The novel mTOR inhibitor RAD001 (Everolimus) induces antiproliferative effects in human pancreatic neuroendocrine tumor cells

Background/Aim: Tumors exhibiting constitutively activated PI(3) K/Akt/mTOR signaling are hypersensitive to mTOR inhibitors such as RAD001 (everolimus) which is presently being investigated in clinical phase II trials in various tumor entities, including neuroendocrine tumors (NETs). However, no preclinical data about the effects of RAD001 on NET cells have been published. In this study, we aimed to evaluate the effects of RAD001 on BON cells, a human pancreatic NET cell line that exhibits constitutively activated PI(3) K/Akt/mTOR signaling. Methods: BON cells were treated with different concentrations of RAD001 to analyze its effect on cell growth using proliferation assays. Apoptosis was examined by Western blot analysis of caspase-3/PARP cleavage and by FACS analysis of DNA fragmentation. Results: RAD001 potently inhibited BON cell growth in a dose-dependent manner which was dependent on the serum concentration in the medium. RAD001-induced growth inhibition involved G0/G1-phase arrest as well as induction of apoptosis. Conclusion: In summary, our data demonstrate antiproliferative and apoptotic effects of RAD001 in NET cells in vitro supporting its clinical use in current phase II trials in NET patients. Copyright (c) 2007 S. Karger AG, Basel

Amino Acid Residues Contributing to Function of the Heteromeric Insect Olfactory Receptor Complex

Olfactory receptors (Ors) convert chemical signals—the binding of odors and pheromones—to electrical signals through the depolarization of olfactory sensory neurons. Vertebrates Ors are G-protein-coupled receptors, stimulated by odors to produce intracellular second messengers that gate ion channels. Insect Ors are a heteromultimeric complex of unknown stoichiometry of two seven transmembrane domain proteins with no sequence similarity to and the opposite membrane topology of G-protein-coupled receptors. The functional insect Or comprises an odor- or pheromone-specific Or subunit and the Orco co-receptor, which is highly conserved in all insect species. The insect Or-Orco complex has been proposed to function as a novel type of ligand-gated nonselective cation channel possibly modulated by G-proteins. However, the Or-Orco proteins lack homology to any known family of ion channel and lack known functional domains. Therefore, the mechanisms by which odors activate the Or-Orco complex and how ions permeate this complex remain unknown. To begin to address the relationship between Or-Orco structure and function, we performed site-directed mutagenesis of all 83 conserved Glu, Asp, or Tyr residues in the silkmoth BmOr-1-Orco pheromone receptor complex and measured functional properties of mutant channels expressed in Xenopus oocytes. 13 of 83 mutations in BmOr-1 and BmOrco altered the reversal potential and rectification index of the BmOr-1-Orco complex. Three of the 13 amino acids (D299 and E356 in BmOr-1 and Y464 in BmOrco) altered both current-voltage relationships and K+ selectivity. We introduced the homologous Orco Y464 residue into Drosophila Orco in vivo, and observed variable effects on spontaneous and evoked action potentials in olfactory neurons that depended on the particular Or-Orco complex examined. Our results provide evidence that a subset of conserved Glu, Asp and Tyr residues in both subunits are essential for channel activity of the heteromeric insect Or-Orco complex

Putative Chemosensory Receptors of the Codling Moth, Cydia pomonella, Identified by Antennal Transcriptome Analysis

The codling moth, Cydia pomonella, is an important fruit pest worldwide. As nocturnal animals, adults depend to a large extent on olfactory cues for detection of food and mates, and, for females, oviposition sites. In insects, odor detection is mediated by odorant receptors (ORs) and ionotropic receptors (IRs), which ensure the specificity of the olfactory sensory neuron responses. In this study, our aim was to identify chemosensory receptors in the codling moth as a means to uncover new targets for behavioral interference. Using next-generation sequencing techniques, we identified a total of 43 candidate ORs, one gustatory receptor and 15 IRs in the antennal transcriptome. Through Blast and sequence similarity analyses we annotated the insect obligatory co-receptor ORco, five genes clustering in a conserved clade containing sex pheromone receptors, one homolog of the Bombyx mori female-enriched receptor BmorOR30 (but no homologs of the other B. mori female-enriched receptors) and one gene clustering in the sugar receptor family. Among the candidate IRs, we identified homologs of the two highly conserved co-receptors IR8a and IR25a, and one homolog of an IR involved in phenylethyl amine detection in Drosophila. Our results open for functional characterization of the chemosensory receptors of C. pomonella, with potential for new or refined applications of semiochemicals for control of this pest insect

A phase II trial of the oral mTOR inhibitor everolimus in relapsed aggressive lymphoma

The phosphatidylinositol 3-kinase signal transduction pathway members are often activated in tumor samples from patients with non-Hodgkin's lymphoma (NHL). Everolimus is an oral agent that targets the raptor mammalian target of rapamycin (mTORC1). The goal of this trial was to learn the antitumor activity and toxicity of single-agent everolimus in patients with relapsed/refractory aggressive NHL. Patients received everolimus 10 mg PO daily. Response was assessed after two and six cycles, and then every three cycles until progression. A total of 77 patients with a median age of 70 years were enrolled. Patients had received a median of three previous therapies and 32% had undergone previous transplant. The overall response rate (ORR) was 30% (95% confidence interval: 20–41%), with 20 patients achieving a partial remission and 3 a complete remission unconfirmed. The ORR in diffuse large B cell was 30% (14/47), 32% (6/19) in mantle cell and 38% (3/8) in follicular grade 3. The median duration of response was 5.7 months. Grade 3 or 4 anemia, neutropenia and thrombocytopenia occurred in 14, 18 and 38% of patients, respectively. Everolimus has single-agent activity in relapsed/refractory aggressive NHL and provides proof-of-concept that targeting the mTOR pathway is clinically relevant

Identification of New Agonists and Antagonists of the Insect Odorant Receptor Co-Receptor Subunit

BACKGROUND: Insects detect attractive and aversive chemicals using several families of chemosensory receptors, including the OR family of olfactory receptors, making these receptors appealing targets for the control of insects. Insect ORs are odorant-gated ion channels, comprised of at least one common subunit (the odorant receptor co-receptor subunit, Orco) and at least one variable odorant specificity subunit. Each of the many ORs of an insect species is activated or inhibited by an unique set of odorants that interact with the variable odorant specificity subunits, making the development of OR directed insect control agents complex and laborious. However, several N-,2-substituted triazolothioacetamide compounds (VUAA1, VU0450667 and VU0183254) were recently shown to act directly on the highly conserved Orco subunit, suggesting that broadly active compounds can be developed. We have explored the chemical space around the VUAA1 structure in order to identify new Orco ligands. PRINCIPAL FINDINGS: We screened ORs from several insect species, using heterologous expression in Xenopus oocytes and an electrophysiological assay, with a panel of 22 compounds structurally related to VUAA1. By varying the nitrogen position in the pyridine ring and altering the moieties decorating the phenyl ring, we identified two new agonists and a series of competitive antagonists. Screening smaller compounds, similar to portions of the VUAA1 structure, also yielded competitive antagonists. Importantly, we show that Orco antagonists inhibit odorant activation of ORs from several insect species. Detailed examination of one antagonist demonstrated inhibition to be through a non-competitive mechanism. CONCLUSIONS: A similar pattern of agonist and antagonist sensitivity displayed by Orco subunits from different species suggests a highly conserved binding site structure. The susceptibility to inhibition of odorant activation by Orco antagonism is conserved across disparate insect species, suggesing the ligand binding site on Orco as a promising target for the development of novel, broadly active insect repellants

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

An Expressed Sequence Tag collection from the male antennae of the Noctuid moth Spodoptera littoralis: a resource for olfactory and pheromone detection research

<p>Abstract</p> <p>Background</p> <p>Nocturnal insects such as moths are ideal models to study the molecular bases of olfaction that they use, among examples, for the detection of mating partners and host plants. Knowing how an odour generates a neuronal signal in insect antennae is crucial for understanding the physiological bases of olfaction, and also could lead to the identification of original targets for the development of olfactory-based control strategies against herbivorous moth pests. Here, we describe an Expressed Sequence Tag (EST) project to characterize the antennal transcriptome of the noctuid pest model, <it>Spodoptera littoralis</it>, and to identify candidate genes involved in odour/pheromone detection.</p> <p>Results</p> <p>By targeting cDNAs from male antennae, we biased gene discovery towards genes potentially involved in male olfaction, including pheromone reception. A total of 20760 ESTs were obtained from a normalized library and were assembled in 9033 unigenes. 6530 were annotated based on BLAST analyses and gene prediction software identified 6738 ORFs. The unigenes were compared to the <it>Bombyx mori </it>proteome and to ESTs derived from Lepidoptera transcriptome projects. We identified a large number of candidate genes involved in odour and pheromone detection and turnover, including 31 candidate chemosensory receptor genes, but also genes potentially involved in olfactory modulation.</p> <p>Conclusions</p> <p>Our project has generated a large collection of antennal transcripts from a Lepidoptera. The normalization process, allowing enrichment in low abundant genes, proved to be particularly relevant to identify chemosensory receptors in a species for which no genomic data are available. Our results also suggest that olfactory modulation can take place at the level of the antennae itself. These EST resources will be invaluable for exploring the mechanisms of olfaction and pheromone detection in <it>S. littoralis</it>, and for ultimately identifying original targets to fight against moth herbivorous pests.</p

Female Behaviour Drives Expression and Evolution of Gustatory Receptors in Butterflies

Secondary plant compounds are strong deterrents of insect oviposition and feeding, but may also be attractants for specialist herbivores. These insect-plant interactions are mediated by insect gustatory receptors (Grs) and olfactory receptors (Ors). An analysis of the reference genome of the butterfly Heliconius melpomene, which feeds on passion-flower vines (Passiflora spp.), together with whole-genome sequencing within the species and across the Heliconius phylogeny has permitted an unprecedented opportunity to study the patterns of gene duplication and copy-number variation (CNV) among these key sensory genes. We report in silico gene predictions of 73 Gr genes in the H. melpomene reference genome, including putative CO2, sugar, sugar alcohol, fructose, and bitter receptors. The majority of these Grs are the result of gene duplications since Heliconius shared a common ancestor with the monarch butterfly or the silkmoth. Among Grs but not Ors, CNVs are more common within species in those gene lineages that have also duplicated over this evolutionary time-scale, suggesting ongoing rapid gene family evolution. Deep sequencing (∼1 billion reads) of transcriptomes from proboscis and labial palps, antennae, and legs of adult H. melpomene males and females indicates that 67 of the predicted 73 Gr genes and 67 of the 70 predicted Or genes are expressed in these three tissues. Intriguingly, we find that one-third of all Grs show female-biased gene expression (n = 26) and nearly all of these (n = 21) are Heliconius-specific Grs. In fact, a significant excess of Grs that are expressed in female legs but not male legs are the result of recent gene duplication. This difference in Gr gene expression diversity between the sexes is accompanied by a striking sexual dimorphism in the abundance of gustatory sensilla on the forelegs of H. melpomene, suggesting that female oviposition behaviour drives the evolution of new gustatory receptors in butterfly genomes

Sequence Comparisons of Odorant Receptors among Tortricid Moths Reveal Different Rates of Molecular Evolution among Family Members

In insects, odorant receptors detect volatile cues involved in behaviours such as mate recognition, food location and oviposition. We have investigated the evolution of three odorant receptors from five species within the moth genera Ctenopseustis and Planotrotrix, family Tortricidae, which fall into distinct clades within the odorant receptor multigene family. One receptor is the orthologue of the co-receptor Or83b, now known as Orco (OR2), and encodes the obligate ion channel subunit of the receptor complex. In comparison, the other two receptors, OR1 and OR3, are ligand-binding receptor subunits, activated by volatile compounds produced by plants - methyl salicylate and citral, respectively. Rates of sequence evolution at non-synonymous sites were significantly higher in OR1 compared with OR2 and OR3. Within the dataset OR1 contains 109 variable amino acid positions that are distributed evenly across the entire protein including transmembrane helices, loop regions and termini, while OR2 and OR3 contain 18 and 16 variable sites, respectively. OR2 shows a high level of amino acid conservation as expected due to its essential role in odour detection; however we found unexpected differences in the rate of evolution between two ligand-binding odorant receptors, OR1 and OR3. OR3 shows high sequence conservation suggestive of a conserved role in odour reception, whereas the higher rate of evolution observed in OR1, particularly at non-synonymous sites, may be suggestive of relaxed constraint, perhaps associated with the loss of an ancestral role in sex pheromone reception

A Family of Chemoreceptors in Tribolium castaneum (Tenebrionidae: Coleoptera)

Chemoperception in invertebrates is mediated by a family of G-protein-coupled receptors (GPCR). To date nothing is known about the molecular mechanisms of chemoperception in coleopteran species. Recently the genome of Tribolium castaneum was sequenced for use as a model species for the Coleoptera. Using blast searches analyses of the T. castaneum genome with previously predicted amino acid sequences of insect chemoreceptor genes, a putative chemoreceptor family consisting of 62 gustatory receptors (Grs) and 26 olfactory receptors (Ors) was identified. The receptors have seven transmembrane domains (7TMs) and all belong to the GPCR receptor family. The expression of the T. castaneum chemoreceptor genes was investigated using quantification real- time RT-PCR and in situ whole mount RT-PCR analysis in the antennae, mouth parts, and prolegs of the adults and larvae. All of the predicted TcasGrs were expressed in the labium, maxillae, and prolegs of the adults but TcasGr13, 19, 28, 47, 62, 98, and 61 were not expressed in the prolegs. The TcasOrs were localized only in the antennae and not in any of the beetles gustatory organs with one exception; the TcasOr16 (like DmelOr83b), which was localized in the antennae, labium, and prolegs of the beetles. A group of six TcasGrs that presents a lineage with the sugar receptors subfamily in Drosophila melanogaster were localized in the lacinia of the Tribolium larvae. TcasGr1, 3, and 39, presented an ortholog to CO2 receptors in D. melanogaster and Anopheles gambiae was recorded. Low expression of almost all of the predicted chemoreceptor genes was observed in the head tissues that contain the brains and suboesophageal ganglion (SOG). These findings demonstrate the identification of a chemoreceptor family in Tribolium, which is evolutionarily related to other insect species