Enhanced immunosuppression by therapy-exposed glioblastoma multiforme tumor cells.

Glioblastoma multiforme (GBM) is a highly malignant brain tumor with an extremely short time to relapse following standard treatment. Since recurrent GBM is often resistant to subsequent radiotherapy and chemotherapy, immunotherapy has been proposed as an alternative treatment option. Although it is well established that GBM induces immune suppression, it is currently unclear what impact prior conventional therapy has on the ability of GBM cells to modulate the immune environment. In this study, we investigated the interaction between immune cells and glioma cells that had been exposed to chemotherapy or irradiation in vitro. We demonstrate that treated glioma cells are more immunosuppressive than untreated cells and form tumors at a faster rate in vivo in an animal model. Cultured supernatant from in vitro-treated primary human GBM cells were also shown to increase suppression, which was independent of accessory suppressor cells or T regulatory cell generation, and could act directly on CD4(+) and CD8(+) T cell proliferation. While a number of key immunosuppressive cytokines were overexpressed in the treated cells, including IL-10, IL-6 and GM-CSF, suppression could be alleviated in a number of treated GBM lines by inhibition of prostaglandin E2. These results reveal for the first time that conventional therapies can alter immunosuppressive pathways in GBM tumor cells, a finding with important implications for the combination of immunotherapy with standard treatment.

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.

FUS pathology defines the majority of tau- and TDP-43-negative frontotemporal lobar degeneration.

Through an international consortium, we have collected 37 tau- and TAR DNA-binding protein 43 (TDP-43)-negative frontotemporal lobar degeneration (FTLD) cases, and present here the first comprehensive analysis of these cases in terms of neuropathology, genetics, demographics and clinical data. 92% (34/37) had fused in sarcoma (FUS) protein pathology, indicating that FTLD-FUS is an important FTLD subtype. This FTLD-FUS collection specifically focussed on aFTLD-U cases, one of three recently defined subtypes of FTLD-FUS. The aFTLD-U subtype of FTLD-FUS is characterised clinically by behavioural variant frontotemporal dementia (bvFTD) and has a particularly young age of onset with a mean of 41 years. Further, this subtype had a high prevalence of psychotic symptoms (36% of cases) and low prevalence of motor symptoms (3% of cases). We did not find FUS mutations in any aFTLD-U case. To date, the only subtype of cases reported to have ubiquitin-positive but tau-, TDP-43- and FUS-negative pathology, termed FTLD-UPS, is the result of charged multivesicular body protein 2B gene (CHMP2B) mutation. We identified three FTLD-UPS cases, which are negative for CHMP2B mutation, suggesting that the full complement of FTLD pathologies is yet to be elucidated.

Disruption of endocytic trafficking in frontotemporal dementia with CHMP2B mutations.

Mutations in CHMP2B cause frontotemporal dementia (FTD) in a large Danish pedigree, which is termed FTD linked to chromosome 3 (FTD-3), and also in an unrelated familial FTD patient. CHMP2B is a component of the ESCRT-III complex, which is required for function of the multivesicular body (MVB), an endosomal structure that fuses with the lysosome to degrade endocytosed proteins. We report a novel endosomal pathology in CHMP2B mutation-positive patient brains and also identify and characterize abnormal endosomes in patient fibroblasts. Functional studies demonstrate a specific disruption of endosome-lysosome fusion but not protein sorting by the MVB. We provide evidence for a mechanism for impaired endosome-lysosome fusion whereby mutant CHMP2B constitutively binds to MVBs and prevents recruitment of proteins necessary for fusion to occur, such as Rab7. The fusion of endosomes with lysosomes is required for neuronal function and the data presented therefore suggest a pathogenic mechanism for FTD caused by CHMP2B mutations.

Odorant receptors from the light brown apple moth (Epiphyas postvittana) recognize important volatile compounds produced by plants.

Moths recognize a wide range of volatile compounds, which they use to locate mates, food sources, and oviposition sites. These compounds are recognized by odorant receptors (OR) located within the dendritic membrane of sensory neurons that extend into the lymph of sensilla, covering the surface of insect antennae. We have identified 3 genes encoding ORs from the tortricid moth, Epiphyas postvittana, a pest of horticulture. Like Drosophila melanogaster ORs, they contain 7 transmembrane helices with an intracellular N-terminus, an orientation in the plasma membrane opposite to that of classical GPCRs. EpOR2 is orthologous to the coreceptor Or83b from D. melanogaster. EpOR1 and EpOR3 both recognize a range of terpenoids and benzoates produced by plants. Of the compounds tested, EpOR1 shows the best sensitivity to methyl salicylate [EC(50) = 1.8 x 10(-12) M], a common constituent of floral scents and an important signaling compound produced by plants when under attack from insects and pathogens. EpOR3 best recognizes the monoterpene citral to low concentrations [EC(50) = 1.1 x 10(-13) M]. Citral produces the largest amplitude electrophysiological responses in E. postvittana antennae and elicits repellent activity against ovipositing female moths. Orthologues of EpOR3 were found across 6 families within the Lepidoptera, suggesting that the ability to recognize citral may underpin an important behavior.

DNA barcoding of the endemic New Zealand leafroller moth genera, Ctenopseustis and Planotortrix.

Molecular techniques such as DNA barcoding have become popular in assisting species identification especially for cryptic species complexes. We have analysed data from a 468-bp region of the mitochondrial cytochrome oxidase subunit I (COI) gene from 200 specimens of 12 species of endemic New Zealand leafroller moths (Tortricidae) from the genera Planotortrix and Ctenopseustis to assess whether the DNA barcoding region can distinguish these species. Among the 200 sequences analysed, 72 haplotypes were recovered, with each genus forming a separate major clade. Maximum likelihood phylogenetic methods were used to test whether species fell into reciprocally monophyletic clades. The optimal phylogeny showed that four species within the genus Ctenopseustis (C. obliquana, C. herana, C. filicis and C. fraterna) and three within Planotortrix (P. octo, P. excessana and P. avicenniae) are polyphyletic. Shimodaira-Hasegawa tests rejected a null hypothesis of monophyly for the species C. obliquana, C. herana, P. octo and P. excessana. Comparisons of within and between species levels of sequence divergence for the same set of seven species showed cases where maximum levels of within-species divergence were greater than some levels of between-species divergence. DNA barcoding using this region of the COI gene is able to distinguish the two genera and some species within each genus; however, many species cannot be identified using this method. Finally, we discuss the possible reasons for this polyphyly, including incomplete lineage sorting, introgression, horizontal gene transfer and incorrect taxonomy.

Functional analysis of a Drosophila melanogaster olfactory receptor expressed in Sf9 cells.

Olfactory receptors (ORs) are seven transmembrane proteins that are responsible for the transduction of volatiles into neuronal signals. Their low sequence homology means that the prediction of ligands for ORs based on extrapolation from empirical data of other ORs is difficult, so an experimental approach must be used. Here, we report a functional assay for insect ORs using calcium-imaging in Sf9 cells. We find that the interaction of the odorant, ethyl butyrate, with the Drosophila melanogaster olfactory receptor Or22a is both dose-dependent and highly sensitive, with Or22a responding to ethyl butyrate with an EC(50) of (1.58+/-0.82)x10(-11)M. This degree of sensitivity does not require the addition of odorant binding proteins or downstream signal transduction elements. Furthermore, we demonstrate that Or22a expressed in Sf9 cells has a similar response profile to a range of odorants previously tested in vivo. This functional assay system will provide a useful tool for the de-orphaning of ORs from a wide range of insect species that are yet to have ligands assigned, and will help provide insight into OR specificity and mechanism of activation.