Influence of human peripheral blood samples preprocessing on the quality of Hi-C libraries.

The genome-wide variant of the chromatin conformation capture technique (Hi-C) is a powerful tool for revealing patterns of genome spatial organization, as well as for understanding the effects of their disturbance on disease development. In addition, Hi-C can be used to detect chromosomal rearrangements, including balanced translocations and inversions. The use of the Hi-C method for the detection of chromosomal rearrangements is becoming more widespread. Modern high-throughput methods of genome analysis can effectively reveal point mutations and unbalanced chromosomal rearrangements. However, their sensitivity for determining translocations and inversions remains rather low. The storage of whole blood samples can affect the amount and integrity of genomic DNA, and it can distort the results of subsequent analyses if the storage was not under proper conditions. The Hi-C method is extremely demanding on the input material. The necessary condition for successfully applying Hi-C and obtaining high-quality data is the preservation of the spatial chromatin organization within the nucleus. The purpose of this study was to determine the optimal storage conditions of blood samples for subsequent Hi-C analysis. We selected 10 different conditions for blood storage and sample processing. For each condition, we prepared and sequenced Hi-C libraries. The quality of the obtained data was compared. As a result of the work, we formulated the requirements for the storage and processing of samples to obtain high-quality Hi-C data. We have established the minimum volume of blood sufficient for conducting Hi-C analysis. In addition, we have identified the most suitable methods for isolation of peripheral blood mononuclear cells and their long-term storage. The main requirement we have formulated is not to freeze whole blood.

Hi-C analysis of genomic contacts revealed karyotype abnormalities in chicken HD3 cell line.

BACKGROUND: Karyotype abnormalities are frequent in immortalized continuous cell lines either transformed or derived from primary tumors. Chromosomal rearrangements can cause dramatic changes in gene expression and affect cellular phenotype and behavior during in vitro culture. Structural variations of chromosomes in many continuous mammalian cell lines are well documented, but chromosome aberrations in cell lines from other vertebrate models often remain understudied. The chicken LSCC-HD3 cell line (HD3), generated from erythroid precursors, was used as an avian model for erythroid differentiation and lineage-specific gene expression. However, karyotype abnormalities in the HD3 cell line were not assessed. In the present study, we applied high-throughput chromosome conformation capture to analyze 3D genome organization and to detect chromosome rearrangements in the HD3 cell line. RESULTS: We obtained Hi-C maps of genomic interactions for the HD3 cell line and compared A/B compartments and topologically associating domains between HD3 and several other cell types. By analysis of contact patterns in the Hi-C maps of HD3 cells, we identified more than 25 interchromosomal translocations of regions ≥ 200 kb on both micro- and macrochromosomes. We classified most of the observed translocations as unbalanced, leading to the formation of heteromorphic chromosomes. In many cases of microchromosome rearrangements, an entire microchromosome together with other macro- and microchromosomes participated in the emergence of a derivative chromosome, resembling "chromosomal fusions'' between acrocentric microchromosomes. Intrachromosomal inversions, deletions and duplications were also detected in HD3 cells. Several of the identified simple and complex chromosomal rearrangements, such as between GGA2 and GGA1qter; GGA5, GGA4p and GGA7p; GGA4q, GGA6 and GGA19; and duplication of the sex chromosome GGAW, were confirmed by FISH. CONCLUSIONS: In the erythroid progenitor HD3 cell line, in contrast to mature and immature erythrocytes, the genome is organized into distinct topologically associating domains. The HD3 cell line has a severely rearranged karyotype with most of the chromosomes engaged in translocations and can be used in studies of genome structure-function relationships. Hi-C proved to be a reliable tool for simultaneous assessment of the spatial genome organization and chromosomal aberrations in karyotypes of birds with a large number of microchromosomes.

Comparison and critical assessment of single-cell Hi-C protocols.

Advances in single-cell sequencing technologies make it possible to study the genome architecture in single cells. The rapid growth of the field has been fueled by the development of innovative single-cell Hi-C protocols. However, the protocols vary considerably in their efficiency, bias, scale and costs, and their relative advantages for different applications are unclear. Here, we compare the two most commonly used single-cell Hi-C protocols. We use long-read sequencing to analyze molecular products of the Hi-C assay and show that whole-genome amplification step results in increased number of artifacts, larger coverage biases, and increased amount of noise compared to PCR-based amplification. Our comparison provides guidance for researchers studying chromatin architecture in individual cells.

FastContext: A tool for identification of adapters and other sequence patterns in next generation sequencing (NGS) data.

The development of next generation sequencing (NGS) methods has created the need for detailed analysis and control of each protocol step. NGS library preparation protocols may include steps with incorporation of various service sequences, such as sequencing adapters, primers, sample-, cell-, and molecule-specific barcodes. Despite a fairly high level of current knowledge, during the protocol development process researches often have to deal with various kinds of unexpected experiment outcomes, which result either from lack of information, lack of knowledge, or defects in reagent manufacturing. Detection and analysis of service sequences, their distribution and linkage may provide important information for protocol optimization. Here we introduce FastContext, a tool designed to analyze NGS read structure, based on sequence features found in reads, and their relative position in the read. The algorithm is able to create human readable read structures with user-specified patterns, to calculate counts and percentage of every read structure. Despite the simplicity of the algorithm, FastContext may be useful in read structure analysis and, as a result, can help better understand molecular processes that take place at different stages of NGS library preparation. The project is open-source software, distributed under GNU GPL v3, entirely written in the programming language Python, and based on well-maintained packages and commonly used data formats. Thus, it is cross-platform, may be patched or upgraded by the user if necessary. The FastContext package is available at the Python Package Index (https://pypi.org/project/FastContext), the source code is available at GitHub (https://github.com/regnveig/FastContext).

Here and there: the double-side transgene localization.

Random transgene integration is a powerful tool for developing new genome-wide screening approaches. These techniques have already been used for functional gene annotation by transposon-insertion sequencing, for identif ication of transcription factor binding sites and regulatory sequences, and for dissecting chromatin position effects. Precise localization of transgenes and accurate artifact f iltration are essential for this type of method. To date, many mapping assays have been developed, including Inverse-PCR, TLA, LAM-PCR, and splinkerette PCR. However, none of them is able to ensure localization of both transgene's f lanking regions simultaneously, which would be necessary for some applications. Here we proposed a cheap and simple NGS-based approach that overcomes this limitation. The developed assay requires using intentionally designed vectors that lack recognition sites of one or a set of restriction enzymes used for DNA fragmentation. By looping and sequencing these DNA fragments, we obtain special data that allows us to link the two f lanking regions of the transposon. This can be useful for precise insertion mapping and for screening approaches in the f ield of chromosome engineering, where chromosomal recombination events between transgenes occur in a cell population. To demonstrate the method's feasibility, we applied it for mapping SB transposon integration in the human HAP1 cell line. Our technique allowed us to eff iciently localize genomic transposon integrations, which was conf irmed via PCR analysis. For practical application of this approach, we proposed a set of recommendations and a normalization strategy. The developed method can be used for multiplex transgene localization and detection of rearrangements between them.

C-InterSecture-a computational tool for interspecies comparison of genome architecture.

MOTIVATION: Recent development of Hi-C technique, a biochemical method to study 3D genome architecture, provided large amount of information describing spatial organization of chromosomes in different cell types and species. While multiple tools are available for analysis and comparison of Hi-C data of different cell types, there are almost no resources for systematic interspecies comparison. RESULTS: To fill this gap, we developed C-InterSecture, a computational pipeline allowing systematic comparison of genome architecture between species. C-InterSecture allows statistical comparison of contact frequencies of individual pairs of loci, as well as interspecies comparison of contacts pattern within defined genomic regions, i.e. topologically associated domains. We employed C-InterSecture to compare mammalian and avian genome organization and showed how evolutionary changes of genomic distance affect 3D architecture of vertebrate's genome. AVAILABILITY AND IMPLEMENTATION: C-InterSecture is implemented as a collection of python scripts freely available on GitHub repository at https://github.com/NuriddinovMA/C-InterSecture. Jucebox-compatible .hic files produced by C-InterSecture are available at http://genedev.bionet.nsc.ru/site/CIntersecture.html. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Interpreting Chromosomal Rearrangements in the Context of 3-Dimentional Genome Organization: A Practical Guide for Medical Genetics.

In this exciting era of "next-gen cytogenetics", the use of novel molecular methods such as comparative genome hybridization and whole genome and whole exome sequencing becomes more and more common in clinics. This results in generation of large amounts of high-resolution patient-specific data and challenges the development of new approaches for interpretation of obtained information. Usually, interpretation of chromosomal rearrangements is focused on alterations of linear genome sequence, underestimating the role of spatial chromatin organization. In this article, we describe the main features of 3-dimentional genome organization, emphasizing their role in normal and pathological development. We highlight some tips to help physicians estimating the impact of chromosomal rearrangements on the patient phenotype. A separate section describes available tools that can be used to visualize and analyze human genome architecture.

Rational design and studies of excimer forming novel dual probes to target RNA.

In this paper, we report structure-based rational design and physico-chemical and biological studies of novel pyrene excimer forming dual probes for visualization of intracellular RNAs. Herein, the probes based on 2'-O-methyl RNA with linkers of different structure and length between pyrene moiety and ribose are studied with respect to their hybridization and spectral properties. We found optimal linkers that provide more intense excimer emission (at ∼480nm) of RNA-bound probes; particularly, the length of the linker arm of the 3'-component of dual probes plays a key role in formation of pyrene excimer. Calculated molecular dynamics trajectories and probability distributions of pyrene-pyrene dimer formation upon hybridization of the dual probes with RNA target are in agreement with the obtained fluorescence spectroscopy data for the corresponding duplexes. Our study demonstrates the excellent binding properties of new dual probes to structured RNA and their feasibility for the visualization of intracellular RNA targets.

Cell divisions are not essential for the direct conversion of fibroblasts into neuronal cells.

Direct lineage conversion is a promising approach for disease modeling and regenerative medicine. Cell divisions play a key role in reprogramming of somatic cells to pluripotency, however their role in direct lineage conversion is not clear. Here we used transdifferentiation of fibroblasts into neuronal cells by forced expression of defined transcription factors as a model system to study the role of cellular division in the direct conversion process. We have shown that conversion occurs in the presence of the cell cycle inhibitors aphidicolin or mimosine. Moreover, overexpression of the cell cycle activator cMyc negatively influences the process of direct conversion. Overall, our results suggest that cell divisions are not essential for the direct conversion of fibroblasts into neuronal cells.

[Reprogramming of somatic cells. Problems and solutions].

An adult mammal is composed of more than 200 different types of specialized somatic cells whose differentiated state remains stable over the life of the organism. For a long time it was believed that the differentiation process is irreversible, and the transition between the two types of specialized cells is impossible. The possibility of direct conversion of one differentiated cell type to another was first shown in the 80s of the last century in experiments on the conversion of fibroblasts into myoblasts by ectopic expression of the transcription factor MyoD. Surprisingly, this technology has remained unclaimed in cell biology for a long time. Interest in it revived after 200 thanks to the research of Novel Prize winner Shinya Yamanaka who has shown that a small set of transcription factors (Oct4, Sox2, Klf4 and c-Myc) is capable of restoring pluripotency in somatic cells which they lost in the process of differentiation. In 2010, using a similar strategy and the tissue-specific transcription factors Vierbuchen and coauthors showed the possibility of direct conversion of fibroblasts into neurons, i. e. the possibility of transdifferentiation of one type of somatic cells in the other. The works of these authoras were a breakthrough in the field of cell biology and gave a powerful impulse to the development of cell technologies for the needs of regenerative medicine. The present review discusses the main historical discoveries that preceded this work, evaluates the status of the problem and the progress in the development of methods for reprogramming at the moment, describes the main approaches to solving the problems of reprogramming of somatic cells into neuronal, and briefly discusses the prospect of application of reprogramming and transdifferentiation of cells for such important application areas as regenerative medicine, cell replacement therapy and drug screening.

Interlaboratory comparison of the determination of chlorinated dibenzo-p-dioxins and dibenzofurans according to regulatory methods EN 1948 and EPA 1613b.

Four laboratories participated in a collaborative study to determine differences in analytical results generated according to two different compliance methods, US EPA Method 1613b and European Union Method EN 1948 for the determination of chlorinated dibenzo-p-dioxins and dibenzofurans (CDD/CDFs). Various sample matrices containing the analytes at levels ranging from parts-per-quadrillion (ppq) to parts-per-billion (ppb) were used to illustrate differences and similarities between the two analytical methods. The choice of the sample matrices analyzed in this study was made to mirror many of the real-world samples that are of interest to Dow and also to test the laboratories on many different, complex matrices. For this reason, commercially available performance evaluation samples were not used. The study results indicate that the 1613b requirement for confirmation of analyte identity and concentration on a second, polar gas chromatographic column for 2378-tetrachlorodibenzofuran (TCDF) only may lead to quantitative results which are biased high compared to EN 1948 which additionally requires confirmation for all 2378-substituted tetra--through hexachlorodibenzo-p-dioxins and dibenzofurans.

Symptomatic improvement in achalasia after botulinum toxin injection of the lower esophageal sphincter.

OBJECTIVES: The aim of this study was to assess the long term clinical outcome of patients with achalasia after treatment with botulinum toxin. METHODS: Sixty five patients with achalasia (60 idiopathic, five secondary) were treated with injection of botulinum toxin at the gastroesophageal junction. Dysphagia, chest pain, and regurgitation were scored (0 = no symptoms, 1 = mild, 2 = moderate, 3 = severe, 4 = very severe), with the sum representing the total symptom score, at 0, 7, 30, 120, 240, and 365 days posttreatment. Responders were defined as patients with a 50% decrease in total symptom score at 1 month posttreatment. RESULTS: The 60 patients with idiopathic achalasia had significant improvement in symptoms of dysphagia, chest pain, and regurgitation at 1 and 4 wk posttreatment. At 1 month posttreatment, 42 of 60 patients (70%) were classified as responders. Of 33 patients with at least 1 yr follow-up, 36% continued to have a good or excellent response, whereas 39% underwent a subsequent treatment with botulinum toxin, pneumatic dilation, or myotomy. When symptoms recurred after an initial response, patients responded to a second injection of botulinum toxin in six of seven cases. In four of five patients with secondary achalasia, there was no response to botulinum toxin. CONCLUSIONS: Botulinum toxin injection at the gastroesophageal junction significantly improved symptoms in 70% of patients with idiopathic achalasia at 1 month. Recurrent symptoms responded to repeat botulinum toxin treatment in initially responsive patients. In contrast, most patients with secondary achalasia did not improve after botulinum toxin injection.

[Effect of rifampicin on the synthesis of antibodies to fraction I of vaccine EV]. / Vliianie rifampitsina na sintez antitel k I fraktsii vaktsiny EV.

Multifactorial analysis was used to study the influence of rifampicin on the dynamics of synthesis of antibodies belonging to IgM and IgG classes in mice immunized by fraction I of the vaccinal strain EV. Equations and quadric surfaces describing individual dynamics of antibody formation within a wide range of antibiotic doses and time of antibody content determination were developed by the experimental data. It was shown that within the range of the doses corresponding to the therapeutic ones in man rifampicin stimulated antibody formation. It had an inhibitory effect on antibody genesis only when used in the doses many times higher than the therapeutic ones.

[Effect of the composition of a nutrient medium on growth and synthesis of ergot alkaloids by Claviceps purpurea in the saprotrophic culture]. / Vliianie sostava pitatel'noi sredy na rost i sintez alkaloidov sporyn'i v saprotrofnoi kul'ture.

The impact of changes in the ratio of the medium-668 components on growth and synthesis of ergot alkaloids in saprotrophic cultures was studied with mathematical design of the experiment. Medium-668 used for cultivation of strain VNIIA-312A-producing peptide +ergot alkaloids was shown to be balanced with respect to the ratio of all the medium components. An important role of phosphate in control of culture growth and alkaloids synthesis was elucidated. It was demonstrated that by changing the ratio of the medium components it was possible to control accumulation and excretion of the alkaloids which permitted development of the conditions required for product isolation.

[Multifactor analysis of parameters of immunity under the combined action of doxycycline and a low molecular weight immunomodulator of microbial origin]. / Mnogofaktornyi analiz pokazatelei immuniteta pri kombinirovannom deistvii doksitsiklina i nizkomolekuliarnogo immunomoduliatora mikrobnogo proiskhozhdeniia.

The effect of doxycycline combination with a low molecular immunomodulator of microbial origin on the primary immune response to the vaccine EV antigens was studied in multifactor experiments. Mathematical processing of the data provided construction of polynomial statistic models of the second order describing increased delayed type hypersensitivity (IDTH) and the antibody titer. Analysis of the quasimonofactor models revealed different character of regulation of the cellular and humoral response. Nomograms were plotted for precise quantitative estimation of the dose-time parameters of the regimens for combined use of the antibiotic and immunomodulator providing the required levels of IDTH and the antibody titer.

[Multifactor immunopharmacological analysis. Effect of rifampicin and low-molecular immunomodulator of microbial origin on the primary immune response to fraction 1 of vaccine EV]. / Mnogofaktornyi immunofarmakologicheskii analiz. Vliianie rifampitsina i nizkomolekuliarnogo immunomoduliatora mikrobnogo proiskhozhdeniia na pervichnyi immunnyi otvet k I fraktsii vaktsiny EV.

Multifactor analysis was applied to combined effect of a low molecular immunomodulator of microbial origin and rifampicin on the primary immune response (increased delayed type hypersensitivity and antibody titer) to the antigens of vaccine EV fraction I. Computer processing of the experimental data provided construction of the 2nd order polynomial models satisfactorily describing cellular and humoral responses in the combined therapy. For increasing the informative capacity of the analysis of the polynomial statistic models it was proposed to develop quasimonofactor relationships reflecting the factor effect on the output with changing of the other factors within the studied ranges. Nomograms (equal level lines at fixed values of one factor) were constructed which provided rapid and correct estimation of optimal values for the regulating parameters (drug doses and administration time). Immunostimulating activity of the microbial immunomodulator was estimated quantitatively and conditions for selective regulation of the cellular and humoral responses were determined.

[Development of a data bank for control of antibiotic resistant causative agents of purulent-inflammatory diseases and complications]. / Razrabotka banka dannykh kontrolia za antibiotikorezistentnost'iu vozbuditelei gnoino-vospalitel'nykh zabolevanii i oslohneii.

To provide constant control of drug resistance in causative agents of surgical infections an automatized data bank based on computer SM-4 was filed at the All-Union Research Institute of Antibiotics. The bank includes information on 1500 objects described by 60 indices referring to the isolated pathogens. For every particular strain there are indicated data on the patients, characteristics of the pathogen biochemical profiles and sensitivity to various antibiotics (28 drugs). The data were obtained during identification of the cultures with automatized microbiological systems. The major functions of the data bank and standard information requests performed on its basis during solving particular epidemiological and clinical problems are described.

[Methodological aspects of an experimental multifactorial study of the combined use of antibiotics and immunomodulators]. / Metodicheskie aspekty éksperimental'nogo mnogofaktornogo izucheniia kombinirovannogo primeneniia antibiotikov i immunomoduliatorov.

Methodological approaches to investigation of the protective effect of natural immunomodulators are described. The use of multifactorial experiment design, a model of infectious processes and immunomodulators alone or in combination with antibiotics is implied. Theoretical preconditions for rational choosing of the experimental factors and their levels, as well as specific designs are discussed. Potentialities of polynomial statistic model analysis in development of optimal regimens for combined chemoimmunotherapy are shown.