Significant scientific results 2021

Silencing the expression of a large number of genes requires interplay between the homologous recombination pathway and the histone chaperone HIRA

Silvia Bágeľová Poláková, Alexandra Piteľová, Ivana Mišová, Karolína Pichlerová, Zsigmónd Benkő

Over the course of evolution, eukaryotes have evolved two pathways responsible for repairing double-strand breaks during mitotic cell division: joining of non-homologous ends and homologous recombination (HR). Our recent research has shown that in yeast S. pombe with HR gene deletion, there is an increase in the expression of more than 500 loci, mainly in the region of subtelomeres, antisense transcripts, and LTR retrotransposons, which represent so-called mobile repetitive elements in the genome. Comparison of the expression profiles showed that HR proteins repress the expression of the same genes as the histone chaperone complex HIRA. Interestingly, the absence of HR proteins can restore the correct level of expression in the centromere region, which is altered in HIRA mutants. In this publication, we provide for the first time evidence for a link between the homologous recombination pathway and the histone chaperone complex of HIRA, which until recently was mainly associated with nucleosome deposition after transcription or with the silencing of retrotransposon activity.

A model for silencing gene expression in hard-to-replicate regions of the genome, such as LTR elements. Such regions in standard-type cells cause replication arrest or collapse of the replication fork during replication, which is accompanied by the formation of single-stranded and double-stranded DNA breaks. Subsequently, HR proteins, in cooperation with the HIRA complex, restore chromatin structure in these regions, leading to silencing of expression.

MIŠOVÁ, Ivana*PITEĽOVÁ, Alexandra* – BUDIŠ, J. – GAZDARICA, Juraj – SEDLÁČKOVÁ, Tatiana – JORDAKOVÁ, Anna – BENKŐ, Zsigmónd – SMONDRKOVÁ, M. – MAYEROVÁ, Nina PICHLEROVÁ, K. – STRIEŠKOVÁ, Lucia – PREVOROVSKY, Martin – GREGAN, Juraj – ČIPÁK, Ľuboš – SZEMES, Tomáš – BÁGEĽOVÁ POLÁKOVÁ, Silvia**. Repression of a large number of genes requires interplay between homologous recombination and HIRA. In Nucleic acids research, 2021, vol. 49, no. 4, p. 1914-1934. (2020: 16.971 – IF, Q1 – JCR, 9.008 – SJR, Q1 – SJR). ISSN 0305-1048.

Some specific changes in leukemia cells associated with the onset of drug resistance

Albert Breier, Viera Boháčová, Ivana Borovská, Ľuboš Janotka, Helena Kavcová, Zuzana Kočibálová, Tomáš Kyca, Lucia Messingerová, Lucia Pavlíková, Alexandra Poturnayová, Zdena Sulová, Mário Šereš, Kristína Šimoničová

We have shown that in acute myeloid leukemia cell lines, expression of the drug transporter P-glycoprotein (P-gp) is associated with a decrease in latrophilin 1 expression. Latrophilin 1, found in the plasma membrane of leukemic myeloid blasts, regulates the expression and secretion of Tim-3 and galectin-9, which allow leukemic cells to evade elimination by the immune system. We are the first to identify all 8 previously only predicted Galectin-9 knockdown variants at the mRNA and protein level. Patients with severe difficult-to-treat acute myeloid leukemia are treated with the DNA-demethylating drugs 5-azacytidine (AZA) and 2-deoxy-5-azacytidine (DAC). In some cases, the leukemia cells are resistant to these treatments. We have generated cellular variants of MOLM-13 cells resistant to either AZA or DAC, in which cross-resistance was not observed, nor did they express increased amounts of typical ABC family drug transporters. Both drugs are activated in cells by sequential phosphorylation on the respective nucleotide triphosphates. Alterations in this activation process may cause a reduced cellular response to AZA and DAC. The proteasome inhibitor bortezomib, is approved for the treatment of severe cases of multiple myeloma. This agent, by blocking proteosomal degradation of poorly folded proteins, leads to their accumulation, subsequent endoplasmic reticulum (SER) stress and programmed death of tumor myeloid blasts. Although the drug transporter P-gp does not transport bortezomib, P-gp-expressing cells respond differently to bortezomib than P-gp-negative cells. This is due to increased expression of BiP, an SER-regulating protein, and the molecular chaperone HSP90α or β. The altered levels of these proteins are accompanied by changes in the expression of the cell cycle and apoptosis regulator p53, the cyclin-dependent kinase inhibitors p15, p21, p27, and the cyclins A1, B1, D1, and E1. All these results point to the complexity of the events involved in the development of drug resistance in leukemic cells.

JANOTKA, ĽubošMESSINGEROVÁ, Lucia**ŠIMONIČOVÁ, KristínaKAVCOVÁ, Helena – ELEFANTOVÁ, Katarína – SULOVÁ, Zdena**BREIER, Albert**. Changes in Apoptotic Pathways in MOLM-13 Cell Lines after Induction of Resistance to Hypomethylating Agents. In International Journal of Molecular Sciences, 2021, vol. 22, no. 4, art. no. 2076. (2020: 5.923 – IF, Q1 – JCR, 1.455 – SJR, Q1 – SJR). ISSN 1422-0067.

KYCA, TomášPAVLÍKOVÁ, LuciaBOHÁČOVÁ, Viera – MIŠÁK, Anton – POTURNAYOVÁ, AlexandraBREIER, Albert**SULOVÁ, Zdena**ŠEREŠ, Mário**. Insight into Bortezomib Focusing on Its Efficacy against P-gp-Positive MDR Leukemia Cells. In International Journal of Molecular Sciences, 2021, vol. 22, no. 11, art. no. 5504. (2020: 5.923 – IF, Q1 – JCR, 1.455 – SJR, Q1 – SJR). ISSN 1422-0067.

KOČIBÁLOVÁ, Zuzana – GUZYOVÁ, Martina – BOROVSKÁ, IvanaMESSINGEROVÁ, Lucia – COPÁKOVÁ, Lucia – SULOVÁ, Zdena** – BREIER, Albert**. Development of Multidrug Resistance in Acute Myeloid Leukemia Is Associated with Alterations of the LPHN1/GAL-9/TIM-3 Signaling Pathway. In Cancers, 2021, vol. 13, no. 14, art. no. 3629. (2020: 6.639 – IF, Q1 – JCR, 1.818 – SJR, Q1 – SJR). ISSN 2072-6694.

Dead cells in mouse embryos are eliminated by neighboring cells that function as non-professional phagocytes

Jozef Pisko, Alexandra Špirková, Štefan Čikoš, Veronika Kovaříková, Zuzana Šefčíková, Dušan Fabian

The aim of the study was to increase the knowledge of the physiological patterns of embryo development during the period from embryo formation to embryo implantation in the uterus. It is known that the embryo during this period is sensitive to changes in the maternal environment (civilization diseases, improper lifestyle, etc.), which leads to a higher incidence of developmental disorders. The resulting disorders are resolved by the embryo through various self-repair mechanisms, such as apoptosis (destruction of damaged cells by activation of the genetic programme of cell death). The unresolved question remains what is the fate of these dead cells. The results of experiments on mouse blastocysts have shown that a minor fraction of the dead cells are expelled into the embryo cavity (up to 10%), but the majority are eliminated by neighbouring cells through phagocytosis. We have shown that dead embryonic cells express surface markers that are required for their recognition by non-professional phagocytes, and that low-differentiated cells of both basic cell lineages of the preimplantation embryo are able to phagocytose and degrade dead cells. We have also described some aspects of the molecular mechanism of embryonic phagocytosis, providing insights into key, evolutionarily conserved features of the process.

PISKO, JozefŠPIRKOVÁ, AlexandraČIKOŠ, Štefan – OLEXIKOVÁ, Lucia – KOVAŘÍKOVÁ, VeronikaŠEFČÍKOVÁ, ZuzanaFABIAN, Dušan**. Apoptotic cells in mouse blastocysts are eliminated by neighbouring blastomeres. In Scientific Reports, 2021, vol. 11, no. 1, art. no. 9228. (2020: 4.379 – IF, Q1 – JCR, 1.240 – SJR, Q1 – SJR, karentované – CCC). (2021 – Current Contents WOS, SCOPUS). ISSN 2045-2322.

Traditional Slovak sheep lump cheese, source of a beneficial strain of Enterococcus durans

Andrea Lauková, Martin Tomáška, Vladimír Kmeť, Viola Strompfová, Monika Pogány Simonová, Emília Dvorožňáková

