Publications by authors named "Jan Bednar"

51 Publications

Quasi-collinear IR AOTF based on mercurous halide single crystals for spatio-spectral hyperspectral imaging.

Opt Express 2021 Apr;29(9):12813-12832

The paper aims to show the advantages of the infrared-optimised quasi-collinear AOTF (acousto-optic tunable filter) for the spatio-spectral hyperspectral imaging system. The optimisation process is presented based on the selected tetragonal anisotropic materials with exceptional optical and acousto-optical properties in IR (infrared) spectral region. These materials are further compared in terms of their features and suitability for AOTF design. The spectral resolution is considered as the main optimising parameter. Resulting from the analysis, the mercurous chloride (HgCl) single crystal is selected as a representative of the mercurous halide family for the presentation of the quasi-collinear AOTF model operating in LWIR (long-wave infrared) spectral band. The overall parameters of the AOTF model such as spectral resolution, chromatic field of view, acoustic frequency, and operational power requirements are estimated and discussed in results.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1364/OE.420571DOI Listing
April 2021

The micronucleus cytome assay - A fast tool for DNA damage screening in human conjunctival epithelial cells.

Ocul Surf 2021 04 4;20:195-198. Epub 2021 Mar 4.

Laboratory of the Biology and Pathology of the Eye, Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Czech Republic.

Purpose: To assess whether the micronucleus cytome assay (MCyt) reliably detects DNA damage occurring in control and pathological superficial epithelial cells from human conjunctiva.

Methods: Impression cytology samples from the bulbar conjunctiva of 33 healthy controls, eight patients with conjunctival intraepithelial neoplasia (CIN) and eight with mucous membrane pemphigoid (MMP) were examined using the MCyt modified for the ocular surface.

Results: The mean number of micronuclei (MNi) in control samples was 0.94 MNi/1000 epithelial cells, with no significant difference between conjunctival quadrants and independent of sex and age. The MCyt assay applied to CIN-affected eyes showed a significantly higher frequency of MNi (18.63/1000 cells), apoptotic cells, nuclear enlargement, multinucleated cells, and keratolysis compared with the corresponding unaffected paired eyes and with the control value. Although the mean MNi frequency in MMP eyes was also higher (1.73 MNi/1000 cells), it did not prove to be statistically different from the control samples. On the other hand, the MMP-affected eyes revealed significantly elevated percentages of cells with snake-like chromatin, multinucleated cells, apoptotic cells, and nuclear buds compared with controls.

Conclusions: Micronucleus cytome assay was adapted as a rapid screening test for genomic instability on the ocular surface. We have determined reference levels for MNi and other nuclear alterations on healthy conjunctiva and demonstrated that particularly frequencies of MNi are significantly elevated in conjunctiva affected by CIN. We demonstrate that MNi are more specific than other nuclear abnormalities and thus can be used for screening of ocular surface neoplasia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jtos.2021.02.011DOI Listing
April 2021

The NANOTUMOR consortium - Towards the Tumor Cell Atlas.

Biol Cell 2021 Jun 26;113(6):272-280. Epub 2021 Feb 26.

INSERM UMR_S1109, Tumor Biomechanics Lab, Université de Strasbourg, Fédération de Médecine Translationnelle de Strasbourg (FMTS), CNRS SNC5055, Strasbourg, France.

Cancer is a multi-step disease where an initial tumour progresses through critical steps shaping, in most cases, life-threatening secondary foci called metastases. The oncogenic cascade involves genetic, epigenetic, signalling pathways, intracellular trafficking and/or metabolic alterations within cancer cells. In addition, pre-malignant and malignant cells orchestrate complex and dynamic interactions with non-malignant cells and acellular matricial components or secreted factors within the tumour microenvironment that is instrumental in the progression of the disease. As our aptitude to effectively treat cancer mostly depends on our ability to decipher, properly diagnose and impede cancer progression and metastasis formation, full characterisation of molecular complexes and cellular processes at play along the metastasis cascade is crucial. For many years, the scientific community lacked adapted imaging and molecular technologies to accurately dissect, at the highest resolution possible, tumour and stromal cells behaviour within their natural microenvironment. In that context, the NANOTUMOR consortium is a French national multi-disciplinary workforce which aims at a providing a multi-scale characterisation of the oncogenic cascade, from the atomic level to the dynamic organisation of the cell in response to genetic mutations, environmental changes or epigenetic modifications. Ultimately, this program aims at identifying new therapeutic targets using innovative drug design.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/boc.202000135DOI Listing
June 2021

Drug-loading capacity of polylactide-based micro- and nanoparticles - Experimental and molecular modeling study.

Int J Pharm 2020 Dec 31;591:120031. Epub 2020 Oct 31.

Jagiellonian University, Faculty of Chemistry, Gronostajowa 2, 30-387 Kraków, Poland. Electronic address:

Micro- and nanostructures prepared from biodegradable homopolymers and amphiphilic block copolymers (AmBCs) have found application as drug-delivery systems (DDSs). The ability to accumulate a drug is a very important parameter characterizing a given DDS. This work focuses on the impact of DDS size, the packing of polymer chains in the DDS, and drug - polymer matrix compatibility on the hydrophobic drug - loading capacity (DLC) of nano/microcarriers prepared from a biodegradable polymer or its copolymer. Using experimental measurements in combination with atomistic molecular dynamics simulations, an analysis of curcumin encapsulation in microspheres (MSs) from polylactide (PLA) homopolymer and nanoparticles (NPs) from PLA-block-poly(2-methacryloyloxyethylphosphorylcholine) AmBC was performed. The results show that curcumin has good affinity for the PLA matrix due to its hydrophobic nature. However, the DLC value is limited by the fact that curcumin only accumulates in the peripheral part of these structures. Such uneven drug distribution in the PLA matrix results from the non-homogeneous density of MSs (non-uniform packing of the polymer chains in the coil). The results also indicate that the MSs can retain a greater amount of hydrophobic drug compared to the NPs, which is associated with the formation of drug aggregates inside the PLA microparticles.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ijpharm.2020.120031DOI Listing
December 2020

Nanoscale Dynamic Readout of a Chemical Redox Process Using Radicals Coupled with Nitrogen-Vacancy Centers in Nanodiamonds.

ACS Nano 2020 10 19;14(10):12938-12950. Epub 2020 Aug 19.

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Flemingovo namesti 2, 166 10 Prague, Czechia.

Biocompatible nanoscale probes for sensitive detection of paramagnetic species and molecules associated with their (bio)chemical transformations would provide a desirable tool for a better understanding of cellular redox processes. Here, we describe an analytical tool based on quantum sensing techniques. We magnetically coupled negatively charged nitrogen-vacancy (NV) centers in nanodiamonds (NDs) with nitroxide radicals present in a bioinert polymer coating of the NDs. We demonstrated that the spin relaxation time of the NV centers is very sensitive to the number of nitroxide radicals, with a resolution down to ∼10 spins per ND (detection of approximately 10 mol in a localized volume). The detection is based on shortening upon the radical attachment, and we propose a theoretical model describing this phenomenon. We further show that this colloidally stable, water-soluble system can be used dynamically for spatiotemporal readout of a redox chemical process (oxidation of ascorbic acid) occurring near the ND surface in an aqueous environment under ambient conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acsnano.0c04010DOI Listing
October 2020

Cryo-electron microscopy of the chromatin fiber.

Curr Opin Struct Biol 2020 10 24;64:97-103. Epub 2020 Jul 24.

Université Grenoble Alpes, CNRS UMR 5309, INSERM U1209, Institute for Advanced Biosciences (IAB), Site Sante´ - Allée des Alpes, 38700 La Tronche, France; Laboratory of the Biology and Pathology of the Eye, Institute of Biology and Medical Genetics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Albertov 4, 128 00 Prague 2, Czech Republic. Electronic address:

The three-dimensional (3D) organization of chromatin plays a crucial role in the regulation of gene expression. Chromatin conformation is strongly affected by the composition, structural features and dynamic properties of the nucleosome, which in turn determine the nature and geometry of interactions that can occur between neighboring nucleosomes. Understanding how chromatin is spatially organized above the nucleosome level is thus essential for understanding how gene regulation is achieved. Towards this end, great effort has been made to understand how an array of nucleosomes folds into a regular chromatin fiber. This review summarizes new insights into the 3D structure of the chromatin fiber that were made possible by recent advances in cryo-electron microscopy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.sbi.2020.06.016DOI Listing
October 2020

Phase-plate cryo-EM structure of the Widom 601 CENP-A nucleosome core particle reveals differential flexibility of the DNA ends.

Nucleic Acids Res 2020 06;48(10):5735-5748

Institute for Advanced Biosciences, Inserm U 1209, CNRS UMR 5309, Université Grenoble Alpes, 38000 Grenoble, France.

The histone H3 variant CENP-A marks centromeres epigenetically and is essential for mitotic fidelity. Previous crystallographic studies of the CENP-A nucleosome core particle (NCP) reconstituted with a human α-satellite DNA derivative revealed both DNA ends to be highly flexible, a feature important for CENP-A mitotic functions. However, recent cryo-EM studies of CENP-A NCP complexes comprising primarily Widom 601 DNA reported well-ordered DNA ends. Here, we report the cryo-EM structure of the CENP-A 601 NCP determined by Volta phase-plate imaging. The data reveal that one ('left') 601 DNA end is well ordered whereas the other ('right') end is flexible and partly detached from the histone core, suggesting sequence-dependent dynamics of the DNA termini. Indeed, a molecular dynamics simulation of the CENP-A 601 NCP confirmed the distinct dynamics of the two DNA extremities. Reprocessing the image data using two-fold symmetry yielded a cryo-EM map in which both DNA ends appeared well ordered, indicating that such an artefact may inadvertently arise if NCP asymmetry is lost during image processing. These findings enhance our understanding of the dynamic features that discriminate CENP-A from H3 nucleosomes by revealing that DNA end flexibility can be fine-tuned in a sequence-dependent manner.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkaa246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261176PMC
June 2020

Generation of Remosomes by the SWI/SNF Chromatin Remodeler Family.

Sci Rep 2019 10 2;9(1):14212. Epub 2019 Oct 2.

Université Grenoble Alpes, CNRS UMR 5309, INSERM U1209, Institute for Advanced Biosciences (IAB), Site Santé - Allée des Alpes, 38700, La Tronche, France.

