Interaction of Hsp90 with the nascent form of the mutant epidermal growth factor receptor EGFRvIII

EGFRvIII is a mutant epidermal growth factor that promotes aggressive growth of glioblastomas. We made a plasmid that directed the expression of an EGFRvIII with three copies of the Flag epitope at its amino terminus. Flag-tagged EGFRvIII was expressed at the same levels as unmodified EGFRvIII, and showed the same subcellular localization. However, the Flag epitope could only be detected on EGFRvIII present in the endoplasmic reticulum; the epitope was covalently modified during trafficking of the receptor through the Golgi so that it was no longer recognized by anti-Flag antibody. This property was exploited to selectively purify nascent EGFRvIII from glioblastoma cells. Nascent EGFRvIII was found to copurify with a set of other proteins, identified by mass spectrometry as the two endoplasmic reticulum chaperones Grp94 and BiP, and the two cytosolic chaperones Hsc70 and Hsp90. The Hsp90-associated chaperone Cdc37 also co-purified with EGFRvIII, suggesting that Hsp90 binds EGFRvIII as a complex with this protein. Geldanamycin and radicicol, two chemically unrelated inhibitors of Hsp90, decreased the expression of EGFRvIII in glioblastoma cells. These studies show that nascent EGFRvIII in the endoplasmic reticulum associates with Hsp90 and Cdc37, and that the Hsp90 association is necessary to maintain expression of EGFRvIII.     

Methane production using whole and screened dairy manure in conventional and fixed-film reactors

The technical feasibility of adopting the fixed-film reactor concept for biogas production from screened dairy manure was investigated. The methane production capability of laboratory-scale 4-L anaerobic reactors (conventional and fixed-film) receiving screened dairy manure and operated at 35 degrees C was compared. Dairy manure filtrate with 4.4% total solids (TS) and 3.4% volatile solids (VS) (average value) was prepared from 1:1 manure-water slurry. The feed material was added intermittently at loading rates ranging from 2.34 to 25 and 2.25 to 785 g VS/L d, respectively, for the conventional and fixed-film reactors. Maximum methane production rate (L CH(4)/L d) for the conventional reactor was 0.63 L CH(4)/L d achieved at a 6-day hydraulic retention time (HRT). For the fixed-film reactor the maximum production rate was 3.53 L CH(4)/L d when operated at a loading rate of 262 g VS/L d (3 h HRT). The fixed-film reactor was capable of sustaining a loading of 785 g VS/L d (1 h HRT). The fixed-film reactor performed much better than the conventional reactors. These results indicate that a large reduction of required reactor volume is possible through application of a fixed-film concept combined with a liquid-solid separation pretreatment of dairy manure.     

Intergroup variation in stable isotope ratios reflects anthropogenic impact on the Barbary macaques (Macaca sylvanus) of Gibraltar

Interactions with humans impact many aspects of behavior and ecology in nonhuman primates. Because of the complexities of the human–nonhuman primate interface, methods are needed to quantify the effects of anthropogenic interactions, including their intensity and differential impacts between nonhuman primate groups. Stable isotopes can be used to quickly and economically assess intergroup dietary variation, and provide a framework for the development of specific hypotheses about anthropogenic impact. This study uses stable carbon and nitrogen isotope analysis to examine intraspecific variation in diet between five groups of Barbary macaques, Macaca sylvanus, in the Upper Rock Nature Reserve, Gibraltar. Analysis of hair from 135 macaques showed significant differences in δ¹³C and δ¹⁵N values between a group with minimal tourist contact and groups that were main tourist attractions. Because we observed no overt physiological or substantial behavioral differences between the groups, feeding ecology is the most likely cause of any differences in stable isotope ratios. Haphazard provisioning by tourists and Gibraltarians is a likely source of dietary variation between groups. Stable isotope analysis and observational data facilitate a deeper understanding of the feeding ecology of the Barbary macaques relevant to the role of an anthropogenic ecology for the species.     

Ku and DNA-dependent Protein Kinase Dynamic Conformations and Assembly Regulate DNA Binding and the Initial Non-homologous End Joining Complex

DNA double strand break (DSB) repair by non-homologous end joining (NHEJ) is initiated by DSB detection by Ku70/80 (Ku) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) recruitment, which promotes pathway progression through poorly defined mechanisms. Here, Ku and DNA-PKcs solution structures alone and in complex with DNA, defined by x-ray scattering, reveal major structural reorganizations that choreograph NHEJ initiation. The Ku80 C-terminal region forms a flexible arm that extends from the DNA-binding core to recruit and retain DNA-PKcs at DSBs. Furthermore, Ku- and DNA-promoted assembly of a DNA-PKcs dimer facilitates trans-autophosphorylation at the DSB. The resulting site-specific autophosphorylation induces a large conformational change that opens DNA-PKcs and promotes its release from DNA ends. These results show how protein and DNA interactions initiate large Ku and DNA-PKcs rearrangements to control DNA-PK biological functions as a macromolecular machine orchestrating assembly and disassembly of the initial NHEJ complex on DNA.     

