Lysophosphatidylcholine (LPC) is a bioactive lipid present at high concentrations in inflamed and injured tissues where it contributes to the initiation and maintenance of pain. One of its important molecular effectors is the transient receptor potential canonical 5 (TRPC5), but the explicit mechanism of the activation is unknown. Using electrophysiology, mutagenesis and molecular dynamics simulations, we show that LPC-induced activation of TRPC5 is modulated by xanthine ligands and depolarizing voltage, and involves conserved residues within the lateral fenestration of the pore domain. Replacement of W577 with alanine (W577A) rendered the channel insensitive to strong depolarizing voltage, but LPC still activated this mutant at highly depolarizing potentials. Substitution of G606 located directly opposite position 577 with tryptophan rescued the sensitivity of W577A to depolarization. Molecular simulations showed that depolarization widens the lower gate of the channel and this conformational change is prevented by the W577A mutation or removal of resident lipids. We propose a gating scheme in which depolarizing voltage and lipid-pore helix interactions act together to promote TRPC5 channel opening.

• MeSH
• gating iontového kanálu účinky léků   MeSH
• HEK293 buňky MeSH
• kationtové kanály TRPC * metabolismus   genetika   chemie   MeSH
• lidé MeSH
• lysofosfatidylcholiny * metabolismus   farmakologie   MeSH
• lysofosfolipidy metabolismus   farmakologie   MeSH
• membránové potenciály účinky léků   MeSH
• mutace MeSH
• simulace molekulární dynamiky * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Pancreatic cancer has the worst prognosis among all cancers. Cancer screening of body fluids may improve the survival time prognosis of patients, who are often diagnosed too late at an incurable stage. Several studies report the dysregulation of lipid metabolism in tumor cells, suggesting that changes in the blood lipidome may accompany tumor growth. Here we show that the comprehensive mass spectrometric determination of a wide range of serum lipids reveals statistically significant differences between pancreatic cancer patients and healthy controls, as visualized by multivariate data analysis. Three phases of biomarker discovery research (discovery, qualification, and verification) are applied for 830 samples in total, which shows the dysregulation of some very long chain sphingomyelins, ceramides, and (lyso)phosphatidylcholines. The sensitivity and specificity to diagnose pancreatic cancer are over 90%, which outperforms CA 19-9, especially at an early stage, and is comparable to established diagnostic imaging methods. Furthermore, selected lipid species indicate a potential as prognostic biomarkers.

• MeSH
• antigen CA-19-9 krev   MeSH
• ceramidy krev   MeSH
• lidé MeSH
• lipidomika metody   MeSH
• lysofosfatidylcholiny krev   MeSH
• metabolismus lipidů genetika   MeSH
• multivariační analýza MeSH
• nádorové biomarkery krev   genetika   MeSH
• nádory slinivky břišní krev   diagnóza   mortalita   patologie   MeSH
• proporcionální rizikové modely MeSH
• senzitivita a specificita MeSH
• sfingomyeliny krev   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• studie případů a kontrol MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Lipid catabolism and anabolism changes play a role in stemness acquisition by cancer cells, and cancer stem cells (CSCs) are particularly dependent on the activity of the enzymes involved in these processes. Lipidomic changes could play a role in CSCs' ability to cause disease relapse and chemoresistance. The exploration of lipid composition and metabolism changes in CSCs in the context of hepatocellular cancer (HCC) is still incomplete and their lipidomic scenario continues to be elusive. We aimed to evaluate through high-throughput mass spectrometry (MS)-based lipidomics the levels of the members of the six major classes of sphingolipids and phospholipids in two HCC cell lines (HepG2 and Huh-7) silenced for the expression of histone variant macroH2A1 (favoring stemness acquisition), or silenced for the expression of focal adhesion tyrosine kinase (FAK) (hindering aggressiveness and stemness). Transcriptomic changes were evaluated by RNA sequencing as well. We found definite lipidomic and transcriptomic changes in the HCC lines upon knockdown (KD) of macroH2A1 or FAK, in line with the acquisition or loss of stemness features. In particular, macroH2A1 KD increased total sphingomyelin (SM) levels and decreased total lysophosphatidylcholine (LPC) levels, while FAK KD decreased total phosphatidylcholine (PC) levels. In conclusion, in HCC cell lines knocked down for specific signaling/epigenetic processes driving opposite stemness potential, we defined a lipidomic signature that hallmarks hepatic CSCs to be exploited for therapeutic strategies.

• MeSH
• buňky Hep G2 MeSH
• fokální adhezní kinasa 1 antagonisté a inhibitory   nedostatek   genetika   MeSH
• fosfatidylcholiny metabolismus   MeSH
• genový knockdown MeSH
• hepatocelulární karcinom genetika   metabolismus   patologie   MeSH
• histony antagonisté a inhibitory   nedostatek   genetika   MeSH
• lidé MeSH
• lipidomika MeSH
• lysofosfatidylcholiny metabolismus   MeSH
• metabolismus lipidů * genetika   MeSH
• nádorové biomarkery genetika   metabolismus   MeSH
• nádorové buněčné linie MeSH
• nádorové kmenové buňky metabolismus   patologie   MeSH
• nádory jater genetika   metabolismus   patologie   MeSH
• regulace genové exprese u nádorů MeSH
• sekvenování transkriptomu MeSH
• sfingomyeliny metabolismus   MeSH
• stanovení celkové genové exprese MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Ultrahigh-performance supercritical fluid chromatography - mass spectrometry (UHPSFC/MS), ultrahigh-performance liquid chromatography - mass spectrometry (UHPLC/MS), and matrix-assisted laser desorption/ionization (MALDI) - MS techniques were used for the lipidomic characterization of exosomes isolated from human plasma. The high-throughput methods UHPSFC/MS and UHPLC/MS using a silica-based column containing sub-2 μm particles enabled the lipid class separation and the quantitation based on exogenous class internal standards in <7 minute run time. MALDI provided the complementary information on anionic lipid classes, such as sulfatides. The nontargeted analysis of 12 healthy volunteers was performed, and absolute molar concentration of 244 lipids in exosomes and 191 lipids in plasma belonging to 10 lipid classes were quantified. The statistical evaluation of data included principal component analysis, orthogonal partial least square discriminant analysis, S-plots, p-values, T-values, fold changes, false discovery rate, box plots, and correlation plots, which resulted in the information on lipid changes in exosomes in comparison to plasma. The major changes were detected in the composition of triacylglycerols, diacylglycerols, phosphatidylcholines, and lysophosphatidylcholines, whereby sphingomyelins, phosphatidylinositols, and sulfatides showed rather similar profiles in both biological matrices.

• MeSH
• diglyceridy krev   izolace a purifikace   metabolismus   MeSH
• dospělí MeSH
• exozómy chemie   metabolismus   MeSH
• fosfatidylcholiny krev   izolace a purifikace   metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lipidomika metody   MeSH
• lysofosfatidylcholiny krev   izolace a purifikace   metabolismus   MeSH
• metabolismus lipidů * MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• superkritická fluidní chromatografie metody   MeSH
• triglyceridy krev   izolace a purifikace   metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• zdraví dobrovolníci pro lékařské studie MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

To enable the early diagnosis of pancreatic cancer, the search for and definition of reliable biomarkers remain a subject of great interest, with the specificity and sensitivity of the currently used biomarkers being below the required values. We tested a novel diagnostic approach for pancreatic cancer based on the specific molecular signature of blood plasma components. To acquire more detailed structural information, structure-sensitive chiroptical methods (electronic circular dichroism and Raman optical activity) were supplemented by conventional Raman and infrared spectroscopies. The obtained spectra were subsequently processed by linear discriminant analysis yielding high values of specificity and sensitivity. In addition, to monitor not only large biomolecules as potential biomarkers but also those of low molecular weight, we conducted an analysis of blood plasma samples by using metabolomics. The achieved results suggest a panel of promising biomarkers for a reliable detection of pancreatic cancer. Copyright © 2018 Wiley Periodicals, Inc.

