Konference CEEPC/IPM/CMSC 2022 (Den 2)

Po, 3.10.2022
| Originální článek z: Česká konference hmotnostní spektrometrie (ČSHS)
Druhý den společné konference o hmotnostní spektrometrii a proteomice v roce 2022 přinesl 17 přednášek, posterovou sekci a předání ceny Zdeňka Hermana.
ČSHS: Konference CEEPC/IPM/CMSC 2022 (Den 2)

ČSHS: Konference CEEPC/IPM/CMSC 2022 (Den 2)

Program CEEPC/IPM/CMSC 2022

Kompletní program včetně abstraktů ke stažení

ČSHS: Konference CEEPC-IPM-CMSC 2022: Den 2 (Lászlo Drahos)

Pátek 30. září 2022

8:50 - 9:40 Plenární přednáška II: An FT-ICR-MS metabolomic journey Chairperson: Michael Volný

  • Carlos Cordeiro

Untargeted metabolomics provides a broad survey of the instant composition of living cells or biological fluids. Dynamic range and chemical diversity of the metabolome are the main challenges for its meaningful in depth characterization. Extreme resolution and mass accuracy MS decoupled from chromatographic separation allow the simultaneous analysis of the most diverse range of metabolites in a short period of time, enabling high throughput studies. Here we show the application of FT-ICR-MS to several biological, environmental and forensic problems, highlight the role of computational methods to transform MS spectra into biological information.

ČSHS: Konference CEEPC-IPM-CMSC 2022: An FT-ICR-MS metabolomic journey (Carlos Cordeiro)

9:40 - 11:20 Sekce II - Nová instrumentace a přístupy

  • Chairperson: Lászlo Drahos
9:40 - 10:00 In a flash of light: Native mass spectrometry for single particle imaging with X ray free electron lasers
  • Alan Kádek

Native mass spectrometry enables the ionization and transfer of intact non-covalent protein complexes into the gas phase. As such, it is a great tool to study protein assemblies in a mass and conformation specific manner, probing structural transitions they undergo. Such transient states are highly interesting for structural biology, but generally cannot be purified and are very hard to access for complementary methods such as crystallography or electron microscopy.

Despite its sensitivity and selectivity, the structural resolution in native MS alone is limited. The amount of structural information could be vastly increased by its combination with powerful hard X ray free electron lasers (XFELs) such as the LCLS II in Stanford (USA) or the European XFEL, the world’s brightest light source in Hamburg (Germany). One of the science drivers for these facilities has been enabling structural studies on single particles. However, these efforts have been hindered by the lack of efficient ways to introduce samples into the X-ray beam. (1) In this regard, native MS offers huge benefits in producing and transmitting molecular ions ranging in size from single proteins and non-covalent protein complexes up to multi-megadalton viral particles. Also, profiting from ion charges, MS can select and separate protein complexes based on their mass and conformation and possibly also pre-orient particles to simplify subsequent data processing and structure determination. (2,3)

This contribution will present the work and current development status of the highly interdisciplinary MS SPIDOC consortium, which works toward a native MS-based sample introduction system for use at beamlines of the European XFEL and other light sources.

10:00 - 10:20 Qualitative and quantitative advancements of QTOF performance: the SCIEX ZenoTOF 7600 system
  • Volker Kruft

A novel hybrid collision cell is at the heart of the technological innovations introduced with the SCIEX ZenoTOF 7600 system. In the past, QTOF mass spectrometers have suffered from duty cycle losses; that is, losses in ion transmission in the ion path. This was mainly due to the mating the continuous beam coming from the quadrupole ion path with time-of-flight (TOF) analysis, a pulsed, discontinuous measurement technique. A series of ion-staging events and reverse-mass sequential ion release, with high-capacity ion traps, have been introduced just after the CID collision cell (Q2) and before the pusher region of the TOF. This allows the duty cycle losses to be mitigated leading to MS/MS sensitivity gains of 4-20 fold (1).

The newly engineered collision cell also has the ability to perform both collision induced dissociation (CID) and electron activated dissociation (EAD) experiments for high-resolution, high sensitivity MS/MS flexibility. Electron kinetic energies can be tuned from 0-25 eV without the use of chemical transfer reagents. This precise tunability means EAD can be performed on a wide range of analytes , from multiply charged peptides to singly-charged small molecules (2).

