RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E)

Activated RAS promotes dimerization of members of the RAF kinase family. ATP-competitive RAF inhibitors activate ERK signalling by transactivating RAF dimers. In melanomas with mutant BRAF(V600E), levels of RAS activation are low and these drugs bind to BRAF(V600E) monomers and inhibit their activity. This tumour-specific inhibition of ERK signalling results in a broad therapeutic index and RAF inhibitors have remarkable clinical activity in patients with melanomas that harbour mutant BRAF(V600E). However, resistance invariably develops. Here, we identify a new resistance mechanism. We find that a subset of cells resistant to vemurafenib (PLX4032, RG7204) express a 61-kDa variant form of BRAF(V600E), p61BRAF(V600E), which lacks exons 4-8, a region that encompasses the RAS-binding domain. p61BRAF(V600E) shows enhanced dimerization in cells with low levels of RAS activation, as compared to full-length BRAF(V600E). In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signalling is resistant to the RAF inhibitor. Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to vemurafenib. Finally, we identified BRAF(V600E) splicing variants lacking the RAS-binding domain in the tumours of six of nineteen patients with acquired resistance to vemurafenib. These data support the model that inhibition of ERK signalling by RAF inhibitors is dependent on levels of RAS-GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.     

Forecasting international bandwidth capability

M‐competition studies provide a set of stylized recommendations to enhance forecast reliability. However, no single method dominates across series, leading to consideration of the relationship between selected data characteristics and the reliability of alternative forecast methods. This study conducts an analysis of predictive accuracy in relation to Internet bandwidth loads. Extrapolation techniques that perform best in M‐competitions perform relatively poorly in predicting Internet bandwidth loads. Such performance is attributed to Internet bandwidth data exhibiting considerably less structure than M‐competition data. Copyright © 2005 John Wiley & Sons, Ltd.     

Brown-headed Cowbird parasitism on endangered species: relationships with neighboring avian species

Loss of reproductive productivity in hosts of the Brown-headed Cowbird ( Molothrus ater ), a nest parasite, may limit the success of conservation and recovery efforts that focus on restoring avian habitat. We investigated the relationships between parasitism frequency of 2 vireos—the endangered Black-capped Vireo ( Vireo atricapillus ) and the White-eyed Vireo ( Vireo griseus )—and characteristics of the neighboring avian assemblage. Our objective was to identify assemblage characteristics that are useful for predicting parasitism frequency. We hypothesized that parasitism frequency of the vireos would decrease with increasing alternate host abundance and increase with increasing abundance of cowbirds. All Black-capped Vireo nests ( n = 20) and 81% of White-eyed Vireo nests ( n = 27) were parasitized. Parasitism frequency of White-eyed Vireos was positively correlated with the number of cowbirds detected ( r = 0.84, df = 4, P = 0.018). Parasitism frequency of White-eyed Vireos was negatively correlated with abundance of the Black-and white Warbler ( Mniotilta varia; r = 0.891, df = 4, P = 0.009) but was not correlated with abundance of other cowbird hosts. The relationship between Black-and-white Warbler abundance and parasitism frequency may be due to chance, given the small sample size and narrow range of parasitism frequencies, but the relationship may also reflect an interaction (requiring further investigation) between parasitism and the vegetation characteristics associated with Black-and white Warblers. Our results imply that reducing cowbird abundance will reduce parasitism frequency of Black-capped Vireos. When the number of cowbirds in an area predicts parasitism, land managers would benefit from identifying a target cowbird abundance based on the levels of parasitism the host population can tolerate while still meeting productivity and population objectives for the species. La disminución de productividad debido al parasitismo por el tordo negro ( Molothrus ater ) podría limitar el éxito de los esfuerzos de conservación y recuperación de las aves que se centran en la restauración del hábitat. Investigamos la relación entre la frecuencia de parasitismo en el vireo gorrinegro ( Vireo atricapilla ), que está en peligro de extinción, y en otra especie de vireo, el vireo ojiblanco ( Vireo griseus ), así como su relación con las características de la población circunvecina de aves para identificar las características que sirven para predecir la frecuencia del parasitismo. Planteamos la hipótesis de que la frecuencia de parasitismo en los vireos disminuiría con el aumento de la abundancia de huéspedes alternativos y aumentaría al incrementarse la abundancia de tordos. Todos los nidos del vireo gorrinegro ( n = 20) y el 81% de los nidos del vireo ojiblanco ( n = 27) fueron parasitados. La frecuencia de parasitismo en los vireos ojiblancos estaba correlacionada positivamente con el número de tordos detectados ( r = 0.84, gdl = 4, P = 0.018). Estaba correlacionada negativamente con la abundancia ( r = 0.891, gdl = 4, P = 0.009) del chipe trepador ( Mniotilta varia ), pero no estaba correlacionada con la abundancia de otros huéspedes de los tordos. La relación entre la abundancia de chipe trepador y la frecuencia da parasitismo podría ser aleatoria, dado lo pequeño de la muestra y la poca variación en las frecuencias de parasitismo, pero también podría reflejar una interacción entre el parasitismo y las características de la vegetación asociada con el chipe trepador que requiere más investigación. Nuestros resultados indican que la disminución de la abundancia de tordos reduce la frecuencia de parasitismo en el vireo gorrinegro. Cuando el número de tordos en una área predice el parasitismo, los administradores de recursos naturales podrían beneficiarse de la identificación de una abundancia meta del tordo, basada en los niveles de parasitismo que la población huésped pueda sostener y todavía alcanzar los objetivos de productividad y población de la especie.     

