BACKGROUND: RT-qPCR quantification of miRNAs expression may play an essential role in pancreatic ductal adenocarcinoma (PDAC) diagnostics. RT-qPCR-based experiments require endogenous controls for the result normalization and reliability. However, expression instability of reference genes in tumors may introduce bias when determining miRNA levels. METHODS: We investigated expression of 6 miRNAs, isolated from FFPE samples of pancreatic adenocarcinomas. Four internal controls were utilized for RT-qPCR result normalization: artificial miR-39 from C. elegans, U6 snRNA, miR-16 and snoRNA U91. RESULTS: We found miR-21, miR-155 or miR-217 expression values in tumors may differ up to several times, depending on selected internal controls. Moreover, different internal controls can produce controversial results for miR-96, miR-148a or miR-196a quantification. Also, expression of our endogenous controls varied significantly in tumors. U6 demonstrated variation from -1.03 to 8.12-fold, miR-16 from -2.94 up to 7.38-fold and the U91 from -3.05 to 4.36-fold respectively. On the other hand, the most stable gene, determined by NormFinder algorithm, was U91. Each miRNA normalized relatively to the spike or U91, demonstrated similar expression values. Thus, statistically significant and insignificant differences between tumors and normal tissues for miRNAs were equal for the spike and the U91. Also, the differences between the spike and U91 were statistically insignificant for all of miRs except miR-217. Among three endogenous controls, U91 had the lowest average expression values and standard deviation in cancer tissues. CONCLUSIONS: We recommend U91 as a new normalizer for miRNA quantification in PDACs.

• MeSH
• adenokarcinom genetika   MeSH
• analýza rozptylu MeSH
• dospělí MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• lidé středního věku MeSH
• lidé MeSH
• malá jadérková RNA metabolismus   MeSH
• mikro RNA analýza   metabolismus   MeSH
• nádorové biomarkery genetika   MeSH
• nádory slinivky břišní genetika   MeSH
• regulace genové exprese u nádorů MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• stanovení celkové genové exprese MeSH
• studie případů a kontrol MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

PURPOSE: While the recommended analysis method for magnetic resonance spectroscopy data is linear combination model (LCM) fitting, the supervised deep learning (DL) approach for quantification of MR spectroscopy (MRS) and MR spectroscopic imaging (MRSI) data recently showed encouraging results; however, supervised learning requires ground truth fitted spectra, which is not practical. Moreover, this work investigates the feasibility and efficiency of the LCM-based self-supervised DL method for the analysis of MRS data. METHOD: We present a novel DL-based method for the quantification of relative metabolite concentrations, using quantum-mechanics simulated metabolite responses and neural networks. We trained, validated, and evaluated the proposed networks with simulated and publicly accessible in-vivo human brain MRS data and compared the performance with traditional methods. A novel adaptive macromolecule fitting algorithm is included. We investigated the performance of the proposed methods in a Monte Carlo (MC) study. RESULT: The validation using low-SNR simulated data demonstrated that the proposed methods could perform quantification comparably to other methods. The applicability of the proposed method for the quantification of in-vivo MRS data was demonstrated. Our proposed networks have the potential to reduce computation time significantly. CONCLUSION: The proposed model-constrained deep neural networks trained in a self-supervised manner can offer fast and efficient quantification of MRS and MRSI data. Our proposed method has the potential to facilitate clinical practice by enabling faster processing of large datasets such as high-resolution MRSI datasets, which may have thousands of spectra.

• MeSH
• deep learning * MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• magnetická rezonanční tomografie metody   MeSH
• mozek diagnostické zobrazování   metabolismus   MeSH
• neuronové sítě MeSH
• počítačové zpracování obrazu metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The concept of personalized medicine includes novel protein biomarkers that are expected to improve the early detection, diagnosis and therapy monitoring of malignant diseases. Tissues, biofluids, cell lines and xenograft models are the common sources of biomarker candidates that require verification of clinical value in independent patient cohorts. Targeted proteomics - based on selected reaction monitoring, or data extraction from data-independent acquisition based digital maps - now represents a promising mass spectrometry alternative to immunochemical methods. To date, it has been successfully used in a high number of studies answering clinical questions on solid malignancies: breast, colorectal, prostate, ovarian, endometrial, pancreatic, hepatocellular, lung, bladder and others. It plays an important role in functional proteomic experiments that include studying the role of post-translational modifications in cancer progression. This review summarizes verified biomarker candidates successfully quantified by targeted proteomics in this field and directs the readers who plan to design their own hypothesis-driven experiments to appropriate sources of methods and knowledge.

• MeSH
• časná detekce nádoru metody   MeSH
• hmotnostní spektrometrie metody   MeSH
• lidé MeSH
• nádorové biomarkery chemie   metabolismus   MeSH
• proteom chemie   metabolismus   MeSH
• proteomika metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

A new CZE method was developed for the determination of 12 purine and pyrimidine nucleotides, two adenine coenzymes and their reduced forms, and acetyl coenzyme A in various cell extracts. As the concentration levels of these metabolites in living cells are low; CZE was combined with field-enhanced sample stacking. As a result, the separation conditions were optimised to achieve a suitable resolution at the relatively high sample volume provided by this on-line pre-concentration technique. The optimum BGE was 150 mM glycine buffer (pH 9.5). Samples were introduced hydrodynamically using a pressure of 35 mbar (3.5 kPa) for 25 s, and data were collected at a detection wavelength of 260 nm. An applied voltage of 30 kV (positive polarity) and capillary temperature of 25°C gave the best separation of these compounds. The optimised method was validated by determining the linearity, sensitivity and repeatability and it was successfully applied for the analysis of extracts from Paracoccus denitrificans bacteria and from stem cells.

• MeSH
• acetylkoenzym A analýza   MeSH
• adenosintrifosfát analýza   MeSH
• chemické techniky analytické metody   normy   MeSH
• cytidintrifosfát analýza   MeSH
• embryonální kmenové buňky chemie   MeSH
• guanosintrifosfát analýza   MeSH
• lidé MeSH
• limita detekce MeSH
• Paracoccus denitrificans chemie   MeSH
• reprodukovatelnost výsledků MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Hippocampal dysfunction is known to be associated with several neurological and neuropsychiatric disorders such as Alzheimer's disease, epilepsy, schizophrenia and depression; therefore, there has been significant clinical interest in studying hippocampal neurochemistry. However, the hippocampus is a challenging region to study using (1) H MRS, hence the use of MRS for clinical research in this region has been limited. Our goal was therefore to investigate the feasibility of obtaining high-quality hippocampal spectra that allow reliable quantification of a neurochemical profile and to establish inter-session reproducibility of hippocampal MRS, including reproducibility of voxel placement, spectral quality and neurochemical concentrations. Ten healthy volunteers were scanned in two consecutive sessions using a standard clinical 3 T MR scanner. Neurochemical profiles were obtained with a short-echo (T(E) = 28 ms) semi-LASER localization sequence from a relatively small (~4 mL) voxel that covered about 62% of the hippocampal volume as calculated from segmentation of T1 -weighted images. Voxel composition was highly reproducible between sessions, with test-retest coefficients of variation (CVs) of 3.5% and 7.5% for gray and white matter volume fraction, respectively. Excellent signal-to-noise ratio (~54 based on the N-acetylaspartate (NAA) methyl peak in non-apodized spectra) and linewidths (~9 Hz for water) were achieved reproducibly in all subjects. The spectral quality allowed quantification of NAA, total choline, total creatine, myo-inositol and glutamate with high scan-rescan reproducibility (CV ≤ 6%) and quantification precision (Cramér-Rao lower bound, CRLB < 9%). Four other metabolites, including glutathione and glucose, were quantified with scan-rescan CV below 20%. Therefore, the highly optimized, short-echo semi-LASER sequence together with FASTMAP shimming substantially improved the reproducibility and number of quantifiable metabolites relative to prior reports. In addition, the between-session variation in metabolite concentrations, as well as CRLB, was lower than the between-subject variation of the concentrations for most metabolites, indicating that the method has the sensitivity to detect inter-individual differences in the healthy brain.

• MeSH
• algoritmy MeSH
• biopolymery metabolismus   MeSH
• dospělí MeSH
• hipokampus anatomie a histologie   metabolismus   MeSH
• lidé MeSH
• molekulární zobrazování metody   MeSH
• protonová magnetická rezonanční spektroskopie metody   MeSH
• reprodukovatelnost výsledků MeSH
• senzitivita a specificita MeSH
• studie proveditelnosti MeSH
• tkáňová distribuce MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

BACKGROUND: Different types of analytical methods, with different characteristics, are applied in metabolomics and lipidomics research and include untargeted, targeted and semi-targeted methods. Ultra High Performance Liquid Chromatography-Mass Spectrometry is one of the most frequently applied measurement instruments in metabolomics because of its ability to detect a large number of water-soluble and lipid metabolites over a wide range of concentrations in short analysis times. Methods applied for the detection and quantification of metabolites differ and can either report a (normalised) peak area or an absolute concentration. AIM OF REVIEW: In this tutorial we aim to (1) define similarities and differences between different analytical approaches applied in metabolomics and (2) define how amounts or absolute concentrations of endogenous metabolites can be determined together with the advantages and limitations of each approach in relation to the accuracy and precision when concentrations are reported. KEY SCIENTIFIC CONCEPTS OF REVIEW: The pre-analysis knowledge of metabolites to be targeted, the requirement for (normalised) peak responses or absolute concentrations to be reported and the number of metabolites to be reported define whether an untargeted, targeted or semi-targeted method is applied. Fully untargeted methods can only provide (normalised) peak responses and fold changes which can be reported even when the structural identity of the metabolite is not known. Targeted methods, where the analytes are known prior to the analysis, can also report fold changes. Semi-targeted methods apply a mix of characteristics of both untargeted and targeted assays. For the reporting of absolute concentrations of metabolites, the analytes are not only predefined but optimized analytical methods should be developed and validated for each analyte so that the accuracy and precision of concentration data collected for biological samples can be reported as fit for purpose and be reviewed by the scientific community.

• MeSH
• hmotnostní spektrometrie * metody   MeSH
• lidé MeSH
• metabolomika * metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Liquid-liquid extraction methods are widely used for sample treatment in bioanalysis, although their implementation poses a common speed-limiting step in the analytical process when fast separation and detection methods such as UHPLC-MS are used. This study aimed to develop high-throughput salting-out assisted liquid-liquid extraction on a 96-well plate in combination with fast LC-MS analysis of ibrutinib and its active metabolite PCI-45227 (dihydrodiol ibrutinib) in human serum. A specially designed 3D printed extraction device developed in our laboratory allowed for the precise and rapid collection of the organic phase from the 96-well plate using a multichannel pipette, without the risk of aspiration of the bottom aqueous layer. The application of this device significantly accelerated sample preparation and allowed the processing of up to 96 samples in 1 h. The method was successfully validated according to EMA guidelines in the concentration range of 0.1-200 ng/mL for both analytes, providing lower limit of quantification at 100 pg/mL. The intra-day accuracy for IBT was in the range of - 1.67-5.67 %, while the inter-day accuracy was in the range of 0.20-6.90 %. The intra-day precision for IBT was in the range of 3.20-4.37 % and 3.13-4.73 % for the inter-day measurement. PCI-45227 showed intra-day accuracy in the range of - 11.2-3.71 % and the inter-day accuracy in the range of - 5.76-0.92 %. The intra-day precision for PCI was in the range of 3.49-7.64 % and 3.63-8.61 % for the measurement between days. In addition to increasing the speed of sample preparation, this method also offers low consumption of the sample and extraction solvent and can be utilized for other similar small-volume in-well plate extractions where organic solvents of lower density than water are used. The method was successfully applied to the analysis of serum samples (n = 5) of patients with chronic lymphoblastic leukaemia at the trough level (ibrutinib concentration range: 1.63-3.78 ng/mL, PCI-45227 concentration range: 1.84-14.02 ng/mL) and 2 h postdose (ibrutinib concentration range: 7.34-89.0 ng/mL, PCI-45227 concentration range: 5.64-124 ng/mL).

• MeSH
• 3D tisk MeSH
• adenin analogy a deriváty   MeSH
• extrakce kapalina-kapalina metody   MeSH
• koronární angioplastika * MeSH
• lidé MeSH
• piperidiny MeSH
• pyrazoly MeSH
• rozpouštědla MeSH
• tandemová hmotnostní spektrometrie * metody   MeSH
• vysokoúčinná kapalinová chromatografie metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

