Tick-borne encephalitis virus (TBEV) causes serious, potentially fatal neurological infections that affect humans in endemic regions of Europe and Asia. Neurons are the primary target for TBEV ...infection in the central nervous system. However, knowledge about this viral infection and virus-induced neuronal injury is fragmental. Here, we directly examined the pathology that occurs after TBEV infection in human primary neurons. We exploited the advantages of advanced high-pressure freezing and freeze-substitution techniques to achieve optimal preservation of infected cell architecture. Electron tomographic (ET) reconstructions elucidated high-resolution 3D images of the proliferating endoplasmic reticulum, and individual tubule-like structures of different diameters in the endoplasmic reticulum cisternae of single cells. ET revealed direct connections between the tubule-like structures and viral particles in the endoplasmic reticulum. Furthermore, ET showed connections between cellular microtubules and vacuoles that harbored the TBEV virions in neuronal extensions. This study was the first to characterize the 3D topographical organization of membranous whorls and autophagic vacuoles in TBEV-infected human neurons. The functional importance of autophagy during TBEV replication was studied in human neuroblastoma cells; stimulation of autophagy resulted in significantly increased dose-dependent TBEV production, whereas the inhibition of autophagy showed a profound, dose-dependent decrease of the yield of infectious virus.
We present a powerful method for the simultaneous detection of Au nanoparticles located on both sides of ultrathin sections. The method employs a high-resolution scanning electron microscope (HRSEM) ...operating in scanning transmission electron microscopy (STEM) mode in combination with the detection of backscattered electrons (BSE). The images are recorded simultaneously during STEM and BSE imaging at the precisely selected accelerating voltage. Under proper imaging conditions, the positions of Au nanoparticles on the top or bottom sides can be clearly differentiated, hence showing this method to be suitable for multiple immunolabelling using Au nanoparticles (NPs) as markers. The difference between the upper and lower Au NPs is so large that it is possible to apply common software tools (such as ImageJ) to enable their automatic differentiation. The effects of the section thickness, detector settings and accelerating voltage on the resulting image are shown. Our experimental results correspond to the results modelled in silico by Monte Carlo (MC) simulations.
The mitochondrial contact site and cristae organization system (MICOS) is a multiprotein complex responsible for cristae formation. Even though cristae are found in all mitochondria capable of ...oxidative phosphorylation, only Mic10 and Mic60 appear to be conserved throughout eukaryotes. The remaining 4 or 5 known MICOS subunits are specific to the supergroup Opisthokonta, which includes yeast and mammals that are the only organisms in which this complex has been analyzed experimentally. We have isolated the MICOS from Trypanosoma brucei, a member of the supergroup Excavata that is profoundly diverged from opisthokonts. We show that it is required for the maintenance of the unique discoidal cristae that typify excavates, such as euglenids and kinetoplastids, the latter of which include trypanosomes. The trypanosome MICOS consists of 9 subunits, most of which are essential for normal growth. Unlike in opisthokonts, it contains two distinct Mic10 orthologs and an unconventional putative Mic60 that lacks a mitofilin domain. Interestingly, one of the essential trypanosomatid-specific MICOS subunits called TbMic20 is a thioredoxin-like protein that appears to be involved in import of intermembrane space proteins, including respiratory chain complex assembly factors. This result points to trypanosome MICOS coordinating cristae shaping and population of its membrane with proteins involved in respiration, the latter via the catalytic activity of TbMic20. Thus, trypanosome MICOS allows us to define which of its features are conserved in all eukaryotes and decipher those that represent lineage-specific adaptations.
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•First study of MICOS outside opisthokonts verifies a conserved role in shaping cristae•Trypanosome MICOS novelties include two distinct Mic10s and an atypical Mic60•TbMICOS features a novel thioredoxin-like subunit called TbMic20•TbMic20 appears to be a catalyst for intermembrane space protein import
The mitochondrial contact site and cristae organizing system (MICOS) is a conserved feature of mitochondria. Kaurov et al. identify and characterize MICOS proteins outside of opisthokonts in the kinetoplastid parasite Trypanosoma brucei. TbMICOS plays a conserved role in mitochondrial cristae shaping and harbors several unique features as well.
