Immediate chromatin immunoprecipitation and on-bead quantitative PCR analysis: a versatile and rapid ChIP procedure

Genome-wide chromatin immunoprecipitation (ChIP) studies have brought significant insight into the genomic localization of chromatin-associated proteins and histone modifications. The large amount of data generated by these analyses, however, require approaches that enable rapid validation and analysis of biological relevance. Furthermore, there are still protein and modification targets that are difficult to detect using standard ChIP methods. To address these issues, we developed an immediate chromatin immunoprecipitation procedure which we call ZipChip. ZipChip significantly reduces the time and increases sensitivity allowing for rapid screening of multiple loci. Here we describe how ZipChIP enables detection of histone modifications (H3K4 mono- and trimethylation) and two yeast histone demethylases, Jhd2 and Rph1, which were previously difficult to detect using standard methods. Furthermore, we demonstrate the versatility of ZipChIP by analyzing the enrichment of the histone deacetylase Sir2 at heterochromatin in yeast and enrichment of the chromatin remodeler, PICKLE, at euchromatin in Arabidopsis thaliana.     

Running with the Red Queen: the role of biotic conflicts in evolution

What are the causes of natural selection? Over 40 years ago, Van Valen proposed the Red Queen hypothesis, which emphasized the primacy of biotic conflict over abiotic forces in driving selection. Species must continually evolve to survive in the face of their evolving enemies, yet on average their fitness remains unchanged. We define three modes of Red Queen coevolution to unify both fluctuating and directional selection within the Red Queen framework. Empirical evidence from natural interspecific antagonisms provides support for each of these modes of coevolution and suggests that they often operate simultaneously. We argue that understanding the evolutionary forces associated with interspecific interactions requires incorporation of a community framework, in which new interactions occur frequently. During their early phases, these newly established interactions are likely to drive fast evolution of both parties. We further argue that a more complete synthesis of Red Queen forces requires incorporation of the evolutionary conflicts within species that arise from sexual reproduction. Reciprocally, taking the Red Queen''s perspective advances our understanding of the evolution of these intraspecific conflicts.     

3D models of human ERα and ERβ complexed with 5-androsten-3β,17β-diol

Recently, binding of 5-androsten-3β,17β-diol (Δ(5)-androstenediol) to human estrogen receptor-beta (ERβ) was found to repress microglia-mediated inflammation, which is associated with various neurodegenerative diseases, such as multiple sclerosis. In contrast, binding of estradiol to ERβ resulted in little or no repression of microglia-mediated inflammation. Binding of Δ(5)-androstenediol to ERβ, as well as to ERα, is unexpected because unlike estradiol, Δ(5)-androstenediol has a saturated A ring and a C19 methyl group. To begin to elucidate the interaction of Δ(5)-androstenediol with both ERs, we constructed 3D models of Δ(5)-androstenediol with human ERα and ERβ for comparison with the crystal structures of estradiol in ERα and ERβ. Conformational flexibility in human ERα and ERβ accommodates the C19 methyl on Δ(5)-androstenediol. This conformational flexibility may be relevant for binding of other Δ(5)-steroids with C19 methyl substituents, such as 25-hydroxycholesterol and 27-hydroxycholesterol, to ERs.     

Secreted phosphoprotein 1 binds integrins to initiate multiple cell signaling pathways,including FRAP1/mTOR,to support attachment and force-generated migration of trophectoderm cells

Attachment and migration of trophectoderm (Tr) cells, hallmarks of blastocyst implantation in mammals, are unique uterine events. Secreted phosphoprotein 1 (SPP1) in the uterus binds integrins on conceptus Tr and uterine luminal epithelium (LE), affecting cell–cell and cell–matrix interactions. The signal transduction pathways activated by SPP1 and integrins in conceptuses have not been elucidated. Results of this study demonstrate that SPP1 binds αvβ3 and α5β1 integrins to induce focal adhesion assembly, a prerequisite for adhesion and migration of Tr, through activation of: 1) P70S6K via crosstalk between FRAP1/mTOR and MAPK pathways; 2) mTOR, PI3K, MAPK3/MAPK1 (Erk1/2) and MAPK14 (p38) signaling to stimulate Tr cell migration; and 3) focal adhesion assembly and myosin II motor activity to induce migration of Tr cells. These cell signaling pathways, acting in concert, mediate adhesion, migration and cytoskeletal remodeling of Tr cells essential for expansion and elongation of conceptuses and attachment to uterine LE for implantation.     

Inhibitors of foot and mouth disease virus targeting a novel pocket of the RNA-dependent RNA polymerase

Background

Foot-and-Mouth Disease Virus (FMDV) is a picornavirus that infects cloven-hoofed animals and leads to severe losses in livestock production. In the case of an FMD outbreak, emergency vaccination requires at least 7 days to trigger an effective immune response. There are currently no approved inhibitors for the treatment or prevention of FMDV infections.

Methodology/Principal Findings

Using a luciferase-based assay we screened a library of compounds and identified seven novel inhibitors of 3Dpol, the RNA-dependent RNA polymerase of FMDV. The compounds inhibited specifically 3Dpol (IC₅₀s from 2-17 µM) and not other viral or bacterial polymerases. Enzyme kinetic studies on the inhibition mechanism by compounds 5D9 and 7F8 showed that they are non-competitive inhibitors with respect to NTP and nucleic acid substrates. Molecular modeling and docking studies into the 3Dpol structure revealed an inhibitor binding pocket proximal to, but distinct from the 3Dpol catalytic site. Residues surrounding this pocket are conserved among all 60 FMDV subtypes. Site directed mutagenesis of two residues located at either side of the pocket caused distinct resistance to the compounds, demonstrating that they indeed bind at this site. Several compounds inhibited viral replication with 5D9 suppressing virus production in FMDV-infected cells with EC₅₀ = 12 µM and EC₉₀ = 20 µM).

Significance

We identified several non-competitive inhibitors of FMDV 3Dpol that target a novel binding pocket, which can be used for future structure-based drug design studies. Such studies can lead to the discovery of even more potent antivirals that could provide alternative or supplementary options to contain future outbreaks of FMD.     

Livelihood Dynamics Across a Variable Flooding Regime

Variability in environmental phenomena such as fire, flooding, and weather-related events can have significant impacts for social and environmental systems and their coupled interactions. Livelihoods systems reliant on the natural environment can be disrupted or eliminated, while associated governance regimes require negotiation to ensure equitable and sustainable management responses. These patterns can be particularly pronounced within areas prone to flooding, as these sites can experience variability in the location, timing, amount, and duration of flooding events. While research within the social and natural sciences has evaluated these dynamics within flooding regimes, the coupled interactions can be underemphasized even though they are integral in producing livelihood systems and possibilities for environmental management. This paper details research conducted from 2011 to 2016 in five villages located in different locations within the Okavango Delta of Botswana. We report the findings from qualitative interviewing and livelihood mapping activities that are integrated with remote sensing analysis to provide concrete empirical detail on the variability of flooding and resulting variations in perception and livelihood responses. The paper demonstrates that flooding dynamics vary at discrete locations and produce diverse perceptions that are tied to livelihood adjustments in place-specific ways. These patterns are also embedded in regional and global processes that have significant implications for household vulnerability within socio-ecological systems strongly impacted by local and distant climatic and hydrological drivers of change.