F1 hybrids between mouse inbred strains PWD and C57BL/6 represent the most thoroughly genetically defined model of hybrid sterility in vertebrates. Hybrid male sterility can be fully reconstituted from three components of this model, the Prdm9 gene, intersubspecific homeology of Mus musculus musculus and Mus musculus domesticus autosomes, and the X-linked Hstx2 locus. Hstx2 modulates the extent of Prdm9-dependent meiotic arrest and harbors two additional factors responsible for intersubspecific introgression-induced oligospermia (Hstx1) and meiotic recombination rate (Meir1). To facilitate positional cloning and to overcome the recombination suppression within the 4.3 Mb encompassing the Hstx2 locus, we designed Hstx2-CRISPR and SPO11/Cas9 transgenes aimed to induce DNA double-strand breaks specifically within the Hstx2 locus. The resulting recombinant reduced the Hstx2 locus to 2.70 Mb (chromosome X: 66.51-69.21 Mb). The newly defined Hstx2 locus still operates as the major X-linked factor of the F1 hybrid sterility, and controls meiotic chromosome synapsis and meiotic recombination rate. Despite extensive further crosses, the 2.70 Mb Hstx2 interval behaved as a recombination cold spot with reduced PRDM9-mediated H3K4me3 hotspots and absence of DMC1-defined DNA double-strand-break hotspots. To search for structural anomalies as a possible cause of recombination suppression, we used optical mapping and observed high incidence of subspecies-specific structural variants along the X chromosome, with a striking copy number polymorphism of the microRNA Mir465 cluster. This observation together with the absence of a strong sterility phenotype in Fmr1 neighbor (Fmr1nb) null mutants support the role of microRNA as a likely candidate for Hstx2.
- MeSH
- X Chromosome genetics MeSH
- Histone-Lysine N-Methyltransferase genetics MeSH
- Homologous Recombination MeSH
- Meiosis MeSH
- MicroRNAs genetics MeSH
- Genes, Modifier * MeSH
- Infertility, Male genetics MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Polymorphism, Genetic * MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
U velikostních frakcí chloridu sodného, chloridu draselného, citronanu sodného a citronanu draselnéhoje sledována použitelnost modifikované penetrometrie a jsou hledány významné souvislostis obvykle používanými charakteristikami sypnosti a struktury práškových látek. Dosažení dostatečněreprodukovatelných výsledků bez nutnosti konsolidace umožnilo systematické studium vzájemnýchvztahů mezi vlastnostmi. Pomocí faktorové analýzy byla prokázána souvislost mezipenetrometrií, rychlostí sypání v objemovém vyjádření a Kawakita-konstantou. Pro vícerozměrnoulineární regresi k odhadu penetrometrie klesá význam sledovaných vlastností v pořadí Kawakitakonstanta> sypná hmotnost volná > celková porozita > rychlost sypání v objemovém vyjádření >rychlost sypání v hmotnostním vyjádření. Nejdůležitějším poznatkem je závislost penetrometrie nakombinaci sypné hustoty volné a Kawakita-konstanty.
Usability of modified penetrometry is examined and significant relationships with routinely usedcharacteristics of flowability and structure of powdered substances are searched for in size fractionsof sodium chloride, potassium chloride, sodium citrate, and potassium citrate. A systematic study ofinterrelationships between properties made it possible to achieve sufficiently reproducible resultswithout a necessity of consolidation. By means of factor analysis, a connection between penetrometry,bulk flow rate, and Kawakita-constant was demonstrated. For multidimensional linear regresssionfor estimation of penetrometry, the significance of examined properties decreases in the orderKawakita-constant > loose bulk density > total porosity > bulk flow rate > mass flow rate. The mostimportant itemof knowledge gained in the study is the dependence of penetrometry on a combinationof loose bulk density and Kawakita-constant.
Oxidative stress in humans is related to various pathophysiological processes, which can manifest in numerous diseases including cancer, cardiovascular diseases, and Alzheimer's disease. On the atomistic level, oxidative stress causes posttranslational modifications, thus inducing structural and functional changes into the proteins structure. This study focuses on fibrinogen, a blood plasma protein that is frequently targeted by reagents causing posttranslational modifications in proteins. Fibrinogen was in vitro modified by three reagents, namely sodium hypochlorite, malondialdehyde, and 3-morpholinosydnonimine that mimic the oxidative stress in diseases. Newly induced posttranslational modifications were detected via mass spectrometry. Electron microscopy was used to visualize changes in the fibrin networks, which highlight the extent of disturbances in fibrinogen behavior after exposure to reagents. We used molecular dynamics simulations to observe the impact of selected posttranslational modifications on the fibrinogen structure at the atomistic level. In total, 154 posttranslational modifications were identified, 84 of them were in fibrinogen treated with hypochlorite, 51 resulted from a reaction of fibrinogen with malondialdehyde, and 19 were caused by 3-morpholinosydnonimine. Our data reveal that the stronger reagents induce more posttranslational modifications in the fibrinogen structure than the weaker ones, and they extensively alter the architecture of the fibrin network. Molecular dynamics simulations revealed that the effect of posttranslational modifications on fibrinogen secondary structure varies from negligible alternations to serious disruptions. Among the serious disruptions is the oxidation of γR375 resulting in the release of Ca2+ ion that is necessary for appropriate fibrin fiber formation. Folding of amino acids γE72-γN77 into a short α-helix is a result of oxidation of γP76 to glutamic acid. The study describes behaviour of fibrinogen coiled-coil connecter in the vicinity of plasmin and hementin cleavage sites.
Mycobacterium tuberculosis O(6)-methylguanine-DNA methyltransferase (MtOGT) contributes to protect the bacterial GC-rich genome against the pro-mutagenic potential of O(6)-methylated guanine in DNA. Several strains of M. tuberculosis found worldwide encode a point-mutated O(6)-methylguanine-DNA methyltransferase (OGT) variant (MtOGT-R37L), which displays an arginine-to-leucine substitution at position 37 of the poorly functionally characterized N-terminal domain of the protein. Although the impact of this mutation on the MtOGT activity has not yet been proved in vivo, we previously demonstrated that a recombinant MtOGT-R37L variant performs a suboptimal alkylated-DNA repair in vitro, suggesting a direct role for the Arg(37)-bearing region in catalysis. The crystal structure of MtOGT complexed with modified DNA solved in the present study reveals details of the protein-protein and protein-DNA interactions occurring during alkylated-DNA binding, and the protein capability also to host unmodified bases inside the active site, in a fully extrahelical conformation. Our data provide the first experimental picture at the atomic level of a possible mode of assembling three adjacent MtOGT monomers on the same monoalkylated dsDNA molecule, and disclose the conformational flexibility of discrete regions of MtOGT, including the Arg(37)-bearing random coil. This peculiar structural plasticity of MtOGT could be instrumental to proper protein clustering at damaged DNA sites, as well as to protein-DNA complexes disassembling on repair.
- MeSH
- Bacterial Proteins chemistry genetics MeSH
- Point Mutation genetics MeSH
- Crystallography MeSH
- Mycobacterium tuberculosis genetics MeSH
- O(6)-Methylguanine-DNA Methyltransferase chemistry genetics MeSH
- DNA Damage genetics MeSH
- Protein Structure, Secondary MeSH
- Protein Structure, Tertiary MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The physiological role of proteins is frequently linked to interactions with non-protein ligands or posttranslational modifications. Structural characterization of these complexes or modified proteins by NMR may be difficult as the ligands are usually not available in an isotope-labeled form and NMR spectra may suffer from signal overlap. Here, we present an optimized approach that uses specific NMR isotope-labeling schemes for overcoming both hurdles. This approach enabled the high-resolution structure determination of the farnesylated C-terminal domain of the peroxisomal protein PEX19. The approach combines specific 13C, 15N and 2H isotope labeling with tailored NMR experiments to (i) unambiguously identify the NMR frequencies and the stereochemistry of the unlabeled 15-carbon isoprenoid, (ii) resolve the NMR signals of protein methyl groups that contact the farnesyl moiety and (iii) enable the unambiguous assignment of a large number of protein-farnesyl NOEs. Protein deuteration was combined with selective isotope-labeling and protonation of amino acids and methyl groups to resolve ambiguities for key residues that contact the farnesyl group. Sidechain-labeling of leucines, isoleucines, methionines, and phenylalanines, reduced spectral overlap, facilitated assignments and yielded high quality NOE correlations to the unlabeled farnesyl. This approach was crucial to enable the first NMR structure of a farnesylated protein. The approach is readily applicable for NMR structural analysis of a wide range of protein-ligand complexes, where isotope-labeling of ligands is not well feasible.
Fused deposition modelling (FDM) is a process of additive manufacturing allowing creating of highly precise complex three-dimensional objects for a large range of applications. The principle of FDM is an extrusion of the molten filament and gradual deposition of layers and their solidification. Potential applications in pharmaceutical and medical fields require the development of biodegradable and biocompatible thermoplastics for the processing of filaments. In this work, the potential of production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)) filaments for FDM was investigated in respect to its thermal stability. Copolymer P(3HB-co-4HB) was biosynthesised by Cupriavidus malaysiensis. Rheological and mechanical properties of the copolymer were modified by the addition of plasticizers or blending with poly(lactic acid). Thermal stability of mixtures was studied employing thermogravimetric analysis and rheological analyses by monitoring the time-dependent changes in the complex viscosity of melt samples. The plasticization of P(3HB-co-4HB) slightly hindered its thermal degradation but the best stabilization effect was found in case of the copolymer blended with poly(lactic acid). Overall, rheological, thermal and mechanical properties demonstrated that the plasticized P(3HB-co-4HB) is a potential candidate of biodegradable polymer for FDM processes.
The saliva of blood-feeding parasites is a rich source of peptidase inhibitors that help to overcome the host's defence during host-parasite interactions. Using proteomic analysis, the cystatin OmC2 was demonstrated in the saliva of the soft tick Ornithodoros moubata, an important disease vector transmitting African swine fever virus and the spirochaete Borrelia duttoni. A structural, biochemical and biological characterization of this peptidase inhibitor was undertaken in the present study. Recombinant OmC2 was screened against a panel of physiologically relevant peptidases and was found to be an effective broad-specificity inhibitor of cysteine cathepsins, including endopeptidases (cathepsins L and S) and exopeptidases (cathepsins B, C and H). The crystal structure of OmC2 was determined at a resolution of 2.45 A (1 A=0.1 nm) and was used to describe the structure-inhibitory activity relationship. The biological impact of OmC2 was demonstrated both in vitro and in vivo. OmC2 affected the function of antigen-presenting mouse dendritic cells by reducing the production of the pro-inflammatory cytokines tumour necrosis factor alpha and interleukin-12, and proliferation of antigen-specific CD4+ T-cells. This suggests that OmC2 may suppress the host's adaptive immune response. Immunization of mice with OmC2 significantly suppressed the survival of O. moubata in infestation experiments. We conclude that OmC2 is a promising target for the development of a novel anti-tick vaccine to control O. moubata populations and combat the spread of associated diseases.
- MeSH
- Immunologic Factors chemistry physiology MeSH
- Crystallization MeSH
- Crystallography, X-Ray MeSH
- Molecular Sequence Data MeSH
- Mice, Inbred C3H MeSH
- Mice, Inbred C57BL MeSH
- Mice, Transgenic MeSH
- Mice MeSH
- Ornithodoros chemistry immunology MeSH
- Amino Acid Sequence MeSH
- Salivary Cystatins chemistry physiology MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
Tato studie zkoumá latentní strukturu vnímaných dopadů na stát. Vnímaná ovladatelnost kontextu dotváří jednotlivé plány a formuje zdroje pro vyrovnávání se se změnami. Přímé a nepřímé vystavení globálním výzvám (např. teroristickým útokům) může změnit individuální pohled na kontrolovatelnost kontextu a vést k negativním psychologickým důsledkům. Na základě této analýzy byla do šetření zahrnuta sada zkoumaných vnímaných dopadů – na danou osobu, obyvatele Lotyšska, politické instituce a globální aktéry a hrozby, coby faktory reprezentující různé úrovně vnějších systémů. Studie se zúčastnilo 647 univerzitních studentů z Rigy, Valmiery a Daugavpils. K hodnocení vnímaných vlivů na Lotyšsko byl použit modifikovaný dotazník Budoucnost země. Exploratorní a konfirmační faktorová analýza podpořila čtyři hlavní faktory reprezentující vnímané dopady: osobní kontrolu, politickou moc, globalizaci a globální hrozby, přičemž poslední faktor zahrnoval vojenské napětí a terorismus. Zjištěné souvislosti přesvědčení o kontrolovatelnosti potvrdily provázanost mezi podřízenými a nadřízenými vnějšími systémy. Spolupráce s ostatními lidmi může posílit pocit vlivu (kontroly).
The study explores a latent structure of perceived impacts on the country. Perceived controllability of the context shapes individual plans and forms resource for dealing with changes. The direct and indirect exposure to global challenges (e.g., terrorist attacks) can change individual views of the controllability of the context and lead to negative psychological consequences. On the basis of the analysis, a set of perceived impacts was included in the investigation – impacts on a person, people of Latvia, its political institutions, and global players and threats – as factors representing different levels of ecological systems. Six hundred forty-seven university students from Riga, Valmiera, and Daugavpils participated in the study. The Future of Country Questionnaire was modified and used for the assessment of perceived impacts on Latvia. Exploratory and confirmatory factor analyses provided evidence for four main factors representing perceived impacts: personal control, political power, globalization, and global threats. The latter factor involved military tension and terrorism. Revealed links between control beliefs confirmed the interconnectedness of sub- and super-ordinated ecological systems. Cooperation with other people can facilitate the sense of controllability of the macro-societal context.
- MeSH
- Adaptation, Psychological * MeSH
- Internationality MeSH
- Humans MeSH
- Stress, Psychological MeSH
- Students MeSH
- Anxiety * psychology MeSH
- Research MeSH
- Check Tag
- Humans MeSH
- Geographicals
- Latvia MeSH