The increase in MS/MS sensitivity, the high speed of acquisition (133Hz) and the choice of fragmentation regimes enables improvements in data quality and depth on analysis in proteomics, metabolomics and lipidomics workflows. Protein IDs from cell lines exceeds 5000 protein groups with up to 95% of these reliably quantitated (CV <20%) at minimal protein loads. Lipids can be fully characterized at an LC time scale (around 30ms EAD reaction time) including their lipid class, acyl group structure, and the location of double bond(s) (3).

ČSHS: Konference CEEPC-IPM-CMSC 2022: Sponzoři konference: BRUKER

10:20 - 10:40 Synergistic success of proteo-genomics in profiling human tear fluid for disease biomarkers using a novel high sensitivity Immunoassay
  • Suresh Jivan Gadher

Quest for key disease biomarker(s) is one of the biggest challenges of today. Human tears, with their comprehensive biomolecule repertoire, are a good source of biomarkers and offer an excellent opportunity for patient stratification in precision medicine. Tear fluid is a potential medium for biomarker discovery in ocular, metabolic and systemic diseases such as diabetic retinopathy, cystic fibrosis and cancers (1). The proximity of the tear fluid to the brain, makes it an ideal fluid for studying neurological disorders such as multiple sclerosis, Parkinson’s disease and brain tumors. Tear collection is fast and noninvasive using a Schirmer's strip (2).

Researchers studying the multifaceted etiology of various diseases require powerful investigational tools with greater sensitivity, better dynamic range, miniscule micro-litre volume consumption and robust data analysis software. ProQuantum™ High Sensitivity Immunoassays incorporating analyte specificity of high affinity antibody-antigen binding with the signal detection and amplification of real-time PCR technologies, offers a flexible and scalable qPCR assay platform enabling multidimensional analysis of DNA, RNA, and now biological proteins. It can play an integral role in screening large number of tear samples for molecular signature of human diseases at protein level in eye clinics and for establishing biomarker profiles of disease status (3).

It is anticipated that an established ‘tear test’ would be a very powerful prognostic and / or diagnostic test in eye clinics in the near future for systemic, neuronal and ocular diseases. ProQuantum™ Immunoassay with its unique features, can benefit precision medicine by maximizing patient’s data outcome while minimizing potential health disparities.

10:40 - 11:00 Back to Basics with Antibodies
  • Stanislav Kukla

In this sponsor talk, a couple of selected overlapping areas between proteomics research and mass spectrometry (MS) techniques will be highlighted and shown on antibodies and their manufacturing, characterization and usage in various antibody-based techniques as examples. You will learn about enhanced antibody validation during which new strategies and recently developed technologies are being used (including MS techniques) as a concept to bring more reproducibility and predictability to Life Science. Part of the talk will be also dedicated to describing different LC-MS approaches for middle-up, intact mass, glyco-profiling and peptide mapping analysis of protein therapeutics, such as monoclonal antibodies (mAbs). All discussed topics will be illustrated by our in-house R&D data that helped develop all the new reagents and tools that will be presented.

ČSHS: Konference CEEPC-IPM-CMSC 2022: Back to Basics with Antibodies (Stanislav Kukla)

11:00 - 11:20 Two-dimensional mass spectrometry for top-down analysis and structural characterization of proteins
  • Maria van Agthoven

Two-dimensional mass spectrometry (2DMS) is a method for tandem mass spectrometry that relies on ion radius modulation instead of ion isolation to correlate between precursor and fragment ion peaks. 2D mass spectra show all the fragmentation patterns of the analytes in a sample. Signal multiplexing yields high signal-to-noise ratios and therefore complete sequence coverage (e.g. for biomolecules) (1). Modifications can easily be assigned and located visually with precursor ion scans and dissociation lines. 2DMS has also successfully been used for label-free relative quantification of modified histone peptides (2).

Acetylation is a covalent labelling method to probe tertiary and quaternary structures of proteins which has successfully been used in combination with top-down analysis to probe the structure of ubiquitin (3). In this study, we analyse acetylated ubiquitin with 2DMS. We use the accuracy of the precursor-fragment correlation to identify and locate the acetylations in the sequence and we use fragment ion abundances for label-free relative quantification. We show that acetylation combined with 2DMS yields accurate information on ubiquitin tertiary structure.

11:20 - 11:40 Přestávka na kávu

Sponzoři akce

ČSHS: Sponzoři akce: CEEPC/IPM/CMSC 2022

11:40 - 13:00 Sekce III - MS a SW nástroje
  • Chairperson: Eva Csosz
11:40 - 12:00 Rapid ion mobility-resolved phosphoproteomics with dia-PASEF
  • Florian Meier

Although protein phosphorylation is one of the best-studied post-translational modifications, cellular function and kinase-substrate relationships remain enigmatic for the vast majority of all identified modification sites. To decipher cellular signaling networks, functional mass spectrometry-based phosphoproteomics is an increasingly attractive strategy that benefits directly from further technological advances. Here, we explore the analytical merits of trapped ion mobility mass spectrometry and data-independent acquisition (dia-PASEF). Using an optimized data acquisition scheme, we quantified over 12,000 phosphopeptides in one hour from low sample amounts equivalent to ~20 ug protein extract per analysis without a spectral library. Strikingly, in 7 min gradients we still quantified about 80% of the class I sites with high accuracy and reproducibility. Our data shows that this is at least partly due to the ion mobility separation compensating for the increased spectral density at shorter gradients. We thus conclude that dia-PASEF offers great potential for scaling up phosphoproteomics both in terms of throughput and sensitivity.

ČSHS: Konference CEEPC-IPM-CMSC 2022: Sponzoři konference: Pragolab (Ladislav Náměstek a Michael Volný)

12:00 - 12:20 Prediction of Intact N-Glycopeptide Retention Time Windows in Hydrophilic Interaction Liquid Chromatography
  • Tomáš Ječmen

Analysis of protein glycosylation is challenging due to micro- and macro-heterogeneity of the attached glycans. Mass spectrometry is a key tool for structural characterization of intact glycopeptides. However, to achieve high-confidence identification, information-rich MS/MS spectra are needed. If such spectra are not available for all analyzed glycopeptides, the confidence of their identification can be improved by including orthogonal information, such as those derived from chromatographic parameters into the search engine algorithms. Here, we propose a simple model predicting relative retention time (RRT) windows for glycopeptides in hydrophilic interaction liquid chromatography (HILIC), which is a mode of choice for separation of this type of analytes as they are inadequately resolved by reversed phase chromatography.

We determined chromatographic parameters for 6 differently glycosylated tryptic peptides of 3 plasma proteins – haptoglobin, hemopexin, and sex hormone-binding globulin – separated by HILIC. For all of them, we calculated retention times of different glycoforms attached to the same peptide relative to the respective bi-antennary form, which is typically found in high yield. We showed that the RRT differences between the glycoforms do not depend greatly on the character of the peptide moiety, and based on the observed variance of the RRTs of individual glycoforms we derived an easy-to-use mathematical model. To prove the concept, we used it to accurately predict the retention time windows, within which fetuin glycopeptides are eluted. The model could therefore be included into post-search filtering of the glycopeptide assignments when HILIC-MS approach is selected for site-specific protein glycosylation characterization. (1)

12:20 - 12:40 Quantification of small molecules in liquid chromatography/mass spectrometry datasets by CycloBranch
  • Jiří Novák

CycloBranch is our open-source, cross-platform, and stand-alone tool originally dedicated to the analysis of accurate tandem mass spectra of cyclic and branched peptides (1). Recently, the tool was extended to support the dereplication and de novo molecular formula determination of compounds in high-resolution conventional mass spectra, liquid chromatography/mass spectrometry (LC/MS) datasets, and imaging mass spectrometry datasets (2, 3). Here, we show how the tool can be used for the quantification of small molecules in LC/MS data. For this purpose, the latest version of CycloBranch processes multiple LC/MS data files in a batch. Shapes of peaks in automatically constructed extracted ion chromatograms are approximated with Gaussian and exponentially modified Gaussian functions. If an input calibration dataset is available, CycloBranch calculates concentrations of compounds from the areas under chromatographic peaks upon an automated calibration curve construction. The tool supports the community standard mzML file format as well as several vendors’ native file formats. A custom database of compounds and a list of ion types to be found can be defined in a user-friendly graphical interface.

ČSHS: Konference CEEPC-IPM-CMSC 2022: Quantification of small molecules in liquid chromatography/mass spectrometry datasets by CycloBranch (Jiří Novák)

12:40 - 13:00 Mass spectrometric analysis of intact proteins: the dark side of deconvolution
  • Lászlo Drahos

One of the most commonly used technique for the study of intact proteins, including monoclonal antibodies (MABs), is the HPLC-MS. Study of pure, large amounts of protein is fairly easy, but for mixtures the situation is more complicated. However, the real challenge is to study small amounts of protein mixtures having large molecular mass. In this case, automatic evaluation (e.g. deconvolution of mass spectra) can give incorrect results and HPLC separation of the mixture is becoming increasingly important.

In this presentation, I would like to illustrate the process, problems and difficulties of evaluation from simple proteins to the study of complex antibody-drug conjugates. In the latter case, when the mixture is too complex, a reliable result can only be obtained using a detailed manual evaluation of the ion chromatograms and mass spectra.

13:00 - 15:30 Oběd + Posterová sekce II (Postery sudé)

ČSHS: Konference CEEPC-IPM-CMSC 2022: Posterová sekce II (Postery sudé)

15:30 - 17:30 Sekce IV - Aplikace v biomedicíně 1

  • Chairperson: Maria van Agthoven
15:30 - 15:50 Identification of potential biomarkers for azoospermia by human testis proteomic analysis
  • Katarina Davalieva

Azoospermia, as the most severe form of male infertility, no longer indicates sterility due to medical advancements. As the current diagnosis is based on testicular biopsy, there is a high need for non-invasive testing. The key point here is the identification of testis-specific proteins that could accurately pinpoint the stage of spermatogenesis failure.

The aim of this study was the identification of proteome differences in human testicular tissues among obstructive azoospermia (OA) and non-obstructive (NOA) subtypes hypospermatogenesis (Hyp) and Sertoli cell-only syndrome (SCO).

We have analyzed 27 FFPE testicular tissues using highly efficient extraction/digestion procedure (1) and label-free data-independent LC-MS/MS acquisition coupled with ion mobility. Validation was done on additional 49 FFPE testicular tissues using qPCR. Out of 2044 proteins identified based on ≥2 peptides, 61 proteins had the power to quantitatively discriminate OA from NOA and 30 to quantitatively discriminate SCO from Hyp and OA. Among these, H1-6, RANBP1 and TKTL2 showed superior potential for quantitative discrimination among OA, Hyp and SCO. Bioinformatics enrichment analysis revealed an association with several GO annotations and pathways. Comparison with 2 transcriptome datasets revealed 278 and 55 co-differentially expressed proteins/genes with statistically significant positive correlation. Gene expression analysis by qPCR of 6 genes with the highest discriminatory power on protein level and the same regulation trend with transcriptomic datasets, confirmed the proteomics results.

Data from our study provides deep insights into the proteins involved in spermatogenesis failure and gives a number of potential candidates for discrimination between OA and NOA.

15:50 - 16:10 Proteotype classification of renal cell carcinoma for prognosis and therapy response

Renal cell carcinoma (RCC) represents a serious oncological disease with one of the highest incidences in the Czech Republic across the world. Reliable molecular prognostic and predictive biomarkers for RCC are mostly unavailable, namely at protein level. To quantify proteins associated with pro-tumorigenic and pro-metastatic mechanisms in RCC, we first generated a comprehensive RCC-specific spectral library of targeted proteomic assays for 7960 protein groups (FDR=1%) [1]. Second, we have applied data independent acquisition mass spectrometry (DIA-MS) on QExactive HF-X LC-MS system to analyze a well-characterized set of initially localized RCC tumors (n=86) of which a half exhibited a relapse in <5 years after diagnosis. We have identified a single potential biomarker and two protein classifiers able to predict the relapse, for which we have developed selected reaction monitoring assay for further validation and routine quantification. CRISPR/Cas9 knockdown confirmed the role of the key protein in cell migration in 786-0 cells, supporting its role in metastatic potential of RCC. Third, we have analyzed a well-characterized set of metastatic RCC tumors (training set n=53, validation set n=22) and adjacent non-cancerous tissues (n=17) a part of which responded and a part did not respond to tyrosine kinase inhibitor (TKI) treatment. We have identified and validated a single protein biomarker and one classifier associated with a poor response to TKI but not with tumor grade and lymph node status. Functional assays using CRISPR/Cas9 knockdown confirmed its role in metastatic potential of 786-0 cells. In a summary, next generation proteomics based on DIA-MS is a powerful tool to classify RCC tissues, to identify prognostic biomarkers and alternative therapeutic targets.

