Multifunctional poly(organosiloxane) nanoparticles as a model system for biomedical applications

Polymere Nanomaterialien finden bereits ein sehr breites Anwendungsspektrum in der Biomedizin, speziell in Bereichen des Wirkstofftransports und der Krebsforschung. Dennoch ist die Synthese geeigneter Modellsysteme für ein besseres Verständnis der Wechselwirkungen von Nanopartikeln mit biologischen Systemen notwendig. Diese Studie berichtet über die Synthese und Charakterisierung multifunktioneller, fluoreszenzmarkierter Poly(organosiloxan)-Kern-Schale-Nanopartikel mit veränderbaren Oberflächeneigenschaften. Die Kern-Schale-Architektur erlaubt eine unabhängige Funktionalisierung unterschiedlicher Partikelkompartimente. So wurde der Kern der Partikel während der Polykondensation mit RhodaminB-Monomer markiert. Durch Einführung geladener Gruppen in der Partikelschale wurden elektrostatisch stabilisierte Partikel erhalten. Alternativ führt das Aufpfropfen von Polymeren (thermoresponsives Poly(2-isopropyl-2- oxazolin) oder Poly(ethylenglykol)) auf die Oberfläche zu sterisch stabilisierten, biokompatiblen Nanopartikeln. Schließlich wurde Biotin an die Partikeloberfläche gekuppelt, um die Immobilisierung weiterer Liganden wie Antikörper oder Enzyme auf der Partikeloberfläche zu ermöglichen. Die Nanopartikel wurden mittels Elektronenmikroskopie, winkelabhängiger dynamischer Lichtstreuung (DLS), asymmetrischer Fluss Feld-Fluss Fraktionierung (AF-FFF) und ζ-Potential-Messungen charakterisiert. Die spektroskopischen Eigenschaften wurden mit der Fluoreszenzspektroskopie untersucht, einschließlich der Bestimmung der absoluten Fluoreszenzquantenausbeuten. Auf Grund ihrer modularen Struktur und Vielfalt an möglichen Funktionalisierungen eignet sich das Poly(organosiloxan)-System für diverse biomedizinische Anwendungen, wie z.B. Abbildungsmethoden, spezifisches Targeting und Wirkstofftransport. Die zelluläre Aufnahme der Nanopartikel in vivo wird oft durch die Bildung einer Proteinkorona beeinflusst, deshalb wurde weiterhin das Verhalten unterschiedlich funktionalisierter Partikel unter physiologischen Bedingungen und in Anwesenheit von Serumproteinen mittels DLS und AF-FFF untersucht. Neben der Synthese und physikochemischen Charakterisierung wurden auch Zellaufnahmeexperimente durchgeführt, in denen die Wechselwirkung der Nanopartikel mit Lungengewebemodellen erforscht wurde. Diese Versuche zeigen Unterschiede in der zellulären Aufnahme der elektrostatisch und der sterisch stabilisierten Partikel.Polymeric nanomaterials offer a wide range of biomedical applications especially in the fields of drug delivery and cancer research. However, suitable model systems are still required to gain deeper knowledge of interactions of nanoparticles with biological systems. This study reports on the synthesis and characterization of multifunctional, fluorescent poly(organosiloxane) core-shell nanoparticles with tunable surface properties. The core-shell approach allows the independent functionalization of different compartments. Thus, rhodamine b-labeled monomer was efficiently incorporated into the core during polycondensation. By introduction of charged groups on the surface, electrostatic stabilization of the particles was achieved. Alternatively, grafting of polymers (e.g. thermo-responsive poly(2-isopropyl-2-oxazoline) or poly(ethylene glycol)) leads to sterically stabilized, biocompatible nanoparticles. Finally biotin was introduced on the surface allowing the immobilization of further ligands such as antibodies or enzymes on the particle’s surface. The nanoparticles were characterized by electron microscopy, multi-angle dynamic light scattering (DLS), asymmetrical flow field-flow fractionation (AF-FFF) and zeta potential measurements. The spectroscopic properties were studied by fluorescence spectroscopy, including the determination of absolute fluorescence quantum yields. Due to their modular structure and the variety of possible functionalizations, the poly(organosiloxane) system is suitable for diverse biomedical applications, e.g. imaging methods, specific targeting and drug delivery. The cellular uptake of nanoparticles in vivo is often determined by the formation of a protein corona. Therefore, the behavior of nanoparticles with different surface characteristics was compared under physiological conditions and in presence of serum proteins using DLS and AF-FFF. Besides the synthesis and the physico-chemical characterization, cell experiments are performed where nanoparticles are applied to lung tissue models. These experiments demonstrate differences in the uptake behavior of electrostatically and sterically stabilized nanoparticles

Vitamin D₃ Modulates NF-kB/p65, 17β-Estradiol, and Vitamin D Receptors Expression at Estrogen Deficiency

The aim of the present study was to focus on the effects of Vitamin D3 (VD3) supplementation (5.0 mg/kg, s.c.) on the NF-kB/p65, 17β-estradiol (17β-E2)/VD3 receptors expression in the hippocampus in the long-term ovariectomized (OVX) rats treated with low dose of 17β-E2 (0.5 μg/rat, s.c.) submitted for the chronic unpredictable mild stress (CUMS) for 28 days. Sucrose preference (SPT), forced swimming (FST), and open-field (OFT) tests were conducted to estimate the anhedonia-/depression-like states. NF-kB/p65, 17β-E2/VD3 receptors levels in the hippocampus were evaluated by ELISA and Western blot assays. The findings demonstrated that VD3 at high dose (5.0 mg/kg, s.c.) in a combination with low dose of 17β-E2 decreased anhedonia in the SPT and depression-like behavior in the FST of the long-term OVX rats submitted to CUMS. VD3 (5.0 mg/kg) resulted in significant decreased levels of hippocampal NF-kB/p65 protein expression, as well as to the normalization of hippocampal 17β-E2/VD3 receptors levels in long-term OVX rats treated with 17β-E2 exposed to CUMS. In conclusion, VD3 (5.0 mg/kg, s.c.) in a combination with low dose of 17β-E2 had a synergic antianhedonic- and antidepressant-like effects in the adult female rats following long-term ovariectomy submitted to CUMS

Abstract P-24: Microscopic Analyses of Liquid-Liquid Phase Separation Induced by Linker Histone H1.0

Background: Liquid-liquid phase separation (LLPS) that leads to the formation of temporary functional domains in cells plays an important role in the processes of chromatin condensation and gene regulation. Earlier, it was demonstrated that histone H1.4 can form LLPS droplets with DNA. In the present work, LLPS was studied for histone H1.0, which is mainly expressed in differentiated and non-dividing cells. H1.0 is involved in cancer development: its amount decreases with the progression of tumor cells to malignancy. Methods: LSM710 confocal microscope (Zeiss) equipped with the 40x/1.2W objective was used to image mixtures of H1.0 with Cy3/Cy5 labeled DNA or nucleosomes in fluorescent and transmitted-light channels at the excitation of 514 nm. The formation of condensates as a result of LLPS was confirmed by salt-jump and FRAP/FLIP experiments. Results: Condensates were not observed when the ratio of negative to positive charges (N/P) in the samples was >1. At N/P~0.7, optically homogeneous droplet-like condensates were found. The appearance of condensates, their size and shape depended on concentrations of H1.0 and DNA. LLPS condensates but not aggregates disappeared by salt-jump to 650 mM NaCl. FRAP/FLIP experiments revealed a moderate rate of fluorescence recovery (τ½22s) indicating moderate DNA mobility of the H1.0-mediated condensates. The appearance of condensates was also observed in the mixtures of H1.0, DNA and Cy3/Cy5-labeled nucleosomes. Nucleosomes were involved in the condensate formation and found to be 2-fold more mobile (τ½10 s) than DNA. Conclusion: LLPS-related properties of H1.0 were studied for DNA and nucleosomes in vitro. Comparison with H1.4 shows that H1.0 forms liquid condensates of approximately the same size. Our result also may indicate that chromatin retains pronounced dynamic properties in H1.0-induced droplets despite the fact that H1.0 induces the formation of more compact chromatin

Selective footprints and genes relevant to cold adaptation and other phenotypic traits are unscrambled in the genomes of divergently selected chicken breeds

Background: The genomes of worldwide poultry breeds divergently selected for performance and other phenotypic traits may also be affected by, and formed due to, past and current admixture events. Adaptation to diverse environments, including acclimation to harsh climatic conditions, has also left selection footprints in breed genomes. Results: Using the Chicken 50K_CobbCons SNP chip, we genotyped four divergently selected breeds: two aboriginal, cold tolerant Ushanka and Orloff Mille Fleur, one egg-type Russian White subjected to artificial selection for cold tolerance, and one meat-type White Cornish. Signals of selective sweeps were determined in the studied breeds using three methods: (1) assessment of runs of homozygosity islands, (2) FST based population differential analysis, and (3) haplotype differentiation analysis. Genomic regions of true selection signatures were identified by two or more methods or in two or more breeds. In these regions, we detected 540 prioritized candidate genes supplemented them with those that occurred in one breed using one statistic and were suggested in other studies. Amongst them, SOX5, ME3, ZNF536, WWP1, RIPK2, OSGIN2, DECR1, TPO, PPARGC1A, BDNF, MSTN, and beta-keratin genes can be especially mentioned as candidates for cold adaptation. Epigenetic factors may be involved in regulating some of these important genes (e.g., TPO and BDNF). Conclusion: Based on a genome-wide scan, our findings can help dissect the genetic architecture underlying various phenotypic traits in chicken breeds. These include genes representing the sine qua non for adaptation to harsh environments. Cold tolerance in acclimated chicken breeds may be developed following one of few specific gene expression mechanisms or more than one overlapping response known in cold-exposed individuals, and this warrants further investigation

INVESTIGATION OF MAGNETIZATION REVERSAL PROCESSES IN TWO-LAYER FeNiCo/Ta/CoW AND THREE-LAYER FeNiCo/FeNiCo FILMS

The aim is to study the nature of interacting multilayer film structures. It has been discovered that the heterophase character of one layer can lead to the asymmetry of other layer behaviour in the external magnetic field. The qualitative analysis of the hysteresis loop shift field value coincides with relation of the shift field for two-layer FeNiCo/CoW film. The experimental model was a base of the patent "Procedure of Making Magnetostatic-Connected Films". In three-layer FeNiCo/Ta/FeNiCo the difference in the motion rate relations of the domen boundaries in the different layers has been explained from conceptions about dependence of the effective bond field of the layers upon the external magnetic field. The observed domen transfers from layer into layer have been explained by a combined action of the scattering fields from domen tops and external magnetic field. The investigation results are used at development of the aids in the magnetoelectronicsAvailable from VNTIC / VNTIC - Scientific & Technical Information Centre of RussiaSIGLERURussian Federatio

Analysis of Homozygous-by-Descent (HBD) Segments for Purebred and Crossbred Pigs in Russia

Intensive selection raises the efficiency of pig farming considerably, but it also promotes the accumulation of homozygosity, which can lead to an increase in inbreeding and the accumulation of deleterious variation. The analysis of segments homozygous-by-descent (HBD) and non-HBD segments in purebred and crossbred pigs is of great interest. Research was carried out on 657 pigs, of which there were Large White (LW, n = 280), Landrace (LR, n = 218) and F1 female (♂LR × ♀LW) (F1, n = 159). Genotyping was performed using the GeneSeek® GGP Porcine HD Genomic Profiler v1 (Illumina Inc., USA). To identify HBD segments and estimate autozygosity (inbreeding coefficient), we used the multiple HBD classes model. LW pigs exhibited 50,420 HBD segments, an average of 180 per animal; LR pigs exhibited 33,586 HBD segments, an average of 154 per animal; F1 pigs exhibited 21,068 HBD segments, an average of 132 per animal. The longest HBD segments in LW were presented in SSC1, SSC13 and SSC15; in LR, in SSC1; and in F1, in SSC15. In these segments, 3898 SNPs localized in 1252 genes were identified. These areas overlap with 441 QTLs (SSC1—238 QTLs; SSC13—101 QTLs; and SSC15—102 QTLs), including 174 QTLs for meat and carcass traits (84 QTLs—fatness), 127 QTLs for reproduction traits (100 QTLs—litter traits), 101 for production traits (69 QTLs—growth and 30 QTLs—feed intake), 21 QTLs for exterior traits (9 QTLs—conformation) and 18 QTLs for health traits (13 QTLs—blood parameters). Thirty SNPs were missense variants. Whilst estimating the potential for deleterious variation, six SNPs localized in the NEDD4, SEC11C, DCP1A, CCT8, PKP4 and TENM3 genes were identified, which may show deleterious variation. A high frequency of potential deleterious variation was noted for LR in DCP1A, and for LW in TENM3 and PKP4. In all cases, the genotype frequencies in F1 were intermediate between LR and LW. The findings presented in our work show the promise of genome scanning for HBD as a strategy for studying population history, identifying genomic regions and genes associated with important economic traits, as well as deleterious variation

Phylogenetic Analysis of Russian Native Sheep Breeds Based on mtDNA Sequences

Eurasia is represented by all climatic zones and various environments. A unique breed variety of farm animals has been developed in Russia, whose territory covers a large area of the continent. A total of 69 local breeds and types of dairy, wool, and meat sheep (Ovis aries) are maintained here. However, the genetic diversity and maternal origin of these local breeds have not been comprehensively investigated. In this study, we describe the diversity and phylogeny of Russian sheep breeds inhabiting different geographical regions based on the analysis of complete sequences of mitochondrial genomes (mtDNA). Complete mtDNA sequences of the studied sheep were obtained using next-generation sequencing technology (NGS). All investigated geographical groups of sheep were characterized by high haplotype (Hd = 0.9992) and nucleotide diversity (π = 0.00378). Analysis of the AMOVA results showed that genetic diversity was majorly determined by within-population differences (77.87%). We identified 128 haplotypes in all studied sheep. Haplotypes belonged to the following haplogroups: B (64.8%), A (28.9%), C (5.5%), and D (0.8%). Haplogroup B was predominant in the western part of Russia. A high level of mtDNA polymorphism in the studied groups of local sheep indicates the presence of a significant reserve of unique genotypes in Russia, which is to be explored

Multibranched-Based Fluorinated Materials: Tailor-Made Design of 19F-MRI Probes

ConspectusFuture medicine is primarily aiming at the development of novel approaches for an early diagnosis of diseases and a personalized therapy for patients. For achieving these objectives, a key role is played by medical imaging. Among available noninvasive imaging techniques, Fluorine-19 (19F) Magnetic Resonance Imaging (MRI) is emerging as a powerful quantitative detection modality for clinical use both for molecular imaging and for cell tracking.The strength of using 19F-MRI is mainly related to the lack of endogenous organic fluorine in tissues, with no background, enabling the visualization of fluorinated tracers as hot-spot images, adding secondary independent information to the anatomical features provided by the grayscale 1H-MRI. The main challenge for 19F-MRI clinical application is the intrinsic reduced sensitivity of MRI. To improve sensitivity, undoubtedly the use of a high field MRI scanner and cryogenic radiofrequency probes is advantageous, but there is a clear need of developing increasingly effective fluorinated tracers.The ideal tracer should bear as many as possible magnetically equivalent fluorine atoms and show optimal magnetic resonance relaxivity properties (i.e., T1 and T2), which enable reduced acquisition time with the possibility to apply fast imaging methods. Moreover, it should be biocompatible with reduced tendency to bioaccumulate in tissues, which is one of the main drawbacks in using perfluorocarbons (PFCs), together with their difficulty to be chemically modified with functional groups. In fact, PFCs such as perfluorooctyl bromide (PFOB), perfluoro-15-crown-5-ether (PFCE), and linear perfluoropolyethers (PFPE) are currently the most used tracers in 19F-MRI preclinical and clinical studies, with the above-mentioned limitations. In this regard, molecules bearing short branched fluorinated chains gained a lot of attention for their high number of equivalent fluorines and expected capability of reducing bioaccumulation concerns. A valuable building block for branched fluorinated tracers is perfluoro-tert-butanol (PFTB), with nine magnetically equivalent fluorines and easy availability and modification.In this Account we will discuss the main challenges that 19F-MRI has to overcome for increasing its clinical use, highlighting on one hand the need of developing customized fluorinated materials for increasing sensitivity and enabling multimodal properties, and on the other hand, the importance of the ultrastructure of the final formulation for the final biological response (i.e., clearance). In this context, our group has been focusing on the synthesis and development of branched fluorinated tracers, for which the originator is a molecule called PERFECTA (from suPERFluorinatEdContrasT Agent), bearing 36 equiv 19F atoms, which showed not only optimal relaxometry properties but also a very specific and intense Raman signal. Thus, PERFECTA and its derivatives represent a new family of multimodal tracers enabling multiscale analysis, from whole body imaging (19F-MRI) to microscopic detection at the cellular/tissue level (Raman microscopy). We believe that our proposed PFTB strategy can strongly promote the production of increasingly effective 19F-MRI materials with additional functionalities, facilitating the clinical translation of this imaging modality