Loss of DNA methyltransferase activity in primed human ES cells triggers increased cell-cell variability and transcriptional repression.

Maintenance of pluripotency and specification towards a new cell fate are both dependent on precise interactions between extrinsic signals and transcriptional and epigenetic regulators. Directed methylation of cytosines by the methyltransferases DNMT3A and DNMT3B plays an important role in facilitating proper differentiation, whereas DNMT1 is essential for maintaining global methylation levels in all cell types. Here, we generated single-cell mRNA expression data from wild-type, DNMT3A, DNMT3A/3B and DNMT1 knockout human embryonic stem cells and observed a widespread increase in cellular and transcriptional variability, even with limited changes in global methylation levels in the knockouts.

Crossover Phenomena in Motor Evoked Potentials During Intraoperative Neurophysiological Monitoring of Cranial Surgeries.

Purpose: Transcranial motor evoked potentials (TcMEPs) are used to assess the corticospinal tract during surgery. Transcranial motor evoked potentials are elicited by preferentially activating the anode over the target cortex. Crossover occurs when stimulation also induces activation of ipsilateral motor evoked responses.

Global delay in nascent strand DNA methylation.

Cytosine methylation is widespread among organisms and essential for mammalian development. In line with early postulations of an epigenetic role in gene regulation, symmetric CpG methylation can be mitotically propagated over many generations with extraordinarily high fidelity. Here, we combine BrdU labeling and immunoprecipitation with genome-wide bisulfite sequencing to explore the inheritance of cytosine methylation onto newly replicated DNA in human cells.

Recommendations of the Oligonucleotide Safety Working Group's Formulated Oligonucleotide Subcommittee for the Safety Assessment of Formulated Oligonucleotide-Based Therapeutics.

The use of lipid formulations has greatly improved the ability to effectively deliver oligonucleotides and has been instrumental in the rapid expansion of therapeutic development programs using oligonucleotide drugs. However, the development of such complex multicomponent therapeutics requires the implementation of unique, scientifically sound approaches to the nonclinical development of these drugs, based upon a hybrid of knowledge and experiences drawn from small molecule, protein, and oligonucleotide therapeutic drug development. The relative paucity of directly applicable regulatory guidance documents for oligonucleotide therapeutics in general has resulted in the generation of multiple white papers from oligonucleotide drug development experts and members of the Oligonucleotide Safety Working Group (OSWG).

A qPCR ScoreCard quantifies the differentiation potential of human pluripotent stem cells.

Research on human pluripotent stem cells has been hampered by the lack of a standardized, quantitative, scalable assay of pluripotency. We previously described an assay called ScoreCard that used gene expression signatures to quantify differentiation efficiency. Here we report an improved version of the assay based on qPCR that enables faster, more quantitative assessment of functional pluripotency.

Integrative Analyses of Human Reprogramming Reveal Dynamic Nature of Induced Pluripotency.

Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here, we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution.

Targeted disruption of DNMT1, DNMT3A and DNMT3B in human embryonic stem cells.

DNA methylation is a key epigenetic modification involved in regulating gene expression and maintaining genomic integrity. Here we inactivated all three catalytically active DNA methyltransferases (DNMTs) in human embryonic stem cells (ESCs) using CRISPR/Cas9 genome editing to further investigate the roles and genomic targets of these enzymes. Disruption of DNMT3A or DNMT3B individually as well as of both enzymes in tandem results in viable, pluripotent cell lines with distinct effects on the DNA methylation landscape, as assessed by whole-genome bisulfite sequencing.

Transcription factor binding dynamics during human ES cell differentiation.

Pluripotent stem cells provide a powerful system to dissect the underlying molecular dynamics that regulate cell fate changes during mammalian development. Here we report the integrative analysis of genome-wide binding data for 38 transcription factors with extensive epigenome and transcriptional data across the differentiation of human embryonic stem cells to the three germ layers. We describe core regulatory dynamics and show the lineage-specific behaviour of selected factors.

The GCTM-5 epitope associated with the mucin-like glycoprotein FCGBP marks progenitor cells in tissues of endodermal origin.

Monoclonal antibodies against cell surface markers are powerful tools in the study of tissue regeneration, repair, and neoplasia, but there is a paucity of specific reagents to identify stem and progenitor cells in tissues of endodermal origin. The epitope defined by the GCTM-5 monoclonal antibody is a putative marker of hepatic progenitors. We sought to analyze further the distribution of the GCTM-5 antigen in normal tissues and disease states and to characterize the antigen biochemically.

[Damage of macular ganglion cell complex and peripapillary retinal nerve fiber layer in multiple sclerosis].

The aim of this study was to evaluate macular ganglion cell complex (GCC) characteristics and peripapillary retinal nerve fiber layer (RNFL) thickness in patients with multiple sclerosis (MS) and to investigate the associations between these parameters and clinical characteristics of patients for evaluation perspectives of using this method for monitoring of disability and neurodegenerative processes. We examined a total of 113 participants (analysis of 211 eyes), divided into three groups: 1. 48 MS patients (66 eyes) with a history of optic neuritis (ON); 2.

Epigenomics and chromatin dynamics.

A report of the 'Joint Keystone Symposium on Epigenomics and Chromatin Dynamics', Keystone, Colorado, 17-22 January 2012.

[Effect of nootropes on quantitative changes in the rat cerebral cortex GABA(A) receptor complexes under experimental hypokinesia conditions].

The influence of picamilon and piracetam on the quantitative changes in the central GABA(A) macromolecular receptor complexes in the rat brain has been investigated under the experimental conditions of hypokinesia. It was found that the injection of these nootropes under the conditions of 7-day hypokinesia and 4-day recovery period did not show visible changes in the amount of active GABA(A) receptors. However, the injection of picamilon under the conditions of 15-day hypokinesia and 8-day recovery period showed a tendency to restoration of the number of active GABA(A) receptors.

Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells.

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry.

[Changes in free amino acid contents in the liver and heart under the conditions of restricted movement activity].

The contents of the free amino acid in the liver and heart tissues were investigated under conditions of experimental hypokinesia on 15th, 30th and 45th days by means of HPLC method. The content of glutamate and aspartate in the liver tissue was sharply decreased with the increase of the hypokinesia period. The degree of the decrease of aspartate was more pronounced than glutamate.

[The effect of hypokinesia on depression and on the central GABA-A receptor complexes in the rat brain].

The influence of a 15-day hypokinesia on the development of anxiety-depression state and quantitative changes in the central GABA-A macromolecular receptor complexes in the rat brain has been investigated under conditions of the despair (forced swim) test. Simultaneously, the effects of well-known nootropic drugs picamilon and piracetam on the dynamics of state parameters in the experimental animals have been evaluated. It was found that hypokinesia led to the development of anxiety and depression accompanied by reduction in the amount of active GABA-A receptors.

[Effect of afobazole on brain-ischemia-induced anxiety in rats].

The ability of afobazole, a selective anxiolytic to prevent the brain local ischemia-induced anxiety has been studied in experiments on rats. It is established that middle cerebral artery occlusion (MCAO) is accompanied by the development of anxiety, the degree of which increases with the duration of occlusion. Afobazole in a doze of 1 mg/kg administrated during 6 days after MCAO prevents the development of anxiety.

[The effect of colchicine on the periodicity of the spontaneous and induced respiratory burst of granulocytes and monocytes during familial Mediterranean fever].

The influence of colchicine on the spontaneous and chemotaxis-, protein kinase C-, and phagocytosis-induced respiratory burst of neutrophils and monocytes in the peripheral blood of patients with familial Mediterranean fever (FMF) has been studied. The transient activation of neutrophils and monocytes on the level of a single cell has been monitored by means of flow cytofluorimetry. It is shown that colchicine blocks the induction of chemotaxis-, phagocytosis-, and proteinkinase C-dependent respiratory burst in vitro, as well as the increased pro-oxidant transient activation of neutrophils and monocytes of FMF patients, both in the period of remission and during the FMF attack.

[The effect of colcichine and iodine-lithium-alpha-dextrin on the phagocytosis of granulocytes and monocytes in patients with familial Mediterranean fever].

Disturbances in the regulation of phagocytic activity of neutrophils and monocytes (PANM) in whole peripheral blood of patients with familial Mediterranean Fever (FMF), which had not received treatment with colchicines, were determined by quantitative flow cytofluorimetry. The effect of iodine-lithium-alpha-dextrin (armenicum) and colchicine on the PANM in the blood of FMF patients was studied in vitro. The intensity of phagocytosis in populations of neutrophils and monocytes in FMF patients (n = 6) during the remission period is higher than that during the FMF attack (n = 6) and higher than in healthy donors (n = 9).

[Effect of GABA on phosphoinositide cycle and membrane lipid modification in the brain synaptosomes of rats in hypokinesia].

The influence of a 30-day hypokinesia on the phosphoinositide cycle (PIC) initiation and on the inclusion of [14C]-arachidonic acid into cerebral synaptosome phospholipids was studied in rats. The results show that the catabolism of phospholipids prevails on the background of signal transduction in synaptosomes on the 30th day of hypokinesia. The effect of gamma-aminobutyric acid (GABA) is manifested by a sharp increase in the PIC activity 5 sec upon initiation and leads to normalization of the PIC activity in the late stage (5 min).

[Leptospirillum-like bacteria and their role in pyrite oxidation].

Two strains of Leptospirillum-like bacteria isolated from dumps of Alaverdi and Akhtala sulfide ore deposits in Armenia were studied. The optimum and maximum temperatures for the growth of both strains were 37 and 40 degrees C, respectively. The pH optimum was 2.

[Three-dimensional computerized sonography in evaluation of the vascular system of the eye and orbit. I. Methodological approach and principles of analysis of results].

An examination of 40 healthy persons (80 eyes) enabled a refinement and unification of the ultrasound techniques applicable to investigations of a. ophthalmica, a. and v.

[Participation of the GABA system in tha adaptive rearrangement of the cerebral circulation in hypokinesia].

Hypokinesia involves a number of significant biochemical, histochemical, and pathophysiological changes in brain tissues, vessels, and neurocytes, including glucose metabolism disturbances, calcium level violation and transduction in synaptosomes, changes, on the levels of neuroactive amino acids, nitric oxide, and lipid peroxidation products. All these variations depend on the terms of hypokinesia. It is established that GABA, together with other neuromediators, play an important role in the adaptation process.

[Effect of GABA and pyracetam on mitochondrial ADP phosphorylation in experimental hypokinesia].

Long-term limitation of motor activity of male rats is accompanied by impairments of mitochondrial ADP phosphorylation in liver and heart. The most significant changes were observed after 15 and 45 days of hypokinesia. Administration of GABA and pyracetam to animals subjected to hypokinesia induced a tendency to "normalization" of ADP phosphorilation processes.

Characterization of a new in vivo hollow fiber model for the study of progression of prostate cancer to androgen independence.

Research investigating the molecular events underlying progression of prostate cancer to androgen independence has been impeded by the lack of an appropriate in vivo model that yields "pure" populations of prostate cancer cells that are not contaminated with host cells. Here we characterize a new in vivo model that uses hollow fibers and allows for the retrieval of uncontaminated prostate cancer cells during various stages of endocrine progression to androgen independence in male immunocompromised mice. Prostate-specific antigen (PSA) gene expression, proliferation of cells, and histology were examined in these mice before and after castration.