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Find video protocols related to scientific articles indexed in Pubmed.
Highly automated nano-LC/MS-based approach for thousand cell-scale quantification of side chain-hydroxylated oxysterols.
J. Lipid Res.
PUBLISHED: 05-02-2014
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Iso-octyl chain-hydroxylated oxysterols were determined in attomoles per 10,000 cells concentrations in 10,000-80,000 cultured pancreatic adenocarcinoma cells, using a sensitive, highly automated nano-LC-ESI-MS-based method. Identified oxysterols included 24S hydroxycholesterol (24S-OHC), 25 hydroxycholesterol (25-OHC), and 27 hydroxycholesterol (27-OHC), while 20S hydroxycholesterol and 22S hydroxycholesterol were not detected. Lower mass limit of quantification was 23 fg (65 amol) for 25-OHC and 27-OHC (100 times lower than our previous method) and 54 fg (135 amol) for 24S-OHC, after derivatization into Girard T hydrazones and online sample cleanup using simplified and robust automatic filtration and filter back flushing solid phase extraction LC/MS/MS. The instrument configuration was easily installed using a commercial nano-LC/MS system. Recoveries in spiked sample were 96, 97, and 77% for 24S-OHC, 25-OHC, and 27-OHC, with within- and between-day repeatabilities of 1-21% and 2-20% relative SD, respectively. The study demonstrates the potential of nano-LC in lipidomics/sterolomics.
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Metabolites in vertebrate Hedgehog signaling.
Biochem. Biophys. Res. Commun.
PUBLISHED: 01-15-2014
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The Hedgehog (HH) signaling pathway is critical in embryonic development, stem cell biology, tissue homeostasis, chemoattraction and synapse formation. Irregular HH signaling is associated with a number of disease conditions including congenital disorders and cancer. In particular, deregulation of HH signaling has been linked to skin, brain, lung, colon and pancreatic cancers. Key mediators of the HH signaling pathway are the 12-pass membrane protein Patched (PTC), the 7-pass membrane protein Smoothened (SMO) and the GLI transcription factors. PTC shares homology with the RND family of small-molecule transporters and it has been proposed that it interferes with SMO through metabolites. Although a conclusive picture is lacking, substantial efforts are made to identify and understand natural metabolites/sterols, including cholesterol, vitamin D3, oxysterols and glucocorticoides, that may be affected by, or influence the HH signaling cascade at the level of PTC and SMO. In this review we will elaborate the role of metabolites in HH signaling with a focus on oxysterols, and discuss advancements in modern analytical approaches in the field.
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Open tubular lab-on-column/mass spectrometry for targeted proteomics of nanogram sample amounts.
PLoS ONE
PUBLISHED: 01-01-2014
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A novel open tubular nanoproteomic platform featuring accelerated on-line protein digestion and high-resolution nano liquid chromatography mass spectrometry (LC-MS) has been developed. The platform features very narrow open tubular columns, and is hence particularly suited for limited sample amounts. For enzymatic digestion of proteins, samples are passed through a 20 µm inner diameter (ID) trypsin + endoproteinase Lys-C immobilized open tubular enzyme reactor (OTER). Resulting peptides are subsequently trapped on a monolithic pre-column and transferred on-line to a 10 µm ID porous layer open tubular (PLOT) liquid chromatography LC separation column. Wnt/ß-catenein signaling pathway (Wnt-pathway) proteins of potentially diagnostic value were digested+detected in targeted-MS/MS mode in small cell samples and tumor tissues within 120 minutes. For example, a potential biomarker Axin1 was identifiable in just 10 ng of sample (protein extract of ?1,000 HCT15 colon cancer cells). In comprehensive mode, the current OTER-PLOT set-up could be used to identify approximately 1500 proteins in HCT15 cells using a relatively short digestion+detection cycle (240 minutes), outperforming previously reported on-line digestion/separation systems. The platform is fully automated utilizing common commercial instrumentation and parts, while the reactor and columns are simple to produce and have low carry-over. These initial results point to automated solutions for fast and very sensitive MS based proteomics, especially for samples of limited size.
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Integrated enzyme reactor and high resolving chromatography in "sub-chip" dimensions for sensitive protein mass spectrometry.
Sci Rep
PUBLISHED: 09-25-2013
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Reliable, sensitive and automatable analytical methodology is of great value in e.g. cancer diagnostics. In this context, an on-line system for enzymatic cleavage of proteins, subsequent peptide separation by liquid chromatography (LC) with mass spectrometric detection has been developed using "sub-chip" columns (10-20??m inner diameter, ID). The system could detect attomole amounts of isolated cancer biomarker progastrin-releasing peptide (ProGRP), in a more automatable fashion compared to previous methods. The workflow combines protein digestion using an 20??m ID immobilized trypsin reactor with a polymeric layer of 2-hydroxyethyl methacrylate-vinyl azlactone (HEMA-VDM), desalting on a polystyrene-divinylbenzene (PS-DVB) monolithic trap column, and subsequent separation of resulting peptides on a 10??m ID (PS-DVB) porous layer open tubular (PLOT) column. The high resolution of the PLOT columns was maintained in the on-line system, resulting in narrow chromatographic peaks of 3-5 seconds. The trypsin reactors provided repeatable performance and were compatible with long-term storage.
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Separation optimization of long porous-layer open-tubular columns for nano-LC-MS of limited proteomic samples.
J Sep Sci
PUBLISHED: 05-11-2013
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The single-run resolving power of current 10 ?m id porous-layer open-tubular (PLOT) columns has been optimized. The columns studied had a poly(styrene-co-divinylbenzene) porous layer (~0.75 ?m thickness). In contrast to many previous studies that have employed complex plumbing or compromising set-ups, SPE-PLOT-LC-MS was assembled without the use of additional hardware/noncommercial parts, additional valves or sample splitting. A comprehensive study of various flow rates, gradient times, and column length combinations was undertaken. Maximum resolution for <400 bar was achieved using a 40 nL/min flow rate, a 400 min gradient and an 8 m long column. We obtained a 2.3-fold increase in peak capacity compared to previous PLOT studies (950 versus previously obtained 400, when using peak width = 2? definition). Our system also meets or surpasses peak capacities obtained in recent reports using nano-ultra-performance LC conditions or long silica monolith nanocolumns. Nearly 500 proteins (1958 peptides) could be identified in just one single injection of an extract corresponding to 1000 BxPC3 beta catenin (-/-) cells, and ~1200 and 2500 proteins in extracts of 10,000 and 100,000 cells, respectively, allowing detection of central members and regulators of the Wnt signaling pathway.
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On-line solid phase extraction-liquid chromatography, with emphasis on modern bioanalysis and miniaturized systems.
J Pharm Biomed Anal
PUBLISHED: 03-24-2013
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On-line solid phase extraction (SPE)-liquid chromatography (LC) allows for automated, sensitive, precise and selective bioanalysis. It is a common feature in miniaturized- or nano LC systems, which are well suited for applications requiring high sensitivity and/or treatment of limited samples (laser micro-dissection samples, rare cancer stem cells, etc.). Traditionally, particles with reversed phase (RP) functional groups are used for the columns in SPE-LC systems. There is however an expanding diversity in SPE-LC combinations applied to meet todays bioanalytical challenges. Current online SPE-LC combinations employ, e.g. porous graphitic carbon (PGC) and hydrophilic interaction liquid chromatography (HILIC) materials for metabolomics and glycomics, restricted access media (RAM) columns coupled with nano LC for peptidomics, immunoaffinity trap columns for targeted proteomics and metal oxide affinity phases for phosphopeptide analysis. However, issues can arise when combining different phases in on-line SPE-LC, e.g. due to solvent incompatibilities between enrichment/separation principles and sample solvent requirements. Consequences can be low recovery and poor resolution, or need for additional instrumentation. On-line SPE-LC with very narrow columns (10-20?m inner diameters) can be appropriate to obtain maximum sensitivity and information. In such highly miniaturized systems, non-particulate columns are arguably more suited (e.g. monolithic or porous layer open tubular (PLOT) columns) as e.g. hardware contributions resulting in extra column volumes are reduced. Basic SPE-LC systems can be configured/modified to perform quite complex analytical operations, and certain columns, configurations and hardware can improve robustness.
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High efficiency, high temperature separations on silica based monolithic columns.
J Chromatogr A
PUBLISHED: 06-30-2011
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The effect of temperature on separation using reversed-phase monolithic columns has been investigated using a nano-LC pumping system for gradient separation of tryptic peptides with MS detection. A goal of this study was to find optimal conditions for high-speed separations. The chromatographic performance of the columns was evaluated by peak capacity and peak capacity per time unit. Column lengths ranging from 20 to 100 cm and intermediate gradient times from 10 to 30 min were investigated to assess the potential of these columns in a final step separation, e.g. after fractionation or specific sample preparation. Flow rates from 250 to 2000 nL/min and temperatures from 20 to 120°C were investigated. Temperature had a significant effect on fast separations, and a flow rate of 2000 nL/min and a temperature of 80°C gave the highest peak capacity per time unit. These settings produced 70% more protein identifications in a biological sample compared to a conventional packed column. Alternatively, an equal amount of protein identifications was obtained with a 40% reduction in run time compared to the conventional packed column.
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Hydrophilic interaction chromatography of nucleoside triphosphates with temperature as a separation parameter.
J Chromatogr A
PUBLISHED: 01-18-2011
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Eight deoxynucleoside triphosphates (dNTPs) and nucleoside triphosphates (NTPs): ATP, CTP, GTP, UTP, dATP, dCTP, dGTP and dTTP, were separated with two 15 cm ZIC-pHILIC columns coupled in series, using LC-UV instrumentation. The polymer-based ZIC-pHILIC column gave significantly better separations and peak shape than a silica-based ZIC-HILIC column. Better separations were obtained with isocratic elution as compared to gradient elution. The temperature markedly affected the selectivity and could be used to fine tune separation. The analysis time was also affected by temperature, as lower temperatures surprisingly reduced the retention of the nucleotides. dNTP/NTP standards could be separated in 35 min with a flow rate of 200 ?L/min. In Escherichia coli cell culture samples dNTP/NTPs could be selectively separated in 7 0min using a flow rate of 100 ?L/min.
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Novel synthetic antagonists of canonical Wnt signaling inhibit colorectal cancer cell growth.
Cancer Res.
PUBLISHED: 01-05-2011
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Canonical Wnt signaling is deregulated in several types of human cancer where it plays a central role in tumor cell growth and progression. Here we report the identification of 2 new small molecules that specifically inhibit canonical Wnt pathway at the level of the destruction complex. Specificity was verified in various cellular reporter systems, a Xenopus double-axis formation assay and a gene expression profile analysis. In human colorectal cancer (CRC) cells, the new compounds JW67 and JW74 rapidly reduced active ?-catenin with a subsequent downregulation of Wnt target genes, including AXIN2, SP5, and NKD1. Notably, AXIN2 protein levels were strongly increased after compound exposure. Long-term treatment with JW74 inhibited the growth of tumor cells in both a mouse xenograft model of CRC and in Apc(Min) mice (multiple intestinal neoplasia, Min). Our findings rationalize further preclinical and clinical evaluation of these new compounds as novel modalities for cancer treatment.
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Hedgehog antagonists cyclopamine and dihydroveratramine can be mistaken for each other in Veratrum album.
J Pharm Biomed Anal
PUBLISHED: 03-16-2010
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A toxic plant, Veratrum album (ssp. viriscens), was found to have an inhibitory effect on Hedgehog (Hh), a developmental signaling pathway that has been shown to be active during development, in adult stem cells and in numerous human tumors. Based on earlier studies it was believed that the known Hh inhibitor cyclopamine was present in V. album (ssp. viriscens). Here we show that instead of cyclopamine, dihydroveratramine (DHV) was found in V. album (ssp. viriscens). These compounds are easily mistaken for each other, as both substances share the same molecular weight, and the same main MS/MS fragments. DHV was found to be a less potent Hh inhibitor compared to cyclopamine. This is the first reported occurrence of DVH in nature.
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Separation of intact proteins on porous layer open tubular (PLOT) columns.
J Chromatogr A
PUBLISHED: 02-09-2010
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Porous layer open tubular (PLOT) polystyrene divinylbenzene columns have been used for separating intact proteins with gradient elution. The 10 microm I.D. x 3 m columns were easily coupled to standard liquid chromatography-mass spectrometry (LC-MS) instrumentation with commercially available fittings. Standard proteins separated on PLOT columns appeared as narrow and symmetrical peaks with good resolution. Average peak width increased linearly with gradient time (tG) from 0.14 to 0.33 min (tG 20 and 120 min, respectively) using a 3 m column. With shorter columns, peak widths were larger and increased more steeply with gradient time. Theoretical peak capacity (nc) increased with column length (tested up to 3 m). The nc increased with tG until a plateau was reached. The highest peak capacity achieved (nc=185) was obtained with a 3 m column, where a plateau was reached with tG 90 min. The within- and between column retention time repeatabilities were below 0.6% and below 2.5% (relative standard deviation, RSD), respectively. The carry-over following injection of 0.5 ng per protein was less than 1.1%. The retention time dependence on column temperature was investigated in the range 20-50 degrees C. Proteins in a skimmed milk sample were separated using the method.
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Hedgehog antagonist cyclopamine isomerizes to less potent forms when acidified.
J Pharm Biomed Anal
PUBLISHED: 02-08-2010
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The effect of acid treatment of cyclopamine, a natural antagonist of the hedgehog (Hh) signaling pathway and a potential anti-cancer drug, has been studied. Previous reports have shown that under acidic conditions, as in the stomach, cyclopamine is less effective. Also, it has been stated that cyclopamine converts to veratramine, which has side effects such as hemolysis. In this study, we examined in detail the influence of acidification on structure and activity of cyclopamine. We found that of acidified cyclopamine converts to two previously unreported isomers, which we have called cyclopamine (S) and cyclopamine (X). These have likely gone undetected because cyclopamine is often analyzed with fast and hence lower resolving chromatographic methods. Compared to natural cyclopamine, these cyclopamine isomers have a significantly reduced effect on the ciliary transport of the Hh receptor smoothened, and reduced inhibition on the Hedgehog signaling pathway. The side effects of these isomers are unknown. Our findings can partly explain a reduced efficiency of cyclopamine in a gastric environment, and may help with the rational design of more pH independent cyclopamine analogues.
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A novel tankyrase inhibitor decreases canonical Wnt signaling in colon carcinoma cells and reduces tumor growth in conditional APC mutant mice.
Cancer Res.
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Increased nuclear accumulation of ?-catenin, a mediator of canonical Wnt signaling, is found in numerous tumors and is frequently associated with tumor progression and metastasis. Inhibition of Wnt/?-catenin signaling therefore is an attractive strategy for anticancer drugs. In this study, we have identified a novel small molecule inhibitor of the ?-catenin signaling pathway, JW55, that functions via inhibition of the PARP domain of tankyrase 1 and tankyrase 2 (TNKS1/2), regulators of the ?-catenin destruction complex. Inhibition of TNKS1/2 poly(ADP-ribosyl)ation activity by JW55 led to stabilization of AXIN2, a member of the ?-catenin destruction complex, followed by increased degradation of ?-catenin. In a dose-dependent manner, JW55 inhibited canonical Wnt signaling in colon carcinoma cells that contained mutations in either the APC (adenomatous polyposis coli) locus or in an allele of ?-catenin. In addition, JW55 reduced XWnt8-induced axis duplication in Xenopus embryos and tamoxifen-induced polyposis formation in conditional APC mutant mice. Together, our findings provide a novel chemotype for targeting canonical Wnt/?-catenin signaling through inhibiting the PARP domain of TNKS1/2.
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High sensitivity measurements of active oxysterols with automated filtration/filter backflush-solid phase extraction-liquid chromatography-mass spectrometry.
J Chromatogr A
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Oxysterols are important in numerous biological processes, including cell signaling. Here we present an automated filtration/filter backflush-solid phase extraction-liquid chromatography-tandem mass spectrometry (AFFL-SPE-LC-MS/MS) method for determining 24-hydroxysterol and the isomers 25-hydroxycholesterol and 22S-hydroxycholesterol that enables simplified sample preparation, high sensitivity (~25 pg/mL cell lysis sample) and low sample variability. Only one sample transfer step was required for the entire process of cell lysis, derivatization and determination of selected oxysterols. During the procedure, autoxidation of cholesterol, a potential/common problem using standard analytical methods, was found to be negligible. The reversed phase AFFL-SPE-LC-MS/MS method utilizing a 1mm inner diameter column was validated, and used to determine levels of the oxysterol analytes in mouse fibroblast cell lines SSh-LII and NIH-3T3, and human cancer cell lines, BxPC3, HCT-15 and HCT-116. In BxPC3 cells, the AFFL-SPE-LC-MS/MS method was used to detect significant differences in 24S-OHC levels between vimentin+ and vimentin- heterogenous sub-populations. The methodology also allowed monitoring of significant alterations in 24S-OHC levels upon delivery of the Hedgehog (Hh) antagonist MS-0022 in HCT-116 colorectal carcinoma cell lines.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.