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Background/purpose Eosinophils are recruited to the brain of mice after infection with Angiostrongylus canonensis. Several factors produced by infected mice are well known playing the role to chemoattract eosinophils from the blood into the brain. The purpose of this study is to investigate whether Angiostronylus cantonensis young-adult worms (AcYA) have components which have eosinophilic chemotactic activity. Methods Eosinophil chemotactic activity of AcYA was tested by Boyden blind-well chamber technique. The components of AcYA were analysed by SDS-PAGE and Mass spectrometry. Furthermore, galectin-9 in the cerebrospinal fluid (CSF) of infected mice and galectin-9-like in AcYA were measured by ELISA technic and also were recognized by western blot analysis respectively. Results Excretory-secretory products of AcYA did not show eosinophil chemotactic activity. However, the extracts of AcYA showed protein concentration-dependent eosinophil chemotactic activity and reached the peak at the 24 μg/ml. The eosinophil chemotactic activity was significantly reduced by lactose. The components of AcYA at molecular weights of approximatively 15 kDa and 35 kDa showed several galectins component in Mass spectrometric analysis. Furthermore, galectin-9-like in AcYA was recognized by ELISA and western blot analysis. In parallel with increase of galectin-9 in the CSF, eosinophils were also significantly increased in mouse after infected with A. cantonensis. Conclusion Galectin-9-like in AcYA and galectin-9 in mouse CSF were confirmed demonstrating eosinophil chemotactic activity both in vitro study and in the infection of mouse in this study.

Pulmonary artery hypertension (PAH) pathology involves extracellular matrix (ECM) remodeling in cardiac tissues, thus promoting cardiac fibrosis progression. miR-29a-3p reportedly inhibits lung progression and liver fibrosis by regulating ECM protein expression, however, its role in PAH-induced fibrosis remains unclear. In this study, we aimed to investigate the role of miR-29a-3p in cardiac fibrosis progression in PAH and its influence on ECM protein thrombospondin-2 (THBS2) expression. The diagnostic and prognostic values of miR-29a-3p and THBS2 in PAH were evaluated. The expressions and effects of miR-29a-3p and THBS2 were assessed in cell culture, monocrotaline-induced PAH mouse model, and patients with PAH. The levels of circulating miR-29a-3p and THBS2 in patients and mice with PAH decreased and increased, respectively. miR-29a-3p directly targets THBS2 and regulates THBS2 expression via a direct anti-fibrotic effect on PAH-induced cardiac fibrosis. The circulating levels of miR-29a-3p and THBS2 were correlated with PAH diagnostic parameters, suggesting their independent prognostic value. miR-29a-3p targeted THBS2 expression via a direct anti-fibrotic effect on PAH-induced cardiac fibrosis, indicating miR-29a-3p acts as a messenger with promising therapeutic effects.

Sensors that can specifically and accurately detect glycosaminoglycans are rare. Here, a dual-mode platform for fluorescence intensity and lifetime sensing of plasma heparin and fluorescence imaging of heparan sulfate proteoglycan-expressed cancer cells was developed by stabilizing the intramolecular charge transfer (ICT) state of dansyl acid-labeling AG73 (DA-AG73) peptide with glutathione-capped gold nanoclusters (GSH-AuNCs). DA-AG73 peptides, including an electron-donor dimethylamino group and an electron-withdrawing sulfonamide moiety in the labeled DA molecules, emitted weak fluorescence due to the formation of the twisted ICT excited state. The complexation of heparin with DA-AG73 peptides followed by interacting with the GSH-AuNCs could restrict the rotation of the dimethylamino groups of the labeled DA molecules, triggering the transition from their twisted ICT state to ICT excited state. As a result, the fluorescence intensity and lifetime of the labeled DA molecules in DA-AG73 peptides were gradually enhanced with increasing the heparin concentration. The proposed platform provided excellent selectivity toward heparin and heparan sulfate and exhibited two linear calibration curves for quantifying 20–800 nM and 20–1000 nM heparin in the fluorescence intensity and lifetime modes, respectively. The proposed platform was practically applied for the fluorescence intensity and lifetime determination of plasma heparin and for the selective imaging of heparan sulfate proteoglycan-expressed cells.

Context Small extracellular vesicles (sEVs) have emerged as modulators of the disease microenvironment, thereby supporting disease progression. However, the potential role of EVs and their content to the pathophysiology of endometriosis remain unclear. Objective This work aimed to investigate whether the EVs from eutopic (Eu) and ectopic (Ec) endometrial stromal cells (ESCs) differ with respect to protein composition and role in endometriosis. Methods Human Eu and Ec endometrium–derived ESCs were isolated from samples of the same patients (n = 3). sEVs were isolated from ESCs via ultracentrifugation; these sEVs were characterized by Western blotting, transmission electron microscopy, and nanoparticle tracking analysis and analyzed using mass spectrometry. The potential role of EcESCs-derived sEVs (EcESCs-sEVs) in endometriosis was explored by assaying their effects on cell viability/proliferation, migration, and angiogenesis. Results In total, 105 ESCs-sEV–associated proteins were identified from EcESCs-sEVs and EuESCs-sEVs by mass spectrometry analysis. The protein content differed between EcESCs-sEVs and EuESCs-sEVs, with annexin A2 (ANXA2) being the most prominent difference—present in EcESCs-sEVs but not EuESCs-sEVs. We also found that sEVs-ANXA2 regulates the motility, proliferation, and angiogenesis of ESCs via the extracellularly regulated kinase (ERK)/STAT3 pathway. Notably, treatment of ESCs with sEVs-ANXA2 resulted in increased proliferation and motility, suggesting that sEVs-ANXA2 may be involved in regulating endometriosis. Our data suggest that EcESCs-sEVs-ANXA2 regulates the motility and the angiogenic potential of ESCs, implying a role for sEVs-ANXA2 in the pathogenesis of endometriosis. Conclusion The study of sEVs-ANXA2 from Ec endometriotic cells uncovers a new mechanism of endometriosis progression and will inform the development of novel therapeutic strategies.

