Critical parameters for efficient sonication and improved chromatin immunoprecipitation of high molecular weight proteins

Solubilization of cross-linked cells followed by chromatin shearing is essential for successful chromatin immunoprecipitation (ChIP). However, this task, typically accomplished by ultrasound treatment, may often become a pitfall of the process, due to inconsistent results obtained between different experiments under seemingly identical conditions. To address this issue we systematically studied ultrasound-mediated cell lysis and chromatin shearing, identified critical parameters of the process and formulated a generic strategy for rational optimization of ultrasound treatment. We also demonstrated that whereas ultrasound treatment required to shear chromatin to within a range of 100–400 bp typically degrades large proteins, a combination of brief sonication and benzonase digestion allows for the generation of similarly sized chromatin fragments while preserving the integrity of associated proteins. This approach should drastically improve ChIP efficiency for this class of proteins

DNMT inhibitors reverse a specific signature of aberrant promoter DNA methylation and associated gene silencing in AML

<b>Background</b>. Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) are neoplastic disorders of hematopoietic stem cells. DNA methyltransferase inhibitors (DNMTi), 5-azacytidine (AzaC) and 5-aza-2’-deoxycytidine (Decitabine), benefit some MDS/AML patients. However, the role of DNMTi-induced DNA hypomethylation in regulation of gene expression in AML is unclear.<p></p> <b>Results. </b> We compared the effects of AzaC on DNA methylation and gene expression using whole-genome single-nucleotide bisulfite-sequencing (WGBS) and RNA-sequencing in OCI-AML3 (AML3) cells. For data analysis, we used an approach recently developed for discovery of differential patterns of DNA methylation associated with changes in gene expression, that is tailored to single-nucleotide bisulfite-sequencing data (Washington University Interpolated Methylation Signatures (WIMSi)). By this approach, a subset of genes upregulated by AzaC was found to be characterized by AzaC-induced signature methylation loss flanking the transcription start site. These genes are enriched for genes increased in methylation and decreased in expression in AML3 cells compared to normal hematopoietic stem and progenitor cells. Moreover, these genes are preferentially upregulated by Decitabine in human primary AML blasts, and control cell proliferation, death and development. <p></p> <b>Conclusions.</b> Our WGBS and WIMSi data analysis approach has identified a set of genes whose is methylation and silencing in AML is reversed by DNMTi. These genes are good candidates for direct regulation by DNMTi, and their reactivation by DNMTi may contribute to therapeutic activity. This study also demonstrates the ability of WIMSi to reveal relationships between DNA methylation and gene expression, based on single-nucleotide bisulfite-sequencing and RNA-seq data.<p></p&gt

Profiling of transcriptional and epigenetic changes during directed endothelial differentiation of human embryonic stem cells identifies FOXA2 as a marker of early mesoderm commitment

Introduction: Differentiation of vascular endothelial cells (ECs) in clinically relevant numbers for injection into ischaemic areas could offer therapeutic potential in the treatment of cardiovascular conditions, including myocardial infarction, peripheral vascular disease and stroke. While we and others have demonstrated successful generation of functional endothelial-like cells from human embryonic stem cells (hESCs), little is understood regarding the complex transcriptional and epigenetic changes that occur during differentiation, in particular during early commitment to a mesodermal lineage. Methods: We performed the first gene expression microarray study of hESCs undergoing directed differentiation to ECs using a monolayer-based, feeder-free and serum-free protocol. Microarray results were confirmed by quantitative RT-PCR and immunocytochemistry, and chromatin immunoprecipitation (ChIP)-PCR analysis was utilised to determine the bivalent status of differentially expressed genes. Results: We identified 22 transcription factors specific to early mesoderm commitment. Among these factors, FOXA2 was observed to be the most significantly differentially expressed at the hESC–EC day 2 timepoint. ChIP-PCR analysis revealed that the FOXA2 transcription start site is bivalently marked with histone modifications for both gene activation (H3K4me3) and repression (H3K27me3) in hESCs, suggesting the transcription factor may be a key regulator of hESC differentiation. Conclusion: This enhanced knowledge of the lineage commitment process will help improve the design of directed differentiation protocols, increasing the yield of endothelial-like cells for regenerative medicine therapies in cardiovascular disease

Спасительная брахитерапия высокой мощности дозы при локальном рецидиве рака предстательной железы после различных методов радикального лечения

Objective: at the primary analysis of prostate cancer treatment referring to the patients who have experienced high dose rate brachytherapy after a confirmed locally recurrent tumor having been treated with diverse treatment modes.Materials and methods. The research includes 28 patients aged 53 to 78 years old (average age is 66 years old) with histologically verified prostate cancer recurrence. Within the period 2015–2017 all the patients got a salvage high dose rate brachytherapy in the Russian Scientific Center of Roentgenradiology. Brachytherapy was carried out as 2 fractions with the single tumor dose of 12.5 Gy. There was a two-week gap between fractions with the total tumor dose of 25 Gy. The follow-up period is 9 to 36 months.Results and conclusion. Overall biochemical free survival rate is 82.1 %. There are 5 people having a growing prostatic specific antigen (PSA) while the case follow-up. There is 1 case of a confirmed local tumor recurrence. Significant factors of the disease progression after salvage brachytherapy are: risk group, pretreatment PSA high, time line from the background therapy to biochemical recurrence uprise and PSA level if there’s prostate cancer recurrence after the background therapy.Цель исследования – первичный анализ лечения больных раком предстательной железы, которым была проведена брахитерапия высокой мощности дозы по поводу подтвержденного локального рецидива после различных методов лечения.Материалы и методы. В исследование вошли 28 пациентов в возрасте 53–78 лет (средний возраст 66 лет) с гистологически подтвержденным локальным рецидивом рака предстательной железы после различных вариантов лечения. В период с 2015 по 2017 г. в Российском научном центре рентгенорадиологии всем пациентам проведена спасительная брахитерапия высокой мощности дозы. Брахитерапию проводили в виде 2 фракций с разовой очаговой дозой 12,5 Гр с двухнедельным перерывом между фракциями, суммарной очаговой дозой 25 Гр. Период наблюдения составил 9–36 мес.Результаты и заключение. Общая ПСА (простатический специфический антиген) специфическая выживаемость составила 82,1 %. У 23 из 28 больных достигнут биохимический контроль опухоли. У 5 больных при динамическом наблюдении отмечался рост уровня ПСА. В 1 случае подтвержден локальный рецидив опухоли. Значимыми факторами прогрессирования после спасительной брахитерапии явились группа риска, максимальный уровень ПСА до лечения, время до развития биохимического рецидива после основного вида лечения и максимальный уровень ПСА при развитии рецидива после основного вида лечения

