BACKGROUND: The Human Cell Differentiation Molecules (HCDM) organizes Human Leukocyte Differentiation Antigen (HLDA) workshops to test and name clusters of antibodies that react with a specific antigen. These cluster of differentiation (CD) markers have provided the scientific community with validated antibody clones, consistent naming of targets and reproducible identification of leukocyte subsets. Still, quantitative CD marker expression profiles and benchmarking of reagents at the single-cell level are currently lacking. OBJECTIVE: To develop a flow cytometric procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets that is standardized across multiple research laboratories. METHODS: A high content framework to evaluate the titration and reactivity of Phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) was created. Two flow cytometry panels were designed: an innate cell tube for granulocytes, dendritic cells, monocytes, NK cells and innate lymphoid cells (12-color) and an adaptive lymphocyte tube for naive and memory B and T cells, including TCRγδ+, regulatory-T and follicular helper T cells (11-color). The potential of these 2 panels was demonstrated via expression profiling of selected CD markers detected by PE-conjugated antibodies and evaluated using 561 nm excitation. RESULTS: Using automated data annotation and dried backbone reagents, we reached a robust workflow amenable to processing hundreds of measurements in each experiment in a 96-well plate format. The immunophenotyping panels enabled discrimination of 27 leukocyte subsets and quantitative detection of the expression of PE-conjugated CD markers of interest that could quantify protein expression above 400 units of antibody binding capacity. Expression profiling of 4 selected CD markers (CD11b, CD31, CD38, CD40) showed high reproducibility across centers, as well as the capacity to benchmark unique clones directed toward the same CD3 antigen. CONCLUSION: We optimized a procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets. The workflow, bioinformatics pipeline and optimized flow panels enable the following: 1) mapping the expression patterns of HLDA-approved mAb clones to CD markers; 2) benchmarking new antibody clones to established CD markers; 3) defining new clusters of differentiation in future HLDA workshops.

• Klíčová slova
• CD marker, cluster of differentiation (CD), expression profiling, flow cytometry, surfaceome,
• MeSH
• antigeny povrchové * metabolismus   MeSH
• buňky NK metabolismus   MeSH
• CD antigeny metabolismus   MeSH
• leukocyty MeSH
• lidé MeSH
• monoklonální protilátky MeSH
• přirozená imunita * MeSH
• průběh práce MeSH
• průtoková cytometrie metody   MeSH
• referenční standardy MeSH
• reprodukovatelnost výsledků MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny povrchové * MeSH
• CD antigeny MeSH
• monoklonální protilátky MeSH

BACKGROUND: Yeast infections are often connected with formation of biofilms that are extremely difficult to eradicate. An excellent model system for deciphering multifactorial determinants of yeast biofilm development is the colony biofilm, composed of surface ("aerial") and invasive ("root") cells. While surface cells have been partially analyzed before, we know little about invasive root cells. In particular, information on the metabolic, chemical and morphogenetic properties of invasive versus surface cells is lacking. In this study, we used a new strategy to isolate invasive cells from agar and extracellular matrix, and employed it to perform genome wide expression profiling and biochemical analyses of surface and invasive cells. RESULTS: RNA sequencing revealed expression differences in 1245 genes with high statistical significance, indicating large genetically regulated metabolic differences between surface and invasive cells. Functional annotation analyses implicated genes involved in stress defense, peroxisomal fatty acid β-oxidation, autophagy, protein degradation, storage compound metabolism and meiosis as being important in surface cells. In contrast, numerous genes with functions in nutrient transport and diverse synthetic metabolic reactions, including genes involved in ribosome biogenesis, biosynthesis and translation, were found to be important in invasive cells. Variation in gene expression correlated significantly with cell-type specific processes such as autophagy and storage compound accumulation as identified by microscopic and biochemical analyses. Expression profiling also provided indications of cell-specific regulations. Subsequent knockout strain analyses identified Gip2p, a regulatory subunit of type 1 protein phosphatase Glc7p, to be essential for glycogen accumulation in surface cells. CONCLUSIONS: This is the first study reporting genome wide differences between surface and invasive cells of yeast colony biofilms. New findings show that surface and invasive cells display very different physiology, adapting to different conditions in different colony areas and contributing to development and survival of the colony biofilm as a whole. Notably, surface and invasive cells of colony biofilms differ significantly from upper and lower cells of smooth colonies adapted to plentiful laboratory conditions.

