GLUTX1, a novel mammalian glucose transporter expressed in the central nervous system and insulin-sensitive tissues.

Based on homology with GLUT1-5, we have isolated a cDNA for a novel glucose transporter, GLUTX1. This cDNA encodes a protein of 478 amino acids that shows between 29 and 32% identity with rat GLUT1-5 and 32-36% identity with plant and bacterial hexose transporters. Unlike GLUT1-5, GLUTX1 has a short extracellular loop between transmembrane domain (TM) 1 and TM2 and a long extracellular loop between TM9 and TM10 that contains the only N-glycosylation site. When expressed in Xenopus oocytes, GLUTX1 showed strong transport activity only after suppression of a dileucine internalization motif present in the amino-terminal region. Transport activity was inhibited by cytochalasin B and partly competed by D-fructose and D-galactose. The Michaelis-Menten constant for glucose was approximately 2 mM. When translated in reticulocytes lysates, GLUTX1 migrates as a 35-kDa protein that becomes glycosylated in the presence of microsomal membranes. Western blot analysis of GLUTX1 transiently expressed in HEK293T cells revealed a diffuse band with a molecular mass of 37-50 kDa that could be converted to a approximately 35-kDa polypeptide following enzymatic deglycosylation. Immunofluorescence microscopy detection of GLUTX1 transfected into HEK293T cells showed an intracellular staining. Mutation of the dileucine internalization motif induced expression of GLUTX1 at the cell surface. GLUTX1 mRNA was detected in testis, hypothalamus, cerebellum, brainstem, hippocampus, and adrenal gland. We hypothesize that, in a similar fashion to GLUT4, in vivo cell surface expression of GLUTX1 may be inducible by a hormonal or other stimulus.

Automatic front-crawl temporal phase detection using adaptive filtering of inertial signals

This study introduces a novel approach for automatic temporal phase detection and inter-arm coordination estimation in front-crawl swimming using inertial measurement units (IMUs). We examined the validity of our method by comparison against a video-based system. Three waterproofed IMUs (composed of 3D accelerometer, 3D gyroscope) were placed on both forearms and the sacrum of the swimmer. We used two underwater video cameras in side and frontal views as our reference system. Two independent operators performed the video analysis. To test our methodology, seven well-trained swimmers performed three 300 m trials in a 50 m indoor pool. Each trial was in a different coordination mode quantified by the index of coordination. We detected different phases of the arm stroke by employing orientation estimation techniques and a new adaptive change detection algorithm on inertial signals. The difference of 0.2 +/- 3.9% between our estimation and video-based system in assessment of the index of coordination was comparable to experienced operators' difference (1.1 +/- 3.6%). The 95% limits of agreement of the difference between the two systems in estimation of the temporal phases were always less than 7.9% of the cycle duration. The inertial system offers an automatic easy-to-use system with timely feedback for the study of swimming.

A robust genomic signature for the detection of colorectal cancer patients with microsatellite instability phenotype and high mutation frequency.

Microsatellite instability (MSI) occurs in 10-20% of colorectal tumours and is associated with good prognosis. Here we describe the development and validation of a genomic signature that identifies colorectal cancer patients with MSI caused by DNA mismatch repair deficiency with high accuracy. Microsatellite status for 276 stage II and III colorectal tumours has been determined. Full-genome expression data was used to identify genes that correlate with MSI status. A subset of these samples (n = 73) had sequencing data for 615 genes available. An MSI gene signature of 64 genes was developed and validated in two independent validation sets: the first consisting of frozen samples from 132 stage II patients; and the second consisting of FFPE samples from the PETACC-3 trial (n = 625). The 64-gene MSI signature identified MSI patients in the first validation set with a sensitivity of 90.3% and an overall accuracy of 84.8%, with an AUC of 0.942 (95% CI, 0.888-0.975). In the second validation, the signature also showed excellent performance, with a sensitivity 94.3% and an overall accuracy of 90.6%, with an AUC of 0.965 (95% CI, 0.943-0.988). Besides correct identification of MSI patients, the gene signature identified a group of MSI-like patients that were MSS by standard assessment but MSI by signature assessment. The MSI-signature could be linked to a deficient MMR phenotype, as both MSI and MSI-like patients showed a high mutation frequency (8.2% and 6.4% of 615 genes assayed, respectively) as compared to patients classified as MSS (1.6% mutation frequency). The MSI signature showed prognostic power in stage II patients (n = 215) with a hazard ratio of 0.252 (p = 0.0145). Patients with an MSI-like phenotype had also an improved survival when compared to MSS patients. The MSI signature was translated to a diagnostic microarray and technically and clinically validated in FFPE and frozen samples.

