O. S. Strelkova

Disclosure of a structural milieu for the proximity ligation reveals the elusive nature of an active chromatin hub

The current progress in the study of the spatial organization of interphase chromosomes became possible owing to the development of the chromosome conformation capture (3C) protocol. The crucial step of this protocol is the proximity ligation—preferential ligation of DNA fragments assumed to be joined within nuclei by protein bridges and solubilized as a common complex after formaldehyde cross-linking and DNA cleavage. Here, we show that a substantial, and in some cases the major, part of DNA is not solubilized from cross-linked nuclei treated with restriction endonuclease(s) and sodium dodecyl sulphate and that this treatment neither causes lysis of the nucleus nor drastically affects its internal organization. Analysis of the ligation frequencies of the mouse β-globin gene domain DNA fragments demonstrated that the previously reported 3C signals were generated predominantly, if not exclusively, in the insoluble portion of the 3C material. The proximity ligation thus occurs within the cross-linked chromatin cage in non-lysed nuclei. The finding does not compromise the 3C protocol but allows the consideration of an active chromatin hub as a folded chromatin domain or a nuclear compartment rather than a rigid complex of regulatory elements.

Solid state synthesis of carbon-encapsulated iron carbide nanoparticles and their interaction with living cells

Superparamagnetic carbon-encapsulated iron carbide nanoparticles (NPs), Fe7C3@C, with unique properties, were produced from pure ferrocene by high pressure-high temperature synthesis. These NPs combine the merits of nanodiamonds and SPIONs but lack their shortcomings which limit their use for biomedical applications. Investigation of these NPs by X-ray diffraction, electron microscopy techniques, X-ray spectroscopic and magnetic measurement methods has demonstrated that this method of synthesis yields NPs with perfectly controllable physical properties. Using magnetic and subsequent fractional separation of magnetic NPs from residual carbon, the aqueous suspensions of Fe7C3@C NPs with an average particle size of ∼25 nm were prepared. The suspensions were used for in vitro studies of the interaction of Fe7C3@C NPs with cultured mammalian cells. The dynamics of interaction of the living cells with Fe7C3@C was studied by optical microscopy using time-lapse video recording and also by transmission electron microscopy. Using novel highly sensitive cytotoxicity tests based on the cell proliferation assay and long-term live cell observations it was shown that the internalization of Fe7C3@C NPs has no cytotoxic effect on cultured cells and does not interfere with the process of their mitotic division, a fundamental property that ensures the existence of living organisms. The influence of NPs on the proliferative activity of cultured cells was not detected as well. These results indicate that the carbon capsules of Fe7C3@C NPs are air-tight which could offer great opportunities for future use of these superparamagnetic NPs in biology and medicine.

Magnetocontrollability of Fe7C3@C superparamagnetic nanoparticles in living cells.

BackgroundA new type of superparamagnetic nanoparticles with chemical formula Fe7C3@C (MNPs) showed higher value of magnetization compared to traditionally used iron oxide-based nanoparticles as was shown in our previous studies. The in vitro biocompatibility tests demonstrated that the MNPs display high efficiency of cellular uptake and do not affect cyto-physiological parameters of cultured cells. These MNPs display effective magnetocontrollability in homogeneous liquids but their behavior in cytoplasm of living cells under the effect of magnetic field was not carefully analyzed yet.ResultsIn this work we investigated the magnetocontrollability of MNPs interacting with living cells in permanent magnetic field. It has been shown that cells were capable of capturing MNPs by upper part of the cell membrane, and from the surface of the cultivation substrate during motion process. Immunofluorescence studies using intracellular endosomal membrane marker showed that MNP agglomerates can be either located in endosomes or lying free in the cytoplasm. When attached cells were exposed to a magnetic field up to 0.15 T, the MNPs acquired magnetic moment and the displacement of incorporated MNP agglomerates in the direction of the magnet was observed. Weakly attached or non-attached cells, such as cells in mitosis or after cytoskeleton damaging treatments moved towards the magnet. During long time cultivation of cells with MNPs in a magnetic field gradual clearing of cells from MNPs was observed. It was the result of removing MNPs from the surface of the cell agglomerates discarded in the process of exocytosis.ConclusionsOur data allow us to conclude for the first time that the magnetic properties of the MNPs are sufficient for successful manipulation with MNP agglomerates both at the intracellular level, and within the whole cell. The structure of the outer shells of the MNPs allows firmly associate different types of biological molecules with them. This creates prospects for the use of such complexes for targeted delivery and selective removal of selected biological molecules from living cells.

Magnet-induced behavior of Iron Carbide (Fe7C3@C) Nanoparticles in the Cytoplasm of Living Cells

I. Alieva, I. Kireev, A. Rakhmanina, A. Garanina, O. Strelkova, O. Zhironkina, V. Cherepaninets, V. Davydov, V. Khabashesku, V. Agafonov, R. Uzbekov Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia L. F. Vereshchagin Institute for High Pressure Physics of the RAS, Troitsk, Russia Center for Technology Innovation, Baker Hughes Inc., Houston, TX, 77040, USA GREMAN, UMR CNRS 7347, Universite Francois Rabelais, 37200 Tours, France Faculte de Medecine, Universite Francois Rabelais, Tours, France Faculty of Bioengineering and Bioinformatics, Moscow State University, Moscow, Russia rustem.uzbekov@univ-tours.fr, viatcheslav.agafonov@univ-tours.fr

Cohesion peculiarities in eu- and heterochromatin in human cells

Sister chromatids are considered to be held together from just after replication until the beginning of compaction in prophase via a specific complex—cohesin consisting of Smc1-Smc3 dimer and two additional subunits Scc1 and Scc3, the process being referred to as “cohesion.” We have characterized peculiarities of binding of the cohesin complex with early and late replicating chromatin at various stages of the cell cycle in human cell lines HeLa and HT1080 using structured illumination microscopy and immune electron microscopy. It has been shown that cohesion in heterochromatic domains is evidently provided without the participation of cohesins, while the majority of cohesins bound to chromatin provides cohesion and subdomain organization in euchromatin.

Overcoming steric hindrances during replication of peripheral heterochromatin

Due to a tight attachment of peripheral heterochromatin to the nuclear lamina its replication is connected with inevitable topological hindrances. Additional hindrances are caused by high stability of the lamina that complicates the access of replication factors to DNA duplication sites under conditions of highly condensed matrix with limited mobility. The work focuses on detailed study of structural organization and dynamics of the lamina in respect to replication of peripheral heterochromatin that is attached to it. The study of mobile properties of lamins at various stages of the S-phase using live cell imaging and super-resolution microscopy showed the absence of the dependence of lamins’ mobility on replicative status of attached heterochromatin. These data confirm the hypothesis on regulation of linkage between chromatin and lamina at the level of molecular intermediates. It has been shown at the ultrastructural level that possible temporary disruption in molecular bonds between the lamina and peripheral chromatin during replication does not cause movement of replicated domains from the nuclear periphery.

Mitochondria-targeted antioxidant SkQ1 suppresses fibrosarcoma and rhabdomyosarcoma tumour cell growth

ABSTRACT Mitochondria are important regulators of tumour growth and progression due to their specific role in cancer metabolism and modulation of apoptotic pathways. In this paper we describe that mitochondria-targeted antioxidant SkQ1 designed as a conjugate of decyl-triphenylphosphonium cation (TPP+) with plastoquinone, suppressed the growth of fibrosarcoma HT1080 and rhabdomyosarcoma RD tumour cells in culture and tumour growth of RD in xenograft nude mouse model. Under the same conditions, no detrimental effect of SkQ1 on cell growth of primary human subcutaneous fibroblasts was observed. The tumour growth suppression was shown to be a result of the antioxidant action of low nanomolar concentrations of SkQ1. We have revealed significant prolongation of mitosis induced by SkQ1 in both tumour cell cultures. Prolonged mitosis and apoptosis could be responsible for growth suppression after SkQ1 treatment in RD cells. Growth suppression in HT1080 cells was accompanied by the delay of telophase and cytokinesis, followed by multinuclear cells formation. The effects of SkQ1 on the cell cycle were proved to be at least partially mediated by inactivation of Aurora family kinases. Abbreviations: TPP+: Triphenylphosphonium cation; ROS: Reactive oxygen species; mtROS: Mitochondrial reactive oxygen species; NAC: N-acetyl-L-cysteine; DCFH-DA: Dichlorodihydrofluorescein diacetate; APC: Anaphase promoting complex; ABPs: Actin-binding proteins; DMEM: Dulbecco’s modified Eagle media; SDS: sodium dodecyl sulfate; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

Interstitial telomeric repeats-associated DNA breaks

ABSTRACT During a cells lifespan, DNA break formation is a common event, associated with many processes, from replication to apoptosis. Most of DNA breaks are readily repaired, but some are meant to persist in time, such as the chromosome ends, protected by telomeres. Besides them, eukaryotic genomes comprise shorter stretches of interstitial telomeric repeats. We assumed that the latter may also be associated with the formation of DNA breaks meant to persist in time. In zebrafish and mouse embryos, cells containing numerous breakage foci were identified. These breaks were not associated with apoptosis or replication, nor did they seem to activate DNA damage response machinery. Unlike short-living, accidental sparse breaks, the ones we found seem to be closely associated, forming discrete break foci. A PCR-based method was developed, allowing specific amplification of DNA regions located between inverted telomeric repeats associated with breaks. The cloning and sequencing of such DNA fragments were found to denote some specificity in their distribution for different tissue types and development stages.

Regulation of cyclooxygenase 2 mRNA degradation by rosiglitazone in C6 glioma cells in the presence of inflammation inductors

It has recently become clear that regulation of mRNA stability plays an important role in the development of cell responses to stimulation of toll-like receptors during inflammation. This discovery motivated a search for low molecular weight substances modulating the stability of mRNA encoding proteins involved in inflammatory responses. In this work, regulation of the cyclooxygenase 2 (COX-2) expression by rosiglitazone–a promising drug for regulation of inflammation–was studied on rat glioma cell line C6. Inflammatory response was induced by lipopolysaccaride (LPS). The concentration of prostaglandin E2 (PGE2) was measured by ELISA, the level of COX-2 mRNA was determined by real-time PCR. It was shown that treatment with LPS caused a 6-fold increase in the PGE2 synthesis, which correlated with an increase in the COX-2 mRNA expression. Rosiglitazone induced a 2-fold decrease of the LPS-stimulated PGE2 release and reduced the levels of COX-2 transcripts. To explore the molecular mechanisms of the rosiglitazone effect, we estimated the stability of COX-2 mRNA. Cells were incubated in the presence of LPS for 1 h, and then de novo mRNA transcription was blocked with actinomycin D. The levels of mRNA were determined at various time points. Treatment with rosiglitazone was carried out for 30 min before the LPS addition. The COX-2 mRNA half-life in native cells was found to be 75 min. LPS stimulation slowed down the mRNA degradation so that its half-life time was 120 min, and the treatment with rosiglitazone restored this process back to the normal level. The results suggest that rosiglitazone regulates the stability of COX-2 mRNA. This opens up new perspectives for therapeutic applications of this drug.

High expression levels and nuclear localization of novel Danio rerio ncRNA transcribed from a genomic region containing repetitive elements

Noncoding and repetitive sequences make up a large part of the genome of high eukaryotes, but the elucidation of their roles and mechanisms of action are poorly understood. In this work, we found that interstitial telomeric repeats in the genome of Danio rerio colocalize with repetitive elements, including hAT and EnSpm, which are widely represented in vertebrate genomes. The investigation of a genomic region containing two pairs of these repeats located in close proximity showed that the area is transcribed. RNA-dependent structures containing this sequence were identified in D. rerio fibroblast nuclei, which indicates the functional importance of genomic repetitive elements or their transcripts.