Chromatin remodelers are complexes able to both alter histone-DNA interactions and to mobilize nucleosomes. The mechanism of their action and the conformation of remodeled nucleosomes remain a matter of debates. In this work we compared the type and structure of the products of nucleosome remodeling by SWI/SNF and ACF complexes using high-resolution microscopy combined with novel biochemical approaches. We find that SWI/SNF generates a multitude of nucleosome-like metastable particles termed "remosomes". Restriction enzyme accessibility assay, DNase I footprinting and AFM experiments reveal perturbed histone-DNA interactions within these particles. Electron cryo-microscopy shows that remosomes adopt a variety of different structures with variable irregular DNA path, similar to those described upon RSC remodeling. Remosome DNA accessibility to restriction enzymes is also markedly increased. We suggest that the generation of remosomes is a common feature of the SWI/SNF family remodelers. In contrast, the ACF remodeler, belonging to ISWI family, only produces repositioned nucleosomes and no evidence for particles associated with extra DNA, or perturbed DNA paths was found. The remosome generation by the SWI/SNF type of remodelers may represent a novel mechanism involved in processes where nucleosomal DNA accessibility is required, such as DNA repair or transcription regulation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-50572-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6775096PMC
October 2019

Structure of an H1-Bound 6-Nucleosome Array Reveals an Untwisted Two-Start Chromatin Fiber Conformation.

Mol Cell 2018 12 1;72(5):902-915.e7. Epub 2018 Nov 1.

Université Grenoble Alpes, CNRS UMR 5309, INSERM U1209, Institute for Advanced Biosciences (IAB), Site Santé - Allée des Alpes, 38700 La Tronche, France; "Roumen Tsanev" Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria. Electronic address:

Chromatin adopts a diversity of regular and irregular fiber structures in vitro and in vivo. However, how an array of nucleosomes folds into and switches between different fiber conformations is poorly understood. We report the 9.7 Å resolution crystal structure of a 6-nucleosome array bound to linker histone H1 determined under ionic conditions that favor incomplete chromatin condensation. The structure reveals a flat two-start helix with uniform nucleosomal stacking interfaces and a nucleosome packing density that is only half that of a twisted 30-nm fiber. Hydroxyl radical footprinting indicates that H1 binds the array in an on-dyad configuration resembling that observed for mononucleosomes. Biophysical, cryo-EM, and crosslinking data validate the crystal structure and reveal that a minor change in ionic environment shifts the conformational landscape to a more compact, twisted form. These findings provide insights into the structural plasticity of chromatin and suggest a possible assembly pathway for a 30-nm fiber.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2018.09.027DOI Listing
December 2018

Antimicrobial efficiency and stability of two decontamination solutions.

Cell Tissue Bank 2018 Dec 30;19(4):581-589. Epub 2018 Jul 30.

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.

Two decontamination solutions, commercially produced BASE•128 and laboratory decontamination solution (LDS), with analogous content of antibiotic and antimycotic agents, were compared in their antimicrobial efficiency and stability (pH and osmolarity). Both solutions were compared immediately after thawing aliquots frozen for 1, 3 or 6 months. Agar well diffusion method was used to test their antimicrobial efficiency against five human pathogens: Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, Escherichia coli and Enterococcus faecalis. The difference in the inhibition of growth between the two decontamination solutions was mostly not statistically significant, with few exceptions. The most pronounced difference between the LDS and BASE•128 was observed in their decontamination efficacy against E. coli and E. faecalis, where the LDS showed to be more efficient than BASE•128. The osmolarity value of LDS decreased with cold-storage, the osmolarity values of the BASE•128 could not be measured as they were below the range of the osmometer. Slight changes were found in pH of the less stable LDS solution, whose pH increased from initial value 7.36 ± 0.07 to 7.72 ± 0.19 after 6 m-storage. We verified that BASE•128 and LDS are similarly efficient in elimination of possible placental bacterial contaminants and may be used for decontamination of various tissues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10561-018-9707-0DOI Listing
December 2018

Characterization and comparison of human limbal explant cultures grown under defined and xeno-free conditions.

Exp Eye Res 2018 11 19;176:20-28. Epub 2018 Jun 19.

Laboratory of the Biology and Pathology of the Eye, Institute of Biology and Medical Genetics, 1st Faculty of Medicine, Charles University and General University Hospital in Prague, Albertov 4, 128 00 Prague 2, Czech Republic; Ophthalmology Department of 3rd Medical Faculty and University Hospital Kralovske Vinohrady, Šrobárova 1150/50, 100 34 Prague 10, Czech Republic.

