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
• Adenine analogs & derivatives   pharmacology   MeSH
• Models, Anatomic MeSH
• Autophagy * drug effects   MeSH
• Inclusion Bodies pathology   ultrastructure   MeSH
• Corneal Dystrophies, Hereditary pathology   physiopathology   MeSH
• Adult MeSH
• Extracellular Matrix metabolism   ultrastructure   MeSH
• Fluorescent Antibody Technique MeSH
• Cells, Cultured MeSH
• Middle Aged MeSH
• Humans MeSH
• Mesenchymal Stem Cells MeSH
• Cadaver MeSH
• Cornea metabolism   MeSH
• Aged MeSH
• Corneal Stroma pathology   physiopathology   ultrastructure   MeSH
• Microscopy, Electron, Transmission MeSH
• Imaging, Three-Dimensional MeSH
• Check Tag
• Adult MeSH
• Middle Aged MeSH
• Humans MeSH
• Aged MeSH
• Publication type
• Journal Article 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
• X-Ray Diffraction MeSH
• Humans MeSH
• Microscopy, Electron, Scanning MeSH
• Molar chemistry   ultrastructure   MeSH
• Dental Enamel chemistry   ultrastructure   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• Animal Structures chemistry   ultrastructure   MeSH
• Biometry MeSH
• Cercaria chemistry   ultrastructure   MeSH
• Hydrogen-Ion Concentration MeSH
• Schistosomatidae chemistry   ultrastructure   MeSH
• Microscopy, Electron, Transmission MeSH
• Organ Size MeSH
• Imaging, Three-Dimensional MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study 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
• Cilia metabolism   MeSH
• Fluorescent Antibody Technique methods   MeSH
• Humans MeSH
• Cattle MeSH
• Microscopy, Electron, Transmission methods   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Cattle MeSH
• Animals MeSH
• Publication type
• Video-Audio Media MeSH
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• Astrocytes pathology   physiology   virology   MeSH
• Cytokines genetics   metabolism   MeSH
• Endoplasmic Reticulum, Rough pathology   MeSH
• Glial Fibrillary Acidic Protein biosynthesis   MeSH
• Host-Pathogen Interactions MeSH
• Encephalitis, Tick-Borne etiology   pathology   physiopathology   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Matrix Metalloproteinase 9 biosynthesis   MeSH
• Virus Replication MeSH
• Microscopy, Electron, Transmission MeSH
• Up-Regulation MeSH
• Encephalitis Viruses, Tick-Borne pathogenicity   physiology   MeSH
• Imaging, Three-Dimensional MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• Actins metabolism   MeSH
• Lipid Droplets metabolism   MeSH
• Odontogenesis physiology   MeSH
• Reptiles anatomy & histology   growth & development   metabolism   MeSH
• Microscopy, Electron, Transmission MeSH
• Gene Expression Regulation, Developmental physiology   MeSH
• Dental Enamel cytology   metabolism   ultrastructure   MeSH
• Tooth MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• Cell Respiration MeSH
• Hep G2 Cells MeSH
• Dimerization MeSH
• Hypoxia MeSH
• Ketoglutaric Acids pharmacology   MeSH
• Humans MeSH
• Mitochondrial Membranes drug effects   ultrastructure   MeSH
• Mitochondrial Proteins metabolism   MeSH
• Mitochondrial Proton-Translocating ATPases metabolism   MeSH
• Mitochondria ultrastructure   MeSH
• Microscopy, Electron, Transmission MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• Leukemia, Promyelocytic, Acute drug therapy   metabolism   pathology   MeSH
• Circular Dichroism MeSH
• Dendrimers administration & dosage   chemistry   metabolism   MeSH
• Dynamic Light Scattering MeSH
• Absorption, Physiological MeSH
• HL-60 Cells MeSH
• Intercalating Agents administration & dosage   chemistry   metabolism   MeSH
• Humans MeSH
• RNA, Small Interfering administration & dosage   chemistry   metabolism   ultrastructure   MeSH
• Molecular Conformation MeSH
• Models, Molecular * MeSH
• Molecular Structure MeSH
• Surface Properties MeSH
• Antineoplastic Agents administration & dosage   chemistry   metabolism   MeSH
• RNA Interference MeSH
• Ruthenium administration & dosage   chemistry   metabolism   MeSH
• Silanes chemistry   metabolism   MeSH
• Molecular Dynamics Simulation MeSH
• Drug Stability MeSH
• RNA Stability MeSH
• Microscopy, Electron, Transmission MeSH
• Particle Size MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study 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 Primers MeSH
• Cryoelectron Microscopy MeSH
• Escherichia coli ultrastructure   virology   MeSH
• Fourier Analysis MeSH
• Molecular Sequence Data MeSH
• Polymerase Chain Reaction MeSH
• Proteolysis MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Virus Assembly MeSH
• Microscopy, Electron, Transmission MeSH
• Virion physiology   MeSH
• Viral Proteins chemistry   metabolism   MeSH
• Leukemia Virus, Murine physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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
• Adaptor Proteins, Signal Transducing genetics   metabolism   MeSH
• beta Catenin antagonists & inhibitors   metabolism   MeSH
• NIH 3T3 Cells MeSH
• Cilia genetics   pathology   MeSH
• Kidney Diseases, Cystic genetics   MeSH
• Zebrafish genetics   MeSH
• Exons MeSH
• Phenotype MeSH
• Phosphoproteins genetics   metabolism   MeSH
• HEK293 Cells MeSH
• Kidney pathology   MeSH
• Humans MeSH
• Mutation MeSH
• Mice MeSH
• Microtubule-Associated Proteins genetics   metabolism   MeSH
• Wnt Signaling Pathway genetics   MeSH
• Microscopy, Electron, Transmission MeSH
• Computational Biology MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH