Publications by authors named "Oldrich Benada"

89 Publications

In Vitro Interactions of TiO Nanoparticles with Earthworm Coelomocytes: Immunotoxicity Assessment.

Nanomaterials (Basel) 2021 Jan 19;11(1). Epub 2021 Jan 19.

Institute of Microbiology of the Czech Academy of Sciences, Videnska 1083, 142 20 Prague 4, Czech Republic.

Titanium dioxide nanoparticles (TiO NPs) are manufactured worldwide. Once they arrive in the soil environment, they can endanger living organisms. Hence, monitoring and assessing the effects of these nanoparticles is required. We focus on the earthworm immune cells exposed to sublethal concentrations of TiO NPs (1, 10, and 100 µg/mL) for 2, 6, and 24 h. TiO NPs at all concentrations did not affect cell viability. Further, TiO NPs did not cause changes in reactive oxygen species (ROS) production, malondialdehyde (MDA) production, and phagocytic activity. Similarly, they did not elicit DNA damage. Overall, we did not detect any toxic effects of TiO NPs at the cellular level. At the gene expression level, slight changes were detected. Metallothionein, fetidin/lysenin, lumbricin and MEK kinase I were upregulated in coelomocytes after exposure to 10 µg/mL TiO NPs for 6 h. Antioxidant enzyme expression was similar in exposed and control cells. TiO NPs were detected on coelomocyte membranes. However, our results do not show any strong effects of these nanoparticles on coelomocytes at both the cellular and molecular levels.
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http://dx.doi.org/10.3390/nano11010250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7832855PMC
January 2021

Gold nanoparticles coated with polyvinylpyrrolidone and sea urchin extracellular molecules induce transient immune activation.

J Hazard Mater 2021 01 30;402:123793. Epub 2020 Aug 30.

Consiglio Nazionale delle Ricerche, Istituto per la Ricerca e l'Innovazione Biomedica (IRIB), Palermo, Italy. Electronic address:

We report that the immunogenicity of colloidal gold nanoparticles coated with polyvinylpyrrolidone (PVP-AuNPs) in a model organism, the sea urchin Paracentrotus lividus, can function as a proxy for humans for in vitro immunological studies. To profile the immune recognition and interaction from exposure to PVP-AuNPs (1 and 10 μg mL), we applied an extensive nano-scale approach, including particle physicochemical characterisation involving immunology, cellular biology, and metabolomics. The interaction between PVP-AuNPs and soluble proteins of the sea urchin physiological coelomic fluid (blood equivalent) results in the formation of a protein "corona" surrounding the NPs from three major proteins that influence the hydrodynamic size and colloidal stability of the particle. At the lower concentration of PVP-AuNPs, the P. lividus phagocytes show a broad metabolic plasticity based on the biosynthesis of metabolites mediating inflammation and phagocytosis. At the higher concentration of PVP-AuNPs, phagocytes activate an immunological response involving Toll-like receptor 4 (TLR4) signalling pathway at 24 hours of exposure. These results emphasise that exposure to PVP-AuNPs drives inflammatory signalling by the phagocytes and the resolution at both the low and high concentrations of the PVP-AuNPs and provides more details regarding the immunogenicity of these NPs.
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http://dx.doi.org/10.1016/j.jhazmat.2020.123793DOI Listing
January 2021

Author Correction: Bacterial nanotubes as a manifestation of cell death.

Nat Commun 2020 Nov 3;11(1):5672. Epub 2020 Nov 3.

Laboratory of Microbial Genetics and Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, 142 20, Prague 4, Czech Republic.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-19618-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609721PMC
November 2020

Bringing SEM and MSI Closer Than Ever Before: Visualizing and Infection in the Rat Lungs.

J Fungi (Basel) 2020 Oct 30;6(4). Epub 2020 Oct 30.

Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague, Czech Republic.

A procedure for processing frozen rat lung tissue sections for scanning electron microscopy (SEM) from deeply frozen samples initially collected and stored for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed. The procedure employed slow thawing of the frozen sections while floating on the surface and melting in a fixative solution. After the float-washing step, the sections were dehydrated in a graded ethanol series and dried in a critical point dryer. The SEM generated images with well-preserved structures, allowing for monitoring of bacterial cells and fungal hyphae in the infected tissue. Importantly, the consecutive nonfixed frozen sections were fully compatible with MALDI-MSI, providing molecular biomarker maps of . The protocol enables bimodal image fusion in the in-house software CycloBranch, as demonstrated by SEM and MALDI-MSI.
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http://dx.doi.org/10.3390/jof6040257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7711807PMC
October 2020

Bacterial nanotubes as a manifestation of cell death.

Nat Commun 2020 10 2;11(1):4963. Epub 2020 Oct 2.

Laboratory of Microbial Genetics and Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, 142 20, Prague 4, Czech Republic.

Bacterial nanotubes are membranous structures that have been reported to function as conduits between cells to exchange DNA, proteins, and nutrients. Here, we investigate the morphology and formation of bacterial nanotubes using Bacillus subtilis. We show that nanotube formation is associated with stress conditions, and is highly sensitive to the cells' genetic background, growth phase, and sample preparation methods. Remarkably, nanotubes appear to be extruded exclusively from dying cells, likely as a result of biophysical forces. Their emergence is extremely fast, occurring within seconds by cannibalizing the cell membrane. Subsequent experiments reveal that cell-to-cell transfer of non-conjugative plasmids depends strictly on the competence system of the cell, and not on nanotube formation. Our study thus supports the notion that bacterial nanotubes are a post mortem phenomenon involved in cell disintegration, and are unlikely to be involved in cytoplasmic content exchange between live cells.
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http://dx.doi.org/10.1038/s41467-020-18800-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7532143PMC
October 2020

The torpedo effect in Bacillus subtilis: RNase J1 resolves stalled transcription complexes.

EMBO J 2020 02 16;39(3):e102500. Epub 2019 Dec 16.

Institute of Microbiology of the Czech Academy of Sciences, Prague 4, Czech Republic.