A simple, cost effective, and fast gas chromatography method with mass spectrometry detection (GC-MS) for simultaneous measurement of formic acid, glycolic acid, methoxyacetic acid, ethoxyacetic acid and 2-hydroxyethoxyacetic acid in serum and urine was developed and validated. This multi-analyte method is highly suitable for clinical and emergency toxicology laboratory diagnostic, allowing identification and quantification of five most common acidosis inducing organic acids present in cases of alcohol intoxication. Furthermore, when patients are admitted to emergency unit at late stage of toxic alcohol intoxication, the concentration of parent compound may be already low or not detectable. This new method employs a relatively less used class of derivatization agents - alkyl chloroformates, allowing the efficient and rapid derivatization of carboxylic acids within seconds. The entire sample preparation procedure is completed within 5 min. The optimal conditions of derivatization procedure have been found using chemometric approach (design of experiment). The calibration dependence of the method was proved to be quadratic in the range of 25-3000 mg L(-1), with adequate accuracy (97.3-108.0%) and precision (<12.8%). The method was successfully applied for identification and quantification of the selected compounds in serum of patients from emergency units.

• MeSH
• acetáty krev   moč   MeSH
• acidóza diagnóza   MeSH
• analýza moči metody   MeSH
• biochemická analýza krve metody   MeSH
• chemické techniky analytické MeSH
• formiáty krev   moč   MeSH
• glykoláty krev   moč   MeSH
• kalibrace MeSH
• lidé MeSH
• limita detekce MeSH
• otrava alkoholem krev   moč   MeSH
• plynová chromatografie s hmotnostně spektrometrickou detekcí metody   MeSH
• reprodukovatelnost výsledků MeSH
• toxikologie metody   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Nuclear Magnetic Resonance (NMR) is an analytical technique extensively used in almost every chemical laboratory for structural identification. This technique provides statistically equivalent signals in spite of using spectrometer with different hardware features and is successfully used for the traceability and quantification of analytes in food samples. Nevertheless, to date only a few internationally agreed guidelines have been reported on the use of NMR for quantitative analysis. The main goal of the present study is to provide a methodological pipeline to assess the reproducibility of NMR data produced for a given matrix by spectrometers from different manufacturers, with different magnetic field strengths, age and hardware configurations. The results have been analyzed through a sequence of chemometric tests to generate a community-built calibration system which was used to verify the performance of the spectrometers and the reproducibility of the predicted sample concentrations.

• MeSH
• kalibrace MeSH
• magnetická rezonanční spektroskopie MeSH
• ovocné a zeleninové šťávy analýza   MeSH
• Vitis chemie   MeSH
• Publikační typ
• časopisecké články MeSH

Mass spectrometry raw data repositories, including Metabolomics Workbench and MetaboLights, have contributed to increased transparency in metabolomics studies and the discovery of novel insights in biology by reanalysis with updated computational metabolomics tools. Herein, we reanalyzed the previously published lipidomics data from nine algal species, resulting in the annotation of 1437 lipids achieving a 40% increase in annotation compared to the previous results. Specifically, diacylglyceryl-carboxyhydroxy-methylcholine (DGCC) in Pavlova lutheri and Pleurochrysis carterae, glucuronosyldiacylglycerol (GlcADG) in Euglena gracilis, and P. carterae, phosphatidylmethanol (PMeOH) in E. gracilis, and several oxidized phospholipids (oxidized phosphatidylcholine, OxPC; phosphatidylethanolamine, OxPE; phosphatidylglycerol, OxPG; phosphatidylinositol, OxPI) in Chlorella variabilis were newly characterized with the enriched lipid spectral databases. Moreover, we integrated the data from untargeted and targeted analyses from data independent tandem mass spectrometry (DIA-MS/MS) acquisition, specifically the sequential window acquisition of all theoretical fragment-ion MS/MS (SWATH-MS/MS) spectra, to increase the lipidomic annotation coverage. After the creation of a global library of precursor and diagnostic ions of lipids by the MS-DIAL untargeted analysis, the co-eluted DIA-MS/MS spectra were resolved in MRMPROBS targeted analysis by tracing the specific product ions involved in acyl chain compositions. Our results indicated that the metabolite quantifications based on DIA-MS/MS chromatograms were somewhat inferior to the MS1-centric quantifications, while the annotation coverage outperformed those of the untargeted analysis of the data dependent and DIA-MS/MS data. Consequently, integrated analyses of untargeted and targeted approaches are necessary to extract the maximum amount of metabolome information, and our results showcase the value of data repositories for the discovery of novel insights in lipid biology.

• Publikační typ
• časopisecké články MeSH