Immunoperoxidase localization of elastin in the human aorta

Summary Use of the peroxidase-antiperoxidase technique with preliminary trypsinization has allowed immunolocalization of elastin in human aorta. By this method it is possible to utilize formalin-fixed and paraffin-embedded tissue. The advantages in studying a strongly-autofluorescent material are discussed.     

Synthetic hosts by monomolecular imprinting inside dendrimers

Synthetic host systems capable of selectively binding guest molecules are of interest for applications ranging from separations and chemical or biological sensing to the development of biomedical materials. Such host systems can be efficiently prepared by 'imprinting' polymers or inorganic materials with template molecules, which, upon removal, leave behind spatially arranged functional groups that act as recognition sites. However, molecularly imprinted polymers have limitations, including incomplete template removal, broad guest affinities and selectivities, and slow mass transfer. An alternative strategy for moulding desired recognition sites uses combinatorial libraries of assemblies that are made of a relatively small number of molecules, interconverting in dynamic equilibrium; upon addition of a target molecule, the library equilibrium shifts towards the best hosts. Here we describe the dynamic imprinting of dendritic macromolecules with porphyrin templates to yield synthetic host molecules containing one binding site each. The process is based on our general strategy to prepare cored dendrimers, and involves covalent attachment of dendrons to a porphyrin core, cross-linking of the end-groups of the dendrons, and removal of the porphyrin template by hydrolysis. In contrast to more traditional polymer imprinting, our approach ensures nearly homogeneous binding sites and quantitative template removal. Moreover, the hosts are soluble in common organic solvents and amenable to the incorporation of other functional groups, which should facilitate further development of this system for novel applications.     

Newly synthesized elastin is associated with neoplastic epithelial cells in human mammary carcinoma

Summary Indirect immunofluorescence with a purified antiserum to human foetal elastin has identified newly synthesized elastin on the membranes of neoplastic epithelial cells in human mammary carcinoma.This research was supported by a project grant from the Medical Research Council, London.     

Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system

Transposons have provided important genetic tools for functional genomic screens in lower eukaryotes but have proven less useful in higher eukaryotes because of their low transposition frequency. Here we show that Sleeping Beauty (SB), a member of the Tc1/mariner class of transposons, can be mobilized in mouse somatic cells at frequencies high enough to induce embryonic death and cancer in wild-type mice. Tumours are aggressive, with some animals developing two or even three different types of cancer within a few months of birth. The tumours result from SB insertional mutagenesis of cancer genes, thus facilitating the identification of genes and pathways that induce disease. SB transposition can easily be controlled to mutagenize any target tissue and can therefore, in principle, be used to induce many of the cancers affecting humans, including those for which little is known about the aetiology. The uses of SB are also not restricted to the mouse and could potentially be used for forward genetic screens in any higher eukaryote in which transgenesis is possible.