Brašno - Kruh '15

Proceedings contains 28 original research articles presented at 8th International Congress Flour – Bread ’15 and 10th Croatian Congress of Cereal Technologists Brašno – Kruh ’1

Stanovení oxylipinl v lidské plzamě pomocí UHPLC / ESI-MS na obrácených fázích: Studie karcinomu prsu u žen

The ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC/MS) method was optimized and validated for the determination of oxylipins in human plasma using the targeted approach with selected reaction monitoring (SRM) in the negative-ion electrospray ionization (ESI) mode. Reversed phase UHPLC separation on an octadecylsilica column enabled the analysis of 63 oxylipins including numerous isomeric species within 12-min run time. The method was validated (calibration curve, linearity, limit of detection, limit of quantification, carry-over, precision, accuracy, recovery rate, and matrix effect) and applied to 40 human female plasma samples from breast cancer patients and age-matched healthy volunteers (control). Thirty-six oxylipins were detected in human plasma with concentrations above the limit of detection, and 21 of them were quantified with concentrations above the limit of quantitation. The concentrations determined in healthy controls are in a good agreement with previously reported data on human plasma. Quantitative data were statistically evaluated by multivariate data analysis (MDA) methods including principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA). S-plot and box plots showed that 13-HODE, 9-HODE, 13-HOTrE, 9-HOTrE, and 12-HHTrE were the most upregulated oxylipin species in plasma of breast cancer patients.Metoda ultra-vysokoúčinné kapalné chromatografie ve spojení s hmotnostní spektrometrií (UHPLC / MS) byla optimalizována a validována pro stanovení oxylipinů v lidské plazmě pomocí Selected Reaction Monitoring (SRM) s ionizací elektrosprejem (ESI) v negativním režimu. UHPLC separace na obrácených fázíxćh s využitím oktadecylsilikagelové kolony umožnila analýzu 63 oxylipinů včetně četných izomerních druhů během 12 minut. Metoda byla validována (kalibrační křivka, linearita, mez detekce, limit kvantifikace, přesah, Preciznost, přesnost, rychlost obnovy a matrixový efekt) a aplikována na 40 vzorků lidské ženské plazmy od pacientů s rakovinou prsu a zdravých dobrovolníků (kontrola). V lidské plazmě bylo zjištěno třicet šest oxylipinů s koncentracemi nad mezí detekce a 21 z nich bylo kvantifikováno s koncentracemi nad mezí kvantity. Koncentrace zjištěné při zdravých kontrolách jsou v dobré shodě s dříve uváděnými údaji o lidské plazmě. Kvantitativní údaje byly statisticky vyhodnoceny metodami multivariate data analysis (MDA), včetně principal component analysis (PCA) a orthogonal partial least square discriminant analysis (OPLS-DA). S-plot a box plots ukázaly, že 13-HODE, 9-HODE, 13-HOTrE, 9-HOTrE a 12-HHTrE jsou nejvíce upregulované oxylipinové druhy v plazmě pacientů s rakovinou prsu

Questioning the appropriateness of sieving for processing indoor settled dust samples

Dust is a widely-used matrix for estimating human exposure to chemicals or as a screening tool for the identification of indoor chemicals of concern. As dust sampling became more common in exposure assessment, techniques used in processing soil have been adapted to dust samples, and separation of dust particles by sieving is common practice. However, there are no defined pore sizes, which results in inconsistent or difficult data interpretation and exposure estimates. Moreover, dust consists of more particle types than soil, particularly fibers, which behave differently during the sieving process. In this study, composite samples from seven microenvironments (homes, apartments, kindergartens, schools, public spaces, offices, and cars) were used to investigate the impact of the separation of dust by sieving on the observed chemical distributions. Dust was sieved to four particle size fractions (1-2 mm, 0.5-1 mm, 0.25-0.5 mm, and < 0.25 mm) and each fraction was analyzed for organic carbon content and polycyclic aromatic hydrocarbons (PAHs), and images of dust samples were taken by optical microscope. We identified irregular distributions across size fractions for carbon and PAHs as well as for fibrous particles. Based on the combination of chemical analyses and microscopy, we recommend careful consideration of pre-processing of dust samples to limit bias in dust exposure assessments, and sieving should be used only when necessary

Korelace lipidomického složení buněčných linií a tkání u pacientů s karcinomem prsu s použitím hydrofilní interakční chromatografie ve spojení s hmotnostně spektrometrickou detekcí

The goal of this work is the comparison of differences in the lipidomic compositions of human cell lines derived from normal and cancerous breast tissues, and tumor vs. normal tissues obtained after the surgery of breast cancer patients. Hydrophilic interaction liquid chromatography/electrospray ionization mass spectrometry (HILIC/ESI-MS) using the single internal standard approach and response factors is used for the determination of relative abundances of individual lipid species from five lipid classes in total lipid extracts of cell lines and tissues. The supplementary information on the fatty acyl composition is obtained by gas chromatography/mass spectrometry (GC/MS) of fatty acid methyl esters. Multivariate data analysis (MDA) methods, such as nonsupervised principal component analysis (PCA), hierarchical clustering analysis (HCA) and supervised orthogonal partial least-squares discriminant analysis (OPLS-DA), are used for the visualization of differences between normal and tumor samples and the correlation of similarity between cell lines and tissues either for tumor or normal samples. MDA methods are used for differentiation of sample groups and also for identification of the most up- and downregulated lipids in tumor samples in comparison to normal samples. Observed changes are subsequently generalized and correlated with data from tumor and normal tissues of breast cancer patients. In total, 123 lipid species are identified based on their retention behavior in HILIC and observed ions in ESI mass spectra, and relative abundances are determined. MDA methods are applied for a clear differentiation between tumor and normal samples both for cell lines and tissues. The most upregulated lipids are phospholipids (PL) with a low degree of unsaturation (e.g., 32:1 and 34:1) and also some highly polyunsaturated PL (e.g., 40:6), while the most downregulated lipids are PL containing polyunsaturated fatty acyls (e.g., 20:4), plasmalogens and ether lipids.Cílem této práce je porovnání rozdílů ve složení lipidomu lidských buněčných linií pocházejících z normálních a rakovinných prsních tkání a nádorů získaným po operaci pacientů s karcinomem prsu. K určení lipidomického složení extraktů buněčných linií a tkání byla použita hydrofilní interakční chromatografie spojená s hmotnostní spektrometrií a ionizací elektrosprejem (HILIC / ESI-MS). Pro vizualizaci rozdílů mezi normálními a nádorovými buňkami byly použity metody multivariační analýzy dat (MDA), analýza hlavních komponent (PCA), hierarchická analýza shluků (HCA) a ortogonální diskriminační analýza (OPLS-DA). Metody MDA se používají pro diferenciaci skupin vzorků a také pro identifikaci up-regulovaných a down-regulovaných lipidů v nádorových vzorcích ve srovnání s normálními vzorky. Pozorované změny jsou následně korelovány s daty z nádorových a normálních tkání pacientů s karcinomem prsu. Celkem bylo identifikováno 123 druhů lipidů

Lipidomická charakterizace exosomů izolovaných z lidské plazmy pomocí různých technik hmotnostní spektrometrie

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 mu m 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.Ultravysokoúčinná superkritická kapalinová chromatografie - hmotnostní spektrometrie (UHPSFC / MS), ultravysokoúčinná kapalinová chromatografie - hmotnostní spektrometrie (UHPLC / MS) a laserová desorpce / ionizace (MALDI) - MS byly použity pro lipidomickou charakterizaci exosomů izolovaných z lidské plazmy. Vysokoúčinné metody UHPSFC / MS a UHPLC / MS využívající kolonu na bázi oxidu křemičitého obsahující částice sub-2 um umožnily separaci lipidové třídy a kvantifikaci na základě interních standardů exogenní třídy v době běhu <7 minut. MALDI poskytl doplňující informace o třídách aniontových lipidů, jako jsou sulfatidy. Byla provedena necílená analýza 12 zdravých dobrovolníků a byla kvantifikována absolutní molární koncentrace 244 lipidů v exosomech a 191 lipidů v plazmě patřících do 10 lipidových tříd. Statistické vyhodnocení dat zahrnovalo analýzu hlavních složek, ortogonální částečnou diskriminační analýzu nejmenších čtverců, S-grafy, p-hodnoty, T-hodnoty, násobné změny, míru falešných objevů, krabicové grafy a korelační grafy, které vyústily v informace o lipidech změny v exosomech ve srovnání s plazmou. Hlavní změny byly detekovány ve složení triacylglycerolů, diacylglycerolů, fosfatidylcholinů a lysofosfatidylcholinů, přičemž sfingomyeliny, fosfatidylinositoly a sulfatidy vykazovaly v obou biologických matricích poměrně podobné profil

Lipidomické profily plazmy pacientů s karcinomem ledvin, prsu a prostaty se liší od zdravých kontrol

Early detection of cancer is one of the unmet needs in clinical medicine. Peripheral blood analysis is a preferred method for efficient population screening, because blood collection is well embedded in clinical practice and minimally invasive for patients. Lipids are important biomolecules, and variations in lipid concentrations can reflect pathological disorders. Lipidomic profiling of human plasma by the coupling of ultrahigh-performance supercritical fluid chromatography and mass spectrometry is investigated with the aim to distinguish patients with breast, kidney, and prostate cancers from healthy controls. The mean sensitivity, specificity, and accuracy of the lipid profiling approach were 85%, 95%, and 92% for kidney cancer; 91%, 97%, and 94% for breast cancer; and 87%, 95%, and 92% for prostate cancer. No association of statistical models with tumor stage is observed. The statistically most significant lipid species for the differentiation of cancer types studied are CE 16:0, Cer 42:1, LPC 18:2, PC 36:2, PC 36:3, SM 32:1, and SM 41:1 These seven lipids represent a potential biomarker panel for kidney, breast, and prostate cancer screening, but a further verification step in a prospective study has to be performed to verify clinical utility.Včasná detekce rakoviny je jednou z nenaplněných potřeb klinické medicíny. Periferní analýza krve je preferovanou metodou pro efektivní populační screening, protože odběr krve je dobře zakotven v klinické praxi a je minimálně invazivní pro pacienty. Lipidy jsou důležité biomolekuly a odchylky v koncentracích lipidů mohou odrážet patologické poruchy. Lipidomická profilace lidské plazmy spojením ultra-vysokoúčinné superkritické kapalinové chromatografie a hmotnostní spektrometrie je zkoumána s cílem odlišit pacienty s karcinomem prsu, ledvin a prostaty od zdravých kontrol. Průměrná citlivost, specifičnost a přesnost při profilování lipidů byla 85 %, 95 % a 92 % u rakoviny ledvin; 91 %, 97 %, a 94 % pro rakovinu prsu; a 87 %, 95 % a 92 % pro rakovinu prostaty. Není pozorována žádná souvislost statistických modelů se stádiem nádoru. Statisticky nejvýznamnějšími druhy lipidů pro diferenciaci zkoumaných typů nádorů jsou CE 16:0, Cer 42:1, LPC 18:2, PC 36:2, PC 36:3, SM 32:1 a SM 41:1. Těchto sedm lipidů představuje potenciální biomarkerový panel pro screening rakoviny ledvin, prsu a prostaty, ale k ověření klinické užitečnosti musí být proveden další ověřovací krok v prospektivní studii

Novel PD-L1- and collagen-expressing patient-derived cell line of undifferentiated pleomorphic sarcoma (JBT19) as a model for cancer immunotherapy

Abstract Soft tissue sarcomas are aggressive mesenchymal-origin malignancies. Undifferentiated pleomorphic sarcoma (UPS) belongs to the aggressive, high-grade, and least characterized sarcoma subtype, affecting multiple tissues and metastasizing to many organs. The treatment of localized UPS includes surgery in combination with radiation therapy. Metastatic forms are treated with chemotherapy. Immunotherapy is a promising treatment modality for many cancers. However, the development of immunotherapy for UPS is limited due to its heterogeneity, antigenic landscape variation, lower infiltration with immune cells, and a limited number of established patient-derived UPS cell lines for preclinical research. In this study, we established and characterized a novel patient-derived UPS cell line, JBT19. The JBT19 cells express PD-L1 and collagen, a ligand of the immune checkpoint molecule LAIR-1. JBT19 cells can form spheroids in vitro and solid tumors in immunodeficient nude mice. We found JBT19 cells induce expansion of JBT19-reactive autologous and allogeneic NK, T, and NKT-like cells, and the reactivity of the expanded cells was associated with cytotoxic impact on JBT19 cells. The PD-1 and LAIR-1 ligand-expressing JBT19 cells show ex vivo immunogenicity and effective in vivo xenoengraftment properties that can offer a unique resource in the preclinical research developing novel immunotherapeutic interventions in the treatment of UPS

Farmers' perception of soil health : The use of quality data and its implication for farm management

Preventing and reversing soil degradation is essential to maintaining the ecosystem services provided by soils and guaranteeing food security. In addition to the scientific community, it is critical to engage multiple stakeholders to assess the degree of soil degradation and mitigation strategies' impact and meet the United Nations' Sustainable Development Goals, European Union's Common Agricultural Policy, and other national and international goals. A semi‐structured questionnaire was distributed across countries participating in the EU Horizon‐2020 “Transforming Unsustainable management of soils in key agricultural systems in E.U. and China. Developing an integrated platform of alternatives to reverse soil degradation (TUdi).” Using farmers' associations and educational institutions as an intermediate to distribute the questionnaires was an effective strategy for gathering a high number of responses. Results from 456 responses to the questionnaire showed that farm country, size, type of agriculture, and educational level of farm managers were significantly associated with the farmers' perception of soil degradation issues. Farm size and type of agriculture were also correlated with applying a nutrient management plan. The implications of the results for soil conservation measures are discussed. Additionally, we highlight the potential of projects such as TUdi for creating collaboration networks to drive widespread adoption by farmers of technologies to reverse the degradation of agricultural soils