Dipeptidyl peptidase 4 (DPP-4) inhibitors are incretin-based medications used as oral antidiabetic agents for the treatment of type 2 diabetes. However, DPP-4 inhibitors produce side effects like acute pancreatitis, upper respiratory tract infection, nasopharyngitis, urinary tract infection, serious allergies, cardiovascular diseases, hemolysis, and retinopathy. Hence, the development of a fast and simple method to detect DPP-4 inhibitors in body fluids is important. In this study, we developed a derivatization-assisted microextraction method to enhance the detection sensitivity for trace levels of a DPP-4 inhibitor, sitagliptin, from a small volume (10 μL) of human plasma by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Subjecting the analyte to 100 W microwave irradiation after derivatization using a quinoline alkylating reagent (8-bromomethyl quinilone, BrMQ) shortened the reaction time to ~120 s and allowed the target analyte to be easily extracted to a small volume of the organic layer (20 μL). The analyte was then detected by MALDI-TOF MS using α-cyano-4-hydroxycinnamic acid as the matrix. The relative standard deviation and relative error were below 10% in intra- and inter-day assays. Using sitagliptin-d4 as an internal standard, the limits of quantitation and detection were found to be 0.03 μg/mL and 0.01 μg/mL, respectively. All the derivatization and extraction procedures described herein were of microliter grade. This method could effectively reduce the use of organic chemicals and solvents, thereby proving to be an eco-friendly strategy that will cause no harm to the environment.

Background Skin cancer is an important environmentally-related health issue. Although sun exposure is closely associated with increasing environmental heat, the effects of environmental heat on the skin, especially in the context of photocarcinogenesis, has not been carefully examined. Objectives This study aimed to explore the effects and interactions of UVB radiation and environmental heat on photocarcinogenesis of the skin using cell and animal models. Methods Cultured keratinocytes and hairless mice were exposed to different treatment conditions including UVB radiation and environmental heat. The effects of treatment on keratinocyte and mice skin were evaluated at indicated time points. Results UVB induced DNA damage was significantly lower in keratinocytes that were pretreated in an environment with slightly elevated temperature followed by UVB treatment (Heat-UVB) as compared to UVB and UVB radiation followed by exposure to equivalent increase in environmental heat (UVB-Heat) groups. Similar phenomenon was observed in terms of keratinocyte viability. In the animal model, it was found that Heat-UVB treated mice showed delayed and reduced tumor formation as compared to the UVB and UVB-Heat treated groups. Quantum simulation analyses demonstrated that the energy required for CPD formation at environment with higher temperature required considerable higher energy as compared to CPD formation at lower temperature. Conclusion Taken together, our results demonstrated that with equivalent UVB exposure, higher temperature environment may protect cells against subsequent UVB-induced DNA damages.

Reactive oxygen species (ROS) can induce oxidative stress and are associated with cell death and chronic diseases in organisms. In the treatment of disease, drugs that induce ROS are associated with many side effects and unpleasant symptoms. Therefore, during the assessment of new drugs and candidate compounds, ROS generation is an issue of concern, because ROS can modify proteins, lipids, and nucleic acids within organisms and alter their biological functions. In this work, we designed a peptide-based probe for the rapid (

Based on the oxidative stress theory, aging derives from the accumulation of oxidized proteins induced by reactive oxygen species (ROS) in the cytoplasm. Hydrogen peroxide (H2O2) elicits ROS that induces skin aging through oxidation of proteins, forming disulfide bridges with cysteine or methionine sulfhydryl groups. Decreased Ca2+ signaling is observed in aged cells, probably secondary to the formation of disulfide bonds among Ca2+ signaling-related proteins. Skin aging processes are modeled by treating keratinocytes with H2O2. In the present study, H2O2 dose-dependently impaired the adenosine triphosphate (ATP)-induced Ca2+ response, which was partially protected via co-treatment with β-mercaptoethanol, resulting in reduced disulfide bond formation in inositol 1, 4, 5-trisphosphate receptors (IP3Rs). Molecular hydrogen (H2) was found to be more effectively protected H2O2-induced IP3R1 dysfunction by reducing disulfide bonds, rather than quenching ROS. In conclusion, skin aging processes may involve ROS-induced protein dysfunction due to disulfide bond formation, and H2 can protect oxidation of this process.

Enterovirus 71 (EV71) infection can cause airway symptoms, brainstem encephalitis, neurogenic shock, and neurogenic pulmonary edema with high morbidity and mortality. There is no proven therapeutic modality. Flos Farfarae is the dried flower bud of Tussilago farfara L. that has been used to manage airway illnesses for thousands of years. It has neuro-protective activity and has been used to manage neuro-inflammatory diseases. However, it is unknown whether Flos Farfarae has activity against EV71-induced neuropathy. The current study used both human foreskin fibroblast (CCFS-1/KMC) and human rhabdomyosarcoma (RD) cell lines to test the hypothesis that a hot water extract of Flos Farfarae could effectively inhibit EV71 infection. The authenticity of Flos Farfarae was confirmed by HPLC-UV fingerprint. Through plaque reduction assays and flow cytometry, Flos Farfarae was found to inhibit EV71 infection ([Formula: see text]). Inhibition of viral replication and protein expression were further confirmed by reverse transcription polymerase chain reaction (RT-PCR) and quantitative RT-PCR (qRT-PCR), and western blot, respectively. The estimated IC[Formula: see text]s were 106.3[Formula: see text][Formula: see text]g/mL in CCFS-1/KMC, and 15.0[Formula: see text][Formula: see text]g/mL in RD cells. Therefore, Flos Farfarae could be beneficial to inhibit EV71 infection by preventing viral replication and structural protein expression.

RATIONALE Understanding drug-drug interactions and predicting the side effects induced by polypharmacy are difficult because there are few suitable platforms that can predict drug-drug interactions and possible side effects. Hence, developing a platform to identify significant protein markers of drug-drug interactions and their associated side effects is necessary to avoid adverse effects. METHODS Human liver cells were treated with ethosuximide in combination with cimetidine, ketotifen, metformin, metronidazole, or phenytoin. After sample preparation and extraction, mitochondrial proteins from liver cells were isolated and digested with trypsin. Then, peptide solutions were detected using a nano ultra-performance liquid chromatographic system combined with tandem mass spectrometry. The Ingenuity Pathway Analysis tool was used to simulate drug-drug interactions and identify protein markers associated with drug-induced adverse effects. RESULTS Several protein markers were identified by the proposed method after liver cells were co-treated with ethosuximide and other drugs. Several of these protein markers have previously been reported in the literature, indicating that the proposed platform is workable. CONCLUSIONS Using the proposed in vitro platform, significant protein markers of drug-drug interactions could be identified by mass spectrometry. This workflow can then help predict indicators of drug-drug interactions and associated adverse effects for increased safety in clinical prescriptions.

X-inactive-specific transcript (XIST), a long non-coding RNA, is essential for the initiation of X-chromosome inactivation. However, little is known about other roles of XIST in the physiological process in eukaryotic cells. In this study, the bioinformatics approaches revealed XIST could be processed into a small non-coding RNA XPi2. The XPi2 RNA was confirmed by a northern blot assay; its expression was gender-independent, suggesting the role of XPi2 was beyond X-chromosome inactivation. The pull-down assay combined with LC-MS-MS identified two XPi2-associated proteins, nucleolin and hnRNP A1, connected to the formation of G-quadruplex. Moreover, the microarray data showed the knockdown of XPi2 down-regulated the KRAS pathway. Consistently, we tested the expression of ten genes, including KRAS, which was correlated with a G-quadruplex formation and found the knockdown of XPi2 caused a dramatic decrease in the transcription level of KRAS among the ten genes. The results of CD/NMR assay also supported the interaction of XPi2 and the polypurine-polypyrimidine element of KRAS. Accordingly, XPi2 may stimulate the KRAS expression by attenuating G-quadruplex formation. Our present work sheds light on the novel role of small RNA XPi2 in modulating the G-quadruplex formation which may play some essential roles in the KRAS- associated carcinogenesis.

Valproic acid (VA) is the drug of choice for managing epilepsy syndrome and is an effective treatment for various seizures. In recent years, new applications of VA have been developed for clinical treatment of other diseases. In the degradation pathway, cytochrome P450 participates in the biotransformation of VA to other metabolites. The resulting compounds then contribute to apoptosis induced by oxidative stress. Hence, clinical applications of VA medication should be selected carefully to avoid potentially severe side effects such as hepatotoxicity, hypersensitivity syndrome reactions, neurological toxicity and teratogenicity. This work established a simple method of using a microwave assisted derivatization procedure for improved detection and monitoring of VA derivatives by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The calibration curve was 1–100 μM for VA analysis, and good linearity was obtained. In intra- and inter-day analyses, all relative standard deviation (RSD) and relative error (RE) values were below 7.5%.

In the clinic, ethosuximide is commonly used to treat generalized absence seizures but has recently been repurposed for other diseases. Because of adverse effects and drug interactions, high-throughput therapeutic drug monitoring of ethosuximide is necessary. Microextraction is a simple, effective, rapid, and low consumption of organic solvents method for sample preparation. In this study, microderivatization-increased detection (MDID)-combined microextraction was used to detect ethosuximide by mass spectrometry. Ethosuximide is a difficult to retain and ionize compound in the C18 nano-flow column and ionization interface, respectively. Hence, we developed a fast method for detecting ethosuximide in human plasma by using the MDID strategy (within 2 min). Chemical microderivatization parameters were studied and optimized to increase the sensitivity of ethosuximide detection at trace levels. The linear range for the analysis of ethosuximide in 10 μL plasma was 5–500 μg/mL with a coefficient of determination (r2) ≥ 0.995. The precision and accuracy of intraday and interday analyses of ethosuximide were below 13.0%. Furthermore, modifications of major proteins in plasma and blood cells, induced by ethosuximide, were identified. The proposed method effectively utilizes microliter samples to detect drug plasma concentrations under suitable microextraction procedures toward the eco-friendly goal of low consumption of organic solvents.

Exosomes are implicated in cancer cell development, migration and invasion. Pigment epithelium-derived factor (PEDF) is a secreted anticancer protein that can regulate lung cancer progression; however, the role of PEDF in non-small cell lung cancer (NSCLC), including metastasis and cancer cell-derived exosome secretion, is unclear. In this study, we analyzed the effects of PEDF on exosome-mediated migration, invasion, and tumorigenicity of cultured NSCLC cells. The results showed that PEDF overexpression significantly reduced NSCLC invasion and migration, while inducing cell aggregation, whereas PEDF knockdown had the opposite effects. Exosomes from NSCLC cells treated with recombinant PEDF had a significantly reduced ability to promote cancer cell motility, migration, and invasion compared to exosomes from untreated cells. Exosomes from PEDF-treated cells contained thrombospondin 1 (THBS1), which inhibited cytoskeletal remodeling and exosome-induced lung cancer cell motility, migration, and invasion. Furthermore, PEDF-overexpressing NSCLC cells formed smaller xenograft tumors with higher THBS1 expression compared to control tumors. Our findings indicate that PEDF decreases the metastatic potential of NSCLC cells through regulation of THBS1 release in cancer cell-derived exosomes, thus uncovering a new mechanism of lung cancer progression.