ČSHS: Konference CEEPC-IPM-CMSC 2022: Sponzoři konference: AMEDIS/SCIEX

16:10 - 16:30 Stability Study of Triacetylfusarinine C and Gliotoxin in bodily fluids
  • Jiří Houšť

Introduction: During invasive pulmonary aspergillosis (IPA), Aspergillus fumigatus (Af) produces secondary metabolites supporting proliferation within a neutropenic host. Of note, extracellular siderophore triacetylfusarinine C (TafC) captures ferric cation [1], and mycotoxin gliotoxin (Gtx) fights host immunity [2]. Detection of these specific biomarkers in bodily fluids can improve early IPA diagnosis [3]. Methods: We tested the stability of TafC/Gtx in human urine (pH 6.44) and serum and rat urine (pH 7.86) at 37 °C in a one-week due course. We collected all data with Dionex UltiMate 3000 HPLC system coupled to a SolariX 12T FTICR and evaluated with DataAnalysis 5.0 and OriginPro 2021b. We calculated the TafC/Gtx half-life (t1/2) after data fitting with linear and exponential decay functions. Results: In human urine, TafC decay followed the zero-order kinetics with a t1/2 = 29.8 days. On the other hand, TafC decomposed 58.4x (t1/2 = 12.2 h) and 48.9x (t1/2 = 14.6 h) faster in the basic pH rat urine and human serum, respectively. The TafC degraded to triacetylfusarinine B and N2-acetyl-N5-anhydromevalonyl-N5-hydroxy-L-ornithine residues, which are simultaneously detected in bodily fluids for diagnostic purposes. Similarly, the most stable matrix for Gtx was human urine (t1/2 = 5.8 days) compared to rat urine and human serum, where 80 and 94% of Gtx decomposed within one day into unknown metabolites, respectively. Conclusion: A slightly acidic human urine represents a suitable matrix for preserving the clinically valuable Af virulence factors secreted during proliferation in a host. The simultaneous detection of secreted secondary metabolites and their degradation products in urine could play an essential role in IPA diagnostics even in the early stages of infection.

16:30 - 16:50 MALDI-TOF mass spectrometry: A powerful method for blood meal identification in insect vectors
  • Petr Halada

Determination of blood meal sources of hematophagous arthropods is crucial for understanding transmission cycles of vector-borne diseases in endemic areas. Most of currently used methods are nevertheless laborious and challenged by tiny volumes of rapidly degraded host blood. A promising approach towards blood meal identification was recently developed for sand flies employing MALDI-TOF MS of host-specific haemoglobin peptides generated by trypsin digestion of the engorged blood (1).

The method was first tested on lab-reared sand flies, allowing correct host identification of 100% females until 36h post blood meal (PBM) and for 80% of samples even 48h PBM and thus providing longer reliable blood source determination than other nowadays used methods. Blind study using sand flies collected during a field survey in Greece yielded unambiguous host identification for 96% of females. Moreover, the method successfully determined blood meals of engorged females collected in Bosnia & Herzegovina and Croatia and stored frozen in ethanol for several years prior to the analysis. The approach also works on blood meals spotted on a filter paper that represents a simple and low-cost alternative of sample storage enabling easy shipment at ambient temperatures from regions of collection to MS facilities for the analysis. Furthermore, it allows correct identification of mixed blood meals as was demonstrated on both experimentally fed and field-collected sand flies and reliable differentiation of closely related host species within the same genus.

MALDI-TOF MS was shown as an accurate method for blood meal identification with a minimal sample input. Besides sand flies, it was applied also on mosquitoes and may be universally applicable to various hematophagous arthropods.

ČSHS: Konference CEEPC-IPM-CMSC 2022: MALDI-TOF mass spectrometry: A powerful method for blood meal identification in insect vectors (Petr Halada)

16:50 - 17:10 Characterization of glycosphingolipids in iPSC-derived cerebral organoids and its application in neurodegenerative disorders
  • Durga Jha

Gangliosides are sialylated glycosphingolipids, highly abundant in the neuronal lipid membrane. Utilizing 3D cerebral models, like cerebral organoids (COs), can provide an opportunity to understand the role of these lipids in aging and neurodegenerative diseases. The study aimed to develop a novel UHPLC/ SRM method for identifying eight sub-classes of gangliosides with information on the composition of fatty acid composition and the sphingoid bases for each sub-class. The method was applied to understand the effect of known risk factors of Alzheimer's disease on these lipids, such as apolipoprotein E and secretases. More than 70 gangliosides were identified in the cerebral organoids. In the organoids, fatty acids were characterized by a prevalence of chain lengths in sizes from C14 to C22. Overall the profiles of different chain length lipids were similar within the same sub-class of gangliosides.

Interestingly the C16 and C18 fatty acids were present in the highest concentrations in the organoids, in contrast to C18 and C20, which are more abundant in an aging human brain. There was a global upregulation in the level of gangliosides in the presence of ApoE4. A similar effect was observed in the treatment of these organoids with secretases. This method can be helpful for the analysis of healthy and neuropathological changes associated with aging and neurodegenerative diseases in the cerebral organoids.

ČSHS: Konference CEEPC-IPM-CMSC 2022: Sponzoři konference: MERCK

17:10 - 17:30 Redox resetting by modulation of redox sensor Kelch-like ECH-associated protein 1 restores leukemic cells sensitivity to Azacytidine
  • Kristýna Pimková

A hypomethylating drug 5-azacytidine (AZA) is used to treat patients with myeloid malignancies who are in a high risk of progression to leukemia. Although AZA significantly prolongs patient survival, the efficacy of treatment is often hampered by the early development of resistance. The mechanisms by which leukemic cells overcome AZA toxicity are not yet fully understood, but there is considerable evidence that the molecular events leading to the loss of response to AZA are driven by redox mechanisms. In this study, we focused on investigating the role of redox homeostasis in leukemic cell resistance to AZA.

We used a quantitative mass spectrometry-based proteomics approach to identify protein targets of oxidative modifications in a leukemia cell model of AZA resistance developed in our laboratory. Analysis showed that treatment of AZA-sensitive cells (AZA-S) resulted in altered cysteine oxidation in 20% of proteins (578 of 2853). We identified key cysteine sites of proteins that regulate apoptosis. We hypothesize that AZA-induced oxidative stress is a key factor in its cytotoxic effect. AZA resistance was associated with changes in the oxidative state of 14% of cysteine proteins involved in glutathione metabolism and the antioxidant defense system, such as the key redox sensor Kelch-like ECH-associated protein 1 (KEAP1). The data suggest that AZA-R are under chronic oxidative stress and have adapted to redox stressors. We show that inhibition of KEAP1 blocks redox adaptation and restores AZA-R cells' sensitivity to AZA.

In conclusion, we demonstrated that the mechanism of AZA resistance involves cellular adaptation to oxidative stress and that modulation of the KEAP1 cellular antioxidant response pathway can re-sensitize AZA-resistant cells in vitro.

17:30 - 18:10 Předání ceny Zdeňka Hermana Nadačním fondem Resonance a prezentace vítězné práce

ČSHS: Konference CEEPC-IPM-CMSC 2022: Předání ceny Zdeňka Hermana Nadačním fondem Resonance (Patrik Španěl)

18:10 - 18:30 Přestávka na kávu

18:30 - 19:00 Schůze ČSHS

Czech Mass Spectrometry Conferences

Mohlo by Vás zajímat

Characterization of carbon materials with Raman spectroscopy

RAMAN Spektrometrie
Materiálová analýza

Single-Injection Analysis of Formic Acid and Free Volatile Fatty Acids Using Flame Ionization Detector with In-jet Methanizer

Shimadzu, ARC
Životní prostředí, Průmysl a chemie

Analysis of Phytosanitary Products in Surface Water and Groundwater Using GCxGC-TOFMS

Životní prostředí

Differences in Metabolic Profiles of Individuals with Heart Failure Using High-Resolution GC/Q-TOF

Agilent Technologies
Klinická analýza

Analysis of Ethylene Oxide and 2-Chloroethanol in Food

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První den letošní společné konference o hmotnostní spektrometrii a proteomice jsme viděli 16 odborných přednášek, diskusi u posterů a předání ceny Josefa Chmelíka.