Endogenous protein "barcode" for data validation and normalization in quantitative MS analysis.

Quantitative proteomic experiments with mass spectrometry detection are typically conducted by using stable isotope labeling and label-free quantitation approaches. Proteins with housekeeping functions and stable expression level such actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase are frequently used as endogenous controls. Recent studies have shown that the expression level of such common housekeeping proteins is, in fact, dependent on various factors such as cell type, cell cycle, or disease status and can change in response to a biochemical stimulation. The interference of such phenomena can, therefore, substantially compromise their use for data validation, alter the interpretation of results, and lead to erroneous conclusions. In this work, we advance the concept of a protein "barcode" for data normalization and validation in quantitative proteomic experiments. The barcode comprises a novel set of proteins that was generated from cell cycle experiments performed with MCF7, an estrogen receptor positive breast cancer cell line, and MCF10A, a nontumorigenic immortalized breast cell line. The protein set was selected from a list of ~3700 proteins identified in different cellular subfractions and cell cycle stages of MCF7/MCF10A cells, based on the stability of spectral count data generated with an LTQ ion trap mass spectrometer. A total of 11 proteins qualified as endogenous standards for the nuclear and 62 for the cytoplasmic barcode, respectively. The validation of the protein sets was performed with a complementary SKBR3/Her2+ cell line.

Apamin inhibits hepatic fibrosis through suppression of transforming growth factor ß1-induced hepatocyte epithelial-mesenchymal transition.

Apamin is an integral part of bee venom, as a peptide component. It has long been known as a highly selective block Ca(2+)-activated K(+) (SK) channels. However, the cellular mechanism and anti-fibrotic effect of apamin in TGF-ß1-induced hepatocytes have not been explored. In the present study, we investigated the anti-fibrosis or anti-EMT mechanism by examining the effect of apamin on TGF-ß1-induced hepatocytes. AML12 cells were seeded at ∼60% confluence in complete growth medium. Twenty-four hours later, the cells were changed to serum free medium containing the indicated concentrations of apamin. After 30 min, the cells were treated with 2 ng/ml of TGF-ß1 and co-cultured for 48 h. Also, we investigated the effects of apamin on the CCl4-induced liver fibrosis animal model. Treatment of AML12 cells with 2 ng/ml of TGF-ß1 resulted in loss of E-cadherin protein at the cell-cell junctions and concomitant increased expression of vimentin. In addition, phosphorylation levels of ERK1/2, Akt, Smad2/3 and Smad4 were increased by TGF-ß1 stimulation. However, cells treated concurrently with TGF-ß1 and apamin retained high levels of localized expression of E-cadherin and showed no increase in vimentin. Specifically, treatment with 2 µg/ml of apamin almost completely blocked the phosphorylation of ERK1/2, Akt, Smad2/3 and Smad4 in AML12 cells. In addition, apamin exhibited prevention of pathological changes in the CCl4-injected animal models. These results demonstrate the potential of apamin for the prevention of EMT progression induced by TGF-ß1 in vitro and CCl4-injected in vivo.

Glycoproteomics on the rise: established methods, advanced techniques, sophisticated biological applications.

Glycosylation is the most complex form of protein PTMs. Affected proteins may carry dozens of glycosylation sites with tens to hundreds of glycan residues attached to every site. Glycosylated proteins have many important functions in biology, from cellular to organismal levels, being involved in cell-cell signaling, cell adhesion, immune response, host-pathogen interactions, and development and growth. Glycosylation, however, expands the biological functional diversity of proteins at the expense of a tremendous increase in structural heterogeneity. Aberrant glycosylation of cell surface proteins, as well as their detectable fingerprint in plasma samples, has been associated with cancer, inflammatory and degenerative diseases, and congenital disorders of glycosylation. Therefore, there are on-going efforts directed toward developing new technologies and approaches for glycan sequencing and high-throughput analysis of glycosylated proteins in complex samples with simultaneous characterization of both the protein and glycan moieties. This work is aimed primarily at pinpointing the challenges associated with the large-scale analysis of glycoproteins and the latest developments in glycoproteomic research, with focus on recent advancements (2011-2012) in microcolumn separations and MS detection.

Protective effect of melittin on inflammation and apoptosis in acute liver failure.

Acute hepatic failure remains an extremely poor prognosis and still results in high mortality. Therefore, better treatment is urgently needed. Melittin, a major component of bee venom, is known to inhibit inflammatory reactions induced by lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-α in various cell types. However, there is no evidence of the anti-inflammatory and anti-apoptotic effect of melittin on liver cells. In the present study, we investigated the effects of melittin on D: -galactosamine (GalN)/lipopolysaccharide (LPS)-induced acute hepatic failure. Acute liver injury was induced with GalN/LPS to determine in vivo efficacy of melittin. Mice were randomly divided into four groups: sterile saline treated group (NC), melittin only treated group (NM), GalN/LPS-treated group (GalN/LPS), and GalN/LPS treated with melittin group (M+GalN/LPS). Mice were given intraperitoneal GalN/LPS with or without melittin treatment. Liver injury was assessed biochemically and histologically. Inflammatory cytokines in the serum, apoptosis of hepatocytes, and cleavage of caspase-3 in the liver were determined. The expression of TNF-α and interleukin (IL)-1ß were increased in the GalN/LPS group. However, treatment of melittin attenuated the increase of inflammatory cytokines. The M+GalN/LPS group showed significantly fewer apoptotic cells compared to the GalN/LPS group. Melittin significantly inhibited the expression of caspase and bax protein levels as well as cytochrome c release in vivo. In addition, melittin prevented the activation of the transcription factor nuclear factor-kappa B (NF-κB) induced by GalN/LPS. These results clearly indicate that melittin provided protection against GalN/LPS-induced acute hepatic failure through the inhibition of inflammatory cytokines and apoptosis.

Apamin inhibits THP-1-derived macrophage apoptosis via mitochondria-related apoptotic pathway.

The development of atherosclerotic lesions is mainly due to macrophage death. The oxidative stresses of monocytes/macrophages play a vital role in the initiation and amplification of atherosclerosis. Apamin, a component of bee venom, exerts an anti-inflammatory effect, and selectively inhibits the Ca(2+)-activated K(+) channels. The mechanisms involved in the inhibition of macrophage apoptosis have been fully elucidated. We induced oxidized low-density lipoprotein (oxLDL) in THP-1-derived macrophage and studied the effect of apamin on intercellular lipid levels, mitochondria-related apoptotic pathway and numbers of apoptotic cells. Oil-red O staining indicates that the inhibition of apamin in the condition significantly prevents intracellular lipid deposition. Treatment with apamin significantly decreased the apoptotic macrophages by decreasing the expression of pro-apoptotic genes Bax, caspase-3 and PARP protein levels, as well as through increasing expression of anti-apoptotic genes Bcl-2 and Bcl-xL protein levels in the absence and presence of oxLDL. In vivo, with apamin treatment reduced apoptotic cells death by TUNEL staining. These results indicate that apamin plays an important role in monocyte/macrophage apoptotic processing, which may provide a potential drug for preventing atherosclerosis.

Alpha-lipoic acid attenuates atherosclerotic lesions and inhibits proliferation of vascular smooth muscle cells through targeting of the Ras/MEK/ERK signaling pathway.

An infectious burden has been suggested to be associated with atherosclerosis in humans, based on the shared and underlying inflammatory responses during infection and atherosclerosis. However, the efficacy of anti-atherogenic drugs is yet to be tested against atherosclerosis in a scenario involving an infectious burden. We have examined alpha-lipoic acid (ALA) for anti-atherogenic effects in a hypercholesterolemic diet-induced atherosclerotic mouse model with inflammatory stimulation. C57BL/6 mice were fed with a hypercholesterolemic diet for 12 weeks to induce atherosclerosis. Lipopolysaccharide was intraperitoneally injected for the 1st week of study to simulate underlying infectious burden during development of atherosclerosis. ALA treatment alleviated atherosclerotic pathologies and reduced serum cholesterol and inflammatory cytokines. Consistently, atherosclerotic markers were improved by ALA treatment. In addition, ALA attenuated the proliferation and migration of vascular smooth muscle cells upon platelet-derived growth factor stimulation through the targeting of the Ras-MEK1/2-ERK1/2 pathway. This study demonstrates the efficacy of ALA on atherosclerosis with immunological complication, by showing that ALA modulates multiple pathogenic aspects of atherosclerosis induced by a hypercholesterolemic diet with inflammatory stimulation consisting of hypercholesterolemia, inflammation and VSMC activation.

Gene silencing by cell-penetrating, sequence-selective and nucleic-acid hydrolyzing antibodies.

Targeting particular mRNAs for degradation is a fascinating approach to achieve gene silencing. Here we describe a new gene silencing tool exploiting a cell-penetrating, nucleic-acid hydrolyzing, single-domain antibody of the light-chain variable domain, 3D8 VL. We generated a synthetic library of 3D8 VL on the yeast surface by randomizing residues located in one of two beta-sheets. Using 18-bp single-stranded nucleic acids as target substrates, including the human Her2/neu-targeting sequence, we selected 3D8 VL variants that had approximately 100-1000-fold higher affinity and approximately 2-5-fold greater selective hydrolyzing activity for target substrates than for off targets. 3D8 VL variants efficiently penetrated into living cells to be accumulated in the cytosol and selectively decreased the amount of target sequence-carrying mRNAs as well as the proteins encoded by these mRNAs with minimal effects on off-target genes. In particular, one 3D8 VL variant targeting the Her2 sequence showed more efficient downregulation of Her2 expression than a small-interfering RNA targeting the same Her2 sequence, resulting in apoptotic cell death of Her2-overexpressing breast cancer cells. Our results demonstrate that cell-penetrating 3D8 VL variants with sequence-selective, nucleic-acid-hydrolyzing activity can selectively degrade target mRNAs in the cytosol, providing a new gene silencing tool mediated by antibody.

The Protective Effect of Apamin on LPS/Fat-Induced Atherosclerotic Mice.

Apamin, a peptide component of bee venom (BV), has anti-inflammatory properties. However, the molecular mechanisms by which apamin prevents atherosclerosis are not fully understood. We examined the effect of apamin on atherosclerotic mice. Atherosclerotic mice received intraperitoneal (ip) injections of lipopolysaccharide (LPS, 2 mg/kg) to induce atherosclerotic change and were fed an atherogenic diet for 12 weeks. Apamin (0.05 mg/kg) was administered by ip injection. LPS-induced THP-1-derived macrophage inflammation treated with apamin reduced expression of tumor necrosis factor (TNF)-α, vascular cell adhesion molecule (VCAM)-1, and intracellular cell adhesion molecule (ICAM)-1, as well as the nuclear factor kappa B (NF-κB) signaling pathway. Apamin decreased the formation of atherosclerotic lesions as assessed by hematoxylin and elastic staining. Treatment with apamin reduced lipids, Ca(2+) levels, and TNF-α in the serum from atherosclerotic mice. Further, apamin significantly attenuated expression of VCAM-1, ICAM-1, TGF-ß1, and fibronectin in the descending aorta from atherosclerotic mice. These results indicate that apamin plays an important role in monocyte/macrophage inflammatory processing and may be of potential value for preventing atherosclerosis.

Protective effects of melittin on transforming growth factor-ß1 injury to hepatocytes via anti-apoptotic mechanism.

Melittin is a cationic, hemolytic peptide that is the main toxic component in the venom of the honey bee (Apis mellifera). Melittin has multiple effects, including anti-bacterial, anti-viral and anti-inflammatory, in various cell types. However, the anti-apoptotic mechanisms of melittin have not been fully elucidated in hepatocytes. Apoptosis contributes to liver inflammation and fibrosis. Knowledge of the apoptotic mechanisms is important to develop new and effective therapies for treatment of cirrhosis, portal hypertension, liver cancer, and other liver diseases. In the present study, we investigated the anti-apoptotic effect of melittin on transforming growth factor (TGF)-ß1-induced apoptosis in hepatocytes. TGF-ß1-treated hepatocytes were exposed to low doses (0.5 and 1 µg/mL) and high dose (2 µg/mL) of melittin. The low doses significantly protected these cells from DNA damage in TGF-ß1-induced apoptosis compared to the high dose. Also, melittin suppressed TGF-ß1-induced apoptotic activation of the Bcl-2 family and caspase family of proteins, which resulted in the inhibition of poly-ADP-ribose polymerase (PARP) cleavage. These results demonstrate that TGF-ß1 induces hepatocyte apoptosis and that an optimal dose of melittin exerts anti-apoptotic effects against TGF-ß1-induced injury to hepatocytes via the mitochondrial pathway. These results suggest that an optimal dose of melittin can serve to protect cells against TGF-ß1-mediated injury.

Mechanical properties of porous Al2O3 composite with surface modified multi-walled carbon nanotubes (MWCNTs).

Multi-walled carbon nanotubes (MWCNTs) have been reinforced in alumina (Al2O3) matrix to overcome the inherent brittleness of the Al2O3 matrix. In this work, MWCNTs were treated by acid to provide hydrophilicity to hydrophobic MWCNTs, inducing the homogeneous dispersion of MWCNTs in an aqueous solution. Aluminum hydroxide (Al(OH)3) as a Al2O3 precursor was added in the solution with the modified MWCNTs, and then this mixture solution was filtered at room temperature. The prepared powders were calcinated at 800-1000 degrees C to reduce the gas pocket in the matrix by decomposition of Al(OH)3. Then the calcinated powders were formed, and heat-treated. The porous MWCNTs-Al2O3 composites show higher mechanical properties in flexure strength and hardness than the porous Al2O3 without the reinforcement phase, which is attributed to the high mechanical properties of MWCNTs. However, higher MWCNTs contents in the composites decrease the mechanical properties due to the aggregation of MWCNTs in the composites. Therefore, control of the MWCNTs content and its dispersibility in the matrix are key factors to be considered for the fabrication of the porous MWCNT-Al2O3 composites.

Heavy metal adsorption capacity of powdered Chlorella vulgaris biosorbent: effect of chemical modification and growth media.

This study investigates the effect of chemical modification and growth medium on the surface characteristics and heavy metal adsorption capacities of Chlorella vulgaris biosorbents, which are prepared in a powder form for the ease of their transport and application. NaOH treatment partially lyses surface cells on cell aggregates, producing rough microscale structures on the biosorbent surface, which enhances the specific surface area by 19-fold and the heavy metal adsorption capacity by factors of 2.4-4.1. Autotrophic C. vulgaris incubation using nitrogen- and phosphorus-rich medium is even a more effective strategy for enhancing the adsorption capacity, showing factors of 1.6-9.4 increase compared to the use of a minimal medium. High phosphorus content of cell residues on the biosorbent surface obtained by luxury phosphorus uptake is responsible for the substantial enhancement. This study suggests a potential of utilizing nitrogen- and phosphorus-rich waste streams to produce a highly efficient microalgal biosorbent for heavy metal adsorption.

Effect of bee venom on transforming growth factor-beta1-treated hepatocytes.

Bee venom (BV) has been used as treatment against a wide variety of ailments, including inflammatory diseases. Various studies have demonstrated anti-inflammatory and anticancer effects of BV. Transforming growth factor (TGF)-beta1 induces hepatocyte apoptosis via the mitochondrial permeability transition. However, there is no evidence or information regarding the antiapoptotic effect of BV on hepatocytes. The authors investigated the antiapoptotic effect of BV on TGF-beta1-treated hepatocytes. The results showed significant protection from DNA damage by BV treatment compared to corresponding TGF-beta1-treated hepatocytes without BV. BV suppressed TGF-beta1-induced activation of the bcl-2 family and caspase family of proteins, which resulted in inhibition of poly ADP-ribose polymerase (PARP) cleavage. Furthermore, BV is not cytotoxic in the low concentrations used in this study. Low concentrations of BV potently suppress the apoptotic response in TGF-beta1-treated hepatocytes; therefore, BV may have therapeutic potential for the treatment of liver diseases.

Applying steel slag leachate as a reagent substantially enhances pH reduction efficiency for humidification treatment.

A cost-effective, easy-to-implement, and sustainable approach is needed to mitigate the production of alkaline leachate from steel slags that are reused or disposed in the environment. To address this issue, a humidification treatment process, which is operated by wetting a stack of steel slag using aqueous reagents and letting atmospheric CO2 to be passively diffused into the slag pores to induce slag carbonation reaction, was previously developed. In this study, we demonstrate that the leachate of raw steel slag can be recycled and used as a humidification reagent to substantially enhance the treatment efficiency as well as to enable operating the process with neither synthetic chemical consumption nor wastewater discharge. In a 24-h study, a 0.61-unit reduction in slag pH is achieved using a raw slag leachate as a reagent, which is substantially greater than a 0.28-unit reduction using deionized water. The net amount of CaCO3 produced during an extended humidification duration of 4 weeks is increased by 2.7-fold when the leachate is used instead of deionized water. A series of systematically designed experiments demonstrates that the pH (11.0) and ionic strength (0.0048) are the two major characteristics of the raw slag leachate that contribute to the enhanced efficiency of humidification treatment. With further demonstration at larger scales in follow-up studies, the novel humidification process that utilizes the leachate generated on-site as a reagent is expected to be a feasible alternative for alkali waste treatment prior to its reuse or disposal.

Structure-tunable supraparticle assemblies of hollow cupric oxide sheathed with nanographenes.

Self-assembled supraparticles (SPs), a secondary structure of clustered nanoparticles, have attracted considerable interest owing to their highly tunable structure, composition, and morphology from their primary nanoparticle constituents. In this study, hierarchically assembled hollow Cu2O SPs were prepared using a cationic polyelectrolyte poly(diallyl dimethylammonium chloride) (PDDA) during the formation of Cu2O nanoparticles. The concentration-dependent structural transformation of PDDA from linear chains to assembled droplets plays a crucial role in forming a hollow colloidal template, affording the self-assembly of Cu2O nanoparticles as a secondary surfactant. The use of the positively charged PDDA also affords negatively charged nanoscale graphene oxide (NGO), an electrical and mechanical supporter to uniformly coat the surface of the hollow Cu2O SPs. Subsequent thermal treatment to enhance the electrical conductivity of NGO within the NGO/Cu2O SPs allows for the concomitant phase transformation of Cu2O to CuO, affording reduced NGO/CuO (RNGO/CuO) SPs. The uniquely structured hollow RNGO/CuO SPs achieve improved electrochemical properties by providing enhanced electrical conductivity and electroactive surface area.

Antifibrotic effect through the regulation of transcription factor using ring type-Sp1 decoy oligodeoxynucleotide in carbon tetrachloride-induced liver fibrosis.

BACKGROUND: Liver fibrosis is characterized by the excessive accumulation of extracellular matrix (ECM). Recent advances in the knowledge about the cellular, molecular and genetic aspects of fibrosis have opened a new era of research on liver cirrhosis. A transcription factor, Sp1, originally described as a ubiquitous transcription factor, is involved in the basal expression of ECM genes and may be important in the fibrotic processes. METHODS: The chronic hepatic damage received intraperitoneal injection of carbon tetrachloride (2 mg/kg) dissolved in corn oil (1 : 3 ratio) three times a weekly for 8 weeks. The delivery of decoy oligodeoxynucleotide (ODN) was performed by injection of 10 microg of scrambled decoy ODN or 10 microg of ring type (R)-Sp1 decoy ODN through the mouse tail vein. All animals of each group were sacrificed, DNA binding activity, expression of cytokines and histological analysis were measured. RESULTS: We have generated a R-Sp1 decoy ODN that effectively blocks Sp1 binding to the promoter region for transcription regulation of transforming growth factor (TGF)-beta1. The expression of fibrotic cytokines and inflammatory cytokines was decreased by using the R-Sp1 decoy ODN in liver cirrhosis. CONCLUSIONS: The present study demonstrates that the R-Sp1 decoy ODN inhibits TGF-beta1 expression in liver cirrhosis. These results indicate that targeting Sp1 can efficiently block ECM expression, and suggest that such an approach may represent an interesting therapeutic alternative towards the treatment of cirrhosis.

The biological effects of topical alginate treatment in an animal model of skin wound healing.

Wound healing is a dynamic and complex process of tissue repair that involves a number of cellular and molecular events. It proceeds from inflammatory response to reepithelialization and finally to formation of a permanent scar. Alginate is a polymer of guluronic and mannuronic acid that is used as a scaffolding material in biomedical applications. For the purpose of studying wound healing, full-thickness skin defects were produced on the dorsal area in rats. We measured the relative sizes of the wounds on days 3, 5, 7, 14, and 28. The wound sizes were decreased in the alginate-treated group compared with the control group and the vaseline-treated group. The expressions of transforming growth factor-beta1, fibronectin, and vascular endothelial growth factor were significantly decreased in the alginate-treated group compared with the control group, while the expression of collagen-I was increased in the alginate-treated group, as indicated by Western blotting and immunohistochemical staining. These data suggest that alginate has significant wound healing promoting activity. The results from the present study indicate that the effect of alginate on wound healing may involve biological mechanisms associated with the expression of transforming growth factor-beta1, fibronectin, vascular endothelial growth factor, and collagen-I.

Anionic surfactant modification of activated carbon for enhancing adsorption of ammonium ion from aqueous solution.

This study investigates the effect of anionic surfactant modification on activated carbon (AC) to enhance the adsorption of ammonium ion in aqueous solution. Sodium dodecyl sulfate (SDS), sodium dodecyl benzene sulfonate (SDBS) or sodium octanoate (SO) was used for the modification. At the initial aqueous concentration of 55 mg NH4-N/L and the adsorbent dose of 50 g/L, the SDS-modified AC showed the highest ammonium removal efficiency of 82% among the modified ACs studied. The hydrophobic group of SDS was strongly attached to AC showing almost negligible desorption after the modification. At the same time, the sulfate functional group of SDS provided ion exchange sites favorable for the ammonium ion adsorption. By maximizing SDS loading to the AC, ammonium removal efficiency can further be improved (5% increase). When Na+, K+ or Ca2+ coexisted in the ammonium solution at the concentration of 55 mg/L, the inhibition effect of these cations on ammonium removal efficiency was negligible (<5%). This study shows the potential of anionic surfactant-modified ACs as the excellent adsorbents for ammonium removal from water.

Demonstration of a magnetic and catalytic Co@Pt nanoparticle as a dual-function nanoplatform.

Co@Pt nanoparticles as a bifunctional nanoplatform system for the hydrogenation of various unsaturated organic molecules under mild conditions and also for magnetic separation and recycling are demonstrated.

Surface to bulk Fermi arcs via Weyl nodes as topological defects.

A hallmark of Weyl semimetal is the existence of surface Fermi arcs. An intriguing question is what determines the connectivity of surface Fermi arcs, when multiple pairs of Weyl nodes are present. To answer this question, we show that the locations of surface Fermi arcs are predominantly determined by the condition that the Zak phase integrated along the normal-to-surface direction is . The Zak phase can reveal the peculiar topological structure of Weyl semimetal directly in the bulk. Here, we show that the winding of the Zak phase around each projected Weyl node manifests itself as a topological defect of the Wannier-Stark ladder, energy eigenstates under an electric field. Remarkably, this leads to bulk Fermi arcs, open-line segments in the bulk spectra. Bulk Fermi arcs should exist in conjunction with surface counterparts to conserve the Weyl fermion number under an electric field, which is supported by explicit numerical evidence.

A plasmonic liquid junction photovoltaic cell with greatly improved power conversion efficiency.

A plasmonic liquid junction photovoltaic cell with greatly improved power conversion efficiency is described. When illuminated with simulated sunlight, the device (Au-TiO2/V3+(0.018 M), V2+(0.182 M)/Pt) reproducibly and sustainably produces an VOC of 0.50 V and a JSC of 0.5 mA cm-2, corresponding to a power conversion efficiency of 0.095%.