Enhancement of visible light response of TiO2 photocatalyst by 3D-deposited Ag nanowires and its charge separation mechanism.

In 3D-deposited AgNW/TiO2, which is prepared by spray-applying titanium dioxide suspension to deposited silver nanowire sheets, the synergistic effects of increased crossing points of AgNWs to enhance localized surface plasmon resonance excitation and longer-lived electrons in the conduction band of TiO2 generated by plasmon-induced charge transfer have successfully resulted in photocatalytic activity in the visible light range. We have developed photocatalytic sheets in which TiO2 particles are uniformly attached to 3D-deposited AgNWs. Regarding the prepared sheet, it was confirmed that TiO2 was indeed well adhered to the AgNWs, and electron transfer was efficient at the interface. This sheet solves the problem that the response wavelength range of the photocatalytic reaction using TiO2 is only in the ultraviolet region and exhibits sufficient photocatalytic effect in the visible light region. Transient absorption spectroscopy measurements in the diffuse reflectance configuration confirmed that the electrons of AgNWs actually move into the conduction band of TiO2 under visible light and that the interaction is independent of the excitation light intensity, thereby extending the lifetime of the electrons.

Mass++: A Visualization and Analysis Tool for Mass Spectrometry.

We have developed Mass++, a plug-in style visualization and analysis tool for mass spectrometry. Its plug-in style enables users to customize it and to develop original functions. Mass++ has several kinds of plug-ins, including rich viewers and analysis methods for proteomics and metabolomics. Plug-ins for supporting vendors' raw data are currently available; hence, Mass++ can read several data formats. Mass++ is both a desktop tool and a software development platform. Original functions can be developed without editing the Mass++ source code. Here, we present this tool's capability to rapidly analyze MS data and develop functions by providing examples of label-free quantitation and implementing plug-ins or scripts. Mass++ is freely available at http://www.first-ms3d.jp/english/ .

High expression of CD109 antigen regulates the phenotype of cancer stem-like cells/cancer-initiating cells in the novel epithelioid sarcoma cell line ESX and is related to poor prognosis of soft tissue sarcoma.

Epithelioid sarcoma (ES) is a relatively rare, highly malignant soft tissue sarcoma. The mainstay of treatment is resection or amputation. Currently other therapeutic options available for this disease are limited. Therefore, a novel therapeutic option needs to be developed. In the present study, we established a new human ES cell line (ESX) and analyzed the characteristics of its cancer stem-like cells/cancer-initiating cells (CSCs/CICs) based on ALDH1 activity. We demonstrated that a subpopulation of ESX cells with high ALDH1 activity (ALDH(high) cells) correlated with enhanced clonogenic ability, sphere-formation ability, and invasiveness in vitro and showed higher tumorigenicity in vivo. Next, using gene expression profiling, we identified CD109, a GPI-anchored protein upregulated in the ALDH(high) cells. CD109 mRNA was highly expressed in various sarcoma cell lines, but weakly expressed in normal adult tissues. CD109-positive cells in ESX predominantly formed spheres in culture, whereas siCD109 reduced ALDH1 expression and inhibited the cell proliferation in vitro. Subsequently, we evaluated the expression of CD109 protein in 80 clinical specimens of soft tissue sarcoma. We found a strong correlation between CD109 protein expression and the prognosis (P = 0.009). In conclusion, CD109 might be a CSC/CIC marker in epithelioid sarcoma. Moreover, CD109 is a promising prognostic biomarker and a molecular target of cancer therapy for sarcomas including ES.

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations: from tension sensing to channel opening.

One of the ultimate goals of the study on mechanosensitive (MS) channels is to understand the biophysical mechanisms of how the MS channel protein senses forces and how the sensed force induces channel gating. The bacterial MS channel MscL is an ideal subject to reach this goal owing to its resolved 3D protein structure in the closed state on the atomic scale and large amounts of electrophysiological data on its gating kinetics. However, the structural basis of the dynamic process from the closed to open states in MscL is not fully understood. In this study, we performed molecular dynamics (MD) simulations on the initial process of MscL opening in response to a tension increase in the lipid bilayer. To identify the tension-sensing site(s) in the channel protein, we calculated interaction energy between membrane lipids and candidate amino acids (AAs) facing the lipids. We found that Phe78 has a conspicuous interaction with the lipids, suggesting that Phe78 is the primary tension sensor of MscL. Increased membrane tension by membrane stretch dragged radially the inner (TM1) and outer (TM2) helices of MscL at Phe78, and the force was transmitted to the pentagon-shaped gate that is formed by the crossing of the neighboring TM1 helices in the inner leaflet of the bilayer. The radial dragging force induced radial sliding of the crossing portions, leading to a gate expansion. Calculated energy for this expansion is comparable to an experimentally estimated energy difference between the closed and the first subconductance state, suggesting that our model simulates the initial step toward the full opening of MscL. The model also successfully mimicked the behaviors of a gain of function mutant (G22N) and a loss of function mutant (F78N), strongly supporting that our MD model did simulate some essential biophysical aspects of the mechano-gating in MscL.

SYT-SSX breakpoint peptide vaccines in patients with synovial sarcoma: a study from the Japanese Musculoskeletal Oncology Group.

In the present study, we evaluated the safety and effectiveness of SYT-SSX-derived peptide vaccines in patients with advanced synovial sarcoma. A 9-mer peptide spanning the SYT-SSX fusion region (B peptide) and its HLA-A*2402 anchor substitute (K9I) were synthesized. In Protocols A1 and A2, vaccines with peptide alone were administered subcutaneously six times at 14-day intervals. The B peptide was used in Protocol A1, whereas the K9I peptide was used in Protocol A2. In Protocols B1 and B2, the peptide was mixed with incomplete Freund's adjuvant and then administered subcutaneously six times at 14-day intervals. In addition, interferon-α was injected subcutaneously on the same day and again 3 days after the vaccination. The B peptide and K9I peptide were used in Protocols B1 and B2, respectively. In total, 21 patients (12 men, nine women; mean age 43.6 years) were enrolled in the present study. Each patient had multiple metastatic lesions of the lung. Thirteen patients completed the six-injection vaccination schedule. One patient developed intracerebral hemorrhage after the second vaccination. Delayed-type hypersensitivity skin tests were negative in all patients. Nine patients showed a greater than twofold increase in the frequency of CTLs in tetramer analysis. Recognized disease progression occurred in all but one of the nine patients in Protocols A1 and A2. In contrast, half the 12 patients had stable disease during the vaccination period in Protocols B1 and B2. Of note, one patient showed transient shrinkage of a metastatic lesion. The response of the patients to the B protocols is encouraging and warrants further investigation.

SOX2 is overexpressed in stem-like cells of human lung adenocarcinoma and augments the tumorigenicity.

Recently, the SOX2 gene has been reported to be amplified in human lung squamous cell carcinomas. However, its roles in human lung adenocarcinomas are still elusive. In this study, we analyzed the functions of SOX2 in cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) derived from human lung adenocarcinoma. Human lung CSCs/CICs were isolated as higher tumorigenic side population (SP) cells using Hoechst 33342 dye from several lung cancer cell lines. Four of nine lung cancer cell lines were positive for SP cells (LHK2, 1-87, A549, Lc817). The ratios of SP cells ranged from 0.4% for Lc817 to 2.8% for LHK2. To analyze the molecular aspects of SP cells, we performed microarray screening and RT-PCR analysis, and isolated SOX2 as one of a SP cell-specific gene. SOX2 was expressed predominantly in LHK2 and 1-87 SP cells, and was also expressed in several other cancer cell lines. The expression of SOX2 protein in primary human lung cancer tissues were also confirmed by immunohistochemical staining, and SOX2 was detected in more than 80% of primary lung cancer tissues. To address SOX2 molecular functions, we established a SOX2-overexpressed LHK2 and A549 cell line (LHK2-SOX2 and A549-SOX2). LHK2-SOX2 cells showed higher rates of SP cells and higher expression of POU5F1 compared with control cells. LHK2-SOX2 and A549-SOX2 cells showed relatively higher tumorigenicity than control cells. On the other hand, SOX2 mRNA knockdown of LHK2 SP cells by gene-specific siRNA completely abrogated tumorigenicity in vivo. These observations indicate that SOX2 has a role in maintenance of stemness and tumorigenicity of human lung adenocarcinoma CSCs/CICs and is a potential target for treatment.

Autologous CTL response against cancer stem-like cells/cancer-initiating cells of bone malignant fibrous histiocytoma.

Malignant fibrous histiocytoma (MFH) of the bone is an aggressive tumor with high rates of local recurrence and metastasis. The development of novel therapeutic approaches is critical to improve the prognosis of patients with MFH. We reported previously that the side population (SP) cells of the MFH2003 bone MFH cell line have the characteristics of cancer stem-like cells (CSC)/cancer-initiating cells. In the present study, to establish immunotherapy targeting CSC, we analyzed cell surface immune molecules on SP cells of the MHF2003 cell line, as well as autologous CTL responses against these SP cells in the tumor microenvironment and peripheral circulating lymphocytes, using autologous tumor-infiltrating lymphocytes and autologous CTL clones derived from peripheral blood, respectively. We found that the SP cells expressed human leukocyte antigen (HLA) Class I molecules on the cell surface. The autologous tumor-infiltrating lymphocyte line TIL2003 recognized both the SP and main population cells of the MFH2003 cell line. Next, we induced the CTL clone Tc4C-6 by mixed lymphocyte tumor cell culture using autologous peripheral blood mononuclear cells and freshly isolated SP cells, followed by a limiting dilution procedure. The Tc4C-6 clone showed specific cytotoxicity against the SP cells. Moreover, the cytotoxicity against SP cells was blocked by the anti-HLA Class I antibody W6/32. In conclusion, the findings of the present study support the idea that CSC of bone MFH are recognized by autologous CTL in the tumor microenvironment and peripheral circulating lymphocytes. Thus, CTL-based immunotherapy could target CSC of bone sarcoma to help prevent tumor recurrence.

Integrated mass spectrometry-based analysis of plasma glycoproteins and their glycan modifications.

We present a protocol for the identification of glycosylated proteins in plasma followed by elucidation of their individual glycan compositions. The study of glycoproteins by mass spectrometry is usually based on cleavage of glycans followed by separate analysis of glycans and deglycosylated proteins, which limits the ability to derive glycan compositions for individual glycoproteins. The methodology described here consists of 2D HPLC fractionation of intact proteins and liquid chromatography-multistage tandem mass spectrometry (LC-MS/MS(n)) analysis of digested protein fractions. Protein samples are separated by 1D anion-exchange chromatography (AEX) with an eight-step salt elution. Protein fractions from each of the eight AEX elution steps are transferred onto the 2D reversed-phase column to further separate proteins. A digital ion trap mass spectrometer with a wide mass range is then used for LC-MS/MS(n) analysis of intact glycopeptides from the 2D HPLC fractions. Both peptide and oligosaccharide compositions are revealed by analysis of the ion fragmentation patterns of glycopeptides with an intact glycopeptide analysis pipeline.

HLA-A*0201-restricted CTL epitope of a novel osteosarcoma antigen, papillomavirus binding factor.

BACKGROUND: To develop peptide-based immunotherapy for osteosarcoma, we previously identified papillomavirus binding factor (PBF) as a CTL-defined osteosarcoma antigen in the context of HLA-B55. However, clinical application of PBF-based immunotherapy requires identification of naturally presented CTL epitopes in osteosarcoma cells in the context of more common HLA molecules such as HLA-A2. METHODS: Ten peptides with the HLA-A*0201 binding motif were synthesized from the amino acid sequence of PBF according to the BIMAS score and screened with an HLA class I stabilization assay. The frequency of CTLs recognizing the selected PBF-derived peptide was determined in peripheral blood of five HLA-A*0201+ patients with osteosarcoma using limiting dilution (LD)/mixed lymphocyte peptide culture (MLPC) followed by tetramer-based frequency analysis. Attempts were made to establish PBF-specific CTL clones from the tetramer-positive CTL pool by a combination of limiting dilution and single-cell sorting. The cytotoxicity of CTLs was assessed by 51Cr release assay. RESULTS: Peptide PBF A2.2 showed the highest affinity to HLA-A*0201. CD8+ T cells reacting with the PBF A2.2 peptide were detected in three of five patients at frequencies from 2 x 10-7 to 5 x 10-6. A tetramer-positive PBF A2.2-specific CTL line, 5A9, specifically lysed allogeneic osteosarcoma cell lines that expressed both PBF and either HLA-A*0201 or HLA-A*0206, autologous tumor cells, and T2 pulsed with PBF A2.2. Five of 12 tetramer-positive CTL clones also lysed allogeneic osteosarcoma cell lines expressing both PBF and either HLA-A*0201 or HLA-A*0206 and T2 pulsed with PBF A2.2. CONCLUSION: These findings indicate that PBF A2.2 serves as a CTL epitope on osteosarcoma cells in the context of HLA-A*0201, and potentially, HLA-A*0206. This extends the availability of PBF-derived therapeutic peptide vaccines for patients with osteosarcoma.

Prognostic impact and immunogenicity of a novel osteosarcoma antigen, papillomavirus binding factor, in patients with osteosarcoma.