Serum vitamin D is bound to vitamin D-binding protein (DBP). We studied the roles of DBP in streptozotocin-diabetic rats and high glucose (HG)-cultured cells. In diabetic rat sera, there was one upregulated (with a lower isoelectric point [pI], phosphorylated at S268, S270, S464 and T269) and one downregulated (with a higher pI, phosphorylated at S454 and S457) DBP. DBP levels with lower pI were increased in diabetic rat kidney and liver. HG (30 mM) increased DBP protein expression in NRK-49F cells and Clone-9 hepatocytes. HG decreased pI of DBP in Clone-9 hepatocytes. Moreover, DBP short hairpin ribonucleic acid attenuated 1,25-(OH)2D3-induced attenuation of HG-induced renin (but not collagen IV and fibronectin) protein expression in NRK-49F cells. Thus, DBP level is increased whereas DBP is phosphorylated in diabetic rat serum. HG increased DBP protein expression in renal fibroblasts and hepatocytes. Moreover, DBP is required for vitamin D-induced attenuation of HG-induced renin in NRK-49F cells.

S100P, a Ca2+ binding protein, has been shown to be overexpressed in various cancers. However, its functional character in lung cancer remains largely unknown. In this study, we show that S100P increases cancer migration, invasion and metastasis in lung cancer cells. Ectopic expression of S100P increases migration, invasion and EMT in less invasive CL1-0 lung cancer cells. Conversely, knockdown of S100P suppressed migration and invasion, and caused a reversion of EMT in highly invasive lung cancer cells. These effects were transduced by increasing the interaction of S100P with integrin α7, which activated focal adhesion kinase (FAK) and AKT. Blocking FAK significantly decreased S100P-induced migration by decreasing Src and AKT activation, whereas inhibiting AKT reduced S100P upregulation on ZEB1 expression. Further study has indicated that S100P knockdown prevents the spread of highly metastatic human lung cancer in animal models. This study therefore suggests that S100P represents a critical activator of lung cancer metastasis. Detection and targeted treatment of S100P-expressing cancer is an attractive therapeutic strategy in treating lung cancer.

In China, a recent outbreak of the new R294K H7N9 influenza has infected 134 people and killed 45 people since January 2014. Prof. Gao et al. reported that an R294K neuraminidase (Shanghai N9: R294K mutation; Anhui N9: no R294K mutation) results in multi-drug resistance with extreme oseltamivir resistance (over 100 000-fold). Herein, we report the findings of molecular simulations and computational alanine-scanning mutagenesis for cyclic peptide I with the neuraminidases of Shanghai N9 and Anhui N9 A. Our results suggest that cyclic peptide I can inhibit the Shanghai N9 and Anhui N9 influenza A virus neuraminidases. The peptide can provide efficient binding affinities to Shanghai N9 either by the five residues (Arg119, Ile277, Lys294, Arg372 and Tyr406) mutations or by the 13 residues (Val117, Gln137, Thr149, Hie151, Asp152, Ser154, Ile224, Asn296, Asn347, Asn348, Ile429, Pro433 and Lys434) analyzed by binding mode analysis. However, the four residues (Ile150, Arg153, Gln155 and Arg157) can obviously affect the binding interactions among the cyclic peptide.

Valproic acid (VA) is a branch chain fatty acid that is widely used to treat epilepsy and convulsion. Recent studies show that VA can also be used to treat migraine headaches, bipolar disorder, and other diseases such as Alzheimer disease. However, clinical treatment with VA may cause hepatotoxicity, bone marrow suppression, and hyperammonemic encephalopathy. Valproic acid is also a known human teratogen. Because of the potential cytotoxic effects of VA and its major metabolite, 2-propyl 4-pentenoic acid (4-ene VA), VA plasma concentrations must be closely monitored during clinical applications of VA in order to avoid severe side effects. This study developed a derivatization oriented strategy for increasing sensitivity in detecting VA in quantities as low as 20μL and its metabolites in human plasma. After micro-scale liquid-liquid extraction (MLLE) and micro-scale derivatization, VA and 4-ene VA were quantitated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The linear ranges were 10-1000μM for VA and 5-500μM for 4-ene VA. All relative standard deviation (RSD) and relative error (RE) values obtained in intra- and inter-day analyses of VA and 4-ene VA were below 8%. The structures of VA and its metabolite derivatives were further identified by nano ultra performance liquid chromatographic system (nanoUPLC) coupled with tandem mass spectrometry (MS/MS). Since protein modifications induced by VA were also identifiable by nanoUPLC-MS/MS, these modifications may be useful biological indicators of a toxic reaction during clinical applications of VA.

Background Tolerogenic dendritic cells (tDCs) play important roles in immune tolerance, autoimmune disease, tissue transplantation, and the tumor micro-environment. Factors that induce tDCs have been reported, however the intracellular mechanisms involved are rarely discussed. Methods Circulating CD14+CD16+ of breast cancer patients and induced CD14+CD16+ DCs were identified as tDCs by treating CD14+ monocytes with galectin-1 and cancer cell-derived medium combined with IL-4 and GM-CSF. In addition, the 4T1 breast cancer syngeneic xenograft model was used to investigate the effect of galectin-1 in vivo. Results The CD14+CD16+ tDC population in the breast cancer patients was comparatively higher than that in the healthy donors, and both the MDA-MB-231 conditioned medium and galectin-1 could induce tDC differentiation. In a BALB/c animal model, the 4T1 breast cancer cell line enhanced IL-10 expression in CD11c+ DCs which was down-regulated after knocking down the galectin-1 expression of 4T1 cells. Analysis of galectin-1 interacting proteins showed that myosin IIa was a major target of galectin-1 after internalization through a caveolin-dependent endocytosis. Myosin IIa specific inhibitor could diminish the effects of galectin-1 on monocyte-derived tDCs and also block the 4T1 cell induced CD11c+/Ly6G+/IL-10+ in the BALB/c mice. Conclusions Galectin-1 can induce tDCs after internalizing into CD14+ monocytes through the caveolae-dependent pathway and activating myosin IIa. For the breast cancer patients with a high galectin-1 expression, blebbistatin and genistein show potential in immune modulation and cancer immunotherapy. General significance Myosin IIa activation and galectin-1 endocytosis are important in tumor associated tDC development.