Enterococci belong to the community of lactic acid-producing bacteria, which are a controversial group. Although they are bacteria commonly found in dairy products, many species or strains of enterococci are becoming increasingly resistant to antibiotics and virulence factor genes are often detected, which can lead to health problems among consumers of these products. Therefore, it is necessary to know whether to exclude the apathogenicity/harmlessness of the detected enterococci species and strains or, on the contrary, to know their beneficial properties, especially in food commodities that are preferred as so-called healthy foods, which include raw goat milk or traditional Slovak sheep lump cheese because of their well-known properties. In the case of enterococci pathogenicity, it is again important to find an effective way of inactivating them, such as antimicrobial, non-residual substances of a protein nature, the so-called bacteriocins and, within them, the enterocins. Slovak sheep’s lump cheese is made from unpasteurised sheep’s milk without starter culture. As it is a product that is produced in the traditional way and is manually moulded into the required shape, it has been classified as a TSG product in the EU, i.e. a ‘traditional speciality guaranteed’, i.e. a guaranteed/traditional speciality. So far, there have been only very limited studies on the individual microbiota in sheep lump cheese. The detected E. durans strain ED26E/7 (identification by MALDI-TOF mass spectrometry) is a non-haemolytic strain, without virulence factor genes (gelatinase, hyaluronidase or IS 16 element), sensitive to commercially used antibiotics. This strain produces the beneficial enzyme lactase (β-galactosidase), which is used in the production of dairy products for lactose-intolerant humans, and also produces an antimicrobial bacteriocin with inhibitory activity mainly against Gram-positive microbiota with activity up to 102 400 AU/mL (including Staphylococcus aureus strain SA5 and listeria). Additionally, the growth of 16 out of 22 different staphylococci was inhibited, as well as the growth of 36 strains of enterococci out of 51 tested. ED26E/7 is an apathogenic strain; it did not cause mortality in the Balb/c mouse experiment. Both E. durans strain ED26E/7 and strain EM 2/2 (from raw goat milk) represent (after further technological tests) beneficial additive bacteria for dairy use.

LAUKOVÁ, Andrea** – TOMÁŠKA, Martin – KMEŤ, VladimírSTROMPFOVÁ, ViolaPOGÁNY SIMONOVÁ, Monika – DVOROŽŇÁKOVÁ, Emília. Slovak Local Ewe’;s Milk Lump Cheese, a Source of Beneficial Enterococcus durans Strain. In Foods, 2021, vol. 10, no., art. no. 3091. (2020: 4.350 – IF, Q2 – JCR, 0.774 – SJR, Q1 – SJR, karentované – CCC). (2021 – Current Contents). ISSN 2304-8158.

LAUKOVÁ, Andrea**FOCKOVÁ, ValentínaPOGÁNY SIMONOVÁ, Monika. Enterococcal Species Associated with Slovak Raw Goat Milk, Their Safety and Susceptibility to Lantibiotics and Durancin ED26E/7. In Processes, 2021, vol. 9, no. 4, art. no. 681. (2020: 2.847 – IF, Q3 – JCR, 0.414 – SJR, Q2 – SJR). ISSN 2227-9717.

Studying the use of polymeric nanoparticles as a transport system for photodynamic diagnosis and therapy of diseases in a chorioallantoic membrane model

Boris Bilčík, Ivan Čavarga, Barbora Kundeková, Mariana Máčajová, Majlinda Meta

Photodynamic method seems to be promising for cancer diagnosis and therapy. Light-sensitive substances, photosensitizers (PS), are injected into the body where they are primarily taken up by the affected tissue. When the tissue is irradiated, the PS emits fluorescence, thereby aiding in the localization of the affected tissue. During therapy, when activated by high-intensity light, PS generates reactive oxygen species in the tissue, leading to cell death and destruction of the pathological tissue. Photosensitizers, however, are small molecules that circulate briefly in the bloodstream. The drug release profile is enhanced by the use of transport systems, the properties of which we have studied in the chorioallantoic membrane (CAM) model of the avian embryo. One of the agents studied was gradient copolymers based on poly(2-oxazoline) with an embedded hydrophobic hypericin molecule. We investigated their biodistribution, release and phototoxicity in vitro and in vivo in CAM. Our experiments showed the promising potential of polymeric nanoparticles for drug delivery, sequential drug release, photodiagnostics and photodynamic therapeutic approaches.

KUNDEKOVÁ, Barbora**MÁČAJOVÁ, MarianaMETA, MajlindaČAVARGA, IvanBILČÍK, Boris. Chorioallantoic Membrane Models of Various Avian Species Differences and Applications. In Biology-Basel, 2021, vol. 10, no. 4, art. no. 301. (2020: 5.079 – IF, Q1 – JCR, 1.731 – SJR, Q1 – SJR). ISSN 2079-7737.

