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Rationale: The development of eukaryote genome engineering tools based on CRISPR-Cas programmable bacterial nucleases systems opens wide horizons for gene therapies, human disease cell modeling, as well as investigation into manifestation of disease phenotypes and visualization of cellular processes. The safety and approximation of experiments both at the cellular and organismal levels depend on the accuracy of introducing double-stranded breaks into the target DNA regions. The search for new variants of more accurate CRISPR-Cas nucleases and evaluation of their ability to hydrolyze nucleosome DNA in vivo is considered a critical task for the development of the genome engineering technologies.Aim: To analyze the activity of the programmable nuclease AsCpf1 (Cas12a), with low level of off-target activity, in the human genome loci that are safe for the introduction of transgenic constructs (“safe harbor”) and to compare its efficiency with that of the widely used SpCas9 nuclease in HEK293 cells.Materials and methods: We performed the bioinformatics analysis of the association between target regions with nucleosomes and other proteins in the safe harbor loci AAVS1 and GSH-Ch1 and the transcriptionally inactive gene MYBPC3 (cardiac myosin binding protein 3) based on ATAC-seq data for the HEK293FT cells obtained from the NCBI SRA database. Plasmids encoding SpCas9 and AsCpf1 nucleases and guide RNA to the target regions were constructed and transfected into the HEK293FT cells. Events in the target regions of the HEK293FT cell genome were studied in the sequenograms with the TIDE algorithm.Results: The results of the ATAC-seq experiments for HEK293FT cells have shown that the AAVS1 locus can be referred as open chromatin with a low nucleosome density, while the GSH-Ch1 locus can be attributed to closed chromatin. In HEK293FT cells, the cardiac MYBPC3 gene has intermediate chromatin density. Assessment of the efficiency of introducing breaks into the studied HEK293FT cell chromatin loci by nucleases has shown that SpCas9 is able to cope with chromatin of any nucleosome density, while AsCpf1 can effectively introduce DNA breaks only at loci with open chromatin, such as AAVS1 and MYBPC3. Editing events occur at a very low rate at the GSH-Ch1 locus with a high nucleosome density.Conclusion: We have found low efficiency of the AsCpf1 nuclease in the genomic safe harbor locus GSH-Ch1, which is characterized by a high nucleosome density. When planning an experiment on AsCpf1 nuclease genome editing, the epigenetic chromatin landscape and the nucleosome density should be considered, as well as chromatin opening substances should be used.

Relevance. Data on the efficacy of endocrine and chemotherapy regimens in patients with hormone-resistant metastatic breast cancer (mBC) after progression with CDK4/6 inhibitors are limited; the search for an effective therapy regimen in this clinical situation is an urgent task of clinical oncology. Aim. Evaluate the efficacy and safety of eribulin therapy in patients with HR+/HER2- mBC after progression with CDK4/6 inhibitors; compare the results of the Russian study and the EMPOWER observational study in the USA. Materials and methods. The Russian observational study included 54 patients (pts) with HR+/HER2- mBC, who were treated with eribulin after CDK4/6 inhibitors in 24 Russian Cancer hospitals. The median age of pts was 56 years; 75.9% of them had recurrent BC, 24.1% de novo BC stage IV; 51.9% of pts had progression with CDK4/6 inhibitors in the first 6 months of therapy (primary endocrine resistance); 48.1% of patients had progression in the period from 6 to 38 months; 89.1% had visceral site of metastases (liver MTS 65.5%, lung MTS 52.8%, brain MTS in 7.5%). Eribulin was used after anthracyclines and taxanes in 94.4% of cases. The efficacy and safety of eribulin therapy in patients with HR+/HER2- mBC after progression with CDK4/6 inhibitors was studied, as well as subgroup analysis according to age, sites of metastasis, and previously treatment options. Results. Eribulin was prescribed in the standard regimen of 1.4 mg/m2 on days 1 and 8, the interval between cycles was 21 days, the number cyclys of chemotherapy was 144 (median 8, the mean number of cycles 10.5). With a median follow-up of 11.5 months (from 3 to 36 months), 30 patients (55.6%) continue therapy with eribulin at present; therapy was cancelled in 24 patients due to progression in 22 (40.7%) cases, and due to intolerable toxicity in 2 (3.7%) patients. The maximum response to eribulin therapy included partial response (in 11 cases, 24.4%), stable disease (in 30 cases, 66.7%) and progression in 4 (8.9%) patients. Median PFS with eribulin therapy was 10.0 months; the 6-month, 1-year, and 2-year PFS were 79.5%, 44.8% and 26.5%, respectively. Eribulin therapy was equally effective in different subgroups (p0.05) and did not depend on the age of patients, the previously received treatment, the presence of visceral MTS and liver damage. The best response to chemotherapy with eribulin was observed in lung metastases: median PFS 24 months vs 9.1 months, p=0.056. The safety profile was favorable; adverse events were registered in 34.5% of patients, which required dose adjustment in 18.5% of cases. With a median follow-up of 11.5 months, 92.6% of patients remain alive. Conclusion. Eribulin has demonstrated high efficacy and favorable safety profile in hormone-resistant HER2- mBC in patients with progression when receiving CDK4/6 inhibitor.

Acetylene is an effective additive to various rocket fuels. Acetam, an equilibrium highly concentrated solution of acetylene in liquid ammonia, is a promising fuel for use in liquid−propellant jet engines. To gain insights into the processes occurring in such systems and provide means for predicting their characteristics, the self-ignition of model acetylene−oxygen−argon mixtures is experimentally and theoretically studied. The experiments are performed behind reflected shock waves in a temperature range from 1100 to 1870 K at a total gas concentration of [M] ≈ 1.0 × 10−5 mol/cm3, with chemiluminescence signals from electronically excited C2* (λ ≅ 516 nm), CH* (429 nm), CO2*(363 nm), and OH* (308 nm) recorded. Numerical simulations within the framework of various detailed kinetic mechanisms closely reproduce the measured temperature dependences of the ignition delay time for the tested mixtures, but in some cases fail to satisfactorily describe the time profiles of the emission signals from C2*, CH*, CO2*, and OH*. The performed numerical simulations make it possible to explain the observed characteristic features of the emission profiles of electronically excited C2*, CH*, CO2*, and OH* during the self-ignition of C2H2–O2−Ar mixtures. Sensitivity analysis demonstrates that the existence and characteristics of the preignition stage in the CO2* and OH* emission signals are largely determined by the kinetic behavior of intermediate species, such as the ketene molecule and CH2 radical. Based on the results obtained, a new detailed kinetic mechanism has been proposed for describing processes in the combustion products of acetylene-containing propellants, in particular chemiluminescent glow, which are of considerable interest for the development of new technologies for astronautics and aeronautics.

Topoisomerase 1 (TOP1) is an enzyme that regulates DNA topology and is essential for replication, recombination, and other processes. The normal TOP1 catalytic cycle involves the formation of a short-lived covalent complex with the 3′ end of DNA (TOP1 cleavage complex, TOP1cc), which can be stabilized, resulting in cell death. This fact substantiates the effectiveness of anticancer drugs—TOP1 poisons, such as topotecan, that block the relegation of DNA and fix TOP1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is able to eliminate TOP1cc. Thus, TDP1 interferes with the action of topotecan. Poly(ADP-ribose) polymerase 1 (PARP1) is a key regulator of many processes in the cell, such as maintaining the integrity of the genome, regulation of the cell cycle, cell death, and others. PARP1 also controls the repair of TOP1cc. We performed a transcriptomic analysis of wild type and PARP1 knockout HEK293A cells treated with topotecan and TDP1 inhibitor OL9-119 alone and in combination. The largest number of differentially expressed genes (DEGs, about 4000 both up- and down-regulated genes) was found in knockout cells. Topotecan and OL9-119 treatment elicited significantly fewer DEGs in WT cells and negligible DEGs in PARP1-KO cells. A significant part of the changes caused by PARP1-KO affected the synthesis and processing of proteins. Differences under the action of treatment with TOP1 or TDP1 inhibitors alone were found in the signaling pathways for the development of cancer, DNA repair, and the proteasome. The drug combination resulted in DEGs in the ribosome, proteasome, spliceosome, and oxidative phosphorylation pathways.

A problem formulation is proposed for simulating lean premixed hydrogen–air combustion in a closed volume with numerically generated turbulence. Two-dimensional simulations of flame propagation from a small ignition kernel showed that the following sequence of regimes is observed with increasing turbulence intensity and decreasing mixture strength: a corrugated flame with a continuously connected front, a regime with a local loss of front connectivity, and flame extinction through disintegration of a growing kernel into fragments. The transition from steady self-sustained combustion to extinction corresponds to changes in mixture and turbulence parameters leading to a stronger influence of turbulent velocity field on local flame structure. The proposed approach to numerical simulation of the transition regime characterized by loss of front connectivity and fragmentation of the flame under the action of turbulent eddies can be used to evaluate the effect of turbulence intensity on flammability limits.

In experiments using cylindrical and conical shock tubes, the parameters of the shock-wave load transmitted to the rigid wall through the layers of a nonwoven covering made of aramid fibers are determined. The effect of amplifying the amplitude of the pressure wave depending on the layer’s thickness is demonstrated. The developed experimental modeling technique and the results obtained show that a conical shock tube is an effective tool for studying the interaction of spherical shock waves (SWs) with layers of explosion-proof coatings.

Simulations of autoignition of stoichiometric С2Н2/О2 and СН4/О2 mixtures were performed in the temperature and pressure ranges of 800 < T < 2100 K and 0.02 < P < 1.1 MPa using various detailed kinetic mechanisms (DKMs) presented in the literature. The ignition delays predicted by DKMs were compared to choose the optimum value for reduction. The number of species in the DKM was reduced by a trial-and-error method based on an analysis of ignition delay response to the removal of species. The automatic computer code developed in this study made it possible to reduce the number of species more than twofold for C2H2/O2 and threefold for CH4/O2. A comparison of predictions based on the reduced kinetic mechanisms with experimental data presented in the literature showed good agreement for С2Н2/О2 and СН4/О2 mixtures with different argon dilutions.

The paper presents the results of drop tests performed to measure ignition delay for ionic liquids under various experimental conditions. Factors that strongly affect correct determination of ignition delay are indicated. Processes preceding ignition upon contact between various quantities of a fuel and an oxidizer are explained. It is argued that well-defined experimental conditions are required to ensure correct comparison when using benchmark fuels.

An analysis of the results of a three-dimensional numerical simulation of the flow in a conical shock tube (CST) with an angle of 38° revealed that the blast waves formed in such a tube have a pressure profile similar to spherical shock waves (SSWs) with a pronounced rarefaction phase following the compression phase. It is established that the main difference between the flow in conical geometry and the spherical case is related to the change in the flow parameters due to the interaction with the bounding surface(cone wall). The revealed inhomogeneity of the flow manifests itself in the form of pressure pulsations in the rarefaction phase in the absence of a secondary compression phase. It is concluded that a CST is an effective tool for reproducing dynamic loads under a spherical explosion.

Numerical simulations are performed of reaction kinetics in the gaseous products of pyrolysis of polypropylene and waste tires at atmospheric pressure in the temperature range of 800 to 1300 K. The initial conditions are set by using the experimental data available in the literature. The reactions between gaseous pyrolysis products are found to result in a decrease in the mixture’s temperature of polypropylene and a temperature increase for waste tires. The increase in the initial temperature reduces the gaseous product’s conversion for both polypropylene and the waste tires. It is established that the conversion pathways for the C1–C4 alkenes and alkanes strongly depend on the initial temperature and their initial composition.

Mutation in the glucocerebrosidase encoding gene (GBA) is one of the most frequent genetic cause of Parkinson's disease. ICGi034-A induced pluripotent stem cell (iPSC) line obtained by reprogramming peripheral blood mononuclear cells (PBMCs) of a patient with heterozygous c.1226A > G (p.N370S) mutation in the GBA gene can be used for studying the fundamental mechanisms of the pathogenesis of GBA-associated Parkinson's disease, and for potential drug screening. The iPSCs express pluripotency markers (NANOG, SSEA4, TRA-1-60, OCT4, SOX2), have a normal karyotype, and are capable of producing derivatives of three germ layers.

Oxidative stress plays an important role in the development of neurodegenerative diseases, being either the initiator or part of a pathological cascade that leads to the neuron's death. Genetically encoded biosensors of oxidative stress demonstrated their general functionality and overall safety in various systems. However, there is still insufficient data regarding their use in the research of disease-related phenotypes in relevant model systems, such as human cells. Here, we establish an approach for monitoring the redox state of live motor neurons with SOD1 mutations associated with amyotrophic lateral sclerosis. Using CRISPR/Cas9, we insert genetically encoded biosensors of cytoplasmic and mitochondrial H

BackgroundOxidative stress plays an important role in the development of neurodegenerative diseases: it either can be the initiator or part of a pathological cascade leading to the neuron’s death. Although a lot of methods are known for oxidative stress study, most of them operate on non-native cellular substrates or interfere with the cell functioning. Genetically encoded (GE) biosensors of oxidative stress demonstrated their general functionality and overall safety in various live systems. However, there is still insufficient data regarding their use for research of disease-related phenotypes in relevant model systems, such as human cells.MethodsWe applied CRISPR/Cas9 genome editing to introduce mutations (c.272A>C and c.382G>C) in the associated with amyotrophic lateral sclerosis SOD1 gene of induced pluripotent stem cells (iPSC) obtained from a healthy individual. Using CRISPR/Cas9, we modified these mutant iPSC lines, as well as the parental iPSC line, and a patient-specific SOD1D91A/D91A iPSC line with ratiometric GE biosensors of cytoplasmic (Cyto-roGFP2-Orp1) and mitochondrial (Mito-roGFP2-Orp1) H2O2. The biosensors sequences along with a specific transactivator for doxycycline-controllable expression were inserted in the “safe harbor” AAVS1 (adeno-associated virus site 1) locus. We differentiated these transgenic iPSCs into motor neurons and investigated the functionality of the biosensors in such a system. We measured relative oxidation in the cultured motor neurons and its dependence on culture conditions, age, and genotype, as well as kinetics of H2O2 elimination in real-time.ResultsWe developed a cell-based platform consisting of isogenic iPSC lines with different genotypes associated with amyotrophic lateral sclerosis. The iPSC lines were modified with GE biosensors of cytoplasmic and mitochondrial H2O2. We provide proof-of-principle data showing that this approach may be suitable for monitoring oxidative stress in cell models of various neurodegenerative diseases as the biosensors reflect the redox state of neurons.ConclusionWe found that the GE biosensors inserted in the AAVS1 locus remain functional in motor neurons and reflect pathological features of mutant motor neurons, although the readout largely depends on the severity of the mutation.

The development of cell models of human diseases based on induced pluripotent stem cells (iPSCs) and a cell therapy approach based on differentiated iPSC derivatives has provided a powerful stimulus in modern biomedical research development. Moreover, it led to the creation of personalized regenerative medicine. Due to this, in the last decade, the pathological mechanisms of many monogenic diseases at the cell level have been revealed, and clinical trials of various cell products derived from iPSCs have begun. However, it is necessary to reach a qualitatively new level of research with cell models of diseases based on iPSCs for more efficient searching and testing of drugs. Biosensor technology has a great application prospect together with iPSCs. Biosensors enable researchers to monitor ions, molecules, enzyme activities, and channel conformation in live cells and use them in live imaging and drug screening. These probes facilitate the measurement of steady-state concentrations or activity levels and the observation and quantification of in vivo flux and kinetics. Real-time monitoring of drug action in a specific cellular compartment, organ, or tissue type; the ability to screen at the single-cell resolution; and the elimination of the false-positive results caused by low drug bioavailability that is not detected by in vitro testing methods are a few of the benefits of using biosensors in drug screening. Here, we discuss the possibilities of using biosensor technology in combination with cell models based on human iPSCs and gene editing systems. Furthermore, we focus on the current achievements and problems of using these methods.

Usnic acid (UA) is a secondary metabolite of lichens that exhibits a wide range of biological activities. Previously, we found that UA derivatives are effective inhibitors of tyrosyl-DNA phosphodiesterase 1 (TDP1). It can remove covalent complex DNA-topoisomerase 1 (TOP1) stabilized by the TOP1 inhibitor topotecan, neutralizing the effect of the drugs. TDP1 removes damage at the 3′ end of DNA caused by other anticancer agents. Thus, TDP1 is a promising therapeutic target for the development of drug combinations with topotecan, as well as other drugs for cancer treatment. Ten new UA enamino derivatives with variation in the terpene fragment and substituent of the UA backbone were synthesized and tested as TDP1 inhibitors. Four compounds, 11a-d, had IC50 values in the 0.23–0.40 μM range. Molecular modelling showed that 11a-d, with relatively short aliphatic chains, fit to the important binding domains. The intrinsic cytotoxicity of 11a-d was tested on two human cell lines. The compounds had low cytotoxicity with CC50 ≥ 60 μM for both cell lines. 11a and 11c had high inhibition efficacy and low cytotoxicity, and they enhanced topotecan’s cytotoxicity in cancerous HeLa cells but reduced it in the non-cancerous HEK293A cells. This “protective” effect from topotecan on non-cancerous cells requires further investigation.

In the paper, the propagation of the detonation wave in the hydrogen-oxygen microfoam on the aqueous base is considered. Microfoam represents a two-phase system containing micron-sized gas bubbles filled with the hydrogen-oxygen mixture. These bubbles are dispersed in the water solution of surfactant (sodium dodecyl sulfate). The dependencies of detonation speed on the equivalence ratio and on the water content in the foam are obtained with the use of high-speed filming. It is found that the detonation speed slightly increases with the decrease in water content in the foam. Based on the pressure measurements, it is established that the detonation propagation in the foam is driven by relatively weak shock waves, which by themselves are not able to induce ignition of the hydrogen-oxygen mixture. To substantiate the fact of detonation existence in the microfoam a hypothesis of the cumulative collapse of gas bubbles under the shock wave action is proposed. The estimation for detonation speed in microfoam is analytically derived on the basis of simple phenomenological representations.

Interferon-regulated factors play a central role in the activation of the innate immune response. The interferon-regulatory factor 7 (IRF7) is one of the factors that are quickly activated and are involved in a cellular response to a viral infection. In this work, monoclonal lines, based on HEK293FT cells defective in the IRF7 gene, were obtained using a CRISPR-Cas9 genome-editing method. These lines differed in the viability, proliferation rate, and susceptibility to infection with influenza A virus. Transcriptomic analysis of the most susceptible cell clone revealed differential expression of IRF7 factor as well as the other interferon-regulated genes.

Generation of patient-specific induced pluripotent stem cells (iPSCs) and their subsequent differentiation into cardiomyocytes opened new opportunities for studying pathogenesis of inherited cardiovascular diseases. One of these diseases is hypertrophic cardiomyopathy (HCM) for which no efficient therapy methods have been developed so far. In this study, the approach based on patient-specific iPSCs was applied to create a model of the disease. Genetic analysis of a hypertrophic cardiomyopathy patient revealed R326Q mutation in the MYBPC3 gene. iPSCs of the patient were generated and characterized. The cells were differentiated into cardiomyocytes together with the control iPSCs from a healthy donor. The patient's iPSC-derived cardiomyocytes exhibited early HCM features, such as abnormal calcium handling and increased intracellular calcium concentration. Therefore, cardiomyocytes obtained by directed differentiation of iPSCs from the HCM patient can be used as a model system to study HCM pathogenesis.

Genetic analysis plays an important role in diagnostics of cardiovascular diseases. One of the diseases is long QT syndrome that results in an increased risk of ventricular tachycardia and sudden cardiac death. The syndrome may be caused by mutations in genes responsible for cardiomyocyte ionic channel functioning. The aim of this study is to examine genetics of long QT syndrome. Genetic analysis of 16 patients with long QT syndrome or suspicion of the syndrome was carried out. Long QT syndrome causing mutations, p.Ala178Pro, p.Val254Met, p.Gly325Arg in KCNQ1 and p.Thr613Met in KCNH2, and a long QT syndrome-associated polymorphism, p.Asp85Asn in KCNE1, were found in five patients. Family analysis of p.Ala178Pro and p.Val254Met mutations in KCNQ1 revealed the mutations carriers that had not demonstrated any syndrome manifestations before. In addition, a mutation, p.Gly604Ala in KCNH2, was found. The mutation has not been previously described and its role in long QT syndrome needs to be clarified.

ICGi021-A and ICGi022-A iPSC lines were obtained by reprogramming PBMCs of two healthy women of the Siberian population using episomal non-integrating vectors expressing Yamanaka factors. iPSC lines expressed pluripotency markers, had a normal karyotype and demonstrated the ability to differentiate into derivatives of the three germ layers. Clinical exome sequencing data of the original biosamples of the donors are available in the NCBI SRA database. The generated cell lines are useful as "healthy" control in biomedical studies.

Metabolomic profiles of somatic cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) reflect their metabolic phenotypes. The comparative study of metabolomes of these cells is important for understanding the differences in metabolism between somatic and pluripotent cells, and also the possible differences between ESCs and iPSCs. Here, we performed for the first time the metabolomic analysis of rat ESCs, iPSCs, and embryonic fibroblasts (EFs) at both quantitative and semi-quantitative levels using NMR spectroscopy and liquid chromatography with mass spectrometric detection, respectively. The total of 106 metabolites has been identified, and the concentrations of 51 compounds have been measured. It is found that the reprogramming of rat EFs into iPSCs affects virtually all metabolic pathways and causes drastic changes in the cell metabolomic profile. The difference between ESCs and iPSCs is much less pronounced: the concentrations of the majority of metabolites in ESCs and iPSCs are similar, and significant differences were observed for only several compounds, including adenosine, cysteic acid, glycerophosphoglycerol, inositol phosphate, glucose, myo-inositol, phosphoserine, xanthosine, guanosine. The observed differences between the metabolomic compositions of ESCs and iPSCs do not influence the pluripotent ability of iPSCs. Graphical Abstract.

Two novel structural types of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors with hexahydroisobenzofuran 11 and 3-oxabicyclo [3.3.1]nonane 12 scaffolds were discovered. These monoterpene-derived compounds were synthesized through preliminary isomerization of (+)-3-carene to (+)-2-carene followed by reaction with heteroaromatic aldehydes. All the compounds inhibit the TDP1 enzyme at micro- and submicromolar levels, with the most potent compound having an IC50 value of 0.65 &mu, M. TDP1 is an important DNA repair enzyme and a promising target for the development of new chemosensitizing agents. A panel of isogenic clones of the HEK293FT cell line knockout for the TDP1 gene was created using the CRISPR-Cas9 system. Cytotoxic effects of topotecan (Tpc) and non-cytotoxic compounds of the new structures were investigated separately and jointly in the TDP1 gene knockout cells. For two TDP1 inhibitors, 11h and 12k, a synergistic effect was observed with Tpc in the HEK293FT cells but was not found in TDP1 &minus, /&minus, cells. Thus, it is likely that the synergistic effect is caused by inhibition of TDP1. Synergy was also found for 11h in other cancer cell lines. Thus, sensitizing cancer cells using a non-cytotoxic drug can enhance the efficacy of currently used pharmaceuticals and, concomitantly, reduce toxic side effects.

The in vitro cellular models are promising tools for studying normal and pathological conditions. One of their important applications is the development of genetically engineered biosensor systems to investigate the processes occurring in living cells in real time. Today, there are fluorescence protein based sensory systems for detecting various substances in living cells (for example, hydrogen peroxide, ATP, Ca2+ etc.) or for detecting processes such as endoplasmic reticulum stress. Such systems help to study mechanisms underlying the pathogenic processes and diseases and for screening potential therapeutic compounds. It is also necessary to develop new tools for processing and analysis of obtained microimages. Here we present our web-application CellCountCV for automation of microscopy cell images analysis which is based on fully-convolutional deep neural networks. This approach can efficiently deal with non-convex overlapping objects, that are virtually inseparable with conventional image processing methods. The cell counts predicted with CellCountCV were very close to expert estimates (the average error rate was < 4%). CellCountCV was used to analyse large series of microscopy images obtained in experimental studies and it was able to demonstrate the endoplasmic reticulum stress development and to catch the dose-dependent effect of tunicamycin.

Self-renewal of cultured pluripotent stem cells is a complex process, which includes multiple functional and regulatory levels. Transcription factors, their target genes, chromatin modifiers, signaling pathways, and regulatory noncoding RNAs are involved in the maintaining of self-renewal. Studies of molecular and genetic bases of maintaining self-renewal and pluripotency in cultured mammalian cells are important to understand processes in preimplantation embryogenesis and to develop efficient techniques to obtain pluripotent stem cell lines for experimental biology and medicine. MicroRNAs (miRNAs) play an important role in pluripotency maintaining and reprogramming. However, involvement of this class of noncoding RNAs and functions of individual molecules are poorly studied. The goal of this study was the search for the miRNAs potentially involved in the pluripotency maintaining and reprogramming of Rattus norvegicus cells. We analyzed the expression of miRNAs in rat embryonic stem cells, induced pluripotent stem cells and embryonic fibroblasts using bioinformatic methods and data obtained with next generation sequencing. The analysis of differential expression between groups of rat pluripotent cells and fibroblasts, and the analysis of experimentally confirmed target genes of differentially expressed known rat miRNAs revealed novel potential players of pluripotency maintaining and reprogramming processes. In addition, novel members of these processes were revealed among novel rat miRNAs. The use of bioinformatic and systems biology approaches is the first step, which is necessary for choosing candidates for the subsequent experimental studies. The results obtained substantially improve our understanding of the self-renewal regulation system of the laboratory rat, a popular biomedical object, and our knowledge about the system in mammals.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) catalyzes the cleavage of the phosphodiester bond between the tyrosine residue of topoisomerase 1 (TOP1) and the 3′ phosphate of DNA in the single-strand break generated by TOP1. TDP1 promotes the cleavage of the stable DNA–TOP1 complexes with the TOP1 inhibitor topotecan, which is a clinically used anticancer drug. This article reports the synthesis and study of usnic acid thioether and sulfoxide derivatives that efficiently suppress TDP1 activity, with IC50 values in the 1.4–25.2 μM range. The structure of the heterocyclic substituent introduced into the dibenzofuran core affects the TDP1 inhibitory efficiency of the compounds. A five-membered heterocyclic fragment was shown to be most pharmacophoric among the others. Sulfoxide derivatives were less cytotoxic than their thioester analogs. We observed an uncompetitive type of inhibition for the four most effective inhibitors of TDP1. The anticancer effect of TOP1 inhibitors can be enhanced by the simultaneous inhibition of PARP1, TDP1, and TDP2. Some of the compounds inhibited not only TDP1 but also TDP2 and/or PARP1, but at significantly higher concentration ranges than TDP1. Leader compound 10a showed promising synergy on HeLa cells in conjunction with the TOP1 inhibitor topotecan.

The major human apurinic/apyrimidinic (AP) site endonuclease, APEX1, is a central player in the base excision DNA repair (BER) pathway and has a role in the regulation of DNA binding by transcription factors. In vertebrates, APEX1 knockouts are embryonic lethal, and only a handful of knockout cell lines are known. To facilitate studies of multiple functions of this protein in human cells, we have used the CRISPR/Cas9 system to knock out theAPEX1gene in a widely used non-cancer hypotriploid HEK 293FT cell line. Two stable knockout lines were obtained, one carrying two single-base deletion alleles and one single-base insertion allele in exon 3, another homozygous in the single-base insertion allele. Both mutations cause a frameshift that leads to premature translation termination before the start of the protein’s catalytic domain. Both cell lines totally lacked the APEX1 protein and AP site-cleaving activity, and showed significantly lower levels of theAPEX1transcript. The APEX1-null cells were unable to support BER on uracil- or AP site-containing substrates. Phenotypically, they showed a moderately increased sensitivity to methyl methanesulfonate (MMS; ~2-fold lower EC50compared with wild-type cells), and their background level of natural AP sites detected by the aldehyde-reactive probe was elevated ~1.5–2-fold. However, the knockout lines retained a nearly wild-type sensitivity to oxidizing agents hydrogen peroxide and potassium bromate. Interestingly, despite the increased MMS cytotoxicity, we observed no additional increase in AP sites in knockout cells upon MMS treatment, which could indicate their conversion into more toxic products in the absence of repair. Overall, the relatively mild cell phenotype in the absence of APEX1-dependent BER suggests that mammalian cells possess mechanisms of tolerance or alternative repair of AP sites. The knockout derivatives of the extensively characterized HEK 293FT cell line may provide a valuable tool for studies of APEX1 in DNA repair and beyond.

In vole Microtus levis, cells of preimplantation embryo and extraembryonic tissues undergo imprinted X chromosome inactivation (iXCI) which is triggered by a long non-coding nuclear RNA, Xist. At early stages of iXCI, chromatin of vole inactive X chromosome is enriched with the HP1 heterochromatin-specific protein, trimethylated H3K9 and H4K20 attributable to constitutive heterochromatin. In the study, using vole trophoblast stem (TS) cells as a model of iXCI, we further investigated chromatin of the inactive X chromosome of M. levis and tried to find out the role of Xist RNA. We demonstrated that chromatin of the inactive X chromosome in vole TS cells also contained the SETDB1 histone methyltransferase and KAP1 protein. In addition, we observed that Xist RNA did not contribute significantly to maintenance of X chromosome inactive state during iXCI in vole TS cells. Xist repression affected neither transcriptional silencing caused by iXCI nor maintenance of trimethylated H3K9 and H4K20 as well as HP1, KAP1, and SETDB1 on the inactive X chromosome. Moreover, the unique repertoire of chromatin modifications on the inactive X chromosome in vole TS cells could be disrupted by a chemical compound, DZNep, and then restored even in the absence of Xist RNA. However, Xist transcript was necessary for recruitment of an additional repressive histone modification, trimethylated H3K27, to the inactive X chromosome during vole TS cell differentiation.

Pluripotent stem cells have great potential for developmental biology and regenerative medicine. Embryonic stem cells, which are obtained from blastocysts, and induced pluripotent stem cells, which are generated by the reprogramming of somatic cells, are two main types of pluripotent cells. It is important to understand the regulatory network that controls the pluripotency state and reprogramming process. Various types of noncoding RNAs (ncRNAs) have emerged as substantial components of regulatory networks. The most studied class of ncRNAs in the context of pluripotency and reprogramming is microRNAs (miRNAs). In addition to canonical microRNAs, other types of small RNAs with miRNA-like function are expressed in PSCs. Another class of ncRNAs, long ncRNAs, are also involved in pluripotency and reprogramming regulation. Thousands of ncRNAs have been annotated to date, and a significant number of the molecules do not have known function. In this review, we briefly summarized recent advances in this field and described existing genome-editing approaches to study ncRNA functions.

Nowadays we approached the turn, when the molecular genetics and the cell biology with its extensive baggage of methods and data, allow us to work with information about nucleotide sequences of whole genomes, to edit the nucleotide sequence of the genomes of laboratory animals and cultured human cells and also explore functions and interactions of genetic elements in health and in disease. The use of these instruments opens up huge possibilities for the study of severe human genetic abnormalities. In various laboratories around world an extensive work is carried out in this area by searching links between genetic elements and diseases, using the latest technology of genome editing and reprogramming somatic mature cells to a pluripotent stem condition. The most progressively developing area of research is the study of neurodegenerative diseases. In this review we discussed about possibilities and problems of using new techniques and instruments of cell biology, genetics and genomics in studying molecular and genetic basis of the pathogenesis of Parkinson's disease.

The development of new revolutionary technologies for directed gene editing has made it possible to thoroughly model and study NgAgo human diseases at the cellular and molecular levels. Gene editing tools like ZFN, TALEN, CRISPR-based systems, NgAgo and SGN can introduce different modifications. In gene sequences and regulate gene expression in different types of cells including induced pluripotent stem cells (iPSCs). These tools can be successfully used for Huntington’s disease (HD) modeling, for example, to generate isogenic cell lines bearing different numbers of CAG repeats or to correct the mutation causing the disease. This review presents common genome editing technologies and summarizes the progress made in using them in HD and other hereditary diseases. Furthermore, we will discuss prospects and limitations of genome editing in understanding HD pathology.

The induced pluripotent stem cell (iPSC) lines ICGi008-A and ICGi008-B were generated from dermal fibroblasts using episomal vectors expressing pluripotency factors. Dermal fibroblasts were obtained from a 55 year old male Сaucasian familial Alzheimer's disease (AD) patient carrying heterozygous V717I mutation in the APP gene. The generated iPSC lines maintained the original APP genotype, expressed pluripotency markers, exhibited a normal karyotype and retained the ability to differentiate into cell types of the three germ layers. The iPSC lines will be useful for the study of the AD molecular and cellular mechanisms and drug screening.

The self-ignition of a stoichiometric acetone−oxygen mixture diluted with argon was studied both experimentally and numerically. The experiments were performed behind reflected shock waves over a temperature range from 1280 to 1810 K at a total gas concentration of [M]50 ≈ 10−5 mol/cm3. The process was monitored by recording the signals of absorption by methyl radicals (λ = 216.5 nm) and emission from electronically excited OH* radicals (λ = 308 nm) and CO2* molecules (λ = 370 nm). Numerical simulations within the framework of various detailed kinetic mechanisms were performed to reproduce our own and published experimental data on the pyrolysis and self-ignition of acetone behind reflected shock waves, including the concentration profiles of acetone and CH3 in the ground state and electronically excited OH* and CO2*, as well as the temperature dependence of the self-ignition delay time. It has been established that various detailed kinetic mechanisms describe the kinetic characteristics of the pyrolysis of acetone in shock waves with varying degrees of accuracy. At the same time, all of them predicted the measured ignition delays within a factor of two. A sensitivity analysis showed that the reactions determining the pyrolysis of acetone produce only a slight effect on the branched-chain ignition process, so that the relevant rate constants only slightly affect the ignition delay time.

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by deletion or mutation in SMN1 gene. SMA human induced pluripotent stem cells (iPSCs) represent a useful and valid model for the study of the disorder, as they provide in vitro the target cells. We generated iPSCs from a SMA type I patient and SMA type II patient by using non-integrating episomal plasmid vectors. The resulting iPSCs are episomal-free, express pluripotency markers, display a normal karyotype, retain the mutation (homozygous deletion of SMN1) and are able to differentiate into the three germ layers.

The values of ignition delay predicted for stoichiometric C2H2/O2 and CH4/O2 mixtures by various detailed kinetic mechanisms presented in the literature are compared over the temperature range of 1000 < T <2100 K and the pressure range of 0.02 < P < 1.1 MPa. The number of species in detailed kinetic mechanisms is reduced by a trial-and-error method while keeping the response of ignition delay within a relative error range of 5 = 30%. The automatic computer code developed in this study has made it possible to reduce the number of species by a factor of more than 2 for the C2H2/O2 mixture and a factor of 3 for the CH4/O2 mixture, respectively. A comparison of predictions based on the reduced mechanisms with experimental data presented in the literature shows good agreement.

One of the reasons for the death of motor neurons of the brain and spinal cord in patients with amyotrophic lateral sclerosis is known to be formation of subcellular protein aggregates that are caused by mutations in the SOD1 gene. Patient survival time was earlier shown to have limiting correlation with thermostability change of SOD1 mutant forms of patients’ carriers. We hypothesized that aggregation of mutant SOD1 may occur not only due to the protein destabilization, but through formation of novel interatomic bonds which stabilize “pathogenic” conformations of the mutant as well. To estimate these effects in the present paper, we performed statistical analysis of occupancy of intramolecular hydrogen bonds, hydrogen bonds between the protein and water molecules, and water bridges with use of molecular dynamics simulation for 38 mutant SOD1 forms. Multiple regression model based on these kinds of bonds demonstrated correlation with patient survival time significantly better (R = .9, p-value