Human limbal epithelial cells (LECs) intended for treatment of limbal stem cell deficiency are commonly cultivated on a 3T3 feeder layer with complex culture medium supplemented with fetal bovine serum (FBS). However, FBS is a xenogeneic component containing poorly characterised constituents and exhibits quantitative and qualitative lot-to-lot variations. Human limbal explants were plated on untreated or fibrin coated plastic plates and cultured in two non-xenogeneic media (supplemented with either human serum or platelet lysate only). Our aim was to find out whether the characteristics of harvested LEC cultures are comparable to those of LEC cultivated in the gold standard - FBS-supplemented complex medium. The growth kinetics, cell proliferation, differentiation, stemness maintenance, apoptosis and contamination by other cell types were evaluated and compared among these conditions. In all of them LECs were successfully cultivated. Stemness was preserved in both xeno-free media. However, cells cultured with human serum on the fibrin-coated plates had the highest growth rate and cell proliferation and very low fibroblast-like cell contamination. These data suggest that xeno-free cell culture conditions can replace the traditional FBS-supplemented medium and thereby provide a safer protocol for ex vivo cultured limbal stem cell transplants.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.exer.2018.06.019DOI Listing
November 2018

Frequency of Complications During Preparation of Corneal Lamellae Used in Posterior Lamellar Keratoplasty Using the Pneumodissection Technique (Big Bubble).

Cornea 2018 Jul;37(7):904-908

Ophthalmology Department of Third Medical Faculty and University Hospital Kralovske Vinohrady, Prague, Czech Republic.

Purpose: To determine the frequency of formation of various types of bubbles and the potential impact of donor and lamella parameters on this frequency, and to identify possible risk factors of unsuccessful "big-bubble" creation in preparation of pre-Descemet endothelial keratoplasty and Descemet membrane endothelial keratoplasty with peripheral stromal support.

Methods: Donor age and sex, death to preservation time (DPT), storage time, presence of corneal scars (mainly a condition after cataract surgery), and endothelial cell density of 256 donor corneas were assessed before Descemet membrane endothelial keratoplasty with peripheral stromal support or pre-Descemet endothelial keratoplasty lamella preparation using the big-bubble technique.

Results: Mean donor age was 62.3 ± 8.5 years (28.3% women and 71.7% men). Mean endothelial cell density of the donor graft was 2866 ± 255 cells/mm. Mean DPT was 10.12 ± 4.88 hours, and mean storage time of the transplant before surgery was 6.5 ± 4.8 days. Corneal scars were present in 17 donor grafts (6.6%) after cataract surgery. Eleven corneas were devalued because of Descemet membrane rupture during preparation (4.3%). In 182 corneas, standard bubble type I was created (71.7%); in 27 corneas, bubble type II was created; eventually, both types of bubbles formed simultaneously (10.5%); in 47 corneas, no bubble was created (18.4%).

Conclusions: We identified higher endothelial cell density, shorter DPT, and the presence of corneal scars after cataract surgery as risk factors threatening successful bubble formation. The only risk factor for creating type II bubbles was higher donor age in our study.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1097/ICO.0000000000001503DOI Listing
July 2018

Endothelial Wound Repair of the Organ-Cultured Porcine Corneas.

Curr Eye Res 2018 07 13;43(7):856-865. Epub 2018 Apr 13.

b Laboratory of the Biology and Pathology of the Eye, Institute of Biology and Medical Genetics, First Faculty of Medicine , Charles University and General University Hospital in Prague , Prague , Czech Republic.

Purpose: To assess whether injured porcine endothelium of small and large corneoscleral disc differ in its reparative/regenerative capacity under various conditions of organ culture storage.

Material And Methods: 166 paired porcine corneas were trephined to obtain tissues with diameter 12.0 mm and 17.5 mm (with area neighboring endothelial periphery). In tested discs, central endothelium was mechanically wounded. Density of live endothelial cells (LECD), percentage of dead cells (%DC), coefficient of variation and cell hexagonality were assessed in central and paracentral endothelium following 5- or 9-day incubation in medium with 2% or 10% fetal bovine serum. The parameters were assessed also in fresh and intact cultured discs. Dead endothelial cells (EC) were visualized by trypan blue, cell borders by Alizarin Red S dye. Endothelial imprints were immunoassayed for the proliferation marker Ki-67 and the nucleolar marker fibrillarin.

Results: In fresh corneas, the LECD/mm (mean ± standard deviation) were 3998.0 ± 215.4 (central area) and 3888.2 ± 363.1 (paracentral area). Only the length of storage had significant effect on wound repair. Lesion was repaired partially after 5-day and fully after 9-day cultivation. After 9-day storage in medium with 10% serum, the mean LECD detected in small discs were 2409.4 ± 881.8 (central area) and 3949.5 ± 275.5 (paracentral area) and in large discs the mean LECD were 2555.0 ± 347.0 (central area) and 4007.5 ± 261.2 (paracentral area). Ki-67 showed cell proliferation associated with healing of EC of both large and small corneas.

Conclusions: The lesions were completely repaired within 9 days of storage. Presence of the area, where stem cells appear to be located, contributes to stimulation of endothelial reparation less than serum concentration and time of culture. Both cell migration and proliferation contribute to the wound repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/02713683.2018.1458883DOI Listing
July 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194820PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5870984PMC
July 2018

Polyion complex vesicles (PICsomes) from strong copolyelectrolytes. Stability and in vitro studies.

Colloids Surf B Biointerfaces 2017 Oct 19;158:658-666. Epub 2017 Jul 19.

Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland. Electronic address:

Polymer vesicles formed by a pair of oppositely charged diblock copolyelectrolytes (PICsomes) are considered as a good alternative to polymersomes formed by amphiphilic copolymers. Here, we report on inherent stability and in vitro biocompatibility of PICsomes prepared from a pair of oppositely charged zwitterionic-ionic copolymers, in which the ionic block is a strong polyelectrolyte. Our results demonstrated that the PICsomes are highly stable over a wide range of pH and temperatures. Direct microscopic observations revealed that the PICsomes retain their morphology in the presence of human serum. In vitro studies using human skin fibroblasts (HSFs) showed that the polymer vesicles are not cytotoxic and do not affect cell proliferation and adhesion. A model hydrophilic dye was effectively incorporated into the PICsomes by simple mixing. Using confocal microscopy observations, we demonstrated that the dye-loaded PICsomes are efficiently internalized by the cells and are located predominantly in endo/lysosomal compartments. Thus, the PICsomes have promising potential for use as nanocontainers for substances of biomedical interest.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.colsurfb.2017.07.042DOI Listing
October 2017

Effect of Polycation Structure on Interaction with Lipid Membranes.

J Phys Chem B 2017 08 19;121(30):7318-7326. Epub 2017 Jul 19.

Faculty of Chemistry, Jagiellonian University , Ingardena 3, 30-348 Kraków, Poland.

Interaction of polycations with lipid membranes is a very important issue in many biological and medical applications such as gene delivery or antibacterial usage. In this work, we address the influence of hydrophobic substitution of strong polycations containing quaternary ammonium groups on the polymer-zwitterionic membrane interactions. In particular, we focus on the polymer tendency to adsorb on or/and incorporate into the membrane. We used complementary experimental and computational methods to enhance our understanding of the mechanism of the polycation-membrane interactions. Polycation adsorption on liposomes was assessed using dynamic light scattering (DLS) and zeta potential measurements. The ability of the polymers to form hydrophilic pores in the membrane was evaluated using a calcein-release method. The polymer-membrane interaction at the molecular scale was explored by performing atomistic molecular dynamics (MD) simulations. Our results show that the length of the alkyl side groups plays an essential role in the polycation adhesion on the zwitterionic surface, while the degree of substitution affects the polycation ability to incorporate into the membrane. Both the experimental and computational results show that the membrane permeability can be dramatically affected by the amount of alkyl side groups attached to the polycation main chain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jpcb.7b05248DOI Listing
August 2017

Comparison of impact of two decontamination solutions on the viability of the cells in human amnion.

Cell Tissue Bank 2017 Sep 4;18(3):413-423. Epub 2017 Jul 4.

Laboratory of the Biology and Pathology of the Eye, Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and General University Hospital in Prague, Ke Karlovu 2, 128 08, Prague, Czech Republic.

Human amniotic membrane (HAM) is used as an allograft in regenerative medicine or as a source of pluripotent cells for stem cell research. Various decontamination protocols and solutions are used to sterilize HAM before its application, but little is known about the toxicity of disinfectants on HAM cells. In this study, we tested two decontamination solutions, commercial (BASE·128) and laboratory decontamination solution (LDS), with an analogous content of antimycotic/antibiotics for their cytotoxic effect on HAM epithelial (EC) and mesenchymal stromal cells (MSC). HAM was processed in a standard way, placed on nitrocellulose scaffold, and decontaminated, following three protocols: (1) 6 h, 37 °C; (2) 24 h, room temperature; (3) 24 h, 4 °C. The viability of EC was assessed via trypan blue staining. The apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). The mean % (±SD) of dead EC (%DEC) from six fresh placentas was 12.9 ± 18.1. Decontamination increased %DEC compared to culture medium. Decontamination with BASE·128 for 6 h, 37 °C led to the highest EC viability (81.7%). Treatment with LDS at 24 h, 4 °C resulted in the lowest EC viability (55.9%) in the set. MSC were more affected by apoptosis than EC. Although the BASE·128 expresses lower toxicity compared to LDS, we present LDS as an alternative decontamination solution with a satisfactory preservation of cell viability. The basic formula of LDS will be optimised by enrichment with nutrient components, such as glucose or vitamins, to improve cell viability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10561-017-9636-3DOI Listing
September 2017

Structure and Dynamics of a 197 bp Nucleosome in Complex with Linker Histone H1.

Mol Cell 2017 May;66(3):384-397.e8

Institut for Advanced Biosciences, Inserm U 1209, CNRS UMR 5309, Université Grenoble Alpes, 38000 Grenoble, France. Electronic address:

Linker histones associate with nucleosomes to promote the formation of higher-order chromatin structure, but the underlying molecular details are unclear. We investigated the structure of a 197 bp nucleosome bearing symmetric 25 bp linker DNA arms in complex with vertebrate linker histone H1. We determined electron cryo-microscopy (cryo-EM) and crystal structures of unbound and H1-bound nucleosomes and validated these structures by site-directed protein cross-linking and hydroxyl radical footprinting experiments. Histone H1 shifts the conformational landscape of the nucleosome by drawing the two linkers together and reducing their flexibility. The H1 C-terminal domain (CTD) localizes primarily to a single linker, while the H1 globular domain contacts the nucleosome dyad and both linkers, associating more closely with the CTD-distal linker. These findings reveal that H1 imparts a strong degree of asymmetry to the nucleosome, which is likely to influence the assembly and architecture of higher-order structures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2017.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5508712PMC
May 2017

Effects of Membrane PEGylation on Entry and Location of Antifungal Drug Itraconazole and Their Pharmacological Implications.

Mol Pharm 2017 04 7;14(4):1057-1070. Epub 2017 Mar 7.

Department of Physics, Tampere University of Technology , P.O. Box 692, FI-33101 Tampere, Finland.

Itraconazole (ITZ) is an antifungal agent used clinically to treat mycotic infections. However, its therapeutic effects are limited by low solubility in aqueous media. Liposome-based delivery systems (LDS) have been proposed as a delivery mechanism for ITZ to alleviate this problem. Furthermore, PEGylation, the inclusion in the formulation of a protective "stealth sheath" of poly(ethylene glycol) around carrier particles, is widely used to increase circulation time in the bloodstream and hence efficacy. Together, these themes highlight the importance of mechanistic and structural understanding of ITZ incorporation into liposomes both with and without PEGylation because it can provide a potential foundation for the rational design of LDS-based systems for delivery of ITZ, using alternate protective polymers or formulations. Here we have combined atomistic simulations, cryo-TEM, Langmuir film balance, and fluorescence quenching experiments to explore how ITZ interacts with both pristine and PEGylated liposomes. We found that the drug can be incorporated into conventional and PEGylated liposomes for drug concentrations up to 15 mol % without phase separation. We observed that, in addition to its protective properties, PEGylation significantly increases the stability of liposomes that host ITZ. In a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer without PEGylation, ITZ was found to reside inside the lipid bilayer between the glycerol and the double-bond regions of POPC, adopting a largely parallel orientation along the membrane surface. In a PEGylated liposome, ITZ partitions mainly to the PEG layer. The results provide a solid basis for further development of liposome-based delivery systems.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.molpharmaceut.6b00969DOI Listing
April 2017

Extra views on structure and dynamics of DNA loops on nucleosomes studied with molecular simulations.

Nucleus 2016 Nov;7(6):554-559

a Université de Lyon/CNRS UMR 5086, MMSB, Institut de Biologie et Chimie des Protéines , Lyon , France.

It has been shown experimentally that the action of the RSC chromatin remodeler leads to the formation of an irregular, partially remodeled nucleosome, termed a remosome. The remosome contains an extra 30-40 base pairs of DNA compared to a canonical nucleosome. Large-scale molecular simulations have provided information on the probable structure of remosomes and have explained why they remain stable in the absence of RSC. Here we explain how these simulations were carried out and what the resulting remosome models imply in terms of the mechanism of action of RSC. We notably show that local kinks within DNA are key in explaining how extra DNA can be in added to nucleosomes without unduly disturbing DNA-histone binding.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/19491034.2016.1260800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5214536PMC
November 2016

The N-terminal domain plays a crucial role in the structure of a full-length human mitochondrial Lon protease.

Sci Rep 2016 09 16;6:33631. Epub 2016 Sep 16.

Institute of Cellular Biology and Pathology, First Faculty of Medicine, Charles University in Prague, Albertov 4, 128 01 Prague 2, Czech Republic.

Lon is an essential, multitasking AAA(+) protease regulating many cellular processes in species across all kingdoms of life. Altered expression levels of the human mitochondrial Lon protease (hLon) are linked to serious diseases including myopathies, paraplegia, and cancer. Here, we present the first 3D structure of full-length hLon using cryo-electron microscopy. hLon has a unique three-dimensional structure, in which the proteolytic and ATP-binding domains (AP-domain) form a hexameric chamber, while the N-terminal domain is arranged as a trimer of dimers. These two domains are linked by a narrow trimeric channel composed likely of coiled-coil helices. In the presence of AMP-PNP, the AP-domain has a closed-ring conformation and its N-terminal entry gate appears closed, but in ADP binding, it switches to a lock-washer conformation and its N-terminal gate opens, which is accompanied by a rearrangement of the N-terminal domain. We have also found that both the enzymatic activities and the 3D structure of a hLon mutant lacking the first 156 amino acids are severely disturbed, showing that hLon's N-terminal domains are crucial for the overall structure of the hLon, maintaining a conformation allowing its proper functioning.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep33631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025710PMC
September 2016

The Flexible Ends of CENP-A Nucleosome Are Required for Mitotic Fidelity.

Mol Cell 2016 08 4;63(4):674-685. Epub 2016 Aug 4.

Université de Grenoble Alpes/INSERM U1209/CNRS UMR 5309, 38042 Grenoble Cedex 9, France. Electronic address:

CENP-A is a histone variant, which replaces histone H3 at centromeres and confers unique properties to centromeric chromatin. The crystal structure of CENP-A nucleosome suggests flexible nucleosomal DNA ends, but their dynamics in solution remains elusive and their implication in centromere function is unknown. Using electron cryo-microscopy, we determined the dynamic solution properties of the CENP-A nucleosome. Our biochemical, proteomic, and genetic data reveal that higher flexibility of DNA ends impairs histone H1 binding to the CENP-A nucleosome. Substituting the 2-turn αN-helix of CENP-A with the 3-turn αN-helix of H3 results in compact particles with rigidified DNA ends, able to bind histone H1. In vivo replacement of CENP-A with H3-CENP-A hybrid nucleosomes leads to H1 recruitment, delocalization of kinetochore proteins, and significant mitotic and cytokinesis defects. Our data reveal that the evolutionarily conserved flexible ends of the CENP-A nucleosomes are essential to ensure the fidelity of the mitotic pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2016.06.023DOI Listing
August 2016

H1-nucleosome interactions and their functional implications.

Biochim Biophys Acta 2016 Mar 23;1859(3):436-43. Epub 2015 Oct 23.

INSERM/UJF, Institut Albert Bonniot, U823, Site Santé-BP 170, 38042 Grenoble Cedex 9, France.

Linker histones are three domain proteins and consist of a structured (globular) domain, flanked by two likely non-structured NH2- and COOH-termini. The binding of the linker histones to the nucleosome was characterized by different methods in solution. Apparently, the globular domain interacts with the linker DNA and the nucleosome dyad, while the binding of the large and rich in lysines COOH-terminus results in "closing" the linker DNA of the nucleosome and the formation of the "stem" structure. What is the mode of binding of the linker histones within the chromatin fiber remains still elusive. Nonetheless, it is clear that linker histones are essential for both the assembly and maintenance of the condensed chromatin fiber. Interestingly, linker histones are post-translationally modified and how this affects both their binding to chromatin and functions is now beginning to emerge. In addition, linker histones are highly mobile in vivo, but not in vitro. No explanation of this finding is reported for the moment. The higher mobility of the linker histones should, however, have strong impact on their function. Linker histones plays an important role in gene expression regulation and other chromatin related process and their function is predominantly regulated by their posttranslational modifications. However, the detailed mechanism how the linker histones do function remains still not well understood despite numerous efforts. Here we will summarize and analyze the data on the linker histone binding to the nucleosome and the chromatin fiber and will discuss its functional consequences.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbagrm.2015.10.012DOI Listing
March 2016

OFT sectorization approach to analysis of optical scattering in mercurous chloride single crystals.

Opt Express 2015 Aug;23(16):21509-26

This paper is devoted to the application of the optical Fourier transform (OFT) for the study and evaluation of optical scattering in the latest generation of calomel single crystals ready for application in several possible devices such as IR polarizers and acoustooptic tunable filters (AOTF). There are numerous effects that are responsible for the scattering of optical wave passing through the crystal sample volume and surface layers because they affect the optical crystal quality. The scattering level is a crucial and limiting parameter in many technical applications of the evaluated crystal. The proposed approach is based upon the high dynamic range optical FT configuration, creating the amplitude spectrum in the focal plane and its spatial angular distribution analysis based on the spectrum sectorization. The optical scattering pattern was tested in nine locations within each crystal sample volume and on numerous crystal samples. The experimental results are presented and discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1364/OE.23.021509DOI Listing
August 2015

Stable polymersomes based on ionic-zwitterionic block copolymers modified with superparamagnetic iron oxide nanoparticles for biomedical applications.

J Mater Chem B 2015 Jul 12;3(27):5523-5531. Epub 2015 Jun 12.

Faculty of Chemistry, Jagiellonian University in Kraków, Ingardena 3, Kraków 30-060, Poland.

Stable polymersomes with semipermeable membranes were prepared by simple mixing of two oppositely charged diblock copolymers containing zwitterionic and cationic (PMPC-b-PMAPTAC) or anionic (PMPC-b-PAMPS) blocks. The formation of vesicular structures in the mixed solution of the block copolymers was confirmed by direct observation using the cryo-TEM technique. Superparamagnetic iron oxide nanoparticles coated with a cationic chitosan derivative (SPION/CCh) and decorated with a fluorescent probe molecule were next incorporated into the polymersome structure. The average diameter of SPION/CCh-polymersomes estimated using cryo-TEM was about 250 nm. Surface topography of the SPION/CCh-loaded vesicles was imaged using AFM and the magnetic properties of these objects were confirmed by MFM and MRI measurements. The ability of SPION/CCh-polymersomes to affect T relaxation time in MRI was evaluated based on the measurements of r relaxivity. The obtained value of r (573 ± 10 mM s) was quite high. The cytotoxicity and intracellular uptake of the SPION/CCh-loaded vesicles into EA.hy926 cells were studied. The results indicate that the SPION/CCh-polymersomes seem to be internalized by vascular endothelium and are not cytotoxic to endothelial cells up to 1 μg Fe per mL. Therefore, it can be suggested that SPION/CCh-polymersomes could prove useful as T contrast agents in the MRI of endothelium.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c5tb00182jDOI Listing
July 2015

Interactions of serum with polyelectrolyte-stabilized liposomes: Cryo-TEM studies.

Colloids Surf B Biointerfaces 2014 Aug 27;120:152-9. Epub 2014 May 27.

Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland. Electronic address:

Liposomes are used for in vitro or in vivo vectorization of drugs, proteins, or nucleic acids. However, the main problem with the application of liposomes for this purpose is their low stability in contact with blood serum. In this article, interactions between the whole serum and anionic liposomes, both bare and covered with strong polycations, were studied. The polycations of different chemical structures were prepared by the modification of poly(allylamine hydrochloride) (PAH). Dynamic light scattering (DLS), zeta potential and transmission cryo-electron microscopy (cryo-TEM) measurements showed that the adsorption of the polycations on the anionic liposomes induced a reversible aggregation of vesicles. The stable isolated polyelectrolyte-covered vesicles were obtained after the addition of sufficient amounts of the polycations. The effect of full serum on the morphology and stability of the polycation-coated liposomes was studied using cryo-TEM and a fluorescence method. The cryo-TEM analysis revealed that the introduction of serum caused the osmotic-driven destabilization of the bare liposomes or formation of twinned vesicles. Due to these processes the liposomes lost most of their content immediately after serum addition. The polycation-covered liposomes showed improved stability in the presence of serum. Partial deflation of the vesicles was observed, however, the loss of the content was significantly limited. The effect of the polymer structure, especially the position of the charged groups with respect to the main polymer backbone, on the stabilization of the polycation-covered liposomes in the presence of serum was discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.colsurfb.2014.02.040DOI Listing
August 2014

Rapid cooling of the amniotic membrane as a model system for the vitrification of posterior corneal lamellae.

Cell Tissue Bank 2014 Mar 27;15(1):165-73. Epub 2013 Jul 27.

Laboratory of the Biology and Pathology of the Eye, 1st Faculty of Medicine and General Teaching Hospital in Prague, Institute of Inherited Metabolic Disorders, Charles University in Prague, Ke Karlovu 2, 128 08, Prague 2, Czech Republic.

To vitrify human amniotic membrane specimens so that the maximum of epithelial cells survives in order to develop a procedure for the eventual vitrification of posterior corneal lamellae without using cryoprotective agents. To assess different methods of tissue sample preparation preceding vitrification. In group 1, the amniotic membrane specimens were stretched on nitrocellulose support. In group 2, mechanical pressure was used to remove the excess culture medium between the support and the membrane. The samples were frozen in liquid ethane (-183 °C) and stored in liquid nitrogen. The specimens in the control group were not vitrified. Re-warming was performed at 40 °C. The epithelial cell survival rate was assessed after 1, 3 and 7 days of storage following re-warming using calcein and ethidium homodimer-1 fluorescence. A wide range of values was observed among the different groups and among individual specimens within the groups. Resulting average survival rate was 41 % for group 1 and 53 % for group 2; in several samples the cell survival rate exceeded 70 %. The storage period did not significantly affect the survival rates. The results of the rapid cooling of amniotic membranes in liquid ethane indicate that significant percentage of epithelial cells remain viable after the re-warming.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10561-013-9388-7DOI Listing
March 2014

Curcumin-containing liposomes stabilized by thin layers of chitosan derivatives.

Colloids Surf B Biointerfaces 2013 Sep 22;109:307-16. Epub 2013 Apr 22.

Faculty of Chemistry, Jagiellonian University, Kraków, Poland.

Stable vesicles for efficient curcumin encapsulation, delivery and controlled release have been obtained by coating of liposomes with thin layer of newly synthesized chitosan derivatives. Three different derivatives of chitosan were obtained and studied: the cationic (by introduction of the stable, quaternary ammonium groups), the hydrophobic (by attachment of N-dodecyl groups) and cationic-hydrophobic one (containing both quaternary ammonium and N-dodecyl groups). Zeta potential measurements confirmed effective coating of liposomes with all these chitosan derivatives. The liposomes coated with cationic-hydrophobic chitosan derivative are the most promising curcumin carriers; they can easily penetrate cell membrane and release curcumin in a controlled manner. Biological studies indicated that such systems are non-toxic for murine fibroblasts (NIH3T3) while toxic toward murine melanoma (B16F10) cell line.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.colsurfb.2013.03.059DOI Listing
September 2013

Nucleosomes stacked with aligned dyad axes are found in native compact chromatin in vitro.

J Struct Biol 2012 May 22;178(2):207-14. Epub 2011 Nov 22.

European Molecular Biology Laboratory, Meyerhofstr. 1, 69117 Heidelberg, Germany.

In this study, electron tomograms of plunge-frozen isolated chromatin in both open and compacted form were recorded. We have resolved individual nucleosomes in these tomograms in order to provide a 3D view of the arrangement of nucleosomes within chromatin fibers at different compaction states. With an optimized template matching procedure we obtained accurate positions and orientations of nucleosomes in open chromatin in "low-salt" conditions (5 mM NaCl). The mean value of the planar angle between three consecutive nucleosomes is 70°, and the mean center-to-center distance between consecutive nucleosomes is 22.3 nm. Since the template matching approach was not effective in crowded conditions, for nucleosome detection in compact fibers (40 mM NaCl and 1 mM MgCl(2)) we developed the nucleosome detection procedure based on the watershed algorithm, followed by sub-tomogram alignment, averaging, and classification by Principal Components Analysis. We find that in compact chromatin the nucleosomes are arranged with a predominant face-to-face stacking organization, which has not been previously shown for native isolated chromatin. Although the path of the DNA cannot be directly seen in compact conditions, it is evident that the nucleosomes stack with their dyad axis aligned in forming a "double track" conformation which is a consequence of DNA joining adjacent nucleosome stacks. Our data suggests that nucleosome stacking is an important mechanism for generating chromatin compaction in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jsb.2011.11.020DOI Listing
May 2012
-->