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Advanced Nanotechnologies and Microtechnologies research programme
Chemical Communications
Convergent and divergent two-dimensional coordination networks formed through substrate-activated or quenched alkynyl ligation
Čechal J.,Kley C., Kumagai T., Schramm F., Ruben M., Stepanow S., Kern K.
Research Group: Fabrication and Characterisation of Nanostructures
Abstract
Metal coordination assemblies of the symmetric bi-functional 4,4’-di-(1,4-buta-1,3-diynyl)-benzoic acid are investigated by scanning tunnelling microscopy on metal surfaces. The formation of long-range ordered, short-range disordered and random phases depends on the competition between the convergent and divergent coordination motifs of the individual functional groups and is crucially influenced by the substrate.
Materials and Design
Finite element analysis of bone loss around failing implants.
Wolff J., Narra N., Antalainen A., Valášek J., Kaiser J., Sándor G., Marcián P.
Research Group: Materials Characterization and Advanced Coatings
Abstract
Dental implants induce diverse forces on their surrounding bone. However, when excessive unphysiological forces are applied, resorption of the neighbouring bone may occur. The aim of this study was to assess possible causes of bone loss around failing dental implants using finite element analysis. A further aim was to assess the implications of progressive bone loss on the strains induced by dental implants.
Between 2003 and 2009 a total of 3700 implant operations were performed in a private clinic. Ten patients with 16 fixtures developed severe marginal bone defects. Finite element analysis was used to assess the effective strains produced at the bone-implant interface under unidirectional axial loading. These simulations were carried out on 4 specific implant types – Camlog Plus, Astra Osseo Speed, Straumann BL and Straumann S/SP. All implant types exhibited degraded performance under circular and horizontal bone loss conditions. This is evidenced by increased distribution of pathological strain intensities (>3000 με), in accordance with the mechanostat hypothesis, in the surrounding bone. Among the implants, the Camlog design seemed to have performed poorly, especially at the chamfer in the implant collar (>25000με). Implants are designed to perform under nearly ideal conditions from insertion till osseointegration. However, when the surrounding bone undergoes remodelling, implant geometries can have varied performance, which in some cases can exacerbate bone loss. The results of this study indicate the importance of evaluating implant geometries under clinically observed conditions of progressive bone loss.
Advanced Materials research programme
Ceramics International
Consolidation of nanoparticle suspensions by centrifugation in non-porous moulds
Trunec M., Mišák J.
Research Group: Advanced Ceramic Materials
Abstract
Alumina and zirconia nanosuspensions with a mean particle size of 100 nm and 15nm, respectively, were consolidated by centrifugal compaction in non-porous moulds. The nano suspensions consolidated by high-speed centrifugation were deposited irregularly, resulting in a powder deposit with density profile. The homogeneity of the powder deposits was described and homogeneous and well packed deposit regions were identified. Plate-like bodies were prepared from the homogeneous regions of the deposit. The advantage of regular and dense nanoparticle packing by centrifugal compaction was demonstrated by fabricating transparent alumina and tetragonal zirconia ceramics. The transparent alumina had an in-line transmission of 55% in the visible light at a thickness of 0.8mm. The transparent tetragonal zirconia reached a dense microstructure with an average grain size of 65 nm and an in-line transmission of 25% at a thickness of 0.5mm.
Structural Biology research programme
Molecular Cell
Molecular Basis for Coordinating Transcription Termination with Noncoding RNA Degradation
Tudek A., Porrua O., Kabzinski T., Lidschreiber M.,Kubicek K., Fortova A., Lacroute F., Vanacova S., Cramer P., Stefl R., Libri D.
Research Group: Structural Biology of Gene Regulation
Abstract
The Nrd1-Nab3-Sen1 complex controls pervasive transcription in yeast by terminating transcription of cryptic transcripts and directing these RNAs to degradation by the exosome. Tudek et al. show that the same domain of Nrd1p mediates mutually exclusive interactions with RNAPII or the exosome cofactor TRAMP to coordinate termination and RNA degradation.
Chemical Science
Azidophenyl as a click-transformable redox label of DNA suitable for electrochemical detection of DNA–protein interactions
Balintová J., Špaček J., Pohl R., Brázdová M., Havran L., Fojta M. and Hocek M.
Research Group: Structure and Interaction of Biomolecules at Surfaces
Abstract:
A new approach for the detection of DNA-protein binding has been proposed. The technique uses electrochemically active azidophenyl group, attached to cytosine or 7-deazaadenine nucleobases incorporated into a DNA probe. 5-(4-Azidophenyl)-2′-deoxycytidine and 7-(4-azidophenyl)-7-deaza-2′-deoxyadenosine nucleosides were prepared by aqueous-phase Suzuki cross-coupling and converted to nucleoside triphosphates (dNTPs) which served as substrates for incorporation into DNA by DNA polymerase. The azidophenyl-modified nucleotides and azidophenyl-modified DNA yield a strong voltammetric signal at −0.9 V due to reduction of the azido function. The Cu-catalyzed click reaction of azidophenyl-modified nucleosides or azidophenyl-modified DNA with 4-nitrophenylacetylene give nitrophenyl-substituted triazoles, exerting a reduction peak at −0.4 V in voltammetry. The click reaction with phenylacetylene yield electrochemically silent phenyltriazoles. Thus, conversion of the azidophenyl moiety to the triazole derivatives can be monitored selectively by voltammetry. The transformation of the azidophenyl label to nitrophenyltriazole was used for electrochemical detection of p53 protein-DNA interactions. Our study has shown that azidophenyl groups in the parts of the DNA not shielded by the bound protein protein were transformed to nitrophenyltriazoles, whereas those covered by the protein were not.
Genomics and Proteomics of Plant Systems research programme
Journal of Experimental Botany
Identification of AHK2- and AHK3-like cytokinin receptors in Brassica napus reveals two subfamilies of AHK2 orthologues
Kuderová A., Gallová L., Kuricová K., Nejedlá E., Čurdová A., Micenková L., Plíhal O., Šmajs D., Spíchal L., Hejátko J.
Research Group: Functional Genomics and Proteomics of Plants
Abstract:
Cytokinin (CK) signalling is known to play key roles in the regulation of plant growth and development, crop yields, and tolerance to both abiotic stress and pathogen defences, but the mechanisms involved are poorly characterized in dicotyledonous crops. Here the identification and functional characterization of sensor histidine kinases homologous to Arabidopsis CK receptors AHK2 and AHK3 in winter oilseed rape are presented. Five HASE-containing His kinases were identified in Brassica napus var. Tapidor (BnCHK1–BnCHK5) by heterologous hybridization of its genomic library with gene-specific probes from Arabidopsis. Using a bioinformatic approach and cDNA cloning, the precise gene and putative protein domain structures were determined. Based on phylogenetic analysis, four AHK2 (BnCHK1–BnCHK4) homologues and one AHK3 (BnCHK5) homologue were defined. It is further suggested that BnCHK1 and BnCHK3, and BnCHK5 are orthologues of AHK2 and AHK3, originally from the B. rapa A genome, respectively. BnCHK1, BnCHK3, and BnCHK5 displayed high affinity for trans-zeatin (1–3 nM) in a live-cell competitive receptor assay, but not with other plant hormones (indole acetic acid, GA3, and abscisic acid), confirming the prediction that they are genuine CK receptors. It is shown that BnCHK1 and BnCHK3, and BnCHK5 display distinct preferences for various CK bases and metabolites, characteristic of their AHK counterparts, AHK2 and AHK3, respectively.
Journal of Chromatography A
Double opposite end injection capillary electrophoresis with contactless conductometric detection for simultaneous determination of chloride, sodium and potassium in cystic fibrosis diagnosis.
Kubáň P., Greguš M., Pokojová E., Skřičková J., Foret F.
Research Group: Bioanalytical Instrumentation
Abstract:
A novel approach for diagnosis of cystic fibrosis is presented. A simple and fast procedure to obtain sweat sample was developed. It consists of repeatedly wiping the skin of the forearm with deionized water moisturized cotton swab and extraction in 1 mL of deionized water. Double opposite end injection capillary electrophoresis with contactless conductometric detection is used for the analysis of the extract. Chloride, sodium and potassium as the three target ions that participate in the ion transfer across the cellular membranes, and are affected by CF, are simultaneously determined in approximately 3 min in a background electrolyte containing 20 mM 2-(N-morpholino)ethanesulfonic acid, 20 mM L-histidineand 2 mM 18-crown-6. By using the target ion ratios rather than the concentrations of each individual ion combined with principal component analysis, the diagnosis of CF can be made more accurately and greatly reduce the number of false positive or negative results as is often the case when single ion (chloride) is analyzed.
Cell Host Microbes
Nonsense-mediated mRNA decay modulates TNL immune receptor levels and plant innate immunity
Gloggnitzer J., Akimcheva S., Srinivasan A., Kusenda B., Riehs N., Stampfl H., Bautor J., Dekrout B., Jonak C., Jiménez-Gómez J.M., Parker J.E., Riha K.
Research group: Plant Molecular Biology
Abstract:
Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic RNA surveillance mechanism that degrades aberrant mRNAs. NMD impairment in Arabidopsisis linked to constitutive immune response activation and enhanced antibacterial resistance, but the underlying mechanisms are unknown. Here we show that NMD contributes to innate immunity in Arabidopsisby controlling the turnover of numerous TIR domain-containing, nucleotide-binding, leucine-rich repeat (TNL) immune receptor-encoding mRNAs. Autoimmunity resulting from NMD impairment depends on TNL signaling pathway components and can be triggered through deregulation of a single TNL gene, RPS6. Bacterial infection of plants causes host-programmed inhibition of NMD, leading to stabilization of NMD-regulated TNL transcripts. Conversely, constitutive NMD activity prevents TNL stabilization and impairs plant defense, demonstrating that host-regulated NMD contributes to disease resistance. Thus, NMD shapes plant innate immunity by controlling the threshold for activation of TNL resistance pathways.
Molecular Medicine research programme
Leukemia
Detailed analysis of therapy-driven clonal evolution of TP53 mutations in chronic lymphocytic leukemia
Malcikova J., Stano-Kozubik K., Tichy B., Kantorova B., Pavlova S., Tom N., Radova L., Smardova J., Pardy F., Doubek M., Brychtova Y., Mraz M., Plevova K., Diviskova E., Oltova A., Mayer J., Pospisilova S., Trbusek M.
Research Group: Medical Genomics & Genomics Core Facility
Abstract:
In chronic lymphocytic leukemia (CLL), the worst prognosis is associated with TP53 defects with the affected patients being potentially directed to alternative treatment. Therapy administration was shown to drive the selection of new TP53 mutations in CLL. Using ultra-deep next-generation sequencing (NGS) we performed a detailed analysis of TP53 mutations' clonal evolution. We retrospectively analyzed samples assessed as TP53-wild-type (wt) by FASAY from 20 patients with a new TP53 mutation detected in relapse and 40 patients remaining TP53-wt in relapse. Minor TP53-mutated subclones were disclosed in 18/20 patients experiencing later mutation selection, while only one minor-clone mutation was observed in those patients remaining TP53-wt (n=40). We documented that (i) minor TP53 mutations may be present before therapy and may occur in any relapse; (ii) the majority of TP53-mutated minor clones expand to dominant clone under the selective pressure of chemotherapy, while persistence of minor-clone mutations is rare; (iii) multiple minor-clone TP53 mutations are common and may simultaneously expand. In conclusion, patients with minor-clone TP53 mutations carry a high risk of mutation selection by therapy. Deep sequencing can shift TP53 mutation identification to a period before therapy administration, which might be of particular importance for clinical trials.
Blood
miR-150 influences B-cell receptor signaling in chronic lymphocytic leukemia by regulating expression of GAB1 and FOXP1
Mraz M., Chen L., Rassenti L.Z., Ghia E.M., Li H., Jepsen K., Smith E.N., Messer K., Frazer K.A., Kipps T.J.
Research Group: Medical Genomics
Abstract:
We examined the microRNAs (miRNAs) expressed in chronic lymphocytic leukemia (CLL) and identified miR-150 as the most abundant, but with leukemia cell expression levels that varied among patients. CLL cells that expressed ζ-chain-associated protein of 70 kDa (ZAP-70) or that used unmutated immunoglobulin heavy chain variable (IGHV) genes, each had a median expression level of miR-150 that was significantly lower than that of ZAP-70-negative CLL cells or those that used mutated IGHV genes. In samples stratified for expression of miR-150, CLL cells with low-level miR-150 expressed relatively higher levels of forkhead box P1 (FOXP1) and GRB2-associated binding protein 1 (GAB1), genes with 3' untranslated regions having evolutionary-conserved binding sites for miR-150. High-level expression of miR-150 could repress expression of these genes, which encode proteins that enhance B-cell receptor signaling, a putative CLL-growth/survival signal. Also, high-level expression of miR-150 was a significant independent predictor of longer treatment-free survival or overall survival, whereas an inverse association was observed for high-level expression of GAB1 or FOXP1 for overall survival. This study demonstrates that expression of miR-150 can influence the relative expression of GAB1 and FOXP1 and the signaling potential of the B-cell receptor, thereby possibly accounting for the noted association of expression of miR-150 and disease outcome.
Brain and Mind research programme
Progress in Neuro-psychopharmacology & Biological Psychiatry 2014
Repetitive transcranial magnetic stimulation reduces cigarette consumption in schizophrenia patients
Prikryl R., Ustohal L., Prikrylova Kucerova H., KasparekT., JarkovskyJ., Hublova V., Vrzalova M., Ceskova E.
Research group: Applied Neuroscience
Abstract:
INTRODUCTION
High-frequency repetitive transcranial magnetic stimulation (rTMS) over the left dorsolateral prefrontal cortex (DLPFC) seemed to decrease tobacco consumption and craving in nicotine-dependent people without psychiatric disorder or otherwise healthy people. Even if the prevalence of cigarette smoking in schizophrenia patients is high and estimated to be between 45% and 88%, this technique has not been systematically studied in this indication in schizophrenia yet.
THE AIM OF THE STUDY
The aim of this study was to test the ability of high-frequency (10Hz) rTMS over the left DLPFC to decrease cigarette consumption in schizophrenia patients.
METHODS
The study included 35 male schizophrenia patients on stable antipsychotic medication. The patients were divided into two groups: the first (18 patients) were actively stimulated and the second (17 patients) underwent sham (placebo) stimulation. The sham rTMS was administered using a purpose-built sham coil that was identical in appearance to the real coil and made the same noise but did not deliver a substantial stimulus. The rTMS was administered at the stimulation parameters: location (left dorsolateral prefrontal cortex: DLPFC), intensity of magnetic stimulation in % of motor threshold (110%), stimulation frequency (10Hz), number of trains (20), single train duration (10s), inter-train interval (30s), and total number of stimulation sessions (21). In each stimulation session, 2000 TMS pulses were given, with a total of 42,000 pulses per treatment course. Patients noted the number of cigarettes smoked in the 7days before treatment, during the whole stimulation treatment (21 days), and again for a 7-day period after treatment.
RESULTS
Cigarette consumption was statistically significantly lower in the actively stimulated patients than in the sham rTMS group as early as the first week of stimulation. No statistically relevant correlations were found in the changes of ongoing negative or depressive schizophrenia symptoms and the number of cigarettes smoked.
CONCLUSION
High-frequency rTMS over the left DLPFC has the ability to decrease the number of cigarettes smoked in schizophrenia patients.
BioMed Research International 2014
Inflammatory Profiling of Schwann Cells in Contact with Growing Axons Distal to Nerve Injury
Dubový P., Klusáková I., Hradilová Svíženská I.
Research Group: Cellular and Molecular Neurobiology
Abstract:
Activated Schwann cells distal to nerve injury upregulate inflammatory mediators, including cytokines. The goal of the present study was to investigate expression of proinflammatory (IL-1β, TNFα) and anti-inflammatory cytokines (IL-4, IL-10) in activated Schwann cells in relation to growing axons distal to crush injury of rat sciatic nerves. Seven days from sciatic nerve crush, transverse cryostat sections were cut 5 mm distal to lesion and incubated for double immunostaining to indicate Schwann cells (GFAP or S100b) and individual investigated cytokines or to demonstrate growing axons (GAP43). The Schwann cells of naïve sciatic nerves and those removed from sham-operated rats displayed similar weak immunoreactivity for the investigated cytokines. In contrast, increased intensity of cytokine immunofluorescence was found in Schwann cells distal to crush lesion. The cytokine positive Schwann cells were found in close contact with growing axons detected by immunostaining for GAP43. The results of immunohistochemical analysis distal to nerve crush injury suggest that inflammatory profiling of Schwann cells including upregulation of both pro- and anti-inflammatory cytokines does not prevent growth of axons distal to nerve crush injury.
Molecular Veterinary Medicine research programme
PROTIST
Hemolivia and Hepatozoon: Haemogregarines with Tangled Evolutionary Relationships
Kvičerová J., Hypša V., Dvořáková N.,Mikulíček P., Jandzik D., Gardner M.G., Javanbakht H., Tiar G., Široký, P.
Research Group: Molecular Bacteriology
Abstract:
The generic name Hemolivia has been used for haemogregarines characterized by morphological and biological features. The few molecular studies, focused on other haemogregarine genera but involving Hemolivia samples, indicated its close relationship to the genus Hepatozoon. Here we analyze molecular data for Hemolivia from a broad geographic area and host spectrum and provide detailed morphological documentation of the included samples. Based on molecular analyses in context of other haemogregarines, we demonstrate that several sequences deposited in GenBank from isolates described as Hepatozoon belong to the Hemolivia cluster. This illustrates the overall difficulty with recognizing Hemolivia and Hepatozoon without sufficient morphological and molecular information. The close proximity of both genera is also reflected in uncertainty about their precise phylogeny when using 18S rDNA. They cluster with almost identical likelihood either as two sister taxa or as monophyletic Hemolivia within paraphyletic Hepatozoon. However, regardless of these difficulties, the results presented here provide a reliable background for the unequivocal placement of new samples into the Hemolivia/ Hepatozoon complex.
Chromosome Research
The frequency of precocious segregation of sister chromatids in mouse female meiosis I is affected by genetic background
Danylevska A., Kovacovicova K., Awadova T., Anger M.
Research group: Mammalian Reproduction
Abstract:
Mammalian female gametes frequently suffer from numerical chromosomal aberrations, the main cause of miscarriages and severe developmental defects. The underlying mechanisms responsible for the development of aneuploidy in oocytes are still not completely understood and remain a subject of extensive research. From studies focused on prevalence of aneuploidy in mouse oocytes, it has become obvious that reported rates of aneuploidy are strongly dependent on the method used for chromosome counting. In addition, it seems likely that differences between mouse strains could influence the frequency of aneuploidy as well; however, up till now, such a comparison has not been available. Therefore, in our study, we measured the levels of aneuploidy which has resulted from missegregation in meiosis I, in oocytes of three commonly used mouse strains—CD-1, C3H/HeJ, and C57BL/6. Our results revealed that, although the overall chromosomal numerical aberration rates were similar in all three strains, a different number of oocytes in each strain contained prematurely segregated sister chromatids (PSSC). This indicates that a predisposition for this type of chromosome segregation error in oocyte meiosis I is dependent on genetic background.
Chromosome Research
Illegitimate recombination between T cell receptor genes in humans and pigs (Sus scrofa domestica)
Musilova P., Drbalova J., Kubickova S., Cernohorska H., Stepanova H., Rubes J.
Research Group: Animal Cytogenomics
Abstract:
T cell receptor (TCR) genes (TRA/TRD, TRB andTRG) reside in three regions on human chromosomes (14q11.2, 7q34 and 7p14, respectively) and pig chromosomes (7q15.3-q21, 18q11.3-q12 and 9q21-22, respectively). During the maturation of T cells, TCR genes are rearranged by site-specific recombination. Occasionally, interlocus recombination of different TCR genes takes place, resulting in chromosome rearrangements. It has been suggested that the absolute number of these “innocent” trans-rearrangements correlates with the risk of lymphoma. The aims of this work were to assess the frequencies of rearrangements with breakpoints in TCR genes in domestic pig lymphocytes and to compare these with the frequencies of corresponding rearrangements in human lymphocytes by using fluorescence in situ hybridization with chromosome painting probes. We show that frequencies of trans-rearrangements involving TRA/TRD locus in pigs are significantly higher than the frequency of translocations with breakpoints in TRB and TRG genes in pigs and the frequencies of corresponding trans-rearrangements involving TRA/TRD locus in humans. Complex structure of the pig TRA/TRD locus with high number of potential V(D)J rearrangements compared to the human locus may account for the observed differences. Furthermore, we demonstrated that trans-rearrangements involving pig TRA/TRD locus occur at lower frequencies in γδ T cells than in αβ T lymphocytes. The decrease of the frequencies in γδ T cells is probably caused by the absence of TRA recombination during maturation of this T cell lineage. High numbers of innocent trans-rearrangements in pigs may indicate a higher risk of T-cell lymphoma than in humans.
