Fully automated high-throughput chromatin immunoprecipitation for ChIP-seq: Identifying ChIP-quality p300 monoclonal antibodies

Chromatin immunoprecipitation coupled with DNA sequencing (ChIP-seq) is the major contemporary method for mapping in vivo protein-DNA interactions in the genome. It identifies sites of transcription factor, cofactor and RNA polymerase occupancy, as well as the distribution of histone marks. Consortia such as the ENCyclopedia Of DNA Elements (ENCODE) have produced large datasets using manual protocols. However, future measurements of hundreds of additional factors in many cell types and physiological states call for higher throughput and consistency afforded by automation. Such automation advances, when provided by multiuser facilities, could also improve the quality and efficiency of individual small-scale projects. The immunoprecipitation process has become rate-limiting, and is a source of substantial variability when performed manually. Here we report a fully automated robotic ChIP (R-ChIP) pipeline that allows up to 96 reactions. A second bottleneck is the dearth of renewable ChIP-validated immune reagents, which do not yet exist for most mammalian transcription factors. We used R-ChIP to screen new mouse monoclonal antibodies raised against p300, a histone acetylase, well-known as a marker of active enhancers, for which ChIP-competent monoclonal reagents have been lacking. We identified, validated for ChIP-seq, and made publicly available a monoclonal reagent called ENCITp300-1

Dynamic DNA methylation across diverse human cell lines and tissues

As studies of DNA methylation increase in scope, it has become evident that methylation has a complex relationship with gene expression, plays an important role in defining cell types, and is disrupted in many diseases. We describe large-scale single-base resolution DNA methylation profiling on a diverse collection of 82 human cell lines and tissues using reduced representation bisulfite sequencing (RRBS). Analysis integrating RNA-seq and ChIP-seq data illuminates the functional role of this dynamic mark. Loci that are hypermethylated across cancer types are enriched for sites bound by NANOG in embryonic stem cells, which supports and expands the model of a stem/progenitor cell signature in cancer. CpGs that are hypomethylated across cancer types are concentrated in megabase-scale domains that occur near the telomeres and centromeres of chromosomes, are depleted of genes, and are enriched for cancer-specific EZH2 binding and H3K27me3 (repressive chromatin). In noncancer samples, there are cell-type specific methylation signatures preserved in primary cell lines and tissues as well as methylation differences induced by cell culture. The relationship between methylation and expression is context-dependent, and we find that CpG-rich enhancers bound by EP300 in the bodies of expressed genes are unmethylated despite the dense gene-body methylation surrounding them. Non-CpG cytosine methylation occurs in human somatic tissue, is particularly prevalent in brain tissue, and is reproducible across many individuals. This study provides an atlas of DNA methylation across diverse and well-characterized samples and enables new discoveries about DNA methylation and its role in gene regulation and disease

Stillbirth Classification-Developing an International Consensus for Research Executive Summary of a National Institute of Child Health and Human Development Workshop

Stillbirth is a major obstetric complication, with 3.2 million stillbirths worldwide and 26,000 stillbirths in the United States every year. The Eunice Kennedy Shriver National Institute of Child Health and Human Development held a workshop from October 22-24, 2007, to review the pathophysiology of conditions underlying stillbirth to define causes of death. The optimal classification system would identify the pathophysiologic entity initiating the chain of events that irreversibly led to death. Because the integrity of the classification is based on available pathologic, clinical, and diagnostic data, experts emphasized that a complete stillbirth workup should be performed. Experts developed evidence-based characteristics of maternal, fetal, and placental conditions to attribute a condition as a cause of stillbirth. These conditions include infection, maternal medical conditions, antiphospholipid syndrome, heritable thrombophilias, red cell alloimmunization, platelet alloimmunization, congenital malformations, chromosomal abnormalities including confined placental mosaicism, fetomaternal hemorrhage, placental and umbilical cord abnormalities including vasa previa and placental abruption, complications of multifetal gestation, and uterine complications. In all cases, owing to lack of sufficient knowledge about disease states and normal development, there will be a degree of uncertainty regarding whether a specific condition was indeed the cause of death. (Obstet Gynecol 2009,114:901-14

Angular momentum and an invariant quasilocal energy in general relativity

Owing to its transformation property under local boosts, the Brown-York quasilocal energy surface density is the analogue of E in the special relativity formula: E^2-p^2=m^2. In this paper I will motivate the general relativistic version of this formula, and thereby arrive at a geometrically natural definition of an `invariant quasilocal energy', or IQE. In analogy with the invariant mass m, the IQE is invariant under local boosts of the set of observers on a given two-surface S in spacetime. A reference energy subtraction procedure is required, but in contrast to the Brown-York procedure, S is isometrically embedded into a four-dimensional reference spacetime. This virtually eliminates the embeddability problem inherent in the use of a three-dimensional reference space, but introduces a new one: such embeddings are not unique, leading to an ambiguity in the reference IQE. However, in this codimension-two setting there are two curvatures associated with S: the curvatures of its tangent and normal bundles. Taking advantage of this fact, I will suggest a possible way to resolve the embedding ambiguity, which at the same time will be seen to incorporate angular momentum into the energy at the quasilocal level. I will analyze the IQE in the following cases: both the spatial and future null infinity limits of a large sphere in asymptotically flat spacetimes; a small sphere shrinking toward a point along either spatial or null directions; and finally, in asymptotically anti-de Sitter spacetimes. The last case reveals a striking similarity between the reference IQE and a certain counterterm energy recently proposed in the context of the conjectured AdS/CFT correspondence.Comment: 54 pages LaTeX, no figures, includes brief summary of results, submitted to Physical Review

Decision rules for determining terrestrial movement and the consequences for filtering high-resolution global positioning system tracks: a case study using the African lion ( Panthera leo )

The combined use of global positioning system (GPS) technology and motion sensors within the discipline of movement ecology has increased over recent years. This is particularly the case for instrumented wildlife, with many studies now opting to record parameters at high (infra-second) sampling frequencies. However, the detail with which GPS loggers can elucidate fine-scale movement depends on the precision and accuracy of fixes, with accuracy being affected by signal reception. We hypothesized that animal behaviour was the main factor affecting fix inaccuracy, with inherent GPS positional noise (jitter) being most apparent during GPS fixes for non-moving locations, thereby producing disproportionate error during rest periods. A movement-verified filtering (MVF) protocol was constructed to compare GPS-derived speed data with dynamic body acceleration, to provide a computationally quick method for identifying genuine travelling movement. This method was tested on 11 free-ranging lions (Panthera leo) fitted with collar-mounted GPS units and tri-axial motion sensors recording at 1 and 40 Hz, respectively. The findings support the hypothesis and show that distance moved estimates were, on average, overestimated by greater than 80% prior to GPS screening. We present the conceptual and mathematical protocols for screening fix inaccuracy within high-resolution GPS datasets and demonstrate the importance that MVF has for avoiding inaccurate and biased estimates of movement

Dead-reckoning animal movements in R: a reappraisal using Gundog.Tracks

BackgroundFine-scale data on animal position are increasingly enabling us to understand the details of animal movement ecology and dead-reckoning, a technique integrating motion sensor-derived information on heading and speed, can be used to reconstruct fine-scale movement paths at sub-second resolution, irrespective of the environment. On its own however, the dead-reckoning process is prone to cumulative errors, so that position estimates quickly become uncoupled from true location. Periodic ground-truthing with aligned location data (e.g., from global positioning technology) can correct for this drift between Verified Positions (VPs). We present step-by-step instructions for implementing Verified Position Correction (VPC) dead-reckoning in R using the tilt-compensated compass method, accompanied by the mathematical protocols underlying the code and improvements and extensions of this technique to reduce the trade-off between VPC rate and dead-reckoning accuracy. These protocols are all built into a user-friendly, fully annotated VPC dead-reckoning R function; Gundog.Tracks, with multi-functionality to reconstruct animal movement paths across terrestrial, aquatic, and aerial systems, provided within the Additional file 4 as well as online (GitHub).ResultsThe Gundog.Tracks function is demonstrated on three contrasting model species (the African lion Panthera leo, the Magellanic penguin Spheniscus magellanicus, and the Imperial cormorant Leucocarbo atriceps) moving on land, in water and in air. We show the effect of uncorrected errors in speed estimations, heading inaccuracies and infrequent VPC rate and demonstrate how these issues can be addressed.ConclusionsThe function provided will allow anyone familiar with R to dead-reckon animal tracks readily and accurately, as the key complex issues are dealt with by Gundog.Tracks. This will help the community to consider and implement a valuable, but often overlooked method of reconstructing high-resolution animal movement paths across diverse species and systems without requiring a bespoke application

How often should dead-reckoned animal movement paths be corrected for drift?

Background Understanding what animals do in time and space is important for a range of ecological questions, however accurate estimates of how animals use space is challenging. Within the use of animal-attached tags, radio telemetry (including the Global Positioning System, ‘GPS’) is typically used to verify an animal’s location periodically. Straight lines are typically drawn between these ‘Verified Positions’ (‘VPs’) so the interpolation of space-use is limited by the temporal and spatial resolution of the system’s measurement. As such, parameters such as route-taken and distance travelled can be poorly represented when using VP systems alone. Dead-reckoning has been suggested as a technique to improve the accuracy and resolution of reconstructed movement paths, whilst maximising battery life of VP systems. This typically involves deriving travel vectors from motion sensor systems and periodically correcting path dimensions for drift with simultaneously deployed VP systems. How often paths should be corrected for drift, however, has remained unclear. Methods and results Here, we review the utility of dead-reckoning across four contrasting model species using different forms of locomotion (the African lion Panthera leo, the red-tailed tropicbird Phaethon rubricauda, the Magellanic penguin Spheniscus magellanicus, and the imperial cormorant Leucocarbo atriceps). Simulations were performed to examine the extent of dead-reckoning error, relative to VPs, as a function of Verified Position correction (VP correction) rate and the effect of this on estimates of distance moved. Dead-reckoning error was greatest for animals travelling within air and water. We demonstrate how sources of measurement error can arise within VP-corrected dead-reckoned tracks and propose advancements to this procedure to maximise dead-reckoning accuracy. Conclusions We review the utility of VP-corrected dead-reckoning according to movement type and consider a range of ecological questions that would benefit from dead-reckoning, primarily concerning animal–barrier interactions and foraging strategies.Additional co-authors: Angela Bruns, O. Louis van Schalkwyk, Nik C. Cole, Vikash Tatayah, Luca Börger, James Redcliffe, Stephen H. Bell, Nikki J. Marks, Nigel C. Bennett, Mariano H. Tonini, Hannah J. Williams, Carlos M. Duarte, Martin C. van Rooyen, Mads F. Bertelsen, Craig J. Tambling & Rory P. Wilso

Triple-Antiretroviral Prophylaxis to Prevent Mother-To-Child HIV Transmission through Breastfeeding—The Kisumu Breastfeeding Study, Kenya: A Clinical Trial

Timothy Thomas and colleagues report the results of the Kisumu breastfeeding study (Kenya), a single-arm trial that assessed the feasibility and safety of a triple-antiretroviral regimen to suppress maternal HIV load in late pregnancy

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy