Thermal polymorphism, usually represented by thermal melanism (darker coloration in cooler habitats), is a well-known phenomenon in animals. In Cetoniinae, several species in captivity tend to become darker after several generations of breeding, which is probably caused by a lower temperature than is typical for their native habitats. Pachnoda iskuulka is a beetle species occurring in Somaliland. This species is easy to breed in captivity, and it is colorful and variable in the proportions of yellow, red, and black coloration. We kept this species from the first instar larva to the adult stage at three different temperatures. Elytra and pronotum of the adults were photographed, and proportions of the three main colors were measured. The proportion of black coloration significantly increased with size and decreased with temperature, while the proportion of yellow color increased. This species is certainly thermally polymorphic, which can be an adaptation for activation even at lower temperatures. The possible mimicry with beetles of the genus Hycleus is discussed. It is the first confirmation of thermal polymorphism in Cetoniinae and one of a few in Coleoptera.

• Klíčová slova
• beetle breeding, color ratio, coloration, habitat, mimicry, polymorphism,
• Publikační typ
• časopisecké články MeSH

Sand fly transmitted Leishmania species are responsible for severe, wide ranging, visceral and cutaneous leishmaniases. Genetic exchange can occur among natural Leishmania populations and hybrids can now be produced experimentally, with limitations. Feeding Phlebotomus orientalis or Phlebotomus argentipes on two strains of Leishmania donovani yielded hybrid progeny, selected using double drug resistance and fluorescence markers. Fluorescence activated cell sorting of cultured clones derived from these hybrids indicated diploid progeny. Multilocus sequence typing of the clones showed hybridisation and nuclear heterozygosity, although with inheritance of single haplotypes in a kinetoplastid target. Comparative genomics showed diversity of clonal progeny between single chromosomes, and extraordinary heterozygosity across all 36 chromosomes. Diversity between progeny was seen for the HASPB antigen, which has been noted previously as having implications for design of a therapeutic vaccine. Genomic diversity seen among Leishmania strains and hybrid progeny is of great importance in understanding the epidemiology and control of leishmaniasis. As an outcome of this study we strongly recommend that wider biological archives of different Leishmania species from endemic regions should be established and made available for comparative genomics. However, in parallel, performance of genetic crosses and genomic comparisons should give fundamental insight into the specificity, diversity and limitations of candidate diagnostics, vaccines and drugs, for targeted control of leishmaniasis.

• MeSH
• genomika MeSH
• křížení genetické MeSH
• Leishmania donovani * genetika   MeSH
• leishmanióza kožní * MeSH
• leishmanióza viscerální * diagnóza   prevence a kontrola   epidemiologie   MeSH
• Phlebotomus * genetika   MeSH
• Psychodidae * genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Classical models of gene expression were built using genetics and biochemistry. Although these approaches are powerful, they have very limited consideration of the spatial and temporal organization of gene expression. Although the spatial organization and dynamics of RNA polymerase II (RNAPII) transcription machinery have fundamental functional consequences for gene expression, its detailed studies have been abrogated by the limits of classical light microscopy for a long time. The advent of super-resolution microscopy (SRM) techniques allowed for the visualization of the RNAPII transcription machinery with nanometer resolution and millisecond precision. In this review, we summarize the recent methodological advances in SRM, focus on its application for studies of the nanoscale organization in space and time of RNAPII transcription, and discuss its consequences for the mechanistic understanding of gene expression.

• Klíčová slova
• cell nucleus, gene expression, photoactivation, stimulated emission depletion, stochastic optical reconstruction, structured illumination, super-resolution microscopy, transcription factors, transcription foci,
• MeSH
• fluorescenční mikroskopie * metody   MeSH
• genetická transkripce * MeSH
• lidé MeSH
• regulace genové exprese * MeSH
• RNA-polymerasa II metabolismus   MeSH
• transkripční faktory metabolismus   MeSH
• vazba proteinů MeSH
• zobrazení jednotlivé molekuly metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• RNA-polymerasa II MeSH
• transkripční faktory MeSH

Current models of gene expression, which are based on single-molecule localization microscopy, acknowledge protein clustering and the formation of transcriptional condensates as a driving force of gene expression. However, these models largely omit the role of nuclear lipids and amongst them nuclear phosphatidylinositol phosphates (PIPs) in particular. Moreover, the precise distribution of nuclear PIPs in the functional sub-nuclear domains remains elusive. The direct stochastic optical reconstruction microscopy (dSTORM) provides an unprecedented resolution in biological imaging. Therefore, its use for imaging in the densely crowded cell nucleus is desired but also challenging. Here we present a dual-color dSTORM imaging and image analysis of nuclear PI(4,5)P2, PI(3,4)P2 and PI(4)P distribution while preserving the context of nuclear architecture. In the nucleoplasm, PI(4,5)P2 and PI(3,4)P2 co-pattern in close proximity with the subset of RNA polymerase II foci. PI(4,5)P2 is surrounded by fibrillarin in the nucleoli and all three PIPs are dispersed within the matrix formed by the nuclear speckle protein SON. PI(4,5)P2 is the most abundant nuclear PIP, while PI(4)P is a precursor for the biosynthesis of PI(4,5)P2 and PI(3,4)P2. Therefore, our data are relevant for the understanding the roles of nuclear PIPs and provide further evidence for the model in which nuclear PIPs represent a localization signal for the formation of lipo-ribonucleoprotein hubs in the nucleus. The discussed experimental pipeline is applicable for further functional studies on the role of other nuclear PIPs in the regulation of gene expression and beyond.

• Klíčová slova
• Cell nucleus, Nuclear speckles, Nucleolus, Phosphatidylinositol phosphates, RNA polymerase II, STORM,
• MeSH
• buněčné jadérko metabolismus   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• fosfatidylinositolfosfáty metabolismus   MeSH
• lidé MeSH
• mikroskopie MeSH
• nádorové buněčné linie MeSH
• RNA-polymerasa II metabolismus   MeSH
• vedlejší histokompatibilní antigeny metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• fosfatidylinositolfosfáty MeSH
• RNA-polymerasa II MeSH
• SON protein, human MeSH Prohlížeč
• vedlejší histokompatibilní antigeny MeSH

Single molecule localization microscopy (SMLM) provided an unprecedented insight into the sub-nuclear organization of proteins and nucleic acids but apart from the nuclear envelope the role of the nuclear lipids in the functional organization of the cell nucleus was less studied. Nevertheless, nuclear lipids and specifically phosphatidylinositol phosphates (PIPs) play increasingly evident roles in gene expression. Therefore, here we provide the SMLM-based approach for the quantitative evaluation of the nuclear PIPs distribution while preserving the context of nuclear architecture. Specifically, on the example of phosphatidylinositol 4,5-bisphosphate (PIP2) we have:•Implemented and optimized the dual-color dSTORM imaging of nuclear PIP2.•Customized the Nearest Neighbor Distance analysis using ImageJ2 plug-in ThunderSTORM to quantitatively evaluate the spatial distribution of nuclear PIP2.•Developed an ImageJ2 tool for the visualization of the Nearest Neighbor Distance analysis results in cellulo.Our customization of the dual-color dSTORM imaging and quantitative analysis provide a tool that is independent of but complementary to the biochemical and lipidomic analyses of the nuclear PIPs. Contrary to the biochemical and lipidomic analyses, the advantage of our analysis is that it preserves the spatial context of the nuclear PIP distribution.

• Klíčová slova
• Cell nucleus, Fibrillarin, ImageJ, Immunofluorescence, Nearest neighbor distance, Nuclear architecture, Nuclear speckles, RNA polymerase II, SON, Super-resolution microscopy, Wide-field microscopy,
• Publikační typ
• časopisecké články MeSH

The cytotoxicity of mouse natural killer (NK) cells in response to pathological changes in target cells is regulated via the Nkrp1b receptor. Here, we characterized the Nkrp1b structure and structural features (stalk, loop, and oligomerization state) that affect its interactions. To study the Nkrp1b protein structure and the functional importance of its stalk, two Nkrp1b protein variants differing by the presence of the stalk were prepared. These variants were studied using a combination of structural mass spectrometry approaches with computational modeling to derive structural models. In addition, information about biological activity and localization in mammalian cells was acquired using scanning microscopy techniques and western blotting. Based on these methods, we obtained the structure of Nkrp1b ectodomain in its monomeric and dimeric conformations, identified the dimerization interface, and determined disulfide connections within the molecule. We found that Nkrp1b occurs as a mixture of monomers and homodimers, both in vitro and in vivo. SIGNIFICANCE: Despite the long-standing assumption that Nkrp1 proteins are homodimers connected by disulfide bonds in the stalk region, our data showed that both Nkrp1b protein variants form monomers and homodimers irrespective of the presence of the stalk. We demonstrated that the stalk is not crucial for protein dimerization or ligand binding and that Nkrp1b interacts with its natural ligands only in its monomeric conformation; therefore, dimers may have another regulatory function. Using a unique combination of computational, biochemical, and biological methods, we revealed the structural conformation and behavior of Nkrp1b in its native state. In addition, it is a first report utilizing the intermolecular chemical cross-linking of light- and heavy-labeled protein chains together with ion mobility-mass spectrometry to design the structural models of protein homodimers.

