Significant scientific results 2019

Is neurogenesis in two songbird species related to variability in their song?

Justína Polomová, Kristína Lukáčová, Boris Bilčík, Ľubica Kubíková

Neurogenesis also takes place in the adult brain, contrary to ideas in the past. In songbirds, new neurons in adulthood are also integrated into areas mediating song. It is hypothesized that these neurons allow for variability in singing. In our work, we have established a baseline level of neurogenesis in two species of songbirds that differ in sequence variability in song as adults. We found that the Japanese foster warbler has a more variable sequence of song parts and has a higher number of new cells in the neurogenic zone than the red-backed zebra finch. Moreover, within species in foster moths, we found that song variability is positively correlated with the number of new neurons incorporated into the vocal areas of the brain controlling song. These results support the hypothesis that newly generated neurons in the adult brain mediate behavioral variability.

POLOMOVÁ, JustínaLUKÁČOVÁ, KristínaBILČÍK, BorisKUBÍKOVÁ, Ľubica. Is neurogenesis in two songbird species related to their song sequence variability? In Proceedings of the Royal Society : B – Biological Sciences, 2019, vol. 286, no. 1895, art. no. 20182872. (4.304 – IF2018). ISSN 0962-8452.

Glucocorticoids can affect embryonic development in the earliest stages of embryogenesis through different isoforms of the glucocorticoid receptor

Štefan Čikoš, Janka Babeľová, Alexandra Špirková, Ján Burkuš, Veronika Kovaříková, Zuzana Šefčíková, Dušan Fabian, Juraj Koppel

Glucocorticoids are released into the bloodstream based on circadian rhythms or in response to stress stimuli. In our work, we have shown in a mouse model that embryos produce glucocorticoid receptors (GRs) from the earliest developmental stages (before implantation into the uterus), with distinct GR isoforms present at different developmental stages (ovulated eggs, 4 – 8 – 16 cell embryos, blastocysts). Comparison of the action of corticosterone (a natural glucocorticoid) and dexamethasone (a synthetic glucocorticoid also used for therapeutic purposes) showed different effects of these two glucocorticoids on preimplantation embryos, which may also be related to the activation of different GR isoforms.

Stress response and expression of glucocorticoid receptor isoforms (GRα, GRβ, GRγ and GR-P) in mouse preimplantation embryos (Ooc: oocytes, 4c: 4-cell embryos, 8-16c: 8-16-cell embryos, Blast: blastocysts; PC: positive control tissue, B: negative control – blank) at the mRNA and protein levels.

ČIKOŠ, ŠtefanBABEĽOVÁ, JankaŠPIRKOVÁ, AlexandraBURKUŠ, JánKOVAŘÍKOVÁ, VeronikaŠEFČÍKOVÁ, ZuzanaFABIAN, DušanKOPPEL, Juraj. Glucocorticoid receptor isoforms and effects of glucocorticoids in ovulated mouse oocytes and preimplantation embryos. In Biology of Reproduction, 2019, vol. 100, no. 2, p. 351-364. (2.960 – IF2018). ISSN 0006-3363.

The transmembrane protein Grina/TMBIM3 plays an important role in synaptic signal transduction from the periphery to the CNS

Lucia Moravčíková, Katarína Ondáčová, Ľubica Lacinová

Grina/TMBIM3 is a transmembrane protein with little known function. We found that it directly interacts with the major CaV2.2 subunit of the neuronal calcium channel. This voltage-dependent calcium channel plays an important role in synaptic signal transduction from the periphery to the CNS, so it is important to know the ways in which it is physiologically regulated. We demonstrated the direct interaction of Grina/TMBIM3 protein and the major subunit of the CaV2.2 channel by co-immunoprecipitation. We investigated current modulation by the patch clamp method in a whole-cell configuration. The presence of Grina/TMBIM3 protein statistically significantly modulated calcium current through the CaV2.2 calcium channel. Current density was reduced by approximately 50% and the kinetics of current activation was slowed. Inhibition of current amplitude could be eliminated by applying a short depolarizing prepulse of high amplitude. This mechanism of de-inhibition is similar to the mechanism of inhibition and de-inhibition of the CaV2.2 channel by G-proteins. Indeed, expression of Gβγ subunits mimicked the effect of Grina/TMBIM3 protein, and simultaneous expression of Grina/TMBIM3 and Gβγ did not enhance the effects. The kinetics of disinhibition by the depolarizing prepulse and the kinetics of restoration of inhibition were the same for both proteins. Thus, the Grina/TMBIM3 protein modulates current through the CaV2.2 channel by a mechanism similar to that described for the Gβγ subunit of G-proteins. Since CaV2.2 channel activation is essential at certain synapses, modulation of synaptic transmission may be a novel physiological function of the Grina/TMBIM3 protein.

MALLMANN, Robert T.* – MORAVČÍKOVÁ, Lucia* – ONDÁČOVÁ, KatarínaLACINOVÁ, Ľubica – KLUGBAUER, Norbert**. Grina/TMBIM3 modulates voltage-gated Ca V 2.2 Ca 2+ channels in a G-protein-like manner. In Cell Calcium, 2019, vol. 80, p. 71-78. ISSN 0143-4160. IF 3,932.

Application of selected strains of potentially probiotic bacteria to dogs improves their immunological parameters

Viola Strompfová, Ivana Kubašová, Jana Ščerbová, Andrea Lauková

Gastrointestinal diseases and immune system disorders are among the most common health problems in dogs. Therefore, the aim of this work was to select a safe potential probiotic bacterium (meeting EFSA requirements) with the possibility of application in these diseases in the form of supportive therapy. Two bacteria differing in their ability to produce bacteriocin, namely Enterococcus faecium D7 and enterocin B producing E. faecium strain IK25, were selected for testing in dogs. Despite the demonstration of inhibitory activity of enterocin B against several undesirable bacteria from the genera Listeria, Staphylococcus, Enterococcus, Micrococcus and Burkholderia , the results of their application for 14 days to dogs showed differences in the tolerance and effects of these strains. Application of the IK25 strain at a dose of 109 KTJ/pes/day to clinically healthy dogs for 14 days resulted in a watery consistency of faeces, an increase in Gram-negative bacterial counts and a decrease in blood haemoglobin concentration. Conversely, supplementation of dogs with strain D7 led to stimulation of phagocytic activity and respiratory burst of leukocytes, a decrease in blood serum glucose concentration, while maintaining a solid consistency of faeces. Based on these results, it can be concluded that strain D7 appears to be a suitable candidate for preventive application in terms of promoting innate immunity in dogs.

STROMPFOVÁ, ViolaKUBAŠOVÁ, IvanaŠČERBOVÁ, Jana – MAĎARI, Aladár – GANCARČÍKOVÁ, Soňa – MUDROŇOVÁ, Dagmar – MILTKO, Renata – BELZECKI, Grzegorz – LAUKOVÁ, Andrea. Oral administration of bacteriocin-producing and non-producing strains of Enterococcus faecium in dogs. In Applied Microbiology and Biotechnology, 2019, vol. 103, no. 12, p. 4953-4965. (3.670 – IF2018). ISSN 0175-7598.

Detection of tetraspanins CD9 and CD81 on bovine and porcine oocytes and embryos

Jana Jankovičová, Petra Sečová, Ľubica Horovská, Katarína Michalková, Jana Antalíková

Tetraspanins are membrane-associated molecules involved in many physiological cellular processes, but also in the pathogenesis of various infectious diseases. The interaction of gametes leading to the formation of the zygote is equally impossible without their participation. In our work, tetraspanins CD9 and CD81 were detected in the plasma membrane of oocytes at different stages of maturity (GV, MI and MII) in addition to embryos in the form of vesicles organized in clusters in the perivitelline space in both animal species (pig, bovine). We also detected an unchanged distribution of both molecules in parthenogenetically activated bovine oocytes. In contrast to porcine oocytes, in cow oocytes, CD9 was also localized in the zona pellucida, as part of the so-called “transzonal projections”, structures important in cumulus cell communication with the oocyte. We did not observe an effect of specific antibodies against CD9 and CD81 molecules on in vitro fertilization of cow oocytes. We hypothesize that CD9 and CD81 do not act as fusogenic proteins but participate in membrane reorganization and curvature, thereby affecting protein interactions within the tetraspanin site, essential for successful fertilization. The species-specific response pattern of CD9 and CD81 likely reflects their distinct roles in processes related to cell adhesion and intercellular communication during fertilization. The expression of tetraspanins, their localization and distribution can potentially be applied as one of the markers of gamete quality in biotechnological methods in the field of reproduction.

Tetraspanin CD9 on bovine ZP-inactive oocytes detected by monoclonal antibody IVA50 (green) and transzonal projections visualized by phalloidin (red), DNA (blue) (a); localization of tetraspanin CD9 on the plasma membrane of bovine embryos detected by antibody IVA50 (green), DNA (blue) (b). Scale bar 50 µm.

JANKOVIČOVÁ, JanaSEČOVÁ, Petra – MAŇÁSKOVÁ-POSTLEROVÁ, Pavla – ŠIMONÍK, Ondřej – FROLÍKOVÁ, Michaela – CHMELÍKOVÁ, Eva – HOROVSKÁ, ĽubicaMICHALKOVÁ, Katarína – DVOŘÁKOVÁ-HORTOVÁ, Kateřina – ANTALÍKOVÁ, Jana. Detection of CD9 and CD81 tetraspanins in bovine and porcine oocytes and embryos. In International Journal of Biological Macromolecules, 2019, vol. 123, p. 931-938. (4.784 – IF2018). ISSN 0141-8130.

Electrophysiological and behavioral testing of the novel molecule SMe1EC2M3 as a representative of a new generation of trial-effect reuptake inhibitors

Romana Koprdová, Kristína Csatlosová, Barbora Durišová, Eszter Bogi, Magdaléna Majeková, Eliyahu Dremencov, Mojmír Mach

SMe1EC2M3 is one of 82 pyridoindole derivatives that have been designed at the Centre for Experimental Medicine (CEM) of the SAS. The original intention was to develop a substance with an antioxidant effect for the treatment of brain injury. However, we also considered the andiprepressive effect of pyridoindoles. In collaboration with CEM SAV, we have conducted comprehensive preclinical testing of SMe1EC2M3 as a potential antidepressant. Adequate intestinal absorption, binding to plasma proteins, permeability across the blood-brain barrier and potential antidepressant effect of SMe1EC2M3 were predicted in silico. Behavioral tests in rats showed an effect comparable to that of known antidepressants. In vivo electrophysiological experiments (single-unit) suggested that the effect of SMe1EC2M3 is probably based on inhibition of serotonin, noradrenaline and dopamine reuptake. Our study suggests that SMe1EC2M3 appears to be a promising triple-effect reuptake inhibitor with antidepressant-like properties. Further studies and assays are needed to obtain a detailed pharmacological characterization of this prospective substance.

KOPRDOVÁ, Romana – CSATLÓSOVÁ, Kristína – ĎURIŠOVÁ, Barbora – BÖGI, Eszter – MÁJEKOVÁ, Magdaléna – DREMENCOV, Eliyahu – MACH, Mojmír**. Electrophysiology and Behavioral Assessment of the New Molecule SMe1EC2M3 as a Representative of the Future Class of Triple Reuptake Inhibitors. In Molecules, 2019, vol. 24, no. 23, art. no. 4218. (3.060 – IF2018).

Determination of mechanical properties in biology using Brillouin microscopy

Silvia Bágeľová Poláková

Brillouin microscopy is used for non-contact mapping of mechanical properties of samples, with possible applications in cell biology. In collaboration with Dr. Gregan from the PriF UK and Kareem Elsayad from Vienna (Advanced Microscopy Facility, Vienna Biocenter Core Facilities, Vienna Biocenter), we have recently published an overview of the basic principles of this technology, as well as its suitability for use in biology and medicine. Many processes in cell biology, from embryogenesis to metastasis, are governed not only by biochemical signals but also by mechanical properties and physical forces. Measuring mechanical properties (how soft or hard a particular subcellular structure may be) is not trivial. Although there are many techniques to measure the mechanical properties of cells, they often come with limitations. Brillouin microscopy is a well-established technique in materials physics and research. Recent applications of Brillouin microscopy have recently expanded into cell biology and biomedical research. A key advantage of Brillouin microscopy is that it is a non-contact optical technique without the need for labeling of the subcellular structures under study, allowing spatial mapping of the elastic module in 3D or 4D.

