Tissue factor in antiphospholipid antibody-induced pregnancy loss:a pro-inflammatory molecule

Fetal loss in patients with antiphospholipid antibodies (aPL) has been ascribed to thrombosis of placental vessels. However, we have shown that inflammation, specifically complement activation with generation of the anaphylotoxin C5a, is an essential mediator of fetal injury. We have analysed the role of tissue factor (TF) in a mouse model of aPL-induced pregnancy loss. TF is the major cellular activator of the coagulation cascade but also has cell signaling activity. Mice that received aPL-IgG showed strong TF staining throughout the decidua and on embryonic debris. This TF staining was not associated with either fibrin staining or thrombi in deciduas. The absence of fibrin deposition and thrombi suggests that TF-dependent activation of coagulation does not mediate aPL-induced pregnancy loss. We found that either blockade of TF with a monoclonal antibody in wild type mice or a genetic reduction of TF prevented aPL-induced inflammation and pregnancy loss indicated a pathogenic role for TF in aPL-induced pregnancy complications. In response to aPL-generated C5a, neutrophils express TF potentiating inflammation in the deciduas and leading to miscarriages. Importantly, we showed that TF in myeloid cells, but not fetal-derived cells (trophoblasts), was associated with fetal injury, suggesting that the site for pathologic TF expression is neutrophils. We found that TF expression in neutrophils contributes to respiratory burst and subsequent trophoblast injury and pregnancy loss induced by aPL. The identification of TF, acting as an important pro-inflammatory mediator in aPL-induced fetal injury, provides a new target for therapy to prevent pregnancy loss in the aPL syndrome

Diversity of Eukaryotic DNA Replication Origins Revealed by Genome-Wide Analysis of Chromatin Structure

Eukaryotic DNA replication origins differ both in their efficiency and in the characteristic time during S phase when they become active. The biological basis for these differences remains unknown, but they could be a consequence of chromatin structure. The availability of genome-wide maps of nucleosome positions has led to an explosion of information about how nucleosomes are assembled at transcription start sites, but no similar maps exist for DNA replication origins. Here we combine high-resolution genome-wide nucleosome maps with comprehensive annotations of DNA replication origins to identify patterns of nucleosome occupancy at eukaryotic replication origins. On average, replication origins contain a nucleosome depleted region centered next to the ACS element, flanked on both sides by arrays of well-positioned nucleosomes. Our analysis identified DNA sequence properties that correlate with nucleosome occupancy at replication origins genome-wide and that are correlated with the nucleosome-depleted region. Clustering analysis of all annotated replication origins revealed a surprising diversity of nucleosome occupancy patterns. We provide evidence that the origin recognition complex, which binds to the origin, acts as a barrier element to position and phase nucleosomes on both sides of the origin. Finally, analysis of chromatin reconstituted in vitro reveals that origins are inherently nucleosome depleted. Together our data provide a comprehensive, genome-wide view of chromatin structure at replication origins and suggest a model of nucleosome positioning at replication origins in which the underlying sequence occludes nucleosomes to permit binding of the origin recognition complex, which then (likely in concert with nucleosome modifiers and remodelers) positions nucleosomes adjacent to the origin to promote replication origin function

Dissecting Nucleosome Free Regions by a Segmental Semi-Markov Model

BACKGROUND: Nucleosome free regions (NFRs) play important roles in diverse biological processes including gene regulation. A genome-wide quantitative portrait of each individual NFR, with their starting and ending positions, lengths, and degrees of nucleosome depletion is critical for revealing the heterogeneity of gene regulation and chromatin organization. By averaging nucleosome occupancy levels, previous studies have identified the presence of NFRs in the promoter regions across many genes. However, evaluation of the quantitative characteristics of individual NFRs requires an NFR calling method. METHODOLOGY: In this study, we propose a statistical method to identify the patterns of NFRs from a genome-wide measurement of nucleosome occupancy. This method is based on an appropriately designed segmental semi-Markov model, which can capture each NFR pattern and output its quantitative characterizations. Our results show that the majority of the NFRs are located in intergenic regions or promoters with a length of about 400-600bp and varying degrees of nucleosome depletion. Our quantitative NFR mapping allows for an investigation of the relative impacts of transcription machinery and DNA sequence in evicting histones from NFRs. We show that while both factors have significant overall effects, their specific contributions vary across different subtypes of NFRs. CONCLUSION: The emphasis of our approach on the variation rather than the consensus of nucleosome free regions sets the tone for enabling the exploration of many subtler dynamic aspects of chromatin biology

Quantification of Epithelial Cell Differentiation in Mammary Glands and Carcinomas from DMBA- and MNU-Exposed Rats

Rat mammary carcinogenesis models have been used extensively to study breast cancer initiation, progression, prevention, and intervention. Nevertheless, quantitative molecular data on epithelial cell differentiation in mammary glands of untreated and carcinogen-exposed rats is limited. Here, we describe the characterization of rat mammary epithelial cells (RMECs) by multicolor flow cytometry using antibodies against cell surface proteins CD24, CD29, CD31, CD45, CD49f, CD61, Peanut Lectin, and Thy-1, intracellular proteins CK14, CK19, and FAK, along with phalloidin and Hoechst staining. We identified the luminal and basal/myoepithelial populations and actively dividing RMECs. In inbred rats susceptible to mammary carcinoma development, we quantified the changes in differentiation of the RMEC populations at 1, 2, and 4 weeks after exposure to mammary carcinogens DMBA and MNU. DMBA exposure did not alter the percentage of basal or luminal cells, but upregulated CD49f (Integrin α6) expression and increased cell cycle activity. MNU exposure resulted in a temporary disruption of the luminal/basal ratio and no CD49f upregulation. When comparing DMBA- or MNU-induced mammary carcinomas, the RMEC differentiation profiles are indistinguishable. The carcinomas compared with mammary glands from untreated rats, showed upregulation of CD29 (Integrin β1) and CD49f expression, increased FAK (focal adhesion kinase) activation especially in the CD29hi population, and decreased CD61 (Integrin β3) expression. This study provides quantitative insight into the protein expression phenotypes underlying RMEC differentiation. The results highlight distinct RMEC differentiation etiologies of DMBA and MNU exposure, while the resulting carcinomas have similar RMEC differentiation profiles. The methodology and data will enhance rat mammary carcinogenesis models in the study of the role of epithelial cell differentiation in breast cancer

Contribution of histone sequence preferences to nucleosome organization: proposed definitions and methodology

We propose definitions and procedures for comparing nucleosome maps and discuss current agreement and disagreement on the effect of histone sequence preferences on nucleosome organization in vivo

Zebrafish brd2a and brd2b are paralogous members of the bromodomain-ET (BET) family of transcriptional coregulators that show structural and expression divergence

<p>Abstract</p> <p>Background</p> <p>Brd2 belongs to the bromodomain-extraterminal domain (BET) family of transcriptional co-regulators, and functions as a pivotal histone-directed recruitment scaffold in chromatin modification complexes affecting signal-dependent transcription. Brd2 facilitates expression of genes promoting proliferation and is implicated in apoptosis and in egg maturation and meiotic competence in mammals; it is also a susceptibility gene for juvenile myoclonic epilepsy (JME) in humans. The <it>brd2 </it>ortholog in <it>Drosophila </it>is a maternal effect, embryonic lethal gene that regulates several homeotic loci, including Ultrabithorax. Despite its importance, there are few systematic studies of <it>Brd2 </it>developmental expression in any organism. To help elucidate both conserved and novel gene functions, we cloned and characterized expression of <it>brd2 </it>cDNAs in zebrafish, a vertebrate system useful for genetic analysis of development and disease, and for study of the evolution of gene families and functional diversity in chordates.</p> <p>Results</p> <p>We identify cDNAs representing two paralogous <it>brd2 </it>loci in zebrafish, <it>brd2a </it>on chromosome 19 and <it>brd2b </it>on chromosome 16. By sequence similarity, syntenic and phylogenetic analyses, we present evidence for structural divergence of <it>brd2 </it>after gene duplication in fishes. <it>brd2 </it>paralogs show potential for modular domain combinations, and exhibit distinct RNA expression patterns throughout development. RNA <it>in situ </it>hybridizations in oocytes and embryos implicate <it>brd2a </it>and <it>brd2b </it>as maternal effect genes involved in egg polarity and egg to embryo transition, and as zygotic genes important for development of the vertebrate nervous system and for morphogenesis and differentiation of the digestive tract. Patterns of <it>brd2 </it>developmental expression in zebrafish are consistent with its proposed role in <it>Homeobox </it>gene regulation.</p> <p>Conclusion</p> <p>Expression profiles of zebrafish <it>brd2 </it>paralogs support a role in vertebrate developmental patterning and morphogenesis. Our study uncovers both maternal and zygotic contributions of <it>brd2</it>, the analysis of which may provide insight into the earliest events in vertebrate development, and the etiology of some forms of epilepsy, for which zebrafish is an important model. Knockdowns of <it>brd2 </it>paralogs in zebrafish may now test proposed function and interaction with homeotic loci in vertebrates, and help reveal the extent to which functional novelty or partitioning has occurred after gene duplication.</p

Autoacetylation of the Ralstonia solanacearum Effector PopP2 Targets a Lysine Residue Essential for RRS1-R-Mediated Immunity in Arabidopsis

Type III effector proteins from bacterial pathogens manipulate components of host immunity to suppress defence responses and promote pathogen development. In plants, host proteins targeted by some effectors called avirulence proteins are surveyed by plant disease resistance proteins referred to as “guards”. The Ralstonia solanacearum effector protein PopP2 triggers immunity in Arabidopsis following its perception by the RRS1-R resistance protein. Here, we show that PopP2 interacts with RRS1-R in the nucleus of living plant cells. PopP2 belongs to the YopJ-like family of cysteine proteases, which share a conserved catalytic triad that includes a highly conserved cysteine residue. The catalytic cysteine mutant PopP2-C321A is impaired in its avirulence activity although it is still able to interact with RRS1-R. In addition, PopP2 prevents proteasomal degradation of RRS1-R, independent of the presence of an integral PopP2 catalytic core. A liquid chromatography/tandem mass spectrometry analysis showed that PopP2 displays acetyl-transferase activity leading to its autoacetylation on a particular lysine residue, which is well conserved among all members of the YopJ family. These data suggest that this lysine residue may correspond to a key binding site for acetyl-coenzyme A required for protein activity. Indeed, mutation of this lysine in PopP2 abolishes RRS1-R-mediated immunity. In agreement with the guard hypothesis, our results favour the idea that activation of the plant immune response by RRS1-R depends not only on the physical interaction between the two proteins but also on its perception of PopP2 enzymatic activity

Varieties of living things: Life at the intersection of lineage and metabolism

publication-status: Publishedtypes: Articl

Genetic Diversity among Enterococcus faecalis

Enterococcus faecalis, a ubiquitous member of mammalian gastrointestinal flora, is a leading cause of nosocomial infections and a growing public health concern. The enterococci responsible for these infections are often resistant to multiple antibiotics and have become notorious for their ability to acquire and disseminate antibiotic resistances. In the current study, we examined genetic relationships among 106 strains of E. faecalis isolated over the past 100 years, including strains identified for their diversity and used historically for serotyping, strains that have been adapted for laboratory use, and isolates from previously described E. faecalis infection outbreaks. This collection also includes isolates first characterized as having novel plasmids, virulence traits, antibiotic resistances, and pathogenicity island (PAI) components. We evaluated variation in factors contributing to pathogenicity, including toxin production, antibiotic resistance, polymorphism in the capsule (cps) operon, pathogenicity island (PAI) gene content, and other accessory factors. This information was correlated with multi-locus sequence typing (MLST) data, which was used to define genetic lineages. Our findings show that virulence and antibiotic resistance traits can be found within many diverse lineages of E. faecalis. However, lineages have emerged that have caused infection outbreaks globally, in which several new antibiotic resistances have entered the species, and in which virulence traits have converged. Comparing genomic hybridization profiles, using a microarray, of strains identified by MLST as spanning the diversity of the species, allowed us to identify the core E. faecalis genome as consisting of an estimated 2057 unique genes

Gastrointestinal decontamination in the acutely poisoned patient

ObjectiveTo define the role of gastrointestinal (GI) decontamination of the poisoned patient.Data sourcesA computer-based PubMed/MEDLINE search of the literature on GI decontamination in the poisoned patient with cross referencing of sources.Study selection and data extractionClinical, animal and in vitro studies were reviewed for clinical relevance to GI decontamination of the poisoned patient.Data synthesisThe literature suggests that previously, widely used, aggressive approaches including the use of ipecac syrup, gastric lavage, and cathartics are now rarely recommended. Whole bowel irrigation is still often recommended for slow-release drugs, metals, and patients who "pack" or "stuff" foreign bodies filled with drugs of abuse, but with little quality data to support it. Activated charcoal (AC), single or multiple doses, was also a previous mainstay of GI decontamination, but the utility of AC is now recognized to be limited and more time dependent than previously practiced. These recommendations have resulted in several treatment guidelines that are mostly based on retrospective analysis, animal studies or small case series, and rarely based on randomized clinical trials.ConclusionsThe current literature supports limited use of GI decontamination of the poisoned patient