• Klíčová slova
• Chemical cross-linking, Homodimers, Ion mobility, Natural killer cells, Nkrp1b, Structural mass spectrometry,
• MeSH
• lektinové receptory NK-buněk - podrodina B chemie   metabolismus   MeSH
• molekulární modely * MeSH
• multimerizace proteinu * MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• proteomika * MeSH
• sekundární struktura proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• lektinové receptory NK-buněk - podrodina B MeSH

Derivation of periosteal and endosteal contours taken from transversal long bone cross-sections limits the accuracy of calculated biomechanical properties. Although several techniques are available for deriving both contours, the effect of these techniques on accuracy of calculated cross-sectional properties in non-adults is unknown. We examine a sample of 86 non-adult femora from birth to 12 years of age to estimate the effect of error in deriving periosteal and endosteal contours on cross-sectional properties. Midshaft cross-sections were taken from microCT scans and contours were derived using manual, fully automatic, spline, and ellipse techniques. Agreement between techniques was assessed against manually traced periosteal and endosteal contours using percent prediction error (%PE), reduced major axis analysis, and limits of agreement. The %PEs were highest in the medullary area and lowest in the total area. Mean %PEs were sufficiently below the 5% level of acceptable error, except for medullary areas, but individual values can greatly exceed this 5% boundary given the high standard deviation of %PE means and wide minimum-maximum range of %PEs. Automatic processing produces greater errors than does combination with manual, spline, and ellipse processing. Although periosteal contour is estimated with stronger agreement compared with endosteal contour, error in deriving periosteal contour has a substantially greater effect on calculated section moduli than does error in deriving endosteal contours. We observed no size effect on the resulting bias. Nevertheless, cross-sectional properties in a younger age category may be estimated with greater error compared with in an older age category. We conclude that non-adult midshaft cross-sectional properties can be derived from microCT scans of femoral diaphyses with mean error of < 5% and that derivation of endosteal contour can be simplified by the ellipse technique because fully automatic derivation of endosteal contour may increase the resulting error, especially in small samples.

• Klíčová slova
• EPmacroJ, ImageJ, biomechanics, femora, microCT,
• Publikační typ
• časopisecké články MeSH

Remodeling of the cellular distribution of gap junctions formed mainly by connexin-43 (Cx43) can be related to the increased incidence of cardiac arrhythmias. It has been shown that adaptation to chronic intermittent hypobaric hypoxia (IHH) attenuates the incidence and severity of ischemic and reperfusion ventricular arrhythmias and increases the proportion of anti-arrhythmic n-3 polyunsaturated fatty acids (n-3 PUFA) in heart phospholipids. Wistar rats were exposed to simulated IHH (7,000 m, 8-h/day, 35 exposures) and compared with normoxic controls (N). Cx43 expression, phosphorylation, localization and n-3 PUFA proportion were analyzed in left ventricular myocardium. Compared to N, IHH led to higher expression of total Cx43, its variant phosphorylated at Ser368 [p-Cx43(Ser368)], which maintains "end to end" communication, as well as p-Cx43(Ser364/365), which facilitates conductivity. By contrast, expression of non-phosphorylated Cx43 and p-Cx43(Ser278/289), attenuating intercellular communication, was lower in IHH than in N. IHH also resulted in increased expression of protein kinase A and protein kinase G while casein kinase 1 did not change compared to N. In IHH group, which exhibited reduced incidence of ischemic ventricular arrhythmias, Cx43 and p-Cx43(Ser368) were more abundant at "end to end" gap junctions than in N group and this difference was preserved after acute regional ischemia (10 min). We further confirmed higher n-3 PUFA proportion in heart phospholipids after adaptation to IHH, which was even further increased by ischemia. Our results suggest that adaptation to IHH alters expression, phosphorylation and distribution of Cx43 as well as cardioprotective n-3PUFA proportion suggesting that the anti-arrhythmic phenotype elicited by IHH can be at least partly related to the stabilization of the "end to end" conductivity between cardiomyocytes during brief ischemia.

• Klíčová slova
• arrhythmia, brief ischemia, chronic hypoxia, connexin-43, heart, n-3 PUFA,
• Publikační typ
• časopisecké články MeSH

Pathogenic and non-pathogenic related microorganisms differ in secondary metabolite production. Here we show that riboflavin overproduction by a fungal pathogen and its hyperaccumulation in affected host tissue exacerbates a skin infection to necrosis. In white-nose syndrome (WNS) skin lesions caused by Pseudogymnoascus destructans, maximum riboflavin concentrations reached up to 815 μg ml(-1), indicating bioaccumulation and lack of excretion. We found that high riboflavin concentrations are cytotoxic under conditions specific for hibernation, affect bats' primary fibroblasts and induce cell detachment, loss of mitochondrial membrane potential, polymerization of cortical actin, and cell necrosis. Our results explain molecular pathology of WNS, where a skin infection becomes fatal. Hyperaccumulation of vitamin B2 coupled with reduced metabolism and low tissue oxygen saturation during hibernation prevents removal of excess riboflavin in infected bats. Upon reperfusion, oxygen reacts with riboflavin resulting in dramatic pathology after arousal. While multiple molecules enable invasive infection, riboflavin-associated extensive necrosis likely contributes to pathophysiology and altered arousal pattern in infected bats. Bioaccumulation of a vitamin under natural infection represents a novel condition in a complex host-pathogen interplay.

• MeSH
• Ascomycota klasifikace   genetika   patogenita   MeSH
• buněčná adheze MeSH
• Chiroptera mikrobiologie   MeSH
• dermatomykózy mikrobiologie   MeSH
• elektronová mikroskopie MeSH
• faktory virulence metabolismus   MeSH
• fibroblasty cytologie   metabolismus   mikrobiologie   MeSH
• fylogeneze MeSH
• interakce hostitele a patogenu MeSH
• křídla zvířecí cytologie   mikrobiologie   ultrastruktura   MeSH
• kultivované buňky MeSH
• membránový potenciál mitochondrií MeSH
• riboflavin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• faktory virulence MeSH
• riboflavin MeSH

SCA2 transgenic mice are thought to be a useful model of human spinocerebellar ataxia type 2. There is no effective therapy for cerebellar degenerative disorders, therefore neurotransplantation could offer hope. The aim of this work was to assess the survival and morphology of embryonic cerebellar grafts transplanted into the cerebellum of adult SCA2 mice. Four month-old homozygous SCA2 and negative control mice were treated with bilateral intracerebellar injections of an enhanced green fluorescent protein-positive embryonic cerebellar cell suspension. Graft survival and morphology were examined three months later. Graft-derived Purkinje cells and the presence of astrocytes in the graft were detected immunohistochemically. Nissl and hematoxylin-eosin techniques were used to visualize the histological structure of the graft and surrounding host tissue. Grafts survived in all experimental mice; no differences in graft structure, between SCA2 homozygous and negative mice, were found. The grafts contained numerous Purkinje cells but long distance graft-to-host axonal connections to the deep cerebellar nuclei were rarely seen. Relatively few astrocytes were found in the center of the graft. No signs of inflammation or tissue destruction were seen in the area around the grafts. Despite good graft survival and the presence of graft-derived Purkinje cells, the structure of the graft did not seem to promise any significant specific functional effects. We have shown that the graft is available for long-term experiments. Nevertheless, it would be beneficial to search for ways of enhancement of connections between the graft and host.

• Klíčová slova
• ATXN2(Q127), Cerebellar degeneration, DNA, Deoxyribonucleic acid, EGFP, GAFP, Neurotransplantation, PC, PCR, Purkinje cell, Purkinje cell degeneration, Purkinje cells, SCA, ataxin 2 with 127 glutamine repeats, enhanced green fluorescent protein, glial fibrillary acidic protein, pcd, polymerase chain reaction, spinocerebellar ataxia,
• MeSH
• mozeček patologie   transplantace   MeSH
• myši transgenní MeSH
• přežívání štěpu MeSH
• sexuální faktory MeSH
• spinocerebelární ataxie terapie   MeSH
• transplantace fetální tkáně * MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH