INTRODUCTION: Multilamellar bodies (MLBs) are concentric cytoplasmic membranes which form through an autophagy-dependent mechanism. In the cornea, the presence of MLBs is associated with Schnyder corneal dystrophy (SCD). Ex vivo 3D modelling of the corneal stroma and SCD can help study pathogenesis and resolution of the disorder. METHODS: Corneal stroma explants were isolated from cadavers and cultivated long-term for more than 3 months to achieve spontaneous 3D outgrowth of corneal stroma-derived mesenchymal stem-like cells (CSMSCs). The 3D tissues were then examined by transmission electron microscopy (TEM) for presence of MLBs, and by immunofluorescent labelling against markers for autophagy (p62, LC3). Autophagy was induced by classical serum starvation or rapamycin (RAP) treatment (50 nM), and inhibited by the autophagy inhibitor 3-methyladenine (3-MA, 10 mM) for 24 hours. RESULTS: CSMSCs can form spontaneously 3D outgrowths over a 3-4 weeks period, depositing their own extracellular matrix containing collagen I. TEM confirmed the presence of MLBs in the long-term (>3 months) 3D cultures, which became more abundant under starvation and RAP treatment, and decreased in number under autophagy inhibition with 3-MA. The presence of autophagy and its disappearance could be confirmed by an inversely related increase and decrease in the expression of LC3 and p62, respectively. CONCLUSIONS: MLB formation in long-standing CSMSC cultures could serve as a potential ex vivo model for studying corneal stroma diseases, including SCD. Inhibition of autophagy can decrease the formation of MLBs, which may lead to a novel treatment of the disease in the future.

• MeSH
• adenin analogy a deriváty   farmakologie   MeSH
• anatomické modely MeSH
• autofagie * účinky léků   MeSH
• buněčná inkluze patologie   ultrastruktura   MeSH
• dědičné dystrofie rohovky patologie   patofyziologie   MeSH
• dospělí MeSH
• extracelulární matrix metabolismus   ultrastruktura   MeSH
• fluorescenční protilátková technika MeSH
• kultivované buňky MeSH
• lidé středního věku MeSH
• lidé MeSH
• mezenchymální kmenové buňky MeSH
• mrtvola MeSH
• rohovka metabolismus   MeSH
• senioři MeSH
• stroma rohovky patologie   patofyziologie   ultrastruktura   MeSH
• transmisní elektronová mikroskopie MeSH
• zobrazování trojrozměrné MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• senioři MeSH
• Publikační typ
• časopisecké články MeSH

The tribosphenic molar is a dental apomorphy of mammals and the molar type from which all derived types originated. Its enamel coat is expected to be ancestral: a thin, evenly distributed layer of radial prismatic enamel. In the bat Myotis myotis, we reinvestigated the 3D architecture of the dental enamel using serial sectioning combined with scanning electron microscopy analyses, biometrics of enamel prisms and crystallites, and X-ray diffraction. We found distinct heterotopies in enamel thickness (thick enamel on the convex sides of the crests, thin on the concave ones), angularity of enamel prisms, and in distribution of particular enamel types (prismatic, interprismatic, aprismatic) and demonstrated structural relations of these heterotopies to the cusp and crest organization of the tribosphenic molar. X-ray diffraction demonstrated that the crystallites composing the enamel are actually the aggregates of much smaller primary crystallites. The differences among particular enamel types in degree of crystallite aggregation and the variation in structural microstrain of the primary crystallites (depending upon the duration and the mechanical context of mineralization) represent factors not fully understood as yet that may contribute to the complexity of enamel microarchitecture in a significant way.

• MeSH
• difrakce rentgenového záření MeSH
• lidé MeSH
• mikroskopie elektronová rastrovací MeSH
• moláry chemie   ultrastruktura   MeSH
• zubní sklovina chemie   ultrastruktura   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Cercariae of schistosomes employ bioactive molecules for penetration into their hosts. These are released from specialized unicellular glands upon stimuli from host skin. The glands were previously well-described in the human pathogen Schistosoma mansoni. As bird schistosomes can also penetrate human skin and cause cercarial dermatitis, our aim was to characterize the architecture and ultrastructure of glands in the neurotropic bird schistosome Trichobilharzia regenti and compare it with S. mansoni. In the context of different histolytic enzymes used by these two species, we focused also on the estimations of gland volumes and pH in T. regenti. RESULTS: The architecture and 3-D models of two types of acetabular penetration glands, their ducts and of the head gland are shown here. We characterized secretory vesicles in all three gland types by means of TEM and confirmed accuracy of the models obtained by confocal microscopy. The results of two independent approaches showed that the glands occupy ca. one third of cercarial body volume (postacetabular glands ca. 15%, circumacetabular 12% and head gland 6%). The inner environment within the two types of acetabular glands differed significantly as evidenced by dissimilar ability to bind fluorescent markers and by pH value which was higher in circumacetabular (7.44) than in postacetabular (7.08) glands. CONCLUSIONS: As far as we know, this is the first presentation of a 3-D model of cercarial glands and the first exact estimation of the volumes of the three gland types in schistosomes. Our comparisons between T. regenti and S. mansoni implied that the architecture and ultrastructure of the glands is most likely conserved within the family. Only minor variations were found between the two species. It seems that the differences in molecular composition have no effect on general appearance of the secretory cells in TEM. Fluorescent markers employed in this study, distinguishing between secretory vesicles and gland types, can be useful in further studies of mechanisms used by cercariae for host invasion. Results of the first attempts to estimate pH within schistosome glands may help further understanding of regulation of enzymatic activities present within the glands.

• MeSH
• anatomické struktury zvířat chemie   ultrastruktura   MeSH
• biometrie MeSH
• cerkárie chemie   ultrastruktura   MeSH
• koncentrace vodíkových iontů MeSH
• Schistosomatidae chemie   ultrastruktura   MeSH
• transmisní elektronová mikroskopie MeSH
• velikost orgánu MeSH
• zobrazování trojrozměrné MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to mitosis. Primary cilia can be visualized with highly sophisticated methods, including transmission electron microscopy, 3D imaging, or using software for the automatic detection of primary cilia. However, immunofluorescent staining of primary cilia is needed to perform these methods. This publication describes a protocol for the easy detection of primary cilia in vitro by staining acetylated alpha tubulin (axoneme) and gamma tubulin (basal body). This immunofluorescent staining protocol is relatively simple and results in high-quality images. The present protocol describes how four cell lines (C2C12, MEF, NHLF, and skin fibroblasts) expressing primary cilia were fixed, immunostained, and imaged with a fluorescent or confocal microscope.

• MeSH
• cilie metabolismus   MeSH
• fluorescenční protilátková technika metody   MeSH
• lidé MeSH
• skot MeSH
• transmisní elektronová mikroskopie metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• audiovizuální média MeSH
• časopisecké články MeSH
• práce podpořená grantem MeSH

Tick-borne encephalitis (TBE), a disease caused by tick-borne encephalitis virus (TBEV), represents the most important flaviviral neural infection in Europe and north-eastern Asia. In the central nervous system (CNS), neurons are the primary target for TBEV infection; however, infection of non-neuronal CNS cells, such as astrocytes, is not well understood. In this study, we investigated the interaction between TBEV and primary human astrocytes. We report for the first time, to the best of our knowledge, that primary human astrocytes are sensitive to TBEV infection, although the infection did not affect their viability. The infection induced a marked increase in the expression of glial fibrillary acidic protein, a marker of astrocyte activation. In addition, expression of matrix metalloproteinase 9 and several key pro-inflammatory cytokines/chemokines (e.g. tumour necrosis factor α, interferon α, interleukin (IL)-1β, IL-6, IL-8, interferon γ-induced protein 10, macrophage inflammatory protein, but not monocyte chemotactic protein 1) was upregulated. Moreover, we present a detailed description of morphological changes in TBEV-infected cells, as investigated using three-dimensional electron tomography. Several novel ultrastructural changes were observed, including the formation of unique tubule-like structures of 17.9 ±0.15 nm diameter with associated viral particles and/or virus-induced vesicles and located in the rough endoplasmic reticulum of the TBEV-infected cells. This is the first demonstration that TBEV infection activates primary human astrocytes. The infected astrocytes might be a potential source of pro-inflammatory cytokines in the TBEV-infected brain, and might contribute to the TBEV-induced neurotoxicity and blood-brain barrier breakdown that occurs during TBE. The neuropathological significance of our observations is also discussed.

• MeSH
• astrocyty patologie   fyziologie   virologie   MeSH
• cytokiny genetika   metabolismus   MeSH
• drsné endoplazmatické retikulum patologie   MeSH
• gliový fibrilární kyselý protein biosyntéza   MeSH
• interakce hostitele a patogenu MeSH
• klíšťová encefalitida etiologie   patologie   patofyziologie   MeSH
• kultivované buňky MeSH
• lidé MeSH
• matrixová metaloproteinasa 9 biosyntéza   MeSH
• replikace viru MeSH
• transmisní elektronová mikroskopie MeSH
• upregulace MeSH
• viry klíšťové encefalitidy patogenita   fyziologie   MeSH
• zobrazování trojrozměrné MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: In mammals, odontogenesis is regulated by transient signaling centers known as enamel knots (EKs), which drive the dental epithelium shaping. However, the developmental mechanisms contributing to formation of complex tooth shape in reptiles are not fully understood. Here, we aim to elucidate whether signaling organizers similar to EKs appear during reptilian odontogenesis and how enamel ridges are formed. RESULTS: Morphological structures resembling the mammalian EK were found during reptile odontogenesis. Similar to mammalian primary EKs, they exhibit the presence of apoptotic cells and no proliferating cells. Moreover, expression of mammalian EK-specific molecules (SHH, FGF4, and ST14) and GLI2-negative cells were found in reptilian EK-like areas. 3D analysis of the nucleus shape revealed distinct rearrangement of the cells associated with enamel groove formation. This process was associated with ultrastructural changes and lipid droplet accumulation in the cells directly above the forming ridge, accompanied by alteration of membranous molecule expression (Na/K-ATPase) and cytoskeletal rearrangement (F-actin). CONCLUSIONS: The final complex shape of reptilian teeth is orchestrated by a combination of changes in cell signaling, cell shape, and cell rearrangement. All these factors contribute to asymmetry in the inner enamel epithelium development, enamel deposition, ultimately leading to the formation of characteristic enamel ridges.

• MeSH
• aktiny metabolismus   MeSH
• lipidová tělíska metabolismus   MeSH
• odontogeneze fyziologie   MeSH
• plazi anatomie a histologie   růst a vývoj   metabolismus   MeSH
• transmisní elektronová mikroskopie MeSH
• vývojová regulace genové exprese fyziologie   MeSH
• zubní sklovina cytologie   metabolismus   ultrastruktura   MeSH
• zuby MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Hypoxia causes mitochondrial cristae widening, enabled by the ~20% degradation of Mic60/mitofilin, with concomitant clustering of the MICOS complex, reflecting the widening of crista junctions (outlets) (Plecitá-Hlavatá et al. FASEB J., 2016 30:1941-1957). Attempting to accelerate metabolism by the addition of membrane-permeant dimethyl-2-oxoglutarate (dm2OG) to HepG2 cells pre-adapted to hypoxia, we found cristae narrowing by transmission electron microscopy. Glycolytic HepG2 cells, which downregulate hypoxic respiration, instantly increased respiration with dm2OG. Changes in intracristal space (ICS) morphology were also revealed by 3D super-resolution microscopy using Eos-conjugated ICS-located lactamase-β. Cristae topology was resolved in detail by focused-ion beam/scanning electron microscopy (FIB/SEM). The spatial relocations of key cristae-shaping proteins were indicated by immunocytochemical stochastic 3D super-resolution microscopy (dSTORM), while analyzing inter-antibody-distance histograms: i) ATP-synthase dimers exhibited a higher fraction of shorter inter-distances between bound F1-α primary Alexa-Fluor-647-conjugated antibodies, indicating cristae narrowing. ii) Mic60/mitofilin clusters (established upon hypoxia) decayed, restoring isotropic random Mic60/mitofilin distribution (a signature of normoxia). iii) outer membrane SAMM50 formed more focused clusters. Less abundant fractions of higher ATP-synthase oligomers of hypoxic samples on blue-native electrophoresis became more abundant fractions at the high dm2OG load and at normoxia. This indicates more labile ATP-synthase dimeric rows established at crista rims upon hypoxia, strengthened at normoxia or dm2OG-substrate load. Hypothetically, the increased Krebs substrate load stimulates the cross-linking/strengthening of rows of ATP-synthase dimers at the crista rims, making them sharper. Crista narrowing ensures a more efficient coupling of proton pumping to ATP synthesis. We demonstrated that cristae morphology changes even within minutes.

• MeSH
• buněčné dýchání MeSH
• buňky Hep G2 MeSH
• dimerizace MeSH
• hypoxie MeSH
• kyseliny ketoglutarové farmakologie   MeSH
• lidé MeSH
• mitochondriální membrány účinky léků   ultrastruktura   MeSH
• mitochondriální proteiny metabolismus   MeSH
• mitochondriální protonové ATPasy metabolismus   MeSH
• mitochondrie ultrastruktura   MeSH
• transmisní elektronová mikroskopie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Dendrimers, which are considered as one of the most promising tools in the field of nanobiotechnology due to their structural organization, showed a great potential in gene therapy, drug delivery, medical imaging and as antimicrobial and antiviral agents. This article is devoted to study interactions between new carbosilane-based metallodendrimers containing ruthenium and anti-cancer small interfering RNA (siRNA). Formation of complexes between anti-cancer siRNAs and Ru-based carbosilane dendrimers was evaluated by transmission electron microscopy, circular dichroism and fluorescence. The zeta-potential and the size of dendriplexes were determined by dynamic light scattering. The internalization of dendriplexes were estimated using HL-60 cells. Results show that ruthenium dendrimers associated with anticancer siRNA have the ability to deliver siRNA as non-viral vectors into the cancer cells. Moreover, dendrimers can protect siRNA against nuclease degradation. Nevertheless, further research need to be performed to examine the therapeutic potential of ruthenium dendrimers as well as dendrimers complexed with siRNA and anticancer drugs towards cancer cells.

• MeSH
• akutní promyelocytární leukemie farmakoterapie   metabolismus   patologie   MeSH
• cirkulární dichroismus MeSH
• dendrimery aplikace a dávkování   chemie   metabolismus   MeSH
• dynamický rozptyl světla MeSH
• fyziologická absorpce MeSH
• HL-60 buňky MeSH
• interkalátory aplikace a dávkování   chemie   metabolismus   MeSH
• lidé MeSH
• malá interferující RNA aplikace a dávkování   chemie   metabolismus   ultrastruktura   MeSH
• molekulární konformace MeSH
• molekulární modely * MeSH
• molekulární struktura MeSH
• povrchové vlastnosti MeSH
• protinádorové látky aplikace a dávkování   chemie   metabolismus   MeSH
• RNA interference MeSH
• ruthenium aplikace a dávkování   chemie   metabolismus   MeSH
• silany chemie   metabolismus   MeSH
• simulace molekulární dynamiky MeSH
• stabilita léku MeSH
• stabilita RNA MeSH
• transmisní elektronová mikroskopie MeSH
• velikost částic MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Immature retroviral particles are assembled by self-association of the structural polyprotein precursor Gag. During maturation the Gag polyprotein is proteolytically cleaved, yielding mature structural proteins, matrix (MA), capsid (CA), and nucleocapsid (NC), that reassemble into a mature viral particle. Proteolytic cleavage causes the N terminus of CA to fold back to form a β-hairpin, anchored by an internal salt bridge between the N-terminal proline and the inner aspartate. Using an in vitro assembly system of capsid-nucleocapsid protein (CANC), we studied the formation of virus-like particles (VLP) of a gammaretrovirus, the xenotropic murine leukemia virus (MLV)-related virus (XMRV). We show here that, unlike other retroviruses, XMRV CA and CANC do not assemble tubular particles characteristic of mature assembly. The prevention of β-hairpin formation by the deletion of either the N-terminal proline or 10 initial amino acids enabled the assembly of ΔProCANC or Δ10CANC into immature-like spherical particles. Detailed three-dimensional (3D) structural analysis of these particles revealed that below a disordered N-terminal CA layer, the C terminus of CA assembles a typical immature lattice, which is linked by rod-like densities with the RNP.

• MeSH
• DNA primery MeSH
• elektronová kryomikroskopie MeSH
• Escherichia coli ultrastruktura   virologie   MeSH
• Fourierova analýza MeSH
• molekulární sekvence - údaje MeSH
• polymerázová řetězová reakce MeSH
• proteolýza MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie aminokyselin MeSH
• sestavení viru MeSH
• transmisní elektronová mikroskopie MeSH
• virion fyziologie   MeSH
• virové proteiny chemie   metabolismus   MeSH
• virus myší leukemie fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

Nephronophthisis-related ciliopathies (NPHP-RC) are recessive diseases characterized by renal dysplasia or degeneration. We here identify mutations of DCDC2 as causing a renal-hepatic ciliopathy. DCDC2 localizes to the ciliary axoneme and to mitotic spindle fibers in a cell-cycle-dependent manner. Knockdown of Dcdc2 in IMCD3 cells disrupts ciliogenesis, which is rescued by wild-type (WT) human DCDC2, but not by constructs that reflect human mutations. We show that DCDC2 interacts with DVL and DCDC2 overexpression inhibits β-catenin-dependent Wnt signaling in an effect additive to Wnt inhibitors. Mutations detected in human NPHP-RC lack these effects. A Wnt inhibitor likewise restores ciliogenesis in 3D IMCD3 cultures, emphasizing the importance of Wnt signaling for renal tubulogenesis. Knockdown of dcdc2 in zebrafish recapitulates NPHP-RC phenotypes, including renal cysts and hydrocephalus, which is rescued by a Wnt inhibitor and by WT, but not by mutant, DCDC2. We thus demonstrate a central role of Wnt signaling in the pathogenesis of NPHP-RC, suggesting an avenue for potential treatment of NPHP-RC.

• MeSH
• adaptorové proteiny signální transdukční genetika   metabolismus   MeSH
• beta-katenin antagonisté a inhibitory   metabolismus   MeSH
• buňky NIH 3T3 MeSH
• cilie genetika   patologie   MeSH
• cystická onemocnění ledvin genetika   MeSH
• dánio pruhované genetika   MeSH
• exony MeSH
• fenotyp MeSH
• fosfoproteiny genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• ledviny patologie   MeSH
• lidé MeSH
• mutace MeSH
• myši MeSH
• proteiny asociované s mikrotubuly genetika   metabolismus   MeSH
• signální dráha Wnt genetika   MeSH
• transmisní elektronová mikroskopie MeSH
• výpočetní biologie MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH