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Real Meat Air Dried 90% Meat Turkey
Real Meat Air Dried 90% Meat Turkey
If your pets like our treats they will love our air dried 90% Real Meat pet foods! The recipe is simple, meat and lots of it! Gently mixed with natural vitamins and minerals,
air dried into the most meaty scrumptous dry food available. Using only the finest free range grass fed meats with no added hormones or antibiotics.
All our Real Meat Pet Foods are formulated to meet the nutritional levels established by theAssociation of American Feed Control Officials (AAFCO) for Cat and Dog Food Nutrient Profiles for all life stages
These diets make a great alternative to raw and frozen diets, easy to travel with, easy to serve and always ready!
Can be fed as is or mixed with water for improved hydration. Always keep plenty of fresh water available.
Ingredients: Turkey, Turkey Liver, Turkey Heart, Turkey Gizzard, Turkey Necks, Parsley, Pumpkin, Rosemary, Inulin (from Chicory Root), Vegetable Glycerin, Choline Chloride, Mixed Tocopherols (Vitamin E Supplement), Zinc Proteinate, Zinc Sulfate, Iron Sulfate, Vitamin E, Iron Proteinate, Selenium Yeast, Copper Sulfate, Nicotinic Acid, Calcium Pantothenate, Copper Proteinate, Manganese Proteinate, Vitamin A Supplement, Vitamin B12 Supplement, Manganous Oxide, Vitamin D3 Supplement, Ethylenediamine Dihydriodide (source of iodine), Riboflavin Supplement (Vitamin B2), Thiamine Mononitrate (Vitamin B1), Pyridoxine Hydrochloride (Vitamin B6), Folic Acid.
The Real Meat Company does NOT use any beef gullets or glands.
Guaranteed Analysis
Crude Protein (min) 35%
Crude Fat (min) 20%
Crude Fiber (max) 2%
Moisture (max) 15%
Calories 4250kcal/kg 181kcal/ 1/4cup (1/4cup=1.5oz) Phosphorus level=1.6% DM Calcium level =1.8% DM Salt/Sodium =.14% DM Chloride =.47% DM


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by john bobey
photography by william brinson | January 23, 2012 | Food & Drink
Crescendo martini glass, Luigi Bormioli (set of four, $46). bloomingdales.com
|The Algonquin, where Dorothy Parker ruled the Round Table
|The Four Seasons Restaurant, as classic as the martini
With the resurgence of supper clubs and speakeasies like The Darby and 1920 Bunker Club, Art Deco ruling the runways, and the success of retro filmed-in-Manhattan series such as Mad Men, New Yorkers are feeling a certain vintage romanticism. "Our tag line at The Campbell Apartment is 'Cocktails from Another Era,' and when we opened the place that was our mandate," says Mark Grossich, owner and CEO of Hospitality Holdings, which oversees the famed Grand Central establishment. "We wanted to bring back the classic cocktail lounge and the drinks associated with that. All of the mixology that has been going on over the past couple of years, with drinks having multiple ingredients, is certainly intriguing, but you can't beat the classics."
Yusef Austin, a Stoli Elit ambassador and creator of cocktail programs for such noted New York establishments as the Boom Boom Room, concurs. "Cocktail programs, which have been on the upswing for the last three or four years, are the latest development in terms of the classic martini. Today, you know that if a place has great food, the cocktail bar will be a complement to what is happening in the kitchen."
Quality and simplicity are the keys to a good martini, and when it is done perfectly, New Yorkers show their appreciation. "In terms of cocktails at our bar, I would say it's a 60-40 split of martinis to everything else," says Pasquale Trunzo, beverage director for The Palm on West 50th Street. "We are known for the generous pours of our martinis, and in a nutshell, that's what The Palm is all about: generosity, opulence, and a nice martini in your hand, in a place to see and be seen."
After all, this is the town that not only incited the modern mixology movement, but also, according to some, invented the martini in the first place. Legend has it that the first martini was stirred up approximately a century ago at the bar at John Jacob Astor's Knickerbocker Hotel, a nowlandmarked building on the corner of Broadway and West 42nd Street. In a 2003 "Morning News" article on the hotel, The New York Times editor Clay Risen wrote, "That bar is where, in 1912, an immigrant bartender named Martini di Arma di Taggia allegedly mixed gin and dry vermouth, perfecting the martini. One of his first tasters was John D. Rockefeller, who liked it so much that he recommended it to his Wall Street buddies, and the drink quickly became a national favorite."
The same concoction has been fueling New York's captains of industry and finance ever since. "I started drinking martinis when I was 24; I had a friend who turned me onto them," says Ward Morgenthau, a stockbroker at Kern, Suslow Securities and a financial services professional for more than 40 years. "In my early days, Fridays and Mondays were big for the three-martini lunch, and there were many lunches where there were many more than three.
"A lot of big producers were very white-shoe," continues Morgenthau, "and would hang out at the hotel bars on the Upper East Side, such as The Drake, where they'd hold court and cultivate wealthy clients. A friend of mine was a member of a place called The Bankers Club, and we always had at least three drinks. They had a Cuban guy at the bar who could bury people, he had such a heavy hand."
Those days are gone except for the rare few that can still get away with it—on television. "They didn't hide it; they drank during the day and then went back to the office and actually were productive or, in the case of Don Draper, reproductive," quips Bryan Batt, who played Salvatore Romano on AMC's award-winning Mad Men. "I personally love a bone-dry vodka martini with a twist. That is my drink of choice, but I seem to think that Sal would have been more classic and had a gin martini. I also think he would have been an olive man, something to pick up and nosh on."
Our city's cultural love affair with the martini predates Don Draper's fondness for women. New York in the roaring '20s witnessed an unsurpassed collection of writers, journalists, and critics, the best of whom were seated at the Algonquin Hotel's Round Table. Dorothy Parker noted her feelings on her favorite drink as, "I love to drink martinis, two at the very most. After three I'm under the table, after four I'm under my host." H.L. Mencken offered that the martini is "the only American invention as perfect as the sonnet."
Mini serveware 12-piece set, including six bowls and six spoons, Macy's ($19.99).
|The Knickerbocker Hotel, birthplace of the martini
Thanks to the infamous imbibers of their day, the martini was inextricably linked with the good life. "The martini became the embodiment of a generation," says Francesco Lafranconi, spirits expert and developer of cocktail programs for restaurateurs such as Daniel Boulud, Thomas Keller, and Wolfgang Puck. "It became part of a lifestyle, and this is why the martini is synonymous with sophistication, good living, and luxury. It represents a state of being."
While many of that generation's favorite haunts are long gone, there are a few places where Parker and Mencken would still feel quite comfortable settling in and having a few. Since opening in 1959, The Four Seasons Restaurant has been the epicenter of the New York power lunch. Nothing has changed in the architecture, design, or service, so a visit to the famed Grill Room or Pool Room is like stepping back in time. "There aren't many places you can walk into 50 years later and it still looks the same," says Greg Connolly, head bartender at the Four Seasons and coauthor of The Four Seasons Book of Cocktails, of the famed East 52nd Street restaurant. "The original martini was gin, and then some of the old-timers let vodka sneak into the category, sometimes with a twist instead of with olives. When someone new starts here, we train them to make a martini the way it was made 40 years ago."
Whether it snuck in or not, the vodka martini is definitely here to stay, thanks in large part to Bond, James Bond. Along with his preferred method of preparation, "shaken, not stirred," as he first uttered in 1964's Goldfinger, Bond wasn't alone in ordering his martinis made with vodka. The 1960s saw a number of vodka brands being imported from Russia, and ever since the vodka martini has been every bit as legitimate as one made with gin.
These days, the demand for premium spirits makes a modern classic even better, and New York's most upscale establishments are all too happy to oblige. "We are a vodka bar, so the expectation is always toward the vodka martini," explains Ana Opitz, co-owner of the swank caviar bar Pravda on Lafayette Street in Soho. "When I watch someone order a martini, it's a much different thing than ordering another cocktail. With a martini, somebody really knows what he or she wants."
And whether it's the frosty evergreen taste of a fine gin or the smooth distilled clarity of icy premium vodka the connoisseur wants, the New York martini drinker will inevitably be treated to a sip of what E.B. White referred to as "the elixir of quietude." "With the economy where it is these days, it is a treat to have something fabulous," says Grossich of a top-shelf martini. "A terrific drink is an affordable luxury. It is a small price to pay to taste a link to the heritage."
photography by Bettmann/corbis (algonquin, knickerbocker); courtesy of the four seasons restaurant; drink stylist: ed gabriels for halley resources

subset: commoncrawl


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Increased levels of brain amyloid-β, a secreted peptide cleavage product of amyloid precursor protein (APP), is believed to be critical in the aetiology of Alzheimer's disease1. Increased amyloid-β can cause synaptic depression2,3, reduce the number of spine protrusions (that is, sites of synaptic contacts)4,5 and block long-term synaptic potentiation (LTP)6,7, a form of synaptic plasticity; however, the receptor through which amyloid-β produces these synaptic perturbations has remained elusive. Laurén et al.8 suggested that binding between oligomeric amyloid-β (a form of amyloid-β thought to be most active5,6,9-11) and the cellular prion protein (PrPC)8 is necessary for synaptic perturbations. Here we show that PrPC is not required for amyloid-β-induced synaptic depression, reduction in spine density, or blockade of LTP; our results indicate that amyloid-β-mediated synaptic defects do not require PrPc.
To test whether PrPc is required for amyloid-β-induced synaptic depression we infected organotypic hippocampal slice neurons with a Sindbis virus driving expression of APPct100, a precursor of amyloid-β, which leads to increased neuronal production and secretion of amyloid-β that does not perturb the health of the neuron, as assessed electrophysiologically3. Synaptic transmission was depressed in neurons expressing APPct100, in both wild-type and Prnp−/− mouse slices (where Prnp is the gene that encodes PrPC) 24 h after infection (Fig. 1a). As previously shown for wild-type tissue3, this depression was blocked if Prnp−/− slices were maintained during the APPct100 expression period with an inhibitor of NMDA (N-methyl-d-aspartate) receptors (Fig. 1a). Thus, synaptic depression after expression of APPct100, an effect of elevated amyloid-β3, is intact in animals lacking PrPC.
We next examined the effects of amyloid-β on dendritic spines, sites of excitatory synapses. Overexpression of APP4 (or exposure to oligomeric amyloid-β5) leads to loss of dendritic spines in organotypic slices prepared from wild-type animals, which can be visualized by co-expression of a cytoplasmic marker. The same APP-induced decrease of spines was seen in slices made from mice lacking PrPC (Fig. 1b). We next determined which species of amyloid-β is responsible for the observed effects. Synthetic amyloid-β(1–40) (Aβ40) peptides remain predominantly in monomeric form, and did not affect spine density (Fig. 1c). Amyloid-β(1–42) peptides (Aβ42) form oligomers, and when exogenously applied they produced a similar loss of dendritic spines in slices prepared from either wild-type or PrPC-lacking mice (Fig. 1c). Thus, the loss of dendritic spines produced by oligomeric Aβ42 exposure does not depend on PrPC.
We repeated the experiments as described in Laurén et al.8 that studied PrPC's role in the Aβ-mediated blockade of LTP. Acutely prepared hippocampal slices from mature wild-type and Prnp−/− mice were exposed to either species of amyloid-β and LTP was induced by theta burst stimulation. We found that Aβ42 (but not Aβ40) blocked LTP irrespective of whether PrPC is present or absent (Fig. 1d).
We show that amyloid-β-induced synaptic depression, loss of dendritic spines and blockade of LTP are present in hippocampal slices prepared from PrPC-deficient animals. In line with our results, amyloid-β oligomers were shown to impair long-term memory equally in PrPC-lacking and PrPC-expressing mice12. Thus, although oligomeric amyloid-β can bind PrPC, it does not seem to be the receptor responsible for synaptic perturbations caused by oligomeric amyloid-β. Elucidation of the molecular mechanisms by which amyloid-β produces synaptic perturbations remains as a major goal in finding therapeutic treatments of Alzheimer's disease.
C57/BL10 and C57/BL10 Prnp−/− mice were provided by M. Oldstone (Scripps Research Institute, grant AG04342). Organotypic slice cultures were infected at 7–12 days in vitro with Sindbis virus expressing APPct1003. After 24h simultaneous whole-cell paired recordings were obtained as described previously13. At 12–17 days in vitro slices were infected with Sindbis virus expressing tdTomato or APP plus tdTomato for 48 h, or infected with eGFP and incubated with 1 μM monomeric Aβ40 or oligomerized Aβ4214 for 24 h. Two-photon laser scanning images were taken of dendrites at the site of primary apical dendrite bifurcation. Spine densities were counted as described previously4. The protocol for the LTP experiments was as described in Laurén et al.8.
This work was supported by NIH grant AG032132 and the Shiley-Marcos Endowment for Alzheimer's Disease Research to R.M.
Competing financial interests: declared none.
Arising from: J. Laurén et al. Nature 457, 1128-1132 (2009)
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Steve's Auto Repair Inc
Review for Steve's Auto Repair Inc
Ashley B. on 04/24/2013
Ashley said this business told her that the clutch in her vehicle was going out and would cost her $700, but when they did a diagnostic test on her vehicle, they found out that a bolt in the clutch was loose and would cost her only $5. She is glad they did not charge her for the diagnostic test, but was not happy about the initial diagnosis.
Vehicle: Mazda Protege
Category: Service
Service Date: 04/16/2013
Review Created: 04/24/2013
This review was collected via phone interview by Customer Research Inc.


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Fixing the Foundation: Local Solutions for Infill Housing
Will we choose to protect our greenbelt of natural landscapes, farms, and ranches as we grow, or sprawl ever outward?
Barriers to infill housing development arise in cities across the region, state, and nation. Greenbelt Alliance's infill housing research team chose to study 12 key cities across the Bay Area with high potential for infill housing development: Concord, Fremont, Mountain View, Oakland, Palo Alto, Redwood City, Santa Clara, Santa Rosa, San Francisco, San Jose, Sunnyvale, and Walnut Creek.
Infill development poses multiple challenges. Greenbelt Alliance's Fixing the Foundation report provides a practical and insightful discussion of barriers and solutions for cities and towns.
—Ken Alex, Director of the Governor's Office of Planning and Research
Fixing the Foundation identifies the top 10 barriers to infill housing development across these cities and provides local solutions for overcoming these barriers. Our City by City analysis applies the report findings to each of our 12 cities, offering specific recommendations for local, tangible impact in the Bay Area.
Download Fixing the Foundation (PDF, 32 pp, November 2013)
Download Fixing the Foundation City by City (PDF, 24 pp, November 2013)


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The ENCODE Project has generated a wealth of experimental information mapping diverse chromatin properties in several human cell lines. Although each such data track is independently informative toward the annotation of regulatory elements, their interrelations contain much richer information for the systematic annotation of regulatory elements. To uncover these interrelations and to generate an interpretable summary of the massive datasets of the ENCODE Project, we apply unsupervised learning methodologies, converting dozens of chromatin datasets into discrete annotation maps of regulatory regions and other chromatin elements across the human genome. These methods rediscover and summarize diverse aspects of chromatin architecture, elucidate the interplay between chromatin activity and RNA transcription, and reveal that a large proportion of the genome lies in a quiescent state, even across multiple cell types. The resulting annotation of non-coding regulatory elements correlate strongly with mammalian evolutionary constraint, and provide an unbiased approach for evaluating metrics of evolutionary constraint in human. Lastly, we use the regulatory annotations to revisit previously uncharacterized disease-associated loci, resulting in focused, testable hypotheses through the lens of the chromatin landscape.
The sequencing of the human genome produced the complete recipe for a human being encoded in digital form, and much of the past decade of molecular biology has been devoted to deciphering the meaning of this code. On this premise, the ENCODE Project Consortium sought to discover a complete catalog of all functional elements in the human genome (1), analogous to delineating sentences and words that comprise the human genome, and understanding the type of function each element plays. Such a catalog will undoubtedly never be complete, given the diversity of cell types where elements may activate, the diversity of experimental assays needed to probe them, and the specific conditions and stimuli to which they may respond. The scale-up phase of the ENCODE project, however, has made substantial advances toward the goal of a comprehensive catalog. It has carried out a daunting 1640 total experiments in 147 cell types, using multiple distinct biochemical assays, including ChIP-seq, DNase-seq, FAIRE-seq and RNA-seq. Interpreting the resulting information is arguably more complex than interpreting the primary sequence of the human genome. The four nucleotides of the sequence have been replaced by a large vector of numerical values, each representing the result of a different biochemical assay in a given condition and a given cell type, at each position of each chromosome. The challenge at hand is thus to turn these vectors of numerical values into an interpretable annotation, namely, the list of functional elements that the ENCODE project set out to annotate.
To address this challenge, we and others have developed a variety of computational techniques that seek to identify functional elements from high-throughput genomic datasets. These techniques fall into two groups: supervised learning methods that attempt to find instances of one or more pre-determined classes of elements, and unsupervised learning methods that seek to simultaneously discover functional classes and annotate their instances de novo. Supervised learning methods have been widely used for automatic gene finding methods that can recognize protein-coding transcripts using sequence features, cDNA sequence and evolutionary conservation of known examples (2,3). Supervised models have also been successfully used to recognize promoters (4), enhancers (5) and microRNAs (6), based on known examples. As supervised learning methods require a training set of known examples, they are incapable of discovering novel types of functional elements. Unsupervised methods, in contrast, identify candidate functional elements without the need for previously defined classes or known examples, thereby avoiding biases toward well-understood phenomena. Despite their generality, peak-finding algorithms for ChIP-seq analysis can be seen as supervised learning methods, seeking to recognize 'peak-like' behavior. Moreover, peak-finding methods have difficulty generalizing to the joint analysis of dozens of tracks of functional genomics data, where the diversity of possibly interesting patterns is very high. Such integrative analyses are central to the mission of ENCODE, which aims not only to produce such data but also to make sense of the resulting collection of datasets.
In this work, we apply unsupervised chromatin state annotation methods that simultaneously discover the locations of functional elements in the human genome and assign to each element one of a small number of labels, which can be interpreted as functional annotations. As input, our methods receive a collection of functional genomics datasets and a user-specified parameter for the number of distinct labels that the method should discover. The input datasets consist of ChIP-seq assays for multiple histone modifications, general transcription factors and chromatin accessibility assays. We restrained ourselves to chromatin-level information in the initial annotation stage, and did not use RNA-seq information as an input to our models, instead reserving it for later validation. Our computational analysis provides as output an annotation of the human genome. This annotation consists of a segmentation into non-overlapping segments, and a labeling of each segment using one of a small set of labels, which we refer to as chromatin states. The goal of the chromatin state annotation is to capture the similarities of segments that show the same patterns across many experiments by assigning them the same label, thus summarizing a very large collection of data into a more meaningful form. The resulting segment labels typically correspond to an intuitive, human interpretable biological function, which we use to summarize them, even though we recognize that the underlying biology is usually more complex. Other times, the segment labels may remain uninterpreted until we learn more about additional functions that may be distinguishable by their specific combinations of chromatin marks, but whose biological roles may not yet be understood until additional biological processes become elucidated, or until additional datasets become available. The unsupervised nature of these chromatin state annotations may thus identify novel instances of known classes of functional elements, suggest novel subdivisions of classes into subclasses, or hypothesize the existence of entirely new types of functional elements.
During the pilot phase of ENCODE, Thurman et al. combined a hidden Markov model (HMM) with wavelet smoothing to produce a two-label segmentation of the ENCODE pilot regions into 'active' and 'repressed' regions (7,8). A variety of segmentation models have been described subsequently, employing HMMs with flat (9,10) or hierarchical (11) structures, or generalizing the HMM to a hierarchical change-point model (12).
For the second phase of ENCODE, two research groups within the consortium independently developed chromatin state annotation algorithms, ChromHMM (13,14) and Segway (15). Although the methods were designed and initially implemented independently of one another, they share many key features. Most significantly, the methods employ closely related probabilistic models. ChromHMM is implemented as an HMM, in which the 'time' axis is the chromosomal coordinate and the various ENCODE datasets are the observed variables. Similarly, Segway employs a dynamic Bayesian network (DBN) approach, which is a generalization of the HMM framework. The HMM/DBN approach offers multiple important advantages, including efficient algorithms for carrying out inference and a modeling paradigm in which the model's internal variables have well-defined semantics.
Key differences between the two chromatin state annotation methods are summarized in Table 1. Broadly speaking, ChromHMM aims to take more of a birds-eye view of the data, opting to compress each data track to a single Boolean value for each 200-bp segment of the genome. This approach makes ChromHMM computationally efficient, enabling training on the entire genome, and reduces the chances that artifacts related to scaling of the data or local patterns of missing data due to mapping problems will mislead ChromHMM. The 200-bp resolution also ensures that ChromHMM produces reasonably large nucleosome-sized segments without having to implement complex constraints on segment length distributions. Segway, in contrast, operates on the full data matrix at 1-bp resolution. Segway handles missing data in a principled fashion by marking each missing data point as a hidden variable and marginalizing over all possible values. To ensure that Segway produces segments of a reasonable size, we employ both hard constraints (for example, enforcing a minimum length of 100 bp per segment) and soft constraints (length priors). For efficiency, we trained the Segway models described here only on 1% of the human genome, but training on the entire genome is possible. Finally, ChromHMM and Segway differ in the choice of algorithm used to assign the final labeling: ChromHMM assigns to each segment the label with maximum posterior probability, whereas Segway selects the series of labels that jointly achieves the highest probability over the entire segmentation path.
The work presented here constitutes the first systematic integration of chromatin elements across the entire ENCODE project. The two methods used reveal functional chromatin elements at different levels of resolution, making it possible to study both the transitions between different types of chromatin states at single-nucleotide resolution, and to obtain a robust annotation that can tolerate small variations in large chromatin domains. These two annotations form the basis of the integrative analysis for the ENCODE project, and they provide a systematic view of the chromatin landscape. This integrated viewpoint will be of great value to epigenomics research. In addition, this work describes a manually curated chromatin annotation that synthesizes the two complementary methodologies.
The resulting annotations capture the remarkable diversity of genomic functions encoded by distinct chromatin states, are robust across different cell types, and are reliably recovered by the two methods used here. We also created a combined segmentation that contains features of both. Our systematic annotation of chromatin elements has important implications for the study of the human genome.
For the coordinated segmentations, we selected all the available combined ENCODE histone modification, Pol2 and CTCF ChIP-seq (plus control) and open chromatin (DNase-seq and FAIRE-seq) tracks that were available in the January 2011 data freeze for each of the six Tier 1–2 cell types. A full list of tracks used is in Supplementary Table S1.
We used a uniform signal-processing pipeline to generate genome-wide normalized signal coverage tracks for different types of ENCODE datasets (Supplementary Figures S1–S3, Supplementary Table S2). We used different subsets of these tracks as input to the segmentation algorithms. We combined signal from multiple replicates of each experiment. In addition, for a select subset of experiments that had equivalent datasets from multiple labs, we combined signal across all datasets. We downloaded read alignment files from the ENCODE portal (http://genome.ucsc.edu/ENCODE/downloads.html) and filtered them to remove multi-mapping reads. For each of the replicates (datasets) that we combined to produce a unified signal track, we estimated a characteristic read-shift parameter from the data and shifted reads by this estimated value in the 5′–3′ direction. We computed the effective fragment coverage for each position in the genome by first computing the read-count coverage followed by a smooth aggregation (using kernel smoothing) in a pre-specified window around each position. We then normalized the fragment coverage for the total number of mapped reads over all replicates, effective read-extension and smoothing lengths, local mappability around each location and the overall mappable size of the genome. We expressed the final signal values at each position in terms of a fold-change over the expected signal from an equivalent uniform distribution of reads over all mappable locations in the genome. We explicitly represented as missing values those signal values at unreliable locations consisting of genomic positions surrounded by a large number of unmappable locations and those in assembly gaps. Supplementary Methods contain a complete description of this procedure.
Before applying ChromHMM, we converted the normalized signal tracks into binarized data at a 200-bp resolution. We used the maximum signal for a mark in each 200-bp interval to represent the mark in that interval. The threshold for each mark was the maximum of 4.0 and the value corresponding to a Poisson tail distribution probability of 0.0001. Requiring a fold threshold, in addition to the tail distribution threshold, enabled more meaningful binarization of some of the most deeply sequenced datasets. We excluded regions that associated with repetitive elements such as α- and β-satellite repeats, ribosomal and mitochondrial DNA (http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeMapability/wgEncodeDukeMapabilityRegionsExcludable.bed.gz).
For Segway, we excluded the ENCODE Data Analysis Consortium Blacklisted Regions (http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeMapability/wgEncodeDacMapabilityConsensusExcludable.bed.gz), comprising a comprehensive set of regions in the human genome that exhibit anomalous or unstructured read-counts in next gen sequencing experiments, independent of cell line and type of experiment. To identify these regions, we used 80 open chromatin tracks (DNase and FAIRE datasets) and 20 ChIP-seq input/control tracks spanning ~60 human tissue types/cell lines in total. The regions tend to have a very high ratio of multi-mapping to unique mapping reads and high variance in mappability. Some of these regions overlap pathological repeat elements such as satellite, centromeric and telomeric repeats. However, simple filters based on mappability do not account for most of these regions.
We trained ChromHMM in concatenated mode on the six cell types using a two-stage nested parameter initialization approach, considering models of up to 30 states, using Euclidean distance for the state pruning distance, and setting the emission and transition smoothing parameters to 0.01 and 0.5, respectively, for the second stage parameter initialization (13,17). For each pass, we completed 200 training iterations. ChromHMM mnemonics and brief state descriptions are in Supplementary Table S3.
We trained Segway on the 1% of the genome selected as the ENCODE pilot regions (7). We performed expectation maximization training until either (i) the log likelihood of the model minus the log likelihood from the previous iteration divided by the log likelihood was <10−5, or (ii) until 100 training iterations. We assigned mnemonic labels according to Supplementary Table S3.
The ChromHMM and Segway chromatin state annotations can be accessed by loading the ENCODE Analysis Hub at http://encodeproject.org/cgi-bin/hgHubConnect.
We applied both chromatin state annotation methods to a set of chromatin signal tracks from the ENCODE Tier 1 (GM12878, H1 hESC, K562) and Tier 2 (HeLa-S3, HepG2, HUVEC) cell types. For this analysis, we included data from the kinds of experiments that have the potential to reveal the most about chromatin state—the open chromatin assays DNase-seq and FAIRE, and ChIP-seq on histone modifications, RNA polymerase 2 (Pol2) and CTCF. Generally, we used only the signal tracks that were available for all six Tier 1–2 cell types, combining data from multiple laboratories when appropriate. Figure 1 displays the full list of signal tracks used, with data sources in Supplementary Table S1.
We trained Segway on each of the six Tier 1–2 cell types independently giving a separate Segway model for each cell type, and ChromHMM jointly using a virtual concatenation of the six cell types giving a single ChromHMM model applicable to all six cell types. We specified that the methods should find 25 chromatin states. We picked this number because it is large enough to describe many interesting functional elements while still being small enough for a biologist to interpret easily, given our previous experience with Segway (15) and ChromHMM (13,17).
The chromatin state procedure is semi-automated in that the two algorithms assign to each segment an integer label, but one must ascertain the functional semantics of these labels in a post hoc analysis step. Based on a variety of types of evidence, including investigation of known genomic loci and examination of the model parameters, we assigned names such as 'TSS' or 'enhancer' to each of the integer labels. These names are summarized in Figure 1, and the basis for these naming assignments is shown in Figure 1, Supplementary Figures S4–S7 and described more fully below. The proportion of the genome covered by each label is shown in Supplementary Figure S8, and the distribution of segment lengths is shown in Supplementary Figure S9. Figure 2 shows a sample locus on chromosome 13 with the two chromatin state annotations displayed along the top. For ease of visualization, we have colored all the chromatin states using a reduced palette of 10 colors derived from Ernst et al. (2011).
In addition to the separate chromatin state annotation produced by Segway and ChromHMM, we considered it desirable to produce a simpler summary-level classification to provide more immediate interpretability and data display for a more general audience. Toward this end, we identified closely related states both within and across methods, and then defined a rule-based metric (Supplementary Methods, Supplementary Results, Supplementary Tables S4 and S5, Supplementary Figure S10) to classify them into seven classes, emphasizing biologically meaningful differences: Transcription Start Site (TSS), Promoter Flanking (PF), Enhancer (E), Weak Enhancer (WE), CTCF binding (CTCF), Transcribed Region (T) and Repressed or Inactive Region (R). Based on these rules, we produced a combined annotation of each of the Tier 1 and Tier 2 cell lines that achieved high coverage of the assayable genome (94.4–96.5%, see Supplementary Table S6 and Supplementary Figures S11 and S12) in each cell type. For the rest of the article we primarily consider the detailed primary chromatin state segmentations, both in aggregate and at individual loci such as ENC1 (Figure 2), NOD2 (Figure 3) and HBB (Supplementary Results and Supplementary Figure S13).
Perhaps the best-understood type of functional element in the human genome is the protein-coding gene. Given that these genes account for a large proportion of the evolutionary constraint observed in multi-species alignments, it is reassuring that both ChromHMM and Segway devote a considerable proportion of their model parameters to identifying and characterizing protein-coding genes. Both types of model contain approximately eight labels that strongly correlate with various genic components. Figure 1 illustrates how these gene-related labels aggregate around protein-coding genes.
Segway and ChromHMM both learn labels associated with promoters generally, and more specifically with regions of varying proximity to the TSS (Supplementary Figure S14). Both methods show excellent recall of TSSs, in a manner dependent on the cell type and data (Table 2). The high-resolution Segway Tss labels have higher log2 fold enrichment of TSSs (Segway: 7.2–8.0; ChromHMM: 6.9–7.3), and the high-continuity ChromHMM Tss label has better base-level recall (Segway: 53–91%; ChromHMM: 79–94%). In particular, Segway has the ability to find high-resolution patterns at TSSs localized to the level of stable nucleosome-free promoter regions, whereas ChromHMM finds larger TSS-associated segments (Supplementary Figure S15).
Additionally, both methods identify other labels likely to occur upstream and downstream of the TSS. These TSS-flanking patterns occur to a certain extent around other transcriptionally active regions such as enhancers. Often, the Segway TSS-flanking TssF label can be tightly associated with well-positioned nucleosomes, flanking a narrow Tss label of a consistent nucleosome-free region. The promoter-associated segments can lead to new discoveries. For example, the ENC1 gene in Figure 2 is annotated with promoter-associated segments not only at the canonical TSS, but also in intron 2. The GENCODE group has identified this as the start site for an internal, non-coding RNA (green gene model).
Some Segway models (H1 hESC and HepG2) and the ChromHMM model contain a 'poised promoter' PromP label associated with both the activating H3K4me3 and the repressing H3K27me3 modifications. This matches a previously identified bivalent chromatin structure that silences genes but keeps them ready for activation (18).
The chromatin state annotations present a more sophisticated and nuanced view of the chromatin landscape than focusing on observations from a single assay. For example, the Gen3′ label discovered by both ChromHMM and Segway has a high frequency for H3K36me3, H4K20me1 and Pol2, with low frequency of other marks (Figure 1). Consistent with previous findings (13), we observe that these states show enrichment around 3′ ends of protein-coding genes (Supplementary Figure S16). Interestingly, ChromHMM also discovers a Pol2 label (associated with high frequency of RNA polymerase II and low frequency of all other signals) that displays a peak in enrichment 1–2 kbp after the 3′ end of the genes. The accumulation of Pol2 could be explained by transcriptional pausing in this region, part of the mechanism of transcription termination (19–21).
Both chromatin state annotations contain labels associated with enhancer type sequences: ChromHMM states Enh and EnhW, and Segway state Enh. These have emission parameters associated with chromatin features particularly suggestive of enhancers (Figure 1). This includes the presence of DNase hypersensitivity and a relatively higher H3K4me1 signal compared to H3K4me3 (7,22). The Enh states show a strong enrichment for transcription factor binding (Supplementary Table S7), while having minimal enrichment with regions proximal to annotated starts of genes (Supplementary Figure S17). The EnhF and EnhWF states also have histone modifications similar to Enh and EnhW states, respectively, and frequently transition to them, but had substantially lower DNase hypersensitivity as well as lower enrichment for transcription factor binding peaks. Such an arrangement is consistent with a central region with no or highly remodeled nucleosomes, flanked by nucleosomes with highly modified histone tails (23,24). The ChromHMM and Segway Enh states show the strongest enrichment for the transcriptional coactivator p300, often found at enhancers (25) (Supplementary Figure S18).
The histone modification H3K27me3, generated by the Polycomb repressor complex 2, covers some repressed genes. Hence, the chromatin states labeled with the prefix Repr, enriched in H3K27me3 signal (Figure 1), are strong indicators that the genes within them have been silenced. ChromHMM state ReprD has a relatively high frequency of H3K27me3 and DNase sensitivity (Duke DNase), and Segway state Repr2 (in GM12878) has some similar features. ChromHMM state Repr as well as Segway states Repr3 and Repr4 (in GM12878) have strong Polycomb repression signal but lack the DNase signal. While ChromHMM state ReprW has low emission probabilities, it frequently transitions to state Repr, suggesting that it is part of broader repressed regions. In contrast, ChromHMM state Low is also associated with low signal but more frequently transitions to active elements, therefore representing low activity domains of the genome near active elements.
Insulators are cis-regulatory modules that restrict the effect of long-range regulatory modules, such as enhancers, so that they act on the appropriate promoter target (26,27). One way to do this is via an enhancer-blocking activity, which requires the protein CTCF (28). Both methods produce one or two states enriched for CTCF occupancy, one of which (CtcfO) is also enriched for DNase-sensitive, open chromatin (Figure 1, Supplementary Figure S19).
The extensive transcriptome mapping, largely by RNA-seq, from the ENCODE consortium was not used as input in our chromatin state annotations. Thus, we can evaluate the transcriptional activity of the DNA segments in each state, both for interpretation of the chromatin states and for discovery of novel relationships. As expected, the chromatin states associated with genes are highly enriched for transcripts in GM12878 cells (Figure 4). This is the case for almost all the biotypes, i.e. RNAs from different categories of genes; the only exception is for RNA from retrotransposed elements in the Segway states in GM12878. When protein-coding genes in each chromatin state are examined, we find that almost all (80–90%) of the genes overlapping the classes with labels for promoters and gene bodies are transcribed (Supplementary Figure S20), and the expression levels within these genes are high (Figure 4). Also as expected, the DNA segments in states associated with repression are depleted for transcripts (Figure 4), only a minority of the protein-coding genes overlapping these states are transcribed (Supplementary Figure S20), and the expression levels (29) for these genes are low (Figure 4).
Notably, the DNA segments in states associated with enhancers are also transcribed, indicating that the transcription of enhancers (30,31) is widespread. Interestingly, states with a high frequency of CTCF are also enriched for transcripts; further work could evaluate how often CTCF-bound regions of different categories (e.g. insulator versus non-insulator) are transcribed.
An unexpected result is that many of the chromatin states with low frequency of most of the epigenetic marks (Low classes) are also enriched for transcripts. Most (75–80%) of the protein-coding genes within the ChromHMM Low class are transcribed, i.e., captured by RNA-seq contigs (Supplementary Figure S20), but they tend to be transcribed at a lower level than the protein-coding genes overlapping the promoter and gene body-associated classes (Figure 4). Thus, the chromatin-based annotations reveal a subset of a genome with a low frequency of histone modifications, but that nonetheless supports transcription. This observation contrasts starkly with the quiescent states described in the next section. Future work should examine the classes of genes and other elements in these regions with transcriptional activity but infrequent appearance of the histone modifications and chromatin features examined here.
Both ChromHMM and Segway collect a large portion of the genome into states with very little signal for any of the features used as inputs, which we term quiescent. We label the quiescent states as Quies in both ChromHMM and Segway. Furthermore, the Low states have emission parameters that are nearly as low as the quiescent states, which have near-zero emission parameters for most signals (Figure 1). Together, the Quies and Low states comprise a majority of the genome for most cell types in both annotations (Supplementary Figure S8). The Quies states showed a consistent enrichment for the nuclear lamina domains mapped previously in human lung fibroblasts (32) (Supplementary Figure S21).
The low signal for epigenetic marks in these quiescent regions is not a result of a failure to map sequencing reads to them. The mappability and repeat content of the quiescent states is not dramatically different from that in other states (Supplementary Figure S22). In addition, we find that the nucleosome content is not significantly lower from that of other segmentation classes. Thus, we interpret these quiescent segments as being in a chromatin structure that is largely devoid of the histone modifications included in the segmentation. The quiescent regions are also strikingly depleted for all annotated transcript categories examined here, including coding and non-coding transcripts (Figure 4). Restricting the analysis to protein-coding genes, we find that slightly more than half of these genes in the Quies states overlap with RNA-seq contigs, much fewer than for genes overlapping promoter and gene body-associated labels (Supplementary Figure S20). Moreover, transcription factors bind much less frequently in the quiescent states than in more active states, showing a level of depletion of TF occupancy comparable to or more depleted than that seen in most of the states associated with repression (Supplementary Table S7). These results indicate that the quiescent segments are inactive for dynamic histone modifications and are transcribed infrequently. Thus, the DNA in these regions is largely inactive.
The apparent reduced functionality of these regions, as indicated by the limited transcriptional activity and absence of histone modifications, suggests that most of these sequences are evolving like neutral DNA, and indeed, we find that the DNA in quiescent states is depleted of constrained sequences (Figure 5). It is likely that the DNA in this low activity, quiescent state is similar to the 'black' state previously described in Drosophila melanogaster (9).
One would expect that some DNA regions are quiescent only in specific cell types or conditions, being activated in other cell types in response to appropriate signals. Other regions could become dormant and not used again, such as for terminally differentiated cells. Still other DNA could be quiescent in all cell types. Indeed, we find that 635 Mbp of genomic DNA (21% of the genome) is in the ChromHMM quiescent state in all 6 cell types examined, including the embryonic stem cell line H1, which may be expected to have a large fraction of its genome active. Of course, some of this DNA may be activated in the many cell types that have not yet been interrogated. It will be informative to observe how small a fraction of genomic DNA remains classified in an apparently constitutively inactive state as the histone modification and transcription profiles are thoroughly examined in a broad set of cell types.
The annotations provide an unbiased view of candidate functional non-coding regulatory elements, enabling us to evaluate different methods for measuring human evolutionary constraint. Accordingly, we overlapped the 25-state ChromHMM annotation defined across 6 cell types with 4 different conserved element sets: PhastCons based on 46 mammals (16,33), GERP based on 33 mammals (16,34), SiPhy-ω and SiPhy-π elements defined based on 29 mammals (16,35). GERP identifies conserved elements by identifying locations of highly rejected substitutions. PhastCons and SiPhy-ω detect elements based on the overall substitution rate. SiPhy-π uses a novel strategy to detect conserved elements based on substitution patterns that deviate from the neutral pattern found in bases evolving without selective pressure. SiPhy-π enables detection of constraint for pairs of nucleotides, rather than individual nucleotides at each position. The PhastCons and SiPhy-ω sets are the smallest of the four, covering 3.8% and 4.2% of the bases considered in the ChromHMM annotation, whereas SiPhy-π and GERP are larger, covering 5.8% and 6.4%, respectively.
It was previously noted that many additional bases detected by SiPhy-π were found in non-coding regions (16) without additional supporting evidence for the biological function of these bases. We observe here that PhastCons, SiPhy-ω and SiPhy-π enrich in most of the ChromHMM states that are suggestive of function with no appreciable decline of enrichments in the larger SiPhy-π set (Figure 5, Supplementary Figure S23). This observation demonstrates at a genome-wide scale the functional relevance of the additional constrained elements detected by SiPhy-π (16). In contrast, for the larger GERP element set, we see substantially lower enrichment in most states on average across cell types, suggesting the additional bases in this set may be less functionally relevant (Figure 5). These phenomena were also observed based on a similar analysis using Segway (Figure 5, Supplementary Figure S23).
The majority of genetic variants associated with phenotypes by GWASs are not in protein-coding regions (36). The large-scale annotation of the human genome, including the non-coding portion, by the ENCODE project showed promise as a guide to interpretation of phenotype-associated SNPs (1), and indeed the regions of the genome associated with function by ENCODE data are enriched in phenotype-associated SNPs (17,37,38). Here, we show that the 25-state annotations from both ChromHMM and Segway provide a higher resolution view of the types of function-associated regions that are enriched for genetic variants with potential phenotypic consequences.
The SNPs on a genotyping array that exhibit the most significant associations with a phenotype of interest are called the lead SNPs in a GWAS. These lead SNPs are not expected necessarily to be the functional SNPs, but they should be in linkage disequilibrium with the functional SNP (39). However, the SNPs on the genotyping arrays are enriched for function-associated regions, and many examples are being found of lead SNPs either being a functional SNP or at least very close to it. For example, the ENCODE Project found such an example in the 8q24 locus (1). We have therefore examined the enrichment of the GWAS lead SNPs in the various chromatin states and compared them to a range of null models, i.e., other SNP sets either not expected to be in functional regions or not currently implicated in function. We use the chromatin state annotations based on epigenetic features in six Tier 1–2 human cell types, regardless of the cell types most likely to be involved in disease susceptibility. While it is preferable to use epigenetic data in cell types of close physiological relevance when available, the studies described here and elsewhere (17,37,38) show that phenotype-associated SNPs are enriched in DNA segments associated with function in the Tier 1–2 cell types, perhaps reflecting regions functional in multiple cell types (39). It is possible that an even stronger enrichment would be found in more relevant cell types, and the analysis presented here may reflect a lower bound estimate.
The phenotype-associated lead SNPs were compiled from the GWAS Catalog in the summer of 2011 (36). This collection was filtered to produce a non-redundant set of 4492 SNPs associated with 362 phenotypes (4860 SNP-phenotype associations, some SNPs are associated with more than one phenotype). The distribution of these SNPs in different chromatin states was compared to the distribution of SNPs not expected to be functional, i.e., two largely unbiased genome-wide collections of SNPs generated by whole-genome sequencing of multiple humans (40) and a compilation of SNPs in 24 published individual human genomes (41). The distribution of SNPs on the 1M Illumina genotyping array was also analysed, along with SNPs from the genotyping array matched to the GWAS SNPs for allele frequency in CEU, distance from a transcription start site and location with respect to gene structure (components such as intronic, exonic and intergenic) (37). This latter set of GWAS-matched SNPs is a particularly rigorous comparison, and we determined the enrichment statistics for 1000 random samplings from the GWAS-matched SNP set. These SNPs are not currently associated with phenotype by GWAS, but they could be in or close to functional regions, e.g., those close to transcription start sites.
In an analysis carried out in the GM12878 cell line, we find that the SNPs in the GWAS Catalog are depleted in the quiescent segments compared to the population and individual SNPs (1000 Genomes and 24 personal genomes) (Figure 3). In contrast, the phenotype-associated SNPs are enriched in many function-associated chromatin states compared to the population and individual SNPs. The genotyping SNPs show a similar pattern to that of the GWAS SNPs, likely reflecting a bias in the latter set for functional regions. We therefore focused on comparing the GWAS-matched SNPs to the GWAS SNPs as a stringent test for significance of the enrichment or depletion. We sampled these matched SNPs from genic regions and the matched SNPs show an even greater enrichment in function-associated chromatin states than do the SNPs on the genotyping arrays (Figure 3). Because 1000 random samplings of the GWAS-matched SNPs were evaluated, we determined whether the observed enrichment for the GWAS SNPs exceeded that found for the lower 950 samplings of the matched SNPs (or conversely, was less than that found for the upper 950 samplings in the case of depletion), establishing an empirical P-value threshold of 0.05. The Segway chromatin states passing this threshold (denoted by red bars in the figure) for significant enrichment for GWAS SNPs are Enh, EnhF, EnhWf, Repr2 and Repr3. The categories showing significant depletion are Quies, ElonW, Low1 and Low6. For ChromHMM, states Enh, EnhF, EnhW, EnhWF, TssF and ReprW showed significant enrichment while states Quies and ElonW showed significant depletion.
The NOD2 locus provides an illustrative example of the use of chromatin states for interpretation of phenotype-associated SNPs. This gene has been associated with Crohn's disease (42,43), but several of the SNPs map into non-coding regions (Figure 3). Three of these SNPs fall into chromatin states associated with enhancers in GM12878 (leftmost), enhancers in HUVEC (middle) and a transcribed region in GM12878 (rightmost outlined in blue). The three highlighted GWAS SNP clusters are close to DNase hypersensitive sites in a lymphoblastoid cell line (GM12878), T-cells (Th2 and Treg cells) and HUVECs, among others. Specific transcription factors binding to these sites are NF-YA, NF-YB, PU.1, SRF, GATA2 and c-Fos, with distinct factors binding to different sites in specific cell lines. All of these observations can be used to formulate testable hypotheses about the genetic variants associated with Crohn's disease. For example, a SNP can affect the affinity for one of these transcription factors in an allele-specific manner, leading to alterations in the level of expression of NOD2 and affecting the inflammatory response associated with Crohn's disease. We note that the DNase sensitivity data include T-cells, which could be implicated in an autoimmune disease such as Crohn's disease. The GATA2 binding data from HUVEC could be pointing to potential sites of occupancy by GATA3, which regulates expression of many genes in T-cells. This is an example of a hypothesis derived from data from the currently studied ENCODE lines that can be readily tested by direct experiments in cell types more relevant to the phenotype.
The chromatin state annotations provided here provide a foundation for interpreting the non-coding portion of the human genome that has so far been difficult to comprehend. For example, while promoter and enhancer regulatory elements contain within them regulatory sequence motifs, their purely sequence-driven identification has remained an unsolved challenge in genomics, while our segmentations provide their systematic annotation independent of the motifs they contain. Similarly, while genomic regions showing extreme sequence conservation across related species frequently show enhancer activity, such extreme conservation is not a general property of enhancer regions, while our systematic annotation contains both conserved and non-conserved elements. Chromatin signatures provide a general approach for discovering active regulatory elements based on their biochemical properties, and beyond regulatory elements, an unbiased genome-wide view of the likely functional roles of every region of the genome. By capturing both high-continuity segments and high-resolution transitions between them, we provide a summary of dozens of genome-wide datasets into a directly interpretable and information-rich resource.
A reader might wonder which chromatin state annotation to use. One significant difference, evident in Supplementary Figure S9, is the relative sizes of the segments, with Segway producing smaller segments on average compared to ChromHMM. Beyond this distinction—high resolution for Segway and high continuity for ChromHMM—the chromatin state annotations exhibit many subtle differences, and each has distinct advantages in different applications. We therefore recommend that the user examine both annotations in regions of interest, because each might capture a different aspect of the underlying biology. As for the joint segmentation, it is not meant to replace the two primary annotations, because their differences can be important and should not be overlooked. Instead, the joint segmentation is meant to help introduce a new user to our chromatin states in a simple and approachable way. Once the user is familiar with our annotations, however, we recommend browsing the joint segmentation in parallel with the two primary chromatin state annotations, to exploit the full richness of the chromatin landscape.
It is important to note that the number of chromatin states presented here was chosen as a compromise between capturing all of the potential complexity of chromatin mark combinations (which requires very large numbers of states) and generating models that are easily interpretable and maximally useful for interpreting genomic features (which requires maintaining a small number of states). In our experience, model selection methods such as the use of the Bayesian information criterion or Akaike information criterion invariably suggest models with higher number of states beyond the point of feasibility for human interpretation. The penalties imposed by these criteria prove insufficient to overcome the increase in likelihood due to increased numbers of states and the consequent increased numbers of parameters. As such, we focused on a relatively small number of 25 states, that is well suited to the number of chromatin marks and other input data tracks that were available, and that allows us to annotate the likely functional roles of each chromatin state. However, as the number of chromatin properties that can be elucidated on a genome-wide scale increases, we expect that additional chromatin states will be discovered. These states will likely provide further functional subdivisions of the states presented here or reveal new types of chromatin elements that we may not even suspect yet.
In addition to the combinations of chromatin marks summarized in our models' emission parameters, the topology of the graph representing allowed transitions between labels contains important information about their genomic relationships. The transition probability matrices (Supplementary Figure S7) capture the structure of these connections. For example, transitions between Tss and TssF or Prom labels occur more frequently, revealing their genomic proximity. In ChromHMM, Low and Quies states both have similar emission profiles with low probabilities of histone modifications, but show distinct transition probabilities and also distinct biological enrichments. In contrast, the transition probabilities have a relatively small impact on the final segmentation for Segway, relative to the emission distributions. This is primarily because the minimum segment length is 100 bp, and thus the Segway Viterbi path probability includes >100 emission probabilities for every transition probability. Indeed, we have observed that various manipulations of a model's transition probability distribution have relatively small impact on the resulting segmentation (data not shown).
Continued methodological improvements will likely be needed to capture additional types of chromatin information, such as three-dimensional interactions between distal regions, looping of chromatin domains or larger-scale region behavior. Thus, the chromatin states presented here may constitute the words of much more complex chromatin sentences whose grammar remains to be elucidated, as new technologies enable deeper and more complex epigenomic maps.
We have tried to illustrate in this article, and in related companion papers, the many applications of the chromatin state annotations, ranging from revealing new genes and functional elements, to interpreting disease datasets, to measuring allele-specific activity or human selection. As genome data have become personalized, we expect that the epigenomes of the future will be genotype and individual specific, and not just cell-type specific. These types of data can have profound implications for understanding the epigenomic consequences of disease, not just its genomic predisposition, and chromatin states will form a necessary component of personal epigenomes. Beyond the applications that we have explicitly listed, however, there are many others that we have not even begun to explore, and we expect these applications to be as rich and diverse as the countless uses of an encyclopedia of genomic knowledge, or a map for navigating our genome.
Supplementary Data are available at NAR Online: Supplementary Tables 1–7, Supplementary Figures 1–23, Supplementary Methods, Supplementary Results and Supplementary References [44–62].
National Institutes of Health [HG004695 to E.B., HG006259 to M.M.H., HG005334 and HG004570 to M.K., DK065806 and HG005573 to R.C.H.]; National Science Foundation [0905968 to J.E.]. Funding for open access charge: National Human Genome Research Institute.
Conflict of interest statement. None declared.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Today we have a new trailer for the upcoming Russian superhero movie Guardians.
It stars Valeriya Shkirando, Alina Lanina, Anton Pampushnyy, Sanzhar Madiyev, and Sebastien Sisak, and is directed by Sarik Andreasyan.
Plot: During the Cold War, an organization called "Patriot" created a super-hero squad, which includes members of multiple soviet republics. For years, the heroes had to hide their identities, but in hard times they must show themselves again.
Guardians hits Russian theaters on February 23rd, 2017.


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Amnesty International on Wednesday accused world leaders at a UN summit of damaging the fight against global poverty through their failure to combat human rights violations.
A greater focus on rights would boost efforts to reach a set of key United Nations development goals, which are under the spotlight at the New York summit, said the London-based group.
"It is completely unacceptable that world leaders have still not agreed to take concrete action to address discrimination and other human rights violations," said Amnesty's Secretary General, Salil Shetty.
Such violations "prevent the MDGs (Millennium Development Goals) from benefiting those who need them most," he added.
Amnesty said there was much evidence that millions could not access basic services due to "discrimination and other human rights violations", which meant they were left out of initiatives to achieve development targets.
Despite this, there was no commitment for future concrete actions on human rights at the three-day summit, said the group in a statement.
Governments were not obliged to show if they were achieving their commitments on development targets or to ensure their efforts were consistent with human rights obligations, said Amnesty.
"In effect, world leaders are asking us to trust them, an incredible demand when we see the gap between what they are required to do and what they have delivered," said Shetty.
The summit aims to find ways to finance and give new political impetus to the eight development goals. They were set in 2000 but most experts now predict they will not be reached by the 2015 target date.
The goals include cutting extreme poverty by half, reducing by two-thirds the number of children who die before the age of five and spreading technological progress.


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MOVIE SYNOPSIS [ +16 years-old ] An American wife works in London for the US State Department. When offices are bombed, she turns out to be the only superiviviente also found in the middle of a conspiracy synopsis makes it suspect. You can watch Survivor online on TV cable or theaters with original audio in English at different dates. This film premiered in theaters in 2014.
Rating: 5 by 220 users./
Runtime: 90 minutes
Katherine Heigl and Clive Owen.
90-minute film (full movie) directed by James McTeigue.
The original audio of Survivor has dialogues in Dolby Digital, it was produced in USA.
It premiered in cinemas or television in 2014.
This film was produced and / or financed by Millennium Films.
Without commercial breaks, the full movie Survivor has a duration of 90 minutes; the official trailer can be watched in streaming on the Internet. You can watch this full movie free in EnglishDolby Digital in movie TV channels, renting the DVD or with VoD services (Video On Demand player, Hulu) and PPV (Pay Per View, Netflix).
See the page of this full movie, Survivor in FULLTV Movies.


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There are so many things wrong with this story.
A notorious Italian gangster has been caught stealing men's underwear and gardening equipment from a supermarket in Milan.
But Renato Vallanzasca, 64, is supposed to be serving four life sentences and nearly 300 years for violent crimes including murder, kidnapping and robbing banks. Despite all that, he was apparently allowed to take a weekend leave, which he used to rob a supermarket of some scratchy, low-value underpants.
Furto da 70 euro all'#Esselunga, #Vallanzasca torna in carcere http://t.co/5OKJabxVyH pic.twitter.com/gOKDIBovav
— TMNews (@TMNewsAgenzia) June 14, 2014
At this point you might be wondering three things: a) What sort of self-respecting mobster steals men's briefs? b) Did they look like these?
#Milano: Furto di mutande, arrestato Renato #Vallanzasca. Che vergogna... come un #Cota qualsiasi! pic.twitter.com/0l8x0EMpdC
— The Ultimate Warrior (@DavideRicci1973) June 15, 2014
And c) Why in the hell was someone with such a violent criminal record as Vallanzasca allowed out of prison? This is what Italian authorities had to say on the matter:
"We are really surprised," said Bollate Prison Director Massimo Parisi.
What? That a dangerous criminal was on the loose? Nooooo. That he tried to steal underwear, garden shears and fertilizer. "No one would have imagined such an illogical move from him."
Vallanzasca, who was the leader of the Comasina gang that terrorized Milan in the 1970s, may lose his hard-won weekend leave pass if convicted of the alleged offense.
While it might sound implausible that a mobster with a rap sheet like Vallanzasca's would shoplift, it wouldn't be the first time he had stolen stuff from a store.
Vallanzasca was first arrested in 1972 after robbing another supermarket.


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While her husband performed during his and Kanye West's 'Watch The Throne' tour stop at American Airline Arena's in Miami, Tuesday night, a six months pregnant Beyonce was spotted getting down during Jay Z's verse on 'Gotta Have It'. A must see.
It's always good to see such a free spirited and down to earth side to her. She's clearly having a good time.


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The Big Call for Tuesday April 29th is at 9PM ET
We will discuss the latest about the dinar and dong. We are hearing some good things...Join us tonight to be part of the precelebration...
Participant Line is 559-726-1150
Listener Only line 530-881-1150
No access code is needed for either line. Q and A is still working fine
Playback number: (559)726-1159 access code 123456#
Link will be given to Dinar Recaps following the call.
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The Big Call
4/29/2014 11:13:24 am
Bruce, as always you remain a steady and guiding light in this challenging investment. We appreciate your wisdom and positivity each and every week. Thank you for all you so un-selfishly give of yourself to we Dinarians, each and every week.
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Hands-on networking experience, without the lab!
The best way to learn about network protocols is to see them in action. But that doesn't mean that you need a lab full of networking equipment. This revolutionary text and its accompanying CD give readers realistic hands-on experience working with network protocols, without requiring all the routers, switches, hubs, and PCs of an actual network.
Computer Networking: Internet Protocols in Action provides packet traces of real network activity on CD. Readers open the trace files using Ethereal, an open source network protocol analyzer, and follow the text to perform the exercises, gaining a thorough understanding of the material by seeing it in action.


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Download FlightAware Flight Tracker 5.2.137 APK for Android
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text: Today I would like to feature one of my resin based pieces. This lovely bracelet is a steampunk inspired piece using multiple cast resin pieces. I use Easy Cast resin and various molds and am willing to encase pretty much anything that will stand still. In this case, I used gears, cogs and other miscellaneous watch parts. I then drilled holes into the them and used my wire skills to link them together. This fun bracelet is a chunky, steamy bauble that any gear head is sure to enjoy! $25


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Hello, my name is Brook and I would like to apologise that this product is not fully translated. I am working hard to remedy this. Would you like to help?
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If your kids do not have the ordinary jigsaw puzzle, surprise them with this 3D kit with 54 pieces, from which they can build a practical and beautiful pencil stand or other needs. Not only will your kids have great fun in building this stand, but building these puzzles in them will develop fine motoring and fantasy. The individual pieces are precisely machined to fit perfectly into each other to form a stand that will hold firmly even without the use of an adhesive . The rigid plastic from which the pieces are made guarantees the stability of the stand. When finished, the stand becomes both a practical helper and a wonderful decoration in every girl's room.
Specifications can be changed without notice. Images are for illustrative purposes only. Retail Price (MSRP) means the manufacturer's/supplier's suggested retail price.
Link to Manufacturer's Website: Enter link to the external review


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News / Arnolfini Arts
Preview: The Bristol Artisan
Sponsored Feature
Designers / Makers bring 50 Bristol and South West designer-makers to the Arnolfini for a free, two-day selling event just in time for Mother's Day.
What can you expect?
The Bristol Artisan presents a beautiful selection of jewellery, ceramics, glassware, textiles, homewares, illustration and furniture – a fantastic place to find the perfect gift or unique items for the home.
Launching for the first time this year at Arnolfini, the event offers visitors the opportunity to meet new designers and view, buy or commission their work. It will be a great way to spend the day for anyone interested in contemporary design and craft – or shopping.
About the organisers:
Designers / Makers is an agency for contemporary design, we offer our members business advice, selling events, PR and opportunities to develop their brand. Founded in 2010, Designers / Makers has grown from a local design market in London into an agency supporting freelance designers – an essential resource to help create a strong and supportive design community across the UK.
The Pocket Pirate
The Bristol Artisan
25 & 26 March / 11am-6pm / Arnolfini
Presenting 50 local Designer Makers for a free, two day selling event.
For more information visit www.thebristolartisan.com or follow on Instagram, Twitter or Facebook.
Top image: Karina Gill, 'Anemone'


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Bully-Busters offers a highly engaging, practical program aimed at preventing bullying in primary schools.
Bully Prevention Challenge Course Curriculum
The Bully Prevention Challenge Course Curriculum utilizes a challenge ropes course to address bully behaviors in seventh-grade middle school classes.
This program works by rewarding children for good behavior rather than punishing them for bad behavior. 1) 3 buddies are assigned to ride each bus. One sits in the front; one sits in the middle, and the other is charged with the back. 2) The buddies monitor the behaviors of students around them. Those who are obeying the seven student-created rules are rewarded tickets. Buddies distribute four or five tickets per ride. 3) The receiver of the ticket then turns it in for a prize. Some of the rewards include pencils, erasers, stress balls and apples. 4) The rules are: obey the bus driver; respect others; let others sit with you; keep the bus clean; keep the aisle clear of things and self; sit in your seat, and talk quietly.
This program gives students tools for solving conflicts at school and home and teaches them better and more effective coping skills.
This program is for children to use in social and personal education where feelings and attitudes count more than knowledge and the aim is to change children's behavior. FearNot! helps in education against bullying and ORIENT is aimed at developing intercultural empathy. This software is freely available.
This program provides individual, group, family and school community level actions to address and prevent bullying in its social context. The Program assists with the design, development, implementation, dissemination and evaluation of a social skill building and comprehensive anti-bullying program.
This program addresses five critical children's health issues - nutrition, physical activity, self-esteem, grieving and bullying - through education, communications, volunteerism, grants and programming.
In KiVa, there is a strong emphasis on influencing the onlookers, who are neither bullies nor victims, to make them show that they are against bullying and to make them support the victim, rather than encourage the bully.
This program is a school-based lunchtime mentoring program where bullied children are paired with college student mentors who visited twice each week.
Implemented at the school, classroom, and individual levels, this program is designed to reduce bullying, prevent future bullying, and improve relationships at every level.
This is a school based physical activity program with the goals and research support that has shown an increase children's physical activity level, decrease in bullying and decrease in playground injuries.
Peaceful Schools programs, products and services are developed to promote a school community that engages students and staff in positive productive conflict resolution.
The RWsA emphasizes the setting up of restorative goals, clear instructions, team building, and good relationships among students, parents, and teachers.
The TA Center on Positive Behavioral Interventions and Supports has been established by the Office of Special Education Programs, US Department of Education to give schools capacity-building information and technical assistance for identifying, adapting, and sustaining effective school-wide disciplinary practices.
Social skills training is a type of psychotherapy that works to help people improve their social skills so they can become socially competent. SST is predominantly a behavioural therapy but cognitive therapy can also be used in some situations to maximise the success of SST. This psychotherapy can be done one-on-one or in a group situation.
Solution Team is a series of structured meetings that any educator or counselor can learn to help a student that is being bullied.
This program teaches elementary students to recognize, refuse, and report bullying, be assertive, and build friendships.
This program used a strengths-based approach to improve students' school experiences.
Student Success Skills is a K-12 evidence-based model that helps students develop key cognitive, social and self-management skills.
The Teasing and Bullying Unacceptable Behavior (TAB) program is taught province-wide to students in grades 3 to 6 to reduce teasing and bullying directed at children with differences—particularly children who stutter.
This program is an intervention procedure for working on bully/victim problems in schools. Instead of punishing the bully, this intervention involves mediation sessions where a teacher expresses sympathy and concern for the victim to show the bully the harm they have done.
This curriculum-based bullying prevention program reduces incidents of bullying by 20%.
This program is a multifaceted change process that brings together a community of caring adults (administrators, teachers, staff, parents) with students to work together to change the social culture in a school or school district.
This program is a collaborative parent-professional partnership, which builds protective factors using a multi-family group process for pre-school, elementary and middle school youth, ages 3-14, to reduce risk for alcohol and other drug abuse, school failure, and juvenile delinquency.
Fight-Free Schools violence prevention process.
This is an evidence-based prevention program for schools, families, and communities.
This is a gender violence, bullying, and school violence prevention approach that encourages young men and women from all socioeconomic, racial and ethnic backgrounds to take on leadership roles in their schools and communities.
A school curriculum designed to improve student health, knowledge, skills, attitudes and behaviors.
This is a nationally recognized, evidence-based program that improves academics, behavior, and character.
This program is an empirically validated, function-based approach to eliminate challenging behaviors and replace them with prosocial skills.
PRAISE is a 20-session classroom-based universal prevention program, designed to be appropriate and responsive to the needs of youth within the urban school context.
Morningside Center's 4Rs Program integrates conflict resolution into the language arts curriculum for pre-K through middle school.
This is an evidence-based classroom program that has shown dramatic effect in reducing levels of aggression among school children by raising social/emotional competence and increasing empathy.
The Second Step violence prevention program is a prevention program designed for use with children in schools. The impulse control and problem solving component of the Second Step program includes the identification of one's own feelings, understanding what the problem is, identifying solutions, friendship making, and positive communication strategies, which purports to have a positive effect on children's behavior.
This program is a multimedia, computer-based violence-prevention intervention that uses games, simulations, graphics, cartoons, and interactive interviews to engage young adolescents in learning new skills to resolve conflicts without violence.
This is an interactive program for young people that uses improvisational theater workshops to teach violence prevention, conflict resolution, and decision-making.
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Tagged: From Social Network to Mobile Discovery
Tagged may just be the biggest social network you've never heard of. Currently the second-highest-grossing social app on Android, Tagged is built by a company called if(we) (which recently rebranded from simply Tagged, Inc.) that's completely dedicated to social discovery apps, and was one of the first social networks to become profitable. Its previous experiments have included Sidewalk, a cool city-based app for serendipitous discovery in cities. if(we) also owns Hi5, a former network for social gaming that now also focuses on social discovery.
As Facebook dominates the social landscape in terms of sheer number of members, if(we) is focusing on social apps that fulfill other human needs – like meeting new people online, finding dates, playing games, and other niches Facebook doesn't quite cover. By building features for specific tasks that match specific human needs, if(we) aims to help everyone cultivate more meaningful relationships – no small feat in an increasingly digital world.
We recently spoke with Steve Sarner, Vice President of Marketing at if(we), to learn a bit more about the company's journey from a single social app to an entire enterprise dedicated to promoting social discovery and other social products. Here's what we found out.
How Tagged Came to Be
Tagged was one of the original social networks, designed to let people connect with others online. Formed in October 2004, right around the same time Facebook started to climb the social networking ladder, Tagged looked at conquering the high school market: "While Facebook was good for college, we'd get them even younger," Tagged co-founder at CEO Greg Tseng told Jason Calacanis on This Week in Startups a few years ago. But an internal study quickly revealed that about 75 percent of connections on Tagged had actually been created through Tagged, not in real life (unlike Facebook connections, typically driven by real-life relationships). Armed with this insight, the company decided to start owning social "discovery" – basically, meeting new people.
Rather than simply curating online lists of people you know offline, if(we) focuses on building social networks that actually connect you with new people. Tagged allows users to find and message one another, and includes original features like PETS, a "stock market for people," where users can buy and sell others' profiles, betting on whose headlines and images will become most popular. if(we) also offers dedicated dating app, MeetMe, and social gaming services.
In fact, in 2010, Tagged was the third largest partner (behind Facebook and MySpace) for Zynga social games. As Zynga increasingly focused its efforts on the Facebook platform, Tagged transferred focus to developing its own games internally. The company quickly found that focusing on a few high quality games was preferable to trying to churn out a ton of titles, and has honed in on the aforementioned PETS game where users buy and sell profiles. Whether it's despite or because of its highly personal nature, PETS delivers remarkably high average revenue per user.
Serving a Diverse User Base
Tagged members are diverse in many ways. There's not a lot of overlap in how they use the social networks: about 20 percent of people are focused on social gaming, 20 percent use MeetMe for dating, and 60 percent browse and use the tool to meet new friends as well as dates. Less than 10 percent of users complete activities in all of these areas. As a result, if(we) needs to think about appealing to all of these different groups with unique apps and features.
In addition to gravitating to different features, if(we) users come from various countries and backgrounds. About 25 percent are from the US, with others all over the world – the UK, Netherlands, Malaysia, Philippines, Mexico, Brazil, and more, with a growing contingent in the Middle East. There are over 200 countries and 20 languages represented throughout the company's services. About 60 percent of users are men and 40 percent are women.
These diverse groups of people have discovered if(we) products in many different ways. The company was one of the first to let users invite their existing networks to the platform, and continues to use viral hooks in the registration process to promote growth. Although the company has done some paid promotion, its growth is largely organic.
In 2011, if(we) acquired Hi5, a social network with a large concentration of users from South America and southeast Asia, in particular Malaysia. It was a challenge to begin to understand how users in Asia interacted with the platform compared with users in the US, and build new features accordingly. By now, if(we) has largely transitioned Hi5 from social games into a social discovery platform, a shift that has resonated with users and driven them to interact more with the service.
Mirroring its experience with Asian users of Hi5, if(we) sees significant regional differences in the ways its apps are used, with some countries going gaga for games and others focusing more on dating. Still other areas simply use if(we) services to find friends online, in tune with the social discovery track. Right now, much of the company's revenue comes from from the US, but the company is looking into opportunities to grow elsewhere financially despite its rather impressive $50 million annual take.
Transitioning from Web to Mobile
As a more established social network, Tagged was originally built for the web. That has made for an educational transition into the world of mobile apps, especially given the company's diverse customer base, which uses many different mobile platforms and devices. Initially, there were attempts to "force" an iOS app into the Android platform, but it quickly became clear that this didn't provide an ideal environment for users, and a native Android app was created.
"Whereas mobile was the biggest threat to our IPO goal, it is also the biggest opportunity for growing our social products," if(we) CEO Greg Tseng noted recently when discussing the company's shift in mission and platform to focus more strictly on social, and the tension between mobile's risks and opportunities is familiar to many companies.
Aside from deepening its social experimentation, another current priority for the company is to knit mobile monetization more tightly into its social apps. As consumers increasingly transition to mobile payments, making these work seamlessly will become more and more vital not only to providing a good experience for those using the app, but also to building revenue for the app creator. By making it as easy as possible for people to stay within the app and pay for additional features, if(we) builds its user base and revenue simultaneously.
What's Next for All of Us
Although Google Glass has yet to take off, if(we) sees potential for wearables to influence the social media experience. Because wearables will be very personal devices, it's only natural that they will also house very personal connections – though these may need to be of a different nature than the large friend or follower lists that many people curate on networks like Facebook, Instagram or Twitter. It's possible that certain social networks will begin to dominate different wearable devices – or that a device will drive the growth of a new network. if(we) says there's still tremendous opportunity on mobile, too, especially in terms of messaging and video, areas where wearables may influence the trajectory of trends as well.
Whatever comes next, the people at if(we) will be hard at work making sure that there's a strong social discovery layer to it. Sarner says that working at if(we) means your code will be used by millions of people to connect with others. And while if(we) is a large, established company, it also offers employees "the freedom to act like a startup and try new things" – but with the "stability and scale that a startup wouldn't have," according to Sarner.
All told, just like its members, if(we) is always looking for the next new thing. So pay attention to what they come up with next as the company moves into a new era of experimentation, one that matches its people-centric and exploration-minded name.


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Nice work Mike. I like the one with the house. The water is so clear you can see the bottom. Where were these taken?
Nikon D600, Nikon D80, AF-S Nikkor VR 18-55mm F/3.5-5.6G DX, AF-S Nikkor VR 55-200mm F/4-5.6G ED DX, AF Nikkor 50mm 1.8D,
AF-S Nikkor 18-105mm f/3.55.6 G ED VR DX, AF-S Nikkor 70-300mm f/4.5-5.6 G ED VR, AF-S Nikkor 24-85mm f/3.5-4.5 G ED VR, AF Micro Nikkor 105mm f/2.8 D, Sigma 150-500mm f/5-6.3 APO DG OS, Sigma 10-20mm f/4-5.6 EX DC HSM, Quantaray AF LD 70-300mm F/4-5.6 Tele-Macro(1:2) , Nikon SB 800, SB 600, Expodisc
Hey guys,
Thanks so much for your positive comments. It's really encouraging. Especially after looking at your albums.
Anyway, as for where the photos were taken.
1st: This was taken near San Pedro de Atacama, Chile. It's a tour you can to do some high altitude lagoons (about 4200 meters). These lagoons are perfect for taking pictures as it's hard to take a bad shot at them.
2nd: This is called Lago Huechulaufquen (sp?) in Argentina. Again, this was taken on a tour to a volcano called Volcan Lanin, it's the snow caped main peak to the right. Beautiful place and perfect time of year.
3rd: This is at a place called Los Saltos De Petrohue, north of Puerto Montt in Chile. A beautiful cascades in front of Volcan Osorno in Southern Chile.
4th: This is from a peak called Poon Hill in Nepal. If you ever get a chance, go there. This was part of a larger trek, but the view from here was incredible of the Himalayas. From this hill you can see some beautiful peaks like Dhaulagiri (10th highest), Annapurna, and Macchapucchre (fish tail peak).
Well, again, thanks guys for all your positive comments. I look forward to posting some more of what I've done here eventually. I also look forward to reading these forums more and learning more stills and abilities from all of you.
This image was created at sunrise today at Garden of the Gods.
Tricky shot digital. Nicely done.
Do you have a polariser? I'm curious if that would have darkened the sky and proportanately lightened the forground.
D800, D300, D90, 24-70 f2.8, 70-200VR f2.8, 300 F4, 105 micro, 16-85VR, 50mm 1.8, Tammy 90 macro, 70-300VR, SB900, 2xSB600, MB-D10, 055XPROB 322RC2. New computers to run photoshop faster. C&C always appreciated. PhotoGallery
Pressing the shutter is the start of the process - Joe McNally ... Buying the body is the start of the process - Dread Pirate
Posted this in the Nikon section few weeks ago, decided to post it here aswell . Haven't checked out this thread for a while, nice pictures guys keep them coming.
We don't usually get this much snow here, it all came down in 2 days but melted away during the next week. At the moment we got no snow here, looks like we wont get any for christmas either.
I live in Finland btw forgot to mention.
D40+Sigma 17-70-2.8-4.5 DC MACRO
No filters on my Kodak P&S. I lightened the image by increasing midtones about 10% and this made the foreground more visible but also lightened the sky. I am starting to play with Gimp to see if it can do selective editing with layers.
mellinger, that last one is truly stunning. That sort of magical light falling on a mountain is the sort of thing I would love to see, especially with a camera. I have seen lots of magical light on mountains, but nothing quite like that. I'm going back to China soon, maybe I can arrange a 2000 mile or so detour.
Aldor88, dig-it-all, great shots. I'll have to raise my standards.
Lake Tahoe
Here are few shots from Natural Bridges SB. I've been meaning to go out there again. I finally got around to it right before storm rolled in.
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Camera: Anonymous
I could tell you but I wouldn't want you to get all pissy if it's the wrong brand
Hey all.....
Hadn't picked up the camera for over three weeks as I had some surgery that really sent me for a loop. Everything that could go wrong, did. I'm on the mend now and picked up the camera yesterday. It felt good to look at the world through the viewfinder once again. Looking out over Ma Natures gifts, and breathing in a lung full of fresh chilled air...is all the therapy I need!
I hope you enjoy what I brought to share. I sure know I enjoyed capturing it
Here's a few shots from this evening taken right after sunset at Seacliff SB.
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