Molecular Mapping of Sebaceous Squalene by Ambient Mass Spectrometry.

Squalene (SQ), a highly unsaturated sebaceous lipid, plays an important role in protecting human skin. To better understand the role of SQ in clinical medicine, an efficient analytical approach is needed to comprehensively study the distribution of SQ on different parts of the skin. In this study, sebaceous lipids were collected from different epidermal areas of a volunteer with sampling probes. Thermal desorption-electrospray ionization/mass spectrometry (TD-ESI/MS) was then used to characterize the lipid species on the probes, and each TD-ESI/MS analysis was completed within a few seconds without any sample pretreatment. The molecular mapping of epidermal squalene on whole-body skin was rendered by scaling the peak area of the extracted ion current (EIC) of SQ based on a temperature color gradient, where colors were assigned to the 1357 sampling locations on a 3D map of the volunteer. The image showed a higher SQ distribution on the face than any other area of the body, indicating the role of SQ in protecting facial skin. The results were in agreement with previous studies using SQ as a marker to explore sebaceous activity. The novelty and significance of this work are concluded as two points: (1) direct and rapid detection of all major classes of sebaceous lipids, including the unsaturated hydrocarbons (SQ) and nonpolar lipids (e.g., cholesterol). The results are unique compared to other conventional and ambient ionization mass spectrometry methods and (2) this is the first study to analyze SQ distribution on the whole-body skin by a high-throughput approach.

Molecular cartography of residue pesticides on grape surface in 3D by ambient ionization tandem mass spectrometry.

Thermal desorption-electrospray ionization tandem mass spectrometry (TD-ESI/MS/MS) was used to characterize the residual pesticides that were collectedfromthe surface of a grapewithmetallic sampling probes. Fungicides, insecticides, and miticides were detected, where results were validated by simple solvent extraction followed by gas chromatography tandem mass spectrometry and liquid chromatography tandem mass spectrometry analyses. To explore the distribution of pesticide residues on grape surfaces, 149 locations of a grape surface were collected and followed by TD-ESI/MS/MS analysis. The molecular cartography was then generated from analysis of residual pesticides on the grape surface in 3D.

Obtaining molecular imagings of pesticide residues on strawberry surfaces with probe sampling followed by ambient ionization mass spectrometric analysis.

Thermal desorption-electrospray ionization tandem mass spectrometry (TD-ESI/MS/MS) was used to rapidly characterize the residual pesticides collected on the surface of a strawberry with a metallic probe. Twelve pesticides, including nine fungicides and three miticides, were detected; the results were validated by comparison with results that used solvent extraction followed by gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry analyses. The distribution of pesticide residues on a strawberry's surface was explored by collecting multiple samples using probes from 40 positions on the strawberry, with the collected samples being analyzed with TD-ESI/MS/MS. The obtained molecular information was used to construct mass spectrometry imaging of the strawberry's pesticide residues.

Vernonia patula (Dryand.) Merr. and Leucas chinensis (Retz.) R. Brown exert anti-inflammatory activities and relieve oxidative stress via Nrf2 activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Vernonia patula (Dryand.) Merr. and Leucas chinensis (Retz.) R. Brown have anti-inflammatory properties and are popularly used as complementary and alternative medicine in Asia. AIM OF THE STUDY: To investigate the underlying molecular mechanism and active chemicals in the ethanol extracts of V. patula (VP) and L. chinensis (LC). MATERIALS AND METHODS: The inhibitory activities of VP and LC on lipopolysaccharide (LPS)-stimulated nitric oxide (NO) and interleukin-6 (IL-6) production were investigated in RAW264.7 macrophages and BV2 microglia. Downregulation of pro-inflammatory genes and upregulation of Nrf2 (NF-E2 p45-related factor 2)-ARE (antioxidant response element) pathway were investigated using RT-Q-PCR and Western blotting. Direct antioxidant capacities were measured using free radical scavenging and Folin-Ciocalteu assays. The flavonoids and triterpenes in VP and LC were identified by HPLC-ESI-MS. RESULTS: VP and LC inhibited NO and IL-6 production and suppressed iNOS, IL-6, IL-1ß and CCL2 gene expression. VP and LC were potent direct antioxidants and effective indirect antioxidants assayed by Nrf2 activation and induction of heme oxygenase (HO)-1, glutamate-cysteine ligase modifier subunit (GCLM) and NAD(P)H quinone oxidoreductase 1 (NQO1). Three flavonoids including apigenin (1), luteolin (2) and chryseriol (3), and one triterpene betulinic acid (4) were found in VP; while compounds 1-4 and oleanolic acid (5) were in LC. CONCLUSION: Anti-inflammatory and antioxidant activities of VP and LC may be in great part attributed to the identified Nrf2 activating compounds, which induce expression of Phase II enzymes and attenuate the upregulation of pro-inflammatory genes.

