Significant scientific results 2018

Disrupted activation of 3′ splice sites due to inherited mutations in the RRM motif of the PUF60 splicing factor

Jana Královičová, Ivana Ševčíková

Eukaryotic genes contain introns that must be removed from the mediator RNA precursor by a large and highly dynamic spliceosome complex. The spliceosome is formed on each intron, and includes U1, U2, and U4/5/6 small nuclear ribonucleoproteins and a number of other proteins. A critical step in the formation of the spliceosome apparatus is the interaction of U1 with the 5′ splice site and U2 with the branching point, which is assisted by a U2 auxiliary factor (U2AF) that binds to the 3′ splice site (3′ss).

PUF60 is one of the assembly facts that are homologous to U2AF. The PUF60 molecule contains two RNA recognition motifs (RRMs) and, in cooperation with U2AF, facilitates the association of U2 with the primary transcript. PUF60 deficiency (PD) is associated with developmental delay, intellectual disability, and renal, spinal cord, and cardiac dysfunctions, but the pathogenesis of the disease has not yet been elucidated.

Using RNA NGS, we identified and characterized genes/exons regulated by the PUF60 protein, which acts predominantly as their activator (panel A). Using co-transfection of knockdown minigenes with PUF60 constructs, we investigated the functional consequences of PD-associated PUF60 mutations. We found that the mutations affect the selection of competent PUF60-regulated 3′ knockdown sites, and that heterogeneity of germline mutations localized in the same PUF60 RRM can increase phenotypic variability at the level of knockdown site selection (panel B).

KRÁLOVIČOVÁ, JanaŠEVČÍKOVÁ, Ivana – STEJSKALOVÁ, Eva – OBUCA, Mina – HILLER, Michael – STANĚK, David – VOŘECHOVSKÝ, Igor. PUF60-activated exons uncover altered 3 ‘; splice-site selection by germline missense mutations in a single RRM. In Nucleic acids research, 2018, vol. 46, no. 12, p. 6166-6187. (11.561 – IF2017). ISSN 0305-1048.

Cardiolipin synthase of the yeast Schizosaccharomyces pombe and its regulation

Veronika Virčíková, Lucia Pokorná, Dana Tahotná, Mária Balážová, Peter Griač

Disturbances in the homeostasis of the mitochondrial phospholipid cardiolipin result in several diseases in humans, some of them hereditary. We focused on the study of the cardiolipin biosynthetic pathway in the yeast Schizosaccharomyces pombe. We found that CL synthase is part of a mitochondrial tandem protein together with a hydrolase of unknown function. We have discovered a novel and unique mechanism by which the eukaryotic cell ensures the abundance of individual partners of this fusion protein and thus regulates the relative expression of two proteins that have evolved during evolution into a tandem fusion protein controlled by a common promoter.

VIRČÍKOVÁ, VeronikaPOKORNÁ, LuciaTAHOTNÁ, Dana – DŽUGASOVÁ, Vladimíra – BALÁŽOVÁ, MáriaGRIAČ, Peter. Schizosaccharomyces pombe cardiolipin synthase is part of a mitochondrial fusion protein regulated by intron retention. In Biochimica et Biophysica Acta : molecular and cell biology of lipids, 2018, vol. 1863, iss. 10, p. 1331-1344. (4.966 – IF2017). ISSN 1388-1981.

High doses of manganese affect the rumen microbiome

Svetlana Kišidayová, Peter Pristaš, Klaudia Čobanová, Ľubomíra Grešáková, Zora Váradyová

The effect of increased intake of organic and inorganic manganese on the ruminal ecosystem of ruminants is poorly studied. In our study, we investigated the effect of increased manganese intake on hydrolytic activities and microbial (protozoal, eubacterial and archaeal) species diversity and abundance in the rumen of lambs. We found that the type of manganese had no effect on the parameters studied. However, increased manganese dose affected the diversity and abundance of eubacterial population, which was also reflected in the observed hydrolytic activities.

KIŠIDAYOVÁ, SvetlanaPRISTAŠ, Peter – ZIMOVČÁKOVÁ, Michaela – WENCELOVÁ, MonikaHOMOĽOVÁ, LuciaMIHALIKOVÁ, KatarínaČOBANOVÁ, KlaudiaGREŠÁKOVÁ, ĽubomíraVÁRADYOVÁ, Zora. The effects of high dose of two manganese supplements (organic and inorganic) on the rumen microbial ecosystem. In PLoS ONE, 2018, vol. 13, iss. 1, art. no. e0191158. (2.766 – IF2017).

Isolation, identification and characterization of lactic acid bacteria for their application in the dairy industry

Anna Kopčáková, Martin Šuľák, Peter Javorský, Vladimír Kmeť, Andrea Lauková, Peter Pristaš

ITMS 26220220065

“Bryndza” is a traditional Slovak cheese made from unpasteurised milk. Enterococci are a key component of the microbiota in the maturation of cheese in raw sheep’s milk, but their survival in the fermentation process is affected by phage infections. Currently, very few data are available on the occurrence of restriction-modification systems (RMS) in enterococci from sheep milk. In our experiments, we characterized 8 types of type II RMS in 61 identified enterococcal isolates. We found that the frequency of RMS in enterococci from sheep milk is low and RMS are not the main defense mechanism against phage infection.

KOPČÁKOVÁ, Anna – DUBÍKOVÁ, Katarína – ŠUĽÁK, MartinJAVORSKÝ, PeterKMEŤ, VladimírLAUKOVÁ, AndreaPRISTAŠ, Peter. Restriction-modification systems and phage resistance of enterococci from ewe milk. In LWT – Food Science and Technology, 2018, vol. 93, p. 131-134. (3.129 – IF2017). ISSN 0023-6438.

Modeling CD9 and CD81 interactions on sperm prior to fertilization.

Jana Jankovičová, Petra Sečová, Jana Antalíková

The processes preceding sperm-oocyte fusion are associated with a radical reorganization of the membranes of both gametes, regulated, among other things, by cholesterol efflux not only in lipid rafts but also in microdomains rich in tetraspanins, molecules capable of forming a tetraspanin network by cross-linking and interacting with other proteins. Based on the detection and localization of the tetraspanins CD9 and CD81 on bovine, mouse and human spermatozoa, in collaboration with the Institute of Biotechnology of the CAS, we have developed a structural model of homologous and heterologous interactions of these two molecules within the sperm plasma membrane (Cis-interactions) and during gamete fusion (Trans-interactions). CD81, due to its ability to bind and release cholesterol, probably acts as a regulator of the dynamic mechanism of the tetraspanin network within the sperm membrane, while CD9 stabilizes the membrane and allows trans-interaction upon sperm-oocyte contact.

Proposed arrangement of the CD81/CD9 tetraspanin network. The CD9 and CD81 complex can be formed by combining a CD81 oligomer formed by alternating non-covalent (yellow-orange) and disulfide bridge-stabilized (orange-orange) dimers. The non-covalent CD9 dimer (shades of blue) can interact with two such oligomers. (A, B) Side and frontal views. (C) Top view.

FROLÍKOVÁ, Michaela – MAŇÁSKOVÁ-POSTLEROVÁ, Pavla – ČERNÝ, Jiří – JANKOVIČOVÁ, Jana – ŠIMONÍK, Ondřej – POHLOVÁ, Alžběta – SEČOVÁ, PetraANTALÍKOVÁ, Jana – DVOŘÁKOVÁ-HORTOVÁ, Kateřina. CD9 and CD81 Interactions and Their Structural Modelling in Sperm Prior to Fertilization. In International Journal of Molecular Sciences, 2018, vol. 19, iss. 4, art. n. 1236. (3.687 – IF2017). ISSN 1422-0067.

Investigation of the possibility to effectively chemotherapeutically target leukemic cells with developed multidrug resistance based on P-gp efflux activity

Viera Boháčová, Mário Šereš, Lucia Pavlíková, Szilvia Kontár, Martin Cagala, Zdena Sulová, Albert Breier

We have shown that triorganocin derivatives, known as xenobiotic ligands of RXR nuclear receptors, at submicromolar concentrations induce apoptosis of L1210 cells independently of P-gp expression. The significance of this result can be underscored by the fact that in applied and translational research associated with the rational development of anticancer drugs, there is increasing interest in those that cannot eliminate proteins associated with multidrug resistance from cells. It seems that such agents could include stannans (organocin derivatives), as they show a more pronounced effect on neoplastically transformed than on normal cells. In cells with increased P-gp expression it is difficult to use analytical methods based on the measurement of retention and specific fluorescence of intracellular markers as these are often substrates of P-gp. This is also the situation when measuring mitochondrial membrane potential with JC-1. Therefore, we have developed a rapid method based on the specific inhibition of P-gp with tariquidar that allows detection of mitochondrial membrane potential even in cells with high P-gp content. This method may find application in applied research associated with the rapid detection of mitochondrial damage by cytotoxic agents even in neoplastic cells with developed multidrug resistance mediated by P-gp efflux activity.

BOHÁČOVÁ, VieraŠEREŠ, MárioPAVLÍKOVÁ, LuciaKONTÁR, SzilviaCAGALA, Martin – BOBÁĽ, Pavel – OTEVŘEL, Jan – BRTKO, Július – SULOVÁ, Zdena – BREIER, Albert. Triorganotin derivatives induce cell death effects on L1210 leukemia cells at submicromolar concentration independently of P-glycoprotein expression. In Molecules, 2018, vol. 23, iss. 5, art. no. 1053 (3.098 – IF2017). ISSN 1420-3049.

. Detection of the Mitochondrial Membrane Potential by the Cationic Dye JC-1 in L1210 Cells
with Massive Overexpression of the Plasma Membrane ABCB1 Drug Transporter.
In International
Journal of Molecular Sciences, 2018, vol. 19, art. no. 1985. (3.687 – IF2017). ISSN 1422-0067.

