The construction of customized nucleosomal arrays

A simple, efficient, and reliable method is demonstrated for cloning long tandem arrays of the 601 nucleosomal positioning sequence. In addition, it is shown that such long arrays can be ligated together in vitro with high efficiency. By combining these two procedures it becomes straightforward to synthesize customized arrays that contain different (or variable) nucleosomal repeat lengths (NRLs) and monosome units bearing chemical modifications such as fluorophores, methyl groups, and reaction sites. This is, therefore, an enabling technology for the in vitro study of chromatin structure and function.     

Cancer therapy

In recent years a growing recognition that molecularly-targeted therapies face formidable obstacles has revived interest in more generic tumor cell phenotypes that could be exploited for therapy. Two recent reports demonstrate that cancer cell survival is critically dependent on the activity of MTH1, a nucleotide pyrophosphatase that converts the oxidized nucleotides 8-oxo-dGTP and 2-OH-dATP to the corresponding monophosphates, thus preventing their incorporation into genomic DNA. Tumor cells frequently overexpress MTH1, probably because malignant transformation creates oxidative stress that renders the nucleotide pool highly vulnerable to oxidation. As a result, MTH1 inhibition in cancer cells results in accumulation and incorporation of 8-oxo-dGTP and 2-OH-dATP into DNA, leading to DNA damage and cell death. This toxic effect is highly cancer cell-specific, as MTH1 is generally dispensable for the survival of normal, untransformed cells. Importantly, MTH1 proves to be a “druggable” enzyme that can be inhibited both by an existing protein kinase inhibitor drug, crizotinib, and by novel compounds identified through screening. Inhibition of MTH1 leading to toxic accumulation of oxidized nucleotides specifically in tumor cells therefore represents an example of a “non-personalised” approach to cancer therapy.     

Long‐Term Stability of Primary Rat Hepatocytes in Micropatterned Cocultures

Primary hepatocytes display functional and structural instability in standard monoculture systems. We have previously developed a model in which primary hepatocytes are organized in domains of empirically optimized dimensions and surrounded by murine embryonic fibroblasts (HepatoPac?). Here, we assess the long‐term phenotype of freshly isolated and cryopreserved rat hepatocytes in a 96‐well HepatoPac format. The viability, cell polarity (actin microfilaments, bile canaliculi), and functions (albumin, urea, Phase I/II enzymes, transporters) of fresh and cryopreserved rat hepatocytes were retained in HepatoPac at similar levels for at least 4 weeks as opposed to rapidly declining over 5 days in collagen/Matrigel? sandwich cultures. Pulse or continuous exposure of rat HepatoPac to GW‐7647, a selective agonist of PPARα, caused reproducible induction of CYP4A1 and 3‐hydroxy‐3‐methylglutaryl‐CoA synthase over 4 weeks. In conclusion, rat HepatoPac in a 96‐well format can be used for chronic dosing of highly functional hepatocytes and assessment of perturbed hepatocellular pathways. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:204‐212, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21469     

Genome size evolution of the extant lycophytes and ferns

Ferns and lycophytes have remarkably large genomes. However, little is known about how their genome size evolved in fern lineages. To explore the origins and evolution of chromosome numbers and genome size in ferns, we used flow cytometry to measure the genomes of 240 species (255 samples) of extant ferns and lycophytes comprising 27 families and 72 genera, of which 228 species (242 samples) represent new reports. We analyzed correlations among genome size, spore size, chromosomal features, phylogeny, and habitat type preference within a phylogenetic framework. We also applied ANOVA and multinomial logistic regression analysis to preference of habitat type and genome size. Using the phylogeny, we conducted ancestral character reconstruction for habitat types and tested whether genome size changes simultaneously with shifts in habitat preference. We found that 2C values had weak phylogenetic signal, whereas the base number of chromosomes (x) had a strong phylogenetic signal. Furthermore, our analyses revealed a positive correlation between genome size and chromosome traits, indicating that the base number of chromosomes (x), chromosome size, and polyploidization may be primary contributors to genome expansion in ferns and lycophytes. Genome sizes in different habitat types varied significantly and were significantly correlated with habitat types; specifically, multinomial logistic regression indicated that species with larger 2C values were more likely to be epiphytes. Terrestrial habitat is inferred to be ancestral for both extant ferns and lycophytes, whereas transitions to other habitat types occurred as the major clades emerged. Shifts in habitat types appear be followed by periods of genomic stability. Based on these results, we inferred that habitat type changes and multiple whole-genome duplications have contributed to the formation of large genomes of ferns and their allies during their evolutionary history.     

Comparison of the Iowa Reference Algorithm to the Heidelberg Spectralis optical coherence tomography segmentation algorithm

For spectral‐domain optical coherence tomography (SD‐OCT) studies of neurodegeneration, it is important to understand how segmentation algorithms differ in retinal layer thickness measurements, segmentation error locations and the impact of manual correction. Using macular SD‐OCT images of frontotemporal degeneration patients and controls, we compare the individual and aggregate retinal layer thickness measurements provided by two commonly used algorithms, the Iowa Reference Algorithm and Heidelberg Spectralis, with manual correction of significant segmentation errors. We demonstrate small differences of most retinal layer thickness measurements between these algorithms. Outer sectors of the Early Treatment Diabetic Retinopathy Study grid require a greater percent of eyes to be corrected than inner sectors of the retinal nerve fiber layer (RNFL). Manual corrections affect thickness measurements mildly, resulting in at most a 5% change in RNFL thickness. Our findings can inform researchers how to best use different segmentation algorithms when comparing retinal layer thicknesses.     

尊重斯德哥尔摩的历史中心—骑士岛的激活改造

瑞典国家财产委员会拥有骑士岛的所有权与管理权，并计划对该岛上所有的公共空间进行更新和开发，以提高其可达性与吸引力。该项目的核心是找到一种更新和修复岛屿的方法，从而在尊重历史价值的同时满足现代功能需求。对骑士岛南部的改造是岛上公共空间更新的第一部分。设计的关键条件是沿滨水区域创造可以供人步行与停坐的大面积空间，并在保持开放海港氛围的同时，对旧的道路铺装进行管理。设计者设计了一套灵活使用公共空间的综合解决方案，将开放空间与之前的码头一样，沿着水滨的形态进行布局。     

An improved system for expressing pancreatic ribonuclease in Escherichia coli

An improved method for expressing and purifying bovine pancreatic ribonuclease from a synthetic gene using the lambda promoter controlled by a temperature-sensitive repressor is described. The procedure involves isolation in the presence of a refolding buffer containing oxidized and reduced glutathione, under conditions where RNase can refold, but where proteases presumably do not. Yields are approx. 2 mg purified protein per 1 ferment.