• MeSH
• cirkulární dichroismus * metody   MeSH
• diskriminační analýza MeSH
• karnitin analogy a deriváty   krev   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lysofosfatidylcholiny krev   MeSH
• metabolomika * metody   MeSH
• nádorové biomarkery krev   MeSH
• nádory slinivky břišní * krev   MeSH
• pilotní projekty MeSH
• Ramanova spektroskopie * metody   MeSH
• senioři MeSH
• spektroskopie infračervená s Fourierovou transformací * metody   MeSH
• studie případů a kontrol MeSH
• Check Tag
• lidé středního věku MeSH
• lidé MeSH
• senioři MeSH

The comprehensive approach for the lipidomic characterization of human breast cancer and surrounding normal tissues is based on hydrophilic interaction liquid chromatography (HILIC)-electrospray ionization mass spectrometry (ESI-MS) quantitation of polar lipid classes of total lipid extracts followed by multivariate data analysis using unsupervised principal component analysis (PCA) and supervised orthogonal partial least square (OPLS). This analytical methodology is applied for the detailed lipidomic characterization of ten patients with the goal to find the statistically relevant differences between tumor and normal tissues. This strategy is selected for better visualization of differences, because the breast cancer tissue is compared with the surrounding healthy tissue of the same patient, therefore changes in the lipidome are caused predominantly by the tumor growth. A large increase of total concentrations for several lipid classes is observed, including phosphatidylinositols, phosphatidylethanolamines, phosphatidylcholines, and lysophosphatidylcholines. Concentrations of individual lipid species inside the abovementioned classes are also changed, and in some cases, these differences are statistically significant. PCA and OPLS analyses enable a clear differentiation of tumor and normal tissues based on changes of their lipidome. A notable decrease of relative abundances of ether and vinylether (plasmalogen) lipid species is detected for phosphatidylethanolamines, but no difference is apparent for phosphatidylcholines.

• MeSH
• analýza hlavních komponent MeSH
• chromatografie kapalinová metody   MeSH
• fosfatidylcholiny analýza   MeSH
• fosfatidylethanolaminy analýza   MeSH
• fosfatidylinositoly analýza   MeSH
• fosfolipidy analýza   chemie   MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací metody   MeSH
• hydrofobní a hydrofilní interakce MeSH
• lidé MeSH
• lipidy analýza   chemie   MeSH
• lysofosfatidylcholiny analýza   MeSH
• multivariační analýza MeSH
• nádory prsu metabolismus   patologie   MeSH
• referenční hodnoty MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• lysofosfatidylcholiny metabolismus   MeSH
• sodíkové kanály účinky léků   MeSH
• srdce MeSH
• techniky in vitro MeSH
• vápníkové kanály účinky léků   MeSH

• MeSH
• dospělí MeSH
• fosfatidylcholiny MeSH
• fosfolipasy A metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• lysofosfatidylcholiny MeSH
• schizofrenie etiologie   MeSH
• trombocyty MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• přehledy MeSH

• MeSH
• glycerolfosfátdehydrogenasa metabolismus   MeSH
• hnědá tuková tkáň enzymologie   MeSH
• jaterní mitochondrie enzymologie   MeSH
• křečci praví MeSH
• krysa rodu rattus MeSH
• lysofosfatidylcholiny farmakologie   MeSH
• malátdehydrogenasa metabolismus   MeSH
• mitochondrie enzymologie   MeSH
• tuková tkáň enzymologie   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• krysa rodu rattus MeSH
• zvířata MeSH