Opposing ISWI- and CHD-class chromatin remodeling activities orchestrate heterochromatic DNA repair

Heterochromatin is a barrier to DNA repair that correlates strongly with elevated somatic mutation in cancer. CHD class II nucleosome remodeling activity (specifically CHD3.1) retained by KAP-1 increases heterochromatin compaction and impedes DNA double-strand break (DSB) repair requiring Artemis. This obstruction is alleviated by chromatin relaxation via ATM-dependent KAP-1S824 phosphorylation (pKAP-1) and CHD3.1 dispersal from heterochromatic DSBs; however, how heterochromatin compaction is actually adjusted after CHD3.1 dispersal is unknown. In this paper, we demonstrate that Artemis-dependent DSB repair in heterochromatin requires ISWI (imitation switch)-class ACF1–SNF2H nucleosome remodeling. Compacted chromatin generated by CHD3.1 after DNA replication necessitates ACF1–SNF2H–mediated relaxation for DSB repair. ACF1–SNF2H requires RNF20 to bind heterochromatic DSBs, underlies RNF20-mediated chromatin relaxation, and functions downstream of pKAP-1–mediated CHD3.1 dispersal to enable DSB repair. CHD3.1 and ACF1–SNF2H display counteractive activities but similar histone affinities (via the plant homeodomains of CHD3.1 and ACF1), which we suggest necessitates a two-step dispersal and recruitment system regulating these opposing chromatin remodeling activities during DSB repair.     

Antitumor action and immune activation through cooperation of bee venom secretory phospholipase A2 and phosphatidylinositol-(3,4)-bisphosphate

We evaluated tumor cell growth modulation by bee venom secretory phospholipase A2 (bv-sPLA2) and phosphatidylinositol-(3,4)-bisphosphate as well as potential cooperative effects. In addition, the immunomodulatory impact of tumor cell treatment was examined by monitoring changes in phenotype and function of monocyte-derived dendritic cells (moDCs) cocultured with pretreated tumor cells. Bv-sPLA2 or phosphatidylinositol-(3,4)-bisphosphate alone displayed moderate effects on the proliferation of A498 renal cell carcinoma cells, T-47D breast cancer cells, DU145 prostate cancer cells and BEAS-2B transformed lung cells. However, when bv-sPLA2 was coadministered with phosphatidylinositol-(3,4)-bisphosphate a potent inhibition of [³H] thymidine incorporation into all tested cell lines occurred. This inhibition was due to massive cell lysis that reduced the number of cells with proliferative capacity. Importantly, tumor cell lysates generated with bv-sPLA2 plus phosphatidylinositol-(3,4)-bisphosphate induced maturation of human moDCs demonstrated by enhanced expression of CD83 and improved stimulation in allogeneic mixed leukocyte reactions. Our data demonstrate that bv-sPLA2 and phosphatidylinositol-(3,4)-bisphosphate synergistically generate tumor lysates which enhance the maturation of immunostimulatory human monocyte-derived dendritic cells. Such tumor lysates which represent complex mixtures of tumor antigens and simultaneously display potent adjuvant properties meet all requirements of a tumor vaccine.     

Production of a recombinant polyester-cleaving hydrolase from Thermobifida fusca in Escherichia coli

The hydrolase (Thermobifida fusca hydrolase; TfH) from T. fusca was produced in Escherichia coli as fusion protein using the OmpA leader sequence and a His₆ tag. Productivity could be raised more than 100-fold. Both batch and fed-batch cultivations yield comparable cell specific productivities whereas volumetric productivities differ largely. In the fed-batch cultivations final rTfH concentrations of 0.5 g L⁻¹ could be achieved. In batch cultivations the generated rTfH is translocated to the periplasm wherefrom it is completely released into the extracellular medium. In fed-batch runs most of the produced rTfH remains as soluble protein in the cytoplasm and only a fraction of about 35% is translocated to the periplasm. Migration of periplasmic proteins in the medium is obviously coupled with growth rate and this final transport step possibly plays an important role in product localization and efficacy of the Sec translocation process.     

Molecular characterization of microbial populations at two sites with differing reductive dechlorination abilities

This study compares three molecular techniques, including terminal restriction fragment length polymorphism (T-RFLP), RFLP analysis with clone sequencing, and quantitative PCR (Q-PCR) for surveying differences in microbial communities at two contaminated field sites that exhibit dissimilar chlorinated solvent degradation activities. At the Idaho National Engineering and Environmental Laboratory (INEEL), trichloroethene (TCE) was completely converted to ethene during biostimulation with lactate. At Seal Beach, California, perchloroethene (PCE) was degraded only to cis-dichloroethene (cDCE) during biostimulation but was degraded to ethene after bioaugmentation with a dechlorinating culture containing Dehalococcoides strains. T-RFLP analysis showed that microbial community composition differed significantly between the two sites, but was similar within each site among wells that had low or no electron donor exposure. Analysis of INEEL clone libraries by RFLP with clone sequencing revealed a complex microbial population but did not identify any Dehalococcoides strains. Q-PCR targeting the 16S rRNA gene of Dehalococcoides strains – known for their unique capability to dechlorinate solvents completely to ethene – revealed a significant population at INEEL, but no detectable population at Seal Beach prior to bioaugmentation. Detection of Dehalococcoides by Q-PCR correlated with observed dechlorination activity and ethene production at both sites. Q-PCR showed that Dehalococcoides was present in even the pristine well at INEEL, suggesting that the difference in dechlorination ability at the two sites was due to the initial absence of this genus at Seal Beach. Of the techniques tested, Q-PCR quantification of specific dechlorinating species provided the most effective and direct prediction of community dechlorinating potential.     

Quantitative model of cell cycle arrest and cellular senescence in primary human fibroblasts

Primary human fibroblasts in tissue culture undergo a limited number of cell divisions before entering a non-replicative "senescent" state. At early population doublings (PD), fibroblasts are proliferation-competent displaying exponential growth. During further cell passaging, an increasing number of cells become cell cycle arrested and finally senescent. This transition from proliferating to senescent cells is driven by a number of endogenous and exogenous stress factors. Here, we have developed a new quantitative model for the stepwise transition from proliferating human fibroblasts (P) via reversibly cell cycle arrested (C) to irreversibly arrested senescent cells (S). In this model, the transition from P to C and to S is driven by a stress function γ and a cellular stress response function F which describes the time-delayed cellular response to experimentally induced irradiation stress. The application of this model based on senescence marker quantification at the single-cell level allowed to discriminate between the cellular states P, C, and S and delivers the transition rates between the P, C and S states for different human fibroblast cell types. Model-derived quantification unexpectedly revealed significant differences in the stress response of different fibroblast cell lines. Evaluating marker specificity, we found that SA-β-Gal is a good quantitative marker for cellular senescence in WI-38 and BJ cells, however much less so in MRC-5 cells. Furthermore we found that WI-38 cells are more sensitive to stress than BJ and MRC-5 cells. Thus, the explicit separation of stress induction from the cellular stress response, and the differentiation between three cellular states P, C and S allows for the first time to quantitatively assess the response of primary human fibroblasts towards endogenous and exogenous stress during cellular ageing.     

Jointly They Edit: Examining the Impact of Community Identification on Political Interaction in Wikipedia

Background

In their 2005 study, Adamic and Glance coined the memorable phrase ‘divided they blog’, referring to a trend of cyberbalkanization in the political blogosphere, with liberal and conservative blogs tending to link to other blogs with a similar political slant, and not to one another. As political discussion and activity increasingly moves online, the power of framing political discourses is shifting from mass media to social media.

Methodology/Principal Findings

Continued examination of political interactions online is critical, and we extend this line of research by examining the activities of political users within the Wikipedia community. First, we examined how users in Wikipedia choose to display their political affiliation. Next, we analyzed the patterns of cross-party interaction and community participation among those users proclaiming a political affiliation. In contrast to previous analyses of other social media, we did not find strong trends indicating a preference to interact with members of the same political party within the Wikipedia community.

Conclusions/Significance

Our results indicate that users who proclaim their political affiliation within the community tend to proclaim their identity as a ‘Wikipedian’ even more loudly. It seems that the shared identity of ‘being Wikipedian’ may be strong enough to triumph over other potentially divisive facets of personal identity, such as political affiliation.     

Recurrent Chromosome 22 Deletions in Osteoblastoma Affect Inhibitors of the Wnt/Beta-Catenin Signaling Pathway

Osteoblastoma is a bone forming tumor with histological features highly similar to osteoid osteoma; the discrimination between the tumor types is based on size and growth pattern. The vast majority of osteoblastomas are benign but there is a group of so-called aggressive osteoblastomas that can be diagnostically challenging at the histopathological level. The genetic aberrations required for osteoblastoma development are not known and no genetic difference between conventional and aggressive osteoblastoma has been reported. In order to identify recurrent genomic aberrations of importance for tumor development we applied cytogenetic and/or SNP array analyses on nine conventional and two aggressive osteoblastomas. The conventional osteoblastomas showed few or no acquired genetic aberrations while the aggressive tumors displayed heavily rearranged genomes. In one of the aggressive osteoblastomas, three neighboring regions in chromosome band 22q12 were homozygously deleted. Hemizygous deletions of these regions were found in two additional cases, one aggressive and one conventional. In total, 10 genes were recurrently and homozygously lost in osteoblastoma. Four of them are functionally involved in regulating osteogenesis and/or tumorigenesis. MN1 and NF2 have previously been implicated in the development of leukemia and solid tumors, and ZNRF3 and KREMEN1 are inhibitors of the Wnt/beta-catenin signaling pathway. In line with deletions of the latter two genes, high beta-catenin protein expression has previously been reported in osteoblastoma and aberrations affecting the Wnt/beta-catenin pathway have been found in other bone lesions, including osteoma and osteosarcoma.     

Phosphate and HEPES buffers potently affect the fibrillation and oligomerization mechanism of Alzheimer's Aβ peptide

The oligomerization of Aβ peptide into amyloid fibrils is a hallmark of Alzheimer’s disease. Due to its biological relevance, phosphate is the most commonly used buffer system for studying the formation of Aβ and other amyloid fibrils. Investigation into the characteristics and formation of amyloid fibrils frequently relies upon material formed in vitro, predominantly in phosphate buffers. Herein, we examine the effects on the fibrillation and oligomerization mechanism of Aβ peptide that occur due solely to the influence of phosphate buffer. We reveal that significant differences in amyloid fibrillation are observed due to fibrillation being initiated in phosphate or HEPES buffer (at physiological pH and temperature). Except for the differing buffer ions, all experimental parameters were kept constant. Fibril formation was assessed using fluorescently monitored kinetic studies, microscopy, X-ray fiber diffraction and infrared and nuclear magnetic resonance spectroscopies. Based on this set up, we herein reveal profound effects on the mechanism and speed of Aβ fibrillation. The three histidine residues at positions 6, 13 and 14 of Aβ(1–40) are instrumental in these mechanistic changes. We conclude that buffer plays a more significant role in fibril formation than has been generally acknowledged.     

Parvulin 17 promotes microtubule assembly by its peptidyl-prolyl cis/trans isomerase activity

The parvulin-type peptidyl-prolyl cis/trans isomerases (PPIases) have been shown to be involved in tumor progression and the pathogenesis of Alzheimer's disease and were therefore a subject of intense research. Here, we describe a role for parvulin 17 in microtubule assembly. Co-precipitation experiments and sedimentation assays demonstrated that parvulin 17 interacts with tubulin in a GTP-dependent manner and thereby promotes the formation of microtubules, as shown by transmission electron microscopy and a microtubule polymerization assay. The microtubule-assembly-promoting properties of parvulin 17 seem to depend on its PPIase activity. Thus, catalytic deficient variants of parvulin 17 were not able to promote microtubule formation. Accordingly, inhibitors of parvulin 17 activity also prevent parvulin-catalyzed tubulin polymerization. The analysis of tubulin interaction sites on parvulin using peptide microarrays revealed that tubulin interacts with the substrate binding pocket of parvulin. Additionally, β-tubulin peptide scan on microarrays demonstrates interaction of parvulin 17 with an Arg-Pro-Asp motif corresponding to proline residue 87 of β-tubulin. Confocal laser scanning microscopy points to a function of parvulin 17 in microtubule dynamics as well. Parvulin 17 is predominantly found in the cytosol and colocalizes with microtubules.