heterogeneity. The upper part of the aquifer was most affected by the river infiltrate, and the highest groundwater velocities and possible preferential flow occurred at shallow to intermediate depths. Time series of the reconstructed resistivity models matched groundwater electrical resistivity data recorded on borehole loggers in the upper and middle parts of the aquifer, whereas the resistivity models displayed smaller variations and delayed responses with respect to the logging data. in the lower part. This study demonstrated that crosshole ERT monitoring of natural electrical resistivity variations of river infiltrate could be used to image and quantify 3D bank filtration and aquifer dynamics at a high spatial resolution.

Association analyses of 249,796 individuals reveal 18 new loci associated with body mass index.

Obesity is globally prevalent and highly heritable, but its underlying genetic factors remain largely elusive. To identify genetic loci for obesity susceptibility, we examined associations between body mass index and ∼ 2.8 million SNPs in up to 123,865 individuals with targeted follow up of 42 SNPs in up to 125,931 additional individuals. We confirmed 14 known obesity susceptibility loci and identified 18 new loci associated with body mass index (P < 5 × 10⁻⁸), one of which includes a copy number variant near GPRC5B. Some loci (at MC4R, POMC, SH2B1 and BDNF) map near key hypothalamic regulators of energy balance, and one of these loci is near GIPR, an incretin receptor. Furthermore, genes in other newly associated loci may provide new insights into human body weight regulation.

Real-time quantitative PCR assay for measurement of bird telomeres

We present the application of a real-time quantitative PCR assay, previously developed to measure relative telomere length in humans and mice, to two bird species, the zebra finch Taeniopygia guttata and the Alpine swift Apus melba. This technique is based on the PCR amplification of telomeric (TTAGGG)(n) sequences using specific oligonucleotide primers. Relative telomere length is expressed as the ratio (T/S) of telomere repeat copy number (T) to control single gene copy number (S). This method is particularly useful for comparisons of individuals within species, or where the same individuals are followed longitudinally. We used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a single control gene. In both species, we validated our PCR measurements of relative telomere length against absolute measurements of telomere length determined by the conventional method of quantifying telomere terminal restriction fragment (TRF) lengths using both the traditional Southern blot analysis (Alpine swifts) and in gel hybridization (zebra finches). As found in humans and mice, telomere lengths in the same sample measured by TRF and PCR were well correlated in both the Alpine swift and the zebra finch.. Hence, this PCR assay for measurement of bird telomeres, which is fast and requires only small amounts of genomic DNA, should open new avenues in the study of environmental factors influencing variation in telomere length, and how this variation translates into variation in cellular and whole organism senescence.

Results of An International Study of Quantitative BCR/ABL Assays Using Defined RNA Samples

Molecular monitoring of BCR/ABL transcripts by real time quantitative reverse transcription PCR (qRT-PCR) is an essential technique for clinical management of patients with BCR/ABL-positive CML and ALL. Though quantitative BCR/ABL assays are performed in hundreds of laboratories worldwide, results among these laboratories cannot be reliably compared due to heterogeneity in test methods, data analysis, reporting, and lack of quantitative standards. Recent efforts towards standardization have been limited in scope. Aliquots of RNA were sent to clinical test centers worldwide in order to evaluate methods and reporting for e1a2, b2a2, and b3a2 transcript levels using their own qRT-PCR assays. Total RNA was isolated from tissue culture cells that expressed each of the different BCR/ABL transcripts. Serial log dilutions were prepared, ranging from 100 to 10-5, in RNA isolated from HL60 cells. Laboratories performed 5 independent qRT-PCR reactions for each sample type at each dilution. In addition, 15 qRT-PCR reactions of the 10-3 b3a2 RNA dilution were run to assess reproducibility within and between laboratories. Participants were asked to run the samples following their standard protocols and to report cycle threshold (Ct), quantitative values for BCR/ABL and housekeeping genes, and ratios of BCR/ABL to housekeeping genes for each sample RNA. Thirty-seven (n=37) participants have submitted qRT-PCR results for analysis (36, 37, and 34 labs generated data for b2a2, b3a2, and e1a2, respectively). The limit of detection for this study was defined as the lowest dilution that a Ct value could be detected for all 5 replicates. For b2a2, 15, 16, 4, and 1 lab(s) showed a limit of detection at the 10-5, 10-4, 10-3, and 10-2 dilutions, respectively. For b3a2, 20, 13, and 4 labs showed a limit of detection at the 10-5, 10-4, and 10-3 dilutions, respectively. For e1a2, 10, 21, 2, and 1 lab(s) showed a limit of detection at the 10-5, 10-4, 10-3, and 10-2 dilutions, respectively. Log %BCR/ABL ratio values provided a method for comparing results between the different laboratories for each BCR/ABL dilution series. Linear regression analysis revealed concordance among the majority of participant data over the 10-1 to 10-4 dilutions. The overall slope values showed comparable results among the majority of b2a2 (mean=0.939; median=0.9627; range (0.399 - 1.1872)), b3a2 (mean=0.925; median=0.922; range (0.625 - 1.140)), and e1a2 (mean=0.897; median=0.909; range (0.5174 - 1.138)) laboratory results (Fig. 1-3)). Thirty-four (n=34) out of the 37 laboratories reported Ct values for all 15 replicates and only those with a complete data set were included in the inter-lab calculations. Eleven laboratories either did not report their copy number data or used other reporting units such as nanograms or cell numbers; therefore, only 26 laboratories were included in the overall analysis of copy numbers. The median copy number was 348.4, with a range from 15.6 to 547,000 copies (approximately a 4.5 log difference); the median intra-lab %CV was 19.2% with a range from 4.2% to 82.6%. While our international performance evaluation using serially diluted RNA samples has reinforced the fact that heterogeneity exists among clinical laboratories, it has also demonstrated that performance within a laboratory is overall very consistent. Accordingly, the availability of defined BCR/ABL RNAs may facilitate the validation of all phases of quantitative BCR/ABL analysis and may be extremely useful as a tool for monitoring assay performance. Ongoing analyses of these materials, along with the development of additional control materials, may solidify consensus around their application in routine laboratory testing and possible integration in worldwide efforts to standardize quantitative BCR/ABL testing.