Ion-induced folding of a kink turn that departs from the conventional sequence

Kink turns (k-turns) are important structural motifs that create a sharp axial bend in RNA. Most conform to a consensus in which a three-nucleotide bulge is followed by consecutive G•A and A•G base pairs, and when these G•A pairs are modified in vitro this generally leads to a failure to adopt the k-turn conformation. Kt-23 in the 30S ribosomal subunit of Thermus thermophilus is a rare exception in which the bulge-distal A•G pair is replaced by a non-Watson–Crick A•U pair. In the context of the ribosome, Kt-23 adopts a completely conventional k-turn geometry. We show here that this sequence is induced to fold into a k-turn structure in an isolated RNA duplex by Mg2+ or Na+ ions. Therefore, the Kt-23 is intrinsically stable despite lacking the key A•G pair; its formation requires neither tertiary interactions nor protein binding. Moreover, the Kt-23 k-turn is stabilized by the same critical hydrogen-bonding interactions within the core of the structure that are found in more conventional sequences such as the near-consensus Kt-7. T. thermophilus Kt-23 has two further non-Watson–Crick base pairs within the non-canonical helix, three and four nucleotides from the bulge, and we find that the nature of these pairs influences the ability of the RNA to adopt k-turn conformation, although the base pair adjacent to the A•U pair is more important than the other

Molecular dynamics simulations suggest that RNA three-way junctions can act as flexible RNA structural elements in the ribosome

We present extensive explicit solvent molecular dynamics analysis of three RNA three-way junctions (3WJs) from the large ribosomal subunit: the 3WJ formed by Helices 90–92 (H90–H92) of 23S rRNA; the 3WJ formed by H42–H44 organizing the GTPase associated center (GAC) of 23S rRNA; and the 3WJ of 5S rRNA. H92 near the peptidyl transferase center binds the 3′-CCA end of amino-acylated tRNA. The GAC binds protein factors and stimulates GTP hydrolysis driving protein synthesis. The 5S rRNA binds the central protuberance and A-site finger (ASF) involved in bridges with the 30S subunit. The simulations reveal that all three 3WJs possess significant anisotropic hinge-like flexibility between their stacked stems and dynamics within the compact regions of their adjacent stems. The A-site 3WJ dynamics may facilitate accommodation of tRNA, while the 5S 3WJ flexibility appears to be essential for coordinated movements of ASF and 5S rRNA. The GAC 3WJ may support large-scale dynamics of the L7/L12-stalk region. The simulations reveal that H42–H44 rRNA segments are not fully relaxed and in the X-ray structures they are bent towards the large subunit. The bending may be related to L10 binding and is distributed between the 3WJ and the H42–H97 contact

Elastic properties of ribosomal RNA building blocks: molecular dynamics of the GTPase-associated center rRNA

Explicit solvent molecular dynamics (MD) was used to describe the intrinsic flexibility of the helix 42–44 portion of the 23S rRNA (abbreviated as Kt-42+rGAC; kink-turn 42 and GTPase-associated center rRNA). The bottom part of this molecule consists of alternating rigid and flexible segments. The first flexible segment (Hinge1) is the highly anharmonic kink of Kt-42. The second one (Hinge2) is localized at the junction between helix 42 and helices 43/44. The rigid segments are the two arms of helix 42 flanking the kink. The whole molecule ends up with compact helices 43/44 (Head) which appear to be modestly compressed towards the subunit in the Haloarcula marismortui X-ray structure. Overall, the helix 42–44 rRNA is constructed as a sophisticated intrinsically flexible anisotropic molecular limb. The leading flexibility modes include bending at the hinges and twisting. The Head shows visible internal conformational plasticity, stemming from an intricate set of base pairing patterns including dynamical triads and tetrads. In summary, we demonstrate how rRNA building blocks with contrasting intrinsic flexibilities can form larger architectures with highly specific patterns of preferred low-energy motions and geometries

Kidney nanotoxicity studied in human renal proximal tubule epithelial cell line TH1

Progressive expansion of nanomaterials in our everyday life raises concerns about their safety for human health. Although kidneys are the primary organs of xenobiotic elimination, little attention has been paid to the kidneys in terms of nanotoxicological studies up to now. Here we investigate the cytotoxic and genotoxic potential of four solid-core uncoated inorganic nanoparticles (TiO₂NPs, SiO₂NPs, Fe₃O₄NPs and AuNPs) using the human renal proximal tubule epithelial TH1 cells. To mimic the in vivo conditions more realistic, TH1 cells were exposed in vitro to inorganic NPs under static as well as dynamic conditions for 3 h and 24 h. The medium throughput alkaline comet assay (12 minigels per slide) was employed to evaluate the impact of these NPs on genome integrity and their capacity to produce oxidative lesions to DNA. The accumulation and localization of studied inorganic NPs inside the cells was monitored by transmission electron microscopy (TEM) and the efficacy of internalization of particular NPs was determined by atomic absorption spectroscopy (AAS) and inductively coupled plasma mass spectrometry (ICP-MS). From all the tested NPs, only Fe₃O₄NPs induced a slight cytotoxicity in TH1 cells exposed to high concentrations (>700 μg/ml) for 24 h. On the other hand, the inorganic NPs did not increase significantly the level of DNA strand breaks or oxidative DNA damage regardless of the treatment mode (static vs. dynamic conditions). Interestingly, substantial differences were observed in the internalized amount of inorganic NPs in TH1 cells exposed to equivalent (2.2 μg/ml) concentration. FeONPs were most efficiently taken up while the lowest quantity of particles was determined in TiONPs-treated cells. As the particle size and shape of individual inorganic NPs in culture medium was nearly identical, it is reasonable to suppose that the chemical composition may contribute to the differences in the efficacy of NPs uptake

Conformations of Flanking Bases in HIV-1 RNA DIS Kissing Complexes Studied by Molecular Dynamics

Explicit solvent molecular dynamics simulations (in total almost 800 ns including locally enhanced sampling runs) were applied with different ion conditions and with two force fields (AMBER and CHARMM) to characterize typical geometries adopted by the flanking bases in the RNA kissing-loop complexes. We focus on flanking base positions in multiple x-ray and NMR structures of HIV-1 DIS kissing complexes and kissing complex from the large ribosomal subunit of Haloarcula marismortui. An initial x-ray open conformation of bulged-out bases in HIV-1 DIS complexes, affected by crystal packing, tends to convert to a closed conformation formed by consecutive stretch of four stacked purine bases. This is in agreement with those recent crystals where the packing is essentially avoided. We also observed variants of the closed conformation with three stacked bases, while nonnegligible populations of stacked geometries with bulged-in bases were detected, too. The simulation results reconcile differences in positions of the flanking bases observed in x-ray and NMR studies. Our results suggest that bulged-out geometries are somewhat more preferred, which is in accord with recent experiments showing that they may mediate tertiary contacts in biomolecular assemblies or allow binding of aminoglycoside antibiotics

Long-Term Accumulation, Biological Effects and Toxicity of BSA-Coated Gold Nanoparticles in the Mouse Liver, Spleen, and Kidneys

Kristina Jakic,1 Michal Selc,1,2 Filip Razga,3 Veronika Nemethova,3 Petra Mazancova,3 Filip Havel,4,5 Michal Sramek,5 Monika Zarska,5 Jan Proska,4 Vlasta Masanova,6 Iveta Uhnakova,6 Peter Makovicky,7 Marta Novotova,8 Vit Vykoukal,9 Andrea Babelova1,2 1Department of Nanobiology, Cancer Research Institute, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia; 2Centre for Advanced Material Application, Slovak Academy of Sciences, Bratislava, Slovakia; 3Selecta Biotech SE, Bratislava, Slovakia; 4Department of Physical Electronics, Faculty of Nuclear Sciences and Physical Engineering, Czech Technical University in Prague, Prague, Czech Republic; 5Department of Genome Integrity, Institute of Molecular Genetics of the Czech Academy of Sciences, Prague, Czech Republic; 6Department of Metallomics, Faculty of Medicine, Slovak Medical University, Bratislava, Slovakia; 7Department of Molecular Oncology, Cancer Research Institute, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia; 8Department of Cellular Cardiology, Institute of Experimental Endocrinology, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia; 9Department of Chemistry, Faculty of Science, Masaryk University, Brno, Czech RepublicCorrespondence: Andrea Babelova, Department of Nanobiology, Cancer Research Institute, Biomedical Research Center, Slovak Academy of Sciences, Bratislava, Slovakia, Tel +421-2-32295-189, Fax +421-2-5477-4284, Email [email protected]: Gold nanoparticles are promising candidates as vehicles for drug delivery systems and could be developed into effective anticancer treatments. However, concerns about their safety need to be identified, addressed, and satisfactorily answered. Although gold nanoparticles are considered biocompatible and nontoxic, most of the toxicology evidence originates from in vitro studies, which may not reflect the responses in complex living organisms.Methods: We used an animal model to study the long-term effects of 20 nm spherical AuNPs coated with bovine serum albumin. Mice received a 1 mg/kg single intravenous dose of nanoparticles, and the biodistribution and accumulation, as well as the organ changes caused by the nanoparticles, were characterized in the liver, spleen, and kidneys during 120 days.Results: The amount of nanoparticles in the organs remained high at 120 days compared with day 1, showing a 39% reduction in the liver, a 53% increase in the spleen, and a 150% increase in the kidneys. The biological effects of chronic nanoparticle exposure were associated with early inflammatory and fibrotic responses in the organs and were more pronounced in the kidneys, despite a negligible amount of nanoparticles found in renal tissues.Conclusion: Our data suggest, that although AuNPs belong to the safest nanomaterial platforms nowadays, due to their slow tissue elimination leading to long-term accumulation in the biological systems, they may induce toxic responses in the vital organs, and so understanding of their long-term biological impact is important to consider their potential therapeutic applications. Keywords: AuNPs, in vivo, long-term accumulation, biodistribution, inflammation, fibrosi

Molecular Dynamics and Quantum Mechanics of RNA: Conformational and Chemical Change We Can Believe In

Structure and dynamics are both critical to RNA’s vital functions in biology. Numerous techniques can elucidate the structural dynamics of RNA, but computational approaches based on experimental data arguably hold the promise of providing the most detail. In this Account, we highlight areas wherein molecular dynamics (MD) and quantum mechanical (QM) techniques are applied to RNA, particularly in relation to complementary experimental studies

Developments in cell biology for quantitative immunoelectron microscopy based on thin sections: a review

Quantitative immunoelectron microscopy uses ultrathin sections and gold particle labelling to determine distributions of molecules across cell compartments. Here, we review a portfolio of new methods for comparing labelling distributions between different compartments in one study group (method 1) and between the same compartments in two or more groups (method 2). Specimen samples are selected unbiasedly and then observed and expected distributions of gold particles are estimated and compared by appropriate statistical procedures. The methods can be used to analyse gold label distributed between volume-occupying (organelle) and surface-occupying (membrane) compartments, but in method 1, membranes must be treated as organelles. With method 1, gold counts are combined with stereological estimators of compartment size to determine labelling density (LD). For volume-occupiers, LD can be expressed simply as golds per test point and, for surface-occupiers, as golds per test line intersection. Expected distributions are generated by randomly assigning gold particles to compartments and expressing observed/expected counts as a relative labelling index (RLI). Preferentially-labelled compartments are identified from their RLI values and by Chi-squared analysis of observed and expected distributions. For method 2, the raw gold particle counts distributed between compartments are simply compared across groups by contingency table and Chi-squared analysis. This identifies the main compartments responsible for the differences between group distributions. Finally, we discuss labelling efficiency (the number of gold particles per target molecule) and describe how it can be estimated for volume- or surface-occupiers by combining stereological data with biochemical determinations

Regulation of (Pro)Renin Receptor in Renin-Positive Smooth Muscle Cells of Kidney Arterioles in Rats with STZ-Induced Diabetes

Objective. The nephron (pro)renin receptor may play a pathophysiological role in renal disorders in hypertension or diabetes. The aim of this study was to determine the relationship of (pro)renin receptors and transdifferentiation between the renin-negative and renin-positive SMCs in the afferent arteriole by estimating the distribution of (pro)renin receptors in renin-positive and renin-negative SMCs of the afferent arteriole of kidneys in normal and streptozotocin- (STZ-) induced diabetic rats. Therefore in diabetes the renin granulation of afferent arterioles is different as in normal, the diabetes model for finding the differences to normal in distribution of (pro)renin receptors of afferent arterioles was used. Method. To estimate the number of (pro)renin receptors in arteriolar SMCs a special protocol of immunohistochemistry to stereology was followed. Results. Our results showed that on the surface of renin-positive SMCs the number of (pro)renin receptors was upregulated, while in the cytoplasm of SMCs there was downregulation in comparison to renin-negative SMCs. There is a significant difference between the number of (pro)renin receptors on the surface and in the cytoplasm of renin-positive SMCs in normal rats. These differences in the number of (pro)renin receptors were not present in rats with STZ-induced diabetes. Any other differences in the number of (pro)renin receptors between the STZ-induced diabetic and normal rats were not detected. The tissue level of angiotensin II did not change in the kidneys of STZ-induced diabetic rats. Conclusion. The distribution of (pro)renin receptors in afferent arteriolar SMCs is related to renin granulation of SMCs, but independent of angiotensin II plasma or tissue levels in the kidney