The salivary gland of hard ticks is a highly innervated tissue where multiple intertwined axonal projections enter each individual acini. In the present study, we investigated the ultrastructural ...architecture of axonal projections within granular salivary gland type II and III acini of Ixodes ricinus female. Using immunogold labeling, we specifically examined the associations of SIFamide neuropeptide, SIFamide receptor (SIFa_R), neuropeptide pigment dispersing factor (PDF), and the invertebrate-specific D1-like dopamine receptor (InvD1L), with acinar cells. In both acini types, SIFamide-positive axons were found to be in direct contact with either basal epithelial cells or a single adlumenal myoepithelial cell in close proximity to the either the acinar duct or its valve, respectively. Accordingly, SIFa_R staining correlated with SIFamide-positive axons in both basal epithelial and myoepithelial cells. Immunoreactivity for both InvD1L and PDF (type II acini exclusively) revealed positive axons radiating along the acinar lumen. These axons were primarily enclosed by the adlumenal myoepithelial cell plasma membrane and interstitial projections of ablumenal epithelial cells. Our study has revealed the detailed ultrastructure of I. ricinus salivary glands, and provides a solid baseline for a comprehensive understanding of the cell-axon interactions and their functions in this essential tick organ.
Two organic scintillators (EJ309 and stilbene) with their different detection chains and elaborate software were used to measure neutron spectra of AmBe and PuC sources. Method of shadow cone ensures ...minimization of scattered neutron influence. To separate neutrons and γ, a modern method of pulse shape discrimination was employed using fast digitizers. Calibration was performed by γ sources and three different methods for the identification of the Compton edge. Spectrometric systems also used different light output functions and unfolding methods. The same spectra were calculated using the MCNP6 code and the JENDL-5 library. The measured and calculated results were mutually compared, and also with the published results available in the literature. Generally, the results agree on the main peaks of neutron spectra but differ in their abundance. The lower limit for the ability to distinguish between neutrons and γ in spectrometry with organic scintillators was found at about 1.5 MeV.
•Neutron spectra of AmBe and PuC sources were measured and calculated.•The measured/unfolded spectra and calculated ones agree in shape.•Methods of energy calibration and neutron/gamma separation are described.
Abstract
We report for the first time the use of two live-cell imaging agents from the group of luminescent transition metal complexes (IRAZOLVE-MITO and REZOLVE-ER) as cathodoluminescent probes. ...This first experimental demonstration shows the application of both probes for the identification of cellular structures at the nanoscale and near the native state directly in the cryo-scanning electron microscope. This approach can potentially be applied to correlative and multimodal approaches and used to target specific regions within vitrified samples at low electron beam energies.
During their long evolution, anoxygenic phototrophic bacteria have inhabited a wide variety of natural habitats and developed specific strategies to cope with the challenges of any particular ...environment. Expression, assembly, and safe operation of the photosynthetic apparatus must be regulated to prevent reactive oxygen species generation under illumination in the presence of oxygen. Here, we report on the photoheterotrophic
sp. strain KRV36, which was isolated from a cold stream in north-western Iceland, 30 km south of the Arctic Circle. In contrast to most aerobic anoxygenic phototrophs, which stop pigment synthesis when illuminated, strain KRV36 maintained its bacteriochlorophyll synthesis even under continuous light. Its cells also contained between 100 and 180 chromatophores, each accommodating photosynthetic complexes that exhibit an unusually large carotenoid absorption spectrum. The expression of photosynthesis genes in dark-adapted cells was transiently downregulated in the first 2 hours exposed to light but recovered to the initial level within 24 hours. An excess of membrane-bound carotenoids as well as high, constitutive expression of oxidative stress response genes provided the required potential for scavenging reactive oxygen species, safeguarding bacteriochlorophyll synthesis and photosystem assembly. The unique cellular architecture and an unusual gene expression pattern represent a specific adaptation that allows the maintenance of anoxygenic phototrophy under arctic conditions characterized by long summer days with relatively low irradiance.IMPORTANCEThe photoheterotrophic bacterium
sp. KRV36 was isolated from a cold stream in Iceland. It expresses its photosynthesis genes, synthesizes bacteriochlorophyll, and assembles functional photosynthetic complexes under continuous light in the presence of oxygen. Unraveling the molecular basis of this ability, which is exceptional among aerobic anoxygenic phototrophic species, will help to understand the evolution of bacterial photosynthesis in response to changing environmental conditions. It might also open new possibilities for genetic engineering of biotechnologically relevant phototrophs, with the aim of increasing photosynthetic activity and their tolerance to reactive oxygen species.
Anaplasma phagocytophilum is an emerging zoonotic pathogen transmitted by Ixodes scapularis that causes human granulocytic anaplasmosis. Here, a high throughput quantitative proteomics approach was ...used to characterize A. phagocytophilum proteome during rickettsial multiplication and identify proteins involved in infection of the tick vector, I. scapularis. The first step in this research was focused on tick cells infected with A. phagocytophilum and sampled at two time points containing 10-15% and 65-71% infected cells, respectively to identify key bacterial proteins over-represented in high percentage infected cells. The second step was focused on adult female tick guts and salivary glands infected with A. phagocytophilum to compare in vitro results with those occurring during bacterial infection in vivo. The results showed differences in the proteome of A. phagocytophilum in infected ticks with higher impact on protein synthesis and processing than on bacterial replication in tick salivary glands. These results correlated well with the developmental cycle of A. phagocytophilum, in which cells convert from an intracellular reticulated, replicative form to the nondividing infectious dense-core form. The analysis of A. phagocytophilum differentially represented proteins identified stress response (GroEL, HSP70) and surface (MSP4) proteins that were over-represented in high percentage infected tick cells and salivary glands when compared to low percentage infected cells and guts, respectively. The results demonstrated that MSP4, GroEL and HSP70 interact and bind to tick cells, thus playing a role in rickettsia-tick interactions. The most important finding of these studies is the increase in the level of certain bacterial stress response and surface proteins in A. phagocytophilum-infected tick cells and salivary glands with functional implication in tick-pathogen interactions. These results gave a new dimension to the role of these stress response and surface proteins during A. phagocytophilum infection in ticks. Characterization of Anaplasma proteome contributes information on host-pathogen interactions and provides targets for development of novel control strategies for pathogen infection and transmission.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The paper describes neutron field characterisation of low-flux multipurpose educational irradiator developed at Czech Technical University in Prague. The irradiator is aimed for demonstration ...experiments including neutron activation analysis, delayed neutron counting, or studies related to neutron and/or gamma detection and spectrometric devices. It may accommodate various neutron sources including 252Cf or AmBe radionuclide sources, D-D generator, or in-reactor irradiated nuclear material serving as delayed neutron source, or gamma sources including radionuclide ones or short-lived sources produced in the adjacent reactor.
The characterisation was performed based on neutron activation technique using gold foils. It included two experimental parts. The first one verified the level of response symmetry in the four irradiation channels and characterisation of the axial distribution in the irradiation channels. Within the second one, the responses to thermal and epithermal neutrons and the cadmium ratio in the central irradiation position were determined. Whereas the former was determined solely with the 252Cf source, the latter was performed for all available sources: 252Cf, AmBe, and the D-D generator. The experimental results were further compared to calculations by the MCNP Monte Carlo code.
•Polyethylene neutron irradiation system with external neutron sources.•Application of neutron activation technique and gamma-spectrometry for neutron field characterisation.•MCNP simulations of neutron transport in polyethylene neutron irradiator.
•Mitochondrially targeted compounds using the triphenylphosphonium (TPP+) moiety are widely used for research and therapeutic purposes.•Previous studies have shown significant functional effects of ...the targeting moiety itself, without any targeted substance.•This study significantly extends the knowledge about these effects on mitochondrial membrane protein levels and tests several hypotheses about the mechanism.•Triphenylphosphonium-based lipophilic cations exert profound but reversible effects on a mitochondrial ETC proteins, which cannot be explained only by a loss of membrane potential.
Triphenylphosphonium (TPP) derivatives are commonly used to target chemical into mitochondria. We show that alkyl-TPP cause reversible, dose- and hydrophobicity-dependent alterations of mitochondrial morphology and function and a selective decrease of mitochondrial inner membrane proteins including subunits of the respiratory chain complexes, as well as components of the mitochondrial calcium uniporter complex. The treatment with alkyl-TPP resulted in the cleavage of the pro-fusion and cristae organisation regulator Optic atrophy-1. The structural and functional effects of alkyl-TPP were found to be reversible and not merely due to loss of membrane potential. A similar effect was observed with the mitochondria-targeted antioxidant MitoQ.
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