HUNTOŠOVÁ, Veronika** – DATTA, Shubhashis* – LENKAVSKÁ, Lenka – MÁČAJOVÁ, MarianaBILČÍK, BorisKUNDEKOVÁ, BarboraČAVARGA, Ivan – KRONEK, Juraj – JUTKOVÁ, Annamária – MIŠKOVSKÝ, Pavol – JANCURA, Daniel. Alkyl chain length in poly(2-oxazoline)-based amphiphilic gradient copolymers regulates the delivery of hydrophobic molecules: A case of the biodistribution and the photodynamic activity of the photosensitizer hypericin. In Biomacromolecules, 2021, vol. 22, no. 10, p. 4199-4216. (2020: 6.988 – IF, Q1 – JCR, 1.689 – SJR, Q1 – SJR). ISSN 1525-7797.

BEŇOVÁ, Eva – HORNEBECQ, Virginie** – ZELEŇÁK, Vladimír** – HUNTOŠOVÁ, Veronika – ALMÁŠI, Miroslav – MÁČAJOVÁ, Mariana – BERGÉ-LEFRANC, David. pH-responsive mesoporous silica drug delivery system, its biocompatibility and co-adsorption/co-release of 5-Fluorouracil and Naproxen. In Applied Surface Science, 2021, vol. 561, art. no. 150001. (2020: 6.707 – IF, Q1 – JCR, 1.295 – SJR, Q1 – SJR, karentované – CCC). (2021 – Current Contents, WOS, SCOPUS). ISSN 0169-4332.

Investigation of the thiadiazine derivative L -17 with putative neuro- and cardioprotective and antidepressant effects in silico, ex vivo and in vivo

Eliyahu Dremencov, Daniil Grinchii, Ruslan Paliokha

Depression that is associated with general ill health, such as depression after a stroke or myocardial infarction, is also characterised by resistance to conventional antidepressants. Special treatment strategies should therefore be developed for such cases. Our study aims to investigate the mechanism of action of the L-17 molecule, with recently proposed neuro- and cardioprotective as well as antidepressant effects. Evaluation of the molecular properties of L-17 by computer simulation (in silico) implies an interaction with target molecules involved in the depressive state, such as serotonin receptors and transporters. The strong interaction of L-17 with serotonin transporters was also demonstrated by electrophysiological measurements of the excitability of brain serotonergic neurons in vivo before and after L-17 administration. Using in vitro neurochemical evaluations, we found that L-17 strongly alters activity in brain regions mediating emotional behavior, the amygdala and hippocampus. The present study is important both methodologically and conceptually. Methodologically, we have demonstrated that combined in silico, in vitro and in vivo evaluations of newly proposed organic molecules can be a powerful tool for their early preclinical evaluation as future central nervous system (CNS) drugs. Conceptually, our study suggested a possible mechanism explaining the putative antidepressant properties of L-17. Because L-17 is also characterized by putative neuro- and cardioprotective effects, this substance could be beneficial in the treatment of depression after stroke or after myocardial infarction.

SARAPULTSEV, Alexey P.** – VASSILIEV, Pavel M. – GRINCHII, Daniil – KISS, Alexander – MACH, Mojmír – OSACKÁ, Jana – BALLÓOVÁ, Alexandra – PALIOKHA, Ruslan – KOCHETKOV, Andrey – SIDOROVA, Larisa – SARAPULTSEV, Petr A. – CHUPAKHIN, Oleg N. – RANTSEV, Maxim – SPASOV, Alexander – DREMENCOV, Eliyahu**. Combined In Silico, Ex Vivo, and In Vivo Assessment of L-17, a Thiadiazine Derivative with Putative Neuro- and Cardioprotective and Antidepressant Effects. In International Journal of Molecular Sciences, 2021, vol. 22, no. 24, art. no. 13626. (2020: 5.923 – IF, Q1 – JCR, 1.455 – SJR, Q1 – SJR, karentované – CCC). (2021 – Current Contents). ISSN 1422-0067.

Intron length as an important factor determining the assembly of mutually exclusive exons of the OGDH gene. Pathway to the development of endothermy

Ivana Borovská, Jana Královičová

Mutually exclusive exons (MXEs) represent a rare type of alternative splicing (AZ) RNA that represents a fraction of about 855 pairs in the human genome. This type of AZ offers the cell a choice of two or more protein isoforms that are very similar in size and function, thus allowing for less modification of protein activity than other types of AZ. It also ensures continuity of essential cellular functions after exon duplication. One of the mechanisms involved in MXE enforcement in the RNA editing process is steric hindrance due to the proximity of the 5′-cleavage site and the branching point. This type of regulation affects only about 9% of exonic pairs, which are most abundant in genes encoding proteins involved in calcium signaling. Cytosolic calcium is a critical ion in muscle contraction, and MXE downregulation is frequently found in genes related to striated muscle function. In our work, we aimed to characterize the evolutionary process leading to the MXE downregulation in the gene for 2-oxoglutarate dehydrogenase (OGDH), which enabled the establishment of a critical, regulatory point of Krebs cycle activity as a driver of central metabolic conversion in animal evolution. We have investigated the organization and structural requirements of the intron splice sites of the MXE deletion exons 4a and 4b in the context of competitive and cooperative interactions of poly(U) RNA-binding proteins. Our results provide new insights into the regulation of MXE assembly, which influences OGDH complex activation and NADH and ATP supply in response to mitochondrial calcium.

