Formation Of Vesicle-like Structures Research Articles

Characterization of Human Immortalized Keratinocyte Cells Infected by Monkeypox Virus.

Monkeypox virus (MPXV) can induce systemic skin lesions after infection. This research focused on studying MPXV proliferation and the response of keratinocytes. Using transmission electron microscopy (TEM), we visualized different stages of MPXV development in human immortalized keratinocytes (HaCaT). We identified exocytosis of enveloped viruses as the exit mechanism for MPXV in HaCaT cells. Infected keratinocytes showed submicroscopic changes, such as the formation of vesicle-like structures through the recombination of rough endoplasmic reticulum membranes and alterations in mitochondrial morphology. Transcriptome analysis revealed the suppressed genes related to interferon pathway activation and the reduced expression of antimicrobial peptides and chemokines, which may facilitate viral immune evasion. In addition, pathway enrichment analysis highlighted systemic lupus erythematosus pathway activation and the inhibition of the Toll-like receptor signaling and retinol metabolism pathways, providing insights into the mechanisms underlying MPXV-induced skin lesions. This study advances our understanding of MPXV's interaction with keratinocytes and the complex mechanisms leading to skin lesions.

Mesoscale structure–function relationships in mitochondrial transcriptional condensates

In live cells, phase separation is thought to organize macromolecules into membraneless structures known as biomolecular condensates. Here, we reconstituted transcription in condensates from purified mitochondrial components using optimized invitro reaction conditions to probe the structure-function relationships of biomolecular condensates. We find that the core components of the mt-transcription machinery form multiphasic, viscoelastic condensates invitro. Strikingly, the rates of condensate-mediated transcription are substantially lower than in solution. The condensate-mediated decrease in transcriptional rates is associated with the formation of vesicle-like structures that are driven by the production and accumulation of RNA during transcription. The generation of RNA alters the global phase behavior and organization of transcription components within condensates. Coarse-grained simulations of mesoscale structures at equilibrium show that the components stably assemble into multiphasic condensates and that the vesicles formed invitro are the result of dynamical arrest. Overall, our findings illustrate the complex phase behavior of transcribing, multicomponent condensates, and they highlight the intimate, bidirectional interplay of structure and function in transcriptional condensates.

Ultrastructural analysis of the morphological phenotypes of microglia associated with neuroinflammatory cues.

Microglia are the primary resident immune cells of the central nervous system that are responsible for the maintenance of brain homeostasis. There is a plethora of evidence to suggest that microglia display distinct phenotypes that are associated with the alteration of cell morphology under varying environmental cues. However, it has not been fully explored how the varying states of microglial activation are linked to the alteration of microglia morphology, especially in the microdomain. The objective of this study was to quantitatively characterize the ultrastructural morphology of human microglia under neuroinflammatory cues. To address this, a human cell line of microglia was stimulated by antiinflammatory (IL-4), proinflammatory (TNF-α), and Alzheimer's disease (AD)-associated cues (Aβ, Aβ + TNF-α). The resulting effects on microglia morphology associated with changes in microdomain were analyzed using a high-resolution scanning electron microscopy. Our findings demonstrated that microglial activation under proinflammatory and AD-cues were closely linked to changes not only in cell shape but also in cell surface topography and higher-order branching of processes. Furthermore, our results revealed that microglia under proinflammatory cues exhibited unique morphological features involving cell-to-cell contact and the formation of vesicle-like structures. Our study provides insight into the fine details of microglia morphology associated with varying status of microglial activation.

Silica-coated magnetic nanoparticles activate microglia and induce neurotoxic d-serine secretion

BackgroundNanoparticles have been studied for brain imaging, diagnosis, and drug delivery owing to their versatile properties due to their small sizes. However, there are growing concerns that nanoparticles may exert toxic effects in the brain. In this study, we assessed direct nanotoxicity on microglia, the resident macrophages of the central nervous system, and indirect toxicity on neuronal cells exerted by silica-coated magnetic nanoparticles containing rhodamine B isothiocyanate dye [MNPs@SiO2(RITC)].MethodsWe investigated MNPs@SiO2(RITC)-induced biological changes in BV2 murine microglial cells via RNA-sequencing-based transcriptome analysis and gas chromatography-mass spectrometry-based intracellular and extracellular amino acid profiling. Morphological changes were analyzed by transmission electron microscopy. Indirect effects of MNPs@SiO2(RITC) on neuronal cells were assessed by Transwell-based coculture with MNPs@SiO2(RITC)-treated microglia. MNPs@SiO2(RITC)-induced biological changes in the mouse brain in vivo were examined by immunohistochemical analysis.ResultsBV2 murine microglial cells were morphologically activated and the expression of Iba1, an activation marker protein, was increased after MNPs@SiO2(RITC) treatment. Transmission electron microscopy analysis revealed lysosomal accumulation of MNPs@SiO2(RITC) and the formation of vesicle-like structures in MNPs@SiO2(RITC)-treated BV2 cells. The expression of several genes related to metabolism and inflammation were altered in 100 µg/ml MNPs@SiO2(RITC)-treated microglia when compared with that in non-treated (control) and 10 µg/ml MNPs@SiO2(RITC)-treated microglia. Combined transcriptome and amino acid profiling analyses revealed that the transport of serine family amino acids, including glycine, cysteine, and serine, was enhanced. However, only serine was increased in the growth medium of activated microglia; especially, excitotoxic D-serine secretion from primary rat microglia was the most strongly enhanced. Activated primary microglia reduced intracellular ATP levels and proteasome activity in cocultured neuronal cells, especially in primary cortical neurons, via D-serine secretion. Moreover, ubiquitinated proteins accumulated and inclusion bodies were increased in primary dopaminergic and cortical neurons cocultured with activated primary microglia. In vivo, MNPs@SiO2(RITC), D-serine, and ubiquitin aggresomes were distributed in the MNPs@SiO2(RITC)-treated mouse brain.ConclusionsMNPs@SiO2(RITC)-induced activation of microglia triggers excitotoxicity in neurons via D-serine secretion, highlighting the importance of neurotoxicity mechanisms incurred by nanoparticle-induced microglial activation.

Formation of vesicle-like structures by stimulation of colchicine.

Formation of vesicle-like structures by stimulation of colchicine.

Cover Picture: Dinuclear Face-Sharing Bi-octahedral Tungsten(VI) Core and Unusual Thermal Behavior in Complex Th Tungstates (Chem. Eur. J. 21/2015)

A rare case of a confacial bi-octahedral tungsten(VI) core was isolated from a thorium tungstate system. These compounds are constructed from a complex polyoxometalate-like cage and exhibit unexpected thermal behavior. Interestingly, the cage with a threefold rotation axis passing through its center has a cross-section remarkably similar to a six-leafed lily. A comprehensive discussion can be found in the Full Paper by E. V. Alekseev et al. on page 7746 ff. A rare case of a confacial bi-octahedral tungsten(VI) core was isolated from a thorium tungstate system. These compounds are constructed from a complex polyoxometalate-like cage and exhibit unexpected thermal behavior. Interestingly, the cage with a threefold rotation axis passing through its center has a cross-section remarkably similar to a six-leafed lily. A comprehensive discussion can be found in the Full Paper by E. V. Alekseev et al. on page 7746 ff. Trifluoromethylation In the Minireview on page 7648 ff., Y.-M. Liang and co-workers describe the recent efforts in the development of trifluoromethylation of alkyne reactions by radical processes, or transition-metal catalysis using nucleophilic or electrophilic trifluoromethylation reagents. Various diverse C(sp, sp2, sp3)CF3 bonds were easily constructed in moderate to good yields under mild conditions. Although the above achievements have been obtained, several limitations still remain in the trifluoromethylation reactions of alkynes.1 Functionalization of Boron Nitride Platelets of hexagonal boron nitride were functionalized with a silicon oxide rim in a simple room-temperature process. The mechanism of this functionalization has been investigated by first principles calculations. More details of this research are reported by R. Nesper and co-workers in their Communication on page 7662 ff.1 Polyoxometalates The reaction of early lanthanides, GeO2, and Na2WO4 in buffered medium was systematically studied and a series of hexameric and dodecameric polyoxometalates with a closely related crown-shaped framework based on [Ln2GeW10O38]6– Keggin-type subunits was isolated. The dodecamers represent the first macroions with dual behavior in the formation of vesicle-like structures that lead to different supramolecular assemblies in terms of composition and size by a solvent-triggered skeletal modification of the species undergoing self-association. For more details, see the Full Paper by S. Reinoso et al. on page 7736 ff.1

Reversible Transitions between Peptide Nanotubes and Vesicle‐Like Structures Including Theoretical Modeling Studies

Peptide-based self-assembling systems are increasingly attractive because of their wide range of applications in different fields. Peptide nanostructures are flexible with changes in the ambient conditions. Herein, a reversible shape transition between self-assembled dipeptide nanotubes (DPNTs) and vesicle-like structures is observed upon a change in the peptide concentration. SEM, TEM, AFM, and CD spectroscopy were used to follow this transition process. We show that dilution of a peptide-nanotube dispersion solution results in the formation of vesicle-like structures, which can then be reassembled into the nanotubes by concentrating the solution. A theoretical model describing this shape-transition phenomenon is presented to propose ways to engineer assembling molecules in order to devise other systems in which the morphology can be tuned on demand.

PKD1, PKD2, and Their Substrate Kidins220 Regulate Neurotensin Secretion in the BON Human Endocrine Cell Line

Neurotensin (NT) is a gut peptide that plays an important role in gastrointestinal secretion, motility, and growth as well as the proliferation of NT receptor-positive cancers. Protein kinase D (PKD) family members (PKD1, 2, and 3) have been identified as important regulators of secretory transport at the trans-Golgi network. Previously, we showed that PKD1 contributes to stimulated NT secretion; however, the mechanisms are not entirely clear. Here, we show that Kidins220, which is a substrate of PKD proteins in neuroendocrine cells, is localized in the ends of the processes of BON cells, similar to the expression pattern of NT vesicles, and translocates to the membrane and large vesicle-like structures formed in response to phorbol 12-myristate 13-acetate treatment. The short hairpin RNA targeting Kidins220 inhibits NT secretion in parental BON cells or BON cells stably expressing the gastrin-releasing peptide receptor treated with either phorbol 12-myristate 13-acetate or bombesin, respectively. Furthermore, we demonstrate that endogenous PKD1, PKD2, and Kidins220 co-exist with NT-containing vesicles. Overexpression of the kinase-dead PKD1 abrogates Kidins220 expression and NT vesicle formation. Our data establish a physiological link between the PKD/Kidins220 pathway and NT-containing vesicles and suggest the role of this pathway in the regulation of hormone secretion. Because NT is an important gut hormone that affects secretion, inflammation, and both normal and tumor cell growth, our findings identify a novel signaling pathway that may be amenable to drug targeting for clinical applications.

Water rise kinetics in refilling xylem after desiccation in a resurrection plant.

The acropetal water refilling kinetics of the dry xylem of branches (up to 80 cm tall) of the resurrection plant Myrothamnus flabellifolia were determined with high temporal resolution by observation of light refraction at the advancing water front and the associated recurving of the folded leaves. To study the effect of gravity on water rise, data were acquired for cut upright, horizontal and inverted branches. Water rise kinetics were also determined with hydrostatic and osmotic pressure as well as at elevated temperatures (up to 100 degrees C) under laboratory conditions and compared with those obtained with intact (rooted) and cut branches under field conditions. Experiments in which water climbed under its capillary pressure alone, showed that the axial flow occurred only in a very few conducting elements at a much higher rate than in many of the other ones. The onset of transpiration of the unfolded and green leaves did not affect the rise kinetics in the 'prominent' conducting elements. Application of pressure apparently increased the number of elements making a major contribution to axial xylem flow. Analysis of these data in terms of capillary-pressure-driven water ascent in leaky capillaries demonstrated that root pressure, not capillary pressure, is the dominant force for rehydration of rooted, dry plants. The main reasons for the failure of capillary forces in xylem refilling were the small, rate-limiting effective radii of the conducting elements for axial water ascent (c. 1 micrometer compared with radii of the vessels and tracheids of c. 18 micrometers and 3 micrometers, respectively) and the very poor wetting of the dry walls. The contact (wetting) angles were of the order of 80 degrees and decreased on root or externally applied hydrostatic pressure. This supported our previous assumption that the inner walls of the dry conducting elements are covered with a lipid layer that is removed or disintegrates upon wetting. Consistent with this, potassium chloride and, particularly, sugars exerted an osmotic pressure effect on axial water climbing (reflection coefficients > zero, but small). Although the osmotically active solutes apparently suppressed radial water spread through the tissue to the leaf cells, they reduced the axial water ascent rather than accelerating it as predicted by the theory of capillary-driven water rise in leaky capillaries. Killing cells by heat treatment and removal of the bark, phelloderm, cortex and phloem also resulted in a reduction of the axial rise rate and final height. These observations demonstrated that radial water movement driven by the developing osmotic and turgor pressure in the living cells was important for the removal of the lipid layer from the walls of those conducting elements that were primarily not involved in water rise. There is some evidence from field measurements of the axial temperature gradients along rooted branches that interfacial (Marangoni) streaming facilitated lipid removal (under formation of vesicle-like structures and lipid bodies) upon wetting. Grant numbers: 50WB 9643, Zi 99/9-1.

Regeneration of intergeneric somatic hybrids by protoplast fusion betweenOnobrychis viciaefolia andMedicago sativa

Protoplast fusion was induced between sainfoin and alfalfa by an improved polyethyleneglycol (PEG) method. The intergeneric somatic calluses were selected based on complementation of hydroxyproline-resistance of sainfoin and hormone autonomy growth of alfalfa transformation cell line. 17 somatic hybrid plantlets were regenerated. PEG could induce the tight agglutination of protoplasts. During diluting and washing process, cyclization of the linked membrane and formation of vesicle-like structures were observed, resulting in protoplast fusion. 5%-10% glycerol supplemented in the fusion inducing solution markedly increased the frequency of heterogeneous fusion. Better fusion results were obtained when mixed protoplast suspension was dripped in petri dishes in which PEG solution was previously placed. Chromosome number of regenerated hybrid buds varied from 30 to 60. The genome of hybrids included the small chromosome from sainfoin and two chromosomes with two clear constrictions from alfalfa. The hybridity of obtained hybrid calluses was confirmed by their isozyme banding patterns and their nopaline synthetase activity.

Incorporation of macromolecules into cells and vesicles by low electric fields: induction of endocytotic-like processes

The present study shows a very efficient incorporation of polysaccharides and β-galactosidase into membrane vesicles and cells by low pulsed electric fields of much lower amplitudes than the threshold electric field for electroporation. The electric induced uptake of dextran-FITC, in the range of 1–2000 kD, was observed in 85% of the cell population, while preserving the cell's viability. The uptake of β-galactosidase, induced by low electric fields, revealed a 30 fold increase in the enzyme's concentration in the cytosol in comparison with its concentration in the extracellular medium. The underlying mechanism of this uptake, based on the electric field induced endocytotic-like process, emerges from the following observations: (i) The uptake is not driven by metabolic energy, since we obtained the incorporation of macromolecules into membrane vesicles or cells at low temperatures; (ii) The formation of vesicle-like structures containing the macromolecules is displayed by confocal fluorescence microscopy, which reveals the existence of massive vesicle-like structures in the cytosol, following exposure to the pulsed electric field; (iii) The formation of vesicle-like structures is supported by the observed decrease in forward light scattering and the increase in side light scattering from single exposed cells.

Excessive Mortality in Market-Age Turkeys Associated with Cellulitis

Between July, 1993, and October, 1994, seven cases were examined that consisted of increased mortality in commercial turkeys due to cellulitis. The condition started at 13-16 wk of age in toms and persisted until the birds were marketed. The mortality rate was 1-2% per week. Lesions began on the ventrum of the tail and consisted of swelling and the formation of vesiclelike structures. Most of the affected birds also had an accumulation of gelatinous fluid in the subcutis of the tail and breast areas. The underlying musculature was often darkened or petechiated. Clostridium perfringens type A was isolated from two of the cases. Lesions similar to those found in the field were reproduced experimentally in turkeys injected with the subcutaneous fluid obtained from birds in field cases.

Surfactosomes: a novel approach to the reconstitution and 2-D crystallisation of membrane proteins

The formation of vesicle-like structures (termed surfactosomes) and lamellar sheets from solutions containing ammonium perfluoroocanoate (APFO) is illustrated using conventional and cryo-transmission electron microscopy. It is shown how this detergent can be used for the solubilisation, reconstitution, and 2-D crystallisation of membrane proteins as demonstrated for the major protein of the membrane sector of the V-type H +-ATPase (16-kDa protein). Electron microscopical analysis of 2-D crystals of the 16-kDa protein (a = b = 13.0 ± 0.2 nm with γ = 90° and p4 projection symmetry) revealed a unit cell comprising four dimeric complexes of the 16-kDa protein the significance of which is discussed.