The Impact of the Anticoagulant Type in Blood Collection Tubes on Circulating Extracellular Plasma MicroRNA Profiles Revealed by Small RNA Sequencing

Pre-analytical factors have a significant influence on circulating microRNA (miRNA) profiling. The aim of this study was a comprehensive assessment of the impact of the anticoagulant type in blood collection tubes on circulating plasma miRNA profiles using small RNA sequencing. Blood from ten healthy participants (five males and five females from 25 to 40 years old) was taken in collection tubes with four different anticoagulants: acid citrate dextrose (ACD-B), sodium citrate, citrate-theophylline-adenosine-dipyridamole (CTAD) and dipotassium-ethylenediaminetetraacetic acid (K2 EDTA). Platelet-free plasma samples were obtained by double centrifugation. EDTA plasma samples had elevated levels of hemolysis compared to samples obtained using other anticoagulants. Small RNA was extracted from plasma samples and small RNA sequencing was performed on the Illumina NextSeq 500 system. A total of 30 samples had been successfully sequenced starting from ~1 M reads mapped to miRNAs, allowing us to analyze their diversity and isoform content. The principal component analysis showed that the EDTA samples have distinct circulating plasma miRNA profiles compared to samples obtained using other anticoagulants. We selected 50 miRNA species that were differentially expressed between the sample groups based on the type of anticoagulant. We found that the EDTA samples had elevated levels of miRNAs which are abundant in red blood cells (RBC) and associated with hemolysis, while the levels of some platelet-specific miRNAs in these samples were lowered. The ratio between RBC-derived and platelet-derived miRNAs differed between the EDTA samples and other sample groups, which was validated by quantitative PCR. This study provides full plasma miRNA profiles of 10 healthy adults, compares them with previous studies and shows that the profile of circulating miRNAs in the EDTA plasma samples is altered primarily due to an increased level of hemolysis

Genetic and Epigenetic Regulation of Plant Development

Центральной проблемой биологии развития является вопрос о том, каким образом многократное деление всего одной клетки приводит к формированию организма, обладающего многоклеточными системами органов и тканей, не похожих друг на друга. Ответ на этот вопрос, как правило, лежит в дифференциальной экспрессии генов в клетках, составляющих эти ткани и органы. Именно дифференциальная активность генома - основа и структурных, и функциональных различий между разными клетками одного и того же организма, т.е. клеточной дифференцировки. Именно геном служит основным носителем информации о том, как растению пройти путь от семени до семени. Однако ген функционирует в некой среде, которая не может не оказывать влияние на характер его экспрессии. Поэтому реализация информации, заключенной в геноме, будет зависеть не только от нуклеотидных последовательностей конкретных генов, но также и от внешних условий, состояния хроматина, модификаций ДНК и ее транскриптов, т.е. находиться под эпигенетическим контролем. Своеобразными «переключателями» программ развития клеток могут также служить открытые недавно малые РНК.The completion of the Arabidopsis thaliana genome sequencing in 2000 was the key event in plant biology. Over the past 10 years, genomes of another three plant species (rice, alfalfa and poplar) were almost completely sequenced. The sequencing of maize, wheat, barley, vine and tomato genomes is in progress. It is arising a new science - genomics, that investigates nucleotide composition of genome, principles of individual genes and their complexes functioning, as well as genome evolution. Biology turns into so called post-genomic period of its history, in which predominant researches start with genome and protein sequencing and complete with determination of individual genes and proteins functions, and their evolutionary origin too. On the other hand, genes activity depends not only on the transcription factors specific to the given genes but also on the whole complex of factors able to directly or indirectly influence chromatin structure. For this reason, realization of information enclosed in genome depends not only on nucleotide sequence of given genes but also on environment, chromatin state, modifications of DNA and its transcripts, i.e. it is under epigenetic control. Epigenetic state of plant organisms is determined by levels and patterns of DNA methylation, post-translational modifications of histones, the presence of histone variants and chromatin compaction. Recently discovered noncoding microRNAs, playing key role in genes silencing by cleavage of genes transcripts or by repression of mRNA translation, may serve as a kind of «switches» for cell developmental programs. It is becoming evident that small RNAs perform a wealth of key functions in eukaryotic organisms, and therefore it is now impossible to use the simple scheme «DNA-RNA-protein» for description of genome work. In the article, it is discussed the role that transcription factors, small RNAs, modifications of DNA and chromatin play in the regulation of plant growth and development

Genetic and Epigenetic Regulation of Plant Development

Центральной проблемой биологии развития является вопрос о том, каким образом многократное деление всего одной клетки приводит к формированию организма, обладающего многоклеточными системами органов и тканей, не похожих друг на друга. Ответ на этот вопрос, как правило, лежит в дифференциальной экспрессии генов в клетках, составляющих эти ткани и органы. Именно дифференциальная активность генома - основа и структурных, и функциональных различий между разными клетками одного и того же организма, т.е. клеточной дифференцировки. Именно геном служит основным носителем информации о том, как растению пройти путь от семени до семени. Однако ген функционирует в некой среде, которая не может не оказывать влияние на характер его экспрессии. Поэтому реализация информации, заключенной в геноме, будет зависеть не только от нуклеотидных последовательностей конкретных генов, но также и от внешних условий, состояния хроматина, модификаций ДНК и ее транскриптов, т.е. находиться под эпигенетическим контролем. Своеобразными «переключателями» программ развития клеток могут также служить открытые недавно малые РНК.The completion of the Arabidopsis thaliana genome sequencing in 2000 was the key event in plant biology. Over the past 10 years, genomes of another three plant species (rice, alfalfa and poplar) were almost completely sequenced. The sequencing of maize, wheat, barley, vine and tomato genomes is in progress. It is arising a new science - genomics, that investigates nucleotide composition of genome, principles of individual genes and their complexes functioning, as well as genome evolution. Biology turns into so called post-genomic period of its history, in which predominant researches start with genome and protein sequencing and complete with determination of individual genes and proteins functions, and their evolutionary origin too. On the other hand, genes activity depends not only on the transcription factors specific to the given genes but also on the whole complex of factors able to directly or indirectly influence chromatin structure. For this reason, realization of information enclosed in genome depends not only on nucleotide sequence of given genes but also on environment, chromatin state, modifications of DNA and its transcripts, i.e. it is under epigenetic control. Epigenetic state of plant organisms is determined by levels and patterns of DNA methylation, post-translational modifications of histones, the presence of histone variants and chromatin compaction. Recently discovered noncoding microRNAs, playing key role in genes silencing by cleavage of genes transcripts or by repression of mRNA translation, may serve as a kind of «switches» for cell developmental programs. It is becoming evident that small RNAs perform a wealth of key functions in eukaryotic organisms, and therefore it is now impossible to use the simple scheme «DNA-RNA-protein» for description of genome work. In the article, it is discussed the role that transcription factors, small RNAs, modifications of DNA and chromatin play in the regulation of plant growth and development

Design of Conjugates Based on Sesquiterpene Lactones with Polyalkoxybenzenes by “Click” Chemistry to Create Potential Anticancer Agents

Using the methodology of “click” chemistry, a singular method has been developed for the synthesis of unique conjugates based on sesquiterpene lactones: dehydrocostuslactone and alantolactone with polyalkoxybenzenes. To expand the structural range of the resulting conjugates, the length of the 1,2,3-triazole spacer was varied. For all synthesized compounds, the cytotoxic profile was determined on the cell lines of tumor origin (SH-SY5Y, HeLa, Hep-2, A549) and normal Hek 293 cells. It was found that the compounds based on alantolactone 7a–d with a long spacer and substances containing dehydrocostuslactone 10a–d with a short spacer have the greatest toxic effect. The decrease in cell survival under the action of these conjugates may be due to their ability to cause dissipation of the transmembrane potential of mitochondria and inhibit the process of glycolysis, leading to cell death. The obtained results confirm the assumption that the development of conjugates based on sesquiterpene lactones and polyalkoxybenzenes can be considered as a promising strategy for the search for potential antitumor agents

Circulating Extracellular miRNA Analysis in Patients with Stable CAD and Acute Coronary Syndromes

Extracellular circulating microRNAs (miRNAs) are currently a focus of interest as non-invasive biomarkers of cardiovascular pathologies, including coronary artery disease (CAD) and acute coronary syndromes (ACS): myocardial infarction with and without ST-segment elevation (STEMI and NSTEMI) and unstable angina (UA). However, the current data for some miRNAs are controversial and inconsistent, probably due to pre-analytical and methodological variances in different studies. In this work, we fulfilled the basic pre-analytical requirements provided for circulating miRNA studies for application to stable CAD and ACS research. We used quantitative PCR to determine the relative plasma levels of eight circulating miRNAs that are potentially associated with atherosclerosis. In a cohort of 136 adult clinic CAD patients and outpatient controls, we found that the plasma levels of miR-21-5p and miR-146a-5p were significantly elevated in ACS patients, and the level of miR-17-5p was decreased in ACS and stable CAD patients compared to both healthy controls and hypertensive patients without CAD. Within the ACS patient group, no differences were found in the plasma levels of these miRNAs between patients with positive and negative troponin, nor were any differences found between STEMI and NSTEMI. Our results indicate that increased plasma levels of miR-146a-5p and miR-21-5p can be considered general ACS circulating biomarkers and that lowered miR-17-5p can be considered a general biomarker of CAD

An Efficient 2D Protocol for Differentiation of iPSCs into Mature Postmitotic Dopaminergic Neurons: Application for Modeling Parkinson’s Disease

About 15% of patients with parkinsonism have a hereditary form of Parkinson’s disease (PD). Studies on the early stages of PD pathogenesis are challenging due to the lack of relevant models. The most promising ones are models based on dopaminergic neurons (DAns) differentiated from induced pluripotent stem cells (iPSCs) of patients with hereditary forms of PD. This work describes a highly efficient 2D protocol for obtaining DAns from iPSCs. The protocol is rather simple, comparable in efficiency with previously published protocols, and does not require viral vectors. The resulting neurons have a similar transcriptome profile to previously published data for neurons, and have a high level of maturity marker expression. The proportion of sensitive (SOX6+) DAns in the population calculated from the level of gene expression is higher than resistant (CALB+) DAns. Electrophysiological studies of the DAns confirmed their voltage sensitivity and showed that a mutation in the PARK8 gene is associated with enhanced store-operated calcium entry. The study of high-purity DAns differentiated from the iPSCs of patients with hereditary PD using this differentiation protocol will allow for investigators to combine various research methods, from patch clamp to omics technologies, and maximize information about cell function in normal and pathological conditions