Репозиторий Университета

Mesenchymal stem cells (MSCs) are thought to be the most attractive type of cells for bone repair. However, much still remains unknown about MSCs and needs to be clarified before this treatment can be widely applied in the clinical practice. The purpose of this study was to establish the involvement of allogeneic MSCs in the bone formation in vivo, using a model of transgenic mice and genetically labeled cells. Polylactide scaffolds with hydroxyapatite obtained by surface selective laser sintering were used. The scaffolds were sterilized and individually seeded with MSCs from the bone marrow of 5-week-old GFP(+) transgenic С57/Вl6 or GFP(−)С57/Вl6 mice. 4-mm-diameter critical-size defects were created on the calvarial bone of mice using a dental bur. Immediately after the generation of the cranial bone defects, the scaffolds with or without seeded cells were implanted into the injury sites. The cranial bones were harvested at either 6 or 12 weeks after the implantation. GFP(+) transgenic mice having scaffolds with unlabeled MSCs were used for the observation of the host cell migration into the scaffold. GFP(−) mice having scaffolds with GFP(+)MSCs were used to assess the functioning of the seeded MSCs. The obtained data demonstrated that allogeneic MSCs were found on the scaffolds 6 and 12 weeks post-implantation. By week 12, a newly formed bone tissue from the seeded cells was observed, without an osteogenic pre-differentiation. The host cells did not appear, and the control scaffolds without seeded cells remained empty. Besides, a possibility of vessel formation from seeded MSCs was shown, without a preliminary cell cultivation under controlled conditions.

It has been established that treatment of mice with sodium nitrite, S-nitrosoglutathione and the water-soluble nitroglycerine derivative isosorbide dinitrate (ISDN) as NO donors initiates in vivo synthesis of significant amounts of EPR-silent binuclear dinitrosyl iron complexes (B-DNIC) with thiol-containing ligands in the liver and other tissues of experimental mice. This effect is especially apparent if NO donors are administered to mice simultaneously with the Fe²⁺-citrate complex. Similar results were obtained in experiments on isolated liver and other mouse tissues treated with gaseous NО in vitro and during stimulation of endogenous NO synthesis in the presence of inducible NO synthase. B-DNIC appeared in mouse tissues after in vitro treatment of tissue samples with an aqueous solution of diethyldithiocarbamate (DETC), which resulted in the transfer of iron-mononitrosyl fragments from B-DNIC to the thiocarbonyl group of DETC and the formation of EPR-detectable mononitrosyl iron complexes (MNIC) with DETC. EPR-Active MNIC with N-methyl-d-glucamine dithiocarbamate (MGD) were synthesized in a similar way. MNIC-MGD were also formed in the reaction of water-soluble MGD-Fe²⁺ complexes with sodium nitrite, S-nitrosoglutathione and ISDN.

В работе обсуждаются дискуссионные аспекты статьи Дробижева М. Ю., Кикты С. В., Мачильской О. В. “Кардиопсихиатрия. Проблема перевода”, опубликованной в журнале Кардиоваскулярной терапия и профилактика 2016; 15(4): 88-97. Представленная авторами классификация кардиопсихиатрических расстройств основана лишь на анализе литературных данных и на деле представляет собой редукционистскую попытку выяснения соотношения нейрофизиологических механизмов со сложными психопатологическими и психосоматическими образованиями, реализующимися в сфере сердечно-сосудистой системы. Возражения вызывает также раздел публикации, посвященный психофармакотерапии психических расстройств в кардиологической практике, в котором авторская позиция сводится к рекомендации применения лишь одного лекарственного препарата.

In this work, we studied water solubility of ampicillin trihydrate and its solid dispersions (SD) with polyethylene glycol-1500, polyvinylpyrrolidone-10000, and β-cyclodextrin. It was found that SD formation increases the solubility by a factor of 1.34–1.73 and the rate of ampicillin dissolution by a factor of 3.43–7.40. The results of complex physicochemical studies suggest that the improved release of ampicillin from SD is due to its micronization and solubilization by the polymer.

Background: Attention-deficit/hyperactivity disorder (ADHD) is a common, highly heritable neurodevelopmental disorder with profound cognitive, behavioral, and psychosocial impairments with persistence across the life cycle. Our initial genome-wide screening approach for copy number variants (CNVs) in ADHD implicated a duplication of SLC2A3, encoding glucose transporter-3 (GLUT3). GLUT3 plays a critical role in cerebral glucose metabolism, providing energy for the activity of neurons, which, in turn, moderates the excitatory-inhibitory balance impacting both brain development and activity-dependent neural plasticity. We therefore aimed to provide additional genetic and functional evidence for GLUT3 dysfunction in ADHD. Methods: Case-control association analyses of SLC2A3 single-nucleotide polymorphisms (SNPs) and CNVs were conducted in several European cohorts of patients with childhood and adult ADHD (SNP, n = 1,886 vs. 1,988; CNV, n = 1,692 vs. 1,721). These studies were complemented by SLC2A3 expression analyses in peripheral cells, functional EEG recordings during neurocognitive tasks, and ratings of food energy content. Results: Meta-analysis of all cohorts detected an association of SNP rs12842 with ADHD. While CNV analysis detected a population-specific enrichment of SLC2A3 duplications only in German ADHD patients, the CNV + rs12842 haplotype influenced ADHD risk in both the German and Spanish cohorts. Duplication carriers displayed elevated SLC2A3 mRNA expression in peripheral blood cells and altered event-related potentials reflecting deficits in working memory and cognitive response control, both endophenotypic traits of ADHD, and an underestimation of energy units of high-caloric food. Conclusions: Taken together, our results indicate that both common and rare SLC2A3 variation impacting regulation of neuronal glucose utilization and energy homeostasis may result in neurocognitive deficits known to contribute to ADHD risk.

Chronic spontaneous urticaria (CSU) is a mast-cell driven skin disease, characterized by the recurrence of transient wheals, angioedema, or both for more than 6 weeks. Autoimmunity is thought to be one of the most frequent causes of CSU. Type I and type II autoimmunity, i.e. IgE to autoallergens and IgG autoantibodies to IgE or its receptor, respectively, have been implicated in the etiology and pathogenesis of CSU. We analyzed the relevant literature and assessed the existing evidence in support of a role for type I and II autoimmunity in CSU with the help of Hill’s criteria of causality. For each of these criteria, i.e. strength of association, consistency, specificity, temporality, biological gradient, plausibility, coherence, experiment and analogy, we categorized the strength of evidence as “insufficient”, “low”, “moderate” or “high” and then assigned levels of causality for type I and II autoimmunity in CSU, from level 1 (causal relationship) to level 5 (causality not likely). Based on the evidence in support of Hill’s criteria, type I autoimmunity in CSU has level 3 causality (causal relationship suggested) and type II autoimmunity has level 2 causality (causal relationship likely). There are still many aspects of the pathologic mechanisms of CSU that need to be resolved, but it is becoming clear that there are at least two distinct pathways, type I and type II autoimmunity, that contribute to the pathogenesis of this complex disease.

Physical adsorption was used to produce nanometer thick thermoresponsive films with a view to nonenzymatic cell detachment. Two polymers were investigated, poly-(N-isopropylacrylamide) and poly (N-isopropylacrylamide-co-N-tertbutylacrylamide). Substrates were prepared above and below the polymers’ LCST to investigate the effect of polymer conformation on the prepared substrates. Endothelial cells were seeded on the prepared films; cell proliferation was higher on the films produced below the polymers’ LCST than on those prepared above and cells detached from the surfaces upon temperature reduction. Physical adsorption of poly-(N-isopropylacrylamide)–based films is a viable approach to produce substrates compliant with cell growth and temperature modulated detachment.

In recent years, engineering of blood vessels, which can provide the effective transport of nutrients and various metabolites, is one of the major challenges in tissue reconstruction. Many researches are carried out to develop cell-seeded bioconstructs based on natural polymers, particularly on PEGylated fibrin. Therefore, the aim of this study was to reveal the optimal component ratio for modified fibrin hydrogels in order to provide favorable conditions for vascular development of endothelial and mesenchymal stem cell co-culture. It has been found out that the PEGylated fibrin hydrogels can support 3D cell growth in HUVECs and hASCs co-culture. The microporous filamentous hydrogel prepared from PEGylated 5 : 1 fibrinogen and using the 1 : 0.2 protein to thrombin ratio had the most favorable microenvironment for cell distribution, growth and development in the studied co-culture that resulted in high levels of expression of proteins required for angiogenesis.
(PDF) Evaluation of the Vasculogenic Potential of Hydrogels Based on Modified Fibrin.

Conducting, mechanically durable, elastic nanocomposite films were prepared with chitosan (CS) as the polymer matrix, graphene obtained from highly exfoliated graphite as the nanofiller, and poly(vinyl pyrrolidone) (PVP) as the stabilizer of the graphene sheets. The maximum graphene content in the composites without a loss of uniformity and other useful properties increased up to 4.0 wt %. The resulting composites were characterized by scanning electron microscopy, Raman spectroscopy, X-ray diffraction analysis, mechanical testing, and electrical conductivity testing to determine the effects of the addition of graphene on the morphology and mechanical and electrical properties of the CS–PVP–graphene nanocomposite films. In this study, we took an approach to making nanocomposites from the perspectives of green chemistry, environmental protection, regenerative medicine, and low cost. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017, 134, 45038. © 2017 Wiley Periodicals, Inc.

The discovery and development of effective antihypoxic drugs is an important issue in modern experimental and clinical pharmacology. Complexes of zinc containing imidazole cycles as ligands are promising candidates for the design of novel drugs, including pharmacotherapeutic tools for patient’s protection under conditions of oxygen deficiency. Herein, we report antihypoxic activity of zinc complexes with N-alkenylimidazoles, and, specifically, a novel drug Acyzol, which can be used as efficient antidote after poisoning by carbon monoxide and other combustion products.

The present study investigates the hepatoprotective effect of a novel zinc-containing drug acyzol in comparison with silymarin, a medicinal extract of milk thistle (Silybum marianum). The hepatoprotective effect was studied in 40 albino nonlinear male rats in a model of toxic liver injury induced by intragastric administration of carbon tetrachloride. Both drugs were diluted in water and administered intragastrically at doses 10 mg/kg (acyzol) and 100 mg/kg (silymarin) for 10 days twice daily, after development of clinical toxic hepatitis. Biochemical and functional indicators of the liver parenchyma demonstrated that both drugs reduced mortality, normalized the body and relative liver weight, reduced intensity of cytolytic, cholestatic, and mesenchymal inflammatory syndromes, and restored liver function. The study demonstrates that acyzol and silymarin have comparable hepatoprotective effect, thus, providing a rationale for the use of acyzol in complex therapy of toxic hepatitis and hepatosis.

One the current challenges of modern hepatology is to find new approaches to stimulate liver regeneration and to find new methods for liver disease treatment. Cell therapies, which are based on using regional stem cells for disease treatment, are under active development. However, studies, devoted to their transplantation, are currently scarce. In recent years, hepatic stellate cells are considered to be hepatic stem cells. It is known that activated hepatic stellate cells can transdifferentiate into myofibroblasts and lead to liver fibrosis. The aim of our work was to study the influence of native and activated hepatic stellate cells in vivo by lead nitrate injection after transplantation into partial hepatectomized rats, which is considered to be a classical model to study liver regeneration. Injection of 2-acetylaminofluorene (AAF), which selectively eliminates hepatocyte proliferation, was used to understand the hepatic stellate cells role in liver regeneration process better. Our results suggest that transplanted native and activated hepatic stellate cells can differentiate into hepatocyte-like cells and positively influence liver regeneration without inducing liver fibrosis.

Polymerase chain reaction (PCR) is one of the most important laboratory techniques used in molecular biology, genetics and molecular diagnostics. The success of a PCR-based method largely depends on the correct nucleic acid sequence analysis in silico prior to a wet-bench experiment. Here, we report the development of an online Java-based software for virtual PCR on linear or circular DNA templates and multiple primer or probe search from large or small databases. Primer or probe sensitivity and specificity are predicted by searching a database to find sequences with an optimal number of mismatches, similarity and stability. The software determines primer location, orientation, efficiency of binding and calculates primer melting temperatures for standard and degenerate oligonucleotides. The software is suitable for batch file processing, which is essential for automation when working with large amounts of data. The online Java software is available for download at http://primerdigital.com/tools/pcr.html. Accession numbers for the sequences resulting from this study: EU140956 EU177767 EU867815 EU882730 FJ975775-FJ975780 HM481419 HM481420 KC686837-KC686839 KM262797.

(PDF) FastPCR: An in silico tool for fast primer and probe design and advanced sequence analysis. Available from: https://www.researchgate.net/publication/316890940_FastPCR_An_in_silico_tool_for_fast_primer_and_probe_design_and_advanced_sequence_analysis [accessed Dec 25 2018].

Triticain-α is a papain-like cysteine protease from wheat (Triticum aestivum L.) that possesses activity towards toxic gluten-derived peptides, and was thus proposed as a novel therapeutic tool for celiac disease. We report an original approach employing rational design of domain architecture of Triticain-α and selection of the appropriate expression system for development of cheap and efficient protocol yielding active recombinant enzyme. The segregated catalytic domain of Triticain-α did not adopt native structure in bacteria, neither being expressed as a single protein nor upon conjugation or co-expression with extrinsic chaperones. Meanwhile, its attachment to prodomain of the enzyme resulted in generation of insoluble (inclusion bodies) product that can be transformed into active protease upon refolding in vitro. The estimated yield of the product was affected by affinity six-histidine tag required for its single-step purification with the preferable N-terminal position of the tag. Expression of the two-domain Triticain-α construct in yeast (Pichia pastoris) strain GS115 and bacterial (Escherichia coli) strain Rosetta gami B (DE3) led to the accumulation of a soluble protein, which underwent autocatalytic maturation during expression (in yeast)/purification (in bacteria) procedures and exhibited pronounced protease activity. Furthermore, expression and solubility of such construct in Rosetta gami B (DE3) cells was improved by reducing the temperature of the bacterial growth yielding more active enzyme than yeast counterpart presumably due to facilitated formation of a characteristic disulfide bond critical for maintaining the catalytic site. We suggest that these findings are helpful for obtaining active Triticain-α preparations for scientific or medical applications, and can be employed for the design and production of beneficial recombinant products based on other papain-like cysteine proteases.

Bacterial intoxication associated with inflammatory conditions during development can impair brain functions, in particular evolutionarily novel forms of memory, such as explicit learning. Little is known about the dangers of early-life inflammation on more basic forms of learning, for example, the acquisition of motor escape abilities, which are generally better preserved under pathological conditions. To address this limitation in knowledge, an inflammatory response was elicited in Wistar pups by lipopolysaccharide (LPS) injections (25 μg/kg) on postnatal days P15, P18 and P21. The acquisition of escape behaviour was tested from P77 by active avoidance footshock model and water maze. Open-field behaviour and blood corticosterone levels were also measured. Rat brain tissue was collected from pups 2 h post-injection and from adult rats which either underwent escape training on P77-P81 or remained untrained. mRNA levels of developmental brain plasticity factors MMP-9 and TIMP-1 were investigated in the medial prefrontal cortex and ventral/dorsal hippocampus. LPS-challenged rats displayed moderately deficient escape responses in both memory tests, increased freezing behaviour and, surprisingly, reduced blood cortisol levels. Mmp9 and Timp1, and their ratio to one another, were differentially altered in pups versus adult untrained rats but remained unchanged overall in rats trained in either learning task. Together, our data indicate that systemic pro-inflammatory response during early postnatal development has long-lasting effects, including on the acquisition of motor escape abilities and plasticity factor expression, into adulthood. Our data suggest that altered stress response could possibly mediate these deviations and repeated training might generate positive effects on plasticity under the employed conditions.

The presence of human cytomegalovirus (HCMV) and glioblastoma multiforme (GBM), first established in 2002, has developed into an area of considerable interest and controversy. Numerous studies have found evidence of possible HCMV infection of GBM tumor cells as well as myriad onco- and immunomodulatory properties exhibited by HCMV antigens and transcripts, while recent reports have failed to detect HCMV particles in GBM and question the virus’ role in tumor progression. This review highlights the known immunomodulatory properties of HCMV, independent of GBM infection status, that help drive the virus from peripheral blood into the vital tissues and subsequently dampen local immune response, assisting GBM tumors in evading immune surveillance and contributing to the disease’s poor prognosis. Emerging antiviral approaches to treating GBM, including antiviral drugs and immunotherapies directed against HCMV, are also examined.

Aim: To develop a reduced representation bisulfite sequencing (RRBS) approach for rapid and affordable genome-wide DNA methylation analysis. Methods: We have selected restriction endonuclease XmaI to produce RRBS library fragments. After digestion and partial fill-in DNA fragments were ligated to barcoded adapters, bisulfite converted, size-selected, and sequenced on the Ion Torrent Personal Genome Machine. XmaI-RRBS results were compared with the previously published RRBS data. Results: We have developed an XmaI-RRBS method for rapid and affordable genome-wide DNA methylation analysis, with library preparation taking only 4 days and sequencing possible within 4 h. We have also addressed several challenges in order to further improve the RRBS technology. XmaI-RRBS may be performed on degraded DNA samples and is compatible with the bench-top next-generation sequencing machines.

Human mucin MUC1 plays an important role in cancer development. The increased level of this molecule expression during cancer cell progression induces metastasis and is associated with poor prognosis for patients. There is a large body of experimental data on the role of various functional domains of human mucin MUC1 in metastasis. While, the cytoplasmic domain determined to play a definitive role, the influence of extracellular domain on cancer cell invasiveness still remains unclear. The present paper reveals that the extracellular domain of MUC1 molecule consists of two functional subdomains-the region of tandem repeats (TR) and the region of irregular repeats (IR). We demonstrate the ability of each of these subdomains to alter the invasiveness of cancer cells. The presence of the MUC1 molecules containing TR subdomain (MUC1-TR) on the surface of low-invasive cancer cells leads to the increase in their transendothelial migration potency, while the addition of the IR subdomain to the MUC1-TR molecule (MUC1-IR-TR) restores their natural low invasiveness. J. Cell. Biochem. 118: 4002-4011, 2017. © 2017 Wiley Periodicals, Inc.

Despite rapid progress, many problems and limitations persist and limit applicability of gene editing techniques. Making use of meganucleases, TALENs or CRISPR/Cas9-based tools requires an initial step of pre-screening to determine the efficiency and specificity of the designed tools. This step remains time- and material-consuming. Here, we propose a simple, cheap, reliable, time-saving and highly sensitive method to evaluate a given gene editing tool based on its capacity to induce chromosomal translocations when combined with a reference engineered nuclease. In the proposed technique designated as “ENIT” for Engineered Nucleases-Induced Translocations, a plasmid coding for the DNA-editing tool to be tested is co-transfected into carefully chosen target cells along with that for an engineered nuclease of known specificity and efficiency. If the new enzyme efficiently cuts within the desired region, specific chromosomal translocations will be generated between the two targeted genomic regions and be readily detectable by a one-step PCR or qPCR assay. The PCR product thus obtained can be directly sequenced thereby determining the exact position of the double-strand breaks induced by the gene-editing tools. As a proof of concept, ENIT was successfully tested in different cell types and with different meganucleases, TALENs and CRISPR/Cas9-based editing tools.

(PDF) A one-step PCR-based assay to evaluate efficiency and precision of genomic DNA-editing tools. Available from: https://www.researchgate.net/publication/314714362_A_one-step_PCR-based_assay_to_evaluate_efficiency_and_precision_of_genomic_DNA-editing_tools [accessed Dec 25 2018].