Changes in songbird brains revealed by magnetic resonance imaging

Kristína Lukáčová, Ľubica Kubíková

Our laboratory is one of only 3 in the world able to measure changes in the songbird brain using magnetic resonance imaging (MRI). This method has an advantage over conventional histological or electrophysiological methods in that it allows repeated measurements throughout the brain in the same individual and over a long period of time. In collaboration with a site in Belgium (Bio-Imaging Lab, Antwerp), we detected volumetric changes in the vocal regions of the developing brain of songbirds of both sexes during adolescence, with males learning to sing during this period. We also used the diffusion-weighted imaging (DTI) method and its parameters fractional anisotropy (FA), mean diffusivity (MD), axial and radial diffusivity (lambda), which indicate structural tissue remodeling after lesioning of one of the vocal brain regions, Area X. We detected changes not only in the lesion, but also in areas efferently associated with Area X. Surprisingly, we also detected changes in the cerebellum, which could be a new region involved in the control of learned vocal communication. Our results clearly show structural neuroplasticity of the brain both during learning and during brain damage and subsequent recovery. These results also point to the existence of a pathway involving the cerebellum for the mediation and control of learned vocalizations in songbirds, similar to that in humans.

The DTI method detects microstructural changes in the cerebellum-thalamus-basal ganglia pathway. The maps show statistically significant changes in DTI parameters (FA, MD, l1) in the zebrafish red zebrafish brain after Area X lesion. The circled letters show the main changes: a – Area X (lesion), b – micropipette input, c – dorsal thalamus, d – lateral cerebellar nuclei, e – tOM tract, f – CA tract.

HAMAIDE, Julie – LUKÁČOVÁ, Kristína – VAN AUDEKERKE, Johan – VERHOYE, Marleen – KUBÍKOVÁ, Ľubica – VAN DER LINDEN, Annemie. Neuroplasticity in the cerebello-thalamo-basal ganglia pathway: A longitudinal in vivo MRI study in male songbirds. In NEUROIMAGE, 2018, vol. 181, p. 190-202. (5.426 – IF2017). ISSN 1053-8119.

HAMAIDE, Julie – DE GROOF, Geert – VAN RUIJSSEVELT, Lisbeth – LUKÁČOVÁ, Kristína – VAN AUDEKERKE, Johan – VERHOYE, Marleen – VAN DER LINDEN, Annemie. Volumetric development of the zebra finch brain throughout the first 200 days of post-hatch life traced by in vivo MRI. In NEUROIMAGE, 2018, vol. 183, p. 227-238. (5.426 – IF2017). ISSN 1053-8119.

Plant nutraceuticals and zinc as therapy for nematode infection in lambs

Zora Váradyová, Dominika Mravčáková, Ľubomíra Grešáková, Klaudia Čobanová, Svetlana Kišidayová, Iveta Plachá

The gastrointestinal parasitic nematode Haemonchus contortus is a pathogenic organism resistant to several anthelmintics and causes economic losses on sheep farms, especially in lambs. We attempted to evaluate the efficacy of a mixture of selected medicinal plants and organic zinc in lambs experimentally infected with H. contortus. The direct effect of the nutraceuticals used on the viability of H. contortus has not been fully demonstrated, but we found that treatment with plant nutraceuticals and zinc probably contributed indirectly to increase the resistance of lambs to nematode infection.

VÁRADYOVÁ, ZoraMRAVČÁKOVÁ, Dominika – BABJÁK, Michal – BRYSZAK, Magdalena – GREŠÁKOVÁ, ĽubomíraČOBANOVÁ, KlaudiaKIŠIDAYOVÁ, SvetlanaPLACHÁ, Iveta – KÖNIGOVÁ, Alžbeta – CIESLAK, A. – SLUSARCZYK, Sylwester – PECIO, Lukasz – KOWALCZYK, Mariusz – VÁRADY, Marián. Effects of herbal nutraceuticals and/or zinc against Haemonchus contortus in lambs experimentally infected. In BMC Veterinary Research, 2018, vol. 14, no. 1, art. no. 78. (1.958 – IF2017). ISSN 1746-6148.

Molecular mechanism of regulation of the inotropic effect of Omecamtiv mecarbil

Marta Gaburjáková

Omecamtiv mecarbil (OM) is currently being tested as a new prospective drug for heart failure (Phase 3 clinical testing) that directly stimulates the contractile apparatus of the heart and skeletal muscle. At the level of individual cardiac muscle cells, we have shown that the effect of OM is dependent on the concentration and frequency of contraction. Atypical frequency dependence may lead to diastolic stiffening of the cardiac muscle in some patients in case of overdose. This hypothesis is supported by the ability of OM to activate the cardiac ryanodine receptor (RYR2), which is an essential component in the process of coupling excitation with contraction. Our findings highlighted the potential risks of using supratherapeutic concentrations of OM, which should therefore be investigated in detail at the molecular level.

Potential molecular mechanism of Omecamtiv mecarbil side effect.

NÁNÁSI JR., Péter – KOMÁROMI, István – GABURJÁKOVÁ, Marta – ALMÁSSY, János. Omecamtiv mecarbil: a myosin motor activator agent with promising clinical performance and new in vitro results. In Current Medicinal Chemistry, 2018, vol. 25, no. 15, p. 1720-1728. (3.469 – IF2017). (2018 – Current Contents). ISSN 0929-8673.