ELSAYAD, Kareem – POLÁKOVÁ, Silvia – GREGAN, Juraj. Probing Mechanical Properties in Biology Using Brillouin Microscopy. In Trends in Cell Biology, 2019, vol. 29, no. 8, p. 6608-6611. (16.588 – IF2018). ISSN 0962-8924.

Analysis and genotype-phenotype correlation of homogentisate 1,2-dioxygenase (HGD) gene variants in the largest cohort of patients with AKU.

Jana Královičová, Ivana Borovská

Alkaptonuria (AKU) is a rare, autosomal recessive metabolic disorder caused by a deficiency of the enzyme homogentisate 1,2-dioxygenase (HGD), involved in tyrosine degradation. The disease has a very low prevalence in most ethnic groups (1:100,000 to 250,000), but within Slovakia and the Dominican Republic the prevalence has increased to 1:19,000. The enzymatic defect in AKU is caused by homozygous or compound heterozygous mutations in the HGD gene. Mutations that result in an amino acid change (“missense”) are the most abundant (66.37%), followed by small deletions and insertions that cause a reading frame shift, and finally nonsense and RNA splicing affecting variants. Identification of the mutation that causes AKU tends to be the final confirmation of the diagnosis.

Structure of HGD hexamer with marked localization of 15 new “missense” variants (red dots).

The collaboration, which involved several major European centres, resulted in the identification of 28 new HGD gene variants from a total of 172 AKU patients from 39 countries. These variants included, among others, three large genomic deletions discovered using the MPLA (self-designed multiplex ligation-dependent probe amplification) method. In turn, the minigene transfection method indicated that three of the eight variants tested may affect RNA editing. To assess the potential inactivating effect of novel missense mutations, analysis by mCSM proved to be an effective bioinformatics tool. For the first time, a genotype-phenotype correlation study of the three most frequent HGD variants was also performed in AKU. This analysis showed that total protein intake plays a more significant role in the accumulation of homogentisic acid in the body of AKU patients than differences in HGD variants.

ASCHER, David – SPIGA, Ottavia – SEKELSKÁ, Martina – PIRES, Douglas E.V. – BERNINI, Andrea – TIEZZI, Monica – KRÁLOVIČOVÁ, JanaBOROVSKÁ, Ivana – ŠOLTÝSOVÁ, Andrea – OLSSON, Birgitta – GALDERISI, Silvia – CICALONI, Vittoria – RANGANATH, Lakshminarayan R. – SANTUCCI, Annalisa – ZAŤKOVÁ, Andrea. Homogentisate 1,2-dioxygenase (HGD) gene variants, their analysis and genotype-phenotype correlations in the largest cohort of patients with AKU. In European Journal of Human Genetics, 2019, vol. 27, no. 6, p. 888-902. (3.650 – IF2018). ISSN 1018-4813.

Feeding different sources of zinc and fibre in the diet positively influences the mineral and antioxidant status of pigs after weaning

Holodová Monika, Čobanová Klaudia, Šefčíková Zuzana, Grešáková Ľubomíra

The weaning period is one of the most critical periods for animals, when there are increased demands for trace element supply but the absorption capacity of the small intestine is insufficient. To increase the bioavailability of nutrients and trace elements from feed, we enriched pig diets with a source of potato fiber and organic Zn in the form of its glycine chelate. The source of Zn did not affect its absorption from the intestine and utilization in tissues, but did affect the absorption of Fe and Mn from the digestive tract. Dietary supplementation with potato fiber increased active Zn transport in enterocytes and the antioxidant activity of Zn-dependent enzymes in tissues. Based on the activity of antioxidant enzymes, trace element concentrations in tissues, and the content of metal transporters in the intestine, we can say that the combination of both additives improved the antioxidant and mineral status of pigs during the weaning period.

HOLODOVÁ, MonikaČOBANOVÁ, KlaudiaŠEFČÍKOVÁ, Zuzana – BARSZCZ, Marcin – TUSNIO, Anna – TACIAK, Marcin – GREŠÁKOVÁ, Ľubomíra. Dietary Zinc and Fibre Source can Influence the Mineral and Antioxidant Status of Piglets. Animals, 2019, vol. 9, no. 8, art. no. 497, 16 p. (1.832 – IF2018). ISSN 2076-2615.