Hepatocellular carcinoma (HCC) is the most common malignant liver tumor. The purpose of this study is to characterize proteins secreted from the HepG2 cells, which may relate to cell differentiation and tumor metastasis. In the proteomic analysis, the secretome was identified by nano-high–performance liquid chromatography/electrospray ionization tandem mass spectrometry (nano-HPLC/ESIMS/MS) followed by peptide fragmentation pattern analysis. In this study, three proteins, p130Cas-associated protein (p130Cas/BCAR1), TAR DNA-binding protein 43 (TDP43/TARDBP) and translationally controlled tumor protein (TCTP/TPT1), were identified and confirmed by Western blotting, which showed significantly differential expression compared with the normal liver cells. Analyzing differential protein expressions in HepG2 cell by proteomic approaches suggests that p130Cas/BCAR1, TDP43/TARDBP and TCTP/TPT1 as key proteins and may serve as biomarkers for HCC.

Antrodia cinnamomea (AC) is one of the Far East's treasured medicinal mushrooms and a major ingredient in different nutritional supplements targeting several serious ailments. Developing a sensitive simple quality control protocol for AC and its products is an unmet goal which we target in the current investigation through detecting its biomarkers. Among the AC components, benzenoid derivatives are considered therapeutically attractive due their anti-inflammatory and cytotoxic activities. We proposed a convenient method for concentrating the benzenoid-rich fraction (FNH), from AC wild fruiting bodies ethanolic extract (EEAC). Three benzenoids, 4,7-dimethoxy-5-methyl-1,3-benzodioxole (1), antrocamphin A (2) and 4,7-dimethoxy-5-methyl-6-(3-methylbut-3-en-1-ynyl)-1,3-benzodioxole (3) were purified and their structures were elucidated. Different wild AC samples and the separated benzenoids (1–3) exhibited anti-inflammatory activity through inhibiting superoxide anion generation and elastase release by human neutrophils in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP)/cytochalasin B (CB). Compound 1 exhibited the most potent activity against superoxide anion generation (IC50 5.08 μg/ml) while compounds 2 and 3 were less active (IC50 > 10 μg/ml) implying the advantageous use of 1–3 as biomarkers for AC. HPLC-tandem MS analytical protocol was developed for the quantification of the three major benzenoids from different AC commercial products. The results showed that compound 1 is a unique characteristic biomarker for AC quality control. The concentrations of compounds 2 and 3 were higher in certain sources of AC fruiting bodies and were absent in mycelia, suggesting their usefulness as biomarkers for quality control of fruiting bodies products. The HPLC-tandem MS results were compared to the results obtained by quantitative NMR.

A simple and sensitive capillary zone electrophoresis (CZE) with UV detection (214 nm) was developed and validated for the simultaneous determination of the acetylcholinesterase inhibitors (AChEI), donepezil, and rivastigmine, with antipsychotic drugs in plasma. A sample pretreatment by liquid–liquid extraction and subsequent quantification by CZE with field-amplified sample injection (FASI) was used. The optimum separation for these analytes was achieved in

Transforming growth factor-β (TGF-β) is pivotal in diabetic nephropathy (DN). Angiotensin converting enzyme-2 (ACE2) converts angiotensin II (Ang II) to angiotensin 1-7 (Ang-(1-7)), which binds to Mas. Proximal tubular ACE2 is decreased in DN. ACE2 deficiency exacerbates whereas ACE2 overexpression attenuates DN. Thus, we investigated the mechanism of high glucose-decreased ACE2 in terms of the interaction between TGF-β and ACE2-Ang-(1-7)-Mas in NRK-52E cells. We found that high glucose increased TGF-β1. SB431542 attenuated high glucose-inhibited ACE2 and Mas and Ang-(1-7) conversion from Ang II while attenuating high glucose-induced fibronectin. TGF-β1 also decreased ACE2 and Mas and Ang-(1-7) conversion from Ang II. A779 attenuated Ang-(1-7)-decreased TGF-β1 and Ang-(1-7)-activated JAK2-STAT3. Moreover, A779, LY294002 and AG490 attenuated Ang-(1-7)-inhibited TGF-β1. The combination of Ang-(1-7) and Mas attenuated TGF-β1 (but not high glucose)-induced fibronectin. Thus, high glucose decreases ACE2 via TGF-βR in NRK-52E cells. Additionally, there is a negative feedback function between TGF-β and ACE2, and the combined inhibition of TGF-β and activation of the ACE2-Ang-(1-7)-Mas may be useful for treating diabetic renal fibrosis.

Scientific Reports 6: Article number: 2574010.1038/srep25740; published online: May122016; updated: June102016. In this Article, Chia-Lin Chen is incorrectly affiliated with ‘Department of Biomedical Sciences, College of Medicine Sciences and Technology, Chung Shan Medical University, Taichung, Taiwan’. The correct affiliation is listed below: Genomics Research Center, Academia Sinica, Taipei, Taiwan.

Background The purpose of the study was to compare the risk of de novo cardiovascular disease (CVD) between hemodialysis (HD) and peritoneal dialysis (PD) in patients with incident end-stage renal disease (ESRD). Methods From a Taiwanese universal insurance claims database, we identified 45309 incident ESRD patients without preexisting CVD from 2000 to 2010. Using the propensity score matching method, we included 6516 patients in HD and PD groups, respectively. All patients were followed up until the end of 2011. The Cox proportional hazards regression model was employed to calculate the impact of dialysis modality on the risk of new onset cardiovascular events including ischemic heart disease, and congestive heart failure (CHF). Results No difference was observed in the overall risk of de novo ischemic heart disease between the propensity score-matched HD and PD groups (HD versus PD, adjusted hazard ratio [HR]: 1.03, 95% confidence interval [CI]: 0.86–1.22). However, HD was associated with a higher risk of de novo CHF (adjusted HR: 1.29, 95% CI: 1.13–1.47) than PD was. The risk of de novo CHF was particularly high in the first year under dialysis treatment for propensity score-matched HD patients, compared to PD patients. Conclusions No difference was observed in the overall risk of de novo major ischemic heart events between HD and PD patients. However, HD was associated with a higher risk of de novo CHF than PD in the first year under dialysis treatment.

We analyzed a panel of cationic molecules secreted in the culture medium of human respiratory epithelial cells (REC) upon activation by IL-1β and different pathogen-associated molecular patterns. A 9 kDa fragment derived from β2-microglobulin (B2M) was identified and named shed 9 kDa B2M (sB2M-9). The primary structure of sB2M-9 was revealed to increase its pI value that potentially could play an important role in innate defense. sB2M-9 exhibits antibacterial activity against Gram positive Staphylococcus aureus (SA) but not against Gram negative Klebsiella pneumonia (KP). Upon its binding to SA, sB2M-9 induces clumps, a phenomenon not observed with B2M. Migration of THP-1 monocytes exposed to SA clumps was significantly greater than that to SA without clumps. sB2M-9 binds to SA, more likely as a chemokine, to facilitate THP-1 migration. As a whole, we demonstrated that REC release a novel chemokine with antibacterial activity that is shed from B2M to facilitate THP-1 migration.

Betel quid chewing is associated with cytotoxicity, genotoxicity and carcinogenicity in diseases such as oral cancer, liver cirrhosis, hepatocellular carcinoma and diabetes mellitus. Arecoline and arecaidine, which are the main alkaloids in the areca nut, are potential exposure biomarkers in habitual betel quid users. This study developed a method of detecting arecoline- and arecaidine-protein adducts by mass spectrometry (MS). First, bovine serum albumin was used to predict and confirm the binding sites of proteins modified by arecoline or arecaidine. Cells were then treated with arecoline to identify new protein adducts after cellular metabolic processing. Finally, human plasma was used to model long-term exposure to arecoline and arecaidine. Following isolation proteins were tryspin digested. The peptides afforded were separated and analyzed by nano-scale liquid chromatography with MS using an LTQ Orbitrap mass spectrometer. The experimental findings showed that cysteine is the predominant amino acid in protein adduct formation. The goal of this study was to establish a screening platform for identifying novel protein adducts that form covalent bonds with arecoline or arecaidine. Use of this strategy to survey new protein-toxic adducts may help to identify novel biomarkers of betel nut exposure. Copyright © 2012 John Wiley & Sons, Ltd.

Gemcitabine (2'-deoxy-2', 2'-difluorocytidine; Gem) is a nucleoside anti-metabolite and is commonly used for treating various human cancers including human bladder carcinoma. Gemcitabine not only functions as a suicide nucleoside analog but also inhibits DNA polymerase activity and results in the termination of chain elongation. Using 2-dimensional gel electrophoresis analysis, a Gem-induced protein was identified as UBE2M (a.k.a. UBC12), a NEDD8 conjugation E2 enzyme which contributes to protein degradation. Gem induced UBE2M expression at both RNA and protein levels in several human cancer cell lines. The induction of UBE2M by Gem was accompanied by a reduction in p27(Kip1) protein levels, which could be restored by silencing UBE2M expression with siRNA or by treating cells with the proteasome inhibitor MG132, indicating that UBE2M mediates Gem-induced p27(Kip1) protein degradation. The induction of UBE2M and reduction of p27(Kip1) by Gem were prevented by the PI3K inhibitor LY294002. These results indicate that PI3K activity is necessary for Gem-induced UBE2M expression and that UBE2M facilitates degradation of p27(Kip1). Notably, silencing of UBE2M expression reduced Gem sensitivity in NTUB1 cells, suggesting that UBE2M mediates in part cell sensitivity to Gem, possibly by degradation of p27(Kip1). Analysis of Gem-resistant sub lines also showed that loss of UBE2M and increased p27(Kip1) expression were associated with the acquisition of drug resistance. In conclusion, our results demonstrate a role for UBE2M in mediating cytotoxicity of gemcitabine in human urothelial carcinoma cells while also suggesting a potential function of p27(Kip1) in drug resistance.

[Cu Y ((G:2-6)- dendri -PAMAM-(Py) n )] 2Y+ complexes (3) were prepared, and their ability to generate oxygen radical anions was investigated. The maximum catalytic efficiency ( k′ cat / K M ) was found to be 0.32 min –1 ·μM –1 , and a positive dendritic effect was observed. The saturated kinetics revealed that the improved catalytic efficiency resulted from an enhanced binding affinity toward molecular oxygen. From the Clinical Editor In this basic science study, the oxygen radical anion generating ability of specific copper complex of a pyridine-modified poly(amidoamine) dendrimer was investigated and reported in details.

A simple and micro-scale liquid chromatographic (LC) method coupled with mass spectrometry was developed for analyzing major proteins in human urine. After one-step sample preparation, proteins were precipitated, redissolved, and digested. Only micro-liter level (10 μL) of urine was sufficient for major protein identification. This method was applied in clinical study, and urine proteins were monitored after medication with angiotensin-converting enzyme inhibitor within 24 hr. This method can identify many important proteins in human urine. Bioactive peptides associated with blood pressure control can also be identified simultaneously. We hope this simple method may prove useful in clinical research and disease diagnosis.

A rapid and simple capillary zone electrophoresis (CZE) with ultraviolet detection has been developed for simultaneous determination of regular insulin and insulin aspart in bulk and commercial injection dosage forms. The simultaneous analysis of the tested drugs was performed in phosphate buffer (80 mM; pH 6.5). The separation of regular insulin and insulin aspart was achieved at 13 kV and detection at 200 nm within 7 min with RSD for the absolute migration time reproducibility of less than 3.8% ( n = 10). Selectivity, linearity, precision, accuracy, limits of quantification (LOQ) and limits of detection (LOD) were evaluated as the method validation. Calibration plots were linear ( r > 0.999) over a range of 2.0–60.0 μg/mL for regular insulin and insulin aspart. The LOD were all 1.0 μg/mL (signal-to-noise ratio = 3; injection 6.89 kPa, 7 s). The small amount of sample required and the expeditiousness of the procedure can be an advantageous alternative to traditional methodology for the quantification of tested drugs in individual pharmaceutical products.

Although accelerated atherosclerosis and arteriosclerosis are the main causes of cardiovascular morbidity and mortality in chronic kidney disease (CKD) patients, the molecular pathogenesis remains largely obscure. Our study of the aortic function in a typical CKD model of subtotal nephrectomy (SNX) rats demonstrated phenotypes that resemble CKD patients with aortic stiffness. The 2-DE analysis of rat aortas followed by MS identified 29 up-regulated and 53 down-regulated proteins in SNX rats. Further Western blot and immunohistochemistry analyses validated the decreased HSP27 and increased milk fat globule epidermal growth factor-8 (MFG-E8) in SNX rats. Functional classification of differential protein profiles using KOGnitor revealed that the two major categories involved in aortic stiffness are posttranslational modification, protein turnover, chaperones (23%) and cytoskeleton (21%). Ingenuity Pathway Analysis highlighted cellular assembly and organization, and cardiovascular system development and function as the two most relevant pathways. Among the identified proteins, the clinical significance of the secreted protein MFG-E8 was confirmed in 50 CKD patients, showing that increased serum MFG-E8 level is positively related to aortic stiffness and renal function impairment. Drug interventions with an inhibitor of the angiotensin converting enzyme, enalapril, in SNX rats improved aortic stiffness and decreased MFG-E8 depositions. Together, our studies provide a repertoire of potential biomarkers related to the aortic stiffness in CKD.

Arecoline is the main alkaloid present in the areca nut (or betel nut) and it has central nervous system effects. Its pharmacological activities induce the constriction of the bronchial smooth muscles, and stimulation of the lacrimal and intestinal glands. Chewing areca nut is harmful to health because this habit may increase the risk of the development of oral cancer. In this study, a fast method was provided for the determination of areca alkaloids by matrix-assisted laser desorption ionization (MALDI) mass spectrometer with a time-of-flight (TOF) analyzer. Traditionally the MALDI-TOF method was not suitable for the analysis of small molecular weight (m/z600) compounds because of the high background of the matrix. In this study, a new matrix was utilized to decrease the background interference effectively. After simple sample preparation, 1 microL sample supernatant was mixed with 1 microL matrix and then deposited on the target plate. This new matrix was also used to test the MALDI imaging experiment. Application of this MALDI-TOF method for trace analysis of arecoline by this new matrix in human plasma at sub microM level proved workable.

Two new alkaloids, 9-methoxy-18,19-dehydrocamptothecin (1) and 5- hydroxymappicine-20-O-beta-glucopyranoside (2a/2b as a racemic mixture), together with nine known compounds: camptothecin (3), 9-methoxy-camptothecin (4), 5-hydroxycamptothecin (5a/5b racemic mixture), 5-hydroxy-9-methoxycamptothecin (6a/6b racemic mixture), diosmetin (7), apigenin (8), apigenin-7-O-glucopyranoside (9), rosin (cinnamyl-O-beta-D-glucopyranoside) (10) and amarantholidoside IV (11) were isolated from the immature seeds of Nothapodytes foetida (Wight) Sleumer. The structures were elucidated by spectroscopic analyses. In the present research, compounds 1, 3, 4, 5a/5b and 6a/6b, also showed in vitro cytotoxicity against six cancer cell lines (HepG2, Hep3B, MDA-MB- 231, MCF-7, A549, and Ca9-22). Among them, compound 1 exhibited significant cytotoxicity against these cancer cell lines, with IC(50) of 0.24-6.57 microM. Furthermore, HPLC profiles were developed for qualitative and quantitative analysis of these active constituents in different parts of this plant, including mature and immature seeds, leaves, stems and roots. The results revealed that compounds 3 and 4 have the highest concentrations, which are found in the roots part of the plant.

A simple nanoscale liquid chromatographic method with tandem mass spectrometry (LC‐MS‐MS) was developed for the determination of very basic compounds, biguanides, such as metformin and phenformin. Plasma samples were deproteinated and acidified with heptafluorobutyric acid (HFBA) and the resulting supernatant injected into the nanoscale separation system. Chromatography was carried out using a short reversed‐phase C18 column under isocratic elution with methanol‐40 mM aqueous HFBA (75∶25, v/v) at a flow rate of 800 nL/min. Therapeutic monitoring on metformin and phenformin to minimize adverse side effects is important. The main advantage of the nanoscale method is the lower consumption of organic solvent. This nanoscale method is suitable for the investigation and monitoring of the concentration of oral antihyperglycemic drugs in clinical patients.

The bacterium Xanthomonas campestris pathovar campestris (XCC) 17 is a local isolate that causes crucifer black rot disease in Taiwan. In this study, its proteome was separated using 2-DE and the well-resolved proteins were excised, trypsin digested, and analyzed by MS. Over 400 protein spots were analyzed and 281 proteins were identified by searching the MS or MS/MS spectra against the proteome database of the closely related XCC ATCC 33913. Functional categorization of the identified proteins matched 141 (50%) proteins to 81 metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In addition, we performed a comparative proteome analysis of the pathogenic strain 17 and an avirulent strain 11A to reveal the virulence-related proteins. We detected 22 up-regulated proteins in strain 17 including the degrading enzymes EngXCA, HtrA, and PepA, which had been shown to have a role in pathogenesis in other bacteria, and an anti-host defense protein, Ohr. Thus, further functional studies of these up-regulated proteins with respect to their roles in XCC pathogenicity are suggested.