RNase J1 is the major 5'-to-3' bacterial exoribonuclease. We demonstrate that in its absence, RNA polymerases (RNAPs) are redistributed on DNA, with increased RNAP occupancy on some genes without a parallel increase in transcriptional output. This suggests that some of these RNAPs represent stalled, non-transcribing complexes. We show that RNase J1 is able to resolve these stalled RNAP complexes by a "torpedo" mechanism, whereby RNase J1 degrades the nascent RNA and causes the transcription complex to disassemble upon collision with RNAP. A heterologous enzyme, yeast Xrn1 (5'-to-3' exonuclease), is less efficient than RNase J1 in resolving stalled Bacillus subtilis RNAP, suggesting that the effect is RNase-specific. Our results thus reveal a novel general principle, whereby an RNase can participate in genome-wide surveillance of stalled RNAP complexes, preventing potentially deleterious transcription-replication collisions.
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http://dx.doi.org/10.15252/embj.2019102500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996504PMC
February 2020

subsp. Releases Differentially Loaded Outer Membrane Vesicles Under Various Stress Conditions.

Front Microbiol 2019 10;10:2304. Epub 2019 Oct 10.

Department of Molecular Pathology and Biology, Faculty of Military Health Sciences, University of Defense, Hradec Kralove, Czechia.

is a Gram-negative, facultative intracellular bacterium, causing a severe disease called tularemia. It secretes unusually shaped nanotubular outer membrane vesicles (OMV) loaded with a number of virulence factors and immunoreactive proteins. In the present study, the vesicles were purified from a clinical isolate of subsp. strain FSC200. We here provide a comprehensive proteomic characterization of OMV using a novel approach in which a comparison of OMV and membrane fraction is performed in order to find proteins selectively enriched in OMV vs. membrane. Only these proteins were further considered to be really involved in the OMV function and/or their exceptional structure. OMV were also isolated from bacteria cultured under various cultivation conditions simulating the diverse environments of life cycle. These included conditions mimicking the milieu inside the mammalian host during inflammation: oxidative stress, low pH, and high temperature (42°C); and in contrast, low temperature (25°C). We observed several-fold increase in vesiculation rate and significant protein cargo changes for high temperature and low pH. Further proteomic characterization of stress-derived OMV gave us an insight how the bacterium responds to the hostile environment of a mammalian host through the release of differentially loaded OMV. Among the proteins preferentially and selectively packed into OMV during stressful cultivations, the previously described virulence factors connected to the unique intracellular trafficking of were detected. Considerable changes were also observed in a number of proteins involved in the biosynthesis and metabolism of the bacterial envelope components like O-antigen, lipid A, phospholipids, and fatty acids. Data are available via ProteomeXchange with identifier PXD013074.
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http://dx.doi.org/10.3389/fmicb.2019.02304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6795709PMC
October 2019

Titanium dioxide nanoparticles temporarily influence the sea urchin immunological state suppressing inflammatory-relate gene transcription and boosting antioxidant metabolic activity.

J Hazard Mater 2020 02 5;384:121389. Epub 2019 Oct 5.

Istituto per la Ricerca e l'Innovazione Biomedica (IRIB), Consiglio Nazionale delle Ricerche, Palermo, Italy. Electronic address:

Titanium dioxide nanoparticles (TiONPs) are revolutionizing biomedicine due to their potential application as diagnostic and therapeutic agents. However, the TiONP immune-compatibility remains an open issue, even for ethical reasons. In this work, we investigated the immunomodulatory effects of TiONPs in an emergent proxy to human non-mammalian model for in vitro basic and translational immunology: the sea urchin Paracentrotus lividus. To highlight on the new insights into the evolutionarily conserved intracellular signaling and metabolism pathways involved in immune-TiONP recognition/interaction we applied a wide-ranging approach, including electron microscopy, biochemistry, transcriptomics and metabolomics. Findings highlight that TiONPs interact with immune cells suppressing the expression of genes encoding for proteins involved in immune response and apoptosis (e.g. NF-κB, FGFR2, JUN, MAPK14, FAS, VEGFR, Casp8), and boosting the immune cell antioxidant metabolic activity (e.g. pentose phosphate, cysteine-methionine, glycine-serine metabolism pathways). TiONP uptake was circumscribed to phagosomes/phagolysosomes, depicting harmless vesicular internalization. Our findings underlined that under TiONP-exposure sea urchin innate immune system is able to control inflammatory signaling, excite antioxidant metabolic activity and acquire immunological tolerance, providing a new level of understanding of the TiONP immune-compatibility that could be useful for the development in Nano medicines.
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http://dx.doi.org/10.1016/j.jhazmat.2019.121389DOI Listing
February 2020

Sea Urchin Extracellular Proteins Design a Complex Protein Corona on Titanium Dioxide Nanoparticle Surface Influencing Immune Cell Behavior.

Front Immunol 2019 20;10:2261. Epub 2019 Sep 20.

Istituto per la Ricerca e l'Innovazione Biomedica (IRIB), Consiglio Nazionale delle Ricerche, Palermo, Italy.

Extensive exploitation of titanium dioxide nanoparticles (TiONPs) augments rapid release into the marine environment. When in contact with the body fluids of marine invertebrates, TiONPs undergo a transformation and adhere various organic molecules that shape a complex protein corona prior to contacting cells and tissues. To elucidate the potential extracellular signals that may be involved in the particle recognition by immune cells of the sea urchin , we investigated the behavior of TiONPs in contact with extracellular proteins . Our findings indicate that TiONPs are able to interact with sea urchin proteins in both cell-free and cell-conditioned media. The two-dimensional proteome analysis of the protein corona bound to TiONP revealed that negatively charged proteins bound preferentially to the particles. The main constituents shaping the sea urchin cell-conditioned TiONP protein corona were proteins involved in cellular adhesion (-toposome, -galectin-8, -nectin) and cytoskeletal organization (actin and tubulin). Immune cells (phagocytes) aggregated TiONPs on the outer cell surface and within well-organized vesicles without eliciting harmful effects on the biological activities of the cells. Cells showed an active metabolism, no oxidative stress or caspase activation. These results provide a new level of understanding of the extracellular proteins involved in the immune-TiONP recognition and interaction , confirming that primary immune cell cultures from can be an optional model for swift and efficient immune-toxicological investigations.
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http://dx.doi.org/10.3389/fimmu.2019.02261DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6763604PMC
October 2020

Adenylate Cyclase Toxin Inhibits Monocyte-to-Macrophage Transition and Dedifferentiates Human Alveolar Macrophages into Monocyte-like Cells.

mBio 2019 09 24;10(5). Epub 2019 Sep 24.

Laboratory of Molecular Biology of Bacterial Pathogens, Institute of Microbiology of the CAS, Prague, Czech Republic

Monocytes arriving at the site of infection differentiate into functional effector macrophages to replenish the resident sentinel cells. , the pertussis agent, secretes an adenylate cyclase toxin-hemolysin (CyaA) that binds myeloid phagocytes through complement receptor 3 (CD11b/CD18) and swiftly delivers its adenylyl cyclase enzyme domain into phagocytes. This ablates the bactericidal capacities of phagocytes through massive and unregulated conversion of cytosolic ATP into the key signaling molecule cAMP. We show that exposure of primary human monocytes to as low a concentration as 22.5 pM CyaA, or a low (2:1) multiplicity of infection by CyaA-producing bacteria, blocks macrophage colony-stimulating factor (M-CSF)-driven differentiation of monocytes. CyaA-induced cAMP signaling mediated through the activity of protein kinase A (PKA) efficiently blocked expression of macrophage markers, and the monocytes exposed to 22.5 pM CyaA failed to acquire the characteristic intracellular complexity of mature macrophage cells. Neither M-CSF-induced endoplasmic reticulum (ER) expansion nor accumulation of Golgi bodies, mitochondria, or lysosomes was observed in toxin-exposed monocytes, which remained small and poorly phagocytic and lacked pseudopodia. Exposure to 22.5 pM CyaA toxin provoked loss of macrophage marker expression on differentiated macrophages, as well as on primary human alveolar macrophages, which appeared to dedifferentiate into monocyte-like cells with upregulated CD14 levels. This is the first report that terminally differentiated tissue-resident macrophage cells can be dedifferentiated The results suggest that blocking of monocyte-to-macrophage transition and/or dedifferentiation of the sentinel cells of innate immunity through cAMP-elevating toxin action may represent a novel immune evasion strategy of bacterial pathogens. Macrophages are key sentinel cells of the immune system, and, as such, they are targeted by the toxins produced by the pertussis agent The adenylate cyclase toxin (CyaA) mediates immune evasion of by suspending the bactericidal activities of myeloid phagocytes. We reveal a novel mechanism of potential subversion of host immunity, where CyaA at very low (22 pM) concentrations could inhibit maturation of human monocyte precursors into the more phagocytic macrophage cells. Furthermore, exposure to low CyaA amounts has been shown to trigger dedifferentiation of mature primary human alveolar macrophages back into monocyte-like cells. This unprecedented capacity is likely to promote survival of the pathogen in the airways, both by preventing maturation of monocytes attracted to the site of infection into phagocytic macrophages and by dedifferentiation of the already airway-resident sentinel cells.
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http://dx.doi.org/10.1128/mBio.01743-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6759761PMC
September 2019

Specific structure, morphology, and properties of polyacrylonitrile (PAN) membranes prepared by needleless electrospinning; Forming hollow fibers.

Mater Sci Eng C Mater Biol Appl 2019 Dec 30;105:110151. Epub 2019 Aug 30.

Faculty of Science, J. E. Purkyně University, České mládeže 8, 400 96 Ústí nad Labem, Czech Republic.

Polyacrylonitrile (PAN) membranes have been prepared using needleless electrospinning with wire electrode and characterized by a series of methods HRSEM, XRD, air permeability and area weight measurements in dependence of high voltage and electrode distance. HRSEM analysis revealed the tendency to longitudinal rolling of strip-shaped PAN fibers forming hollow fibers. Combination of XRD analysis and molecular modeling explains this phenomenon as the consequence of the specific crystal structure of PAN fibers, where the isotactic PAN chains are arranged in layers forming belt shaped nanofibers with the strong tendency to roll up longitudinally forming hollow fibers. This effect offers the possibility to create hollow nanofibers by electrospinning with the appropriate choice of structure of polymer chains.
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http://dx.doi.org/10.1016/j.msec.2019.110151DOI Listing
December 2019

A comparative analysis of protein virulence factors released via extracellular vesicles in two Candida albicans strains cultivated in a nutrient-limited medium.

Microb Pathog 2019 Nov 11;136:103666. Epub 2019 Aug 11.

Charles University, Faculty of Pharmacy in Hradec Králové, Teaching and Research Center, Czech Republic.

One of the pathways for the delivery of virulence effector molecules into the extracellular environment of Candida albicans relies on the release of membrane-bound carriers which are called extracellular vesicles (EVs). Only a few studies aimed at investigating Candida albicans extracellular vesicles protein cargo and its potential contribution to the pathogenesis of C. albicans infections have been conducted to date. In this study, we mainly focused on a search for proteins with a demonstrated linkage to pathogenesis in EVs isolated from two C. albicans strains, the model strain ATCC 90028 and the clinical isolate from a woman suffering from vulvovaginal candidiasis. For the purpose of mimicking one of many hostile conditions during a host-pathogen interaction, C. albicans strains in a nutrient-limited medium were cultivated. We have hypothesized that this unfavourable, stressful condition could contribute to the induction of virulence effector molecules being released at a more extensive rate. In conclusion, 34 proteins with an undisputed linkage to C. albicans pathogenesis were detected in the extracellular vesicle cargoes of both strains. In case of the clinical isolate strain, no unique virulence-associated proteins were detected. In the C. albicans ATCC 90028 model strain, three unique proteins were detected, namely: agglutinin-like protein 3 (Als3), secreted aspartic protease 8 (Sap8) and cell surface superoxide dismutase [Cu-Zn] 6 (Sod6).
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http://dx.doi.org/10.1016/j.micpath.2019.103666DOI Listing
November 2019

Proteomic analysis revealed the survival strategy of Coxiella burnetii to doxycycline exposure.

J Proteomics 2019 09 5;208:103479. Epub 2019 Aug 5.

Institute of Virology, Biomedical Research Center, Slovak Academy of Sciences, Dúbravská cesta 9, 845 05 Bratislava, Slovakia; Institute of Microbiology, Academy of Sciences of the Czech Republic, Videnská 1083, 142 20 Prague, Czech Republic. Electronic address:

Antibiotic resistance is a global threat with a top concern in healthcare. Doxycycline is an antibiotic highly permeable to cell membrane used for treating a broad variety of bacteria, including Coxiella burnetii. This intracellular pathogen is the causative agent of Q fever, a re-emerging zoonosis found worldwide. Hence, C. burnetii has a considerable impact on the farming industry and public health, it is essential to explore its antibiotic adaptation/tolerance strategy to ensure effective therapy. Herein, we tracked changes in the bacterium induced by doxycycline exposure. Our proteomic analysis detected fifteen significantly altered proteins. Adjustments of some key proteins were verified by gene expression analysis. We also observed an increasing in hydrogen peroxide as a consequence of treatment, indicating deregulation of redox balance. Thus, our data suggests the reduction of protein synthesis to minimal levels, activation of the defense mechanism against oxidative stress and maintenance of cell envelope integrity as the key processes ensuring C. burnetii survival under doxycycline exposure. SIGNIFICANCE: Infection by intracellular microorganisms like C. burnetii requires long periods of treatment, thus antibiotic resistance development is a risk. In this report, 2-DE quantitative proteomics was used to identify changes in the proteome that occurs when C. burnetii is exposed to high concentrations of doxycycline. The identification of pathways impacted by doxycycline could be helpful to understand the mechanism of how C. burnetii is dealing with antibiotic stress.
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http://dx.doi.org/10.1016/j.jprot.2019.103479DOI Listing
September 2019

Silencing of carbonic anhydrase I enhances the malignant potential of exosomes secreted by prostatic tumour cells.

J Cell Mol Med 2019 05 27;23(5):3641-3655. Epub 2019 Mar 27.

Biomedical Research Center SAS, Bratislava, Slovak Republic.

We report results showing that the silencing of carbonic anhydrase I (siCA1) in prostatic (PC3) tumour cells has a significant impact on exosome formation. An increased diameter, concentration and diversity of the produced exosomes were noticed as a consequence of this knock-down. The protein composition of the exosomes' cargo was also altered. Liquid chromatography and mass spectrometry analyses identified 42 proteins significantly altered in PC3 siCA1 exosomes compared with controls. The affected proteins are mainly involved in metabolic processes, biogenesis, cell component organization and defense/immunity. Interestingly, almost all of them have been described as 'enhancers' of tumour development through the promotion of cell proliferation, migration and invasion. Thus, our results indicate that the reduced expression of the CA1 protein enhances the malignant potential of PC3 cells.
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http://dx.doi.org/10.1111/jcmm.14265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6484292PMC
May 2019

Morphology of the vasa vasorum in coronary arteries of the porcine heart: A new insight.

Ann Anat 2019 May 13;223:119-126. Epub 2019 Mar 13.

Department of Anatomy, Third Faculty of Medicine, Charles University, Prague, Czech Republic; Centre of Scientific Information, Third Faculty of Medicine, Charles University, Prague, Czech Republic. Electronic address:

Introduction: The vasa vasorum interna were described during the last decade as a special kind of vessels originating directly from the lumen of the paternal artery and participating in the nourishment of its wall, especially of the aorta and coronary arteries. At the same time, their existence was repeatedly denied/negated by many other authors.

Aim: The purpose of the actual study was the anatomical verification of the existence of the vasa vasorum interna in porcine coronary arteries.

Materials And Methods: The vascular supply was studied on the wall of the anterior interventricular branch of the left coronary artery on 36 hearts taken from healthy pigs. Light microscopy, vascular injections and scanning electron microscopy were used for the analysis of 141 samples.

Results: In only two cases small arteries resembling vasa vasorum interna and originating directly from the lumen of the coronary artery were found. But, in both cases these vessels ran without branching, passed over the whole thickness of adventitia and branched in the wider periarterial space. In contrast to this all feeding arteries of the vasa vasorum arose from the larger branches of the paternal artery, branched entirely in its adventitia and did not enter the media.

Conclusion: Due to the very low incidence of these small arteries originating from the lumen of the paternal artery and the absence of their participation on the nourishment of the arterial wall we came to the conclusion that it is not suitable to use the term "vasa vasorum interna" for their designation.
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http://dx.doi.org/10.1016/j.aanat.2019.02.006DOI Listing
May 2019

Fungi, a neglected component of acidophilic biofilms: do they have a potential for biotechnology?

Extremophiles 2019 May 6;23(3):267-275. Epub 2019 Mar 6.

Department of Biology, Faculty of Science, Jan Evangelista Purkyně University in Ústí nad Labem, Za válcovnou 1000/8, 400 01, Ústí nad Labem, Czech Republic.

Fungi from extreme environments, including acidophilic ones, belong to biotechnologically most attractive organisms. They can serve as a source of enzymes and metabolites with potentially uncommon properties and may actively participate within bioremediation processes. In respect of their biotechnological potential, extremophilic fungi are mostly studied as individual species. Nevertheless, microorganisms rarely live separately and they form biofilms instead. Living in biofilms is the most successful life strategy on the Earth and the biofilm is the most abundant form of life in extreme environments including highly acidic ones. Compared to bacterial fraction, fungal part of acidophilic biofilms represents a largely unexplored source of organisms with possible use in biotechnology and especially data on biofilms of highly acidic soils are missing. The functioning of the biofilm results from interactions between organisms whose metabolic capabilities are efficiently combined. When we look on acidophilic fungi and their biotechnological potential we should take this fact into account as well. The practical problem to be resolved in connection with extensive studies of exploitable properties and abilities of acidophilic fungi is the methodology of isolation of strains from the nature. In this respect, novel isolation techniques should be developed.
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http://dx.doi.org/10.1007/s00792-019-01085-9DOI Listing
May 2019

Stability of antibacterial modification of nanofibrous PA6/DTAB membrane during air filtration.

Mater Sci Eng C Mater Biol Appl 2019 Mar 28;96:807-813. Epub 2018 Nov 28.

Nanovia, s. r. o., Litvínov, Podkrušnohorská 271, 436 03, Chudeřín, Czech Republic.

Stable antimicrobial nanofibrous membrane for air filtration based on polyamide 6 (hereafter PA6) modified by 1-dodecyltrimethylammonium bromide (DTAB) has been prepared by electrospinning using one-step technology, i.e. with modifying antimicrobial agent dissolved in spinning solution. Stability of antibacterial membrane function has been tested by air-blowing test to prove the permanency of chemical composition and antibacterial activity. X-ray diffraction, high-resolution scanning electron microscopy (HRSEM) revealed the effect of modifying agent on structure and morphology of PA6 nanofibres. X-ray photoelectron spectroscopy, electrokinetic analysis and antibacterial tests proved the stability of chemical composition and antibacterial activity after air-blowing tests. Special air-blowing device has been constructed for this purpose. The results prove the applicability so prepared membrane for a long-term air-conditioning.
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http://dx.doi.org/10.1016/j.msec.2018.11.065DOI Listing
March 2019

Soil Matrix Determines the Outcome of Interaction Between Mycorrhizal Symbiosis and Biochar for Growth and Nutrition.

Front Microbiol 2018 27;9:2862. Epub 2018 Nov 27.

Laboratory of Fungal Biology, Institute of Microbiology, Czech Academy of Sciences, Prague, Czechia.

Biochar has been heralded as a multipurpose soil amendment to sustainably increase soil fertility and crop yields, affect soil hydraulic properties, reduce nutrient losses, and sequester carbon. Some of the most spectacular results of biochar (and organic nutrient) inputs are the soils in the Amazon, dark anthropogenic soils with extremely high fertility sustained over centuries. Such soil improvements have been particularly difficult to achieve on a short run, leading to speculations that biochar may need to age (weather) in soil to show its best. Further, interaction of biochar with arbuscular mycorrhizal fungi (AMF), important root symbionts of a great majority of terrestrial plants including most agricultural crops, remains little explored. To study the effect of aged biochar on highly mycotrophic plants and their associated AMF, we made use of softwood biochar, collected from a historic charcoal burning site. This biochar (either untreated or chemically activated, the latter serving as a proxy for freshly prepared biochar) was added into two agricultural soils (acid or alkaline), and compared to soils without biochar. These treatments were further crossed with inoculation with a synthetic AMF community to address possible interactions between biochar and the AMF. Biochar application was generally detrimental for growth and mineral nutrition of our experimental plants, but had no effect on the extent of their root colonized by the AMF, nor did it affect composition of their root-borne AMF communities. In contrast, biochar affected development of two out of five AMF ( and ) in the soil. Establishment of symbiosis with AMF largely mitigated biochar-induced suppression of plant growth and mineral nutrition, mainly by improving plant acquisition of phosphorus. Both mycorrhizal and non-mycorrhizal plants grew well in the acid soil without biochar application, whereas non-mycorrhizal plants remained stunted in the alkaline soils under all situations (with or without biochar). These different and strong effects indicate that response of plants to biochar application are largely dependent on soil matrix and also on microbes such as AMF, and call for further research to enable qualified predictions of the effects of different biochar applications on field-grown crops and soil processes.
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http://dx.doi.org/10.3389/fmicb.2018.02862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277529PMC
November 2018

Preclinical alternative model for analysis of porous scaffold biocompatibility in bone tissue engineering.

ALTEX 2019 23;36(1):121-130. Epub 2018 Nov 23.

Institute of Anatomy, Charles University, Prague, Czech Republic.

Using scaffolds with appropriate porosity represents a potential approach for repair of critical-size bone defects. Vascularization is essential for bone formation and healing. This study investigates methods for monitoring angiogenesis within porous biopolymer scaffolds on the basis of polyhydroxybutyrate (PHB)/chitosan. We use the chick and quail chorioallantoic membrane (CAM) assay as an in vivo model focused on the formation of new blood vessels inside the implant structure. Chemical properties of the surface in biopolymer scaffold matrix were characterized as well as the tissue reaction of the CAM. Implantation of a piece of polymer scaffold results in vascular reaction, documented visually and by ultrasound biomicroscopy. Histological analysis shows myofibroblast reaction (smooth muscle actin-positive cells) without excessive collagen deposition. Cell invasion is observed inside the implant, and QH1 marker, detecting hemangioblasts and endothelial cells of quail origin, confirms the presence of vascular network. The CAM assay is a rapid and easy way to test biocompatibility and vasculogenic potential of new candidate scaffolds for bone tissue bioengineering with respect to the 3R´ s.
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http://dx.doi.org/10.14573/altex.1807241DOI Listing
April 2019

The use of styrene-maleic acid copolymer (SMA) for studies on T cell membrane rafts.

Biochim Biophys Acta Biomembr 2019 01 14;1861(1):130-141. Epub 2018 Aug 14.

Institute of Molecular Genetics of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Praha 4, Czech Republic. Electronic address:

An emerging alternative to the use of detergents in biochemical studies on membrane proteins is apparently the use styrene-maleic acid (SMA) amphipathic copolymers. These cut the membrane into nanodiscs (SMA-lipid particles, SMALPs), which contain membrane proteins possibly surrounded by their native lipid environment. We examined this approach for studies on several types of T cell membrane proteins, previously defined as raft or non-raft associated, to see whether the properties of the raft derived SMALPs differ from non-raft SMALPs. Our results indicate that two types of raft proteins, GPI-anchored proteins and two Src family kinases, are markedly present in membrane fragments much larger (>250 nm) than those containing non-raft proteins (<20 nm). Lipid probes sensitive to membrane fluidity (membrane order) indicate that the lipid environment in the large SMALPs is less fluid (more ordered) than in the small ones which may indicate the presence of a more ordered lipid L phase which is characteristic of membrane rafts. Also the lipid composition of the small vs. large SMALPs is markedly different - the large ones are enriched in cholesterol and lipids containing saturated fatty acids. In addition, we confirm that T cell membrane proteins present in SMALPs can be readily immunoisolated. Our results support the use of SMA as a potentially better (less artifact prone) alternative to detergents for studies on membrane proteins and their complexes, including membrane rafts.
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http://dx.doi.org/10.1016/j.bbamem.2018.08.006DOI Listing
January 2019

Differential Effects of the Flavonolignans Silybin, Silychristin and 2,3-Dehydrosilybin on Larvae (Cestoda) under Hypoxic and Aerobic In Vitro Conditions.

Molecules 2018 Nov 16;23(11). Epub 2018 Nov 16.

Laboratory of Biotransformation, Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, CZ 142 20 Prague, Czech Republic.

larvae represent a suitable model for evaluating the larvicidal potential of various compounds. In this study we investigated the in vitro effects of three natural flavonolignans-silybin (SB), 2,3-dehydrosilybin (DHSB) and silychristin (SCH)-on larvae at concentrations of 5 and 50 μM under aerobic and hypoxic conditions for 72 h. With both kinds of treatment, the viability and motility of larvae remained unchanged, metabolic activity, neutral red uptake and concentrations of neutral lipids were reduced, in contrast with a significantly elevated glucose content. Incubation conditions modified the effects of individual FLs depending on their concentration. Under both sets of conditions, SB and SCH suppressed metabolic activity, the concentration of glucose, lipids and partially motility more at 50 μM, but neutral red uptake was elevated. DHSB exerted larvicidal activity and affected motility and neutral lipid concentrations differently depending on the cultivation conditions, whereas it decreased glucose concentration. DHSB at the 50 μM concentration caused irreversible morphological alterations along with damage to the microvillus surface of larvae, which was accompanied by unregulated neutral red uptake. In conclusion, SB and SCH suppressed mitochondrial functions and energy stores, inducing a physiological misbalance, whereas DHSB exhibited a direct larvicidal effect due to damage to the tegument and complete disruption of larval physiology and metabolism.
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http://dx.doi.org/10.3390/molecules23112999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6278466PMC
November 2018

Diamond nanoparticles suppress lateral growth of bacterial colonies.

Colloids Surf B Biointerfaces 2018 Oct 28;170:544-552. Epub 2018 Jun 28.

Institute of Physics of the Czech Academy of Sciences, v.v.i., Cukrovarnická 10, 162 00, Prague 6, Czech Republic.

Diamond nanoparticles (DNPs) of various types have been recently reported to possess antibacterial properties. Studies have shown a decrease of the colony forming ability on agar plates of the bacteria that had been previously co-incubated with DNPs in the suspension. Before plating, bacteria with DNPs were adequately diluted in order to obtain a suitable number of colony forming units. However, residual DNPs were still present on an agar plate, concentrated on the surface during the plating process; this introduces a potential artifact which might affect colony growth. The effect of DNPs remaining on the surface, alongside growing bacteria, has not been previously investigated. In this work, we present the experiments designed to investigate the effect of DNPs on bacterial survival and on the growth of the bacterial colony on a solid media. We employed Escherichia coli and Bacillus subtilis as models of Gram-negative and Gram-positive bacteria, respectively, and Proteus mirabilis as a model of bacterium exhibiting swarming motility on the surfaces. We analyzed the number, area, and weight of bacterial colonies grown on the agar surface covered with DNPs. We did not observe any bactericidal effect of such applied DNPs. However, in all bacterial species used in this work, we observed the appreciable reduction of colony area, which suggests that DNPs obstruct either bacterial growth or motility. The most obvious effect on colony growth was observed in the case of motile P. mirabilis. We show that DNPs act as the mechanical barrier blocking the lateral colony growth.
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http://dx.doi.org/10.1016/j.colsurfb.2018.06.057DOI Listing
October 2018

σ from Bacillus subtilis: Impact on Gene Expression and Characterization of σ-Dependent Transcription That Requires New Types of Promoters with Extended -35 and -10 Elements.

J Bacteriol 2018 09 10;200(17). Epub 2018 Aug 10.

Laboratory of Microbial Genetics and Gene Expression, Institute of Microbiology of the Czech Academy of Sciences, Prague, Czech Republic

The σ sigma factor from is a σ factor associated with RNA polymerase (RNAP) that was previously implicated in adaptation of the cell to elevated temperature. Here, we provide a comprehensive characterization of this transcriptional regulator. By transcriptome sequencing (RNA-seq) of wild-type (wt) and σ-null strains at 37°C and 52°C, we identified ∼130 genes affected by the absence of σ Further analysis revealed that the majority of these genes were affected indirectly by σ The σ regulon, i.e., the genes directly regulated by σ, consists of 16 genes, of which eight (the and operons) are involved in iron metabolism. The involvement of σ in iron metabolism was confirmed phenotypically. Next, we set up an transcription system and defined and experimentally validated the promoter sequence logo that, in addition to -35 and -10 regions, also contains extended -35 and -10 motifs. Thus, σ-dependent promoters are relatively information rich in comparison with most other promoters. In summary, this study supplies information about the least-explored σ factor from the industrially important model organism In bacteria, σ factors are essential for transcription initiation. Knowledge about their regulons (i.e., genes transcribed from promoters dependent on these σ factors) is the key for understanding how bacteria cope with the changing environment and could be instrumental for biotechnologically motivated rewiring of gene expression. Here, we characterize the σ regulon from the industrially important model Gram-positive bacterium We reveal that σ affects expression of ∼130 genes, of which 16 are directly regulated by σ, including genes encoding proteins involved in iron homeostasis. Detailed analysis of promoter elements then identifies unique sequences important for σ-dependent transcription. This study thus provides a comprehensive view on this underexplored component of the transcription machinery.
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http://dx.doi.org/10.1128/JB.00251-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6088155PMC
September 2018

The enzymatic de-epithelialization technique determines denuded amniotic membrane integrity and viability of harvested epithelial cells.

PLoS One 2018 27;13(3):e0194820. Epub 2018 Mar 27.

