Genome-Wide Screen for Differential DNA Methylation Associated with Neural Cell Differentiation in Mouse

Cellular differentiation involves widespread epigenetic reprogramming, including modulation of DNA methylation patterns. Using Differential Methylation Hybridization (DMH) in combination with a custom DMH array containing 51,243 features covering more than 16,000 murine genes, we carried out a genome-wide screen for cell- and tissue-specific differentially methylated regions (tDMRs) in undifferentiated embryonic stem cells (ESCs), in in-vitro induced neural stem cells (NSCs) and 8 differentiated embryonic and adult tissues. Unsupervised clustering of the generated data showed distinct cell- and tissue-specific DNA methylation profiles, revealing 202 significant tDMRs (p<0.005) between ESCs and NSCs and a further 380 tDMRs (p<0.05) between NSCs/ESCs and embryonic brain tissue. We validated these tDMRs using direct bisulfite sequencing (DBS) and methylated DNA immunoprecipitation on chip (MeDIP-chip). Gene ontology (GO) analysis of the genes associated with these tDMRs showed significant (absolute Z score>1.96) enrichment for genes involved in neural differentiation, including, for example, Jag1 and Tcf4. Our results provide robust evidence for the relevance of DNA methylation in early neural development and identify novel marker candidates for neural cell differentiation

Aberrant septin 9 DNA methylation in colorectal cancer is restricted to a single CpG island.

BACKGROUND: The septin 9 gene (SEPT9) codes for a GTP-binding protein associated with filamentous structures and cytoskeleton formation. SEPT9 plays a role in multiple cancers as either an oncogene or a tumor suppressor gene. Regulation of SEPT9 expression is complex and not well understood; however, hypermethylation of the gene was recently introduced as a biomarker for early detection of colorectal cancer (CRC) and has been linked to cancer of the breast and of the head and neck. Because the DNA methylation landscape of different regions of SEPT9 is poorly understood in cancer, we analyzed the methylation patterns of this gene in distinct cell populations from normal and diseased colon mucosa. METHODS: Laser capture microdissection was performed to obtain homogeneous populations of epithelial and stromal cells from normal, adenomatous, and tumorous colon mucosa. Microdissected samples were analyzed using direct bisulfite sequencing to determine the DNA methylation status of eight regions within and near the SEPT9 gene. Septin-9 protein expression was assessed using immunohistochemistry (IHC). RESULTS: Regions analyzed in SEPT9 were unmethylated in normal tissue except for a methylation boundary detected downstream of the largest CpG island. In adenoma and tumor tissues, epithelial cells displayed markedly increased DNA methylation levels (>80%, p <0.0001) in only one of the CpG islands investigated. SEPT9 methylation in stromal cells increased in adenomatous and tumor tissues (<=50%, p <0.0001); however, methylation did not increase in stromal cells of normal tissue close to the tumor. IHC data indicated a significant decrease (p <0.01) in Septin-9 protein levels in epithelial cells derived from adenoma and tumor tissues; Septin-9 protein levels in stromal cells were low in all tissues. CONCLUSIONS: Hypermethylation of SEPT9 in adenoma and CRC specimens is confined to one of several CpG islands of this gene. Tumor-associated aberrant methylation originates in epithelial cells; stromal cells appear to acquire hypermethylation subsequent to epithelial cells, possibly through field effects. The region in SEPT9 with disease-related hypermethylation also contains the CpGs targeted by a novel blood-based screening test (Epi proColon(R)), providing further support for the clinical relevance of this biomarker

Erforschung von biokompatiblen und korrosionsbestandigen Beschichtungen, die auf Titanlegierung Vt6 durch Elektroerosivemetallbearbeitung aufgetragen werden

В статье рассмотрены фазовый и химико-элементный составы покрытия для ортопедии и дентальной медицины, нанесенного на титановый сплав ВТ6методом электроискровой обработки металлов с целью предотвращения попадания с поверхности сплава в организм человека химических элементов, оказывающих токсическое воздействие

Detection of methylated septin 9 in tissue and plasma of colorectal patients with neoplasia and the relationship to the amount of circulating cell-free DNA

BACKGROUND: Determination of methylated Septin 9 (mSEPT9) in plasma has been shown to be a sensitive and specific biomarker for colorectal cancer (CRC). However, the relationship between methylated DNA in plasma and colon tissue of the same subjects has not been reported. METHODS: Plasma and matching biopsy samples were collected from 24 patients with no evidence of disease (NED), 26 patients with adenoma and 34 patients with CRC. Following bisulfite conversion of DNA a commercial RT-PCR assay was used to determine the total amount of DNA in each sample and the fraction of mSEPT9 DNA. The Septin-9 protein was assessed using immunohistochemistry. RESULTS: The percent of methylated reference (PMR) values for SEPT9 above a PMR threshold of 1% were detected in 4.2% (1/24) of NED, 100% (26/26) of adenoma and 97.1% (33/34) of CRC tissues. PMR differences between NED vs. adenoma and NED vs. CRC comparisons were significant (p<0.001). In matching plasma samples using a PMR cut-off level of 0.01%, SEPT9 methylation was 8.3% (2/24) of NED, 30.8% (8/26) of adenoma and 88.2% (30/34) of CRC. Significant PMR differences were observed between NED vs. CRC (p<0.01) and adenoma vs. CRC (p<0.01). Significant differences (p<0.01) were found in the amount of cfDNA (circulating cell-free DNA) between NED and CRC, and a modest correlation was observed between mSEPT9 concentration and cfDNA of cancer (R2 = 0.48). The level of Septin-9 protein in tissues was inversely correlated to mSEPT9 levels with abundant expression in normals, and diminished expression in adenomas and tumors. CONCLUSIONS: Methylated SEPT9 was detected in all tissue samples. In plasma samples, elevated mSEPT9 values were detected in CRC, but not in adenomas. Tissue levels of mSEPT9 alone are not sufficient to predict mSEPT9 levels in plasma. Additional parameters including the amount of cfDNA in plasma appear to also play a role

Extracorporeal hemoperfusion as a potential therapeutic option for critical accumulation of rivaroxaban

Because of its efficacy, ease of dosing, and safety, the direct oral anticoagulant rivaroxaban is increasingly applied in a number of indications, for example, prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation and treatment of deep vein thrombosis and pulmonary embolism. Median therapeutic peak plasma concentrations range from 46 to 270 µg/L, depending on the respective indication. However, there are concerns regarding the accumulation of the drug in patients with impaired renal clearance or in case of overdosing, potentially leading to an increased risk of bleeding. With its high degree of protein binding of 92–95%, rivaroxaban is regarded as non-dialyzable, as also suggested by results from a clinical study conducted by Dias et al.. Since protein binding is regarded as not a limiting factor in hemoperfusion, the removal of rivaroxaban, as for example by commonly available coated charcoal cartridges, has been deemed possible, but experimental evidence is still lacking. While Andexanet alfa may offer a promising approach to reverse the FXa inhibitor-mediated anticoagulation of rivaroxaban, it has not yet been approved. In case of rivaroxaban-related major bleeding events or emergency interventions with a high bleeding risk, therefore, a fast and effective countermeasure is urgently needed. Here, we present experimental work to remove rivaroxaban from the blood by means of hemoperfusion using an approved adsorption device (CytoSorb®; Cyto¬Sorbents Europe, Germany). Currently, CytoSorb is used mainly in patients with severe infections and sepsis (cytokine storm). We applied a model device containing 60 mL of the adsorbent polyvinylpyrrolidone-coated polystyrene-divinylbenzene copolymer in an in vitro recirculation system to remove high plasma concentrations of rivaroxaban (571 ± 20 µg/L) from citrate-anticoagulated human whole blood (1,000 mL, flow rate 40 mL/min) during 120 min of hemoperfusion (Fig. 1a). Molecules are captured on the internal pore surface of polystyrene-divinylbenzene by nonspecific hydrophobic interactions, whereby solutes with molecular weights equal to and larger than that of albumin, particularly clotting factors, are excluded from adsorption by adjustment of the pore size distribution

Exploring Albumin Functionality Assays: A Pilot Study on Sepsis Evaluation in Intensive Care Medicine

Human serum albumin (HSA) as the most abundant plasma protein carries multifunctional properties. A major determinant of the efficacy of albumin relies on its potent binding capacity for toxins and pharmaceutical agents. Albumin binding is impaired in pathological conditions, affecting its function as a molecular scavenger. Limited knowledge is available on the functional properties of albumin in critically ill patients with sepsis or septic shock. A prospective, non-interventional clinical trial assessed blood samples from 26 intensive care patients. Albumin-binding capacity (ABiC) was determined by quantifying the unbound fraction of the fluorescent marker, dansyl sarcosine. Electron paramagnetic resonance fatty acid spin-probe evaluated albumin’s binding and detoxification efficiencies. Binding efficiency (BE) reflects the strength and amount of bound fatty acids, and detoxification efficiency (DTE) indicates the molecular flexibility of patient albumin. ABiC, BE, and DTE effectively differentiated control patients from those with sepsis or septic shock (AUROC > 0.8). The diagnostic performance of BE showed similarities to procalcitonin. Albumin functionality correlates with parameters for inflammation, hepatic, or renal insufficiency. Albumin-binding function was significantly reduced in critically ill patients with sepsis or septic shock. These findings may help develop patient-specific algorithms for new diagnostic and therapeutic approaches

The Utility of Miniaturized Adsorbers in Exploring the Cellular and Molecular Effects of Blood Purification: A Pilot Study with a Focus on Immunoadsorption in Multiple Sclerosis

Immunoadsorption (IA) has proven to be clinically effective in the treatment of steroid-refractory multiple sclerosis (MS) relapses, but its mechanism of action remains unclear. We used miniaturized adsorber devices with a tryptophan-immobilized polyvinyl alcohol (PVA) gel sorbent to mimic the IA treatment of patients with MS in vitro. The plasma was screened before and after adsorption with regard to disease-specific mediators, and the effect of the IA treatment on the migration of neutrophils and the integrity of the endothelial cell barrier was tested in cell-based models. The in vitro IA treatment with miniaturized adsorbers resulted in reduced plasma levels of cytokines and chemokines. We also found a reduced migration of neutrophils towards patient plasma treated with the adsorbers. Furthermore, the IA-treated plasma had a positive effect on the endothelial cell barrier’s integrity in the cell culture model. Our findings suggest that IA results in a reduced infiltration of cells into the central nervous system by reducing leukocyte transmigration and preventing blood–brain barrier breakdown. This novel approach of performing in vitro blood purification therapies on actual patient samples with miniaturized adsorbers and testing their effects in cell-based assays that investigate specific hypotheses of the pathophysiology provides a promising platform for elucidating the mechanisms of action of those therapies in various diseases