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The characterization of glycosylation is required for many protein therapeutics. The emergence of antibody and antibody-like molecules with multiple glycan attachment sites has rendered glycan analysis increasingly more complicated. Reliance on site-specific glycopeptide analysis is therefore necessary to fully analyze multi-glycosylated biotherapeutics. Established glycopeptide methodologies have generally utilized a priori knowledge of the glycosylation states of the investigated protein(s), database searching of results generated from data-dependent liquid chromatography–tandem mass spectrometry workflows, and extracted ion quantitation of the individual identified species. However, the inherent complexity of glycosylation makes predicting all glycoforms on all glycosylation sites extremely challenging, if not impossible. That is, only the “knowns” are assessed. Here, we describe an agnostic methodology to qualitatively and quantitatively assess both “known” and “unknown” site-specific glycosylation for biotherapeutics that contain multiple glycosylation sites. The workflow uses data-independent, all ion fragmentation to generate glycan oxonium ions, which are then extracted across the entirety of the chromatographic timeline to produce a glycan-specific “fingerprint” of the glycoprotein sample. We utilized both HexNAc and sialic acid oxonium ion profiles to quickly assess the presence of Fab glycosylation in a therapeutic monoclonal antibody, as well as for high-throughput comparisons of multi-glycosylated protein drugs derived from different clones to a reference product. An automated method was created to rapidly assess oxonium profiles between samples, and to provide a quantitative assessment of similarity.

Ethnopharmacological relevance Danshen (Salvia miltiorrhiza Bunge) is widely used in traditional Chinese medicine. However, it's definite clinical effect and mechanism on colon carcinoma is unclear. Aim of the study To test the hypothesis that the protective effect of danshen on colon cancer and discover the bioactive compounds through in vitro study. Materials and methods We conducted a nationwide cohort study by using population-based data from the Taiwan National Health Insurance Research Database (NHIRD). The study cohort comprised patients diagnosed with malignant neoplasm of colon (ICD-9-CM codes:153) in catastrophic illness database between January 1, 2000, and December 31, 2010. We used the Kaplan–Meier method to estimate lung cancer cumulative incidences. Next, human colon cancer cells (HCT 116 cells and HT29 cells) were used to investigate the effect of dihydroisotanshinone I (DT) on the proliferation and apoptosis of human colon cancer cells and the underlying mechanism through XTT assay and flow cytometry. The in vivo effect of DT treatment was investigated through a xenograft nude mouse model. Results In our study, the in vivo protective effect of danshen in the different stage of colon cancer patients was validated through data from the National Health Insurance Research Database in Taiwan. In vitro, we found that dihydroisotanshinone I (DT), a bioactive compound present in danshen, can inhibit the proliferation of colon carcinoma cells, HCT 116 cells and HT-29 cells. Moreover, DT induced apoptosis of colorectal cancer cells. DT also repressed the protein expression of Skp2 (S-Phase Kinase Associated Protein 2) and the mRNA levels of its related gene, Snail1 (Zinc finger protein SNAI1) and RhoA (Ras homolog gene family, member A). In addition, DT also blocked the colon cancer cells recruitment ability of macrophage by decreasing CCL2 secretion in macrophages. DT treatment also significantly inhibited the final tumor volume in a xenograft nude mouse model. Conclusion Danshen has protective effects in colon cancer patients, which could be attributed to DT through blocking the proliferation of colon cancer cells through apoptosis.

// Ching-Yuan Wu 1, 2 , Jong-Yuh Cherng 3 , Yao-Hsu Yang 1, 2 , Chun-Liang Lin 4, 5 , Feng-Che Kuan 6 , Yin-Yin Lin 1 , Yu-Shih Lin 7 , Li-Hsin Shu 1 , Yu-Ching Cheng 1 , Hung Te Liu 1 , Ming-Chu Lu 6 , Jthau Lung 8 , Pau-Chung Chen 9, 17 , Hui Kuan Lin 10, 11, 12, 13 , Kuan-Der Lee 6, 16 and Ying-Huang Tsai 14, 15 1 Department of Chinese Medicine, Chiayi Chang Gung Memorial Hospital, Chiayi, Taiwan 2 School of Chinese medicine, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan 3 Department of Chemistry and Biochemistry, National Chung Cheng University, Taiwan 4 Departments of Nephrology, Chiayi Chang Gung Memorial Hospital, Chiayi, Taiwan 5 Kidney and Diabetic Complications Research Team (KDCRT), Chiayi Chang Gung Memorial Hospital, Chiayi, Taiwan 6 Department of Hematology and oncology, Chiayi Chang Gung Memorial Hospital, Chiayi, Taiwan 7 Department of Pharmacy, Chiayi Chang Gung Memorial Hospital, Chiayi, Taiwan 8 Department of Medical Research and Development, Chang Gung Memorial Hospital, Chiayi branch, Taiwan 9 Institute of Occupational Medicine and Industrial Hygiene, National Taiwan University College of Public Health, Taipei, Taiwan 10 Department of Molecular and Cellular Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 11 Department of Cancer Biology, Wake Forest University School of Medicine, Medical Center Blvd, Winston-Salem, NC, USA 12 Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan 13 Department of Biotechnology, Asia University, Taichung, Taiwan 14 Division of Pulmonary and Critical Care Medicine of Chang Gung Memorial Hospital, Chiayi, Taiwan, Department of Respiratory Therapy, Chang Gung University, Taoyuan, Taiwan 15 Chang-Gung University College of Medicine, Taoyuan, Taiwan 16 Division of Hematology and Oncology, Department of Internal Medicine, Taipei Medical University Hospital, Taiwan 17 Department of Environmental and Occupational Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan Correspondence to: Ying-Huang Tsai, email: chestmed@adm.cgmh.org.tw Ching-Yuan Wu, email: smbepigwu77@gmail.com Keywords: dihydroisotanshinone I, macrophage, lung cancer, Skp2, CCL2 Received: February 15, 2017 Accepted: June 10, 2017 Published: June 28, 2017 ABSTRACT In traditional Chinese medicine, Salvia miltiorrhiza Bunge (danshen) is widely used in the treatment of numerous cancers. However, its clinical effort and mechanism in the treatment of advanced lung cancer are unclear. In our study, the in vivo protective effort of danshen in patients with advanced lung cancer were validated using data from the National Health Insurance Research Database in Taiwan. We observed in vitro that dihydroisotanshinone I (DT), a bioactive compound in danshen, exerts anticancer effects through many pathways. First, 10 μM DT substantially inhibited the migration ability of lung cancer cells in both macrophage and macrophage/lung cancer direct mixed coculture media. Second, 10 μM DT repressed the phosphorylation of signal transducer and activator of transcription 3 (STAT3), the protein expression of S-phase kinase associated protein-2 (Skp2), and the mRNA levels of STAT3-related genes, including chemokine (C–C motif) ligand 2 (CCL2). In addition, 10 μM DT suppressed the macrophage recruitment ability of lung cancer cells by reducing CCL2 secretion from both macrophages and lung cancer cells. Third, 20 μM DT induced apoptosis in lung cancer cells. Furthermore, DT treatment significantly inhibited the final tumor volume in a xenograft nude mouse model. In conclusion, danshen exerts protective efforts in patients with advanced lung cancer. These effects can be attributed to DT-mediated interruption of the cross talk between lung cancer cells and macrophages and blocking of lung cancer cell proliferation.

// Ching-Yuan Wu 1, 2 , Yao-Hsu Yang 1, 2 , Yin-Yin Lin 1 , Feng-Che Kuan 3 , Yu-Shin Lin 4 , Wei-Yu Lin 5, 6 , Ming-Yen Tsai 7 , Jia-Jing Yang 1 , Yu-Ching Cheng 1 , Li-Hsin Shu 1 , Ming-Chu Lu 3 , Yun-Ju Chen 8, 9 , Kuan-Der Lee 2 and Hong-Yo Kang 8, 9 1 Department of Chinese Medicine, Chiayi Chang Gung Memorial Hospital, Chiayi, Taiwan 2 School of Chinese medicine, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan 3 Department of Hematology and oncology, Chiayi Chang Gung Memorial Hospital, Chiayi, Taiwan 4 Department of Pharmacy, Chiayi Chang Gung Memorial Hospital, Chiayi, Taiwan 5 Department of Urology, Chang Gung Memorial Hospital at Chiayi, Puzi City, Taiwan 6 Chang Gung University of Science and Technology, Chia-Yi, Taiwan 7 Department of Chinese Medicine, Chang Gung Memorial Hospital-Kaohsiung Medical Center, Kaohsiung, Taiwan 8 Graduate Institute of Clinical Medical Sciences, Chang Gung University, College of Medicine, Kaohsiung, Taiwan 9 Hormone Research Center, Department of Obstetrics and Gynecology, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University, College of Medicine, Kaohsiung, Taiwan Correspondence to: Kuan-Der Lee, email: kdlee@cgmh.org.tw Hong-Yo Kang, email: hkang3@mail.cgu.edu.tw Keywords: dihydroisotanshinone I, STAT3, prostate cancer, Skp2, CCL2 Received: May 10, 2016 Accepted: January 10, 2017 Published: February 01, 2017 ABSTRACT Danshen (Salvia miltiorrhiza Bunge) is widely used in traditional Chinese medicine. In our study, the in vivo protective effect of danshen in prostate cancer patients was validated through data from the National Health Insurance Research Database in Taiwan. In vitro , we discovered that dihydroisotanshinone I (DT), a bioactive compound present in danshen, can inhibit the migration of both androgen-dependent and androgen-independent prostate cancer cells. In addition, we noted that DT substantially inhibited the migratory ability of prostate cancer cells in both a macrophage-conditioned medium and macrophage/prostate cancer coculture medium. Mechanistically, DT both diminished the ability of prostate cancer cells to recruit macrophages and reduced the secretion of chemokine (C-C motif) ligand 2 (CCL2) from both macrophages and prostate cancer cells in a dose-dependent manner. Moreover, DT inhibited the protein expression of p-STAT3 and decreased the translocation of STAT3 into nuclear chromatin. DT also suppressed the expression of tumor epithelial–mesenchymal transition genes, including RhoA and SNAI1. In conclusion, danshen can prolong the survival rate of prostate cancer patients in Taiwan. Furthermore, DT can inhibit the migration of prostate cancer cells by interrupting the crosstalk between prostate cancer cells and macrophages via the inhibition of the CCL2/STAT3 axis. These results may provide the basis for a new therapeutic approach toward the treatment of prostate cancer progression.

Ethnopharmacological relevance Traditional Chinese medicine (TCM), one of the most commonly used complementary and alternative medicines, has been receiving increasing attention among elderly patients. However, epidemiological reports and prescription patterns of geriatric TCM users are few. The aim of this study is to use data from a nationwide cohort database to analyze TCM use by the geriatric population in Taiwan from 2005 to 2009. Materials and methods TCM outpatient claims data was obtained from the Taiwan National Health Insurance database. Data for elderly patients aged 65 years and older were included in the analysis during the study period. The demographic data, disease distributions, and frequencies and prescription patterns of TCM use by the geriatric population were analyzed. Results The geriatric cohort included 97,210 patients, in which 46,883 patients (48%) had used TCM at least once, with a total of 723,478 TCM outpatient visits. Of these, 175,857 visits (24.3%) were prompted by “diseases of the musculoskeletal system and connective tissue”; more than half of patients with such diseases were treated using acupuncture and traumatology manipulative therapies. Overall, among the 552,835 visits during which Chinese herbal products (CHP) were prescribed, Shu-Jing-Huo-Xie-Tang and Dan Shen ( Radix Salvia Miltiorrhizae ) were the most frequently prescribed herbal formula and single herb, respectively, for elderly patients. In addition, Shu-Jing-Huo-Xie-Tang was also the most prescribed herbal formula for the most common disease categories of “diseases of the musculoskeletal system and connective tissue” among TCM elderly patients, followed by Du-Huo-Ji-Sheng-Tang, and Shao-Yao-Gan-Cao-Tang. Conclusion This study elucidated the TCM utilization patterns of the geriatric population. However, additional studies are warranted to determine the safety and efficacy of these CHPs for use by elderly patients in further clinical trials.

A copper oxide (CuxO) layer was formed by applying plasma oxidization on a copper film grown on a Si substrate. Pt deposited on this CuxO layer then function as the top electrode to form a Pt/CuxO/Cu structure. A device created with this structure exhibited a forming-free bipolar resistive switching property. Conductive atom force microscope (C-AFM) was employed to investigate the nanoscale electrical properties of the device. Based on I–V curve analysis, it was found that the Poole–Frankel and conducting filaments models were suitable for the present device. C-AFM analysis for the sample indicated that the random formation/rupture of conducting paths in the CuxO layer may play a key role for the device instability in high resistance state.

ZrO2 thin film with suitable dielectric constant for MFIS FET's application was prepared by novel metal-organic-decomposition (MOD) method. The dielectric constants of the thin film laid between 10 and 30 and increased with the development of the crystallinity. The magnitude of the leakage current of the thin film with thickness of 27nm and 35nm was 1E-5 and 1E-7 A/cm2 respectively when 5V bias voltage was applied. The C-V characteristics show a memory window of 2.5V for a sweep voltage from -5V to +5V. It indicates that the thin film could satisfy the requirement of MFIS structure.