KRÁLOVIČOVÁ, Jana*BOROVSKÁ, Ivana* – PENGELLY, Reuben – LEE, Eunice – ABAFFY, Pavel – ŠINDELKA, Radek – GRUTZNER, Frank – VOŘECHOVSKÝ, Igor**. Restriction of an intron size en route to endothermy. In Nucleic Acids Research, 2021, vol. 49, no. 5, p. 2460-2487. (2020: 16.971 – IF, Q1 – JCR, 9.008 – SJR, Q1 – SJR). ISSN 0305-1048.

Zinc in combination with medicinal plants in feed can affect rumen fermentation, microbiota and histopathology of lambs

Daniel Petrič, Dominika Mravčáková, Katarína Kucková, Svetlana Kišidayová, Klaudia Čobanová, Zora Váradyová

The study presents unique results of the use of nutraceuticals containing organic zinc and a selected mixture of dried medicinal plants as nutritional supplements in sheep. The combination of organic zinc and medicinal plant mixtures can positively affect sheep health without adversely affecting rumen and intestinal fermentation characteristics. These findings emphasize that the effect of dried medicinal plants depends on the diversity, mutual synergy and combination of plant bioactives. To the best of our knowledge, this is the first study to determine the interactions between organic zinc supplementation and a mixture of dried medicinal plants using phytochemical, physiological, microbiological and histopathological measurements and analyses in the rumen of lambs. The study showed that the lambs’ microbiome can adapt to long-term (70 days) supplementation with organic zinc; however, the combination of zinc with a mixture of four dried medicinal plants, (i.e. wormwood, chamomile, ground elder and wood mallow), which have strong antioxidant activity, may negatively affect the health of the rumen epithelium. The multidisciplinary approach in this study provides a more comprehensive view of the use of nutraceuticals as additives in ruminant nutrition.

PETRIČ, DanielMRAVČÁKOVÁ, DominikaKUCKOVÁ, KatarínaKIŠIDAYOVÁ, Svetlana – CIESLAK, Adam – SZUMACHER-STRABEL, Malgorzata – HUANG, Haihao – KOLODZIEJSKI, Pawel – LUKOMSKA, Anna – SLUSARCZYK, Sylwester – ČOBANOVÁ, KlaudiaVÁRADYOVÁ, Zora**. Impact of Zinc and/or Herbal Mixture on Ruminal Fermentation, Microbiota, and Histopathology in Lambs. In Frontiers in Veterinary Science, 2021, vol. 8, art. no. 630971. (2020: 3.412 – IF, Q1 – JCR, 0.877 – SJR, Q1 – SJR). ISSN 2297-1769.

Are hens with a high frequency of feather pecking more impulsive in their decision making?

Katarína Pichová, Ľubor Košťál

Wageningen Institute of Animal Science fellowship,

Feather picking is a serious problem from the point of view of both the welfarist and the economics of laying hen farming. In collaboration with Wageningen University & Research, we used a judgment bias method to characterize affective states (emotions) in lines selected for increased and decreased prevalence of feather picking. Emotions influence decisions about how to respond to ambiguous stimuli (“optimism” vs “pessimism”). We hypothesized that an increased incidence of feather nibbing in a line with a high frequency of nibbing would lead to negative affective states, which would be reflected in a negative (pessimistic) bias in judgment. However, our results showed the opposite. One possible explanation is that laying hens from a line with a high frequency of feather pecking are more impulsive, which affects their decision making. Our results complement the behavioural characterization of the lineages and point to the need to test individual phenotypes (preening, preening and neutral individuals) within lineages.

PICHOVÁ, Katarína**KOŠŤÁL, Ľubor – DE HAAN, Tara – VAN DER EIJK, Jerine A.J. – RODENBURG, T. Bas. High and low feather pecking selection lines of laying hens differ in response to a judgment bias test. In Applied Animal Behaviour Science, 2021, vol. 238, art. no. 105305. (2020: 2.448 – IF, Q1 – JCR, 0.726 – SJR, Q1 – SJR). ISSN 0168-1591.