Optical mapping discerns genome wide DNA methylation profiles

BACKGROUND: Methylation of CpG dinucleotides is a fundamental mechanism of epigenetic regulation in eukaryotic genomes. Development of methods for rapid genome wide methylation profiling will greatly facilitate both hypothesis and discovery driven research in the field of epigenetics. In this regard, a single molecule approach to methylation profiling offers several unique advantages that include elimination of chemical DNA modification steps and PCR amplification. RESULTS: A single molecule approach is presented for the discernment of methylation profiles, based on optical mapping. We report results from a series of pilot studies demonstrating the capabilities of optical mapping as a platform for methylation profiling of whole genomes. Optical mapping was used to discern the methylation profile from both an engineered and wild type Escherichia coli. Furthermore, the methylation status of selected loci within the genome of human embryonic stem cells was profiled using optical mapping. CONCLUSION: The optical mapping platform effectively detects DNA methylation patterns. Due to single molecule detection, optical mapping offers significant advantages over other technologies. This advantage stems from obviation of DNA modification steps, such as bisulfite treatment, and the ability of the platform to assay repeat dense regions within mammalian genomes inaccessible to techniques using array-hybridization technologies

Movement of the inactivating chromosome to the heterochromatic compartment is coincident with the irreversibility of the inactivation process

X chromosome inactivation is initiated by expression of Xist RNA and occurs through the formation of facultative heterochromatin. Inactivation is necessary to achieve dosage compensation in female mammals. Previous studies have shown that Xist-mediated chromosome inactivation becomes irreversible after 72 hours of ES cell differentiation. Using fluorescent in-situ hybridization and a male mouse ES cell line transgenic for Xist on chromosome 11, we have now shown that during this important time period the inactivating chromosome moves into the heterochromatic compartment of the nucleus

Transgenic animals

Through increase understanding of biology, humanity has gained new and powerful tools for research and medicine. One of the tools is transgenic technology. The use of this technology to create transgenic animals has produced new disease and research models, bioreactors for transpharming pharmaceutical proteins, and has the potential to produce viable xenotransplant organs. Modification of animals raises the ethical dilemma of benefits to society vs. animal suffering. Our recommendations include increasing public awareness about the benefits of transgenic animals, along with explaining the current regulations both governmental and self imposed that guide the scientific community in making ethical decisions about creating transgenic animals

High-throughput screening identifies idarubicin as a preferential inhibitor of smooth muscle versus endothelial cell proliferation.

Intimal hyperplasia is the cause of the recurrent occlusive vascular disease (restenosis). Drugs currently used to treat restenosis effectively inhibit smooth muscle cell (SMC) proliferation, but also inhibit the growth of the protective luminal endothelial cell (EC) lining, leading to thrombosis. To identify compounds that selectively inhibit SMC versus EC proliferation, we have developed a high-throughput screening (HTS) format using human cells and have employed this to screen a multiple compound collection (NIH Clinical Collection). We developed an automated, accurate proliferation assay in 96-well plates using human aortic SMCs and ECs. Using this HTS format we screened a 447-drug NIH Clinical Library. We identified 11 compounds that inhibited SMC proliferation greater than 50%, among which idarubicin exhibited a unique feature of preferentially inhibiting SMC versus EC proliferation. Concentration-response analysis revealed this differential effect most evident over an ∼10 nM-5 µM window. In vivo testing of idarubicin in a rat carotid injury model at 14 days revealed an 80% reduction of intimal hyperplasia and a 45% increase of lumen size with no significant effect on re-endothelialization. Taken together, we have established a HTS assay of human vascular cell proliferation, and identified idarubicin as a selective inhibitor of SMC versus EC proliferation both in vitro and in vivo. Screening of larger and more diverse compound libraries may lead to the discovery of next-generation therapeutics that can inhibit intima hyperplasia without impairing re-endothelialization

High Throughput Small Molecule Screen for Reactivation of <i>FMR1</i> in Fragile X Syndrome Human Neural Cells

Fragile X syndrome (FXS) is the most common inherited cause of autism and intellectual disability. The majority of FXS cases are caused by transcriptional repression of the FMR1 gene due to epigenetic changes that are not recapitulated in current animal disease models. FXS patient induced pluripotent stem cell (iPSC)-derived gene edited reporter cell lines enable novel strategies to discover reactivators of FMR1 expression in human cells on a much larger scale than previously possible. Here, we describe the workflow using FXS iPSC-derived neural cell lines to conduct a massive, unbiased screen for small molecule activators of the FMR1 gene. The proof-of-principle methodology demonstrates the utility of human stem-cell-based methodology for the untargeted discovery of reactivators of the human FMR1 gene that can be applied to other diseases

Bacillimidazoles A&minus;F, Imidazolium-Containing Compounds Isolated from a Marine Bacillus

Chemical investigations of a marine sponge-associated Bacillus revealed six new imidazolium-containing compounds, bacillimidazoles A&ndash;F (1&ndash;6). Previous reports of related imidazolium-containing natural products are rare. Initially unveiled by timsTOF (trapped ion mobility spectrometry) MS data, extensive HRMS and 1D and 2D NMR analyses enabled the structural elucidation of 1&ndash;6. In addition, a plausible biosynthetic pathway to bacillimidazoles is proposed based on isotopic labeling experiments and invokes the highly reactive glycolytic adduct 2,3-butanedione. Combined, the results of structure elucidation efforts, isotopic labeling studies and bioinformatics suggest that 1&ndash;6 result from a fascinating intersection of primary and secondary metabolic pathways in Bacillus sp. WMMC1349. Antimicrobial assays revealed that, of 1&ndash;6, only compound six displayed discernible antibacterial activity, despite the close structural similarities shared by all six natural products

Dose-responses of HuAoSMCs and HuAoECs to idarubicin treatment.

<p>Proliferation of SMCs or ECs in the presence of various concentrations of idarubicin or resveratrol was assayed in a 96-well plate and handled by the same robotic system as described in Materials and Methods. Each data point is a mean ± SD of triplicates, *P<0.05.</p

Lack of effect of idarubicin on re-endothelialization in balloon-injured rat carotid arteries.

<p>Following balloon angioplasty, idarubicin was applied locally around the injured arteries. For determination of re-endothelialization, immunostaining of CD31 was performed on the sections of carotid arteries collected on day 14 post angioplasty, as described in detail in Materials and Methods. Shown in A and B are representative immunostained sections from the arteries treated with vehicle (DMSO) and idarubicin, respectively. Arrow heads point to IEL. A section of uninjured right carotid artery (C) shows CD31 staining of the undisrupted endothelial layer (see the brown circle). The relative score of re-endothelialization (stained versus total circumference) was quantified with the data pooled from 5 rats in each treatment group (D). Each bar represents a mean ± SEM.</p

Inhibitory effect of idarubicin on intimal hyperplasia in balloon-injured rat carotid arteries.

<p>Following balloon angioplasty, idarubicin was applied locally around the injured arteries. Morphometric analysis was performed on the sections of carotid arteries collected on day 14 post angioplasty, as described in detail in Materials and Methods. Shown in A and B are representative H&E-stained sections from the arteries treated with vehicle (DMSO) and idarubicin, respectively. Arrow heads point to IEL. Statistics of the area ratio of intima versus media (C), residual lumen (the ratio of lumen area versus IEL area) (D), and EEL length (E) were calculated with the data pooled from 5 rats in each treatment group. Each bar represents a mean ± SEM (*P<0.05).</p

Differential inhibition of HuAoSMC versus HuAoEC proliferation by the 11 hits selected from the NIH Clinical Collection.

<p><b>A).</b> HTS of the NIH Clinical Collection was performed using SMCs as well as ECs, as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089349#pone-0089349-g002" target="_blank">Figure 2</a>. Percent inhibition of SMC proliferation (Black) by the 11 hits was compared with that of EC proliferation (red). The vertical bar highlights greater inhibition of SMC versus EC proliferation by idarubicin, which is opposite to the effect of most of the other hits. <b>B).</b> Inhibition of cell proliferation by rapamycin was compared between HuAoSMCs and HuAoECs. The experiment was performed using the automated assay system as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089349#pone-0089349-g001" target="_blank">Figure 1</a>. Rapamycin was added to a final concentration of 200 nM. Cell number was assessed by Cell Titer Glo assay. Each bar represents a mean ± SD (*P<0.05).</p