Electron microscopy reveals toroidal shape of master neuronal cell differentiator REST - RE1-silencing transcription factor.

The RE1-Silencing Transcription factor (REST) is essential for neuronal differentiation. Here, we report the first 18.5-angstrom electron microscopy structure of human REST. The refined electron map suggests that REST forms a torus that can accommodate DNA double-helix in the central hole. Additionally, we quantitatively described REST binding to the canonical DNA sequence of the neuron-restrictive silencer element. We developed protocols for the expression and purification of full-length REST and the shortened variant REST-N62 produced by alternative splicing. We tested the mutual interaction of full-length REST and the splicing variant REST-N62. Revealed structure-function relationships of master neuronal repressor REST will allow finding new biological ways of prevention and treatment of neurodegenerative disorders and diseases.

Phosphorylation of TRF2 promotes its interaction with TIN2 and regulates DNA damage response at telomeres.

Protein phosphatase magnesium-dependent 1 delta (PPM1D) terminates the cell cycle checkpoint by dephosphorylating the tumour suppressor protein p53. By targeting additional substrates at chromatin, PPM1D contributes to the control of DNA damage response and DNA repair. Using proximity biotinylation followed by proteomic analysis, we identified a novel interaction between PPM1D and the shelterin complex that protects telomeric DNA. In addition, confocal microscopy revealed that endogenous PPM1D localises at telomeres. Further, we found that ATR phosphorylated TRF2 at S410 after induction of DNA double strand breaks at telomeres and this modification increased after inhibition or loss of PPM1D. TRF2 phosphorylation stimulated its interaction with TIN2 both in vitro and at telomeres. Conversely, induced expression of PPM1D impaired localisation of TIN2 and TPP1 at telomeres. Finally, recruitment of the DNA repair factor 53BP1 to the telomeric breaks was strongly reduced after inhibition of PPM1D and was rescued by the expression of TRF2-S410A mutant. Our results suggest that TRF2 phosphorylation promotes the association of TIN2 within the shelterin complex and regulates DNA repair at telomeres.

Magnetic nanoparticles of Ga-substituted ε-Fe2 O3 for biomedical applications: Magnetic properties, transverse relaxivity, and effects of silica-coated particles on cytoskeletal networks.

Magnetic nanoparticles of ε-Fe1.76 Ga0.24 O3 with the volume-weighted mean size of 17 nm were prepared by thermal treatment of a mesoporous silica template impregnated with metal nitrates and were coated with silica shell of four different thicknesses in the range 6-24 nm. The bare particles exhibited higher magnetization than the undoped compound, 22.4 Am2 kg-1 at 300 K, and were characterized by blocked state with the coercivity of 1.2 T at 300 K, being thus the very opposite of superparamagnetic iron oxides. The relaxometric study of the silica-coated samples at 0.47 T revealed promising properties for MRI, specifically, transverse relaxivity of 89-168 s-1 mmol(f.u.)-1 L depending on the shell thickness was observed. We investigated the effects of the silica-coated nanoparticles on human A549 and MCF-7 cells. Cell viability, proliferation, cell cycle distribution, and the arrangement of actin cytoskeleton were assessed, as well as formation and maturation of focal adhesions. Our study revealed that high concentrations of silica-coated particles with larger shell thicknesses of 16-24 nm interfere with the actin cytoskeletal networks, inducing thus morphological changes. Consequently, the focal adhesion areas were significantly decreased, resulting in impaired cell adhesion.

Quantitative Biology of Human Shelterin and Telomerase: Searching for the Weakest Point.

The repetitive telomeric DNA at chromosome ends is protected from unwanted repair by telomere-associated proteins, which form the shelterin complex in mammals. Recent works have provided new insights into the mechanisms of how human shelterin assembles and recruits telomerase to telomeres. Inhibition of telomerase activity and telomerase recruitment to chromosome ends is a promising target for anticancer therapy. Here, we summarize results of quantitative assessments and newly emerged structural information along with the status of the most promising approaches to telomerase inhibition in cancer cells. We focus on the mechanism of shelterin assembly and the mechanisms of how shelterin affects telomerase recruitment to telomeres, addressing the conceptual dilemma of how shelterin allows telomerase action and regulates other essential processes. We evaluate how the identified critical interactions of telomerase and shelterin might be elucidated in future research of new anticancer strategies.

Human Telomere Repeat Binding Factor TRF1 Replaces TRF2 Bound to Shelterin Core Hub TIN2 when TPP1 Is Absent.

Human telomeric repeat binding factors TRF1 and TRF2 along with TIN2 form the core of the shelterin complex that protects chromosome ends against unwanted end-joining and DNA repair. We applied a single-molecule approach to assess TRF1-TIN2-TRF2 complex formation in solution at physiological conditions. Fluorescence cross-correlation spectroscopy was used to describe the complex assembly by analyzing how coincident fluctuations of differently labeled TRF1 and TRF2 correlate when they move together through the confocal volume of the microscope. We observed, at the single-molecule level, that TRF1 effectively substitutes TRF2 on TIN2. We assessed also the effect of another telomeric factor TPP1 that recruits telomerase to telomeres. We found that TPP1 upon binding to TIN2 induces changes that expand TIN2 binding capacity, such that TIN2 can accommodate both TRF1 and TRF2 simultaneously. We suggest a molecular model that explains why TPP1 is essential for the stable formation of TRF1-TIN2-TRF2 core complex.

Magnetic properties of rare-earth-doped La0.7Sr0.3MnO3.

Rare-earth-doped ferromagnetic manganites La0.63RE0.07Sr0.30MnO3 (RE = Gd, Tb, Dy, and Ho) are synthesized in the form of sintered ceramics and nanocrystalline phases with the mean size of crystallites ≈30 nm. The electronic states of the dopants are investigated by SQUID magnetometry and theoretically interpreted based on the calculations of the crystal field splitting of rare-earth energy levels. The samples show the orthorhombic perovskite structure of Ibmm symmetry, with a complete FM order of Mn spins in bulk and reduced order in nanoparticles. Non-zero moments are also detected at the perovskite A sites, which can be attributed to magnetic polarization of the rare-earth dopants. The measurements in external field up to 70 kOe show a standard Curie-type contribution of the spin-only moments of Gd3+ ions, whereas Kramers ions Dy3+ and non-Kramers ions Ho3+ contribute by Ising moments due to their doublet ground states. The behaviour of non-Kramers ions Tb3+ is anomalous, pointing to singlet ground state with giant Van Vleck paramagnetism. The Tb3+ doping leads also to a notably increased coercivity compared to other La0.63RE0.07Sr0.30MnO3 systems.

The effect of magnetic nanoparticles on neuronal differentiation of induced pluripotent stem cell-derived neural precursors.

INTRODUCTION: Magnetic resonance (MR) imaging is suitable for noninvasive long-term tracking. We labeled human induced pluripotent stem cell-derived neural precursors (iPSC-NPs) with two types of iron-based nanoparticles, silica-coated cobalt zinc ferrite nanoparticles (CZF) and poly-l-lysine-coated iron oxide superparamagnetic nanoparticles (PLL-coated γ-Fe2O3) and studied their effect on proliferation and neuronal differentiation. MATERIALS AND METHODS: We investigated the effect of these two contrast agents on neural precursor cell proliferation and differentiation capability. We further defined the intracellular localization and labeling efficiency and analyzed labeled cells by MR. RESULTS: Cell proliferation was not affected by PLL-coated γ-Fe2O3 but was slowed down in cells labeled with CZF. Labeling efficiency, iron content and relaxation rates measured by MR were lower in cells labeled with CZF when compared to PLL-coated γ-Fe2O3. Cytoplasmic localization of both types of nanoparticles was confirmed by transmission electron microscopy. Flow cytometry and immunocytochemical analysis of specific markers expressed during neuronal differentiation did not show any significant differences between unlabeled cells or cells labeled with both magnetic nanoparticles. CONCLUSION: Our results show that cells labeled with PLL-coated γ-Fe2O3 are suitable for MR detection, did not affect the differentiation potential of iPSC-NPs and are suitable for in vivo cell therapies in experimental models of central nervous system disorders.

Using ferromagnetic nanoparticles with low Curie temperature for magnetic resonance imaging-guided thermoablation.

INTRODUCTION: Magnetic nanoparticles (NPs) represent a tool for use in magnetic resonance imaging (MRI)-guided thermoablation of tumors using an external high-frequency (HF) magnetic field. To avoid local overheating, perovskite NPs with a lower Curie temperature (T c) were proposed for use in thermotherapy. However, deposited power decreases when approaching the Curie temperature and consequently may not be sufficient for effective ablation. The goal of the study was to test this hypothesis. METHODS: Perovskite NPs (T c =66°C-74°C) were characterized and tested both in vitro and in vivo. In vitro, the cells suspended with NPs were exposed to a HF magnetic field together with control samples. In vivo, a NP suspension was injected into a induced tumor in rats. Distribution was checked by MRI and the rats were exposed to a HF field together with control animals. Apoptosis in the tissue was evaluated. RESULTS AND DISCUSSION: In vitro, the high concentration of suspended NPs caused an increase of the temperature in the cell sample, leading to cell death. In vivo, MRI confirmed distribution of the NPs in the tumor. The temperature in the tumor with injected NPs did not increase substantially in comparison with animals without particles during HF exposure. We proved that the deposited power from the NPs is too small and that thermoregulation of the animal is sufficient to conduct the heat away. Histology did not detect substantially higher apoptosis in NP-treated animals after ablation. CONCLUSION: Magnetic particles with low T c can be tracked in vivo by MRI and heated by a HF field. The particles are capable of inducing cell apoptosis in suspensions in vitro at high concentrations only. However, their effect in the case of extracellular deposition in vivo is questionable due to low deposited power and active thermoregulation of the tissue.

The impact of silica encapsulated cobalt zinc ferrite nanoparticles on DNA, lipids and proteins of rat bone marrow mesenchymal stem cells.

Nanomaterials are currently the subject of intense research due to their wide variety of potential applications in the biomedical, optical and electronic fields. We prepared and tested cobalt zinc ferrite nanoparticles (Co0.5Zn0.5Fe2O4+γ [CZF-NPs]) encapsulated by amorphous silica in order to find a safe contrast agent and magnetic label for tracking transplanted cells within an organism using magnetic resonance imaging (MRI). Rat mesenchymal stem cells (rMSCs) were labeled for 48 h with a low, medium or high dose of CZF-NPs (0.05; 0.11 or 0.55 mM); silica NPs (Si-NPs; 0.11 mM) served as a positive control. The internalization of NPs into cells was verified by transmission electron microscopy. Biological effects were analyzed at the end of exposure and after an additional 72 h of cell growth without NPs. Compared to untreated cells, Annexin V/Propidium Iodide labeling revealed no significant cytotoxicity for any group of treated cells and only a high dose of CZF-NPs slowed down cell proliferation and induced DNA damage, manifested as a significant increase of DNA-strand breaks and oxidized DNA bases. This was accompanied by high concentrations of 15-F2t-isoprostane and carbonyl groups, demonstrating oxidative injury to lipids and proteins, respectively. No harmful effects were detected in cells exposed to the low dose of CZF-NPs. Nevertheless, the labeled cells still exhibited an adequate relaxation rate for MRI in repeated experiments and ICP-MS confirmed sufficient magnetic label concentrations inside the cells. The results suggest that the silica-coated CZF-NPs, when applied at a non-toxic dose, represent a promising contrast agent for cell labeling.

Silica-coated La0.75Sr0.25MnO3 nanoparticles for magnetically driven DNA isolation.

Magnetic La(0.75)Sr(0.25)MnO(3) nanoparticles possessing an approximately 20-nm-thick silica shell (LSMO(0.25)@SiO(2) ) were characterised and tested for the isolation of PCR-ready bacterial DNA. The results presented here show that the nanoparticles do not interfere in PCR. DNA was apparently reversibly adsorbed on their silica shell from the aqueous phase system (16% PEG 6000-2 M NaCl). The method proposed was used for DNA isolation from complex food samples (dairy products and probiotic food supplements). The isolated DNA was compatible with PCR. The main advantages of the nanoparticles tested for routine use were their high colloidal stability allowing a more precise dosage and therefore high reproducibility of DNA isolation.