We initially performed exome-sequencing 11 of the two UV S S-A patients, Kps3 and XP24KO (details described in Methods, Supplementary Note, Table 2c). The patients were homozygous for c.367A>T mutation in UVSSA, which led to a premature termination, p.Lys123* (Fig. 1a, b). We identified the same homozygous mutation in Kps2 (sib. of Kps3), and a homozygous c.87delG, causing a frameshift p.Ile31Phefs*9, in an Israeli patient UV S S24TA (Fig. 1b, c, Supplementary Note, Supplementary Fig. 1). The identified mutations are summarized in Fig. 1d. We did not detect the 80kDa UVSSA protein in any of the UV S S-A patients (Fig. 1e). We additionally examined several mild xeroderma pigmentosum (XP) cases; in one such case, XP70TO 12 (Supplementary Table 1), we identified a homozygous p.Cys32Arg, in the UVSSA (Fig. 1c, d), implying that XP70TO is also in the UV S S-A group. The mutant protein was stably expressed in XP70TO cells (Fig. 1f, Supplementary Fig. 2a-d). 4Allele frequencies of the identified mutations in a control population were examined (Supplementary Note, Supplementary Fig. 3a). Haploinsufficiency for UVSSA is negligible as the parents of Kps2/Kps3 showed no symptoms 4 . In parallel with exome-sequencing, we performed whole-genome SNP-genotyping to identify runs-of-homozygosity (ROH) shared among the patients. We identified three overlapping-ROHs (> 1Mbps) on autosomes, one of which encompasses the UVSSA locus (Fig. 1g, Supplementary Table 3a, b, Supplementary Fig. 3b, c). No chromosome copy number variation was detected (Supplementary Fig. 3d).The above findings strongly suggest that the mutations in UVSSA in the UV S S-A patients are causal for the disease; we therefore, next examined the NER-activities in the UV S S-A cells (Fig. 2). Unscheduled-DNA-synthesis (UDS 13 , defective in XP) was nearly normal; however, RNA-synthesis-recovery (RRS 14 , defective in UV S S and in CS) was reduced in all cell-strains mutated in UVSSA ( Fig. 2a, b; UDS and RRS were measured using a recently-developed rapid non-radioactive system 15,16 ). Similarly, siRNA-based depletion of the UVSSA gene (Fig. 2c) caused a drastic reduction of RRS (Fig. 2d, Supplementary Fig. 4), whereas UDS was unaffected (Fig. 2e). Ectopic-expression of the wild-type UVSSA cDNA in UV S S-A cells restored normal RRS ( Fig. 2f; V5-tagged-UVSSA immunofluorescent-staining shown in Fig. 2g), while it did not affect RRS-level in normal, CS-A, or CS-B cells; neither ERCC8 nor ERCC6 cDNA expression in UV S S-A cells restored the RRS-level.We conclude that KIAA1530/UVSSA is the causal gene for UV S S-A.ERCC8 and ERCC6 genes are responsible for both CS and UV S S 7,8 . To evaluate whether UVSSA mutations may also result in CS-phenotypes, we sequenced 5 the UVSSA gene of 61 CS-patients whose genetic defects had not yet been determined (Supplementary Table 4). We found no obvious mutations except for four novel heterozygous changes. These changes as well as the SNPs, also found in control and UV S S-A individuals, do not affect the RRS-activity (Suppleme...

Osteoclast differentiation is critically dependent on calcium (Ca 2þ ) signaling. Transient receptor potential vanilloid 4 (TRPV4), /calmodulin signaling, which involves an association of TRPV4 with myosin IIa, and promotes sufficient osteoclast function. ß

Tanimura et al. demonstrate that SH3P2 binds to and functions as a cytosolic anchor for myosin 1E (Myo1E). ERK signaling–dependent phosphorylation of SH3P2 induces the dissociation of bound Myo1E and its consequent localization to the tips of lamellipodia, where it promotes cell motility.

BACKGROUND Analysis of circulating immune complexes (CICs) produced during an immune response may be useful in elucidating some aspects of this process. Identification of antigens incorporated into CICs provides information that may be helpful in developing diagnostic and treatment strategies for autoimmune diseases, infection, cancer, and transplantation therapy, and such information might be more relevant than information on free antigens. Because CICs may contain many antigens, comprehensive identification and profiling of such antigens is more effective than immunoblotting detection. METHODS We developed a novel proteomic strategy (immune complexome analysis) in which immune complexes (ICs) are separated from serum, digested directly with trypsin, and then subjected to nano-liquid chromatography–tandem mass spectrometry for identifying and profiling antigens in CICs. We applied this strategy to the analysis of CICs in 21 rheumatoid arthritis (RA) patients. Serum samples from 13 healthy donors and 8 osteoarthritis patients were used as controls. RESULTS CICs containing thrombospondin-1 (TSP-1) and platelet factor 4 (PF4) were found in the serum of 81% and 52% of RA patients, respectively, and in none of the controls. CONCLUSIONS The ICs in the serum of a majority of the RA patients contained TSP-1 or PF4, and these ICs may have potential as alternative biomarkers. Our technique for immune complexome analysis uses routine clinical samples, simple protocols, and widely available equipment. This method may be generally applicable to the study of the relationship between CICs and certain diseases associated with the immune response in animals and humans.

In order to evaluate the chemiluminescence (CL) reagents for selective detection of reactive oxygen species (ROS), we comprehensively measured the CL responses of 20 CL reagents (three luminol derivatives, two imidazopyrazinone derivatives, eight lophine derivatives, six acridinium ester derivatives and lucigenin) against six types of ROS (superoxide anion: O(2)(*-), hydroxyl radical: *OH, hydrogen peroxide: H(2)O(2), hypochlorite anion: ClO(-), singlet oxygen: (1)O(2), and nitric oxide: NO). As a result of the screening, it was found that nine CL reagents selectively detected O(2)(*-) while one CL reagent selectively detected *OH. However, no CL reagent had selectivity on the detection of H(2)O(2), ClO(-), (1)O(2) and NO. Our screening results could help to select the most suitable CL reagent for selective determination of different ROS. As an application study, 4-methoxyphenyl-10-methylacridinium-9-carboxylate (MMAC), one of the acridinium ester derivatives, showed high selectivity on the detection of O(2)(*-), and thus was applied to the assay of superoxide dismutase (SOD) activity. The dynamic range and detection limit of the developed CL assay were 0.1-10 and 0.06 U mL(-1), respectively. Significant correlation (r=0.997) was observed between the results by the CL assay using MMAC and the spectrophotometric assay using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt.