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In contrast with advances in massively parallel DNA sequencing (1), high-throughput protein analyses (2-4) are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods (5) is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display (6) or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies) (7) and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables 'library versus library' screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity., To analyse proteins in a massively parallel single-molecule format, we generated proteins that are molecularly coupled to a DNA bearing a barcoding sequence. One barcoding approach is to translate and [...]

[CD4.sup.+] T-helper type 2 ([T.sub.H]2) cells, characterized by their expression of interleukin (IL)-4, IL-5, IL-9 and IL-13, are required for immunity to helminth parasites (1) and promote the pathological inflammation associated with asthma and allergic diseases (2). Polymorphisms in the gene encoding the cytokine thymic stromal lymphopoietin (TSLP) are associated with the development of multiple allergic disorders in humans, indicating that TSLP is a critical regulator of [T.sub.H]2 cytokine-associated inflammatory diseases (3-6). In support of genetic analyses, exaggerated TSLP production is associated with asthma, atopic dermatitis and food allergies in patients, and studies in murine systems demonstrated that TSLP promotes [T.sub.H]2 cytokine-mediated immunity and inflammation (5,7-12). However, the mechanisms through which TSLP induces [T.sub.H]2 cytokine responses remain poorly defined. Here we demonstrate that TSLP promotes systemic basophilia, that disruption of TSLP-TSLPR interactions results in defective basophil responses, and that TSLPR-sufficient basophils can restore [T.sub.H]2-cell-dependent immunity in vivo. TSLP acted directly on bone-marrow-resident progenitors to promote basophil responses selectively. Critically, TSLP could elicit basophil responses in both IL-3-IL-3R-sufficient and -deficient environments, and genome-wide transcriptional profiling and functional analyses identified heterogeneity between TSLP-elicited versus IL-3-elicited basophils. Furthermore, activated human basophils expressed TSLPR, and basophils isolated from eosinophilic oesophagitis patients were distinct from classical basophils. Collectively, these studies identify previously unrecognized heterogeneity within the basophil cell lineage and indicate that expression of TSLP may influence susceptibility to multiple allergic diseases by regulating basophil haematopoiesis and eliciting a population of functionally distinct basophils that promote [T.sub.H]2 cytokine-mediated inflammation., Previous in vitro studies suggested that TSLP could promote [T.sub.H]2 cytokine-mediated inflammation by influencing dendritic cell, mast cell and lymphocyte populations (13). However, the IL-4-expressing cell populations that TSLP targets [...]

Effective clinical management of prostate cancer (PCA) has been challenged by significant intratumoural heterogeneity on the genomic and pathological levels and limited understanding of the genetic elements governing disease progression (1). Here, we exploited the experimental merits of the mouse to test the hypothesis that pathways constraining progression might be activated in indolent Pten-null mouse prostate tumours and that inactivation of such progression barriers in mice would engender a metastasis-prone condition. Comparative transcriptomic and canonical pathway analyses, followed by biochemical confirmation, of normal prostate epithelium versus poorly progressive Pten-null prostate cancers revealed robust activation of the TGFβ/BMP-SMAD4 signalling axis. The functional relevance of SMAD4 was further supported by emergence of invasive, metastatic and lethal prostate cancers with 100% penetrance upon genetic deletion of Smad4 in the Pten-null mouse prostate. Pathological and molecular analysis as well as transcriptomic knowledge-based pathway profiling of emerging tumours identified cell proliferation and invasion as two cardinal tumour biological features in the metastatic Smad4/Pten-null PCA model. Follow-on pathological and functional assessment confirmed cyclin D1 and SPP1 as key mediators of these biological processes, which together with PTEN and SMAD4, form a four-gene signature that is prognostic of prostate-specific antigen (PSA) biochemical recurrence and lethal metastasis in human PCA. This model-informed progression analysis, together with genetic, functional and translational studies, establishes SMAD4 as a key regulator of PCA progression in mice and humans., Adenocarcinoma of the prostate (PCA) is the most common form of cancer and the second leading cause of cancer death in American men (2). Current methods of stratifying tumours to [...]

Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas (1). Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma (2-6)--an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials (7-9) However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance (10-12). Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies (13). Here we expressed ~600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies., To identify kinases capable of circumventing RAF inhibition, we assembled and stably expressed 597 sequence-validated kinase ORF clones representing ~75% of annotated kinases (Center for Cancer Systems Biology (CCSB)/Broad Institute [...]

The exponential growth in the amount of biological data means that revolutionary measures are needed for data management, analysis and accessibility. Online databases have become important avenues for publishing biological [...]

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease caused by expansion of a glutamine-encoding repeat in ataxin 1 (ATXN1). In all known polyglutamine diseases, the glutamine expansion [...]

Author(s): Derek Sieburth [1, 6]; QueeLim Ch'ng [1, 6]; Michael Dybbs [1]; Masoud Tavazoie [1, 5]; Scott Kennedy [2]; Duo Wang [1]; Denis Dupuy [3]; Jean-François Rual [3]; David E. [...]

The various aftershocks produced in remote areas from the mainshock of the 1992 Landers earthquake in Southern California were due to advective overpressure. Aftershocks of large earthquakes occur only near the main region of the earthquake though for the 1992 Landers earthquake, aftershocks occurred in distant regions from the main shock and were near geothermal and volcanic systems. The advective overpressure, which causes these remote aftershocks, is produced by the bubbles in the volcanic magma in the ground.

The article reviews several Web sites including www.genenames.org from HUGO Gene Nomenclature Committee, http://daphnia.cgb.indiana from Dapnia Genomics Consortium and http://iggi.sanbi.ac.za from Glossina Genomics Initiatives.

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease caused by expansion of a glutamine-encoding repeat in ataxin 1 (ATXN1). In all known polyglutamine diseases, the glutamine expansion confers toxic functions onto the protein; however, the mechanism by which this occurs remains enigmatic, in light of the fact that the mutant protein apparently maintains interactions with its usual partners. Here we show that the expanded polyglutamine tract differentially affects the function of the host protein in the context of different endogenous protein complexes. Polyglutamine expansion in ATXN1 favours the formation of a particular protein complex containing RBM17, contributing to SCA1 neuropathology by means of a gain-of-function mechanism. Concomitantly, polyglutamine expansion attenuates the formation and function of another protein complex containing ATXN1 and capicua, contributing to SCA1 through a partial loss-of-function mechanism. This model provides mechanistic insight into the molecular pathogenesis of SCA1 as well as other polyglutamine diseases. [ABSTRACT FROM AUTHOR]