PbZr0.52Ti0.48O3 thin film prepared by metal-organic decomposition method was chemically etched by utilizing HF/HNO3 solution. Typical semiconductor processes were used to pattern the film. The dependence of the conformity of pattern transfer on the microstructure of the film was studied. The results showed that the etching rates were different between the islands of the grains and the surrounding areas within the film. The film with small grains is favored for pattern transfer of fidelity. The dimension of the islands of grains contributes greatly to the tolerance of patterned features. Tolerance of 2 μm has been achieved to fulfill the need of usage in microelectromechanical systems.

The transcription factor RUNX-1 plays a key role in megakaryocyte differentiation and is mutated in cases of myelodysplastic syndrome and leukemia. In this study, we purified RUNX-1-containing multiprotein complexes from phorbol ester-induced L8057 murine megakaryoblastic cells and identified the ets transcription factor FLI-1 as a novel in vivo-associated factor. The interaction occurs via direct protein-protein interactions and results in synergistic transcriptional activation of the c-mpl promoter. Interestingly, the interaction fails to occur in uninduced cells. Gel filtration chromatography confirms the differentiation-dependent binding and shows that it correlates with the assembly of a complex also containing the key megakaryocyte transcription factors GATA-1 and Friend of GATA-1 (FOG-1). Phosphorylation analysis of FLI-1 with uninduced versus induced L8057 cells suggests the loss of phosphorylation at serine 10 in the induced state. Substitution of Ser10 with the phosphorylation mimic aspartic acid selectively impairs RUNX-1 binding, abrogates transcriptional synergy with RUNX-1, and dominantly inhibits primary fetal liver megakaryocyte differentiation in vitro. Conversely, substitution with alanine, which blocks phosphorylation, augments differentiation of primary megakaryocytes. We propose that dephosphorylation of FLI-1 is a key event in the transcriptional regulation of megakaryocyte maturation. These findings have implications for other cell types where interactions between runx and ets family proteins occur.

Study objective Molecular definition of disease has been changing all aspects of medical practice, from diagnosis and screening to understanding and treatment. Acute appendicitis is among many human conditions that are complicated by the heterogeneity of clinical presentation and shortage of diagnostic markers. Here, we sought to profile the urine of patients with appendicitis, with the goal of identifying new diagnostic markers. Methods Candidate markers were identified from the urine of children with histologically proven appendicitis by using high-accuracy mass spectrometry proteome profiling. These systemic and local markers were used to assess the probability of appendicitis in a blinded, prospective study of children being evaluated for acute abdominal pain in our emergency department. Tests of performance of the markers were evaluated against the pathologic diagnosis and histologic grade of appendicitis. Results Test performance of 57 identified candidate markers was studied in 67 patients, with median age of 11 years, 37% of whom had appendicitis. Several exhibited favorable diagnostic performance, including calgranulin A (S100-A8), α-1-acid glycoprotein 1 (orosomucoid), and leucine-rich α-2-glycoprotein (LRG), with the receiver operating characteristic area under the curve and values of 0.84 (95% confidence interval [CI] 0.72 to 0.95), 0.84 (95% CI 0.72 to 0.95), and 0.97 (95% CI 0.93 to 1.0), respectively. LRG was enriched in diseased appendices, and its abundance correlated with severity of appendicitis. Conclusion High-accuracy mass spectrometry urine proteome profiling allowed identification of diagnostic markers of acute appendicitis. Usage of LRG and other identified biomarkers may improve the diagnostic accuracy of clinical evaluations of appendicitis.

Analysis of the possible mechanisms of degradation of Ru(bpy)32+-based OLEDs has led to the idea of quencher formation in the metalloorganic area close to the cathode. It has been suggested that the quencher results from an electrochemical process where one of the bipyridine (bpy) groups is replaced with two water molecules [1] or from reduction of Ru(bpy)32+ to Ru(bpy)30 [2]. We have tested these and other degradation ideas for Ru(bpy)32+-based OLEDs, both prepared and tested with considerable exposure to the ambient environment and using materials and procedures that emphasize cost of preparation rather than overall efficiency. In order to understand the mechanisms involved in these particular devices, we have correlated changes in the devices' electrical and optical properties with MALDI-TOF mass spectra and UV-vis absorption and fluorescence spectra.