• Klíčová slova
• Cell differentiation, Colony biofilms, Invasive cell subpopulation, Regulation of glycogen metabolism, Saccharomyces cerevisiae, Transcriptomics,
• MeSH
• biofilmy * MeSH
• metabolické sítě a dráhy MeSH
• regulace genové exprese u hub * MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   fyziologie   MeSH
• stanovení celkové genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Saccharomyces cerevisiae - proteiny MeSH

Glycosylated sphingolipids (GSLs) are a diverse group of cellular lipids typically reported as being rare in normal mammary tissue. In breast cancer (BCa), GSLs have emerged as noteworthy markers associated with breast cancer stem cells, mediators of phenotypic plasticity, and contributors to cancer cell chemoresistance. GSLs are potential surface markers that can uniquely characterize the heterogeneity of the tumor microenvironment, including cancer cell subpopulations and epithelial-mesenchymal plasticity (EMP). In this study, mass spectrometry analyses of the total sphingolipidome in breast epithelial cells and their mesenchymal counterparts revealed increased levels of Gb3 in epithelial cells and significantly elevated GD2 levels in the mesenchymal phenotype. To elucidate if GSL-related epitopes on BCa cell surfaces reflect EMP and cancer status, we developed and rigorously validated a 12-color spectral flow cytometry panel. This panel enables the simultaneous detection of native GSL epitopes (Gb3, SSEA1, SSEA3, SSEA4, and GD2), epithelial-mesenchymal transition markers (EpCAM, TROP2, and CD9), and lineage markers (CD45, CD31, and CD90) at the single-cell level. Next, the established panel was used for the analysis of BCa primary tumors and revealed surface heterogeneity in SSEA1, SSEA3, SSEA4, GD2, and Gb3, indicative of native epitope presence also on non-tumor cells. These findings further highlighted the phenotype-dependent alterations in GSL surface profiles, with differences between epithelial and stromal cells in the tumor. This study provides novel insights into BCa heterogeneity, shedding light on the potential of native GSL-related epitopes as markers for EMP and cancer status in fresh clinical samples. The developed single-cell approach offers promising avenues for further exploration.

• Klíčová slova
• breast cancer, epithelial cells, glycosphingolipids, phenotypic plasticity, stromal-like cells, surface profiling,
• MeSH
• analýza jednotlivých buněk * metody   MeSH
• epitelo-mezenchymální tranzice * MeSH
• fenotyp MeSH
• glykosfingolipidy * metabolismus   analýza   MeSH
• lidé MeSH
• nádory prsu * metabolismus   patologie   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• glykosfingolipidy * MeSH

Sandflies (Diptera: Psychodidae) (Newstead, 1911) are blood-feeding insects that transmit human pathogens including Leishmania (Trypanosomatida: Trypanosomatidae) parasites, causative agents of the leishmaniases. To elucidate Leishmania transmission cycles, conclusive identification of vector species is essential. Molecular approaches including matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) protein profiling have recently emerged to complement morphological identification. The aim of this study was to evaluate the effect of the trap type used to collect sandflies, specifically Centers for Disease Control (CDC) light or sticky traps, the two most commonly used in sandfly surveys, on subsequent MALDI-TOF MS protein profiling. Specimens of five species (Phlebotomus ariasi, Phlebotomus papatasi, Phlebotomus perniciosus, Phlebotomus sergenti, Sergentomyia minuta) collected in periurban and agricultural habitats in southeast Spain were subjected to protein profiling. Acquired protein spectra were queried against an in-house reference database and their quality assessed to evaluate the trap type effect. The results indicate that trap choice can substantially affect the quality of protein spectra in collected sandflies. Whereas specimens retrieved from light traps produced intense and reproducible spectra that allowed reliable species determination, profiles of specimens from sticky traps were compromised and often did not enable correct identification. Sticky traps should therefore not be used in surveys that deploy MALDI-TOF MS protein profiling for species identification.

• Klíčová slova
• MALDI-TOF MS protein profiling, Phlebotomus, light traps, species identification, sticky traps, trapping methods,
• MeSH
• odběr biologického vzorku metody   MeSH
• Psychodidae klasifikace   genetika   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice * MeSH
• stanovení celkové genové exprese * MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Španělsko MeSH

CD molecules are surface molecules expressed on cells of the immune system that play key roles in immune cell-cell communication and sensing the microenvironment. These molecules are essential markers for the identification and isolation of leukocytes and lymphocyte subsets. Here, we present the results of the first phase of the CD Maps study, mapping the expression of CD1-CD100 (n = 110) on 47 immune cell subsets from blood, thymus, and tonsil using an eight-color standardized EuroFlow approach and quantification of expression. The resulting dataset included median antibody binding capacities (ABCs) and percentage of positivity for all markers on all subsets and was developed into an interactive CD Maps web resource. Using the resource, we examined differentially expressed proteins between granulocyte, monocyte, and dendritic cell subsets, and profiled dynamic expression of markers during thymocyte differentiation, T-cell maturation, and between functionally distinct B-cell subset clusters. The CD Maps resource will serve as a benchmark of antibody reactivities ensuring improved reproducibility of flow cytometry-based research. Moreover, it will provide a full picture of the surfaceome of human immune cells and serves as a useful platform to increase our understanding of leukocyte biology, as well as to facilitate the identification of new biomarkers and therapeutic targets of immunological and hematological diseases.

• Klíčová slova
• B-cell, CD marker, T-cell, expression profiling, flow cytometry, lymphocyte, monocyte, surfaceome,
• MeSH
• B-lymfocyty imunologie   metabolismus   MeSH
• CD antigeny biosyntéza   MeSH
• datové soubory jako téma MeSH
• dendritické buňky imunologie   metabolismus   MeSH
• dítě MeSH
• dospělí MeSH
• granulocyty imunologie   metabolismus   MeSH
• imunofenotypizace MeSH
• internet MeSH
• leukocyty imunologie   metabolismus   MeSH
• lidé MeSH
• lymfopoéza MeSH
• monocyty imunologie   metabolismus   MeSH
• peptidové mapování MeSH
• podskupiny lymfocytů imunologie   metabolismus   MeSH
• předškolní dítě MeSH
• průtoková cytometrie MeSH
• reprodukovatelnost výsledků MeSH
• separace buněk MeSH
• T-lymfocyty imunologie   metabolismus   MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé MeSH
• předškolní dítě MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• CD antigeny MeSH

INTRODUCTION: Microarray-based gene expression profiling represents a major breakthrough for understanding the molecular complexity of breast cancer. cDNA expression profiles cannot detect changes in activities that arise from post-translational modifications, however, and therefore do not provide a complete picture of all biologically important changes that occur in tumors. Additional opportunities to identify and/or validate molecular signatures of breast carcinomas are provided by proteomic approaches. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) offers high-throughput protein profiling, leading to extraction of protein array data, calling for effective and appropriate use of bioinformatics and statistical tools. METHODS: Whole tissue lysates of 105 breast carcinomas were analyzed on IMAC 30 ProteinChip Arrays (Bio-Rad, Hercules, CA, USA) using the ProteinChip Reader Model PBS IIc (Bio-Rad) and Ciphergen ProteinChip software (Bio-Rad, Hercules, CA, USA). Cluster analysis of protein spectra was performed to identify protein patterns potentially related to established clinicopathological variables and/or tumor markers. RESULTS: Unsupervised hierarchical clustering of 130 peaks detected in spectra from breast cancer tissue lysates provided six clusters of peaks and five groups of patients differing significantly in tumor type, nuclear grade, presence of hormonal receptors, mucin 1 and cytokeratin 5/6 or cytokeratin 14. These tumor groups resembled closely luminal types A and B, basal and HER2-like carcinomas. CONCLUSION: Our results show similar clustering of tumors to those provided by cDNA expression profiles of breast carcinomas. This fact testifies the validity of the SELDI-TOF MS proteomic approach in such a type of study. As SELDI-TOF MS provides different information from cDNA expression profiles, the results suggest the technique's potential to supplement and expand our knowledge of breast cancer, to identify novel biomarkers and to produce clinically useful classifications of breast carcinomas.

• MeSH
• biologické modely MeSH
• čipová analýza proteinů metody   MeSH
• diagnostické techniky molekulární MeSH
• komplementární DNA metabolismus   MeSH
• lidé MeSH
• nádorové biomarkery MeSH
• nádory prsu genetika   metabolismus   MeSH
• posttranslační úpravy proteinů MeSH
• proteomika metody   MeSH
• regulace genové exprese * MeSH
• shluková analýza MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• stanovení celkové genové exprese MeSH
• výpočetní biologie metody   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• komplementární DNA MeSH
• nádorové biomarkery MeSH

Multicolor flow cytometry allows for analysis of tens of cellular parameters in millions of cells at a single-cell resolution within minutes. The lack of technologies that would facilitate feasible and relatively cheap profiling of such a number of cells with an antibody-based approach led us to the development of a high-throughput cytometry-based platform for surface profiling. We coupled the fluorescent cell barcoding with preexisting, commercially available screening tools to analyze cell surface fingerprint at a large scale. This powerful approach will help to identify novel biomarkers and druggable targets and facilitate the discovery of new concepts in immunology, oncology, and developmental biology.

• Klíčová slova
• Cell surface phenotyping, Fluorescent cell barcoding, High-throughput screening, Multicolor flow cytometry,
• MeSH
• antigeny povrchové * MeSH
• biologické markery analýza   MeSH
• fluorescenční barviva MeSH
• průtoková cytometrie MeSH
• výzkum * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny povrchové * MeSH
• biologické markery MeSH
• fluorescenční barviva MeSH

Human cytomegalovirus (HCMV) US2, US3, US6 and US11 act in concert to prevent immune recognition of virally infected cells by CD8+ T-lymphocytes through downregulation of MHC class I molecules (MHC-I). Here we show that US2 function goes far beyond MHC-I degradation. A systematic proteomic study using Plasma Membrane Profiling revealed US2 was unique in downregulating additional cellular targets, including: five distinct integrin α-chains, CD112, the interleukin-12 receptor, PTPRJ and thrombomodulin. US2 recruited the cellular E3 ligase TRC8 to direct the proteasomal degradation of all its targets, reminiscent of its degradation of MHC-I. Whereas integrin α-chains were selectively degraded, their integrin β1 binding partner accumulated in the ER. Consequently integrin signaling, cell adhesion and migration were strongly suppressed. US2 was necessary and sufficient for degradation of the majority of its substrates, but remarkably, the HCMV NK cell evasion function UL141 requisitioned US2 to enhance downregulation of the NK cell ligand CD112. UL141 retained CD112 in the ER from where US2 promoted its TRC8-dependent retrotranslocation and degradation. These findings redefine US2 as a multifunctional degradation hub which, through recruitment of the cellular E3 ligase TRC8, modulates diverse immune pathways involved in antigen presentation, NK cell activation, migration and coagulation; and highlight US2's impact on HCMV pathogenesis.

• MeSH
• aktivace lymfocytů imunologie   MeSH
• buněčná membrána metabolismus   MeSH
• buňky NK imunologie   MeSH
• Cytomegalovirus imunologie   MeSH
• hmotnostní spektrometrie MeSH
• imunitní únik imunologie   MeSH
• imunoblotting MeSH
• imunoprecipitace MeSH
• lidé MeSH
• malá interferující RNA MeSH
• membránové glykoproteiny metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• nádorové buněčné linie MeSH
• proteiny virového obalu metabolismus   MeSH
• proteomika metody   MeSH
• průtoková cytometrie MeSH
• transdukce genetická MeSH
• virové proteiny metabolismus   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• malá interferující RNA MeSH
• membránové glykoproteiny MeSH
• membránové proteiny MeSH
• proteiny virového obalu MeSH
• UL141 glycoprotein, human cytomegalovirus MeSH Prohlížeč
• US2 protein, Varicellovirus MeSH Prohlížeč
• virové proteiny MeSH

Numerous cellular functions including respiration require iron. Plants and phytoplankton must also maintain the iron-rich photosynthetic electron transport chain, which most likely evolved in the iron-replete reducing environments of the Proterozoic ocean [1]. Iron bioavailability has drastically decreased in the contemporary ocean [1], most likely selecting for the evolution of efficient iron acquisition mechanisms among modern phytoplankton. Mesoscale iron fertilization experiments often result in blooms dominated by diatoms [2], indicating that diatoms have adaptations that allow survival in iron-limited waters and rapid multiplication when iron becomes available. Yet the genetic and molecular bases are unclear, as very few iron uptake genes have been functionally characterized from marine eukaryotic phytoplankton, and large portions of diatom iron starvation transcriptomes are genes encoding unknown functions [3-5]. Here we show that the marine diatom Phaeodactylum tricornutum utilizes ISIP2a to concentrate Fe(III) at the cell surface as part of a novel, copper-independent and thermodynamically controlled iron uptake system. ISIP2a is expressed in response to iron limitation several days prior to the induction of ferrireductase activity, and it facilitates significant Fe(III) uptake during the initial response to Fe limitation. ISIP2a is able to directly bind Fe(III) and increase iron uptake when heterologously expressed, whereas knockdown of ISIP2a in P. tricornutum decreases iron uptake, resulting in impaired growth and chlorosis during iron limitation. ISIP2a is expressed by diverse marine phytoplankton, indicating that it is an ecologically significant adaptation to the unique nutrient composition of marine environments.

• MeSH
• druhová specificita MeSH
• fytoplankton metabolismus   MeSH
• membránové proteiny metabolismus   MeSH
• mořská biologie MeSH
• mořská voda chemie   MeSH
• rozsivky metabolismus   MeSH
• stanovení celkové genové exprese MeSH
• terciární struktura proteinů MeSH
• železo metabolismus   farmakokinetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• membránové proteiny MeSH
• železo MeSH

Pollen is a cornerstone of life for plants. Its durability, adaptability, and complex design are the key factors to successful plant reproduction, genetic diversity, and the maintenance of ecosystems. A detailed study of its chemical composition is important to understand the mechanism of pollen-pollinator interactions, pollination processes, and allergic reactions. In this study, a multimodal approach involving Fourier transform infrared spectrometry (FTIR), direct mass spectrometry with an atmospheric solids analysis probe (ASAP), matrix-assisted laser desorption/ionization (MALDI) and ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) was applied for metabolite profiling. ATR-FTIR provided an initial overview of the present metabolite classes. Phenylpropanoid, lipidic, and carbohydrate structures were revealed. The hydrophobic outer layer of pollen was characterized in detail by ASAP-MS profiling, and esters, phytosterols, and terpenoids were observed. Diacyl- and triacylglycerols and carbohydrate structures were identified in MALDI-MS spectra. The MALDI-MS imaging of lipids proved to be helpful during the microscopic characterization of pollen species in their mixture. Polyphenol profiling and the quantification of important secondary metabolites were performed by UHPLC-MS in context with pollen coloration and their antioxidant and antimicrobial properties. The obtained results revealed significant chemical differences among Magnoliophyta and Pinophyta pollen. Additionally, some variations within Magnoliophyta species were observed. The obtained metabolomics data were utilized for pollen differentiation at the taxonomic scale and provided valuable information in relation to pollen interactions during reproduction and its related applications.

• Klíčová slova
• Magnoliophyta, Pinophyta, atmospheric solids analysis probe, infrared spectrometry, mass spectrometry, matrix-assisted laser desorption/ionization, metabolite profiling, metabolomics, pollen, secondary metabolite, taxonomic differentiation, ultra-high-performance liquid chromatography,
• MeSH
• metabolom MeSH
• metabolomika * metody   MeSH
• polyfenoly metabolismus   analýza   chemie   MeSH
• pyl * chemie   metabolismus   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice * metody   MeSH
• spektroskopie infračervená s Fourierovou transformací metody   MeSH
• vysokoúčinná kapalinová chromatografie MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• polyfenoly MeSH