A micro-scale liquid chromatography electrospray ionization tandem mass spectrometric method was developed for the identification of polymerized impurities in ampicillin and amoxicillin in aqueous solution. Ampicillin and amoxicillin are broad-spectrum antibiotics and widely used for the treatment of human and animal infections. In this study ampicillin, amoxicillin, and their dimers were trapped in a 5-cm capillary column containing C18 sorbents. The analytes were separated on a reversed-phase column and introduced into the mass spectrometer via a nanospray ion source. An isocratic mobile phase consisting of 1% formic acid-acetonitrile (50 : 50, v/v) was used. For identification, the fragment ions of the analytes were monitored. The aim of the present study was to develop an optimized quality control method for the analysis of high molecular weight impurities of ampicillin and amoxicillin.

This study used national claims data to investigate the technique and patient survival over time in incident peritoneal dialysis (PD) patients. Incident end-stage renal disease patients undergoing PD and older than 18 years were selected from Taiwan health insurance databases. These patients were grouped into three study periods according to year of dialysis initiation: 1997–2001, 2002–2006, and 2007–2011. The study end-points included technique failure and mortality. The patients in the most recent era were older and more likely to have higher levels of comorbidity. Compared with the 1997–2001 group, the risks of technique failure were similar in the in the 2002–2006 (hazard ratio [HR] 1.10, 95 % confidence interval [CI] 0.98–1.24) and 2007–2011 groups (HR 1.11, 95 % CI 0.98–1.26), respectively. Relative to the 1997–2001 group, the risks of mortality were higher in the 2002–2006 group (HR 1.59, 95 % CI 1.26–2.02) and similar in the 2007–2011 group (HR 1.20, 95 % CI 0.93–1.55). Using icodextrin and automated peritoneal dialysis (APD) were associated with lower risks of technique failure (HR 0.62 and 0.86, 95 % CI 0.56–0.68 and 0.77–0.95, respectively) and mortality (HR 0.55 and 0.81, 95 % CI 0.45–0.66 and 0.67–0.99, respectively). Despite increase in disease burden in the most recent era, the outcomes remained relatively stable. The use of APD and icodextrin appears to have significantly ameliorated the impact of the increase in comorbidity burden.

Fluorescent boron dipyrromethene (BODIPY) analogs are often used as sensors for detecting various species because of their relatively high extinction coefficients, outstanding fluorescence quantum yields, photostability, and pH-independent fluorescence. However, there is little-to-no information in the literature that describes the use of BODIPY analogs for detecting alkaline phosphatase (ALP) activity and inhibition. This study discovered that the fluorescence of BODIPY-conjugated adenosine triphosphate (BODIPY-ATP) was quenched by Fe(III) ions through photoinduced electron transfer. The ALP-catalyzed hydrolysis of BODIPY-ATP resulted in the formation of BODIPY-adenosine and phosphate ions. The fluorescence of the generated BODIPY-adenosine was insensitive to the change in the concentration of Fe(III) ions. Thus, the Fe(III)-induced fluorescence quenching of BODIPY-ATP can be paired with its ALP-mediated dephosphorylation to design a turn-on fluorescence probe for ALP sensing. A method detection limit at a signal-to-noise ratio of 3 for ALP was estimated to be 0.02 units/L (~6 pM; 1 ng/mL). This probe was used for the screening of ALP inhibitors, including Na3VO4, imidazole, and arginine. Because ALP is widely used in enzyme-linked immunosorbent assays, the probe was coupled to an ALP-linked immunosorbent assay for the sensitive and selective detection of immunoglobulin G (IgG). The lowest detectable concentration for IgG in this system was 5 ng/mL. Compared with the use of 3,6-fluorescein diphosphate as a signal reporter in an ALP-linked immunosorbent assay, the proposed system provided comparable sensitivity, large linear range, and high stability over temperature and pH changes.

The metabolites of environmental chemicals play key roles in carcinogenesis. Areca nut is strongly associated with the development of oral potentially malignant disorders (OPMD) or cancer. The main alkaloid in the areca nut is arecoline, which is highly cytotoxic and genotoxic. Arecoline N-oxide, a metabolite of areca nut alkaloids, which has been identified in animal urine, has been shown to induce mutagenicity in bacteria. In this study, it was found that its protein adduct could be detected in oral keratinocytes treated with areca nut extract. Increased collagen expression and severity of squamous hyperplasia were observed in arecoline N-oxide treated mice. In cultured oral fibroblasts, arecoline N-oxide showed stronger effects on the increase of fibrotic related genes including TGF-beta1, S100A4, MMP-9, IL-6, and fibronectin and a decrease of E-cadherin as compared with arecoline. Finally, arecoline N-oxide stimulation effectively increased the DNA damage marker, gamma-H2A.X, both in vitro and in vivo. Taken together, these results indicate that arecoline N-oxide shows a high potential for the induction of OPMD.

Alzheimer's disease (AD) is the most common cause of dementia of late life. The aim of this study was to utilize the proteomic approaches to establish serum protein patterns of AD. By using nano-high-performance liquid chromatography/electrospray ionization tandem mass spectrometry followed by peptide fragmentation pattern software to analyze proteome in human serum, the activity-dependent neuroprotector homeobox protein was found to exhibit significant differential expression compared with the control group and was confirmed by Western blotting and enzyme-linked immunosorbent assay. It may play an important role in slowing the progression of clinical symptoms of AD.