Disentangling the paradox of insect phenology: are temporal trends reflecting the response to warming?

The strength and direction of phenological responses to changes in climate have been shown to vary significantly both among species and among populations of a species, with the overall patterns not fully resolved. Here, we studied the temporal and spatial variability associated with the response of several insect species to recent global warming. We use hierarchical models within a model comparison framework to analyze phenological data gathered over 40 years by the Japan Meteorological Agency on the emergence dates of 14 insect species at sites across Japan. Contrary to what has been predicted with global warming, temporal trends of annual emergence showed a later emergence day for some species and sites over time, even though temperatures are warming. However, when emergence data were analyzed as a function of temperature and precipitation, the overall response pointed out an earlier emergence day with warmer conditions. The apparent contradiction between the response to temperature and trends over time indicates that other factors, such as declining populations, may be affecting the date phenological events are being recorded. Overall, the responses by insects were weaker than those found for plants in previous work over the same time period in these ecosystems, suggesting the potential for ecological mismatches with deleterious effects for both suites of species. And although temperature may be the major driver of species phenology, we should be cautious when analyzing phenological datasets as many other factors may also be contributing to the variability in phenology.     

Chancelloriid sclerites from the Lower Cambrian (Meishucunian) of eastern Yunnan,China, and the early history of the group

Chancelloriids are a group of enigmatic sessile animals that are covered with sclerites shaped as rosettes of spines, producing an appearance like that of a barrel cactus. They are known only from Cambrian rocks. Isolated sclerites of chancelloriids are widespread in small shelly faunas, but they have proven difficult to treat taxonomically due to the variation within and between individual animals. We report on large samples of chancelloriid sclerites from the Meishucunian (pre‐trilobitic Lower Cambrian) of eastern Yunnan Province, China, including material from the Dahai Member of the Zhujiaqing Formation (the Zhujiaqing section, Dahai, Huize County) and the Shiyantou Formation (the Xiaotan section, Yongshan County). The material from the Dahai Member appears to come from a single species, Chancelloriella irregularis. Statistical analysis of morphotype co‐occurrences in different samples suggests that several species are represented in the material from the Shiyantou Formation, which we herein tentatively place in four species, Allonnia erromenosa, Allonnia tetrathallis, Archiasterella charma sp. nov. and Archiasterella cf. pentactina. We suggest therefore that careful study of large collections of isolated chancelloriid sclerites permits the identification of different species, opening the possibility of their use in evolutionary or stratigraphical studies. In particular, in South China, it seems that Chancelloriella irregularis is characteristic of rocks of the Paragloborilus subglobosus – Purella squamulosa Assemblage Zone and the Watsonella crosbyi Assemblage Zone, whilst Archiasterella and Allonnia first appear in the overlying Sinosachites flabelliformis – Tannuolina zhangwentangi Assemblage Zone. C. irregularis is thus amongst the oldest well‐characterized chancelloriids, and the irregularity and poor organization of its sclerites relative to those of younger forms support the hypothesis that chancelloriid sclerites are compound structures arising from the fusion of originally separate elements, perhaps homologous with sclerites of halkieriids and other coeloscleritophorans. The similarity between the arrangement of rays in a chancelloriid sclerite and the arrangement of bubbles in small bubble clusters suggests that in many cases, aspects of the form of chancelloriid sclerites are the result of simple physical processes.     

Efficacy model for antibody-mediated pre-erythrocytic malaria vaccines

Antibodies to the pre-erythrocytic antigens, circumsporozoite protein (CSP), thrombospondin-related adhesive protein (TRAP) and liver-stage antigen 1, have been measured in field studies of semi-immune adults and shown to correlate with protection from Plasmodium falciparum infection. A mathematical model is formulated to estimate the probability of sporozoite infection as a function of antibody titres to multiple pre-erythrocytic antigens. The variation in antibody titres from field data was used to estimate the relationship between the probability of P. falciparum infection per infectious mosquito bite and antibody titre. Using this relationship, we predict the effect of vaccinations that boost baseline CSP or TRAP antibody titres. Assuming the estimated relationship applies to vaccine-induced antibody titres, then single-component CSP or TRAP antibody-mediated pre-erythrocytic vaccines are likely to provide partial protection from infection, with vaccine efficacy of approximately 50 per cent depending on the magnitude of the vaccine-induced boost to antibody titres. It is possible that the addition of a TRAP component to a CSP-based vaccine such as RTS,S would provide an increase in infection-blocking efficacy of approximately 25 per cent should the problem of immunological interference between antigens be overcome.