To develop peptide-based immunotherapy for osteosarcoma, we previously identified papillomavirus binding factor (PBF) as a cytotoxic T lymphocytes (CTL)-defined osteosarcoma antigen in the context of human leukocyte antigen (HLA)-B55. In the present study, we analyzed the distribution profile of PBF in 83 biopsy specimens of osteosarcomas and also the prognostic impact of PBF expression in 78 patients with osteosarcoma who had completed the standard treatment protocols. Next, we determined the antigenic peptides from PBF that react with peripheral T lymphocytes of HLA-A24(+) patients with osteosarcoma. Immunohistochemical analysis revealed that 92% of biopsy specimens of osteosarcoma expressed PBF. PBF-positive osteosarcoma conferred significantly poorer prognosis than those with negative expression of PBF (P = 0.025). In accordance with the Bioinformatics and Molecular Analysis Section score, we synthesized 10 peptides from the PBF sequence. Subsequent screening with an HLA class I stabilization assay revealed that peptide PBF A24.2 had the highest affinity to HLA-A24. CD8(+) T cells reacting with a PBF A24.2 peptide were detected in eight of nine HLA-A24-positive patients with osteosarcoma at the frequency from 5 x 10(-7) to 7 x 10(-6) using limiting dilution/mixed lymphocyte peptide culture followed by tetramer-based frequency analysis. PBF A24.2 peptide induced CTL lines from an HLA-A24-positive patient, which specifically killed an osteosarcoma cell line that expresses both PBF and HLA-A24. These findings suggested prognostic significance and immunodominancy of PBF in patients with osteosarcoma. PBF is the candidate target for immunotherapy in patients with osteosarcoma.

Clonal T-cell response against autologous pleomorphic malignant fibrous histiocytoma antigen presented by retrieved HLA-A*0206.

Towards the goal of identifying tumor-rejection antigens on eradicated tumors in bone and soft tissue sarcomas, we evaluated the immune response against antigens presented by lost HLA class I molecules. Tumor specimens and peripheral blood samples were obtained from a 70-year-old woman with pleomorphic malignant fibrous histiocytoma. Over 1-year culture, a tumor cell line (MFH2004) was established. A B-cell line infected with Epstein-Barr virus (B2004-EBV) was developed from the blood samples. HLA genotypes of B2004-EBVcells were A*0206/2402, B*4006/4601, and C*0102/0801, whereas MFH2004 cells were defective for A*0206, B*4006, and C*0102. Loss of HLA-A2 expression was also proved immunohistochemically in the primary tumor tissues. Lost HLA-A2 in MFH2004 cells was retrieved by transfection of HLA-A*0206 cDNA to develop MFH2004-A2. Attempts to induce CTLs by mixed culture with autologous T cells and MFH2004 cells resulted in failure. In contrast, those with MFH2004-A2 induced CTL clones CTL2004-c6 and CTL2004-c17. These CTL clones specifically killed MFH2004-A2 but not MFH2004 or B2004-EBV in an HLA-A2-restricted manner. These findings suggest that CTL2004-c6 and CTL2004-c17 recognize autologous tumor-rejection antigens presented by HLA-A*0206, which may have been expressed by tumor cells that had been eradicated by the host's immunosurveillance system.

Influence of diabetes mellitus on diagnostic potential of iodine-123-BMIPP imaging for coronary artery stenosis in hemodialysis patients.

BACKGROUND: Single-photon emission computed tomography (SPECT) using a fatty acid analogue, iodine-123-beta-methyl iodophenyl-pentadecanoic acid (123I-BMIPP), as a tracer may be effective for detecting coronary artery disease in end-stage renal disease (ESRD) patients. In this study, we investigated whether the presence of diabetes mellitus may affect the diagnostic potential of BMIPP SPECT for detecting coronary stenosis in ESRD patients. METHODS: 123I-BMIPP SPECT was performed in 98 diabetic hemodialysis patients (male to female ratio 66:32; mean age 63.6+/-9.8 years) and 103 nondiabetic hemodialysis patients (68:35; 64.5+/-10.4 years), followed by coronary angiography within 60 days of the SPECT. SPECT imaging was evaluated and graded on a 5-point scale (0=normal, 4=absence of tracer) and assessed as a BMIPP summed score for 17 left ventricular segments. RESULTS: Coronary angiography revealed that 72.4% (71/98) of the diabetic patients and 56.3% (58/103) of the non-diabetic patients had significant coronary stenosis more than 50%; incidences of asymptomatic coronary stenosis were 77.5% in diabetic patients and 72.4% in nondiabetic patients. When a BMIPP summed score of 8 or more was defined as abnormal, sensitivity, specificity and accuracy for detecting coronary stenosis by BMIPP SPECT were 97.2, 63.0 and 87.8% in diabetic patients, and 96.6, 73.3 and 86.4% in nondiabetic patients. In receiver operating characteristic analysis, the areas under the curve of BMIPP SPECT to diagnose coronary stenosis were 0.897 in diabetic and 0.906 in nondiabetic patients. CONCLUSIONS: BMIPP SPECT seems to be able to detect coronary stenosis in diabetic as well as nondiabetic hemodialysis patients.