Fast screening of trace multiresidue pesticides on fruit and vegetable surfaces using ambient ionization tandem mass spectrometry.

An ambient ionization tandem mass spectrometric approach was developed to rapidly screen multiresidue pesticides on fruits and vegetables without sample preparation and chromatographic separation. The residual pesticides on fruits and vegetables were collected by sweeping a metallic probe across the sample surface for 2 cm. The analytes collected on the probe were desorbed and ionized in a thermal desorption electrospray ionization (TD-ESI) source, after which analyte ions were detected by a triple quadruple mass analyzer (QqQ) operated in multiple reaction monitoring (MRM) mode. With this TD-ESI/MS/MS approach, 308 pesticides were monitored, where a mixture containing 15 pesticide standards was successfully identified to demonstrate the capability of this approach to screen trace multiresidue pesticides. The approach had reasonable detection limits (<50 ppb) and reproducibility (RSD: 8.43%, n = 9) from the analysis of a benthiazole standard solution. Real samples including a tomato and bell pepper were analyzed using this TD-ESI/MS/MS approach. After TD-ESI/MS/MS analysis, the organic solvent extracts from the same samples were subjected to TD-ESI/MS/MS, gas chromatography mass spectrometry (GC-MS), and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis for validation.

Antrodia cinnamomea boosts the anti-tumor activity of sorafenib in xenograft models of human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) has been recognized worldwide as one of the major causes of cancer death. The medicinal fungus Antrodia cinnamomea (A. cinnamomea) has been served as a functional food for liver protection. The aim of the present study was to investigate the potential activity of A. cinnamomea extracts as a safe booster for the anticancer activity of sorafenib, a multi-kinase inhibitor approved for the treatment of HCC. The biologically active triterpenoids in the ethanolic extracts of A. cinnamomea (EAC) were initially identified by HPLC/LC/MS then the different extracts and sorafenib were assessed in vitro and in vivo. EAC could effectively sensitize HCC cells to low doses of sorafenib, which was perceived via the ability of the combination to repress cell viability and to induce cell cycle arrest and apoptosis in HCC cells. The ability of EAC to enhance sorafenib activity was mediated through targeting mitogen-activated protein (MAP) kinases, modulating cyclin proteins expression and inhibiting cancer cell invasion. Moreover, the proposed combination significantly suppressed ectopic tumor growth in mice with high safety margins compared to single-agent treatment. Thus, this study highlights the advantage of combining EAC with sorafenib as a potential adjuvant therapeutic strategy against HCC.

Impact of pyrrolidine-bispyrrole DNA minor groove binding agents and chirality on global proteomic profile in Escherichia Coli.

