Detection of specific gene rearrangements by fluorescence in situ hybridization in 16 cases of clear cell sarcoma of soft tissue and 6 cases of clear cell sarcoma-like gastrointestinal tumor

Abstract Background Clear cell sarcoma of soft tissue (CCSST) and clear cell sarcoma-like gastrointestinal tumor (CCSLGT) are malignant mesenchymal tumors that share some pathological features, but they also have several different characteristics. They are well known to express chimeric fusions of Ewing sarcoma breakpoint region 1 (EWSR1) and cAMP response element-binding protein (CREB) family members; namely, EWSR1-activating transcription factor 1 (ATF1) and EWSR1-CREB1. In addition, recent studies have suggested the presence of other fusions. Methods We used fluorescence in situ hybridization to detect specific rearrangements including EWSR1, ATF1, CREB1, and cAMP response element modulator (CREM) in 16 CCSST and 6 CCSLGT cases. We also used reverse transcription polymerase chain reaction (RT-PCR) to detect specific chimeric fusions of EWSR1-ATF1 and EWSR1-CREB1 using fresh tumor samples in available cases. Results A total of 15 of 16 CCSST cases (93.8%) had EWSR1 rearrangement, of which 11 (68.8%) also had ATF1 rearrangement, suggestive of the presence of EWSR1-ATF1 fusions. One CCSST case (6.3%) was found to have EWSR1 and CREM rearrangements, and 4 of 6 CCSLGT cases (66.7%) had EWSR1 rearrangement, of which 2 (33.3%) showed ATF1 rearrangement and the other 2 cases (33.3%) showed CREB1 rearrangement. These cases most likely had EWSR1-ATF1 and EWSR1-CREB1 fusions, respectively. RT-PCR was performed in 8 available cases, including 6 CCSSTs and 2 CCSLGTs. All CCSSTs showed EWSR1-ATF1 fusions. Among the 2 CCSLGT cases, one had EWSR1-ATF1 fusion and the other had EWSR1-CREB1 fusion. Conclusions Rearrangements of EWSR1 and ATF1 or EWSR1-ATF1 fusion were predominantly found in CCSST, whereas those of EWSR1 and CREB1 or EWSR1-CREB1 tended to be detected in CCSLGT. A novel CREM fusion was also detected in a few cases of CCSST and CCSLGT. The cases in which EWSR1 rearrangement was detected without definitive partner genes should be considered for the presence of CREM rearrangement

Characterization of dystroglycan binding in adhesion of human induced pluripotent stem cells to laminin-511 E8 fragment

Human induced pluripotent stem cells (hiPSCs) grow indefinitely in culture and have the potential to regenerate various tissues. In the development of cell culture systems, a fragment of laminin-511 (LM511-E8) was found to improve the proliferation of stem cells. The adhesion of undifferentiated cells to LM511-E8 is mainly mediated through integrin alpha 6 beta 1. However, the involvement of non-integrin receptors remains unknown in stem cell culture using LM511-E8. Here, we show that dystroglycan (DG) is strongly expressed in hiPSCs. The fully glycosylated DG is functionally active for laminin binding, and although it has been suggested that LM511-E8 lacks DG binding sites, the fragment does weakly bind to DG. We further identified the DG binding sequence in LM511-E8, using synthetic peptides, of which, hE8A5-20 (human laminin alpha 5 2688-2699: KTLPQLLAKLSI) derived from the laminin coiled-coil domain, exhibited DG binding affinity and cell adhesion activity. Deletion and mutation studies show that LLAKLSI is the active core sequence of hE8A5-20, and that, K2696 is a critical amino acid for DG binding. We further demonstrated that hiPSCs adhere to hE8A5-20-conjugated chitosan matrices. The amino acid sequence of DG binding peptides would be useful to design substrata for culture system of undifferentiated and differentiated stem cells

Effect of a financial incentive (shopping point) on increasing the number of daily walking steps among community-dwelling adults in Japan: a randomised controlled trial

