Visualizing olfactory receptor expression and localization in Drosophila.

Odor detection and discrimination by olfactory systems in vertebrates and invertebrates depend both on the selective expression of individual olfactory receptor genes in subpopulations of olfactory sensory neurons, and on the targeting of the encoded proteins to the exposed, ciliated endings of sensory dendrites. Techniques to visualize the expression and localization of olfactory receptor gene products in vivo have been essential to reveal the molecular logic of peripheral odor coding and to permit investigation of the developmental and cellular neurobiology of this sensory system. Here, we describe methods for detection of olfactory receptor transcripts and proteins in the antennal olfactory organ of the fruit fly, Drosophila melanogaster, an important genetic model organism. We include protocols both for antennal cryosections and whole-mount antennae. These methods can be adapted for detection of receptor expression in other olfactory and gustatory tissues in Drosophila, as well as in the chemosensory systems of other insects.

Dextramers: new generation of fluorescent MHC class I/peptide multimers for visualization of antigen-specific CD8+ T cells.

Direct identification as well as isolation of antigen-specific T cells became possible since the development of "tetramers" based on avidin-fluorochrome conjugates associated with mono-biotinylated class I MHC-peptide monomeric complexes. In principle, a series of distinct class I MHC-peptide tetramers, each labelled with a different fluorochrome, would allow to simultaneously enumerate as many unique antigen-specific CD8(+) T cells. Practically, however, only phycoerythrin and allophycocyanin conjugated tetramers have been generally available, imposing serious constraints for multiple labeling. To overcome this limitation, we have developed dextramers which are multimers based on a dextran backbone bearing multiple fluorescein and streptavidin moieties. Here we demonstrate the functionality and optimization of these new probes on human CD8(+) T cell clones with four independent antigen specificities. Their applications to the analysis of relatively low frequency antigen-specific T cells in peripheral blood, as well as their use in fluorescence microscopy, are demonstrated. The data show that dextramers produce a stronger signal than their fluoresceinated tetramer counterparts. Thus, these could become the reagents of choice as the antigen-specific T cell labeling transitions from basic research to clinical application.

Indexing strategies for rapid searches of short words in genome sequences.

Searching for matches between large collections of short (14-30 nucleotides) words and sequence databases comprising full genomes or transcriptomes is a common task in biological sequence analysis. We investigated the performance of simple indexing strategies for handling such tasks and developed two programs, fetchGWI and tagger, that index either the database or the query set. Either strategy outperforms megablast for searches with more than 10,000 probes. FetchGWI is shown to be a versatile tool for rapidly searching multiple genomes, whose performance is limited in most cases by the speed of access to the filesystem. We have made publicly available a Web interface for searching the human, mouse, and several other genomes and transcriptomes with oligonucleotide queries.

Synthesis of F-18-labeled cyclic-[RGDfK] peptides for PET imaging of angiogenesis

Objectives: αvβ3 integrin is of great interest for tumor targeting because of its high concentration in tumor tissue. It recognizes ligands containing an arginine-glycine-aspartate motif (RGD), and a number of RGD-containing peptides have been developed as PET imaging probes of angiogenesis. We synthesized a series of 18F-labeled cyclic-[RGDfK] peptides for in vivo imaging of αvβ3 expression. Our F-18 labeled prosthetic groups were attached to the αvβ3 ligand via click chemistry, and the reaction conditions (time, temperature, solvent and pH) were optimized by using single modified amino acids.Methods: Seven amino acids were selected considering their different biochemical properties (polarity, total charge, presence of aromatic ring and heteroatom). All the amino acids were modified by the introduction of azido moiety to allow the interaction with alkyne prosthetic groups. Once the conditions of the click chemistry were optimized, the prosthetic groups were also coupled with the cyclic-[RGDfK] exhibiting an azido function. 4- Trimethylammonium-nitrobenzene triflate was used as precursor for the radiosynthesis of the prosthetic groups. The fluorination was carried out with K2CO3/K2.2.2 in CH3CN at 95 oC, and the nitro group was reduced with NaBH4 and Pd/C in MeOH. The resulting 18F-aniline was subsequently coupled to alkynoic acids to yield the final F-18 labeled prosthetic groups. Finally, the prosthetic groups were attached to the peptides via Huisgen's cycloaddition. Figure 1. F-18 labeled αvβ3 ligand.Results: Our new prosthetic groups were successfully clicked to the modified amino acids and to the cyclic- [RGDfK], and the reactions were almost quantitative within 1 to 3.5 h. The pH of the reaction did not influence the reaction kinetic and yield. The four steps of the F-18 labeling were completely automated providing the final products in quantities and yields practical for PET imaging. IC50 values of our ligands for αvβ3 and α5β1 demonstrated a high selectivity of our compounds towards αvβ3, as well as the negligible effect of the prosthetic groups on the affinity of the ligand to its receptor, as confirmed by the prediction of the molecular modeling.Conclusions: We have successfully synthesized novel F-18 labeled prosthetic groups, as well as novel PET imaging probes of αvβ3 expression. The reaction conditions of the Huisgen's cycloaddition were optimized with selected modified amino acids, and subsequently transposed to the cyclic-[RGDfK] peptide. IC50 data demonstrate that our 18F-labeled ligands were selective for αvβ3. In vivo microPET/CT studies in tumor bearing mice are underway.

Biochemical characterization of FANCD2 and FANCM in the Fanconi Anaemia pathway

Abstract : The maintenance of genome stability is a challenge for all living organisms. DNA is regularly subjected to chemical alterations by both endogenous and exogenous DNA damaging agents. If left unrepaired, these lesions will create mutations or lead to chromosomal instability. DNA crosslinking agents probably bring about the most toxic lesions. By linking covalently the two strands of DNA, crosslinking agents will impede essential cellular processes such as replication and transcription. Cells from Fanconi anaemia patients are extremely sensitive to these agents. Fanconi anaemia (FA) is a rare chromosomal instability disorder that leads to developmental defects, pancytopenia and cancer susceptibility. FA is a genetically heterogeneous disease with thirteen complementation groups identified. Proteins encoded by the FA genes work together in the FA pathway. Eight of these proteins form the FA core complex (FANC-A, B, C,E, F, G, L and -M), whose integrity is required to monoubiquitinate FANCD2 and FANCI in response to DNA damage. The hypersensitivity of FA cells to crosslinking agents, which perturb the progression of replication forks, has led to the hypothesis that FA proteins play a crucial role in the response to replication stress. However, at the molecular level, the functions of the FA pathway remain largely unknown. Our efforts were first focused on the characterization of FANCD2, "the key effector of the FA pathway". Using different substrates, we found that in vitro, purified hFANCD2 preferentially binds single strand DNA and double strand DNA extremities. Concomitantly, FANCM was identified as a new component of the FA core complex. Moreover FANCM was shown to have specific branch migration activities and probably a role as a "landing platform" on DNA for the other components of the core complex. By using FANCM mutants carrying deletions within the internal domain, we investigated the role of FANCM as a DNA anchor protein for the core complex. We observed that indeed, a specific part of the internal domain of FANCM interacts with components of the core complex. Finally, in collaboration with Weidong Wang's lab we characterized two new components of the FA pathway: FAAP10 and FAAP16. As a heterodimer these two proteins show affinity for dsDNA, and anneal complementary oligonucleotides in vitro. Moreover these proteins can associate with FANCM via a part of its internal domain. We find that FANCM, FAAP 10 and FAAP 16 can co-exist on the branch point of replication and recombination intermediates, and that FAAP10 and FAAP16 stimulate replication fork reversal by FANCM. These results suggest that FANCM may function as a landing platform for the core complex. After loading on DNA, the core complex can activate FANCD2 through monoubiquitination leading to its recruitment to the site of damage. Since ssDNA and double strand breaks are intermediates that are generated as a consequence of collapsed replication forks, FANCD2 by binding to ds DNA ends and ssDNA could protect such structures from the recombination repair machinery and prevent unscheduled recombination events. Alternatively, FANCD2 could avoid nucleases from gaining access to collapsed forks, preserving the DNA in state that can be used as a starting point for resumption of DNA synthesis. The overall comprehension of the FA pathway is far from been complete. Our results unravel new aspects of Fanconi Anaemia, which hopefully in the near future will address keys questions leading to a better understanding of the fascinating Fanconi Anaemia. Résumé : Le maintien de l'intégrité du génome est fondamentale chez tous les organismes vivants. L'ADN est constamment altéré par des composés aussi bien endogènes qu'exogènes. Si ces altérations ne sont pas réparées, elles peuvent conduire à l'apparition de mutations, ainsi qu'à une instabilité génomique accrue. Les lésions les plus sévères qui peuvent survenir sur l'ADN, sont les pontages inter caténaires. Des agents pontants en liant de façon covalente les deux brins d'ADN, vont empêcher le déroulement normal de processus cellulaires essentiels tels que la réplication ou la transcription. La compréhension des mécanismes permettant à la cellule de tolérer et réparer ces lésions est primordiale, notamment dans le cas des patients atteints de l'anémie de Fanconi qui présentent une très grande sensibilité à ces composés pontants. L'anémie de Fanconi est une maladie génétique rare appartenant à un groupe de pathologies associées à une grande instabilité chromosomique. Les patients atteints de l'anémie de Fanconi présentent des malformations du squelette, une pancytopénie et une forte propension à la survenue de cancer. L'anémie de Fanconi est génétiquement très hétérogène. À ce jour, 13 gènes codant pour 13 protéines FANC différentes ont été identifiés. Huit de ces protéines fonctionnent ensemble au sein d'un complexe (nommé le complexe FANC) ayant pour but de monoubiquitiner FANCD2 et FANCI en réponse à la formation de lésions sur l'ADN. L'extrême sensibilité des cellules de patients atteints de l'anémie de Fanconi à ces agents pontant l'ADN suggère l'implication des protéines FANC dans la réponse cellulaire suite à une stress réplicatif. Cependant, le rôle moléculaire exact de ces protéines demeure encore inconnu. Après purification, nous avons observé que FANCD2 était capable de lier l'ADN simple brin, ainsi que les extrémités d'ADN in vitro. Dans le même temps, FANCM fut identifié comme appartenant au complexe FANC. FANCM est décrit comme une translocase capable de promouvoir le déplacement de point de jonction dans des structures d'ADN spécifiques in vitro. De plus, en se liant à l'ADN, FANCM peut agir comme une plateforme pour les autres protéines FANC, leur permettant ainsi d'être adressées à l'ADN. En créant des protéines FANCM recombinantes ayant des délétions dans le domaine interne, nous avons pu observer que certaines protéines du complexe FANC se fixent à des sites spécifiques sur le domaine interne de FANCM. Enfin, au travers d'une collaboration, nous avons été amenés à caractériser deux nouvelles protéines appartenant au complexe FANC : FAAP 10 et FAAP16. Elles s'associent à FANCM par l'intermédiaire du domaine interne, et forment ainsi un hétérotrimére. La présence de FAAP10 et FAAP16 n'affecte pas la liaison de FANCM à l'ADN, mais semble potentialiser son activité de régression in vitro. FANCM semble donc fonctionner comme une plateforme pour les autres composants du complexe FANC. Ces derniers, une fois liés à l'ADN permettent la monoubiquitination de FANCD2 et son recrutement au site lésé de l'ADN. FANCD2 en se liant de façon préférentielle à l'ADN simple brin et aux extrémités d'ADN qui sont générés lors de l'arrêt et du démantèlement d'une fourche de réplication, pourrait protéger ces même fourches de réplication arrêtées, d'évènements de recombinaison aléatoires. Nos résultats apportent de nouveaux éléments concernant les mécanismes moléculaires de l'anémie de Fanconi. Enfin, l'étude de l'anémie de Fanconi permet aussi de mieux comprendre les mécanismes mis en place par la cellule pour tolérer des lésions survenant lors de la réplication.

Optimizing immuno-labeling for correlative fluorescence and electron microscopy on a single specimen.

Correlative fluorescence and electron microscopy has become an indispensible tool for research in cell biology. The integrated Laser and Electron Microscope (iLEM) combines a Fluorescence Microscope (FM) and a Transmission Electron Microscope (TEM) within one set-up. This unique imaging tool allows for rapid identification of a region of interest with the FM, and subsequent high resolution TEM imaging of this area. Sample preparation is one of the major challenges in correlative microscopy of a single specimen; it needs to be apt for both FM and TEM imaging. For iLEM, the performance of the fluorescent probe should not be impaired by the vacuum of the TEM. In this technical note, we have compared the fluorescence intensity of six fluorescent probes in a dry, oxygen free environment relative to their performance in water. We demonstrate that the intensity of some fluorophores is strongly influenced by its surroundings, which should be taken into account in the design of the experiment. Furthermore, a freeze-substitution and Lowicryl resin embedding protocol is described that yields excellent membrane contrast in the TEM but prevents quenching of the fluorescent immuno-labeling. The embedding protocol results in a single specimen preparation procedure that performs well in both FM and TEM. Such procedures are not only essential for the iLEM, but also of great value to other correlative microscopy approaches.

On the diurnal soil water content dynamics during evaporation using dielectric methods

The water content dynamics in the upper soil surface during evaporation is a key element in land-atmosphere exchanges. Previous experimental studies have suggested that the soil water content increases at the depth of 5 to 15 cm below the soil surface during evapo- ration, while the layer in the immediate vicinity of the soil surface is drying. In this study, the dynamics of water content profiles exposed to solar radiative forcing was monitored at a high temporal resolution using dielectric methods both in the presence and absence of evaporation. A 4-d comparison of reported moisture content in coarse sand in covered and uncovered buckets using a commercial dielectric-based probe (70 MHz ECH2O-5TE, Decagon Devices, Pullman, WA) and the standard 1-GHz time domain reflectometry method. Both sensors reported a positive correlation between temperature and water content in the 5- to 10-cm depth, most pronounced in the morning during heating and in the afternoon during cooling. Such positive correlation might have a physical origin induced by evaporation at the surface and redistribution due to liquid water fluxes resulting from the temperature- gradient dynamics within the sand profile at those depths. Our experimental data suggest that the combined effect of surface evaporation and temperature-gradient dynamics should be considered to analyze experimental soil water profiles. Additional effects related to the frequency of operation and to protocols for temperature compensation of the dielectric sensors may also affect the probes' response during large temperature changes.

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The expansion of international standardization has reinforced enduring questions on the legitimacy of standards. In that respect, the participation of all stakeholders, including the weakest ones (unions, NGO, consumers' associations) is crucial. Given the recognized role of consumers' associations to express legitimate objectives, the question of their representation becomes central. In order to get a deeper understanding of their participation, this article explores the evolution of their representation within the Swiss national mirror committees of international standardization between 1987 and 2007. It probes the extent to which their participation is determined by the distinctiveness of issues supposedly related to consumers' concerns and by their own use of standards. The empirical findings of our study indicate an underrepresentation of consumers' associations and confirm the topical specificity of their implication in standardization processes. Finally, we found evidence that the use of standards in an association's activities supports and encourages its participation in standardization committees.

On-line desorption of dried blood spots coupled to hydrophilic interaction/reversed-phase LC/MS/MS system for the simultaneous analysis of drugs and their polar metabolites.

An assay for the simultaneous analysis of pharmaceutical compounds and their metabolites from micro-whole blood samples (i.e. 5 microL) was developed using an on-line dried blood spot (on-line DBS) device coupled with hydrophilic interaction/reversed-phase (HILIC/RP) LC/MS/MS. Filter paper is directly integrated to the LC device using a homemade inox desorption cell. Without any sample pretreatment, analytes are desorbed from the paper towards an automated system of valves linking a zwitterionic-HILIC column to an RP C18 column. In the same run, the polar fraction is separated by the zwitterionic-HILIC column while the non-polar fraction is eluted on the RP C18. Both fractions are detected by IT-MS operating in full scan mode for the survey scan and in product ion mode for the dependant scan using an ESI source. The procedure was evaluated by the simultaneous qualitative analysis of four probes and their relative phase I and II metabolites spiked in whole blood. In addition, the method was successfully applied to the in vivo monitoring of buprenorphine metabolism after the administration of an intraperitoneal injection of 30 mg/kg on adult female Wistar rat.

Development of a duplex real-time PCR for the detection of Rickettsia spp. and typhus group rickettsia in clinical samples.

Molecular diagnosis using real-time polymerase chain reaction (PCR) may allow earlier diagnosis of rickettsiosis. We developed a duplex real-time PCR that amplifies (1) DNA of any rickettsial species and (2) DNA of both typhus group rickettsia, that is, Rickettsia prowazekii and Rickettsia typhi. Primers and probes were selected to amplify a segment of the 16S rRNA gene of Rickettsia spp. for the pan-rickettsial PCR and the citrate synthase gene (gltA) for the typhus group rickettsia PCR. Analytical sensitivity was 10 copies of control plasmid DNA per reaction. No cross-amplification was observed when testing human DNA and 22 pathogens or skin commensals. Real-time PCR was applied to 16 clinical samples. Rickettsial DNA was detected in the skin biopsies of three patients. In one patient with severe murine typhus, the typhus group PCR was positive in a skin biopsy from a petechial lesion and seroconversion was later documented. The two other patients with negative typhus group PCR suffered from Mediterranean and African spotted fever, respectively; in both cases, skin biopsy was performed on the eschar. Our duplex real-time PCR showed a good analytical sensitivity and specificity, allowing early diagnosis of rickettsiosis among three patients, and recognition of typhus in one of them.

Expression of the glucagon-like peptide-1 receptor gene in rat brain.

Evidence that glucagon-like peptide-1 (GLP-1) (7-36) amide functions as a novel neuropeptide prompted us to study the gene expression of its receptor in rat brain. Northern blot analysis showed transcripts of similar size in RINm5F cells, hypothalamus, and brain-stem. First-strand cDNA was prepared by using RNA from hypothalamus, brainstem, and R1Nm5F cells and subsequently amplified by PCR. Southern blot analysis of the PCR products showed a major 1.4-kb band in all these preparations. PCR products amplified from hypothalamus were cloned, and the nucleotide sequence of one strand was identical to that described in rat pancreatic islets. In situ hybridization studies showed specific labeling in both neurons and glia of the thalamus, hypothalamus, hippocampus, primary olfactory cortex, choroid plexus, and pituitary gland. In the hypothalamus, ventromedial nuclei cells were highly labeled. These findings indicate that GLP-1 receptors are actually synthesized in rat brain. In addition, the colocalization of GLP-1 receptors, glucokinase, and GLUT-2 in the same areas supports the idea that these cells play an important role in glucose sensing in the brain.

The complex SNP and CNV genetic architecture of the increased risk of congenital heart defects in Down syndrome.

Congenital heart defect (CHD) occurs in 40% of Down syndrome (DS) cases. While carrying three copies of chromosome 21 increases the risk for CHD, trisomy 21 itself is not sufficient to cause CHD. Thus, additional genetic variation and/or environmental factors could contribute to the CHD risk. Here we report genomic variations that in concert with trisomy 21, determine the risk for CHD in DS. This case-control GWAS includes 187 DS with CHD (AVSD = 69, ASD = 53, VSD = 65) as cases, and 151 DS without CHD as controls. Chromosome 21-specific association studies revealed rs2832616 and rs1943950 as CHD risk alleles (adjusted genotypic P-values <0.05). These signals were confirmed in a replication cohort of 92 DS-CHD cases and 80 DS-without CHD (nominal P-value 0.0022). Furthermore, CNV analyses using a customized chromosome 21 aCGH of 135K probes in 55 DS-AVSD and 53 DS-without CHD revealed three CNV regions associated with AVSD risk (FDR ≤ 0.05). Two of these regions that are located within the previously identified CHD region on chromosome 21 were further confirmed in a replication study of 49 DS-AVSD and 45 DS- without CHD (FDR ≤ 0.05). One of these CNVs maps near the RIPK4 gene, and the second includes the ZBTB21 (previously ZNF295) gene, highlighting the potential role of these genes in the pathogenesis of CHD in DS. We propose that the genetic architecture of the CHD risk of DS is complex and includes trisomy 21, and SNP and CNV variations in chromosome 21. In addition, a yet-unidentified genetic variation in the rest of the genome may contribute to this complex genetic architecture.

Auditory-somatosensory multisensory interactions in humans: dissociating detection and spatial discrimination.

Simple reaction times (RTs) to auditory-somatosensory (AS) multisensory stimuli are facilitated over their unisensory counterparts both when stimuli are delivered to the same location and when separated. In two experiments we addressed the possibility that top-down and/or task-related influences can dynamically impact the spatial representations mediating these effects and the extent to which multisensory facilitation will be observed. Participants performed a simple detection task in response to auditory, somatosensory, or simultaneous AS stimuli that in turn were either spatially aligned or misaligned by lateralizing the stimuli. Additionally, we also informed the participants that they would be retrogradely queried (one-third of trials) regarding the side where a given stimulus in a given sensory modality was presented. In this way, we sought to have participants attending to all possible spatial locations and sensory modalities, while nonetheless having them perform a simple detection task. Experiment 1 provided no cues prior to stimulus delivery. Experiment 2 included spatially uninformative cues (50% of trials). In both experiments, multisensory conditions significantly facilitated detection RTs with no evidence for differences according to spatial alignment (though general benefits of cuing were observed in Experiment 2). Facilitated detection occurs even when attending to spatial information. Performance with probes, quantified using sensitivity (d'), was impaired following multisensory trials in general and significantly more so following misaligned multisensory trials. This indicates that spatial information is not available, despite being task-relevant. The collective results support a model wherein early AS interactions may result in a loss of spatial acuity for unisensory information.

Analysis of parapoxvirus genomes.

Eight stomatitis papulosa (SP), four orf and two milker's nodes (MN) virus isolates were compared by restriction enzyme analysis. Considerable genetic heterogeneity was found not only between isolates belonging to the three different taxonomic groups but also between members of the same group. This heterogeneity precludes classification of parapoxviruses simply by comparison of their DNA cleavage patterns. Restriction maps were therefore prepared for 12 parapoxvirus DNAs. Fragments from defined regions of the genome were then selected and used as probes for cross-hybridizations to all other parapoxvirus DNAs. DNA fragments derived from an internal region of the genome hybridized strongly to all parapoxvirus isolates examined. In contrast, cross-hybridization of the end region of the DNA molecule was observed only between members of the same virus group. Molecular hybridization as a means of classifying parapoxvirus isolates is discussed.

Reconstitution of the strand invasion step of double-strand break repair using human Rad51 Rad52 and RPA proteins.

The human Rad51 recombinase is essential for the repair of double-strand breaks in DNA that occur in somatic cells after exposure to ionising irradiation, or in germ line cells undergoing meiotic recombination. The initiation of double-strand break repair is thought to involve resection of the double-strand break to produce 3'-ended single-stranded (ss) tails that invade homologous duplex DNA. Here, we have used purified proteins to set up a defined in vitro system for the initial strand invasion step of double-strand break repair. We show that (i) hRad51 binds to the ssDNA of tailed duplex DNA molecules, and (ii) hRad51 catalyses the invasion of tailed duplex DNA into homologous covalently closed DNA. Invasion is stimulated by the single-strand DNA binding protein RPA, and by the hRad52 protein. Strikingly, hRad51 forms terminal nucleoprotein filaments on either 3' or 5'-ssDNA tails and promotes strand invasion without regard for the polarity of the tail. Taken together, these results show that hRad51 is recruited to regions of ssDNA occurring at resected double-strand breaks, and that hRad51 shows no intrinsic polarity preference at the strand invasion step that initiates double-strand break repair.