BACKGROUND: There is great interest in the design of small molecules that selectively target minor grooves of duplex DNA for controlling specific gene expression implicated in a disease. The design of chiral small molecules for rational drug design has attracted increasing attention due to the chirality of DNA. Yet, there is limited research on the chirality effect of minor groove binders on DNA interaction, especially at the protein expression level. This paper is an attempt to illustrate that DNA binding affinity might not provide a full picture on the biological activities. Drug interacting at the genomic level can be translated to the proteomic level. Here we have illustrated that although the chiral bispyrrole-pyrrolidine-oligoamides, PySSPy and PyRSPy, showed low binding affinity to DNA, their influence at the proteomic level is significant. More importantly, the chirality also plays a role. Two-dimensional proteomic profile to identify the differentially expressed protein in Escherichia coli DH5α (E coli DH5α) were investigated. RESULTS: E coli DH5α incubated with the chiral PySSPy and PyRSPy, diastereomeric at the pyrrolidine ring, showed differential expression of eighteen proteins as observed through two dimensional proteomic profiling. These eighteen proteins identified by MALDI_TOF/TOF MS include antioxidant defense, DNA protection, protein synthesis, chaperone, and stress response proteins. No statistically significant toxicity was observed at the tested drug concentrations as measured via MTT assay. CONCLUSION: The current results showed that the chiral PySSPy and PyRSPy impact on the proteomic profiling of E coli DH5α, implicating the importance of drug chirality on biological activities at the molecular level.

Using high-concentration trypsin-immobilized magnetic nanoparticles for rapid in situ protein digestion at elevated temperature.

We describe an innovative approach - using a high concentration of trypsin-modified magnetic nanoparticles (TMNPs) - for the rapid and efficient digestion of proteins at elevated temperature. The required digestion time could be reduced to less than 10 s. After digestion, the TMNPs were collected magnetically from the sample solution for reuse and the digested peptides were characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Protein digestion was optimized when using the TMNPs (5 microg/microL) at 57 degrees C; a significantly high peptide coverage was achieved for protein identification (e.g., 98% for lysozyme). Although a high concentration of TMNPs was used for digestion, the short digestion time led to much lower amounts of trypsin peptides being produced through self-digestion. As a result, interference in the mass spectrometric detection of the peptide ions was reduced significantly.

Fluorescence and interactions with thiol compounds of Nile Red-adsorbed gold nanoparticles.

We have investigated the interactions between a variety of thiols and Nile Red-adsorbed gold nanoparticles (NRAuNPs). After adding thiols to solutions of NRAuNPs, the solutions fluoresce strongly as a result of the displacement of a Nile Red-derived product from the surface of the AuNPs. We propose a mechanism for the formation of this NR product on the surface of AuNPs by conducting mass spectrometry, fluorescence, and capillary electrophoresis measurements. By recording the fluorescence changes of the NRAuNP solutions after addition of the thiols, we investigated the interactions between the thiols and NRAuNPs. Using the Langmuir isotherm model, we found that the displacement rate constants for thiols having one carboxyl residue, such as 3-mercaptopropionic acid, fall within the range 0.55-1.19 x 10(-2) s(-1). Thiols containing hydroxyl groups [e.g., 2-mercaptoethanol (2-ME)] or amino groups [e.g., N-(2-mercaptopropionyl)glycine (MPG)], or that have flat structures on the AuNP surface, such as mercaptosuccinic acid, exhibit double-exponential kinetics with first rate constants of 0.51-2.83 x 10(-2) s(-1) and second rate constants of 6.0-23.4 x 10(-4) s(-1). Our results reveal that steric effects and the charge density of the thiols both play important roles in determining the interactions with NRAuNPs. The interactions (displacement and/or induced aggregation) are also dependent on the size of NRAuNPs.

Foreign body granuloma caused by monosodium glutamate after BCG vaccination.

We describe a 7-month-old male infant with a foreign body granuloma caused by monosodium glutamate (MSG) after a Bacille Calmette-Guérin (BCG) immunization. A ridged, erythematous, indurated plaque developed over a BCG injection site on his left upper arm 1 month after the first BCG immunization. Biopsy showed multiple noncaseating foreign body granulomas without detectable mycobacteria by both Ziehl-Neelsen stain and polymerase chain reaction assay. Birefringent crystals were identified in the foreign body giant cells with polarized light microscopy. The crystals were further determined to be glutamic acid by the method of fast atom bombardment. Hence, MSG, the only composite of BCG vaccine except the bacillus, was believed to be responsible for the granulomatous foreign body reaction. On review of the literature, we could find no previous report of an adverse reaction of BCG immunization attributable to MSG (glutamic acid).

Direct protein detection from biological media through electrospray-assisted laser desorption ionization/mass spectrometry.

We report here using a novel technology-electrospray-assisted laser desorption ionization (ELDI)/mass spectrometry-for the rapid and sensitive detection of the major proteins that exist in dried biological fluids (e.g., blood, tears, saliva, serum), bacterial cultures, and tissues (e.g., porcine liver and heart) under ambient conditions. This technique required essentially no sample pretreatment. The proteins in the samples were desorbed using a pulsed nitrogen laser without the assistance of an organic matrix. The desorbed protein molecules were then post-ionized through their fusion into the charged solvent droplets produced from the electrospray of an acidic methanol solution; electrospray ionization (ESI) proceeded from the newly formed droplets to generate the ESI-like protein ions. This new ionization approach combines some of the features of electrospray ionization with those of matrix-assisted laser desorption ionization (MALDI), that is, sampling of a solid surface with spatial resolution, generating ESI-like mass spectra of the desorbed proteins, and operating under ambient conditions.

Quercetin inhibition of ROS-dependent and -independent apoptosis in rat glioma C6 cells.

In the present study, we investigated the protective mechanism of quercetin (QUE) and its glycosides, rutin (RUT) and quercitrin (QUI), on reactive oxygen species (ROS)-dependent (H(2)O(2)) and -independent (chemical anoxia) cell death in rat glioma C6 cells. Induction of HO-1 protein expression was detected in QUE- but not RUT- or QUI-treated C6 cells, and this was prevented by cycloheximide and actinomycin D. Incubation of C6 cells with QUE, but not RUT or QUI, protected C6 cells from H(2)O(2)- and chemical anoxia-induced cytotoxicity according to the MTT and LDH release assays. Apoptotic characteristics including chromatin condensation, DNA ladders, and hypodiploid cells appeared in H(2)O(2)-and chemical anoxia-treated C6 cells, and those events were significantly suppressed by adding QUE (but not RUT or QUI). Increases in caspase 3, 8, and 9 enzyme activities with decreases in pro-PARP and pro-caspase 3 protein levels and an increase in cleaved D4-GDI protein were identified in H(2)O(2)-and chemical anoxia-treated C6 cells, and these were blocked by the addition of QUE, but not by RUT or QUI. Intracellular peroxide levels increased with H(2)O(2) and decreased with chemical anoxia, and the addition of QUE reduced the intracellular peroxide levels induced by H(2)O(2). Results of an anti-DPPH radical assay showed that QUE, RUT, and QUI dose-dependently inhibited the production of DPPH radicals in vitro; however, QUE (but not RUT or QUI) prevention of DNA damage induced by OH radicals was identified with a plasmid digestion assay. Increases in phosphorylated ERK and p53 protein expressions were detected in H(2)O(2)- but not chemical anoxia-treated C6 cells, and the addition of QUE significantly blocked H(2)O(2)-induced phosphorylated ERK and p53 protein expressions. Adding the HO-1 inhibitors, SnPP, CoPP, and ZnPP, reversed the protective effect of QUE against H(2)O(2)- and chemical anoxia-induced cell death according to the MTT assay and morphological observations. Additionally, QUE exhibited inhibitory effects on LPS/TPA-induced transformation in accordance with a decrease in MMP-9 enzyme activity and iNOS protein expression in C6 cells. Taken together, the results of this study suggest that QUE exhibits an inhibitory effect on both ROS-dependent and -independent cell death, and induction of HO-1 protein expression is involved.

Electrospray from nanostructured tungsten oxide surfaces with ultralow sample volume.

This study demonstrated the feasibility of performing protein analysis with ultralow sample volume by combining a tungsten oxide nanowire (TON) fiber with a miniaturized electrospray ionization interface. An increase in wettability of the tugsten surface after growing randomly oriented TON on its surface allows strong adhesion of approximately 50 nL of the methanol solution at its tip. Under the influence of a high electric field, electrospray from a Taylor cone on the adhered methanol solution was observed and the multiply charged ions of protein molecules predissolved in the solution were detected.

Induction of apoptosis in macrophages by air oxidation of dioleoylphosphatidylglycerol.

Dioleoylphosphatidylglycerol (DOPG) containing unsaturated sites is the target of oxidation during preparation, storage, or in vivo use of anionic liposomes. We investigated the biological effect of air oxidation of DOPG on RAW 264.7 murine macrophage-like cells. Oxidation was induced by exposing DOPG to air for 24-72 h. The extent of air oxidation was confirmed using Matrix-Assisted Laser Desorption and Ionization with Time-of-Flight (MALDI-TOF) mass spectrometry. The product of the air oxidation of DOPG was identified as the addition of one oxygen atom to one of the symmetrical fatty moieties of DOPG at m/z 814.77. The treatment of DOPG with air oxidation produced dose-dependent cytotoxicity in macrophages. RAW 264.7 cells exposed to oxidized DOPG exhibited morphological features of apoptosis, such as chromatin condensation and cell shrinkage. Typical apoptotic ladders were observed in DNA extracted from RAW 264.7 cells treated with oxidized DOPG. Flow cytometric analysis demonstrated an increase in the hypodiploid DNA population (sub-G1), indicating that DNA cleavage occurred after treatment with oxidized DOPG. In addition, we showed that pretreating RAW 264.7 cells with zVAD-fmk, a general caspase inhibitor, did not prevent apoptosis induced by oxidized DOPG, suggesting that apoptosis in macrophage cells follows a caspase-independent pathway. These results point to a need for precaution in formulating DOPG liposomes for drug delivery and therapeutic purposes.

Use of a water-soluble fullerene derivative as precipitating reagent and matrix-assisted laser desorption/ionization matrix to selectively detect charged species in aqueous solutions.

A starlike water-soluble fullerene derivative, hexa(sulfonbutyl)fullerene (C60[(CH2)4SO3-]6; HSBF), consisting of a C60 cage covalently bonded with six negatively charged sulfonate arms, was synthesized and used to selectively precipitate positively charged surfactants, amino acids, peptides, and proteins. The affinity of HSBF to the analytes depends on the charge, structure, and hydrophobic characteristics of the analytes. The ion pair precipitate was easily removed from the solution by centrifugation. After washing, the precipitate was redissolved in the solvent or buffer solution and the analyte was characterized by laser desorption ionization-time-of-flight mass spectrometry (LD-TOF). HSBF shows strong optical absorbance in the UV range, so no additional organic matrix was required to conduct LD-TOF analysis of small analytes. For the solution that contained five quaternary amines differing only in alkyl chain length, HSBF exhibits the highest affinity to the amine with the longest alkyl chain. Only the arginine signal was detected from the solution that contained 14 amino acids. The peptides with arginine as the end groups interacted most strongly with HSBF and could be selectively precipitated from a solution of a mixture of five peptides. The signals associated with a trace amount of charged peptides derived from the digestion of proteins by trypsin were greatly enhanced after concentration with HSBF. Among eight proteins in the sample solution, insulin had the strongest affinity to the HSBF and exhibited the strongest signal on the matrix-assisted laser desorption/ionization mass spectrum.

Electrospray ionization from a droplet deposited on a surface-modified glass rod.

This paper reports development of a non-mechanical electrospray ionization (ESI) method to generate electrospray from a droplet deposited on an optical fiber coated with a thin gold or Nafion film. Modification of the surface of the optical fiber in this manner increases its wettability, such that a droplet of the aqueous sample solution can adhere sufficiently strongly to the tip of the fiber. The aqueous sample solution was deposited near the tip of the fiber with a micropipette. When a high voltage (2,000 V) was applied to the fiber by electrical connection through the gold film, the sample solution moved and hung at the tip of the fiber. Simultaneously, ESI was generated from the sample droplet. Multiply charged peptide and protein ions were detected by connecting the ESI source to a quadrupole mass analyzer.