Exosome-mediated signal transportation plays a variety of critical roles in cancer progression and metastasis. From the aspect of cancer diagnosis, circulating exosomes are ideal resources of biomarkers because molecular features of tumor cells are transcribed on them. However, isolating pure exosomes from body fluids is time-consuming and still major challenge to be addressed for comprehensive profiling of exosomal proteins and miRNAs. Here we constructed anti-CD9 antibody-coupled highly porous monolithic silica microtips which allowed automated rapid and reproducible exosome extraction from multiple clinical samples. We applied these tips to explore lung cancer biomarker proteins on exosomes by analyzing 46 serum samples. The mass spectrometric quantification of 1,369 exosomal proteins identified CD91 as a lung adenocarcinoma specific antigen on exosomes, which was further validated with CD9-CD91 exosome sandwich ELISA measuring 212 samples. Our simple device can promote not only biomarker discovery studies but also wide range of omics researches about exosomes.

RNA helicase A (RHA) is a member of an ATPase/DNA and RNA helicase family and is a homologue of Drosophila maleless protein (MLE), which regulates X-linked gene expression. RHA is also a component of holo-RNA polymerase II (Pol II) complexes and recruits Pol II to the CREB binding protein (CBP). The ATPase and/or helicase activity of RHA is required for CREB-dependent transcription. To further understand the role of RHA on gene expression, we have identified a 50-amino-acid transactivation domain that interacts with Pol II and termed it the minimal transactivation domain (MTAD). The protein sequence of this region contains six hydrophobic residues and is unique to RHA homologues and well conserved. A mutant with this region deleted from full-length RHA decreased transcriptional activity in CREB-dependent transcription. In addition, mutational analyses revealed that several tryptophan residues in MTAD are important for the interaction with Pol II and transactivation. These mutants had ATP binding and ATPase activities comparable to those of wild-type RHA. A mutant lacking ATP binding activity was still able to interact with Pol II. In CREB-dependent transcription, the transcriptional activity of each of these mutants was less than that of wild-type RHA. The activity of the double mutant lacking both functions was significantly lower than that of each mutant alone, and the double mutant had a dominant negative effect. These results suggest that RHA could independently regulate CREB-dependent transcription either through recruitment of Pol II or by ATP-dependent mechanisms.RNA helicase A (RHA) is a member of the DExH family of ATPases/helicases and catalyzes the displacement of both double-stranded RNA and DNA from 3Ј to 5Ј (32,61,63). Functional domains of RHA include two double-stranded RNA binding domains at the amino terminus known as dsRBD1 and dsRBD2. The catalytic core domain is located within the central region and contains a DExH motif. This core domain contains seven well-conserved motifs; one of them has an ATP binding site with the consensus GCGKT and FILDD, known as the A site the B site, respectively. The carboxyl terminus contains an RGG-rich region that is capable of binding singlestrand nucleic acids (62).RHA was originally isolated as a human homologue of Drosophila maleless protein (MLE), with which it has 50% sequence identity and 90% sequence similarity (33). In Drosophila, MLE colocalizes with acetylated histone H4 (8, 48). MLE is involved in sex-specific gene dosage compensation and elevates the level of transcription derived from a single X chromosome in male flies to a level equivalent to that derived from two X chromosome in the female (25,29). MLE mutants are embryonic lethal to males, indicating that MLE is an essential factor in Drosophila development.In mammals, RHA-knockout mice are embryonic lethal for homozygous RHA mutants (35). Analysis of these mice revealed that RHA is associated with differentiation of the embryonic ectoderm during gastrulation. It is possible that RHA has an i...