The role of Pten in the Hematopoietic System and the Hematopoietic Stem Cells

Résumé Identification, localisation et activation des cellules souches hématopoiétiques dormantes in vivo Les cellules souches somatiques sont présentes dans la majorité des tissus régénératifs comme la peau, l'épithélium intestinal et le système hématopoiétique. A partir d'une seule cellule, elles ont les capacités de produire d'autres cellules souches du même type (auto-renouvellement) et d'engendrer un ensemble défini de cellules progénitrices différenciées qui vont maintenir ou réparer leur tissu hôte. Les cellules souches adultes les mieux caractérisées sont les cellules souches hématopoiétiques (HSC), localisées dans la moelle osseuse. Un des buts de mon travail de doctorat était de caractériser plus en profondeur la localisation des HSCs endogènes in vivo. Pour ce faire, la technique "label retaining assay", se basant sur la division peu fréquentes et sur la dormance des cellules souches, a été utilisée. Après un marquage des souris avec du BrdU (analogue à l'ADN) suivi d'une longue période sans BrdU, les cellules ayant incorporés le marquage ("label retaining cells" LCRs) ont pu être identifiées dans la moelle osseuse. Ces cellules LCRs étaient enrichies 300 fois en cellules de phenotype HSC et, en utilisant de la cytofluorométrie, il a pu être montré qu'environ 15% de toutes les HSCs d'une souris restent dormantes durant plusieures semaines. Ces HSCs dormantes à long terme ne sont probablement pas impliquées dans la maintenance de 'hématopoièse. Par contre, on assiste à l'activation rapide de ces HSCs dormantes lors d'une blessure, comme une ablation myéloide. Elles re-entrent alors en cycle cellulaire et sont essentielles pour une génération rapide des cellules progénitrices et matures qui vont remplacer les cellules perdues. De plus, la détection des LCRs, combinée avec l'utilisation du marqueur de HSCs c-kit, peut être utilisée pour la localisation des HSCs dormantes présentes dans la paroi endostéale de la cavité osseuse. De manière surprenante, les LCRs c-kit+ ont surtout étés trouvées isolées en cellule unique, suggérant que le micro-environement spécifique entourant et maintenant les HSCs, appelé niche, pourrait être très réduit et abriter une seule HSC par niche. Rôles complexes du gène supresseur de tumeur Pten dans le système hématopoiétique La phosphatase PTEN disparaît dans certains cancers héréditaires ou sporadiques humains, comme les gliomes, les cancers de l'utérus ou du sein. Pten inhibe la voie de signalisation de la PI3-kinase et joue un rôle clé dans l'apoptose, la croissance, la prolifération et la migration cellulaire. Notre but était d'étudier le rôle de Pten dans les HSC normale et durant la formation de leucémies. Pour ce faire, nous avons généré un modèle murin dans lequel le gène Pten peut être supprimé dans les cellules hématopoiétiques, incluant les HSCs. Ceci a été possible en croissant l'allèle conditionnelle ptenflox soit avec le transgène MxCre inductible par l'interféron α soit avec le transgène Scl-CreERt inductible par le tamoxifen. Ceci permet la conversion de l'allèle ptenflox en l'allèle nul PtenΔ dans les HSCs et les autres types cellulaires hématopoiétiques. Les souris mutantes Pten développent une splénomégalie massive causée par une expansion dramatiques de toutes les cellules myéloides. De manière interessante, alors que le nombre de HSCs dans la moelle osseuse diminue progressivement, le nombre des HSCs dans la rate augmente de manière proportionnelle. Etrangement, les analyses de cycle cellulaire ont montrés que Pten n'avait que peu ou pas d'effet sur la dormance des HSCs ou sur leur autorenouvellement. En revanche, une augmentation massive du niveau de la cytokine de mobilisation G-CSF a été détéctée dans le serum sanguin, suggérant que la suppression de Pten stimulerait la mobilisation et la migration des HSC de la moelle osseuse vers la rate. Finallement, la transplantation de moelle osseuse délétée en Pten dans des souris immuno-déficientes montre que Pten fonctionnerait comme un suppresseur de tumeur dans le système hématopoiétique car son absence entraîne la formation rapide de leucémies lymphocytaires. Summary Identification, localization and activation of dormant hematopoietic stun cells in vivo Somatic stem cells are present in most self-renewing tissues including the skin, the intestinal epithelium and the hematopoietic system. On a single cell basis they have the capacity to produce more stem cells of the same phenotype (self-renewal) and to give rise to a defined set of mature differentiated progeny, responsible for the maintenance or repair of the host tissue. The best characterized adult stem cell is the hematopoietic stem cell (HSC) located in the bone marrow. One goal of my thesis work was to further characterize the location of endogenous HSCs in vivo. To do this, a technique called "label retaining assay» was used which takes advantage of the fact that stem cells (including HSCs) divide very infrequently and can be dormant for months. After labeling mice with the DNA analogue BrdU followed by a long BrdU free "chase", BrdU "label retaining cells" (CRCs) could be identified in the bone marrow. These CRCs were 300-fold enriched for phenotypic HSCs and by using flow cytometry analysis it could be shown that about 15% of all HSCs in the mouse are dormant for many weeks. Our results suggest that these long-term dormant HSCs are unlikely to be involved in homeostatic maintenance. However they are rapidly activated and reenter the cell cycle in response to injury signals such as myeloid ablation. In addition, detection of LRCs in combination with the HSC marker c-Kit could be used to locate engrafted dormant HSCs close to the endosteal lining of the bone marrow cavities. Most surprisingly, c-Kit+LRCs were found predominantly as single cells suggesting that the specific stem cell maintaining microenvironment, called niche, has limited space and may house only single HSCs. Complex roles of the tumor suppressor gene Pten in the hematopoietic system. The phosphatase PTEN is lost in hereditary and sporadic forms of human cancers, including gliomas, endometrial and breast cancers. Pten inhibits the PI3-kina.se pathway and plays a key role in apoptosis, cell growth, proliferation and migration. Our aim was to study the role of Pten in normal HSCs and during leukemia formation. To do this, we generated a mouse model in which the Pten gene can be deleted in hematopoietic cells including HSCs. This was achieved by crossing the conditional ptenflox allele with either the interferona inducible MxCre or the tamoxifen inducible Scl-CreERT transgene. This allowed the conversion of the ptenflox allele into a pterr' null allele in HSCs and other hematopoietic cell types. As a result Pten mutant mice developed massive splenomegaly due to a dramatic expansion of all myeloid cells. Interestingly, while the number of bone marrow HSCs progressively decreased, the number of HSCs in the spleen increased to a similar extent. Unexpectedly, extensive cell cycle analysis showed that Pten had little or no effect on HSC dormancy or HSC self-renewal. Instead, dramatically increased levels of the mobilizing cytokine G-CSF were detected in the blood serum suggesting that loss-of Pten stimulates mobilization and migration of HSC from the BM to the spleen. Finally, transplantation of Pten deficient BM cells into immuno-compromised mice showed that Pten can function as a tumor suppressor in the hematopoietic system and that its absence leads to the rapid formation of T cell leukemia.