Objective The aim of this study was to investigate the effect of a financial incentive on the number of daily walking steps among community-dwelling adults in Japan.Study design Two-arm, parallel-group randomised controlled trial.Setting/participants We recruited physically inactive community-dwelling adults from Sendai city, Japan. Eligible participants were randomly allocated to an intervention or a wait list control group. Pedometers were used to assess the mean number of daily steps in three periods: baseline (weeks 1–3), intervention (weeks 4–6) and follow-up (weeks 7–9).Intervention The intervention group was offered a financial incentive (shopping points) to meet the target number of increased daily steps in the intervention period.Main outcome measures The primary outcome was an increase in the mean number of daily steps in the intervention and follow-up periods compared with baseline.Results Seventy-two participants (69.4% women; mean age, 61.2±16.2 years; mean number of daily steps at baseline, 6364±2804) were randomised to the intervention (n=36) and control groups (n=36). During the intervention period, the increase in mean daily steps was significantly higher in the intervention group (1650, 95% CI=1182 to 2119) than in the control group (514, 95% CI=136 to 891; p<0.001). However, the difference between groups was not significant at follow-up after the incentives were removed (p=0.311). In addition, compared with controls, a significantly higher proportion of participants in the intervention group showed an increase in mean daily steps of ≥1000 (69.4% vs 30.6%, respectively; OR=5.17, 95% CI=1.89 to 14.08). There were no adverse effects from the intervention.Conclusions The present results suggest that financial incentives are effective in promoting short-term increases in physical activity.Trial registration number UMIN000033276

An Anti-Human Lutheran Glycoprotein Phage Antibody Inhibits Cell Migration on Laminin-511: Epitope Mapping of the Antibody

<div><p>The Lutheran glycoprotein (Lu), also known as basal cell adhesion molecule (B-CAM), is an Ig superfamily (IgSF) transmembrane receptor for laminin α5. Although Lu is not present in normal hepatocytes, its expression is significantly increased in hepatocellular carcinoma (HCC). In this study, we isolated thirteen phage antibodies to Lu from a phage library of peripheral blood from HCC patients, suggesting that these patients produced autoantibodies against endogenous Lu. To characterize the phage antibodies, we determined the Lu domains they recognize. The extracellular domain of Lu contains five IgSF domains, D1-D2-D3-D4-D5. The epitope of one phage antibody (A7) was localized to the D5 domain. The other phage antibodies recognized the D2 domain, which is also recognized by a function blocking mouse monoclonal antibody. One of the antibodies to D2 (C7) inhibited the binding of Lu to ligand, and it also prevented tumor cell migration on laminin-511 (LM-511). However, the C7 scFv purified from the periplasm fraction of bacteria did not exhibit the inhibitory effects, indicating that the scFv form could not sterically inhibit the binding of Lu to LM-511. We also identified the amino acid residues that form the epitope recognized by the C7 phage antibody. Mutagenesis studies showed that Arg²⁴⁷ is necessary for forming the epitope. The C7 phage antibody and its epitope may be useful for developing drugs to prevent HCC progression and/or metastasis.</p></div

Characterization of C7 scFv purified from the <i>E</i>. <i>coli</i> extract.

<p>(A) Purification of C7 scFv from <i>E</i>. <i>coli</i> extracts. The purified protein was subjected to SDS-PAGE on a 10–20% gradient gel under reducing conditions and was stained with Coomassie brilliant blue. (B) ELISA using the purified C7 scFv. C7 scFv diluted at 1 μg/ml was incubated in wells coated with 1 μg/ml of Lu-Fc. (C) SPR analysis of C7 scFv. The sensorgrams represent the association and dissociation upon injecting different concentrations of C7scFv (6.25, 12.5, 25, 50, and 100 nM) to immobilized Lu-Fc. The rate constants, Ka and Kd, and KD are shown in the sensorgram. (D) Migration of A549 cells on LM-511 in the presence of C7 scFv. After the cells adhered to substrata coated with LM-511 (0.8 nM), control or C7 scFv was added to the media (30 μg/ml). Cell movements were evaluated as described above.</p

Mapping the amino acid residue recognized by C7 scFv on a three-dimensional model of Lu D1 and D2 domains.

<p>An amino acid residue of the C7 scFv epitope mapped on the crystal structure of Lu D1 and D2 domains (Protein Data Bank code 2PET) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167860#pone.0167860.ref016" target="_blank">16</a>]. The location of Arg²⁴⁷ is not very close to the epitope of the mAb87207 epitope. The binding sites of laminin α5 on Lu D2 domain are located on the opposite side of Arg²⁴⁷.</p

Isolation of human Lu-specific phage clones.

<p>(A) Biopanning was performed against Sol-Lu immobilized on wells using HCC patient tissues (left) or peripheral blood cells (right) -derived phage libraries. Enrichment for human Lu-specific phages was only observed from the peripheral blood cell-derived phage library using phage ELISA. (B) Cloning of human Lu-specific phage. After the third round of biopanning, phages were cloned by colony formation. The binding specificities of each phage clone were examined by monoclonal phage ELISA. Thirteen phage clones specific to human Lu were obtained.</p