Mapping H4K20me3 onto the chromatin landscape of senescent cells indicates a function in control of cell senescence and tumor suppression through preservation of genetic and epigenetic stability

Background: Histone modification H4K20me3 and its methyltransferase SUV420H2 have been implicated in suppression of tumorigenesis. The underlying mechanism is unclear, although H4K20me3 abundance increases during cellular senescence, a stable proliferation arrest and tumor suppressor process, triggered by diverse molecular cues, including activated oncogenes. Here, we investigate the function of H4K20me3 in senescence and tumor suppression. Results: Using immunofluorescence and ChIP-seq we determine the distribution of H4K20me3 in proliferating and senescent human cells. Altered H4K20me3 in senescence is coupled to H4K16ac and DNA methylation changes in senescence. In senescent cells, H4K20me3 is especially enriched at DNA sequences contained within specialized domains of senescence-associated heterochromatin foci (SAHF), as well as specific families of non-genic and genic repeats. Altered H4K20me3 does not correlate strongly with changes in gene expression between proliferating and senescent cells; however, in senescent cells, but not proliferating cells, H4K20me3 enrichment at gene bodies correlates inversely with gene expression, reflecting de novo accumulation of H4K20me3 at repressed genes in senescent cells, including at genes also repressed in proliferating cells. Although elevated SUV420H2 upregulates H4K20me3, this does not accelerate senescence of primary human cells. However, elevated SUV420H2/H4K20me3 reinforces oncogene-induced senescence-associated proliferation arrest and slows tumorigenesis in vivo. Conclusions: These results corroborate a role for chromatin in underpinning the senescence phenotype but do not support a major role for H4K20me3 in initiation of senescence. Rather, we speculate that H4K20me3 plays a role in heterochromatinization and stabilization of the epigenome and genome of pre-malignant, oncogene-expressing senescent cells, thereby suppressing epigenetic and genetic instability and contributing to long-term senescence-mediated tumor suppression

Asymmetric and symmetric fission of excited nuclei of 180,190Hg and 184,192,202Pb formed in the reactions with 36Ar and 40,48Ca ions

Background: Observation of asymmetric fission of 180Hg has led to intensive theoretical and experimental studies of fission of neutron-deficient nuclei in the lead region. Purpose: The study of asymmetric and symmetric fission modes of 180,190Hg and 184,192,202Pb nuclei. Methods: Mass-energy distributions of fission fragments of 180,190Hg and 184Pb formed in the 36Ar+144,154Sm and 40Ca+144Sm reactions, respectively, at energies near the Coulomb barrier have been measured using the double-arm time-of-flight spectrometer CORSET and compared with previously measured 192,202Pb isotopes produced in the 48Ca+144,154Sm reactions. The mass distributions for 180,190Hg and 184,192,202Pb together with old data for 187Ir, 195Au, 198Hg, 201Tl, 205,207Bi, 210Po, and 213At [J. Nucl. Phys. 53, 1225 (1991)] have been decomposed into symmetric and asymmetric fission modes. The total kinetic-energy distributions for different fission fragment mass regions have been analyzed for 180,190Hg and 184Pb. Results: The stabilization role of proton numbers at Z≈36, 38, Z≈45, 46, and Z=28/50 in asymmetric fission of excited preactinide nuclei has been observed. The high (≈145−MeV) and the low (≈128−MeV) energy components have been found in the total kinetic-energy distributions of 180,190Hg fission fragments corresponding to the fragments with proton numbers near Z≈46 and Z≈36, respectively. In the case of fission of 184Pb only the low-energy component (≈135MeV) for the fragments with masses corresponding to the proton numbers Z≈36 and 46 has been found. Conclusions: The studied properties of asymmetric fission of 180,190Hg and 184,192,202Pb nuclei point out the existence of well deformed proton shell at Z≈36 and less deformed proton shell at Z≈46.peerReviewe

Company town socio-economic transformation: concentration and factors system

In the article the authors suggest evaluating the level of diversification using Herfindahl-Hirschman index that reflects the economic activity concentration within the certain timespan. The study of the index in dynamics for Berezovsky company town located in Kemerovo region shows the downward tendency in economic activity concentration whereas its factors remain ambiguous. The research concludes that, on the one hand, a low level of investments in diversification projects does not act upon the diversification process, on the other hand, socio-economic targets of diversification does not correlate with economic structural changes. Thus the diversification process has low effectiveness