Laboratory of the Biology and Pathology of the Eye, Department of Paediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital, Prague, Czech Republic.

The human amniotic membrane (HAM) is widely used for its wound healing effect in clinical practice, as a feeder for the cell cultivation, or a source of cells to be used in cell therapy. The aim of this study was to find effective and safe enzymatic HAM de-epithelialization method leading to harvesting of both denuded undamaged HAM and viable human amniotic epithelial cells (hAECs). The efficiency of de-epithelialization using TrypLE Express, trypsin/ ethylenediaminetetraacetic (EDTA), and thermolysin was monitored by hematoxylin and eosin staining and by the measurement of DNA concentration. The cell viability was determined by trypan blue staining. Scanning electron microscopy and immunodetection of collagen type IV and laminin α5 chain were used to check the basement membrane integrity. De-epithelialized hAECs were cultured and their stemness properties and proliferation potential was assessed after each passage. The HAM was successfully de-epithelialized using all three types of reagents, but morphological changes in basement membrane and stroma were observed after the thermolysin application. About 60% of cells remained viable using trypsin/EDTA, approximately 6% using TrypLE Express, and all cells were lethally damaged after thermolysin application. The hAECs isolated using trypsin/EDTA were successfully cultured up to the 5th passage with increasing proliferation potential and decreased stem cell markers expression (NANOG, SOX2) in prolonged cell culture. Trypsin/EDTA technique was the most efficient for obtaining both undamaged denuded HAM and viable hAECs for consequent culture.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194820PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5870984PMC
July 2018

γ-Tubulin has a conserved intrinsic property of self-polymerization into double stranded filaments and fibrillar networks.

Biochim Biophys Acta Mol Cell Res 2018 May 27;1865(5):734-748. Epub 2018 Feb 27.

Institute of Microbiology of the Czech Academy of Sciences, Vídeňská 1083, 142 20 Prague 4, Czech Republic. Electronic address:

γ-Tubulin is essential for microtubule nucleation and also plays less understood roles in nuclear and cell-cycle-related functions. High abundancy of γ-tubulin in acentrosomal Arabidopsis cells facilitated purification and biochemical characterization of large molecular species of γ-tubulin. TEM, fluorescence, and atomic force microscopy of purified high molecular γ-tubulin forms revealed the presence of linear filaments with a double protofilament substructure, filament bundles and aggregates. Filament formation from highly purified γ-tubulin free of γ-tubulin complex proteins (GCPs) was demonstrated for both plant and human γ-tubulin. Moreover, γ-tubulin associated with porcine brain microtubules formed oligomers. Experimental evidence on the intrinsic ability of γ-tubulin to oligomerize/polymerize was supported by conservation of α- and β-tubulin interfaces for longitudinal and lateral interactions for γ-tubulins. STED (stimulated emission depletion) microscopy of Arabidopsis cells revealed fine, short γ-tubulin fibrillar structures enriched on mitotic microtubular arrays that accumulated at polar regions of acentrosomal spindles and the outer nuclear envelope before mitosis, and were also present in nuclei. Fine fibrillar structures of γ-tubulin representing assemblies of higher order were localized in cell-cycle-dependent manner at sites of dispersed γ-tubulin location in acentrosomal plant cells as well as at sites of local γ-tubulin enrichment after drug treatment. Our findings that γ-tubulin preserves the capability of prokaryotic tubulins to self-organize into filaments assembling by lateral interaction into bundles/clusters help understanding of the relationship between structure and multiple cellular functions of this protein species and suggest that besides microtubule nucleation and organization, γ-tubulin may also have scaffolding or sequestration functions.
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http://dx.doi.org/10.1016/j.bbamcr.2018.02.009DOI Listing
May 2018

Diethyldithiocarbamate enhanced chemical generation of volatile palladium species, their characterization by AAS, ICP-MS, TEM and DART-MS and proposed mechanism of action.

Anal Chim Acta 2018 Apr 28;1005:16-26. Epub 2017 Dec 28.

Institute of Analytical Chemistry of the Czech Academy of Sciences, Veveří 97, 602 00 Brno, Czech Republic. Electronic address:

Comprehensive investigation of chemical generation of volatile species (VSG) of palladium for detection by analytical atomic and mass spectrometry and, specifically, the mechanistic aspects of their formation and tentative identification are presented. VSG was achieved in a flow injection mode using a generator that permitted rapid mixing of acidified sample with NaBH reductant. Atomization in a diffusion flame with detection by atomic absorption spectrometry was exclusively used for optimization of generation conditions while inductively coupled plasma mass spectrometry was utilized to investigate overall system efficiency and analytical metrics of the VSG system for potential ultratrace analysis. Sodium diethyldithiocarbamate (DDTC) served as a crucial reaction modifier, enhancing overall system efficiency 9-fold. Combinations of modifiers, Triton X-100 and Antifoam B surfactants provided a synergistic effect to yield a further 2-fold enhancement of VSG. The overall system efficiency was in the range 16-22%, with higher efficiencies correlating with higher Pd concentrations. The contribution of co-generated aerosol to the overall system efficiency, determined by means of concurrent measurement of added Cs, was negligible - less than 0.1%. The nature of the volatile species was investigated using several approaches, but principally by transmission electron microscopy (TEM) after their collection on a grid, and by direct analysis in real time (DART) using high resolution orbitrap mass spectrometry. These experiments suggest a parallel but dual-route mechanism of VSG of Pd, one attributed to generation of a volatile DDTC chelate of Pd and a second to nanoparticle formation.
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http://dx.doi.org/10.1016/j.aca.2017.12.013DOI Listing
April 2018

Secondary Metabolites Produced during the Germination of .

Front Microbiol 2017 13;8:2495. Epub 2017 Dec 13.

First Faculty of Medicine, Institute of Immunology and Microbiology, Charles University, Prague, Czechia.

Spore awakening is a series of actions that starts with purely physical processes and continues via the launching of gene expression and metabolic activities, eventually achieving a vegetative phase of growth. In spore-forming microorganisms, the germination process is controlled by intra- and inter-species communication. However, in the clade, which is capable of developing a plethora of valuable compounds, the chemical signals produced during germination have not been systematically studied before. Our previously published data revealed that several secondary metabolite biosynthetic genes are expressed during germination. Therefore, we focus here on the secondary metabolite production during this developmental stage. Using high-performance liquid chromatography-mass spectrometry, we found that the sesquiterpenoid antibiotic albaflavenone, the polyketide germicidin A, and chalcone are produced during germination of the model streptomycete, . Interestingly, the last two compounds revealed an inhibitory effect on the germination process. The secondary metabolites originating from the early stage of microbial growth may coordinate the development of the producer () and/or play a role in competitive microflora repression () in their nature environments.
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http://dx.doi.org/10.3389/fmicb.2017.02495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5733532PMC
December 2017

Plectin controls biliary tree architecture and stability in cholestasis.

J Hepatol 2018 05 20;68(5):1006-1017. Epub 2017 Dec 20.

Laboratory of Integrative Biology, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic. Electronic address:

Background & Aims: Plectin, a highly versatile cytolinker protein, controls intermediate filament cytoarchitecture and cellular stress response. In the present study, we investigate the role of plectin in the liver under basal conditions and in experimental cholestasis.

Methods: We generated liver-specific plectin knockout (Ple) mice and analyzed them using two cholestatic liver injury models: bile duct ligation (BDL) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) feeding. Primary hepatocytes and a cholangiocyte cell line were used to address the impact of plectin on keratin filament organization and stability in vitro.

Results: Plectin deficiency in hepatocytes and biliary epithelial cells led to aberrant keratin filament network organization, biliary tree malformations, and collapse of bile ducts and ductules. Further, plectin ablation significantly aggravated biliary damage upon cholestatic challenge. Coincidently, we observed a significant expansion of A6-positive progenitor cells in Ple livers. After BDL, plectin-deficient bile ducts were prominently dilated with more frequent ruptures corresponding to an increased number of bile infarcts. In addition, more abundant keratin aggregates indicated less stable keratin filaments in Ple hepatocytes. A transmission electron microscopy analysis revealed a compromised tight junction formation in plectin-deficient biliary epithelial cells. In addition, protein profiling showed increased expression of the adherens junction protein E-Cadherin, and inefficient upregulation of the desmosomal protein desmoplakin in response to BDL. In vitro analyses revealed a higher susceptibility of plectin-deficient keratin networks to stress-induced collapse, paralleled by elevated activation of p38 MAP kinase.

Conclusion: Our study shows that by maintaining proper keratin network cytoarchitecture and biliary epithelial stability, plectin plays a critical role in protecting the liver from stress elicited by cholestasis.

Lay Summary: Plectin is a cytolinker protein capable of interconnecting all three cytoskeletal filament systems and linking them to plasma membrane-bound junctional complexes. In liver, the plectin-controlled cytoskeleton mechanically stabilizes epithelial cells and provides them with the capacity to adapt to increased bile pressure under cholestasis.
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http://dx.doi.org/10.1016/j.jhep.2017.12.011DOI Listing
May 2018

Teicoplanin resistance in Staphylococcus haemolyticus is associated with mutations in histidine kinases VraS and WalK.

Diagn Microbiol Infect Dis 2018 Mar 16;90(3):233-240. Epub 2017 Nov 16.

Institute of Microbiology v. v. i., The Czech Academy of Sciences, Průmyslová 595, Vestec 252 50, Czech Republic. Electronic address:

We investigated the genetic basis of glycopeptide resistance in laboratory-derived strains of S. haemolyticus with emphasis on differences between vancomycin and teicoplanin. The genomes of two stable teicoplanin-resistant laboratory mutants selected on vancomycin or teicoplanin were sequenced and compared to parental S. haemolyticus strain W2/124. Only the two non-synonymous mutations, VraS Q289K and WalK V550L were identified. No other mutations or genome rearrangements were detected. Increased cell wall thickness, resistance to lysostaphin-induced lysis and adaptation of cell growth rates specifically to teicoplanin were phenotypes observed in a sequenced strain with the VraS Q289K mutation. Neither of the VraS Q289K and WalK V550L mutations was present in the genomes of 121S. haemolyticus clinical isolates. However, all but two of the teicoplanin resistant strains carried non-synonymous SNPs in vraSRTU and walKR-YycHIJ operons pointing to their importance for the glycopeptide resistance.
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http://dx.doi.org/10.1016/j.diagmicrobio.2017.11.007DOI Listing
March 2018

Biological safety and tissue distribution of (16-mercaptohexadecyl)trimethylammonium bromide-modified cationic gold nanorods.

Biomaterials 2018 Feb 1;154:275-290. Epub 2017 Nov 1.

Department of Genome Integrity, Institute of Molecular Genetics of the CAS, v.v.i., Prague, Czech Republic. Electronic address:

The exceptionally high cellular uptake of gold nanorods (GNRs) bearing cationic surfactants makes them a promising tool for biomedical applications. Given the known specific toxic and stress effects of some preparations of cationic nanoparticles, the purpose of this study was to evaluate, in an in vitro and in vivo in mouse, the potential harmful effects of GNRs coated with (16-mercaptohexadecyl)trimethylammonium bromide (GNRs). Interestingly, even after cellular accumulation of high amounts of GNRs sufficient for induction of photothermal effect, no genotoxicity (even after longer-term accumulation), induction of autophagy, destabilization of lysosomes (dominant organelles of their cellular destination), alterations of actin cytoskeleton, or in cell migration could be detected in vitro. In vivo, after intravenous administration, the majority of GNRs accumulated in mouse spleen followed by lungs and liver. Microscopic examination of the blood and spleen showed that GNRs interacted with white blood cells (mononuclear and polymorphonuclear leukocytes) and thrombocytes, and were delivered to the spleen red pulp mainly as GNR-thrombocyte complexes. Importantly, no acute toxic effects of GNRs administered as 10 or 50 μg of gold per mice, as well as no pathological changes after their high accumulation in the spleen were observed, indicating good tolerance of GNRs by living systems.
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http://dx.doi.org/10.1016/j.biomaterials.2017.10.044DOI Listing
February 2018