A new, automated, four-colour interphase FISH approach for the simultaneous detection of specific aneuploidies of diagnosis and prognosis significance in high hyperdiploid ALL

In hyperdiploid acute lymphoblastic leukaemia (ALL), the simultaneous occurrence of specific aneuploidies confers a more favourable outcome than hyperdiploidy alone. Interphase (I) FISH complements conventional cytogenetics (CC) through its sensitivity and ability to detect chromosome aberrations in non-dividing cells. To overcome the limits of manual I-FISH, we developed an automated four-colour I-FISH approach and assessed its ability to detect concurrent aneuploidies in ALL. I-FISH was performed using centromeric probes for chromosomes 4, 6, 10 and 17. Parameters established for automatic nucleus selection and signal detection were evaluated (3 controls). Cut-off values were determined (10 controls, 1000 nuclei/case). Combinations of aneuploidies were considered relevant when each aneuploidy was individually significant. Results obtained in 10 ALL patients (1500 nuclei/patient) were compared with those by CC. Various combinations of aneuploidies were identified. All clones detected by CC were observed by I-FISH. I-FISH revealed numerous additional abnormal clones, ranging between 0.1 % and 31.6%, based on the large number of nuclei evaluated. Four-colour automated I-FISH permits the identification of concurrent aneuploidies of prognostic significance in hyperdiploid ALL. Large numbers of cells can be analysed rapidly by this method. Owing to its high sensitivity, the method provides a powerful tool for the detection of small abnormal clones at diagnosis and during follow up. Compared to CC, it generates a more detailed cytogenetic picture, the biological and clinical significance of which merits further evaluation. Once optimised for a given set of probes, the system can be easily adapted for other probe combinations.

Detection of promoter activity by flow cytometric analysis of GFP reporter expression.

Low efficiency of transfection is often the limiting factor for acquiring conclusive data in reporter assays. It is especially difficult to efficiently transfect and characterize promoters in primary human cells. To overcome this problem we have developed a system in which reporter gene expression is quantified by flow cytometry. In this system, green fluorescent protein (GFP) reporter constructs are co-transfected with a reference plasmid that codes for the mouse cell surface antigen Thy-1.1 and serves to determine transfection efficiency. Comparison of mean GFP expression of the total transfected cell population with the activity of an analogous luciferase reporter showed that the sensitivity of the two reporter systems is similar. However, because GFP expression can be analyzed at the single-cell level and in the same cells the expression of the reference plasmid can be monitored by two-color fluorescence, the GFP reporter system is in fact more sensitive, particularly in cells which can only be transfected with a low efficiency.

Detection of EPO doping and blood doping: the haematological module of the Athlete Biological Passport.

The increase of the body's capacity to transport oxygen is a prime target for doping athletes in all endurance sports. For this pupose, blood transfusions or erythropoiesis stimulating agents (ESA), such as erythropoietin, NESP, and CERA are used. As direct detection of such manipulations is difficult, biomarkers that are connected to the haematopoietic system (haemoglobin concentration, reticulocytes) are monitored over time (Athlete Biological Passport (ABP)) and analyzed using mathematical models to identify patterns suspicious of doping. With this information, athletes can either be sanctioned directly based on their profile or targeted with conventional doping tests. Key issues for the appropriate use of the ABP are correct targeting and use of all available information (e.g. whereabouts, cross sectional population data) in a forensic manner. Future developments of the passport include the correction of all concentration-based variables for shifts in plasma volume, which might considerably increase sensitivity. New passport markers from the genomic, proteomic, and metabolomic level might add further information, but need to be validated before integration into the passport procedure. A first assessment of blood data of federations that have implemented the passport show encouraging signs of a decreased blood-doping prevalence in their athletes, which adds scientific credibility to this innovative concept in the fight against ESA- and blood doping. Copyright © 2012 John Wiley & Sons, Ltd.

Ultrastructure of the membrane attack complex of complement: detection of the tetramolecular C9-polymerizing complex C5b-8.

The ultrastructure of the membrane attack complex (MAC) of complement had been described as representing a hollow cylinder of defined dimensions that is composed of the proteins C5b, C6, C7, C8, and C9. After the characteristic cylindrical structure was identified as polymerized C9 [poly(C9)], the question arose as to the ultrastructural identity and topology of the C9-polymerizing complex C5b-8. An electron microscopic analysis of isolated MAC revealed an asymmetry of individual complexes with respect to their length. Whereas the length of one boundary (+/- SEM) was always 16 +/- 1 nm, the length of the other varied between 16 and 32 nm. In contrast, poly(C9), formed spontaneously from isolated C9, had a uniform tubule length (+/- SEM) of 16 +/- 1 nm. On examination of MAC-phospholipid vesicle complexes, an elongated structure was detected that was closely associated with the poly(C9) tubule and that extended 16-18 nm beyond the torus of the tubule and 28-30 nm above the membrane surface. The width of this structure varied depending on its two-dimensional projection in the electron microscope. By using biotinyl C5b-6 in the formation of the MAC and avidin-coated colloidal gold particles for the ultrastructural analysis, this heretofore unrecognized subunit of the MAC could be identified as the tetramolecular C5b-8 complex. Identification also was achieved by using anti-C5 Fab-coated colloidal gold particles. A similar elongated structure of 25 nm length (above the surface of the membrane) was observed on single C5b-8-vesicle complexes. It is concluded that the C5b-8 complex, which catalyzes poly(C9) formation, constitutes a structure of discrete morphology that remains as such identifiable in the fully assembled MAC, in which it is closely associated with the poly(C9) tubule.

Detection of coronary stenoses with contrast enhanced, three-dimensional free breathing coronary MR angiography using the gadolinium-based intravascular contrast agent gadocoletic acid (B-22956).

PURPOSE: To determine the diagnostic value of the intravascular contrast agent gadocoletic acid (B-22956) in three-dimensional, free breathing coronary magnetic resonance angiography (MRA) for stenosis detection in patients with suspected or known coronary artery disease. METHODS: Eighteen patients underwent three-dimensional, free breathing coronary MRA of the left and right coronary system before and after intravenous application of a single dose of gadocoletic acid (B-22956) using three different dose regimens (group A 0.050 mmol/kg; group B 0.075 mmol/kg; group C 0.100 mmol/kg). Precontrast scanning followed a coronary MRA standard non-contrast T2 preparation/turbo-gradient echo sequence (T2Prep); for postcontrast scanning an inversion-recovery gradient echo sequence was used (real-time navigator correction for both scans). In pre- and postcontrast scans quantitative analysis of coronary MRA data was performed to determine the number of visible side branches, vessel length and vessel sharpness of each of the three coronary arteries (LAD, LCX, RCA). The number of assessable coronary artery segments was determined to calculate sensitivity and specificity for detection of stenosis > or = 50% on a segment-to-segment basis (16-segment-model) in pre- and postcontrast scans with x-ray coronary angiography as the standard of reference. RESULTS: Dose group B (0.075 mmol/kg) was preferable with regard to improvement of MR angiographic parameters: in postcontrast scans all MR angiographic parameters increased significantly except for the number of visible side branches of the left circumflex artery. In addition, assessability of coronary artery segments significantly improved postcontrast in this dose group (67 versus 88%, p < 0.01). Diagnostic performance (sensitivity, specificity, accuracy) was 83, 77 and 78% for precontrast and 86, 95 and 94% for postcontrast scans. CONCLUSIONS: The use of gadocoletic acid (B-22956) results in an improvement of MR angiographic parameters, asssessability of coronary segments and detection of coronary stenoses > or = 50%.

Evaluation of aggregating brain cell cultures for the detection of acute organ-specific toxicity.

As part of the ACuteTox project aimed at the development of non-animal testing strategies for predicting human acute oral toxicity, aggregating brain cell cultures (AGGR) were examined for their capability to detect organ-specific toxicity. Previous multicenter evaluations of in vitro cytotoxicity showed that some 20% of the tested chemicals exhibited significantly lower in vitro toxicity as expected from in vivo toxicity data. This was supposed to be due to toxicity at supracellular (organ or system) levels. To examine the capability of AGGR to alert for potential organ-specific toxicants, concentration-response studies were carried out in AGGR for 86 chemicals, taking as endpoints the mRNA expression levels of four selected genes. The lowest observed effect concentration (LOEC) determined for each chemical was compared with the IC20 reported for the 3T3/NRU cytotoxicity assay. A LOEC lower than IC20 by at least a factor of 5 was taken to alert for organ-specific toxicity. The results showed that the frequency of alerts increased with the level of toxicity observed in AGGR. Among the chemicals identified as alert were many compounds known for their organ-specific toxicity. These findings suggest that AGGR are suitable for the detection of organ-specific toxicity and that they could, in conjunction with the 3T3/NRU cytotoxicity assay, improve the predictive capacity of in vitro toxicity testing.

Arrested kinetic Li isotope fractionation at the margin of the llimaussaq complex, South Greenland: Evidence for open-system processes during final cooling of peralkaline igneous rocks

Li contents [Li] and isotopic composition (delta Li-7) of mafic minerals (mainly amphibole and clinopyroxene) from the alkaline to peralkaline Ilimaussaq plutonic complex, South Greenland, track the behavior of Li and its isotopes during magmatic differentiation and final cooling of an alkaline igneous system. [Li] in amphibole increase from < 10 ppm in Caamphiboles of the least differentiated unit to >3000 ppm in Na-amphiboles of the highly evolved units. In contrast, [Li] in clinopyroxene are comparatively low (<85 ppm) and do not vary systematically with differentiation. The distribution of Li between amphibole and pyroxene is controlled by the major element composition of the minerals (Ca-rich and Na-rich, respectively) and changes in oxygen fugacity (due to Li incorporation via coupled substitution with ferric iron) during magmatic differentiation. delta(7) Li values of all minerals span a wide range from + 17 to - 8 parts per thousand, with the different intrusive units of the complex having distinct Li isotopic systematics. Amphiboles, which dominate the Li budget of whole-rocks from the inner part of the complex, have constant delta Li-7 of + 1.8 +/- 2.2 parts per thousand (2 sigma, n = 15). This value reflects a homogeneous melt reservoir and is consistent with their mantle derivation, in agreement with published O and Nd isotopic data. Clinopyroxenes of these samples are consistently lighter, with Delta Li-7(amph-cpx). as large as 8 parts per thousand and are thus not in Li isotope equilibrium. These low values probably reflect late-stage diffusion of Li into clinopyroxene during final cooling of the rocks, thus enriching the clinopyroxene in 6 Li. At the margin of the complex delta(7) Li in the syenites increases systematically, from +2 to high values of + 14 parts per thousand. This, coupled with the observed Li isotope systematics of the granitic country rocks, reflects post-magmatic open-system processes occurring during final cooling of the intrusion. Although the shape and magnitude of the Li isotope and elemental profiles through syenite and country rock are suggestive of diffusion-driven isotope fractionation, they cannot be modeled by one-dimensional diffusive transport and point to circulation of a fluid having a high 67 Li value (possibly seawater) along the chilled contact. In all, this study demonstrates that Li isotopes can be used to identify complex fluid- and diffusion-governed processes taking place during the final cooling of such rocks. (c) 2007 Elsevier B.V All rights reserved.

Induction of an immune response through the idiotypic network with monoclonal anti-idiotype antibodies in the carcinoembryonic antigen system.

Anti-idiotype antibodies can mimic the conformational epitopes of the original antigen and act as antigen substitutes for vaccination and/or serological purposes. To investigate this possibility concerning the tumor marker carcinoembryonic antigen (CEA), BALB/c mice were immunized with the previously described anti-CEA monoclonal antibody (MAb) 5.D11 (AB1). After cell fusion, 15 stable cloned cell lines secreting anti-Ids (AB2) were obtained. Selected MAbs gave various degrees of inhibition (up to 100%) of the binding of 125I-labeled CEA to MAb 5.D11. Absence of reactivity of anti-Id MAbs with normal mouse IgG was first demonstrated by the fact that anti-Id MAbs were not absorbed by passage through a mouse IgG column, and second because they bound specifically to non-reduced MAb 5.D11 on Western blots. Anti-5.D11 MAbs did not inhibit binding to CEA of MAb 10.B9, another anti-CEA antibody obtained in the same fusion as 5.D11, or that of several anti-CEA MAbs reported in an international workshop, with the exception of two other anti-CEA MAbs, both directed against the GOLD IV epitope. When applied to an Id-anti-Id competitive radioimmunoassay, a sensitivity of 2 ng/ml of CEA was obtained, which is sufficient for monitoring circulating CEA in carcinoma patients. To verify that the anti-Id MAbs have the potential to be used as CEA vaccines, syngeneic BALB/c mice were immunized with these MAbs (AB2). Sera from immunized mice were demonstrated to contain AB3 antibodies recognizing the original antigen, CEA, both in enzyme immunoassay and by immunoperoxidase staining of human colon carcinoma. These results open the perspective of vaccination against colorectal carcinoma through the use of anti-idiotype antibodies as antigen substitutes.

Prenatal detection of rare chromosomal autosomal abnormalities in Europe.

The aim of the present study was to evaluate the prenatal detection of rare chromosomal autosomal abnormalities by ultrasound (US) examination. Data were obtained from 19 congenital malformation registries from 11 European countries, between 01/07/96 and 31/12/98. A total of 664,340 births were covered and 7,758 cases with congenital malformations were recorded. Rare autosomal abnormalities were diagnosed in 114 cases (6.6%) from a total of 1,738 chromosome abnormalities. There were a wide variety of autosomal abnormalities: the most common were deletions (33 cases), duplications (32 cases), trisomies of chromosomes 8, 9, 10, 14, 15, and 16 (23 cases), and unbalanced rearrangements (19 cases). Out of these cases, 45.6% were detected prenatally by US examination due to the presence of congenital anomaly. As for the types of chromosomal anomaly, unbalanced rearrangements and deletions were the most frequently detected by US. A high percentage of cases with balanced rearrangements were associated with severe congenital anomalies. The most frequent congenital anomalies detected by US were cystic hygroma (20.6%), central nervous system defects (17.6%), cardiac defects (13.2%), and diaphragm defects (10.3%). This large series offers useful information about prenatal diagnosis by US of congenital defects associated with rare autosomal abnormalities and it provides a valuable knowledge about outcome. Fetal anomalies detected by US that were associated with rare autosomal abnormalities were significantly more frequent than those associated with common chromosomal abnormalities (45.6 vs. 34.7%). This study indicates the need to increase the detection of congenital anomalies by US.

Acid sensing by the Drosophila olfactory system.

The odour of acids has a distinct quality that is perceived as sharp, pungent and often irritating. How acidity is sensed and translated into an appropriate behavioural response is poorly understood. Here we describe a functionally segregated population of olfactory sensory neurons in the fruitfly, Drosophila melanogaster, that are highly selective for acidity. These olfactory sensory neurons express IR64a, a member of the recently identified ionotropic receptor (IR) family of putative olfactory receptors. In vivo calcium imaging showed that IR64a+ neurons projecting to the DC4 glomerulus in the antennal lobe are specifically activated by acids. Flies in which the function of IR64a+ neurons or the IR64a gene is disrupted had defects in acid-evoked physiological and behavioural responses, but their responses to non-acidic odorants remained unaffected. Furthermore, artificial stimulation of IR64a+ neurons elicited avoidance responses. Taken together, these results identify cellular and molecular substrates for acid detection in the Drosophila olfactory system and support a labelled-line mode of acidity coding at the periphery.

Ultra high throughput sequencing in human DNA variation detection: a comparative study on the NDUFA3-PRPF31 region.

BACKGROUND: Ultra high throughput sequencing (UHTS) technologies find an important application in targeted resequencing of candidate genes or of genomic intervals from genetic association studies. Despite the extraordinary power of these new methods, they are still rarely used in routine analysis of human genomic variants, in part because of the absence of specific standard procedures. The aim of this work is to provide human molecular geneticists with a tool to evaluate the best UHTS methodology for efficiently detecting DNA changes, from common SNPs to rare mutations. METHODOLOGY/PRINCIPAL FINDINGS: We tested the three most widespread UHTS platforms (Roche/454 GS FLX Titanium, Illumina/Solexa Genome Analyzer II and Applied Biosystems/SOLiD System 3) on a well-studied region of the human genome containing many polymorphisms and a very rare heterozygous mutation located within an intronic repetitive DNA element. We identify the qualities and the limitations of each platform and describe some peculiarities of UHTS in resequencing projects. CONCLUSIONS/SIGNIFICANCE: When appropriate filtering and mapping procedures are applied UHTS technology can be safely and efficiently used as a tool for targeted human DNA variations detection. Unless particular and platform-dependent characteristics are needed for specific projects, the most relevant parameter to consider in mainstream human genome resequencing procedures is the cost per sequenced base-pair associated to each machine.