Lipid Profiles Obtained from MALDI Mass Spectrometric Imaging in Liver Cancer Metastasis Model.

Liver cancer metastasis is known to be a poor prognosis and a leading cause of mortality. To overcome low therapeutic efficacy, understanding the physiological properties of liver cancer metastasis is required. However, the metastatic lesion is heterogeneous and complex. We investigate the distribution of lipids using matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) in an experimental metastasis model. We obtained the differentially expressed mass peaks in comparison between normal sites and metastatic lesions. The relationship of mass to charge ratio (m/z) and intensity were measured, m/z-indicated species were analyzed by MALDI-MS/MS analysis, and identification of these mass species was confirmed using the METASPACEannotation platform and Lipid Maps®. MALDI-MSI at m/z 725.6, 734.6, 735.6, 741.6, 742.6, 744.6, 756.6, and 772.6 showed significantly higher intensity, consistent with the metastatic lesions in hematoxylin-stained tissues. Sphingomyelin SM [d18:0/16:1], phosphatidylcholine (PC) [32:0], PC [31:0], PC [31:1], and PE [36:2] were highly expressed in metastatic lesions. Our results could provide information for understanding metastatic lesions. It suggests that the found lipids could be a biomarker for the diagnosis of metastatic lesions.

Metformin induces muscle atrophy by transcriptional regulation of myostatin via HDAC6 and FoxO3a.

BACKGROUND: Skeletal muscle atrophy is a severe condition that involves loss of muscle mass and quality. Drug intake can also cause muscle atrophy. Biguanide metformin is the first-line and most widely prescribed anti-diabetic drug for patients with type 2 diabetes. The molecular mechanism of metformin in muscle is unclear. METHODS: Myostatin expression was investigated at the protein and transcript levels after metformin administration. To investigate the pathways associated with myostatin signalling, we used real-time polymerase chain reaction, immunoblotting, luciferase assay, chromatin immunoprecipitation assay, co-immunoprecipitation, immunofluorescence, primary culture, and confocal microscopy. Serum analysis, physical performance, and immunohistochemistry were performed using our in vivo model. RESULTS: Metformin induced the expression of myostatin, a key molecule that regulates muscle volume and triggers the phosphorylation of AMPK. AMPK alpha2 knockdown in the background of metformin treatment reduced the myostatin expression of C2C12 myotubes (-49.86 ± 12.03%, P < 0.01) and resulted in increased myotube diameter compared with metformin (+46.62 ± 0.88%, P < 0.001). Metformin induced the interaction between AMPK and FoxO3a, a key transcription factor of myostatin. Metformin also altered the histone deacetylase activity in muscle cells (>3.12-fold ± 0.13, P < 0.001). The interaction between HDAC6 and FoxO3a induced after metformin treatment. Confocal microscopy revealed that metformin increased the nuclear localization of FoxO3a (>3.3-fold, P < 0.001). Chromatin immunoprecipitation revealed that metformin induced the binding of FoxO3a to the myostatin promoter. The transcript-level expression of myostatin was higher in the gastrocnemius (GC) muscles of metformin-treated wild-type (WT) (+68.9 ± 10.01%, P < 0.001) and db/db mice (+55.84 ± 6.62%, P < 0.001) than that in the GC of controls (n = 4 per group). Average fibre cross-sectional area data also showed that the metformin-treated C57BL/6J (WT) (-31.74 ± 0.75%, P < 0.001) and C57BLKS/J-db/db (-18.11 ± 0.94%, P < 0.001) mice had decreased fibre size of GC compared to the controls. The serum myoglobin level was significantly decreased in metformin-treated WT mice (-66.6 ± 9.03%, P < 0.01). CONCLUSIONS: Our results demonstrate that metformin treatment impairs muscle function through the regulation of myostatin in skeletal muscle cells via AMPK-FoxO3a-HDAC6 axis. The muscle-wasting effect of metformin is more evident in WT than in db/db mice, indicating that more complicated mechanisms may be involved in metformin-mediated muscular dysfunction.

A sensitive environmental forensic method that determines bisphenol S and A exposure within receipt-handling through fingerprint analysis.

As human beings have been consistently exposed to bisphenol A (BPA) and bisphenol S (BPS) derived from various products, the intake of BPS/BPA to humans has been extensively studied. However, using conventional biological matrices such as urine, blood, or dissected skin to detect BPS/BPA in the human body system requires longer exposure time to them, hardly defines the pollutant source of the accumulated BPS/BPA, and is often invasive. Herein, our new approach i.e. fingerprint analysis quantitatively confirms the transfer of BPS/BPA from receipts (specific pollution source) to human skin only within receipt-handling of "20 s". When receipts (fingertip region size; ~1 cm2) containing 100-300 µg of BPS or BPA are handled, 20-40 µg fingerprint-1 of BPS or BPA is transferred to human skin (fingertip). This transferred amount of BPS/BPA can still be toxic according to the toxicity test using water fleas. As a visual evidence, a fingerprint map that matches the distribution of the absorbed BPS/BPA is developed using a mass spectrometry imaging tool. This is the first study to analyze fingerprints to determine the incorporation mechanism of emerging pollutants. This study provides an efficient and non-invasive environmental forensic tool to analyze amounts and sources of hazardous substances.

Biglycan reduces body weight by regulating food intake in mice and improves glucose metabolism through AMPK/AKT dual pathways in skeletal muscle.

While biglycan (BGN) is suggested to direct diverse signaling cascades, the effects of soluble BGN as a ligand on metabolic traits have not been studied. Herein, we tested the effects of BGN on obesity in high-fat diet (HFD)-induced obese animals and glucose metabolism, with the underlying mechanism responsible for observed effects in vitro. Our results showed that BGN administration (1 mg/kg body weight, intraperitoneally) significantly prevented HFD-induced obesity, and this was mainly attributed to reduced food intake. Also, intracerebroventricular injection of BGN reduced food intake and body weight. The underlying mechanism includes modulation of neuropeptides gene expression involved in appetite in the hypothalamus in vitro and in vivo. In addition, BGN regulates glucose metabolism as shown by improved glucose tolerance in mice as well as AMPK/AKT dual pathway-driven enhanced glucose uptake and GLUT4 translocation in L6 myoblast cells. In conclusion, our results suggest BGN as a potential therapeutic target to treat risk factors for metabolic diseases.

Molecular-Level Understanding of Continuous Growth from Iron-Oxo Clusters to Iron Oxide Nanoparticles.

The formation of inorganic nanoparticles has been understood based on the classical crystallization theory described by a burst of nucleation, where surface energy is known to play a critical role, and a diffusion-controlled growth process. However, this nucleation and growth model may not be universally applicable to the entire nanoparticle systems because different precursors and surface ligands are used during their synthesis. Their intrinsic chemical reactivity can lead to a formation pathway that deviates from a classical nucleation and growth model. The formation of metal oxide nanoparticles is one such case because of several distinct chemical aspects during their synthesis. Typical carboxylate surface ligands, which are often employed in the synthesis of oxide nanoparticles, tend to continuously remain on the surface of the nanoparticles throughout the growth process. They can also act as an oxygen source during the growth of metal oxide nanoparticles. Carboxylates are prone to chemical reactions with different chemical species in the synthesis such as alcohol or amine. Such reactions can frequently leave reactive hydroxyl groups on the surface. Herein, we track the entire growth process of iron oxide nanoparticles synthesized from conventional iron precursors, iron-oleate complexes, with strongly chelating carboxylate moieties. Mass spectrometry studies reveal that the iron-oleate precursor is a cluster comprising a tri-iron-oxo core and carboxylate ligands rather than a mononuclear complex. A combinatorial analysis shows that the entire growth, regulated by organic reactions of chelating ligands, is continuous without a discrete nucleation step.

Large-Area Graphene Films as Target Surfaces for Highly Reproducible Matrix-Assisted Laser Desorption Ionization Suitable for Quantitative Mass Spectrometry.

Due to the known sweet-spot issues that intrinsically arise from inhomogeneous formation of matrix-analyte crystals utilized as samples in matrix-assisted laser desorption ionization (MALDI) mass spectrometry, its reproducibility and thus its applications for quantification have been somewhat limited. In this paper, we report a simple strategy to improve the uniformity of matrix-analyte crystal spots, which we realized by adapting large-area graphene films, i.e., non-inert, interacting surfaces, as target surfaces. In this example, the graphitic surfaces of the graphene films interact with excess matrix molecules during the sample drying process, which induces spontaneous formation of optically uniform MALDI sample crystal layers on the film surfaces. Further, mass spectrometric imaging reveals that the visible uniformity is indeed accompanied by reproducible MALDI ionization over an entire sample spot, which greatly suppresses the appearance of sweet spots. The results of this study confirm that the proposed method achieves good linear responses of ion intensity to the analyte concentration (R2 > 0.99) with small relative standard deviations (σ < 10%), which is a range applicable for quantitative measurements using MALDI mass spectrometry. Graphical Abstract ᅟ.

Leaching of polycyclic aromatic hydrocarbons (PAHs) from industrial wastewater sludge by ultrasonic treatment.

Ultrasonic treatment for sludge reduction in wastewater treatment plants (WWTPs) can substantially affect the fate of trace pollutants. However, their fates in the different phases of sludge and mass balances have rarely been reported. In this study, wastewater sludge samples were ultrasonicated at 600W for 0-30min. Then, the leaching of the 16 priority polycyclic aromatic hydrocarbons (PAHs) from the sludge solids (sediment) to the liquid phase (supernatant) was investigated. The total concentration of PAHs (∑16 PAHs) in the sludge sediment (2.10µg/g) was comparable with those of previous worldwide studies. Among the 16 PAHs, naphthalene and acenaphthylene were dominant. The total concentrations of PAHs in the supernatant generally increased with sonication time, indicating that PAHs associated with sludge materials, such as microorganisms, were released into the supernatant. Lighter and more water soluble PAHs were released preferentially into the supernatant in dissolved form, whereas heavier and more hydrophobic PAHs were strongly bound to particles. According to mass balance calculations, 21% of the PAHs in the sludge sediment moved to the supernatant without discernible sonodegradation. An additional experiment for degradation of PAHs supported this interpretation, and several reasons for the no significant sonodegradation were discussed. This result suggests that leaching trace pollutants may significantly contaminate the sludge filtrate after ultrasonic treatment, and therefore their fates should be investigated.

Infection status of endoparasites in foreigner workers living in Cheonan City, Chungnam Province, Korea.

At present, more than 500,000 foreigner workers, most of them from Asian countries with high parasitic infection rates, are working in Korea. Since investigation into the prevalence of parasitic infections in foreigner workers has not yet been conducted in Korea, the present study was performed to determine the parasitic infection status of foreigner workers living in Cheonan City, Chungcheongnam-do (Chungnam Province) and to plan, on that basis, effective control measures. From October to December 2013, the parasitic infection status of 231 foreigner workers employed at selected Cheonan-si small businesses was investigated by both stool examination and ELISA. A total of 60 individuals (26.0%) were found to be infected with parasites. The stool examination detected 14 positive cases (6.1%), and ELISA revealed 50 positive people (21.6%), for at least a kind of parasitic disease. The most common infection was cysticercosis (8.7%), followed by toxocariasis (7.8%) and clonorchiasis (7.4%). Since it was proved that parasitic infections were prevalent among foreigner workers living in Cheonan City, more comprehensive study is urgently needed in order to understand the nationwide status of parasitic infections in foreigner workers.

Very high cycle fatigue behavior of SAE52100 bearing steel by ultrasonic nanocrystalline surface modification.

In this paper, the SAE52100 bearing steel contained large quantities of cementite dispersed in ferrite matrix was subjected to the ultrasonic nanocrystalline surface modification (UNSM) treatment that aims for the extension of fatigue life. The microstructure and fatigue life of the untreated and treated specimens were studied by using electron backscattered diffraction (EBSD) and transmission electron microscopy (TEM), and a developed ultra-high cycle fatigue test (UFT). After UNSM treatment, the coarse ferrite grains (- 10 µm) were refined to nanosize (- 200 nm), therefore, nanostructured surface layers were fabricated. Meanwhile, in the deformed layer, the number density and area fraction of cementite were increased up to - 400% and - 550%, respectively, which increased with the decrease in depth from the topmost treated surface. The improvement of hardness (from 200 Hv to 280 Hv) and high cycles fatigue strength by - 10% were considered the contribution of the developed nanostructure in the UNSM treated specimen.

Improving the spatial resolution of atmospheric polycyclic aromatic hydrocarbons using passive air samplers in a multi-industrial city.

The source-receptor relationship of polycyclic aromatic hydrocarbons (PAHs) in the industrial city has been generally investigated using active air samplers (AAS), but they only provide low spatial resolution data. In this study, the spatial resolution of PAHs was improved by the use of polyurethane foam based passive air samplers (PUF-PAS). We deployed 40 passive air samplers in duplicate at 20 sites in the largest industrial city of Ulsan, South Korea during winter (January 7-February 25, 2011). Among the 16 US-EPA priority PAHs, 13 compounds excluding naphthalene, acenaphthylene, and acenaphthene were selected for the calculation of air concentrations. The level of gaseous ∑(13)PAHs in Ulsan (mean: 43 ng/m(3)) was not as high as we expected due to prevailing winds which transported large amounts of PAHs to the East Sea. The spatial distribution of PAHs, principal component analysis, and diagnostic ratios suggested the influence of PAH emissions from industrial complexes to the surrounding areas. This study demonstrated that the source-receptor relationship of PAHs in the industrial area can be more clearly understood using passive air samplers.

The ethyl acetate extract of Cordyceps militaris inhibits IgE-mediated allergic responses in mast cells and passive cutaneous anaphylaxis reaction in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Cordyceps militaris has been used as a traditional herbal medicine for treating allergy in East Asia. AIM OF THE STUDY: We investigated the anti-allergic efficacy of Cordyceps militaris and its mechanism of action. MATERIALS AND METHODS: ß-Hexosaminidase release of mast cells, a key parameter of degranulation, was evaluated. Anti-allergic potential of Cordyceps militaris was studied using passive cutaneous anaphylaxis (PCA) in vivo. The anti-allergic mechanism of Cordyceps militaris was investigated by immunoblotting analysis, RT-PCR and other biological approaches in mast cells. RESULTS: GSCM EtOAc extract (GSCME) inhibited antigen-induced degranulation with a IC50 value (28.5 µg/ml) in RBL-2H3 cells and antigen-induced passive cutaneous anaphylaxis (PCA) response with a ED50 value (665 mg/kg) in vivo. The release of interleukin (IL-4) and tumor necrosis factor (TNF-α were decreased by GSCME in RBL-2H3 cells. In order to elucidate the anti-allergic mechanisms of GSCME in mast cells, we examined the activated levels of signaling molecules. GSCME inhibited the phosphorylation Syk, ERK, p38 and JNK expression. Identified genistein, daidzein, genistein 7-O-ß-d-glucoside 4″-O-methylate, genistein 4'-O-ß-d-glucoside 4″-O-methylate, glycitein 7-O-ß-d-glucoside 4″-O-methylate, daidzein 7-O-ß-d-glucoside 4″-O-methylate and adenosine in GSCME, inhibited antigen-induced degranulation in RBL-2H3 cells. CONCLUSIONS: Our study suggests that GSCME might be used as a therapeutic agent for allergic diseases.

Cordyceps militaris extract suppresses dextran sodium sulfate-induced acute colitis in mice and production of inflammatory mediators from macrophages and mast cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Cordyceps militaris is a well-known medicinal mushroom used for treatment of asthma, and other bronchial and lung inflammatory diseases. AIM OF THE STUDY: To investigate the anti-inflammatory effects and mechanism of Cordyceps militaris extract on a murine model of acute colitis. MATERIALS AND METHODS: We induced colitis using DSS for 1 week. The disease activity index (DAI) took into account body weight loss, diarrhea, and bleeding. Colon length and crypt length were measured using a microscope. Structural changes of the colon were observed by H&E staining. NO, iNOS, and TNF-α were determined using the Griess assay. iNOS protein was determined using western blotting and quantitative reverse-transcriptase polymerase chain reaction, respectively. Degranulated mast cells in colon tissue were stained using toluidine blue. The degree of degranulated RBL-2H3 cells was measured by the ß-hexosaminidase assay. RESULTS: Cordyceps militaris extract significantly attenuated DSS-induced DAI scores (e.g., body weight loss, diarrhea, gross bleeding). Cordyceps militaris extract also effectively prevented shortening of colon length and crypt length. Histological analysis indicated that Cordyceps militaris extract suppressed epithelial damage, loss of goblet cells, loss of crypts, and infiltration of inflammatory cells induced by DSS. In addition, Cordyceps militaris extract inhibited iNOS and TNF-α mRNA expression in colon tissue of DSS-induced colitis and in LPS-stimulated RAW264.7 cells. Cordyceps militaris extract suppressed degranulation of mast cells in the colon of mice with DSS-induced colitis and in antigen-stimulated mast cells. CONCLUSION: These results suggest that Cordyceps militaris extract has anti-inflammatory activity in DSS-induced acute colitis by down-regulating production and expression of inflammatory mediators. These findings suggest that Cordyceps militaris extract might be applied as an agent for prevention or treatment of inflammatory bowel diseases (IBDs).

A systematic and efficient method to estimate the vibrational frequencies of linear peptide and protein ions with any amino acid sequence for the calculation of Rice-Ramsperger-Kassel-Marcus rate constant.

A systematic method to automatically estimate the vibrational frequency sets of linear peptide and protein ions with any amino acid sequence, which is needed in Rice-Ramsperger-Kassel-Marcus (RRKM) calculations for dissociation of these ions, has been developed. The method starts from the frequencies of free amino acids calculated quantum chemically at the DFT/B3LYP/6-31G** level. Some of these were systematically eliminated to get fictitious sets of frequencies for each amino acid at the C-terminus, N-terminus, and inside the chain. By collecting these sets as needed for a specified amino acid sequence and adding vibrations appearing upon peptide bond formation and protonation, a complete set of vibrational frequencies was obtained. Other conditions for RRKM calculations have also been systematically specified. RRKM calculations performed under various conditions have shown that the present method can be useful for an order of magnitude estimation of a statistical rate constant even at low internal energy region. The fact that arbitrariness involved in constructing an entire frequency set simply through spectral correlation can be avoided, and that any protein ion can be handled systematically and rapidly once its sequence and the number of protons attached are specified, are the main advantages of the present method.

Characteristics of photodissociation at 193 nm of singly protonated peptides generated by matrix-assisted laser desorption ionization (MALDI).

Photodissociation (PD) at 193 nm of various singly protonated peptides was investigated. These include peptides with an arginine residue at the C-terminus, N-terminus, at both termini, inside the chain, and those without an arginine residue. Monoisotopomeric selection was made for the precursor ions. Interference from the post-source decay (PSD) product signals was reduced as much as possible by using the deflection system (reported previously) and subtracting the remaining signals from the laser-on signals. The presence of an arginine residue and its position inside the peptide were found to significantly affect the PD spectra, as reported previously. Presence of a proline, aspartic acid, or glutamic acid residue hardly affected the PD spectral patterns. By comparing the PD spectra obtained at a few different wavelengths, it is concluded that the dissociation of the photoexcited ions occurs in their ground electronic states. Tentative explanations for the observed spectral correlations based on the statistical picture for the reactions are also presented.

Chromophore effect in photodissociation at 266 nm of protonated peptides generated by matrix-assisted laser desorption ionization (MALDI).

Chromophore effect in the photodissociation of protonated peptides at 266 nm was investigated using synthetic peptides with the sequence RGGXGGGGGR where X was a phenylalanyl(F), tyrosyl(Y), cysteinyl(C), glycyl(G), seryl(S), or histidyl(H) residue. The peptides with an F or Y residue dissociated efficiently. Fragment ions due to cleavages at either end of the chromophore were especially prominent just as for the peptide with a tryptophanyl residue reported previously.1Photodissociation was observed even for the peptides without any noticeable chromophore at 266 nm. Here, dissociation at all the peptide bonds was almost equally prominent. Photodissociation of the protonated angiotensin I was investigated using the spectral correlation rules observed in the model systems. Role of the chromophores and the plausible mechanisms involved are discussed.

Photodissociation tandem mass spectrometry at 266 nm of an aliphatic peptide derivatized with phenyl isothiocyanate and 4-sulfophenyl isothiocyanate.

Photodissociation (PD) tandem mass spectra have been obtained at 266 nm for the protonated molecules of a tryptic peptide, ASHLGLAR, and of its phenyl isothiocyanate and 4-sulfophenyl isothiocyanate derivatives, generated by matrix-assisted laser desorption/ionization. Derivatization with the aromatic chromophores greatly reduced the intensity of the laser required for efficient PD. Major fragment ions observed in the three spectra are quite similar. General features of the PD tandem mass spectra and their potential utility for analytical purposes are discussed.

Sequence- and site-specific photodissociation at 266 nm of protonated synthetic polypeptides containing a tryptophanyl residue.

Photodissociation at 266 nm of protonated synthetic polypeptides containing a tryptophanyl residue was investigated using a homebuilt tandem time-of-flight mass spectrometer equipped with a matrix-assisted laser desorption/ionization source. Efficient photodissociation of the protonated peptides was demonstrated. Most of the intense peaks in the laser-induced tandem mass spectra were sequence ions. Furthermore, sequence ions due to cleavages at all the peptide bonds were observed; this is a feature of the technique that is particularly useful for peptide sequencing. Fragmentations at both ends of the tryptophanyl residue were especially prevalent, which can be useful for location of the tryptophanyl chromophore in a peptide.

Tandem time-of-flight mass spectrometer for photodissociation of biopolymer ions generated by matrix-assisted laser desorption ionization (MALDI-TOF-PD-TOF) using a linear-plus-quadratic potential reflectron.

A tandem time-of-flight mass spectrometer for the study of photodissociation of biopolymer ions generated by matrix-assisted laser desorption ionization was designed and constructed. A reflectron with linear and quadratic (LPQ) potential components was used. Characteristics of the LPQ reflectron and its utility as the second stage analyzer of the tandem mass spectrometer were investigated. Performance of the instrument was tested by observing photodissociation of [M + H](+) from angiotensin II, a prototype polypeptide. Quality of the photodissociation tandem mass spectrum was almost comparable to that of the post-source decay spectrum. Monoisotopic selection of the parent ion was possible, which was achieved through the ion beam-laser beam synchronization. General theoretical considerations needed for a successful photodissociation of large biopolymer ions are also presented.

Transcaruncular approach to blowout fractures of the medial orbital wall.

Transcutaneous and transconjunctival approaches are still frequently used to repair orbital wall fractures. However, medial orbital wall fracture remains a challenging area for plastic surgeons due to technical difficulties and postoperative scars. The transcaruncular approach is described and we present our experience with this approach to access the medial orbital wall in 10 patients with blowout fracture in the medial orbital region. All patients were corrected satisfactorily without cutaneous scar. The transcaruncular approach is a useful technique to repair medial orbital wall fractures.